Effect of fibroblastic growth factor 23 on parathyroid hormone

The effects of FGF23 on the parathyroid are unclear and may depend on duration of exposure or experimental conditions. Some experimental studies suggest that FGF23 inhibits PTH. But patients with CKD as well as patients with X-linked hypophosphatemic rickets consistently have both high FGF23 and high PTH.

Recently, Kawakami et al. found that mice with conditional knock-out (k/o) FGF receptors in the parathyroid glands had low PTH levels. This was true in mice with or without CKD. In cultured parathyroid cells without FGF receptors, there was no increase in PTH after 4 days of culture. When they added FGF23 to cultures of normal parathyroid cells, they found initial inhibition of PTH secretion, but after 4 days there was increased proliferation of the cells. They concluded that the long-term effect of FGF23 was to enhance circulating levels of PTH by causing parathyroid cells to proliferate.

Some experimental studies, however, have found that FGF23 inhibits PTH secretion . Administration of FGF23 to rats decreased PTH gene expression, and this was also seen in parathyroid cultures together with a slight increase in parathyroid cell proliferation . Another study in rats found that FGF23 inhibited PTH, and that blocking the FGF receptor completely restored PTH levels. But in acute hypocalcemia FGF23 did not inhibit PTH .

In patients with CKD the parathyroid glands appear resistant to any suppression by FGF23. This could be due to lower levels of Klotho , to time-dependent effects of FGF23, or to the fact that PTH secretion is controlled by several pathways.

Conversely, PTH increases secretion of FGF23. This is a consistent finding (discussed in section 54.4.2.3 ).

Fibroblastic growth factor 23 metabolism

FGF23 is a protein with 251 amino acids expressed mainly in the osteocytes . The N-terminal end binds to the FGF1 receptor and the C-terminal end interacts with Klotho. Between are several O -glycosylation sites where FGF23 is cleaved within the Golgi apparatus. The FGF23 is cleaved by the protein furin and other protein convertases. This cleavage is important to maintain physiologic levels of FGF23 . In addition, the C-terminal FGF23 can act as a competitive antagonist to FGF23 .

GALNT3 ( N -acetylgalactosaminyltransferase 3) stabilizes the FGF23 at these sites to maintain higher levels of intact FGF23. High phosphate increases GALNT3 activity and therefore increases intact FGF23. Conversely, FAM20C (family with sequence similarity 20 member C) phosphorylates FGF23 and prevents GALNT3 from stabilizing the FGF23. In patients with CKD, there is dysregulation of the cleavage, so more FGF23 is in the active intact state .

Multiple different factors modify FGF23 levels ( Fig. 54.3 ). Although the main driving factor for FGF23 production is a chronic increase in phosphate load, the phosphate itself does not directly increase FGF23 secretion. However, low phosphate is detected by the PIT-2 transporter, which inhibits FGF23, and high phosphate increases GALNT3 activity .

Factors that regulate FGF23 other hormones are aldosterone, estrogen, TNF alpha and IL-6. 1,25D , 1,25-Dihydroxycholecalciferol; Dmp , dentin matrix protein; ENPP , ectonucleotide pyrophosphatase phosphodiesterase; EPO , erythropoietin; FAM20C , family with sequence similarity 20, member C; FGF23 , fibroblastic growth factor 23; GALNT3 , N-acetylgalactosaminyltransferase 3; HIF , hypoxia-inducible factor; HIF-1α , hypoxia-inducible factor 1-α; IV , intravenous; PHEX , phosphate regulating gene with homologies to endopeptidases on the X chromosome; PIT-2 , Type III sodium-dependent phosphate transporter; PTH , parathyroid hormone; sKlotho , soluble Klotho .

Copyright from Susan M. Ott, used with permission.

Proteins associated with the cell surface, including PHEX and Dmp-1, regulate mineralization and inhibit FGF23 synthesis . ENPP (ectonucleotide pyrophosphatase phosphodiesterase) synthesizes pyrophosphate, a potent extracellular mineralization inhibitor, and also inhibits FGF23.

