Information

Why severely increased Ligand (eg Hormones) concentration downregulates the Receptor?

Why severely increased Ligand (eg Hormones) concentration downregulates the Receptor?



We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

As an example continuous high blood level of GnRH in humans causes a suppression of LH and FSH. This is due to the fact that increased GnRH downregulates GnRH-Receptors . My question is how this is done and why? What are the probable molecular mechanisms underlying this effect?

Kumar P, Sharma A. Gonadotropin-releasing hormone analogs: Understanding advantages and limitations. J Hum Reprod Sci. 2014;7(3):170-174. doi:10.4103/0974-1208.142476 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229791/


Gonadotropins are hormones synthesized and released by the anterior pituitary gland, which act on the testes and ovaries to increase the production of sex hormones and stimulate production of either sperm or ova.

The hypothalamus contains Gonadotropin-releasing hormone (GnRH). Intermittent released of GnRH is responsible for the biosynthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary gland.

LH stimulates interstitial cells to release testosterone, which triggers spermatogenesis. The rising level of testosterone exerts negative feedback control on the hypothalamus and pituitary glands.

GnRH has been shown here in a negative feedback loop

This feedback loop has great interplay with the number receptor levels as you suggest.

(From a quick search) I found one paper (behind a paywall) that states the following information when studying rats:

We observed an acute reduction of both GnRH and GnRH-R mRNAs 24 h after the injection [of GnRH agonist (triptorelin)] (about 38% of control).

Transcription does not equal translation, but it seems that acute increased levels of GnRH cause a reduction and mRNA of GnRH and GnRH-R and therefore potential reduction (down-regulation) of protein levels of GnRH-R.


Drug–Receptor Interactions

Receptors are macromolecules involved in chemical signaling between and within cells they may be located on the cell surface membrane or within the cytoplasm (see table Some Types of Physiologic and Drug-Receptor Proteins). Activated receptors directly or indirectly regulate cellular biochemical processes (eg, ion conductance, protein phosphorylation, DNA transcription, enzymatic activity).

Molecules (eg, drugs, hormones, neurotransmitters) that bind to a receptor are called ligands. The binding can be specific and reversible. A ligand may activate or inactivate a receptor activation may increase or decrease a particular cell function. Each ligand may interact with multiple receptor subtypes. Few if any drugs are absolutely specific for one receptor or subtype, but most have relative selectivity. Selectivity is the degree to which a drug acts on a given site relative to other sites selectivity relates largely to physicochemical binding of the drug to cellular receptors.

Some Types of Physiologic and Drug-Receptor Proteins

Multisubunit ion channels

Cell surface transmembrane

Cell surface transmembrane

alpha- and beta-adrenergic receptor proteins

Cell surface transmembrane

GABA = gamma-aminobutyric acid GDP = guanosine diphosphate GTP = guanosine triphosphate.

A drug’s ability to affect a given receptor is related to the drug’s affinity (probability of the drug occupying a receptor at any given instant) and intrinsic efficacy (intrinsic activity—degree to which a ligand activates receptors and leads to cellular response). A drug’s affinity and activity are determined by its chemical structure.

The pharmacologic effect is also determined by the duration of time that the drug-receptor complex persists (residence time). The lifetime of the drug-receptor complex is affected by dynamic processes (conformation changes) that control the rate of drug association and dissociation from the target. A longer residence time explains a prolonged pharmacologic effect. Drugs with long residence times include finasteride and darunavir . A longer residence time can be a potential disadvantage when it prolongs a drug's toxicity. For some receptors, transient drug occupancy produces the desired pharmacologic effect, whereas prolonged occupancy causes toxicity.

Physiologic functions (eg, contraction, secretion) are usually regulated by multiple receptor-mediated mechanisms, and several steps (eg, receptor-coupling, multiple intracellular 2nd messenger substances) may be interposed between the initial molecular drug–receptor interaction and ultimate tissue or organ response. Thus, several dissimilar drug molecules can often be used to produce the same desired response.

Ability to bind to a receptor is influenced by external factors as well as by intracellular regulatory mechanisms. Baseline receptor density and the efficiency of stimulus-response mechanisms vary from tissue to tissue. Drugs, aging, genetic mutations, and disorders can increase (upregulate) or decrease (downregulate) the number and binding affinity of receptors. For example, clonidine downregulates alpha 2 receptors thus, rapid withdrawal of clonidine can cause hypertensive crisis. Chronic therapy with beta-blockers upregulates beta-receptor density thus, severe hypertension or tachycardia can result from abrupt withdrawal. Receptor upregulation and downregulation affect adaptation to drugs (eg, desensitization, tachyphylaxis, tolerance, acquired resistance, postwithdrawal supersensitivity).

Ligands bind to precise molecular regions, called recognition sites, on receptor macromolecules. The binding site for a drug may be the same as or different from that of an endogenous agonist (hormone or neurotransmitter). Agonists that bind to an adjacent site or a different site on a receptor are sometimes called allosteric agonists. Nonspecific drug binding also occurs—ie, at molecular sites not designated as receptors (eg, plasma proteins). Drug binding to such nonspecific sites, such as binding to serum proteins, prohibits the drug from binding to the receptor and thus inactivates the drug. Unbound drug is available to bind to receptors and thus have an effect.


Abstract

Males and females differ in their immunological responses to foreign and self-antigens and show distinctions in innate and adaptive immune responses. Certain immunological sex differences are present throughout life, whereas others are only apparent after puberty and before reproductive senescence, suggesting that both genes and hormones are involved. Furthermore, early environmental exposures influence the microbiome and have sex-dependent effects on immune function. Importantly, these sex-based immunological differences contribute to variations in the incidence of autoimmune diseases and malignancies, susceptibility to infectious diseases and responses to vaccines in males and females. Here, we discuss these differences and emphasize that sex is a biological variable that should be considered in immunological studies.

Sex is a biological variable that affects immune responses to both self and foreign antigens (for example, those from fungi, viruses, bacteria, parasites and allergens). The sex of an individual is defined by the differential organization of chromosomes, reproductive organs, and sex steroid levels it is distinct from gender, which includes behaviours and activities that are determined by society or culture in humans. Male and female differences in immunological responses may be influenced by both sex and gender, with sex contributing to physiological and anatomical differences that influence exposure, recognition, clearance, and even transmission of microorganisms. By contrast, gender may reflect behaviours that influence exposure to microorganisms, access to healthcare or health-seeking behaviours that affect the course of infection. Although we acknowledge that both sex and gender influence the immune response, the focus of this Review will be on the biological factors that influence immunological differences between the sexes. Despite a growing body of literature illustrating sex-based differences in immune responses, immunology ranks the lowest of ten biological disciplines for reporting the sex of animal or human subjects in published papers, with fewer than 10% of articles analysing data by sex 1 . The field of sex-based biology is undergoing a revolution, in which research funding agencies and journals have launched policies to promote greater consideration, reporting and analyses of sex and gender in the biomedical sciences in an effort to improve rigour and reproducibility (Box 1).

It is increasingly important to acknowledge sex differences in immune responses when we consider the marked differences seen between males in females in various diseases. For instance, 80% of autoimmune disease occurs in females, women with acute HIV infection have 40% less viral RNA in their blood than men, men show an almost twofold higher risk of death from malignant cancer than women and antibody responses to seasonal influenza vaccines are consistently at least twice as strong in women than men. Generally, adult females mount stronger innate and adaptive immune responses than males. This results in faster clearance of pathogens and greater vaccine efficacy in females than in males but also contributes to their increased susceptibility to inflammatory and autoimmune diseases. In this Review, we explain how these immunological differences between the sexes reflect hormonal, genetic and environmental effects on the immune system that can change throughout life in humans.

Box 1: A brief history of sex and gender-based research in the US

The history of excluding females from clinical studies is reflected in the 1977 US Food and Drug Administration (FDA) guidelines advising that women of childbearing potential should be excluded from drug trials. These recommendations resulted in inadequate representation of women in clinical trials for decades. In the early 1990s, the FDA and the National Institutes of Health (NIH) in the US, with advocacy from US Congresswomen, recommended that clinical trials should include female subjects. Although women are now included in clinical trials of drugs, devices and biologics, there remains inadequate analysis of whether outcomes differ between men and women or boys and girls. Of drugs withdrawn from the US market from 1997–2000, the US Government Accountability Office (GAO) reported that 8 out of 10 drugs taken off the market had greater adverse effects in women. In 2015, the US GAO documented that although more women than men currently enrol in NIH-funded clinical research, the NIH does not ensure that these studies are designed to identify differences between men and women in disease processes and responses to treatment. Preclinical studies in animal models and cell culture systems could help to prevent these costly mistakes but, here too, analysis of potential sex effects has been lacking. Following behind policy changes in Canada and Europe, in 2015 the NIH announced new policies to ensure that sex is considered as a biological variable in preclinical research in an effort to increase rigour and reproducibility.


Chemistry and Structural Biology of Androgen Receptor

Androgen receptor (AR) is a member of the steroid and nuclear receptor superfamily, 1 which is composed of over 100 members and continues to grow. Among this large family of proteins, only five vertebrate steroid receptors𠅎strogen, progesterone, androgen, glucocorticoid, and mineralocorticoid receptors𠅊re known. Two subtypes of estrogen receptor have been identified, estrogen receptor α and estrogen receptor β. 2 Like other steroid receptors, AR is a soluble protein that functions as an intracellular transcriptional factor. AR function is regulated by the binding of androgens, which initiates sequential conformational changes of the receptor that affect receptor–protein interactions and receptor𠄽NA interactions.

AR-regulated gene expression is responsible for male sexual differentiation and male pubertal changes. AR ligands are widely used in a variety of clinical applications (i.e., agonists are employed for hypogonadism, while antagonists are used for prostate cancer therapy). Fang et al. 3 recently summarized a large number of chemicals that bind to the AR. The current review focuses on well-characterized AR ligands that bind to the AR with high affinity and integrates discussion regarding the biology, metabolism, and structure-activity relationships for therapeutic and emerging classes of AR ligands. The known AR ligands can be classified as steroidal or nonsteroidal based on their structure or as agonist or antagonist based on their ability to activate or inhibit transcription of AR target genes. Synthetic AR ligands were first developed by modifying the steroidal structure of endogenous androgens. The structure�tivity relationship of these steroidal AR ligands is well documented 4 - 6 and will only be briefly summarized in this review. However, low oral bio-availability, poor pharmacokinetic properties, and side effects have limited the use of many steroidal AR ligands. Until recently, it was considered impossible to separate the androgenic and anabolic effects of AR ligands due to their reliance on a single AR. However, newly discovered nonsteroidal AR ligands may provide a new strategy to achieve tissue selectivity, as is possible with estrogen receptor ligands. Novel nonsteroidal pharmacophores are summarized in this review with discussion of the emerging structure�tivity relationships and examples of their tissue selectivity included. 7

1.1. Physiologic Roles and Clinical Application of Androgens

AR is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver, and central nervous system (CNS), with the highest expression level observed in the prostate, adrenal gland, and epididymis as determined by real-time polymerase chain reaction (PCR). 8 AR can be activated by the binding of endogenous androgens, including testosterone and 5α-dihydrotestosterone (5α-DHT). Physiologically, functional AR is responsible for male sexual differentiation in utero and for male pubertal changes. In adult males, androgen is mainly responsible for maintaining libido, spermato-genesis, muscle mass and strength, bone mineral density, and erythropoisis. 7 , 9 The actions of androgen in the reproductive tissues, including prostate, seminal vesicle, testis, and accessory structures, are known as the androgenic effects, while the nitrogen-retaining effects of androgen in muscle and bone are known as the anabolic effects.

Numerous and varied site mutations in AR have been identified (The Androgen Receptor Gene Mutations Database World Wide Web Server, http://www.androgendb.mcgill.ca/). The majority of these mutations are associated with diseases, like Androgen Insensitivity Syndrome and prostate cancer. The androgen withdrawal syndrome observed in prostate cancer therapy also appeared to be related to certain AR mutations, such as T877A and W741C mutations, which convert some AR antagonists into agonists (see more discussion in section 3.1.1). Besides the site mutations documented, AR gene polymorphism has also been identified, particularly, the poly-Q (CAG)n at exon I. The polymorphic (CAG)10� triplet repeat sequence, starting from codon 58, codes for polyglutamine. The length of the repeat is inversely correlated with the transactivation activity of AR. The correlation between the length of the CAG repeat and disease stage was recently reviewed by Oettel. 10

Classically, testosterone is used to treat male hypogonadism, Klinefelter's syndrome, anemia secondary to chronic renal failure, aplastic anemia, protein wasting diseases associated with cancer, burns, traumas,acquiredimmunodeficiencysyndrome(AIDS), etc., short stature, breast cancer (as an anti-estrogen), and hereditary angioedema. 7 Recently, hormone replacement therapy in aging males has also been proposed to improve body composition, bone and cartilage metabolism, and certain domains of brain function and even decrease cardiovascular risk. 10

1.2. Gene and Protein Structure and Function

1.2.1. Androgen Receptor Gene and Protein Structure

In 1981, Migeon et al. 11 first localized the AR gene to the human X chromosome. In 1998, Lubahn et al. 12 cloned human AR genomic DNA from a human X chromosome library using a consensus nucleotide sequence from the DNA-binding domain of the nuclear receptor family. In the same year, several groups, including Chang et al., 13 Lubahn et al., 14 and Trapman et al., 15 cloned human AR cDNAs. To date, only one AR gene has been identified in humans.

The AR gene is more than 90 kb long and codes for a protein of 919 amino acids that has three major functional domains, as illustrated in Figure 1 . The N-terminal domain (NTD), which serves a modulatory function, is encoded by exon 1 (1586 bp). The DNA-binding domain (DBD) is encoded by exons 2 and 3 (152 and 117 bp, respectively). 16 The ligand-binding domain (LBD) is encoded by five exons, which vary from 131 to 288 bp in size. There is also a small hinge region between the DNA-binding domain and ligand-binding domain. Two transactivation functions have been identified. The N-terminal activation function 1 (AF1) is constitutively active in truncated receptor that does not contain the ligand-binding domain and is not conserved in sequence compared to other steroid receptors ( Figure 2 ), whereas the C-terminal activation function 2 (AF2) functions in a ligand-dependent manner and is relatively more conserved in sequence as compared to other steroid hormone receptors, particularly with regard to the charge-clamp residues. 17 A nuclear localization signal (NLS) spans the region between the DNA-binding domain and the hinge region.

Structural organization of the AR gene and protein.

Amino acid sequence identity among members of the steroid receptor family (adapted from ref 19 ). Sequence alignment was performed using William Pearson's LALIGN program. 20

The human AR amino acid sequence is very similar to the rat AR amino acid sequence with identical sequences in the DNA- and ligand-binding domains and an overall sequence identity of 85% 14 ( Figure 2 ). All steroid receptors share a similar organization with an individual N-terminal domain, conserved DNA-binding domain, and C-terminal ligand-binding domain. The N-terminal domain of different steroid receptors shows the least conservation of sequence (less than 25% identity), while the central DNA-binding domain is well conserved for all steroid receptors (59�%), reflecting the common need to bind to DNA, while the variation is responsible for the selection of different target sequences. The ligand-binding domain of different steroid receptors shows sequence identity ranging from 22% to 55%, reflecting receptor specificity for individual hormones. Among all the steroid receptors, the human AR ligand-binding domain shares more sequence identity with the human progesterone receptor, glucocorticoid receptor, and mineralocorticoid receptor ligand-binding domains (all around 50%) 18 with an overall sequence homology of 88% to the progesterone receptor ligand-binding domain when conservative mutations are included. Even though the ligand-binding domains of steroid receptors share relatively low sequence identity, they all assume a similar three-dimensional structure with certain highly conserved structural features, including a 𠇌harge clamp” and helical features (see subsequent sections for more detailed discussion). These similarities in conformation provide the structural basis for the cross reactivity that is commonly observed with synthetic steroids.

1.2.2. Androgen Receptor Protein Conformation and Function

Similar to the other steroid receptors, unbound AR is mainly located in the cytoplasm and associated with a complex of heat shock proteins (HSPs) through interactions with the ligand-binding domain. 21 Upon agonist binding, 1 AR goes through a series of conformational changes: the heat shock proteins dissociate from AR, and the transformed AR undergoes dimerization, phosphorylation, and translocation to the nucleus, which is mediated by the nuclear localization signal. Translocated receptor then binds to the androgen response element (ARE), which is characterized by the six-nucleotide half-site consensus sequence 5′-TGTTCT-3′ spaced by three random nucleotides and is located in the promoter or enhancer region of AR gene targets. Recruitment of other transcription co-regulators (including co-activators and co-repressors) 22 and transcriptional machinery 23 further ensures the transactivation of AR-regulated gene expression. All of these complicated processes are initiated by the ligand-induced conformational changes in the ligand-binding domain.

Currently, ligand-binding domain and DNA-binding domain crystal structures of many nuclear receptors are solved, but no crystal structure of a full-length receptor is available yet. The first crystal structure of the AR ligand-binding domain was solved by Matias et al. in 2000. 24 The AR ligand-binding domain shares similar three-dimensional structure with other agonist-bound steroid receptors (e.g., estrogen receptor) (Figures ​ (Figures3 3 and ​ and4A). 4A ). The protein contains 11 α-helices (H) and two short β-turns, which are arranged in three layers to form an antiparallel “α-helical sandwich”. Different from other steroid receptors, H2 is not present in the AR ligand-binding domain. However, it is important to note that the same numbering of the other helices was retained for easy comparison. Helices H1 and H3 form one face of the ligand-binding domain, while helices H4 and H5, the first β-turn, and helices H8 and H9 form the central layer of the structure, and helices H6, H7, H10, and H11 constitute the second face. H5, the N-terminal region of H3 and the C-terminal region of H10 and H11 form the main part of the hydrophobic ligand-binding pocket (LBP). Upon agonist binding, H12 is repositioned and serves as the “lid” of the ligand-binding pocket to stabilize the ligand, and the very end of the C-terminal region of the ligand-binding domain forms the second β-turn (next to H8 and H10), which works as a “lock” to further stabilize the “lid” (H12) conformation. The agonist-induced conformational change in the ligand-binding domain allows the formation of a functional activation function 2 (AF2) region on the surface of ligand-binding domain ( Figure 3, highlighted in green ), which is crucial for both the amino/carboxyl-terminal (N/C) interaction of AR and co-regulator recruitment during transcriptional activation ( Figure 5A,B ). Unlike other steroid receptors, agonist-bound AR prefers N-terminal and C-terminal interaction, 25 which could further stabilize the agonist-bound ligand-binding domain.