Circulating hormones increase FGF23. 1,25D acts through osteocyte vitamin D receptors to increase FGF23 transcription . Treatment of patients with vitamin D analogs had a net effect of increasing the FGF23 levels . PTH increases FGF23 both directly and indirectly. PTH directly activates the PKA pathway in osteoblasts, which increases FGF23 mRNA levels. In mice with constitutive activation of PTH receptor signaling in osteocytes, there is elevated FGF23 expression . PTH indirectly increases FGF23 by converting 25(OH)D to 1,25D, which increases FGF23 . On a clinical level, lowering PTH levels in hemodialysis patients with cinacalcet or parathyroidectomy will decrease FGF23 levels.

Wnt-signaling increases the expression of FGF23. Umr-106 osteoblast-like cells stimulated with β-glycerophosphate (β-GP) increase expression of FGF23 as well as markers of intracellular wnt-signaling. The FGF23 increases further with addition of lithium, which enhances wnt-signaling . In UMR 106 osteoblast-like cell culture, adding sclerostin (which inhibits wnt-signaling) abolished the ability of PTH to increase FGF23 . Mice with PTH receptor activation express excess FGF23, but this is inhibited by sclerostin . Sclerostin has dual, opposing effects because it also inhibits PHEX which results in greater secretion of FGF23 .

Soluble Klotho also has dual opposing effects of decreasing FGF23 via inhibiting wnt and increasing FGF23 via the FGF receptor signaling pathway. In UMR 106 cells that were cultured with β-GP, addition of Klotho attenuated the increase in both FGF23 and the markers of wnt signaling. This was reversed with lithium . However, mice that were infected with an adenovirus producing soluble Klotho developed high FGF23 along with hypophosphatemia, severe osteomalacia, hyperparathyroidism, hypocalcemia, and low 1,25D. The investigators thought the increase in FGF23 was potentially through the Klotho–FGFR–FGF23 receptor complex . The same constellation of findings was seen in a patient with a mutation that increased Klotho secretion .

Several other factors play a role in FGF metabolism . Leptin induces FGF23 in rat calvarial cultures and in ob/ob mice . Adiponectin inhibits FGF23. Estrogens also have been shown to dose-dependently increase FGF23 levels through transcriptional upregulation . Aldosterone also increases FGF23. TNF alpha and IL-6 increase FGF23 transcription in oocytes . When an enhancer of FGF23 was deleted from thymus and spleen FGF23 values were lower .

A recently discovered factor that directly increases FGF23 is lipocalin-2, which is a proinflammatory and iron-shuttling molecule expressed by the kidneys in response to acute inflammation .

Fibroblastic growth factor 23 and iron deficiency

Erythropoietin, iron deficiency, and hypoxia-inducible factor also stimulate FGF23 . Iron deficiency increases FGF23 secretion even in normal persons , but the protein is degraded so that the intact molecule levels return toward normal, the fragments are increased, and the serum phosphate remains normal. In CKD G1–4 the serum iron was inversely correlated to serum FGF23 using an assay that measured both intact and C-terminal fragments . In acute kidney injury, erythropoietin increases secretion of FGF23 from bone marrow cells via an erythropoietin receptor .

Paradoxically, iron infusions increase FGF23 in dialysis patients and some degree of hypophosphatemia occurs in 2.1%–32.1% of patients without CKD after intravenous ferric polymaltose or carboxymaltose for treatment of iron deficiency . It is possible that iron infusions inhibit furin, and consequently, stabilize intact FGF23 levels .

Fibroblastic growth factor 23 actions

FGF23-Klotho signaling decreases renal phosphate reabsorption in the proximal tubule by causing internalization and proteolytic degradation of the sodium-phosphate transporters ( Table 54.4 ). FGF23-Klotho also inhibits Cyp27B1 (1-alpha-hydroxylase) as well as increasing the activity of the catabolic 24-hydroxylase enzyme (CYP 24A1). This latter action enhances the conversion of 1,25D to 1,24,25(OH) ₃ D and reduces the availability of substrate to the 1-alpha-hydroxylase enzyme by converting 25(OH)D to 24,25(OH) ₂ D . The low 1,25D levels will decrease the serum phosphate because there is less absorption from the intestine.

Reports about the effects of FGF23 on production of Klotho are inconsistent . Some find increased Klotho and others do not . There may be indirect effects because FGF23 decreases 1,25D which stimulates expression of Klotho.

In the distal nephron , FGF23-Klotho increases calcium and sodium reabsorption by activating ANK4, which increases the epithelial calcium channel transient receptor potential vanilloid-subtype 5 (TRPV5) and decreases angiotensin-converting enzyme. These actions contribute to the left ventricular failure seen in patients with CKD .

In the heart , FGF23 reduces contractility and enhances arrhythmogenicity in cardiomyocytes by mishandling intracellular calcium . Cardiac FGF23 signaling through the FGFR4 activates the calcineurin-NFAT signaling pathway to increase left ventricular hypertrophy (LVH) . Pharmacological interference with cardiac FGF23/FGFR4 signaling can protect or even reverse worsening LVH in a rat model of CKD . Patients with X-linked hypophosphatemia also have high FGF23 levels but do not develop LVH, and recent studies suggest that the hypophosphatemia protects the heart against the actions of FGF23 .

In the bone , FGF23 has an autocrine effect of inhibiting the wnt-signaling pathway ( Fig. 54.4 ). In uremic rats fed a high phosphate diet, FGF23 and sKlotho serum levels increased, and in the bone, there was an increased expression of Dkk1 and Sfrp1. The rats subsequently lost bone. Further investigations in an osteoblast cell line exposed to uremic serum found inhibited mineralization and proliferation . When osteoblast-like cells were exposed separately to serum with high PTH, FGF23 or sKlotho, there was no increase in Dkk1, but the combination of FGF23 and sKlotho was what increased Dkk1 and thereby inhibited wnt-signaling A direct action of FGF23 on bone mineralization is independent of effect on systemic phosphate . Also, FGF23 is a potent suppressor of alkaline phosphatase (ALP) .

Autocrine actions of FGF23 in the osteocyte. The secreted FGF23 binds to the FGF receptor, which is associated with Klotho, leading to signaling that results in expression and secretion of Dkk1 which binds to lipoprotein receptor–related protein 5, thereby interrupting the wnt-signaling pathway . Dkk1 , Dickkopf; FGF , fibroblastic growth factor.

Copyright from Susan M. Ott, used with permission.

Fibroblastic growth factor 23 knock-out mice

FGF23-k/o mice have hyperphosphatemia and multiple abnormalities, including emphysema, thickening of vessel walls with extensive calcifications, amyloid accumulation in heart and kidneys, hypogonadism, atrophic skin, reduction in bone density, and premature lethality . They also have high 1,25 vitamin D levels and high osteopontin levels, with osteomalacia . Mice with double k/o of FGF23 and 1a(OH)ase did not have the atherosclerosis, soft-tissue calcifications or short life span of the FGF23 k/o mice , but the bone density in the double k/o mice was even lower than in the FGF23 k/o mice ( Table 54.5 ).

Table 54.5

Fibroblastic growth factor 23 (FGF23) and Klotho genetic altered mice.

 

Fibroblastic growth factor 23 in chronic kidney disease

An increase in FGF23 levels is detected in very early CKD. In otherwise healthy kidney donors, FGF is increased despite decreased serum phosphate levels . Similar results were seen in another study of 3879 patients with CKD G2–4. The mean serum phosphate and median PTH levels were still in the normal range, but median FGF23 was markedly greater than in healthy populations, and increased significantly with decreasing eGFR . In children with CKD, levels of FGF23 are also reported to increase prior to the development of hyperphosphatemia . In acute kidney injury after cardiac surgery, FGF23 levels rose 15.9-fold in 1 day .

In a mouse model of renal injury the serum FGF23 levels were increased prior to elevations in BUN and serum creatinine, but the gene transcription in osteoblasts was not increased until CKD further declined. The source of the early increase in FGF23 was not clear, but the authors thought it could represent changes in excretion or posttranscriptional processing of the hormone . A different acute kidney injury model also found rapid increase in FGF23 along with increased bone production . Most of the FGF23 in patients with CKD is intact , suggesting that there may be something in uremic serum that inhibits the cleavage of FGF23 .