Crystal structures of wild-type AR ligand-binding domain bound with DHT (1I37.pdb): (A) front view (B) ligand view. Space filled atoms are (black) carbon and (red) oxygen. The activation function 2 region (helices 3, 4, and 12) is highlighted in green.

AF2 antagonist model, as illustrated by crystal structures of wild-type estrogen receptor α ligand-binding domain bound with (A) estradiol (1ERE.pdb) and (B) raloxifene (1ERR.pdb). Estradiol is shown in yellow raloxifene is shown in green. Helix 12 is highlighted in red. Helix 12 folds over the activation function 2 (AF2) region when antagonist (raloxifene) binds to the estrogen receptor α ligand-binding domain.

Interactions of FxxLF or LxxLL motifs with the AR or estrogen receptor ligand-binding domain: upper panels, hydrogen-bonding interactions (shown as yellow dotted line) between peptide and activation function 2 region residues lower panels, surface view of the activation function 2 interface, in which side chains of the hydrophobic residues of FxxLF and LxxLL motifs are shown as spheres (A) FxxLF bound to AR activation function 2 interface (1T7R.pdb) (B) LxxLL bound to AR activation function 2 interface (1T7F.pdb) (C) LxxLL bound to estrogen receptor α activation function 2 interface (1GWQ.pdb).

The activation function 2 region is a surface hydrophobic groove ( Figure 5A,B ) formed by the C-terminal region of H3, loop 3-4, H4, and H12, a region that covers the highly conserved nuclear receptor ligand-binding domain signature motif 26 and the activation function 2 core, which is similar to the structure that is also observed in agonist-bound estrogen receptor 27 ( Figure 5C ). A functional activation function 2 region is believed to be crucial for co-activator recruitment, because the so-called nuclear receptor box “LxxLL” motif 28 from the nuclear-receptor-interacting domain (NID) of co-activators specifically binds to this surface. 29 On the other hand, similar motifs from the AR N-terminal domain, 23 FxxLF 27 and 433 WxxLF 437 ( Figure 1 ), can also interact with the activation function 2 region. 25 Therefore, both interactions could compete for the activation function 2 region upon agonist binding. The detailed topology change induced by different ligands within this region might provide the structural basis for recognition of specific binding motifs. 30 , 31

The steroidal androgen-bound AR ligand-binding domain was cocrystallized with short peptides that contain the LxxLL motif or the FxxLF motif ( Figure 4A,B ). Hydrophobic interactions between the leucine residues in the LxxLL motif or phenylalanine residues in the FxxLF motif and the hydrophobic groove hold the peptide in place, while the hydrogen bonds between peptide backbone atoms and two well conserved residues, a lysine (K720) at the C-terminus of H3 and a glutamate (E897) in H12, form a charge clamp ( Figure 4A,B ) to further stabilize the interaction. 30 , 32 Very similar interactions were also observed in the LxxLL motif bound estrogen receptor α ligand-binding domain activation function 2 region ( Figure 5C ). 33

Despite the overall similarity in peptide binding modes, DHT-bound AR ligand-binding domain prefers the binding of the FxxLF motif to that of the LxxLL motif, suggesting that N/C interaction is preferred over co-activator recruitment in DHT-bound AR. In contrast, agonist-bound estrogen receptor α ligand-binding domain prefers the binding of the LxxLL motif to that of the FxxLF motif. 32 As shown in Figure 5 , DHT-bound AR formed a deeper hydrophobic groove in activation function 2 region (region located between the charge clamp residues, as illustrated in Figure 5A,B lower panels ), which could accommodate the bulky side chain of phenylalanine residues. However, the hydrophobic groove in agonist-bound estrogen receptor α ligand-binding domain is more shallow and accommodates the side chain of leucine residues better. On the other hand, when the LxxLL motif binds to the AR ligand-binding domain ( Figure 5B ), 30 the peptide backbone only forms a hydrogen bond with K720 a shift in peptide position prevents the direct hydrogen bonding with E897, which could explain the relatively lower affinity of AR for this LxxLL motif.

The distinct preferences of AR for N/C interaction could become targets for new drug discovery. 34 Studies 35 , 36 have shown that ligand binding induced AR N/C interaction correlates with its ability to activate transcription, where disruption of the N/C interaction might become an effective strategy to develop antagonists. In comparison, although estrogen receptor α does not share a similar N/C interaction as AR, estrogen receptor α antagonists have been developed to disrupt co-activator recruitment by the activation function 2 (AF2) region by blocking LxxLL motif binding. The � antagonist model” is shown in Figure 4 . When agonist (estradiol) binds estrogen receptor α ( Figure 4A ), H12 (shown in red) adopts a conformation that helps form a functional activation function 2. However, when antagonist (tamoxifene) binds estrogen receptor α ( Figure 4B ), H12 is displaced from agonist conformation and folds over the AF2 region. The 540 LLEML 544 motif in H12 binds to the AF2 region in a similar way as the LxxLL motif and blocks the binding of co-activators.

In AR, the 895 MAEII 899 motif in H12 may also mimic the LxxLL motif of co-activators. In fact, one train of thought is that the AF2-antagonist model might apply to AR as well, and H12 is repositioned to bind to the activation function 2 (AF2) region upon antagonist binding with the MAEII motif blocking the interaction with other binding motifs. 29 However, this hypothesis has not been proved by crystallography studies. Besides the AF2 antagonist model, other mechanisms have been proposed to explain AR antagonist activity. Although the agonist-induced conformational changes in the ligand-binding domain result in the dissociation of the chaperone protein complex from AR, 37 the dissociation does not seem to happen upon antagonist binding in AR, 38 which might also account for the antagonist activity by simply blocking access to the activation function 2 region. On the other hand, some evidence 39 suggested that AR antagonist bicalutamide could stimulate AR nuclear translocation and specific DNA binding, but it could not mediate co-activator recruitment, instead, bicalutamide binding mediated the recruitment of co-repressor, NCoR. 40 Also, similar ligand-specific (agonist vs antagonist) recruitment of coregulators has been characterized with selective estrogen receptor modulators, which appears to be related to the tissue selectivity of selective estrogen receptor modulators. 41 - 43 Therefore, differential recruitment of co-regulators is also considered as a possible mechanism of action for AR antagonist. Despite all the theories proposed above, the precise mechanism of action for AR antagonists remains unclear.

Ligand-induced AR conformational changes provide the structural basis for the recruitment of cofactor proteins and transcriptional machinery, which is also required for the assembly of AR-mediated transcription complexes. 23 Shang et al. 23 showed that the formation of an activation complex involves AR, co-activators, and RNA polymerase II recruitment to both the enhancer and promoter regions of the prostate-specific antigen (PSA) gene, whereas the formation of a repression complex involves factors bound only at the promoter but not the enhancer. Since the formation of a functional activation function 2 region provides a structural basis for ligand-induced protein-protein interaction, ligand-specific recruitment of co-regulators might be crucial for the agonist or antagonist activity of AR ligands. 40 On the other hand, as has been demonstrated with bicalutamide, possible ligand-specific interactions could be directly regulated by the surface topology of the activation function 2 region as shown by Sathya et al. 31 Due to the subtle change in surface topology, formation of a functional activation function 2 does not guarantee effective N/C interaction. In other words, lack of N/C interaction does not correlate with the loss of activation function 2 functionality.

DNA binding is also required for AR-regulated gene expression, which is known as the classic genomic function of AR and has been characterized by DNA microarray studies. 44 - 46 The androgen response element half-site sequence can be arranged as either inverted repeats or direct repeats, 47 , 48 and AR recognizes and binds to the ARE site through two zinc fingers located in the DNA-binding domain. Like other steroid receptors, ligand-bound AR forms homodimers and appears to form “head-to-head’ dimers 49 even when it is bound to the direct repeats of androgen response element. Selective recognition of specific androgen response element sequences could be regulated by ligand binding 50 or the presence of other transcriptional factors, which bind to their own DNA binding sites as well (combinatorial regulation), or both. 51

1.2.3. Nongenomic Pathway

Besides the genomic pathway, the nongenomic pathway of AR has also been reported in oocytes, 52 skeletal muscle cells, 53 osteoblasts, 54 , 55 and prostate cancer cells. 56 , 57 As compared to the genomic pathway, the nongenomic actions of steroid receptors are characterized by the rapidity of action, which varies from seconds to an hour or so, and interaction with plasma membrane-associated signaling pathways. 58 Nevertheless, the structural basis for nongenomic action is direct interactions between AR and cytosolic proteins from different signaling pathways, 59 which could be closely related to the ligand-induced conformational change of the ligand-binding domain or, indirectly, the N-terminal domain. However, the detailed structural basis for these interactions is unclear. Functionally, the nongenomic action of androgen involves either rapid activation of kinase-signaling cascades or modulation of intracellular calcium levels, which could be related to stimulation of gap junction communication, neuronal plasticity, and aortic relaxation. 60 Separation of the genomic and nongenomic functions of steroid receptors using specific ligands was also proposed as a new strategy to achieve tissue selectivity. 58 , 61 However, structural features that are essential for achieving the separation have not been determined.

1.3. Androgen Biochemistry, Endogenous Agonists

1.3.1. Testosterone Synthesis

Endogenous AR ligands include testosterone and its active metabolite, 5α-DHT. Testosterone is primarily synthesized from cholesterol ( Figure 6 ) in Leydig cells in the testes. It is also synthesized in adrenal cortex, liver, and ovary in women. The rate-limiting step in testosterone synthesis, cholesterol side chain cleavage by P450scc, is regulated by luteinizing hormone (LH) from the pituitary, which is controlled by gonadotropin releasing hormone (GnRH) from the hypothalamus and the feedback regulation of testosterone at both the pituitary and hypothalamus levels. Although dehydroepiandrosterone (DHEA) also has weak agonist activity, test-osterone and 5α-DHT are the major endogenous androgens. Besides AR, testosterone also cross-reacts with other steroid receptors at low affinity, such as progesterone receptor and estrogen receptor. In comparison, 5α-DHT binds more specifically to AR.

Testosterone synthesis (adapted from ref 62 ). Abbreviations are as follows: P450scc, cholesterol side-chain cleavage enzyme HSD, hydroxy steroid dehydrogenase.

Healthy adult men typically produce approximately 3� mg of testosterone per day with circulating levels ranging from 300 to 700 ng/dL in eugonadal men. Due to the pulsatile release of gonadotropin releasing hormone, endogenous testosterone secretion is pulsatile and diurnal, the highest concentration occurring at about 8:00 a.m. and the lowest at about 8:00 p.m. Average serum concentrations and diurnal variation in testosterone diminish as men age. 63

Testosterone is highly bound to plasma proteins. About 40% is sequestered with high affinity to sex hormone-binding globulin (SHBG), while almost 60% is bound with low affinity to albumin, leaving only about 2% as free, unbound hormone. 5α-DHT has even greater binding affinity to sex hormone-binding globulin than does testosterone, although 5α-DHT is only about 5% as abundant in the blood as testosterone and is largely derived from peripheral metabolism of testosterone. The unbound testosterone concentration determines its metabolic clearance rate therefore the amount of sex hormone-binding globulin in plasma affects the half-life of circulating testosterone.

Endogenous testosterone levels decline in aging males, while circulating levels of sex hormone-binding globulin increase, which further decreases free testosterone levels. 64 Despite the decrease in free testosterone concentrations, the incidence of prostate cancer and benign prostate hyperplasia (BPH) increases with age, which could be related to the fact that testosterone is almost completely converted to 5α-DHT in the prostate by 5α-reductase. Although there is no direct evidence to suggest that testosterone causes the disease, early-stage prostate cancer is clearly dependent on androgen. Evidence is also accumulating to suggest that residual or adrenal androgens and the AR play a role in “hormone-refractory” prostate cancer. 65

1.3.2. Testosterone Metabolism

Testosterone can be metabolized in either its target tissues or the liver 4 , 66 , 67 ( Figure 7 ). In androgen target tissues, testosterone can be converted to physiologically active metabolites. In the prostate gland, skin, and liver, 68 testosterone is reduced to 5α-DHT by 5α-reductase (type 1 or type 2) 69 in the presence of NADPH. 5α-DHT is the most potent endogenous androgen. On the other hand, a small amount of testosterone (0.2%) can also be converted to estradiol by aromatase through the cleavage of the C19 methyl group and aromatization of ring A, which mainly occurs in adipose tissue. This process also occurs in the ovaries of women. In men, approximately 80% of the circulating estrogen arises from aromatization of testosterone in the adipose tissue 9 with the other 20% secreted by the Leydig cells in the testes. 70

Testosterone metabolism (adapted from ref 72 ). Abbreviationsare as follows: G, glucuronide HSD, hydroxy steroid dehydrogenase UGT, UDP-glucuronosyltransferase.

Both 5α-reduction and aromatization are irreversible processes. Besides these pathways, testosterone can also be further inactivated in the liver through reduction and oxidation, followed by glucuronidation and renal excretion. It can be metabolized to androstenedione through oxidation of the 17β-OH group and androstanedione with 5α-reduction of ring A. Androstanedione can be further converted to androsterone after 3-keto group reduction. Alternatively, androstenedione can also be converted to etiocholanolone through 5β- and 3-keto reduction. Similarly, 5α-DHT can be converted to androstanedione, androsterone, and androstanediol. 71

After the administration of radiolabeled testosterone, about 90% of the radioactivity is found in the urine and 6% is recovered in the feces through enterohepatic circulation. 7 Major urinary metabolites include androsterone and etiocholanolone. Both are inactive metabolites and are excreted mainly as glucuronide conjugates or to a lesser extent as sulfate conjugates. 4 Most of the other metabolites mentioned above undergo extensive glucuronidation of the 3α-or 17β-OH groups as well, either in the target tissues or the liver, 72 and are further excreted in the urine. Therefore, following oral administration, the plasma testosterone half-life is less than 30 min due to the extensive metabolism. Approximately 90% of an oral dose of testosterone is metabolized before it reaches the systemic circulation. To improve the bioavailability, most of the testosterone preparations are delivered through transdermal patch or intramuscular injections. Alkylation or esterification at the 17 position was widely used in structural modification to markedly slow the hepatic metabolism and increase the oral bioavailability or duration of action of testosterone.

1.3.3. Testosterone Tissue Disposition and Function

Animal studies 73 showed that, after intravenous administration, radiolabeled androgens demonstrate higher tissue uptake in androgen target tissues, like the prostate where AR is highly expressed. The tissue uptake efficiency and selectivity of different ligands seemed to be related to their binding affinity to AR and their resistance to metabolism.

As mentioned above, there are three modes of action of testosterone. It may directly act through AR in target tissues where 5α-reductase is not expressed, be converted to 5α-DHT (5�%) by 5α-reductase before binding to AR, or be aromatized to estrogen (0.2%) and act through the estrogen receptor. 7 The formation of 5α-DHT is a natural way for the 𠇍HT-dependent” tissues, such as prostate and seminal vesicle, to amplify the androgenic activity of testosterone. 5α-DHT is a more potent AR ligand than testosterone. It binds to AR with higher affinity ( Table 1 ) and has 2�-fold higher potency than testosterone in androgen-responsive tissues. 10 On the other hand, estrogen plays a major role in regulating metabolic process, 74 , 75 mood and cognition, 76 cardiovascular disease, 77 , 78 sexual function including libido, 79 and bone turnover in men. 80 , 81 Besides these active metabolites, testosterone is the major androgen that acts in the 𠇍HT-independent” tissues, such as skeletal muscle, where 5α-reductase is not expressed or is expressed at a very low level. 68 It directly regulates skeletal muscle growth, bone formation, fat distribution, and sexual function. Free testosterone is considered the most 𠇋iologically active” form, so the circulating level of sex hormone-binding globulin also affects the biologic effects of testosterone.


PHYSIOLOGICAL REGULATION OF TSH SECRETION IN HUMANS

A number of experimental paradigms have been used to mimic clinical situations that affect the hypothalamic-pituitary thyroid axis in man. However, with the exception of the studies of thyroid status and iodine deficiency, such perturbations have limited application to humans due to differences in the more subtle aspects of TSH regulation between species. For example, starvation is a severe stress and markedly reduces TSH secretion in rats, but only marginally in humans. Cold stress increases TSH release in adult rats by alpha-adrenergic stimulation, while this phenomenon is usually not observed in the adult human. Thus, it is more relevant to evaluate the consequences of various pathophysiological influences on TSH concentrations in humans rather than to extrapolate from results in experimental animals. This approach has the disadvantage that, in many cases, the precise mechanism responsible for the alteration in TSH secretion cannot be identified. This deficit is offset by the enhanced relevance of the human studies for understanding clinical pathophysiology.

Table 4.

Common Polymorphisms Related to Serum Thyroid Hormones and TSH Variation (270)

GenePolymorphismEffect on serum
TSHT4T4/T3T3rT3T3/rT3
TSHRrs10149689 A/G*=====
rs12050077 AG=====
DIO1D1a-C/T=
D1b-A/G=
rs2235544 C/A=
DIO2D2-ORFa-Asp3=↑ 1 ====
Thr92Ala====== 2
rs225014 C/T====== 3
THRBTRHB-in9 A/G(↑)=====
PDE8Brs4704397 A/G=====

Alleles associated with the specified trait are reported in bold 1 Only in young subjects

Influence L-T4 dose needed to normalize serum TSH in hypothyroid patients 3 Influence psychological well-being of hypothyroid patients on L-T4 therapy

Normal Physiology

The concentration of TSH can now be measured with exquisite sensitivity using immunometric techniques (see below). In euthyroid humans, this concentration ranges from 0.4-0.5 to 4.0-5.0 mU/L. This normal range is to some extent method-dependent in that the various assays use reference preparations of slightly varying biological potency. The glycosylation of circulating TSH is different from that of standard TSH, thus preventing the calculation of a precise molar equivalent for TSH concentrations (272,273). Recently, a narrower range (0.5-2.5 mU/L) has been proposed in order to exclude subjects with minimal thyroid dysfunction, particularly subclinical hypothyroidism (274), but the issue is still controversial (275). Moreover, data form large epidemiological studies mostly carried out in iodine sufficient countries like the USA, suggest that age together with racial/ethnic factors may significantly affect the respective “normal” TSH range, with higher levels for older Caucasian subjects (276,277). These data differ from the findings previously reported in selected small series of healthy elderly subjects (278) suggesting an age-associated trend to lower serum TSH concentrations (see below). The reason(s) for such discrepancies are still not understood. Independently from the “true” normal range of serum TSH, there is substantial evidence that this is genetically controlled, the heritability being estimated between 40-65% (279). As reported in Table 4, polymorphisms of several genes encoding potentially involved in the control of HPT axis show a significant association with serum TSH concentrations (280) and PDE8B, a gene encoding a high-affinity phosphodiesterase catalyzing the hydrolysis and inactivation of cAMP, has been shown by genome-wide association study to be one of the most important (281).

The free alpha subunit is also detectable in serum with a normal range of 1 to 5 µg/L, but free TSHB is not detectable (4,282). Both the intact TSH molecule and the alpha subunit increase in response to TRH. The alpha subunit is also increased in post-menopausal women thus, the level of gonadal steroid production needs to be taken into account in evaluating alpha subunit concentrations in women. In most patients with hyperthyroidism due to TSH-producing thyrotroph tumors, there is an elevation in the ratio of the alpha subunit to total TSH (4,16,283,182,184). In the presence of normal gonadotropins, this ratio is calculated by assuming a molecular weight for TSH of 28,000 and of 13,600 Da for the alpha subunit. The approximate specific activity of TSH is 0.2 mU/mg. To calculate the molar ratio of alpha subunit to TSH, the concentration of the alpha subunit (in ug/L) is divided by the TSH concentration (in mU/L) and this result multiplied by 10. The normal ratio is ρ.0 and it is usually elevated in patients with TSH-producing pituitary tumors but it is normal in patients with thyroid hormone resistance unless they are post-menopausal (284).

The volume of distribution of TSH in humans is slightly larger than the plasma volume, the half-life is about 1 hour, and the daily TSH turnover between 40 and 150 mU/day (283). Patients with primary hypothyroidism have serum TSH concentrations greater than 5 and up to several hundred mU/L (118). In patients with hyperthyroidism due to Graves' disease or autonomous thyroid nodules, TSH is suppressed with levels which are inversely proportional to the severity and duration of the hyperthyroidism, down to levels as low as π.004 mU/L (285-287).

TSH secretion in humans is pulsatile (288-290). The pulse frequency is slightly less than 2 hours and the amplitude approximately 0.6 mU/L. The TSH pulse is significantly synchronized with PRL pulsatility: this phenomenon is independent of TRH and suggests the existence of unidentified underlying pulse generator(s) for both hormones (291). The frequency and amplitude of pulsations increases during the evening reaching a peak at sleep onset, thus accounting for the circadian variation in basal serum TSH levels (292,293). The maximal serum TSH is reached between 21:00 and 02:00 hours and the difference between the afternoon nadir and peak TSH concentrations is 1 to 3 mU/L. Sleep prevents the further rise in TSH as reflected in the presence of increases in TSH to 5-10 mU/ml during sleep deprivation (294,295). The circadian variation of TSH secretion is probably the consequence of a varying dopaminergic tone modulating the pulsatile TSH stimulation by TRH (296). Interestingly, TSH molecules secreted during the night are less bioactive and differently glycosylated than those circulating in the same individual during the day, thus explaining why thyroid hormone levels do not rise after the nocturnal TSH surge (296). There is convincing evidence seasonal change in basal TSH (297), but there are no gender-related differences in either the amplitude or frequency of the TSH pulses (290). The diurnal rhythmicity of serum TSH concentration is maintained in mild hyper- and hypothyroidism, but it is abolished in severe short-term primary hypothyroidism, suggesting that the complete lack of negative feedback to the hypothalamus or pituitary or both may override the central influences on TSH secretion (298).

Age may have a major effect on circulating serum TSH levels (278). There is a marked increase in serum TSH in neonates which peaks within the first few hours of delivery returning towards normal over the next few days. It is thought to be a consequence of the marked reduction in environmental temperature at birth. Serum TSH concentrations in apparently euthyroid patients over the age of 70 may be somewhat reduced (299,300). However, some studies show an increase of TSH in older adults (301).

TSH in Pathophysiological States

NUTRITION

In the rat, starvation causes a marked decrease in serum TSH and thyroid hormones. While there is an impairment of T4 to T3 conversion in the rat liver due to a decrease in both thiol co-factor and later in the Type 1 deiodinase (302-304), the decrease in serum T3 in the fasted rat is primarily due to the decrease in T4 secretion consequent to TSH deficiency (304,305). In humans, starvation and moderate to severe illness are also associated with a decrease in basal serum TSH, pulse amplitude and nocturnal peak (306-310). In the acutely-fasted man, serum TSH falls only slightly and TRH responsiveness is maintained, although blunted (311,312). This suggests that the thyrotroph remains responsive during short-term fasting and that the decrease in TSH is likely due to changes secondary to decreased TRH release. There is evidence to support this in animal studies, showing reduced TRH gene expression in fasted rats (313,314). Administration of anti-somatostatin antibodies prevents the starvation induced serum TSH falls in rats, suggesting a role for hypothalamic somatostatinergic pathways (315). However, fasting-induced changes in dopaminergic tone do not seem to be sufficient to explain the TSH changes (315,309).

Recent studies provide compelling evidence that the starvation-induced fall in leptin levels (Fig. 15) plays a major role in the decreased TSH and TSH secretion of fasted animals and, possibly, humans (251,316,317). This concept stems from the observation that administration of leptin prevents the starvation-induced fall of hypothalamic TRH (318). The mechanisms involved in this phenomenon include decreased direct stimulation by leptin of TRH production by neurons of the PVN (251,319), as well as indirect effects on distinct leptin-responsive neuroendocrine circuits communicating with TRH neurons (318,320). The direct stimulatory effects of leptin on TRH production are mediated by binding to leptin receptors, followed by STAT3 activation and subsequent binding to the TRH promoter (321,322). One of the latter circuits has been identified in the melanocortin pathway, a major target of leptin action. This pathway involves 2 ligands expressed in distinct populations of arcuate nucleus neurons in the hypothalamus [the alpha-MSH and the Agouti receptor protein (AgRP)] and the melanocortin 4 receptor (MC4R) on which these ligands converge, but exert antagonistic effects (stimulation by alpha-MSH inhibition by AgRP). Leptin activates MC4R by increasing the agonist alpha-MSH and by decreasing the antagonist AgRP and this activation is crucial for the anorexic effect of leptin. The specific involvement of the melanocortin pathway in TRH secretion is suggested by the presence of alpha-MSH in nerve terminals innervating hypothalamic TRH neurons in rat (128) and human (323) brains and by the ability of alpha-MSH to stimulate and of AgRP to inhibit hypothalamus-pituitary thyroid axis both in vitro and in vivo (319). The activities of alpha-MSH and AgRP on the thyroid axis are fully mediated by MCR4, as shown by experiments carried out in MCR4 knock out mice (324). Fasting may inhibit the hypothalamic-pituitary-thyroid axis also via the orexigenic peptide NPY, which inhibits TRH synthesis by activation of Y1 and Y5 receptors in hypophysiotropic neurons of the hypothalamic paraventricular nucleus (325). At least two distinct populations of NPY neurons innervate hypophysiotropic TRH neurons (326), suggesting that NPY is indeed an important regulator of the hypothalamic-pituitary-thyroid axis.

A further contributing cause to the decreased TSH release in fasting may be an abrupt increase in the free fraction of T4 due to the inhibition of hormone binding by free fatty acids (327). This would cause an increase in pituitary T4 and, hence, in pituitary nuclear T3. Fasting causes a decrease in the amplitude of TSH pulses, not in their frequency (328).

Ingestion of food results in an acute decline of the serum TSH concentration: this is the consequence of meal composition, rather than stomach distension (329). Long-term overfeeding is associated to a transient increase of serum T3 concentration and a sustained increased response of TSH to TRH (330).

Taken together, the above data provide compelling evidence that the hypothalamic-pituitary-thyroid axis is tightly related to the mechanisms involved in weight control. In keeping with this concept, several epidemiological studies suggest that small differences in thyroid function may be important for the body mass index and the occurrence of obesity in the general population (331-334).

ILLNESS

The changes in circulating TSH which occur during fasting are more exaggerated during illness. In moderately ill patients, serum TSH may be slightly reduced but the serum free T4 does not fall and is often mildly increased (327,335-337). However, if the illness is severe and/or prolonged, serum TSH will decrease and both serum T4 (and of course T3) decrease during the course of the illness. This may be due to a decreased pulse amplitude and nocturnal TSH secretion (338-341). Since such changes are short-lived, they do not usually cause symptomatic hypothyroidism. They are often associated with an impaired TSH release after TRH (306). However, the illness-induced reductions in serum T4 and T3 will often be followed by a rebound increase in serum TSH as the patient improves. This may lead to a transient serum TSH elevation in association with the still subnormal levels of circulating thyroid hormones and thus be mistaken for primary hypothyroidism (342). On occasion, a transient TSH elevation occurs while the patient is still ill. The pathophysiology of this apparent resistance of the thyroid gland to TSH is not clear (343), although this phenomenon could be the consequence of reduced TSH bioactivity, possibly a consequence of abnormal sialylation (344). The transient nature of these changes is reflected in normalization of the pituitary-thyroid axis after complete recovery. It is currently not clearly established whether the above abnormalities in hypothalamic-pituitary-thyroid axis during critical illness reflect an adaptation of the organism to illness or instead a potentially harmful condition leading to hypothyroidism at the tissue level (345,346).

NEUROPSYCHIATRIC DISORDERS

Certain neuropsychiatric disorders may also be associated with alterations in TSH secretion. In patients with anorexia nervosa or depressive illness, serum TSH may be reduced and/or TRH-induced TSH release blunted (347). Such patients often have decreases in the nocturnal rise in TSH secretion (293). The etiology of these changes is not known although it has been speculated that they are a consequence of abnormal TRH secretion (348,349). The latter is supported by observations that TRH concentrations in cerebrospinal fluid of some depressed patients are elevated (350,351). There may be a parallel in such patients between increases in TRH and ACTH secretion (352). The increased serum T4 and TSH levels sometimes found at the time of admission to psychiatric units is in agreement with this concept (353,349).

MECHANISMS INVOLVED IN THE HYPOTHALAMIC-PITUITARY-THYROID AXIS SUPPRESSION IN NON-THYROIDAL ILLNESSES

The precise mechanism(s) underlying the suppression of the hypothalamic-pituitary-thyroid axis in severe illnesses are only partially known. Evidence for a direct involvement of TRH-producing neurons in humans has been recently provided by the demonstration of low levels of TRH mRNA in the PVN of patients who died of non-thyroidal disease (354). Alterations in neuroendocrine pathways including opioidergic, dopaminergic and somatostatinergic activity have been suggested, but in acutely ill patients the major role appears to be played by glucocorticoids (355) (See below for a more detailed discussion). Activation of pro-inflammatory cytokine pathways is another mechanism potentially involved in the suppression of TSH secretion in nonthyroidal illness. As discussed earlier, IL-1 beta, TNF-alpha and IL-6 exert in vivo and in vitro a marked inhibitory activity on TRH-TSH synthesis/secretion. High levels of pro-inflammatory cytokines (particularly IL-6 and TNF-alpha) have been described in sera of patients with non-thyroidal illnesses (356,357,262,358,359). Serum cytokine concentration is directly correlated with the severity of the underlying disease and to the extent of TSH and thyroid hormone abnormalities observed in these patients. Furthermore, cytokines also affect thyroid hormone secretion, transport and metabolism providing all the characteristics to be considered important mediators of thyroid hormone abnormalities observed in non-thyroidal illness (360-362).

EFFECTS OF HORMONES AND NEUROPEPTIDES

Dopamine and Dopamine Agonists

Dopamine and dopamine agonists inhibit TSH release by mechanisms discussed earlier. Dopamine infusion can overcome the effects of thyroid hormone deficiency in the severely ill patient, suppressing the normally elevated TSH of the patient with primary hypothyroidism nearly into the normal range (235,363). Dopamine causes a reduction of the amplitude of TSH pulsatile release, but not in its frequency (328). However, chronic administration of dopamine agonists, for example in the treatment of prolactinomas, does not lead to central hypothyroidism despite the fact that there is marked decrease in the size of the pituitary tumor and inhibition of prolactin secretion.

Glucocorticoids

The acute administration of pharmacological quantities of glucocorticoids will transiently suppress TSH (364-366). The mechanisms responsible for this effect may act both at the hypothalamic and pituitary level, as discussed above. Direct evidence of suppressed TRH synthesis was provided by an autopsy study showing reduced hypothalamic TRH mRNA expression in subjects treated with corticosteroids before death (367). TSH secretion recovers and T4 production rates are generally not impaired. In Cushing's syndrome, TSH may be normal or suppressed and, in general, there is a decrease in serum T3 concentrations relative to those of T4 (366). High levels of glucocorticoids inhibit basal TSH secretion slightly and may influence the circadian variation in serum TSH (222). Perhaps as a reflection of this, a modest serum TSH elevation may be present in patients with Addison's disease (368,369). TSH normalizes with glucocorticoid therapy alone if primary hypothyroidism is not also present. Similar to patients treated with long-acting somatostatin analogs, patients receiving long-term glucocorticoid therapy do not have a sustained reduction of serum TSH nor does hypothyroidism develop, because of the predominant effect of reduced thyroid hormone secretion in stimulating TSH secretion (370).

Gonadal Steroids

Aside from the well described effects of estrogen on the concentration of thyroxine-binding globulin (TBG), estrogen and testosterone have only minor influences on thyroid economy. In contrast with the mild inhibitory activity on alpha and beta TSH subunits expression described in rats(216), in humans TSH release after TRH is enhanced by estradiol treatment perhaps because estrogens increase TRH receptor number (371,372). Treatment with the testosterone analog, fluoxymesterone, causes a significant decrease in the TSH response to TRH in hypogonadal men (373), possibly due to an increase in T4 to T3 conversion by androgen (374). This and the small estrogen effect may account for the lower TSH response to TRH in men than in women although there is no difference in basal TSH levels between the sexes. This is one of the few instances where there is not a close correlation between basal TSH levels and the response to TRH (see below).

Growth Hormone (GH)

The possibility that central hypothyroidism could be induced by GH replacement in GH-deficient children was raised in early studies (375,376). However, these patients received human pituitary GH which in some cases was contaminated with TSH, perhaps inducing TSH antibodies. Nonetheless, in a cohort of children treated with recombinant hGH (rhGH) and affected with either idiopathic isolated GHD or MPHD, it was demonstrated that in the former the decrease in serum FT4 levels was not of clinical relevance, while in the latter a clear state of central hypothyroidism was seen in more than a half of the children (377). Concerning adults with GHD treated with rhGH, contradictory results have been reported. One study showed no significant changes in TSH concentrations during rhGH therapy of adults with GH deficiency (378). Later on, in two studies, thyroid function was evaluated in a large cohort of patients with adult or childhood onset of severe GHD. In 47% and 36% of euthyroid subjects, independently from rhGH dose, serum FT4 clearly fell into the hypothyroid range and some of these patients reported symptoms of hypothyroidism (375,376). Such results underline that, in adults as well as in children with organic GHD, rhGH therapy unmasks a state of central hypothyroidism, hidden by the condition of GHD itself.

In conclusion, GH does cause an increase in serum free T3, a decrease in free T4, and an increase in the T3 to T4 ratio in both T4-treated and T4 untreated patients. This suggests that the GH-induced increase in IGF-I stimulates T4 to T3 conversion. In keeping with this concept, IGF-I administration in healthy subjects is followed by a fall in serum TSH concentration (379).

Catecholamines

Different from the rat, there is scanty evidence of an adrenergic control of TSH secretion in humans. Acute infusions of alpha or beta adrenergic blocking agents or agonists for short periods of time do not affect basal TSH (380,381), although a small stimulatory activity for endogenous adrenergic pathways is suggested by other studies (382,383). Furthermore, there is no effect of chronic propranolol administration on TSH secretion even though there may be modest inhibition of peripheral T4 to T3 conversion if amounts in excess of 160 mg/day are given (384). Evidence of a tonic inhibition of TSH secretion mediated by endogenous catecholamines has been obtained in women during the early follicular phase of the menstrual cycle (385).

The Response of TSH to TRH in Humans and the Role of Immunometric TSH Assays

More than 4 decades ago, application of ultrasensitive TSH measurements to the evaluation of patients with thyroid disease has undergone a revolutionary change. This is due to the widespread application of the immunometric TSH assay. This assay uses monoclonal antibodies which bind one epitope of TSH and do not interfere with the binding of a second monoclonal or polyclonal antibody to a second epitope. The principle of the test is that TSH serves as the link between an immobilized antibody binding TSH at one epitope and a labelled (radioactive, chemiluminescent or other tag) monoclonal directed against a second portion of the molecule. This approach has improved both sensitivity and specificity by several orders of magnitude. Technical modifications have led to successive "generations" of TSH assays with progressively greater sensitivities (218,316). The first generation TSH assay was the standard radioimmunoassay which generally has lower detection limits of 1-2 mU/L. The "second" generation (first generation immunometric) assay improved the sensitivity to 0.1-0.2 mU/L and “third" generation assays further improved the sensitivity to approximately 0.005 mU/L. From a technical point-of-view, the American Thyroid Association recommendations are that third generation assays should be able to quantitate TSH in the 0.010 to 0.020 mU/L range on an interassay basis with a coefficient of variation of 20% or less (386). As assay sensitivity has improved, the reference range has not changed, remaining between approximately 0.5 and 5.0 mU/L in most laboratories. However, the TSH concentrations in the sera of patients with severe thyrotoxicosis secondary to Graves' disease have been lower with each successive improvement in the TSH assays: using a fourth-generation assay, the serum TSH is π.004 mU/L in patients with severe hyperthyroidism (287,387).