Patients with polycystic kidney disease have higher FGF23 levels than those with other kinds of CKD. Most of the FGF23 is cleaved, so they do not have excess phosphaturia. The source of this FGF23 is the liver, and surgical reduction of the cystic liver mass is associated with a decrease in the FGF23 levels. This is independent of GFR, phosphate, or iron .

Fibroblastic growth factor 23 levels and clinical bone disease

In patients on hemodialysis, FGF23 values did not correlate to bone mass, or to serum levels of bone-specific ALP or C-telopeptide . However, in CKD G 1–4, FGF23 levels were inversely correlated with bone mineral density at the hip . A study of 105 Japanese patients with CKD found that those with vertebral fractures had higher FGF23 levels . In a case-control study from a cohort of 268 Canadian dialysis patients, those with fractures had higher FGF23 . The Health ABC study followed 2234 patients with serial bone density measurements and fracture outcomes. In those without CKD the FGF23 levels were not related to bone density changes, but in people with CKD, higher FGF23 levels were associated with greater bone loss. However, fracture risk was not associated with FGF23 .

Fibroblastic growth factor 23 levels and other clinical outcomes

High FGF23 concentrations are associated with poor clinical outcomes, including cardiovascular disease, both in patients with renal disease and in the general population . Compared with the lowest quartile of FGF23, each subsequent quartile was associated with a progressively higher risk for death. In the MESA study of older adults, FGF23 was associated with heart failure with normal ejection fraction . However, it is not clear if these problems are caused by FGF23 or if it is a surrogate marker for disease. A metaanalysis of studies relating FGF23 to cardiovascular disease did not find dose relationships and the authors concluded that the association was not causative .

In patients with CKD and hyperparathyroidism, a 30% or more reduction in FGF23 induced by cinacalcet was associated with lower rates of cardiovascular mortality than seen with placebo . Within this study, a polymorphism in FGF23 was associated with cardiovascular mortality .

Discovery

In 1997 when Kuro-o was searching for genes that altered the life span of mice, he discovered a gene that increased life span when overexpressed, but with an inactivating mutation, the mice had shortened life span and many features that are also seen with aging (arteriosclerosis, emphysema, thin skin, infertility, kyphosis). He named the gene Klotho after the first of the Moirae, who were the three Greek goddesses controlling man’s fate. Klotho spun the thread of life .

Metabolism

Klotho is a 130-kDa single-pass transmembrane protein expressed in the nephron, the parathyroids, and the choroid plexus. Although expression is highest in the distal nephron, Klotho is also found in the proximal tubule and in the bone at very low levels .

The Klotho promoter contains response elements for vitamin D and for thiazolidinediones. 1,25D increases secretion of Klotho , whereas aldosterone, angiotensin II, and adiponectin suppress transcription . Inflammation or injury can increase micro-RNA34 in the renal tubules, and this results in decreased Klotho . It is not clear whether FGF23 directly decreases Klotho synthesis, or whether there is indirect inhibition due to lower 1,25D. In rats with unilateral obstruction, Klotho expression decreased in the diseased kidney but did not change in the normal kidney, suggesting that the Klotho reduction was due to local factors .

The Klotho ectodomain can be cleaved by ADAM10 and released as a soluble form of Klotho. Insulin may stimulate this process .

Klotho actions

Klotho is both a coreceptor and a systemic hormone. Both membrane Klotho and soluble Klotho associate with FGF1 receptors to allow FGF23 signaling . Soluble Klotho acts as a membrane scaffold to tether FGF23 to the FGF1 receptor , but it is not clear if that complex signals as well as the complex with membrane Klotho .

Klotho-FGF23 signaling in the proximal tubule will affect bone by reducing serum phosphate and 1,25D. Soluble Klotho also has effects that are independent of FGF23. In the distal nephron, Klotho can increase calcium reabsorption. Previously, it was thought that this action was related to glucosidase activity (Klotho would trap the TRPV calcium channels in an open position) but new studies of the molecular structure of Klotho show this is not correct. Instead, soluble Klotho binds to ganglioside-enriched lipid rafts, which allows greater calcium reabsorption .