The primary consequence of the availability of (ultra)sensitive TSH assays is to allow the substitution of a basal TSH measurement for the TRH test in patients suspected of thyrotoxicosis (388,285,389,286,287). Nonetheless, it is appropriate to review the results of TRH tests from the point-of-view of understanding thyroid pathophysiology, particularly in patients with hyperthyroidism or autonomous thyroid function. In healthy individuals, bolus i.v. injection of TRH is promptly followed by a rise of serum TSH concentration peaking after 20 to 30 minutes. The magnitude of the TSH peak is proportional to the logarithm of TRH doses between 6.25 up to � ug, is significantly higher in women than in men, and declines with age (390,391). The individual TSH response to TRH is very variable and declines after repeated TRH administrations at short time intervals (391). In the presence of normal TSH bioactivity and adequate thyroid functional reserve, serum T3 and T4 also increase 120-180 minutes after TRH injection (391). There is a tight correlation between the basal TSH and the magnitude of the TRH-induced peak TSH (Fig. 12) Using a normal basal TSH range of 0.5 to 5 mU/L, the TRH response 15 to 20 minutes after 500 ug TRH (intravenously) ranges between 2 and 30 mU/L. The lower responses are found in patients with lower (but still normal) basal TSH levels (287). These results are quite consistent with older studies using radioimmunoassays (392). When the TSH response to TRH of all patients (hypo-, hyper- and euthyroid) is analyzed in terms of a "fold" response, the highest response (approximately 20-fold) occurs at a basal TSH of 0.5 mU/L and falls to less than 5 at either markedly subnormal or markedly elevated basal serum TSH concentrations (Fig. 16) (287). Thus, a low response can have two explanations. The low response in patients with hyperthyroidism and a reduced basal TSH is due to refractoriness to TRH or depletion of pituitary TSH as a consequence of chronic thyroid hormone excess. In patients with primary hypothyroidism, the low fold-response reflects only the lack of sufficient pituitary TSH to achieve the necessary increment over the elevated basal TSH.

Figure 16.

Relationship between basal and absolute (TRH stimulated-basal TSH) TRH-stimulated TSH response in 1061 ambulatory patients with an intact hypothalamic-pituitary (H-P) axis compared with that in untreated and T4-treated patients with central hypothyroidism. (From Spencer et al. (287) with permission)

Although, as stated before, the clinical relevance of the TRH test is presently limited, there are still some conditions in which the test may still be useful. These include subclinical primary hypothyroidism, central hypothyroidism (25), the syndromes of inappropriate TSH secretion (393) and non-thyroidal illnesses.

In patients with normal serum thyroid hormone concentrations and borderline TSH, an exaggerated TSH response to TRH not followed by an adequate increase in serum thyroid hormone levels may confirm the presence of subtle primary hypothyroidism (391).

An abnormal relationship between the basal TSH and the TRH-response is found in patients with central hypothyroidism. Here the fold TSH response to TRH is lower than normal (371,23,287). Again, however, TRH testing does not add substantially to the evaluation of such patients in that the diagnosis of central hypothyroidism is established by finding a normal or slightly elevated basal TSH in the presence of a significantly reduced free T4 concentration. While statistically (287) lower and sometimes delayed increments in TSH release after TRH infusion are found in patients with pituitary as opposed to hypothalamic hypothyroidism, the overlap in the TSH increments found in patients with these two conditions is sufficiently large (371,23,24,394), so that other diagnostic technologies, such as MRI, must be used to provide definitive localization of the lesion in patients with central hypothyroidism. It should be recalled that the TRH test may be useful in the diagnosis and follow-up of several pituitary disorders, but the discussion of this point is beyond the purpose of this chapter.

The TRH test still provides fundamental information in the differential diagnosis of hyperthyroidism due to TSH-secreting adenomas from syndromes with non-neoplastic TSH hypersecretion due to pituitary selective or generalized thyroid hormone resistance. In all the above conditions, increased or “inappropriately normal” serum TSH concentrations are observed in the presence of elevated circulating thyroid hormone levels. However, in most (㺐%) of TSH-secreting adenomas serum TSH does not increase after TRH, while TRH responsiveness is observed in 㺕% of patients with nontumoral inappropriate TSH secretion (283,213,391).

Perhaps of most interest pathophysiologically is the response to TRH in patients with non-thyroidal illness and either normal or low free T4 indices (Fig. 12). Results from these patients fit within the normal distribution in terms of the relationship between basal TSH (whether suppressed or elevated) and the fold-response to TRH. Thus the information provided by a TRH infusion test adds little to that obtained from an accurate basal TSH measurement (395). With respect to the evaluation of sick patients, while basal TSH values are on average higher than in patients with thyrotoxicosis, there is still some overlap between these groups (396,337,287,397). This indicates that even with second or third generation TSH assays, it may not be possible to establish that thyrotoxicosis is present based on a serum TSH measurement in a population which includes severely ill patients.


Mechanisms/pathophysiology

Inflammation

Sepsis is fundamentally an inflammatory disease mediated by the activation of the innate immune system. Two key findings characterize the innate immune response in sepsis. The first finding is that sepsis is generally initiated by simultaneous recognition of multiple infection-derived microbial products and endogenous danger signals by complement and specific cell-surface receptors on cells whose primary job is surveillance 37 . These cells include immune, epithelial and endothelial populations that are physically located where they can continuously sample their local environment. Binding of both pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to complement, Toll-like receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid-inducible gene (RIG)-like receptors, mannose-binding lectin and scavenger receptors, among others, induces a complex intracellular signalling system with redundant and complementary activities 38 ( FIG. 1 ).

Sepsis is initiated upon host recognition of pathogen- associated molecular patterns (PAMPs) and is characterized by the activation of inflammatory signalling pathways. A large number of cell-associated and intracellular receptors are available to detect PAMPs or damage-associated molecular patterns (DAMPs), a few examples of which are illustrated here. PAMPs and DAMPs can be microbial and host glycoproteins, lipoproteins and nucleic acids. The associated pattern- recognition receptors include Toll-like receptors (TLRs), C-type lectin domain family 7 member A (dectin 1) and C-type lectin domain family 6 member A (dectin 2). At least ten different TLRs are known, and in many cases they exist as either homodimers or heterodimers. Once activated, the ensuing signalling pathways generally converge towards interferon regulatory factor (IRF) signalling and nuclear factor-㮫 (NF-㮫). IRF is responsible for type I interferon (IFN) production. NF-㮫 and activator protein 1 (AP-1) signalling are predominately responsible for the early activation of inflammatory genes, such as TNF, IL1 and those encoding endothelial cell-surface molecules. CARD9, caspase recruitment domain-containing protein 9 dsDNA, double-stranded DNA dsRNA, double-stranded RNA FcRγ, Fcγ receptor HMGB1, high-mobility group protein B1 iE-DAP, d-glutamyl-meso-diaminopimelic acid LGP2, laboratory of genetics and physiology 2 (also known as DHX58) LPL, lipoprotein lipase LPS, lipopolysaccharide LY96, lymphocyte antigen 96 MAPK, mitogen- activated protein kinase MCG, mannose-containing glycoprotein MDA5, melanoma differentiation-associated protein 5 (also known as IFIH1) MDP, muramyl dipeptide MCL, mannose-capped lipoarabinomannan Mincle, also known as CLEC4E MYD88, myeloid differentiation primary response protein 88 NIK, NF-㮫-inducing kinase (also known as MAP3K14) NOD, nucleotide-binding oligomerization domain RAF1, RAF proto-oncogene serine/threonine-protein kinase RAGE, advanced glycosylation end product-specific receptor RIG-I, retinoic acid-inducible gene 1 protein (also known as DDX58) ssRNA, single-stranded RNA STING, stimulator of interferon genes protein SYK, spleen tyrosine kinase TDM, trehalose-6,6′-dimycolate TICAM1, TIR domain-containing adaptor molecule 1.

The second key finding in sepsis is that activation of these multiple signalling pathways ultimately leads to the expression of several common gene classes that are involved in inflammation, adaptive immunity and cellular metabolism. That is, the recognition of many different components of bacteria, viruses and fungi, as well as host products of tissue injury, leads to the recruitment of pro-inflammatory intermediates that in turn result in the phosphorylation of mitogen- activated protein kinases (MAPKs), Janus kinases (JAKs) or signal transducers and activators of transcription (STATs) and nuclear translocation of nuclear factor-κΒ (NF-κΒ), to name simply a few. These intermediates initiate the expression of early activation genes. Taken together, these two characteristics of innate immunity assure a common response pattern, the intensity and direction of which can be finely regulated by the level of and variation in the repertoire of PAMPs and DAMPs and the signalling pathways activated. This complementary nature of the pathways explains the overlapping but unique early inflammatory response to common Gram-negative bacterial, Gram-positive bacterial, fungal and viral infections and tissue injury.

Early activation genes

Nuclear translocation of NF-㮫 and activation of its promoter in particular induce the expression of multiple early activation genes, including cytokines that are associated with inflammation (including tumour necrosis factor (TNF), IL-1, IL-12, IL-18 and type I interferons (IFNs)). These cytokines initiate a cascade of other inflammatory cytokines and chemokines (including IL-6, IL-8, IFNγ, CC-chemokine ligand 2 (CCL2), CCL3 and CXC-chemokine ligand 10 (CXCL10)), as well as the polarization and suppression of components of adaptive immunity. The activation of these inflammatory networks begins within minutes of PAMP or DAMP recognition owing to the existence of preformed inactive and active cytokine pools. Simultaneously, activation of these sentinel innate immune receptors, activation of complement and/or production of inflammatory cytokines have a profound effect on coagulation and the vascular and lymphatic endothelium, resulting in the increased expression of selectins and adhesion molecules 39 . The alteration in the expression of various procoagulant and anticoagulant proteins, including thrombomodulin, tissue factor, von Willebrand factor, plasminogen activator inhibitor 1 (PAI-1) and activated protein C, results in the transition of the endothelium from an anticoagulant state (in health) to a procoagulant state (in sepsis). Pro-inflammatory proteases induce the internalization of the vascular endothelial (VE)-cadherin leading to the loss of endothelial tight junctions and increased vascular permeability 40 .

The C5a� receptor axis

Complement activation is considered to be one of the hallmarks of sepsis and is initiated immediately upon exposure to PAMPs and DAMPs. Complement activation leads to the generation of complement peptides (namely, C3a and C5a). C5a has been shown to be one of the most active inflammatory peptides produced during sepsis 41 and is one of the most potent chemo attractants for neutrophils, monocytes and macro phages. In neutrophils, C5a triggers an oxidative burst leading to the generation of reactive oxygen species and the release of granular enzymes, which are thought to be crucially involved in inflammatory tissue damage. Furthermore, C5a is a stimulant for the synthesis and release of pro-inflammatory cytokines and chemokines, thereby amplifying inflammatory responses. These mechanisms are believed to contribute to vasodilation, tissue damage and multiple organ failure in settings of acute inflammation. The potential role of C5a in the development of sepsis has been linked to neutrophil dysfunction, apoptosis of lymphoid cells, exacerbation of systemic inflammation, cardiomyopathy, disseminated intra vascular coagulation (DIC) and complications associated with multiple organ failure 42 .

Blockade of C5a in experimental models of sepsis has been shown to be beneficial in various models from different groups. For example, inhibition of C5a by rabbit polyclonal antibodies in a primate model of sepsis induced by infusion of live Escherichia coli substantially attenuated evidence of acute sepsis-induced lung injury and failure 43 . Similarly, the blockade of C5a with antibodies in rats or mice with sepsis caused by caecal ligation puncture was highly effective in diminishing the severity of sepsis and improving outcome 44,45 . In addition, severe inflammatory responses and their associated organ damage during avian H5N1 and H1N1 viral infections have also been linked to complement activation, especially the overproduction of C5a 46,47 . Along these lines, a monoclonal antibody raised against human C5a greatly attenuated H7N9-induced lung damage in non-human primates, reducing the viral load and the levels of several different cytokines in this setting 48 . The same antibody is currently being tested in patients with early abdominal or pulmonary septic organ dysfunction 49 .

Immune suppression

Although the early systemic inflammatory response has been considered the hallmark of sepsis, immuno-suppression occurs both early and late in the host sepsis response. Patients who survive sepsis often have protracted clinical trajectories and exhibit both chronic immune suppression and inflammation. This finding has recently been termed the persistent inflammation/immunosuppression and catabolism syndrome (PICS) 50� ( FIG. 2 ). PICS-associated inflammation is characterized by markedly increased C-reactive protein concentrations (an acute phase protein), neutrophilia and the release of immature myeloid cells. Unlike the immediate inflammatory response that is presumed to be predominantly driven by PAMPs and DAMPs, the aetiology behind the persistent inflammation is unknown. PICS is probably driven by DAMPs and alarmins that are produced by injured organs and tissues, such as mitochondrial DNA and nucleosides, histones, high- mobility group protein B1 (HMGB1), protein S100A, ATP, adeno-sine and/or hyaluronan products 53 . Alternative explanations for how PICS progresses include opportunistic infections such as viral reactivation 54 , changes in the host microbiota and mechanical injury secondary to ventilation or catheter placement.

Originally conceived by Bone et al. 165 in the 1990s, the current model of the clinical trajectory that patients traverse in sepsis has evolved to reflect the concurrent inflammatory and immunosuppressive responses, and the observation that fewer patients are dying in the early period owing to earlier recognition and better implementation of best clinical practices 50 . Successful resuscitation is occurring more frequently and the patients recover sufficiently to be discharged from the intensive care unit and hospital (blue lines). Some patients experience a pronounced early inflammatory response to the pathogen or danger signals, leading to multiple organ failure and death (red line). Other patients survive the early inflammatory response but experience chronic critical illness (green lines) that is characterized by persistent inflammation, immunosuppression and catabolism syndrome (PICS) reactivation of latent viral infections nosocomial infections and long-term functional and cognitive declines 52 . DAMP, damage-associated molecular pattern DC, dendritic cell MDSC, myeloid-derived suppressor cell NO, nitric oxide ROS, reactive oxygen species TH2, T helper 2.

The paradoxical immunosuppression and infectious complications in patients with sepsis compound as sepsis progresses, with an increasing frequency of positive blood cultures and a shift to infection by opportunistic organisms 55,56 . Compared with control individuals without sepsis, patients with sepsis have increased rates of reactivation of latent viruses, with viral DNA being detected in the blood of 42% of patients with sepsis (only 5% of critically ill patients without sepsis have detectable viral DNA) 54 . One autopsy study confirmed the immunosuppressed state of patients with sepsis, with persistent foci of infection and microabscesses identified in 80% of cases 57 .

The changes in adaptive immunity in response to sepsis are profound. Lymphopaenia, an immature neutrophil (polymorphonuclear) phenotype 58,59 , loss of monocyte inflammatory cytokine production and antigen presentation 60 and increased numbers of neutrophil- like myeloid-derived suppressor cells (MDSCs) in the circulation 61 are all common consequences of sepsis. Immature myeloid cells in the circulation have characteristically defective antimicrobial activity with decreased expression of adhesion molecules and decreased formation of extracellular traps (networks of extracellular fibres composed of chromatin, DNA and granular proteins) that capture pathogens 62,63 . Both immature blood neutrophils and MDSCs secrete multiple anti-inflammatory cytokines, including IL-10 and transforming growth factor-β (TGFβ), which further suppress immune function. In addition, sepsis causes professional antigen-presenting cells (APCs) — including dendritic cells and macrophages — to lose expression of the activating major histocompatibility complex (MHC) class II molecule human leukocyte antigen-antigen D related (HLA-DR). In addition, loss of HLA-DR by circulating APCs has been associated with decreased responsiveness, and the failure of monocytes to recover HLA-DR levels predicts a poor outcome from sepsis 64 . Sepsis also causes both stromal cells and professional APCs to increase the expression of the T cell protein programmed death ligand 1 (PDL1), which binds to the inhibitory programmed death protein 1 (PD1) receptor that is expressed by T cells, further suppressing T cell function 65 . The combination of the increased surface expression of inhibitory T cell ligands by APCs, loss of activating MHC class II molecules and increased production of anti-inflammatory cytokines skews the T cell phenotype towards an immunosuppressive T helper 2 (TH2) phenotype, increases the suppressor activity of T regulatory cells and causes broad T cell anergy (lack of reaction) ( FIG. 3 ). Lending further support to the notion that immune suppression occurs in sepsis, pro-inflammatory and TH1 cytokine production by lymphocytes from patients with sepsis is 㰐% of that of controls without sepsis 65 . Together, these data provide a mechanism for the well-described loss of the delayed-type hypersensitivity in patients with sepsis, a metric for the profound suppression of the adaptive immune system seen in sepsis 66 .