Soluble Klotho can inhibit important signaling systems at the membrane level, including wnt, TGF-β, and PTH. In vascular smooth muscle cells (VSMCs) Klotho inhibits wnt signaling, thereby preventing arterial calcifications. This effect is reversed with lithium . In cultured bone cells (osteoblast-like UMR-106), soluble Klotho binds to wnt, thus inhibiting the wnt-signaling pathway and decreasing FGF23 secretion .

Klotho can also block wnt signaling in the kidney ( Fig. 54.5 ), thereby preventing renal fibrosis .

Inflammation results in oxidation protein products, which activate the membrane RAGE on the podocytes. This leads to increased nicotinamide adenine dinucleotide phosphate (NADPH) and reactive oxygenation species (ROS) which then will activate NF kβ that increases expression and secretion of wnt. The wnt then acts in an autocrine manner by canonical signaling and increasing the synthesis of actin, fibrinonectin, and renin. Klotho can interrupt this pathological sequence by blocking wnt, thereby preventing renal fibrosis . NF , Nuclear factor; RAGE , receptor of advanced glycation end products.

Copyright from Susan M. Ott, used with permission.

Knock-out mice

Table 54.5 shows phenotypic features of genetically altered mice. 1,25D levels are elevated in Klotho ^−/− mice . Double k/o mice (Klotho and VDR) had hypophosphatemia and low serum FGF23, with no soft-tissue calcifications, emphysema, or skin changes .

The Klotho mutated ( kl/kl ) or k/o mice resemble the FGF23 ^−/− mice in most systems but there are differences in the skeleton. Although the kl/kl mice have kyphosis and thin cortical bone, they have increased trabecular bone. This pattern is typically seen in patients with CKD who have hyperparathyroidism, but in the kl/kl mice the PTH was not increased. Histology showed low numbers of both osteoblasts and osteoclasts . High-resolution micro-computed tomography (microCT) found that the trabecular bone was denser than in wild-type mice . In addition, the bone was resistant to the bone loss that is seen with unloading . When the kl/kl mouse was crossed with mice heterozygous for deletion of osteoprotegerin (OPG), the metaphyseal region no longer showed dense trabecular bone . Further studies demonstrated that this high trabecular bone density was seen because Klotho inhibits wnt-signaling. Therefore the Klotho k/o mice had more β-catenin in the osteoblasts, which leads to increased bone formation, as well as increased OPG .

Conditional knockout of Klotho in the osteocytes results in increased bone formation and increased density . This is also consistent with the findings that Klotho inhibits wnt signaling.

The findings are more complicated when Klotho k/o mice develop uremia. The complex interactions of metabolism between FGF23 and Klotho were explored in a study in which mice were generated with conditional Klotho k/o in mesenchymal cells (which develop into bone of the limbs) who underwent 5/6 nephrectomy. When they developed uremia, they increased transcription of FGF23 from the axial skeleton but not from the limb bones .

Klotho in chronic kidney disease

In early renal failure Klotho is decreased even though there are still enough nephrons to maintain levels. This could be due to the reduced 1,25D caused by rising levels of FGF23. In a study of dialysis patients, those with osteoporosis at the femoral neck had lower serum klotho levels than those with osteopenia or normal bone density, whereas at the lumbar spine there was no difference between those with osteoporosis or osteopenia .

Klotho and cardiovascular disease

In animal models of CKD, enhanced expression of soluble Klotho results in fewer vascular calcifications . Klotho inhibited VSMC transformation and this was partially mediated by inhibition of wnt pathway .

Several studies have shown that soluble Klotho may be cardioprotective. In a 6-year clinical trial that enrolled 3555 subjects with heart disease, those with low Klotho at baseline had more heart failure or mortality. Those with low Klotho and high FGF23 had a 16.9% rate of CV death or heart failure compared to 3.4% in high Klotho-low FGF23 group . In a study of 2010 patients with CKD G 1–5 from Korea, the left ventricular mass showed an inverse association with serum Klotho. The study excluded patients with serious heart disease and adjusted for phosphorus and FGF23 .

Serum sclerostin and Dkk1 levels in chronic kidney disease

Serum sclerostin values increase with reduction in eGFR and in patients on hemodialysis, sclerostin values are approximately fivefold higher than in premenopausal women without CKD, and twofold higher than postmenopausal women . In a cross-sectional study of patients with CKD, urinary excretion of sclerostin was around 10-fold higher in patients with CKD G5 than in patients with CKD G1 , and in older adults from Iceland, those with an eGFR<45 mL/min/1.73 m ² had higher sclerostin than those with eGFR>60 mL/min/1.73 m ² . They also had more bone marrow fat . The increased sclerostin may have a protective effect. A 4-year study of 396 dialysis patients found higher sclerosin levels associated with lower mortality .

PTH decreases sclerostin expression, and as patients develop hyperparathyroidism the sclerostin decreases . In a study of 88 patients with CKD G3–4 who were treated with the vitamin D receptor activator paricalcitol, levels of sclerostin increased, which was likely due to the reduction in PTH .

With progression of CKD-MBD, osteocytes secrete more FGF23, which has an autocrine action to increase osteocytic secretion of Dkk1 . Serum levels of Dkk1, however, are not consistently elevated in clinical studies. In an observational study of 308 patients with CKD the Dkk1 was lower than in controls .

wnt signaling in bone in chronic kidney disease

In a mouse model, an early increase in expression of SOST led to reduced wnt signaling and a consequent increase in the RANKL/OPG ratio .

FGF23 has an autocrine effect to inhibit wnt-signaling in bone. In a study of rats with CKD a high phosphate diet resulted in high FGF23 and PTH. Sclerostin increased at 8 weeks but then decreased. Meanwhile Sfrp1 and Dkk1 increased with time. Further studies in UMR cells exposed to uremic serum, or to FGF23 and Klotho, showed increased expression of Dkk1 and Sfrp1 (see Fig. 54.4 ).

Patients in different grades of CKD show increasing expression of sclerostin by the osteocytes, which is associated with lower bone formation . Higher turnover was seen in patients with high PTH and low sclerostin .

In bone biopsies from 60 patients with CKD G5, Cejka et al. investigated the associations between serum levels of sclerostin or Dkk1 and histomorphometric parameters of bone turnover, mineralization, and volume (TMV). Serum sclerostin levels were higher in CKD patients than in normal women. Sclerostin correlated negatively with histomorphometric parameters of turnover, osteoblastic number, and function in both unadjusted and adjusted analyses. Furthermore, sclerostin was superior to PTH for positive prediction of high bone turnover. However, Dkk1 did not show correlations with any of the parameters of bone turnover measured on bone biopsies.

Sclerostin has been studied in relationship to bone density in CKD patients, with inconsistent results. In 89 dialysis patients, sclerostin correlated inversely with bone mineral density (BMD) and cortical mass changes .

wnt signaling in kidney

Although wnt signaling is beneficial to the skeleton, it plays a negative role in other systems. In the kidney, wnt signaling is active during development but becomes low in adults. In most experimental animal models of kidney injury or CKD, including kidney aging, wnt is upregulated . This response to injury seeks to recapitulate growth to repair damage, but it can result in fibrosis and worsening renal disease. For example, in the kidneys wnt signaling leads to podocyte dysfunction, proteinuria , and increased epithelial damage . When podocytes were exposed to oxidative stress, wnt-signaling was increased. If this signaling is blocked by either Klotho or knockdown of β-catenin, the podocyte damage was reduced ( Fig. 54.5 ). Wnt production in the renal cells has autocrine functions, but the signaling also increases Dkk1 secretion, which can enter the circulation and decrease the bone formation rate.

wnt signaling in vasculature

Wnt signaling also has vascular effects. In VSMCs, activation of wnt signaling promotes the transition to an osteoblast phenotype. The cells react by secreting Sfrp4 . In a rat model, of 5/6 nephrectomy, aortic sclerostin mRNA was increased .

Sclerostin-k/o (SOST ^−/− ) mice with normal kidneys had higher carotid artery calcification than wild-type, but these were not significant (although the mean was five times as high). When the SOST ^−/− mice and controls underwent 5/6 nephrectomy the survival time was similar and the degree of renal failure and increase in PTH and degree of vascular calcifications were also similar, but there was higher cardiac calcium content in the SOST ^−/− mice. In the bones the SOST ^−/− sham and nephrectomized mice had much greater bone area than controls. They also had higher osteoblast perimeter and bone formation rates .