After the transitory acute inflammatory response, sepsis results in an immunocompromised state. Immunosuppressive immature polymorphonuclear leukocytes (PMNs) and myeloid-derived suppressor cells (MDSCs) mobilize from the bone marrow and monocyte differentiation skews to the production of M2 macrophages (which decrease inflammation and promote tissue repair). Although these responses can be considered normal, if the source of infection is not controlled, the continued responses rapidly become pathological and lead to chronic immune suppression. Together, immature PMNs, MDSCs and M2 macrophages produce anti-inflammatory cytokines, such as IL-10 and transforming growth factor-β (TGFβ). Professional antigen-presenting cells, including dendritic cells and macrophages, reduce the expression of the activating major histocompatibility complex (MHC) class II molecule human leukocyte antigen-antigen D related (HLA-DR). T cells and stromal cells upregulate negative co-stimulatory molecules, including programmed death protein 1 (PD1) and programmed death ligand 1 (PDL1), respectively, to drive the expansion of regulatory T (Treg) cells and anergic (unresponsive) T cells. Follicular dendritic cells, B cells and T cells undergo apoptosis, further abrogating the immune response. TCR, T cell receptor TH2, T helper 2.

An autopsy study of patients with sepsis identified apoptotic cell death as an underlying driver of innate and adaptive immunosuppression 67 . Indeed, patients with sepsis demonstrate a profound apoptotic loss of T cells, B cells and dendritic cells, an observation that is recapitulated in animal models of sepsis 68 . Apoptotic loss of lymphocytes is directly immunosuppressive, contributing to the lymphopaenia observed in patients with severe sepsis 69 . The degree of lymphocyte apoptosis correlates with the severity of sepsis and the persistent lymphopaenia predicts sepsis mortality 70 . Apoptotic cells also suppress immune function through interaction with other leukocytes. For instance, phagocytosis of apoptotic lymphocytes causes the release of anti-inflammatory cytokines such as IL-10 and TGFβ from macrophages and dendritic cells. This process also suppresses the production of pro-inflammatory cytokines at the level of gene transcription, thereby contributing to the paralysis of the innate inflammatory response in sepsis 70 . Accordingly, pharmacological or genetic manipulations that decrease sepsis-induced apoptosis improve survival in animal models of sepsis 68,71� . These data demonstrate the functional consequence of sepsis-induced apoptosis. The next generation of treatments being evaluated for sepsis includes therapies that target both lymphocyte apoptosis and sepsis-induced immunosuppression the results of these studies are eagerly anticipated.

Endothelial barrier dysfunction

In addition to profound changes in host protective immunity, endothelial barrier function is an integral component of the sepsis response. A continuous endothelial barrier coats the vascular system and separates the fluid phase of the blood compartment from the tissues ( FIG. 4 ). Under normal resting conditions, the endothelium serves as an anticoagulant surface that regulates the flow of gases, water, solutes, hormones, lipids, proteins and a multitude of other macro molecules within the microcirculation. Sepsis is now viewed as a dysregulation of the interacting and oscillating circuitry networks of cell�ll communication that maintain homeostasis under normal conditions 9 . Along these lines, endothelial barrier dysfunction is a fundamental pathophysiological event that occurs early in sepsis and septic shock in particular. The border between the blood and the interstitium is highly interactive and dynamic in both health and disease, with the endothelial cell as the principal regulatory cell type 11 . The endothelium functions to cover the underlying capillary basement membrane and adventitia to avoid exposing collagen fibres and tissue factor primarily to von Willebrand factor and factor VII. Collagen can immediately fix and polymerize von Willebrand factor, which activates platelets via glycoprotein 1β at the same time, exposing tissue factor to circulating factor VII can initiate clotting via the tissue factor (formerly known as the extrinsic) pathway 77 .

a | The resting vascular endothelium in its natural anticoagulant state. b | Sepsis produces profound changes that convert the endothelium to a procoagulant state. This disrupted endothelium expedites the loss of fluid through disengaged tight junctions and expedites the recruitment, attachment and extravasation of inflammatory cells through the endothelium. Activation of the coagulation cascade potentiates inflammation and completes a vicious cycle in which inflammation induces and exacerbates coagualopathies and endothelial injury. ESL1, E-selectin ligand 1 ICAM1, intercellular adhesion molecule 1 LFA1, lymphocyte function-associated antigen 1 MPO, myeloperoxidase NO, nitric oxide PAF, platelet- activating factor PAI-1, plasminogen activator inhibitor 1 PGI2, prostaglandin I2 PMN, polymorphonuclear leukocyte PSGL1, P-selectin ligand 1 ROS, reactive oxygen species TFPI, tissue factor pathway inhibitor TM, thrombomodulin t-PA, tissue plasminogen activator TXA2, thromboxane A2 VE, vascular endothelial.

The integrity of the endothelium is maintained by the cell cytoskeleton (actin), intercellular adhesion molecules (tight junctions) and an array of supportive proteins. In sepsis, these structures are disrupted primarily in response to platelet and neutrophil adhesion, the release of inflammatory mediators and toxic oxidative and nitrosative intermediates. Combined with the increased expression of selectins and integrins, binding of leukocytes to the endothelial surface results in the leakage of vascular fluid and migration of extravasating leukocytes across the compromised endothelial barrier. This event also provides the opportunity for collagen polymerization and tissue factor-mediated clotting to occur. Although these responses enable platelets and immune cells to reach tissue sites in response to trauma or localized infection, sepsis produces generalized, excessive and prolonged responses that can lead to considerable tissue injury.

In addition, the glycocalyx is a glycoprotein–polysaccharide layer that covers the endothelium and supports the anticoagulant state and maintains tight junctions. Sepsis alters the continuity of the glycocalyx, which also increases endothelial permeability 78 . In sepsis, the glycocalyx is a target for inflammatory mediators and leukocytes because it is imbedded with endothelial cell-surface receptors. The widespread presence of the glycocalyx in organ microvasculature can explain the endothelial activation and damage of tissues distant from the original site of infection via this systemic release of cytokines and other inflammatory mediators during sepsis. Inflammatory-mediated injury to the glycocalyx contributes to acute kidney injury, respiratory failure and hepatic dysfunction.

Numerous factors regulate the expression of tight junction linkers and actin polymer networks. Prominent among these regulators are the relative expression of two competing intracellular, G protein-linked GTPases known as RHOA and RAC1. RHOA generally induces actin filament breakdown and internalizes VE-cadherin, resulting in endothelial barrier breakdown. RAC1 signalling has opposing effects, stabilizing the actin cytoskeleton and preventing apoptosis. The relative concentrations of RHOA and RAC1 can be regulated, at least experimentally, by protease-activated receptors (PARs) on endothelial surfaces. Early thrombin generation in sepsis activates PAR1, which promotes RHOA GTPase signalling and induces endothelial barrier breakdown. Other proteases that activate PAR2 promote RAC1 signalling and support endothelial barrier protection 79 . TABLE 1 lists some of the candidate therapies that might prove to be effective in maintaining and re-acquiring endothelial barrier function in sepsis and septic shock.

Table 1

Candidate treatment options to treat endothelial barrier injury *

DrugStage of developmentProposed mechanism of action
ANG1–TIE2 modulatorsIn clinical trials for cancer 166 Reduce the loss of tight junctions and endothelial tight junction function in sepsis
S1P1 agonistsIn clinical trials for multiple sclerosis 167 and plaque psoriasis 168 Stimulate VE-cadherin and actin polymerization of endothelial junctions, preserving endothelial tight junction function in sepsis
Fibrinopeptide Bβ15�In clinical trials for myocardial infarction 169 Fibrin cleavage product that binds to VE-cadherin and stabilizes interendothelial tight junctions to reduce endothelial permeability
SLIT2N agonistsPreclinical testingStabilize endothelial tight junctions by binding to ROBO4 to reduce p120-catenin phosphorylation and increase p120-catenin association with VE-cadherin
PepducinsPAR1 pepducin in clinical trials for cardiac catheterization 170 Lipidated peptides are super agonists of PAR2, inducing RAC1-mediated endothelial barrier stabilization
HMGB1-specific monoclonal antibodyPreclinical testingBlocks HMGB1-mediated loss of endothelial barrier function, upregulation of cytokine production and of adhesion molecules
Statins and angiotensin receptor blockersIn clinical trials for sepsis 171 Block angiotensin receptor-mediated oxidant stress on endothelial cells, regulate RAC1/RHOA ratio and prevent apoptosis
SelepressinIn clinical trials for sepsis 172 Vasopressin type 1a receptor antagonist 121

ANG1, angiopoietin 1 HMGB1, high-mobility group B1 NF-㮫, nuclear factor-㮫 PAR, protease-activated receptor RAC1, a subfamily of GTPases ROBO4, roundabout homologue 4 S1P1, sphingosine 1-phosphate receptor 1 SLIT2N, slit homologue 2 protein N-product TIE2, angiopoietin 1 receptor VE, vascular endothelial.

The leaky capillary membranes create massive loss of intravascular proteins and plasma fluids into the extravascular space. Diffuse vasodilation throughout the microcirculation alters capillary blood flow, which contributes to poor tissue perfusion and — ultimately — shock. In septic shock, events within tissue capillaries induce distributive shock in which the recovery of blood pressure is not achieved upon the administration of additional intravenous fluids, and requires a vasoconstrictive agent such as noradrenaline and/or vasopressin. The large volumes of crystalloid given to maintain central blood pressure in the presence endothelial injury frequently leads to oedema.

Coagulation

In sepsis and septic shock, the normal anticoagulative state within the vasculature is disrupted. Sepsis results in a hypercoagulable state that is characterized by microvascular thrombi, fibrin deposition, neutrophil extracellular trap (NET) formation and endothelial injury. Inflammatory cytokines as well as other mediators, such as platelet-activating factor and cathepsin G, target the endothelium and platelets. Platelet activation can itself propagate both coagulation and the inflammatory response by forming aggregates that can activate thrombin release. Thrombin is a serine protease that converts fibrinogen into insoluble strands of fibrin, as well as catalysing many other coagulation-related reactions. These strands of fibrin, along with platelets, provide the structural integrity to clot formation. In addition, inflammatory cytokines can promote coagulation by targeting the endothelium and causing endothelial injury ( FIG. 5 ).

Microorganisms and damage -associated molecular patterns (DAMPs), as well as complement activation and the release of inflammatory cytokines or mediators, can initiate the coagulation cascade (involving the coagulation factors designated here as 𠆏’ followed by the requisite Roman numeral). Primarily through the upregulation of procoagulant proteins such as tissue factor (TF), excessive fibrin deposition and reduced plasmin activity lead to thrombus and fibrin deposition and microcirculatory defects. The system is self-activating as complement activation and the exposure of myeloid and endothelial cells to microbial products and inflammatory cytokines increase the expression of TF. Products of complement activation, such as C3a and C5a, induce platelet-activating factor (PAF not shown) and inflammatory cytokines. Cytokines, PAF and thrombi can also damage the endothelium, exposing collagen fibres and activating von Willebrand factor (vWF), which further increases TF expression and inflammatory cytokine production. Although not shown here, inflammatory cytokines also decrease the expression of the fibrinolytic pathway, by increasing plasminogen activator inhibitor 1 (PAI-1) activity and decreasing plasmin activity. DIC, disseminated intravascular coagulation MBL, mannose-binding lectin.

The damaged endothelium and exposure of the underlying collagen activate von Willebrand factor, which further activates platelet aggregation and fibrin formation. Platelets might also trigger inflammation by activating dendritic cells. The activated endothelium also upregulates tissue factor, which can act directly on circulating factor VII, leading to tissue factor�tor VIIa complexes that convert factor X to factor Xa, resulting in thrombin generation, fibrin deposition, contact factor activation, clot formation, bradykinin synthesis and complement activation. Furthermore, complement activation feeds back to promote further clotting through complement-mediated shedding of cell-derived microvesicles. These microvesicles from monocytes and macrophages contain additional tissue factor, thereby exaggerating inflammation and thrombosis 80 .

Complement deposition on erythrocytes triggers haemolysis and the release of erythrocyte-derived microvesicles that are prothrombotic 81 . The resulting interaction between tissue factor and factor VIIa propagates the inflammatory process and leads to fibrin deposition on the endothelium. Microthrombi deposition, especially in the microvasculature, leads to decreased perfusion and thrombus formation. Concordantly, coagulation augments inflammation predominantly through a thrombin-induced secretion of pro- inflammatory cytokines and growth factors. Extracellular tissue factor signalling through PARs elicits cellular activation and inflammatory responses 82 .

Endogenous anticoagulants that inhibit different parts of the coagulation cascade (thereby inhibiting clot formation) are downregulated by the same processes that lead to the upregulation of tissue factor. For example, antithrombin and activated protein C concentrations decrease, as does endothelial glycosaminoglycans, such as heparan sulfate 83 . Inhibition of both thrombomodulin and endothelial cell protein C receptor contributes to the decrease in activated protein C concentrations 84 . Simultaneously, fibrinolysis is dramatically decreased. Increased levels of PAI-1 inhibit both tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). This dysregulation of the PAI-1–t-PA–u-PA network results in a substantial reduction in the concentration of plasmin, which is required for dissolving intravascular fibrin clots. Thrombin generation and its binding to thrombomodulin activate thrombin-activatable fibrinolysis inhibitor — further reducing plasmin generation.

Ultimately, this exaggerated coagulopathy can lead to uncontrolled bleeding. This event might seem inconsistent with the previous statements regarding a sepsis-induced hypercoagulation and fibrin deposition, but the process is thought to occur secondary to a consumptive thrombocytopaenia and depletion of clotting factors 10 . The transition from a hypercoagulable state to DIC is characterized by fibrinolysis with increased circulating fibrin degradation products, thrombocytopaenia and exhaustion of liver-derived prothrombin, fibrinogen, factor X and factor V reserves.

Clot formation

Inflammation and coagulation are tightly linked defence mechanisms following injury and auto-amplify by co-stimulation 85 . In an anticoagulant state during health, endothelial cells generally do not express adhesion molecules that bind to leukocytes and platelets, but will do so in sepsis in response to the early inflammatory response, resulting in the activation of coagulation. The local cytokine milieu in this stage of inflammation induces cell-surface receptors for myeloid cells, lymphocytes and platelets. Platelets bind to fibrin strands and provide a ready source of P-selectin for neutrophil attachment activated neutrophils produce NETs that provide a scaffold for more clot formation and this process self-amplifies 10,86 . This cooperative interaction serves to ‘wall off’ sites of injury from the rest of the host, limiting infection risk. The clot also serves to avoid blood loss and possible exsanguination by plugging the defect in the vascular system. This co-regulated clot formation and innate immune activation has an obvious survival advantage when a limited site of injury can be contained locally. However, if generalized activation of coagulation and inflammation occurs throughout the host, such as during DIC, the consequences can be devastating and lead to potentially lethal septic shock (see below).

Effect on organ systems

Sepsis is also a systemic disorder that can affect all organs of the body, probably owing to the panoply of cytokines and other mediators that are released into the general circulation during the onset of the dis order. The presenting signs and symptoms of sepsis are variable and depend on the particular organ systems that are affected. Six types of organ dysfunction predominate in sepsis: neurological (altered mental status), pulmonary (with hypoxaemia), cardiovascular (shock), renal (oliguria and/or increased creatinine concentration), haematological (decreased platelet count) and hepatic (hyperbilirubinaemia).

Neurological

Patients typically present with altered mental status manifested by lethargy, confusion or delirium. Occasionally, the mental status of the patient is so severely depressed that it is necessary to secure their airway (that is, perform endotracheal intubation). Despite this, the neurological examination at this time is typically without focal neurological findings. In the assessment, other causes of neurological disturbance (for example, hypoxaemia, hypoglycaemia, drug toxicity or central nervous system infection) should be ruled out or if present, addressed.

Pulmonary

One of the most common manifestations of sepsis is increased respiratory rate. Tachypnoea (a hallmark of sepsis-induced adult respiratory distress syndrome) can be associated with abnormal arterial blood gases, typically, a primary respiratory alkalosis. Accompanying hypoxaemia and/or hypercarbia can also occur respiratory muscle fatigue, hypoxaemia or hypercarbia might necessitate endotracheal intubation for therapy. The aetiology of the respiratory failure in sepsis is due to inflammatory mediator- induced damage to alveolar capillary membranes. This cytokine-mediated lung injury results in noncardiogenic pulmonary oedema that can be profound and that causes decreased lung compliance and impaired oxygen uptake and carbon dioxide elimination. Decreased lung compliance and activation of juxtacapillary receptors lead to increased ventilation and are partly responsible for the tachypnoea. Chest X-ray imaging usually shows increased lung water with bilateral pulmonary infiltrates. Left ventricular heart failure must be ruled out as the cause of the pulmonary changes. Although patients with sepsis may have profound, life-threatening hypoxaemia, most patients do not die of hypoxaemia but rather of multiple organ failure.

Cardiovascular

Myocardial depression, which is characterized by hypotension or shock, is a hallmark of severe sepsis 87 . Several cytokines have direct cardiomyocyte toxic effects. Mild increases in circulating cardiac troponins are frequently present in sepsis and are indicative of sepsis severity. Myocardial depression affects both the right and the left ventricles and this finding distinguishes sepsis-induced myocardial depression from coronary atherosclerotic-induced myocardial ischaemic dysfunction. Sepsis-induced myocardial depression can be profound with decreases in the left and right ventricular ejection fractions, necessitating therapy with inotropic agents.

Oxidative and nitrosative stress (the build-up of reactive oxygen and nitrogen species, respectively) also contribute to cardiovascular and other organ failure, which is one of the root causes of tissue hypoxia 88 . Nitrosative stress is a major component of the pathophysiology of sepsis, and upregulation of inducible nitric oxide synthase (iNOS) might provide the link between inflammatory activation and cardio vascular compromise. In this context, the role of hypoxia-induced factor-α (HIFα) in sepsis also plays a major part in defining its pathophysiology 89 .

Renal

Renal dysfunction that progresses to frank renal failure is a major cause of sepsis-induced morbidity 12 . Although the exact mechanisms responsible for sepsis- induced renal failure are unknown, clinicians can reduce the incidence of severe renal failure in sepsis by aggressive and appropriate volume resuscitation in the disorder. Because of loss of intravascular volume in sepsis due to leaky capillary membranes and vasodilation, patients typically require volume resuscitation to replace these losses. Accordingly, clinicians must avoid the use of nephrotoxic agents in patients with sepsis if at all possible. For example, administration of intravenous contrast agents for radiological imaging studies can precipitate new-onset renal failure if given to a patient with sepsis who is intravascularly volume depleted. The absence of full renal recovery in sepsis is associated with poor long-term outcomes, so management of renal function during sepsis is of crucial importance. Even minor increases in the concentrations of serum creatinine are associated with increased mortality 90 .