In a clinical study of 67 patients on dialysis, CACs measured by CT showed significant correlations with serum sclerostin levels. Analysis of calcified aortic valves in 10 dialysis patients revealed strong sclerostin expression which was not seen in noncalcified valves .

Whether the circulating sclerostin has a vascular effect is uncertain, but this could be important, because treatment of bone disease with antisclerostin medications could potentially increase vascular calcifications . A study of 157 kidney transplant patients reported that baseline sclerostin levels were associated with cardiovascular calcification, but sclerostin levels did not predict cardiovascular events, fracture, or mortality over the next 3.7 years .

Activin and the skeleton

The effects in the skeletal system vary depending on the physiological conditions. Activin is embedded within the bone matrix and is released by resorption, as is TGF-β. Giving activin to ovariectomized rats increases bone formation and bone density, but only at low doses . Some studies have found stimulation of osteoblasts, others inhibition of bone formation . Likewise, both increases and decreases in osteoclasts have been reported . In osteoblast cultures, activin A inhibits mineralization . In a mouse model of disuse osteoporosis, activin receptor blockade mitigated the loss of femoral neck bone mass . Primates treated with an inhibitor of activin signaling show both increased bone formation and decreased bone resorption , and a small randomized clinical trial in women with osteoporosis showed increases in markers of bone formation and decreased markers of bone resorption .

Activin signaling in the kidney

In kidney, activin A levels increase, and trigger proliferation of fibroblasts, following ischemia or injury . Ureteral ligation increased activin A plasma levels as well as expression in the obstructed kidney but in the normal kidney there were no changes .

In 104 patients with CKD G 2–5, serum activin A increased significantly in CKD G 2, whereas PTH and sclerostin were not increased until CKD G 3, and FGF23 and 1,25D were not significantly changed until CKD G 4. The activin A levels were higher in those with high bone turnover and levels showed significant correlations to both the bone formation rate and the osteoclast surface ( Fig. 54.6 ).

Changes in serum levels of hormones in patients with progressive grades of CKD. Stars show first grade in which values were different from controls. The serum calcium and phosphate remained normal until grade 4. The serum activin was significantly increased with G2 CKD. Results from Lima . CKD , Chronic kidney disease.

Activin signaling in the vasculature

Activation of the ACRII receptors can increase VSMC transformation into osteoblast-like cells that can cause medial calcifications. This happens early in CKD . Activin-A can inhibit bone morphogenetic protein (BMP) 7 signaling in the vasculature, because there is competition for Smad4 . Since BMP-7 inhibits vascular calcification, the effect of activin-A is to promote the calcification. On the other hand, exogenous BMP-7 can inhibit the aortic expression of activin-A .

Effects of blocking activin signaling

Soluble activin RIIA is a ligand trap, and this mitigates bone loss in a mouse model of disuse osteopenia . In young mice, skeletal muscle hypertrophy was seen with administration of a ligand trap .

Treatment with an ActRIIA ligand trap in Alport mice reversed aortic expression of osteoblast transition markers Runx2 and osterix. The mice did not develop cardiac hypertrophy. Bone histomorphometry showed reductions in resorption as well as increases in bone formation rate. The treated mice and control mice had similar bone-forming surfaces and osteoblast numbers, but the mineral apposition rate was higher in treated mice, indicating greater forming activity per osteoblast. The inhibition of osteoclastogenesis was distal to RANK activation; thus it could block osteoclasts even when PTH was present . Renal function did not deteriorate as quickly as in the control mice. Similar results were seen when the ligand trap was used in other mouse models of CKD . In ldlr ^−/− mice with CKD, sKlotho levels were increased with the ligand trap .

The ligand traps also have been shown to increase red blood cell production in several clinical trials of patients with chronic anemia .