Haematological

DIC is one of the most striking manifestations of severe sepsis. DIC can present in one of two contrasting clinical fashions: with overt bleeding from multiple sites or, conversely, with thrombosis of small and medium blood vessels. The reason for the striking differences in presentation of DIC is attributable to the fact that the coagulation system represents a balance between the clotting and fibrinolytic systems. In individual cases of sepsis, either system can predominate. If the fibrinolytic system is dominant, the patient will present with bleeding from multiple sites. Conversely, if the coagulation system is dominant, the patient will present with cyanotic (discoloured) fingers and toes that may progress to frank gangrene of the digits or upper and lower extremities. It is imperative to rule out heparin-induced DIC, which may masquerade as sepsis-induced DIC.

Hepatic

Liver dysfunction is common in sepsis, whereas sepsis-induced acute liver failure is rare, occurring in ς% of patients 91 . Sepsis-induced liver injury is indicated by increased concentrations of serum alanine transaminase and increased levels of bilirubin. The exact aetiology of liver dysfunction in sepsis is unknown. Undoubtedly, a large part of liver dysfunction in patients with septic shock is due to centrilobular necrosis of the liver secondary to poor hepatic perfusion. Autopsy studies of patients who died of sepsis have shown necrotic hepatocytes in the regions surrounding the central veins 65,67 . In addition to necrotic cell death in the livers of patients with sepsis, hepatocytes have also been observed to be undergo apoptotic cell death 65,67 . Interestingly, electron microscopy has shown that there are increased autophagic vacuoles present within hepatocytes from patients with sepsis. In rare cases, autophagic vacuoles were so extensive as to be consistent with autophagy-induced cell death 92 . Thus, it seems that hepatocytes undergo multiple different types of cell death in sepsis.


Fight-or-flight response

Our editors will review what you’ve submitted and determine whether to revise the article.

Fight-or-flight response, response to an acute threat to survival that is marked by physical changes, including nervous and endocrine changes, that prepare a human or an animal to react or to retreat. The functions of this response were first described in the early 1900s by American neurologist and physiologist Walter Bradford Cannon.

When a threat is perceived, the sympathetic nerve fibres of the autonomic nervous system are activated. This leads to the release of certain hormones from the endocrine system. In physiological terms, a major action of these hormones is to initiate a rapid, generalized response. This response may be triggered by a fall in blood pressure or by pain, physical injury, abrupt emotional upset, or decreased blood glucose levels (hypoglycemia). The fight-or-flight response is characterized by an increased heart rate (tachycardia), anxiety, increased perspiration, tremour, and increased blood glucose concentrations (due to glycogenolysis, or breakdown of liver glycogen). These actions occur in concert with other neural or hormonal responses to stress, such as increases in corticotropin and cortisol secretion, and they are observed in some humans and animals affected by chronic stress, which causes long-term stimulation of the fight-or-flight response.

In addition to increased secretion of cortisol by the adrenal cortex, activation of the fight-or-flight response causes increased secretion of glucagon by the islet cells of the pancreas and increased secretion of catecholamines (i.e., epinephrine and norepinephrine) by the adrenal medulla. The tissue responses to different catecholamines depend on the fact that there are two major types of adrenergic receptors (adrenoceptors) on the surface of target organs and tissues. The receptors are known as alpha-adrenergic and beta-adrenergic receptors, or alpha receptors and beta receptors, respectively (see human nervous system: Anatomy of the human nervous system). In general, activation of alpha-adrenergic receptors results in the constriction of blood vessels, contraction of uterine muscles, relaxation of intestinal muscles, and dilation of the pupils. Activation of beta-receptors increases heart rate and stimulates cardiac contraction (thereby increasing cardiac output), dilates the bronchi (thereby increasing air flow into and out of the lungs), dilates the blood vessels, and relaxes the uterus.

The Editors of Encyclopaedia Britannica This article was most recently revised and updated by Adam Augustyn, Managing Editor, Reference Content.


Renal Klotho Regulation by 1,25(OH)2D Occurs via a Primary Genomic Mechanism

The kidney is both the source of endocrine 1,25(OH)2D and a target for its actions

As we have emphasized in reviews over the past 20 years, ( 32, 62, 64-69 ) the kidney is the nexus of the vitamin D endocrine system: both generating 1,25(OH)2D in response to hypocalcemic signals and engaging as a site for metered bone mineral elimination and/or reabsorption. Fig. 3 illustrates the central role of the kidneys in the metabolic activation of vitamin D catalyzed by CYP27B1 in response to PTH-elicited stimulation of the renal enzyme, normally under conditions such as hypocalcemia. The effects of endocrine 1,25(OH)2D are sufficient to generate normal bone mineral physiology as we now understand it, as well as to promulgate the plethora of other biological actions now attributed to 1,25(OH)2D/VDR–RXR. As also depicted in Fig. 3, 1,25(OH)2D/VDR-RXR drives transcriptional activation of a minimum of six genes in kidney, including Klotho (KL), NPT2a, NPT2c, TRPV5, CaBP28K, and CYP24A1. Developed below is one focus of the current review, namely the relatively recent discovery that 1,25(OH)2D/VDR–RXR induces KL mRNA in the kidney. ( 70 ) We also highlight the progress made in the last decade defining the pathobiological significance of Klotho and characterize the mechanism of its regulation by the vitamin D hormone.

Renal Klotho expression is governed by 1,25(OH)2D/VDR–RXR binding to VDREs

α-Klotho (referred to herein as Klotho) was first reported by Kuro-o and colleagues. ( 71 ) Its disruption in mice is associated with soft tissue calcification, profound hyperphosphatemia, osteoporosis, emphysema, arteriosclerosis, skin atrophy, infertility, hypoglycemia, and a curtailed lifespan. A recessive inactivating mutation in the human KL gene elicits the phenotype of severe tumoral calcinosis. ( 72 ) KL is expressed primarily in the kidneys and brain choroid plexus. ( 73 ) With respect to the regulation of Klotho biosynthesis, Forster and colleagues ( 70 ) reported that 1,25(OH)2D significantly induces mRNA expression of KL in a human renal (HK-2) cell line and kl in a mouse distal convoluted tubule (mpkDCT) cell line. These findings indicate that 1,25(OH)2D is capable of both amplifying FGF23 responsiveness in the kidney by inducing the Klotho membrane coreceptor for FGF23 and of eliciting elaboration of the shed soluble Klotho hormone. To mechanistically probe regulation of KL by 1,25(OH)2D, Forster and colleagues ( 70 ) performed bioinformatic analyses of both the human and mouse Klotho genes, which unveiled numerous candidate VDREs in mouse and human genes. ( 70 ) When assessed for functionality by cotransfection of reporter constructs into HK-2 cells, only one mouse VDRE (AGGTCAgagAGTTCA) located at −35,360 bp and two human VDREs (TGAACTctaCGAACC and TGAACTtctTGAACT) located on the negative strand at −47,293 bp and −32,203 bp, respectively, displayed a potency similar to the established rat osteocalcin VDRE. ( 70 ) Notably, the –35 kb area of the mouse Klotho gene given in Fig. 4A is marked by VDR and RXR in vivo as deciphered by ChIP-seq technology (J. Wesley Pike, personal communication, November 24, 2017). Furthermore, a ChIP-seq map of the human genome ( 74 ) yields an additional pair of VDREs, AGTTGAaagGGTTCC and GGAACTgcaTCCACC (negative strand), in the first intron of KL at +1536 and + 1392 bp, respectively. We thus propose that 1,25(OH)2D-liganded VDR–RXR induces Klotho expression by binding to functional VDREs somewhat remote to the mouse and human KL structural genes, with the human VDREs apparently being separated by as much as 49 kb. Importantly, the VDREs identified in KL genes fit the pattern of possessing nearby cis-elements that bind c/ebpβ and runx2 (Fig. 4A is an illustration of this for the mouse Klotho VDRE), a signature property of many 1,25(OH)2D/VDR-RXR-induced genes, ( 75 ) most recently highlighted by the case of mmp13. ( 76 ) Finally, as presented in Fig. 4B for comparison of VDRE environments, the promoter proximal region of the mouse spp1 gene reveals a classic VDRE that is flanked on the 5′-side by a runx2 site and on the 3′-side by a c/ebpβ element. In combination with the data of Tsujikawa and colleagues, ( 77 ) that 1,25(OH)2D increases steady-state Klotho mRNA levels in mouse kidney in vivo, the results reviewed herein verify that 1,25(OH)2D is the first discovered natural inducer of the Klotho longevity gene. Moreover, Tsujikawa and colleagues ( 77 ) showed that renal Klotho mRNA induction after 1,25(OH)2D injection displayed identical kinetics to those of renal cyp24a1, a well-established primary induction target for 1,25(OH)2D/VDR. In summary, Klotho is controlled by 1,25(OH)2D via a primary VDR–RXR mechanism consisting of direct binding of the hormone-receptor complex to bona fide VDREs remote from the structural gene, but each positioned in the immediate neighborhood of cis-associated c/ebpβ and runx2 cooperating transcription factors that apparently generate multiprotein complexes to drive induction of KL mRNA biosynthesis.

Klotho and its actions on ion transport in the kidney

The gene products for KL consist of multiple Klotho protein forms including a full-length transmembrane Klotho, a proteolytically shed soluble Klotho, and a much less-abundant secreted truncated Klotho with unknown function that may not exist in humans because of degradation of alternatively spliced Klotho mRNA. ( 78 ) The transmembrane Klotho (mKL) form is cleaved by membrane-anchored proteases including ADAM17, liberating the shed soluble Klotho (sKL) that contains both KL1 and KL2 domains functional in FGF23 ligand binding by the coreceptor complex of sKL and FGFR1c. After entering the circulation, this shed soluble Klotho form may function as a circulating hormone-like principle. ( 78 ) This circulating Klotho form has been reported to employ the glycosyl hydrolase catalytic activity to regulate the TRPV5 calcium channel ( 79 ) and the renal outer medullary potassium channel 1 (ROMK1). ( 80 ) However, in a recent review, Erben ( 50 ) summarized the work of Chen and colleagues, ( 81 ) who crystallized the ternary complex of the extracellular domain of FGFR1c, FGF23 ligand, and the Klotho ectodomain to provide compelling experimental evidence that Klotho lacks any biologically relevant glycosidase activity. Thus, there has occurred a recent paradigm shift in our understanding of Klotho to reveal that it is devoid of enzymatic activity and instead increases the abundance of TRPV5 on the renal cell membrane through FGF23 signaling (Fig. 3) via ERK1/2, SGK1, and WNK4 to depress endocytotic removal of TRPV5 from the plasma membrane. ( 50 ) Thus, 1,25(OH)2D, by independently inducing TRPV5 ( 82 ) and Klotho, ( 70 ) apparently elicits FGF23-driven calcium retention at the kidneys. One could speculate, therefore, that 1,25(OH)2D/VDR–RXR chronically initiates calcium conservation at the kidneys over a lifetime, possibly to lower the incidence of osteoporosis. ROMK1 is likely upregulated by Klotho analogously to TRPV5 through enhancement in the apical membrane via ERK1/2, SGK1, and WNK4-signaling, but the outcome would be increased potassium excretion.

Klotho is a FGF23 coreceptor in controlling phosphate and vitamin D metabolism

The most physiologically significant function of full-length, membrane Klotho is to act as a renal coreceptor of FGFR1c in the feedback control of phosphate and vitamin D metabolism by bone-derived FGF23 (Fig. 3). FGF23 signals by binding to renal FGFR1c/Klotho coreceptors and an ERK/SGK1/NHERF1 phosphorylation-transduction pathway to cause degradation of membrane sodium phosphate cotransporter type 2a (Npt2a), with resultant downregulation of phosphate reabsorption to promote phosphaturia. ( 49 ) FGF23 also signals to repress renal specific CYP27B1 ( 51 ) via a pathway that involves the binding of as yet unidentified transcription factors (triggered by ERK1/2) to upstream regions of the mouse cyp27b1 gene centered at approximately −5 kb and −12.5 kb. ( 47 ) Finally, FGF23 further signals to induce renal CYP24A1 by binding to FGFR1c/Klotho coreceptors and promulgating an as yet unknown signal transduction pathway initiated by ERK1/2 and including unidentified transcription factors that bind to a downstream region of the mouse cyp24a1 gene centered at approximately +27 kb and possibly to the promoter proximal sequence immediately upstream of the transcription start site, ( 53 ) resulting in induction of CYP24A1. ( 49, 52 ) The latter two actions serve to curb 1,25(OH)2D levels (Fig. 3). Remarkably, double knockouts of FGF23 (or its Klotho coreceptor) with either VDR ( 83 ) or CYP27B1 ( 84 ) essentially rescue FGF23 null mice, underscoring the role of FGF23 and Klotho as counter-regulatory hormones to 1,25(OH)2D, which appears to be the key to their health and longevity benefits. Shed, soluble Klotho may possess systemic antiaging properties independent of the phosphaturic and 1,25(OH)2D-attenuating actions of transmembrane Klotho, but the mechanism(s) of these actions is not known. ( 80 ) Conversely, although FGF23 is antiaging at the kidney by eliciting phosphate elimination and detoxifying 1,25(OH)2D, its “off-target” actions could actually be pro-aging in terms of coronary artery disease, as well as potential neoplastic actions in the colon, ( 85 ) and it is possible that these off-target FGF23 pathologies are “buffered” by soluble Klotho. ( 86 ) Upregulation of Klotho by 1,25(OH)2D ( 70 ) is thus consistent not only with potentiation of FGF23 signaling in the kidney, but also may offer protection for other cell types (eg, vascular and colon), in which the circulating soluble form of Klotho could exert beneficial actions. ( 87 )

Klotho exerts numerous additional bioprotective and cellular antiaging actions

Klotho exerts antioxidative effects, some of which are reminiscent of 1,25(OH)2D actions. For example, Klotho has been reported to bind to the transient-receptor potential canonical Ca 2+ channel 1 (TRPC-1) through its KL2 domain, and regulates TRPC-1-mediated Ca 2+ entry to maintain endothelial integrity and prevent Ca 2+ -stimulated nitric oxide synthetase formation, which contributes to the formation of potent reactive nitrogen species. ( 88 ) Thus, the maintenance of Ca 2+ and redox signaling at a low resting state by 1,25(OH)2D and its effectors, Nrf2 and Klotho, appears to constitute the mechanism whereby 1,25(OH)2D and Klotho prevent the ravages of oxidation. Also, Wang and colleagues ( 89 ) reported that Klotho downregulates the expression of a catalytic subunit of NADPH oxidase and suppresses angiotensin II-induced superoxide production, oxidative damage, and apoptosis through the cAMP/PKA pathway. In vivo, Klotho gene delivery similarly attenuates NADPH oxidase activity and superoxide production to prevent the progression of spontaneous hypertension and resulting renal damage. ( 87 ) These findings led us to propose that 1,25(OH)2D/VDR–RXR primary induction of Klotho mRNA represents a natural pathway to maintaining healthful aging, with intracellular calcium current regulation and mitigation of oxidation being common themes.

Klotho is also known to impact Wnt signaling ( 73 ) to curtail renal fibrosis and perhaps suppress tumorigenesis. Klotho-mediated regulation of Wnt signaling was reported by Liu and colleagues, ( 90 ) who showed that sKL binds to Wnt ligands to suppress downstream signal transduction, and that KL knockout enhances Wnt signaling in mice. Regarding the disease-related consequences of Wnt signaling suppression by Klotho, activated Wnt3 signaling extends the cell cycle by arresting it at the G2/M phase, and induces fibrogenic cytokines in mouse kidney but Klotho-treated cells circumvent this phase and are protected against renal fibrosis. ( 91 ) The loss of Klotho may therefore contribute to kidney injury by releasing the inhibition of pathogenic Wnt/β-catenin signaling. ( 92 ) Indeed, in vivo expression of Klotho decreases the activation of renal β-catenin and diminishes renal fibrosis in chronic kidney disease. ( 92 ) Conversely, reduced Klotho expression aggravates renal interstitial fibrosis, ( 93 ) and overexpression of sKL abolishes the fibrogenic effects of TGF-β1. ( 92, 94 ) In summary, Klotho overexpression or supplementation protects against fibrosis in several models of renal and cardiac fibrotic disease, with its actions appearing to be rooted in the direct inhibitory effects of circulating/soluble Klotho on TGFβ1 and Wnt signaling. ( 95 )

Regarding antitumor actions of Klotho, Behera and colleagues ( 96 ) found a correlation between loss of Klotho and a gain in Wnt5A expression, leading to progression of melanoma. Similarly, Abramovitz and colleagues ( 97 ) have reported that both membrane and soluble Klotho serve as tumor suppressors by inhibiting tumor cell proliferation through regulation of IGF-1 signaling. Another in vivo experiment showed that soluble Klotho possesses greater inhibitory effects on tumor cell growth than full-length membrane Klotho. ( 97 ) Therefore, through attenuation of TGFβ1-, Wnt-, and IGF1-signaling pathways, Klotho also inhibits tumorigenesis. The promoter proximal region of the Klotho gene is reported to be hypermethylated in cancer, and transgenic overexpression or introduction of Klotho protein is observed to retard tumor growth in several animal models.