Osteocalcin

Osteocalcin (OC), which has also been referred to as bone gamma-carboxyglutamic acid (Gla) protein, is a small water-soluble vitamin K-dependent protein that is one of the most abundant noncollagenous proteins in bone. It is secreted by osteoblasts in response to vitamin D and PTH and undergoes posttranslational vitamin K-dependent gamma-carboxylation. Within bone, gamma-carboxylated OC is bound to hydroxyapatite and affects crystal size and shape and osteoblast and osteoclast activity. However, uncarboxylated OC (ucOC) is not bound to bone and is released into the circulation together with OC that is released during bone resorption. The serum of people with normal renal function contains from 20% to 50% ucOC and 50% or more carboxylated OC .

Many patients with CKD and on dialysis have low values of vitamin K , which may be due to limitations of dietary phosphate and potassium and a corresponding reduction in foods with high vitamin K content. Vitamin K supplementation of patients on dialysis results in a reduction in circulating ucOC and high dietary vitamin K1 intake, or supplementation with vitamin K2, can increase carboxylated OC .

In patients with normal kidney function, serum OC reflects bone turnover, because levels represent a combination of osteoblast and osteoclast activity. However, OC accumulates as CKD progresses, and in ESKD, serum OC ceases to be a reliable turnover marker. Nevertheless, in patients on dialysis, states of high bone turnover have been associated with increased ucOC levels . This was particularly apparent in patients with high bone turnover assessed by levels of PTH and the osteoclast marker TRAcP-5b . Conversely, patients with diabetes, who tend to have low bone formation, are likely to have lower ucOC levels.

Circulating OC is now known to act systemically to increase insulin sensitivity . In some studies, total, but not ucOC concentrations, have been associated with glycemia, glucose metabolism and measures of adiposity . In others, ucOC and carboxylated OC have been associated with improved glucose tolerance, with the ucOC associated with enhanced β-call function and the carboxylated form with improved insulin levels . However, a study of 189 hemodialysis patients found that higher ucOC correlated inversely with lower plasma glucose, hemoglobin A1C, and glycated albumin . Overall, the active endocrine conformation appears to be ucOC, which stimulates insulin secretion directly, increases intestinal glucagon-like peptide-1 secretion and enhances the release of adiponectin from adipose tissue, thereby increasing the sensitivity of muscle to insulin while carboxylated OC mediates important bone effects. Despite these associations, vitamin K1 supplementation given to postmenopausal women to reduce ucOC did not influence blood sugar levels or insulin resistance suggesting that in humans, the endocrine function of OC requires further investigation.

Malnutrition, insulin-like growth factor

Patients with CKD usually have nutritional problems. Their diets must be restricted because they cannot excrete excess phosphate or potassium or sodium. Nausea and anorexia limit the intake of food. Malnutrition, inflammation, metabolic acidosis, and hormonal factors are all likely to contribute to the development of bone disease and muscle loss.

Circulating insulin-like growth factor 1 (IGF-1) is synthesized in the liver in response to growth hormone and is necessary for kidney growth, and the normal development of lean body mass, longitudinal bone growth, and cortical integrity . Defective IGF signaling activates the degradation of muscle protein and muscle loss. On the other hand, the skeletal synthesis of IGF-1 from osteoblast progenitors and fully differentiated osteoblasts is primarily dependent on PTH. This locally produced IGF-1 stimulates bone formation, stabilizes β-catenin, enhances wnt signaling, and plays a more significant role in the maintenance of cancellous bone integrity . IGF-1 is required to obtain an anabolic response to teriparatide, and the stimulatory effect of abaloparatide, a modified 1–34 amino terminal fragment of PTH-related peptide (PTHrp), is mediated at least in part by enhanced local IGF-1 production . Because it is stored in the bone matrix and released during bone resorption, IGF-1 may also be involved in the coupling of bone formation and resorption.

Serum levels of IGF-1 must be interpreted cautiously, because the bioavailability of IGF-1 is altered in kidney disease . In the US population, serum IGF-1 is positively associated with estimated GFR . However, Jehle et al. studied 319 patients with various grades of kidney disease, including dialysis patients, and found that total and free IGF-1 levels remained constant in most patients. The IGF-1 was positively correlated with the bone formation rate.

Studies report various levels in dialysis patients . The bioavailable levels of IGF may be low even when total levels are increased, because the binding proteins are also increased. Han et al. found lower IGF-1 levels in patients on dialysis than in normal subjects. These patients also had abnormalities in their muscle strength.