With respect to the antifibrotic qualities of Klotho, high concentrations of extracellular phosphate are toxic to cells, and impaired urinary phosphate excretion increases serum phosphate levels to induce a premature-aging phenotype. Urinary phosphate levels are increased by dietary phosphate overload and might induce tubular injury and interstitial fibrosis. Extracellular phosphate exerts its cytotoxic effects by forming insoluble nanoparticles with calcium and fetuin-A. These nanoparticles are referred to as calciprotein particles and are capable of inducing various cellular responses, including the osteogenic transformation of vascular smooth muscle cells and cell death of vascular endothelial cells and renal tubular epithelial cells. Calciprotein particles can be detected in the serum of animal models of kidney disease and in patients with chronic kidney disease (CKD) and probably contribute to the pathogenesis of CKD. This important insight provides a mechanism whereby Klotho, by preventing hyperphosphatemia, protects the vascular and renal systems, thereby prolonging lifespan. In addition, 1,25(OH)2D is thought to cooperate with Klotho in retarding vascular calcification by the induction of OPN (Fig. S1C), a powerful antimineralization factor. ( 60 ) Ironically, the two renal hormones that are deficient in patients with chronic renal failure because of renal mass loss, namely 1,25(OH)2D and Klotho, are two vital effectors of renal and vascular health, suggesting a strategy for the prevention and treatment of vascular disease, as well as CKD.

It is controversial whether Klotho affects insulin secretion and sensitivity

It has been observed that Klotho increases the plasma membrane retention of TRPV2, leading to enhanced glucose-triggered insulin secretion from pancreatic β cells. ( 98 ) Vitamin D has long been known to promote insulin secretion, ( 99 ) meaning that insulin release is yet another example of the dual beneficial effects of 1,25(OH)2D and Klotho. Klotho KO mice exhibit less energy storage and expenditure compared with WT mice, ( 100 ) as well as attenuated insulin production and enhanced insulin sensitivity. ( 101, 102 ) However, Anour and colleagues ( 103 ) demonstrated that Klotho lacks a vitamin D-independent physiological role in glucose homeostasis, bone turnover, and steady-state PTH secretion, in vivo, casting doubt on Klotho alone as a bona fide regulator of any of these phenomena. In a sense, this original research by the Erben group ( 103 ) was prescient and consistent with the recent opinion piece ( 50 ) that “Klotho's effects on mineral homeostasis are fibroblast growth factor-23 dependent.” Are all Klotho actions dependent upon FGFR1 signal transduction initiated by FGF23? This is the provocative question regarding Klotho in 2020! Because FGFR1 is almost universally expressed, are all of the influences of Klotho on aging actually promulgated by FGF23 signaling? In an opposing theory, Dalton and colleagues ( 78 ) propose that soluble Klotho targets GM1 and GM3 sialogangliosides clustered in membrane lipid rafts which act as a Klotho “receptor” that signals control of growth factor functions. Regardless of the mechanism by which Klotho functions, the many consequences of Klotho activity, such as antioxidation, antifibrosis, antimalignancy, anticalcium transients, and antiphosphatemia, clearly collectively contribute to the antiaging potential of Klotho. Based on all these observations, Klotho can be considered a 1,25(OH)2D/VDR-induced, organ-protection hormone that promotes healthful aging by delaying chronic diseases, even if exclusively through beneficial signaling via FGFR1.

Accordingly, both FGF23 and Klotho have recently been implicated in maintaining male reproductive function. ( 104 ) Indeed, Hansen and colleagues, ( 104 ) employing mice null for either FGF23 or Klotho, found that global loss of either FGF23 or Klotho compromised testicular weight and reduced sperm count as well as motility, whereas FGF23 enhances testicular weight in WT mice. However, germ-cell–specific knockout of Klotho elicited neither decreased sperm count nor mobility, although fewer pregnancies and Klotho heterozygous pups occurred in this group that was characterized by overexpression of testicular trpv5 and npt2b, ( 104 ) indicating that testicular calcium and phosphate exchange play a role in the actions of FGF23 and Klotho in gonads, as they do in the functions of FGF23 and Klotho in kidney.

Klotho supports synaptic functioning and protects the central nervous system

In recent years, interest has arisen in the potential role of Klotho in the brain, including its possible protection of the central nervous system (CNS) and prevention of neurological diseases such as depression and the decline in cognitive function associated with aging. ( 105, 106 ) Indeed, high levels of Klotho originating in the choroid plexus are postulated to function as a gatekeeper at the interface between the brain and immune system in the choroid plexus. ( 107 ) Klotho depletion in aging or disease may weaken this barrier and promote immunomediated neuropathogenesis. Experimental depletion of Klotho from the choroid plexus enhanced microglial activation in the hippocampus after peripheral injection of mice with lipopolysaccharide. In primary cultures, Klotho suppressed thioredoxin-interacting protein-dependent activation of the NLRP3 inflammasome in macrophages by enhancing FGF23 signaling. ( 107 ) Finally, peripheral delivery of an α-Klotho fragment acutely enhanced cognition and neural resilience in young, aging, and disease-model mice by inducing GluN2B cleavage and increasing NMDAR-dependent synaptic plasticity. ( 108 ) Thus, as 1,25(OH)2D is hypothesized to affect the CNS, Klotho induction by 1,25(OH)2D/VDR appears to be able to accomplish similar enhancement of synaptic functions—leading to the proposal that part of the benefits to brain health afforded by 1,25(OH)2D are mediated by its primary induction of Klotho, analogous to the known improvements in renal and vascular health promulgated by 1,25(OH)2D/VDR action to boost Klotho.

Summary of the significant actions of Klotho that are potentially antipathogenic

In summary, the primary genomic induction of renal Klotho by 1,25(OH)2D/VDR–RXR leads to the following extensive list of biological consequences that benefit the entire organism because Klotho: (i) acts as a renal coreceptor for FGF23 in binding to FGFR isoforms that signal feedback control of hyperphosphatemia and excessive 1,25(OH)2D levels, both of which can lead to vascular calcification via osteogenic transformation of vascular smooth muscle cells and vascular endothelial cell death, as well as to renal tubular epithelial cell death resulting in chronic kidney disease (ii) exerts antioxidative effects and controls intracellular calcium currents to protect tissues from the ravages of aging (iii) blunts TGFβ1 and Wnt signaling to prevent renal and cardiac fibrotic disease, as well as tumorigenesis (iv) acts in the CNS to enhance synaptic functions that mediate plasticity to support neural resiliency and cognition, as well as to protect against immunomediated degenerative neuropathogenesis. Although Klotho and its vitamin D hormone inducer may only play a small part in healthful aging, the sheer number of pathways impacted by this duo of renal hormones leads one to suspect that their pleiotropic actions are certainly a crucial factor in the quality and quantity of life. Because potentiation of FGF23/FGFR1 signaling is the only proven function of Klotho, the next section of this review addresses the induction of the FGF23 ligand by the 1,25(OH)2D renal hormone.


Discussion

Based on extensive time-resolved data sets in primary erythroid progenitor cells and mathematical modeling, we dissected the different contributions of the two feedback regulators CIS and SOCS3 and identified dual transcriptional feedback effective at discrete ligand concentrations as key property of the EpoR-STAT5 signaling system. This result underlines the general principle of negative feedback regulation to increase control and stability of transcriptional responses over a broad range of input values ( Becskei and Serrano, 2000 Freeman, 2000 ). Our observation that multiple feedbacks are necessary to orchestrate tight regulation of transcription factor activity from very low to very high ligand doses suggests a new strategy of SOCS-mediated feedback control.

Most of the previous JAK/STAT models have a complex structure comprising a large number of variables and literature-based parameters ( Yamada et al, 2003 Zi et al, 2005 Soebiyanto et al, 2007 ). In this work, as an alternative modeling strategy, a bottom-up approach was employed, which aims at fully identifiable parameters that are essential to obtain models with high predictive power ( Bruggeman et al, 2002 Aldridge et al, 2006 ). We therefore employed a large set of data measured under different perturbation conditions in combination with a model structure comprising the minimal number of parameters necessary to explain the data. Parameter identifiability was analyzed by the profile likelihood approach ( Raue et al, 2009 ). Applying this method, we could establish a dual negative feedback model of JAK2-STAT5 signaling with structurally and in most cases also practically identifiable parameters.

Qualitatively, the important role of STAT5 as crucial regulator of survival and differentiation of erythroid progenitor cells has been established previously ( Socolovsky et al, 2001 Yao et al, 2006 Grebien et al, 2008 Zhu et al, 2008 ). We demonstrate that the calculated integrated response of pSTAT5 in the nucleus accurately correlates with the experimentally determined survival of CFU-E cells, thereby providing a quantitative link of the dependency of primary CFU-E cells on pSTAT5 activation dynamics. In line with this, a recent study that quantitatively determined pSTAT5 activity levels in IL-2 induced single T cells reported on a causal link between enhanced pSTAT5 levels and decreased cell death ( Feinerman et al, 2010 ). We concluded that fine-tuned changes in STAT5 activation levels are a critical determinant for cell fate decisions. This observation is also true during an earlier differentiation stage of the erythroid lineage. A previous study showed that partial depletion of STAT5 from CD34+ cells by a lentiviral RNAi approach in the presence of thrombopoietin and stem cell factor resulted in a decrease of erythroid progenitors (BFU-E). Conversely, overexpression of an activated STAT5a mutant strongly induced erythroid differentiation ( Olthof et al, 2008 ).

Our finding that the integral of npSTAT5 is a good predictor for survival does not imply that survival is exclusively depending on STAT5. The integral of STAT5 activity in the nucleus transfers quantitative information about extracellular ligand concentrations to downstream signals, i.e., expression of anti-apoptotic target genes such as Pim-1 that contribute to the ultimate cellular response. Additional pro-survival factors such as the PI3K/AKT pathway that have been shown previously to be involved in prevention of apoptosis in CFU-E cells ( Bouscary et al, 2003 ) may also contribute. However, overexpression of constitutively active AKT could not substitute for the apoptosis-suppressing function of the EpoR-STAT5 pathway in JAK2 −/− erythroid cells ( Ghaffari et al, 2006 ). Hence, though we cannot rule out the involvement of other pathways, our study underlines the direct relationship of the integral STAT5 response and survival decisions of primary erythroid progenitor cells.

A major bottle-neck in combining signal transduction events with cellular phenotypes is the discrepancy in the time scale and stimuli concentrations that are applied in the different experiments. The sensitivity of biochemical assays to determine phosphorylation events within minutes or hours after stimulation is usually lower than the threshold of sensitivity in assays to determine the physiological response after one or more days. By employing our mathematical model, we were able to compute the integrated response of STAT5 at Epo concentrations that are beyond the threshold of biochemical experiments. This allowed us to directly link the activation status of the transcription factor with a cellular response. By correlation analysis, we could identify the early signaling phase (⩽1 h) of STAT5 to be most predictive for survival decisions, which was determined ∼24 h later. Interestingly, an earlier study that investigated a compendium of signaling pathways and responses in TNF-, EGF- and insulin-treated HT-29 cells revealed similar results in the predictability of apoptosis-survival cell fate decisions by early signaling events ( Gaudet et al, 2005 ). The authors report that early signaling events between 5 and 90 min immediately downstream of the receptors were more predictive than 2–8 h, presumably because information is forwarded to downstream transcriptional events. Thus, we hypothesize that as a general principle in apoptotic decisions, ligand concentrations translated into kinetic-encoded information of early signaling events downstream of receptors (⩽1 h) can be predictive for survival decisions 24 h later.

We could show by experimental data and mathematical modeling that SOCS3 and CIS reduce the steady-state STAT5 phosphorylation level after ∼1 h of stimulation. Our results at a single Epo concentration are consistent with other studies that investigated the regulation of STAT phosphorylation dynamics by SOCS proteins. As one example, SOCS3 that is induced upon IL-6 stimulation controls the late phosphorylation profile of STAT1 and STAT3 in the liver. This was shown in an experimental study using a conditional SOCS3 knockout, which resulted in enhanced phosphorylation of STATs, although the amplitude was unaffected ( Croker et al, 2003 ). The importance for adjusting the steady-state phase of transcription factor activity by transcriptional feedback regulators was shown also for other signaling pathways. EGF-induced ERK signaling in HeLa cells is increased 1 h after stimulation when translation is blocked by cycloheximide ( Amit et al, 2007 ). This temporal regulation pattern, including short-term deactivation by constitutively expressed phosphatases and late-phase modulation by slow transcriptional feedbacks may evolve as a general paradigm for tightly controlled cytokine-induced signaling pathways ( Legewie et al, 2008 ). The existence of multiple overlapping feedback regulation mechanisms, each with distinct temporal characteristics, ensures the effective control of the signaling dynamics to appropriately fine-tune cellular responses.

The fact that one or two SOCS family members are induced upon stimulation appears to be a general principle in cytokine-induced signaling, although there is no simple relationship between a particular cytokine and the pattern of respectively induced SOCS mRNAs ( Krebs and Hilton, 2001 ). Induced in a cell type-specific and cytokine-dependent manner, SOCS proteins can distinctively coordinate cytokine-induced responses. A prime example for the evidence that two SOCS proteins uniquely attenuate STAT dynamics is the study of Wormald et al (2006) showing that SOCS1 and SOCS3 regulate STAT1 and STAT3 phosphorylation dynamics with different kinetics. Yet, the impact of different ligand concentrations has not been considered so far. By using model simulations to analyze the inhibitory effects of CIS and SOCS3 on the STAT5 phosphorylation level at previously unobserved Epo concentrations, our results revealed that the two feedbacks are most effective at different Epo concentration ranges. Conventional experimental techniques, however, cannot resolve the entire dynamic range of ligand concentrations. According to our mathematical model, the major role of CIS is modulating STAT5 phosphorylation levels at low, basal Epo concentrations, whereas SOCS3 is essential to control the STAT5 phosphorylation levels at high Epo doses.

As potential molecular mechanism of this dose-dependent inhibitory effect, we could identify the quantity of pJAK2 relative to pEpoR that increases with higher Epo concentrations. Since SOCS3 can inhibit JAK2 directly via its KIR domain to attenuate downstream STAT5 activation, SOCS3 becomes more effective with the relative increase of JAK2 activation. These observations are further supported by the CIS and SOCS3 overexpression experiments that showed the strong inhibition of JAK2 phosphorylation only by SOCS3 in primary CFU-E cells. Moreover, in cells overexpressing SOCS3 the STAT5 phosphorylation level was more extensively reduced and survival was more decreased than in CIS overexpressing cells considering similar overexpression levels.

Our findings raise the question why it is important for the cell to tightly control the long-term steady-state signaling level of STAT5 by transcriptional feedback regulators over the entire range of high and low Epo doses although the first hour of STAT5 activation is predictive for the survival decision. We hypothesize that when the decision for survival has occurred, it is essential to constrain signaling to a residual steady-state level in order to prevent aberrant events that could lead to uncontrolled erythroid progenitor growth. Constitutive phosphorylation of the JAK2/STAT5 pathway caused by activating JAK2 mutations has a crucial role in the onset of polycythemia vera (PV), a disease that is characterized by the formation of endogenous colonies with Epo-independent differentiation ( Prchal and Axelrad, 1974 Weinberg et al, 1989 Kota et al, 2008 ). Moreover, human erythroid progenitor cells transduced with a constitutive phosphorylated form of STAT5 were reported to survive, proliferate and differentiate in the absence of Epo and in this way mimic the PV phenotype. The essential requirement for progenitor cells to tightly constrict the Epo input signal after 1 h of stimulation over the broad range of physiological Epo concentrations that can vary over 1000-fold is already apparent at the upstream receptor level. Different studies have shown that activation of EpoR and JAK2 is rapidly terminated within the first hour by dephosphorylation as well as internalization and degradation of Epo ( Gross and Lodish, 2006 Becker et al, 2010 ).

We propose that the transcriptional feedback proteins CIS and SOCS3 are required to tightly adjust the phosphorylation level of STAT5 after 1 h of stimulation. This hypothesis is supported by reports demonstrating the crucial role of SOCS3 in embryonic development. Mice lacking the SOCS3 gene exhibit embryonic lethality at days E12–16. Marine et al (1999) showed that these mice display erythrocytosis with dramatic expansion of erythropoiesis within the fetal liver as well as throughout the embryo. Roberts et al (2001) demonstrated that the death is associated with abnormalities in the placenta. Vice versa, enforced expression of SOCS3 in vivo specifically suppressed fetal liver erythropoiesis ( Marine et al, 1999 ). Moreover, a loss-of-function mutation of SOCS3 has been proposed to contribute to the onset of myeloproliferative disease in PV patients ( Suessmuth et al, 2009 ). In cancer, high input doses are mimicked by aberrant activation of JAKs that are frequently mutated in many tumor cells. Interestingly, there are numerous recent studies showing that in several malignant tumors JAK activating mutations are complemented by gene silencing of SOCS3 and SOCS1, the two SOCS members which contain a KIR domain ( He et al, 2003 Chim et al, 2004 Johan et al, 2005 Jost et al, 2007 ). This underlines the essential role of SOCS3 to control high input doses. Thus, the absence of SOCS3 severely impacts the growth and survival of erythroid progenitor cells and is essential to abrogate signaling downstream of the EpoR at long-term steady-state phosphorylation levels upon high upstream input conditions.

In contrast to SOCS3, our model predicts that CIS acts most efficiently at a single point of the network at low ligand concentrations. This is line with other reports that demonstrated the major role of CIS as a specific competitive binding inhibitor of STAT5 at the pY401 position of the EpoR ( Matsumoto et al, 1997 Verdier et al, 1998 Ketteler et al, 2002 ). The specific inhibition of STAT5-mediated responses by CIS is also supported by transgenic mice that overexpress CIS. These animals display diminished expression of STAT5-mediated responses in growth hormone and prolactin signaling, similar to STAT5 ΔN/ΔN knockout mice ( Matsumoto et al, 1999 ). In contrast to SOCS3 knockout mice, CIS knockout mice are viable, but show an increase in hematopoietic progenitor cells ( Kubo et al, 2003 ), which is in line with our model prediction of CIS as modulator of fine-tuned pSTAT5 responses at basal level Epo input.

In summary, our mathematical approach provided new insights into the specific function of feedback regulation in STAT5-mediated life or death decisions of primary erythroid cells. We dissected the roles of the transcriptionally induced proteins CIS and SOCS3 that operate as dual feedback with divided function thereby facilitating the control of STAT5 activation levels over the entire range of physiological Epo concentrations. The detailed understanding of the molecular processes and control distribution of Epo-induced JAK/STAT signaling can be further applied to gain insights into alterations promoting malignant hematopoietic diseases.


What Does It All Come Down To?

Here you are, at my final say on PHPT.

Look for it in every calcium stone former, and look with diligence and a suspicious eye. For PHPT is a systemic disease with real potential to harm people that can be cured in most by modern and highly effective and safe surgery. Whereas many older women who have PHPT without any apparent consequence may never need a surgery and do well, no one recommends against surgical cure for stone formers.

Even if normocalcemic, as most are, calcium stone formers may harbor among their ranks a few smoldering normocalcemic PHPT patients who will manifests their disease only over time. This means I favor yearly fasting blood tests that no one thus transformed misses their chance for a cure.

Exclusion of all systemic diseases begins all stone prevention efforts. Frankly it is a fool’s errand to treat any calcium stone former without first excluding systemic disease as the cause. This requires serum and 24 hour urine testing and significant clinical ability. True, a majority will have nothing but idiopathic calcium stones, and whatever money and time used was used to no direct gain. But even one PHPT patient left untreated, even one, could in a year or two of unbridled stones mount up surgical costs in the tens of thousands of dollars. And miseries and dangers too. Even more, that patient’s disease can probably be cured.

Worse happens if one through lack of proper testing misses even more damaging diseases like severe hyperoxaluria. Acute kidney injury and even irreversible kidney failure wait on such patients from even incidental dehydration.

For these and other reasons I am opposed utterly to those clinically naive statisticians who advise all calcium stone formers be treated without blood and 24 hour urine testing. No idea is more likely to cause some patients real harm and, although I hesitate to mention the vulgar, some unlucky physicians their unwelcome day in court.

48 Responses to &ldquoPRIMARY HYPERPARATHYROIDISM&rdquo

Carol

Dr Coe, how many days or weeks apart should you retest the blood calcium and PTH to check for Primary Hyperparathyroidism, you mentioned morning fasting and to do at least 3 times. Thank you!

Fredric L Coe, MD

Hi Carol, I tend to get bloods to the convenience of the patient. What matters is fasting, morning blood draw, and calcium and PTH in the same sample. If calcium levels are not entirely within the normal range I keep repeating until it becomes obvious if the average is really high – if so we have a probability of PHPT. Regards, Fred Coe

Carol

Dr. Coe, I was sent to a general surgeon by my primary doctor for a third opinion on primary hyperparathyroidism because of forming stones and my labs being iffy with a couple higher PTH’s but only one high calcium. My endocrinologist sent me to Quest lab to repeat and they were normal and she does not believe I have this. I was dismayed that the surgeon who seems very knowledgeable and told me he does these surgeries often and spent a great amount of time with me told me it did not matter time of day or fasting that I had the PTH and Calcium blood tests done, he said over and over it didn’t matter if you were not fasting which my last two were not because the endocrinologist and urologist did not tell me to do them fasting. I stated to the surgeon after doing research online that they must be done fasting and in the morning, he just kept saying that does not matter. Of note, he also leans toward the endo doc that I do not have this and to get more labs to confirm for sure so that was encouraging that he wasn’t out to just do a surgery. I just wonder why so many physicians that should have knowledge when dealing with this illness don’t see how important this is. Do you come across these same type of physicians that don’t realize these need to be done fasting? I can possibly understand a primary doctor not knowing since it’s not their specialty but these other docs should know. I thank you for telling me this because I will make sure I get them fasting at my next visit.

Fredric L Coe, MD

Hi Carol, I hope I am not making problems for others here. Fasting does matter, as serum calcium rises with meals. However if your serum calcium values are all normal (I usually require less than 10.1 ml/dl to be satisfied about normal, whatever the lab normal range), then fasting does not matter as serum calcium would go up with meals and is still not high. Just be sure your serum calcium values are below 10.1. For PTH, it goes down with meals, because serum calcium rises. This is all normal physiology, and one can find these very small changes in our publications and others. High PTH with normal serum calcium can be low calcium diet, low vitamin D levels, or mild reduction of renal function. It is not treated surgically. Of course PHPT almost always includes high urine calcium excretion absent kidney disease, or some other complication. The crucial element is serum calcium, and I am afraid I have to disagree with any and all who do not believe in standardizing time of day and fasting to compare results to normal people. Regards, Fred Coe

ANGELA SMITH

I am a 69 year old woman. I have been forming stones for over 20 years an have had at least 20 procedures to treat them. About 5 years ago, routine blood tests showed my blood calcium at 10.6. My PTH was not high, but vitamin D was quite low. Sestamibi scan showed a parathyroid adenoma. I had a parathyroidectomy. Just recently I had another BMP and my PTH was 92. I saw my endocrine surgeon and he was pretty sure my parathyroid disease returned. He order another calcium and PTH. PTH came back 47 calcium 9.2. He walked back his diagnosis and said I do not have parathyroid disease. I’ve had a few years of reprieve from kidney stones. Suddenly, I have multiple stones in each kidney and a few weeks ago I ended up in the ED. My calcium was 12.2 at that time. I was treated for the stones and 15 small stones were retrieved. This was only a few weeks ago and I am again having right flank pain. My endocrine surgeon want me back in 6 months. I am not convinced that my parathyroid disease has not returned. Isn’t it unusual for PTH to go from 92 to 47 in such a short time? I know it can fluctuate, but I read the normally the fluctuations are within a few points. Here are my recent stats using parathyroid.com app. 1/2/20: Calcium 10, PTH 92, 1/20/20: Calcium 12, 1/24/20 Calcium 9.2, PTH 47, 2/9/20: Calcium 10.2, 2/11/20: Calcium 10. The app analysis says that is is “very likely” I have parathyroid disease, if the app is indeed reliable. During the 20 years of forming stones, my calcium was under 10. Vit. D and PTH never checked.

Fredric L Coe

Hi Angela, Your serum calcium levels do indeed seem high but they vary a lot. Here is what I would do: Get morning, fasting, serum calcium levels, fasting since midnight before the blood draw, and find out what your serum calcium really is. 12 is very high, and so is 10.2. PTH is regulated by serum calcium, so you cannot interpret a PTH without a serum calcium to use it with. Sometimes in early recurrent PHPT serum PTH is high fasting and serum calcium normal or high normal, but with food serum calcium rises abnormally and PTH falls into the normal range – the calcium is regulating PTH but at an abnormal way. ANyway, the diagnosis depends on fasting morning serum calcium values which will gradually rise into the abnormally high range if you have recurrent disease. You do not mention urine calcium, but often 24 hour urine calcium rises a lot as PHPT recurs. I suspect it has, and I would get a lot of serum calcium measurements. Regards, Fred Coe

Lynette Ackman

I had recurrent kidney stones. Then I was diagnosed with hyperparathyroidism. I am fortunate in that I was referred to an endocrinologist (Dr. Garzia Aleppo) that performed tests before scheduling me for surgery, and found I had calcium in my urine and a vitamin D deficiency. Once the vitamin D deficiency was corrected, the parathyroid levels became normal and no more kidney stones.

How do we inform more urologists about the connection between vitamin D and kidney stones? I’ve seen a urologist since that says I must “misunderstand” and vitamin D deficiency does not lead to stones… likewise, I had to fight with my father’s care providers to get them to test his vitamin D levels and try to find a cause for his recurrent stones.

Fredric L Coe

Hi Lynette, I think the sequence of events had another cause. Your high urine calcium may well be idiopathic hypercalciuria. Your serum calcium was presumably normal and your serum PTH high. This latter, the high PTH, was from vitamin D deficiency. The stones arise from the high urine calcium, and vitamin D deficiency is not the cause of that high urine calcium – genetics is the cause. Your father probably has high urine calcium from the same genetic cause, and you inherited it. Vitamin D metabolism is high in genetic hypercalciuria and vitamin D deficiency common. The high urine calcium may well continue to cause stones and needs its own care – this is detailed in the article. Regards, Fred Coe

Janine Tips

Hello. I have had two uric acid kidney stone surgeries by a urologist. I then went to a parathyroid specialist and she thinks I have hyperparathyrodism and wants to do surgery on my parathyroid glands. My blood calcium levels range from 9 – 10, with my intact PTH of 29. Since I have uric acid kidney stones, is there a connection to these kind of stones and hyperparathyroidism? I’ve not found information on uric acid kidney stones. I have cut way back on animal protein in hopes to help with this stone issue. I also have AFIB, MTHFR, and Osteopenia. I am 66 years old. Your thoughts on my situation would be greatly appreciated. Thank you sir.

Fredric L Coe

Hi Janine, As the article you post on notes, PHPT requires serum calcium be above normal and the values you post are not high. Your PTH is rather on the low side, although that is not a major point. I cannot imagine surgery given a lack of high serum calcium. Uric acid stones occur because of acid urine pH, and can be prevented completely by raising the pH. Of course your physicians are responsible for your care, so you should take my remarks for that they are-comments from a distant outsider. But before surgery they may want to get a second opinion, and that is something I can recommend without much hesitation. Regards, Fred Coe

Sonya Patterson

Dr. Coe, Can I get your help. Here are my labs over the course of the last several months. My doctor had me scheduled for surgery to remove Parathyroid, then decided there was a possibility it could be a renal leak. She put me on a dose of HCTZ on Oct. 3, but there is still great confusion – she tells me I am a mystery. Here are my labs and I have done 3 of the 24 urine quest. I am 48 and very healthy and fit. I had 2 occasions of kidneys stones over this past year and have osteopenia. She increased the HCTZ from 1/2 to a whole pill but it was causing my potassium to be low.
PTH Calcium Ionized Calcium
25-Sep High 119.4 9.6 1.26 then started HCTZ
11-Oct 9.8
16-Oct High 108.2 10 High 1.32
21-Oct 75.1 9.8 High 1.30
23-Oct High 115.0 1.27
31-Oct 87.3 9.8 1.26
14-Nov High 116.6 High 10.2 High 1.33
22-Nov High 125.8 9.7 1.26

24 hour Urine Quest
Calcium Urine Quest Bushite Quest Calcium oxalate Quest Sodium Urate Quest
23-Jul High 347 High 11.42 High 6.18 High 6.85
25-Oct High 331 High 9.08 High 2.93 High 4.34
19-Nov Waiting on these results

Fredric L Coe

Hi Sonya, Let’s count your serum calcium levels. Before the thiazide, 9.6 after going on it, 10, 9.8, 9.8, 10.2, 9.8 ionized calcium levels were 1.26 1.32, 1.3, 1.27, 1.26, 1.33, 1.26. The normal range is 1.3 – 1.5 with some variability among labs. I presume all of the bloods were taken fasting and in the morning, as serum calcium rises with meals. If so, the evidence for high serum calcium is weak indeed. The urine calcium values you give are 347 and 331 mg/d, both high. The SS values have nothing to do with the question of hyperparathyroidism or not. As in the article, I do not think your situation is complex intrinsically, but complicated by the drug. PHPT requires serum calcium be elevated and so if I were doing your work I would stop the drug, wait a week or two, and get more fasting morning serum calcium measurements, each with a PTH. If all of the serum calcium values are like most of those you quote I would presume you have mere idiopathic hypercalciuria and a cause of secondary high PTH such as low serum 25D, or low calcium diet, or perhaps even the slightest reduction of renal function. The thiazide raises serum calcium, and so makes it impossible to be sure if your real values are high – I suspect they are not. Please consider all of this as material to discuss with your physician as she is responsible for your care and I am a distant outsider with no real knowledge of your actual medical situation. My remarks, if scientifically correct, may not be pertinent to your case. Regards, Fred Coe

Michelle

Dr Coe: Thank you for sharing so much information on your website. I am writing about my husband, he is 55 years old, 5𔄀″. 155lb. He had 2 kidney stones in March 2015, 95% calcium oxalate, 5% carbonate apatite. He has had microscopic hematuria for many years, starting before the stones. His urologist said the blood is coming from his kidneys.
He had an episode of kidney/back pain in August, a cat scan was negative for stones, it showed renal cysts.
Our concern now is that his renal function is declining. His eGFR was 62 in August, down from 78 in 2013. His urologist has added a diagnosis of “Chronic kidney disease” .
He had routine fasting blood work on 8/25/19, his calcium was 10.3, vit D 25-OH was 49. He takes vit D 2000 IU daily. Creatinine was 1.29.
Based on the calcium of 10.3, his history of stones, declining renal function and some GI symptoms (GERD, abdominal pain) we asked the urologist to check his PTH. On 9/4/19 his calcium was 9.8, PTH 47. However this was not fasting. I know after reading your articles that this should have been drawn fasting.
Over the past 10 years or so his CO2 level has been high normal or slighty elevated, not sure if that is relevant to his kidney function.
In November 2015 (8 months after his kidney stones) we asked for a 24 hour urine, I will include the results below but I know this needs to be repeated.
Calcium 258 oxalate 11 uric acid 121 citrate 238 pH 5.6 Volume 1L sodium 189 phosphorous 874 magnesium 90
potassium 35 creatinine 1846 sulfate 6 sodium urate 1.2 calcium oxalate 1.42 ss brushite 2.24
Based on the low urine volume he has increased his fluid intake and recently has decreased the sodium in his diet.
We will ask his doctor for a current 24 hour urine. Should we also ask for more fasting calcium/PTH levels? His doctors do not seem concerned about his eGFR because it is still in the normal range, but we would like to figure out what is causing his eGFR to decline and treat the cause if possible. Thank you for any input you may have.

Fredric L Coe

Hi Michelle, The one high serum calcium + stones means what you thought: more fasting serums for calcium and PTH – in the same samples. I always do three, and if all are normal I am done with PHPT as a diagnosis. His urine calcium is high and could cause stones all by itself, but the 24 hour urine concerns me. The oxalate at 11 is very low, low enough I suspect a technical lab failure. Likewise the sulfate of 6. The uric acid of 121 is uninterpretable. If that is the total uric acid in the 24 hour urine it is too low to believe except that some has precipitated during collection. The very low volume and pH of 5.6 makes this a real possibility. Given the creatinine of 1846 I believe it is a reasonably complete collection, and his moderate hypercalciuria and stunning low volume – fluid intake – are more than enough to cause stones and crystals. He is probably crystallizing uric acid all of the time and that can cause hematuria as well as orange – red gravel, if one looks. As for the low GFR, I would worry about blood pressure, and any possibility of impaired urinary drainage. I am sure his physicians are aware of this. You might want to bring some of my suggestions to his physicians for their consideration. They are in charge, and responsible for his care. Regards, Fred Coe

Lauren

Hello Mr. Coe,
I’m not sure where to post this, hopefully this is ok. I have been searching for help for years, don’t know where to turn. While researching I came across this site. I have been forming kidney stones for almost 20 years at this point. I’ve been to hundreds of doctors and no one can figure out why. I’d also lost weight without trying and cannot gain no matter how much I eat. I have hair loss/thinning as well, both on head and body. Aches and pains are the norm now and I’m only 40. I’ve had doctors suggest parathyroid disease but when labs are run, everything is normal. I know you cannot diagnose, but have you heard of situations of parathyroid adenomas in the presence of normal labs? I am open to any and all suggestions you may have, I’m really suffering. Thank you very much for your time.

Fredric L Coe

Hi Lauren, Given so many stones you must have serum and 24 hour urine testing done, which will surely reveal your problem. PHPT is fasting serum calcium above the upper limit of normal and urine calcium not low and serum PTH level not suppressed. It is an easy diagnosis if blood and urine testing are done properly. But you need full studies as I said including the kinds of stones passed. Here is another article on workup. Take a look and see if you have been evaluated properly. Regards, Fred Coe

Mary Knights

I have several issues that are probably linked ?
Atrial Fibrillation, Kidney stones and underfunctioning kidneys , underactive thyroid since about 40 years ago have had a parathyroid adenoma removed in about 2002 and tend to have high parathyroid levels , but normal calcium levels .also have osteoporosis high oxalates and the MTHFR gene .also quite a few food sensitivities .Any suggestions how to manage all this .
regards
Mary

Fredric L Coe

Hi Mary, I can perhaps do a little. The MTHFR gene variants in adults confer such low risks that NIH recommends against screening for them. Given normal serum calcium levels, I presume the increased serum PTH levels reflect the reduced kidney function, or alternatively low calcium diet or low serum 25 D levels. Given reduced bone mineral levels, you may have idiopathic hypercalciuria as a cause of stones, and it can promote bone mineral loss. High urine oxalate is almost always worsened by low calcium diet, so given the increased PTH and high urine oxalate perhaps you should consider if your diet has sufficient calcium. Supplements are not unreasonable but must be taken with meals to lower urine oxalate. Take a look at the kidney stone diet, as a good place to start. The atrial fibrillation is a separate matter, I think. Regards, Fred Coe

Maria Suppa

Hi, I have elevaterd sodium and alanine aminotransferase. In my urine, calcium oxalate crystals are present and my calcium ionized is high. My PTH , erythrocytes and leukocyte esterase are also high. I have been experiencing lower back and right side pain, confusion, fatigue, nausea and vomiting, stomach pains, my bones ache and I feel depressed for no apparent reason. My doctor is trying to figure out what is wrong with me. She gave me anibiotics for sinus infection and a puffer for my caugh. My symptoms are still present. After my third visit to the doctor, she called me to tell me to drink 2L of water a day, no protein and no salt in my food. Any suggestions?

Fredric Coe, MD

Hi Maria, alanine aminotransferase in blood, so I presume you have some liver issues. Sodium is in blood and urine, and I presume it is high in your urine as blood sodium in rarely abnormal. If your blood calcium and PTH are high you may well have primary hyperparathyroidism and that can be cured by modern minimally invasive surgery, so I would focus there. Are your bloods always morning and fasting? They need to be for diagnosis. How many have been fasting, morning, and high calcium? Count it up. Is your urine calcium high? Take another look in the article, and be sure of what is found in you vs. what is found in PHPT. Regards, Fred Coe