Fibroblast Growth Factor 23 and the Bone-Vascular Axis: Lessons Learned From Animal Studies

In Translation Fibroblast Growth Factor 23 and the Bone-Vascular Axis: Lessons Learned From Animal Studies Giacomo Zoppellaro, MD,1 Elisabetta Faggin, PhD,2 Massimo Puato, MD,2 Paolo Pauletto, MD,2 and Marcello Rattazzi, MD, PhD2 Calcification of arteries and cardiac valves is observed commonly in dialysis patients and represents a major determinant of the heightened cardiovascular risk observed during chronic kidney disease (CKD) progression. Recent advances from clinical and basic science studies suggest that vascular calcification should be considered a systemic disease in which pathologic processes occurring in the bone and kidney contribute to calcium deposition in the vasculature. Among the factors potentially involved in the vascular-bone axis dysregulation associated with CKD, there now is increasing interest in the role of the phosphaturic hormone fibroblast growth factor 23 (FGF-23). Increased FGF-23 plasma levels are observed with a decrease in kidney function and predict the risk of future cardiovascular mortality. However, clinical data are still unclear about whether a direct pathogenetic effect of FGF-23 on vascular/kidney/bone health exists. In the last few years, a series of basic science studies, performed using engineered mice, have contributed important pathophysiologic information about FGF-23 activities. This review summarizes findings from these studies and discusses the potential role of FGF-23 during the pathologic interplay between kidney, vessels, and bone in CKD. Am J Kidney Dis. 59(1):135-144. © 2011 by the National Kidney Foundation, Inc. INDEX WORDS: Chronic kidney disease (CKD); fibroblast growth factor 23 (FGF-23); klotho; phosphate; vascular calcification.

BACKGROUND Diffuse calcification of the vascular tree is a common finding in patients with chronic kidney disease (CKD). Structural and functional modifications of the arterial vessels and heart valves due to calcium deposition may affect cardiac function, eventually leading to myocardial ischemia and heart failure. Stiffness of large arterial conduits, together with accelerated atherosclerosis, is among the major determinants of the increased cardiovascular mortality observed in patients with CKD.1,2 Although several players involved in vascular calcification have been recognized, most mechanisms driving calcium deposition in the arteries and cardiac valves are unknown. Of note, some of these factors have been shown to affect not only kidney activity and metabolism, but also bone and vascular function (ie, OPG/RANK/RANKL axis [osteoprotegerin/receptor activator of nuclear factor ␬B/ receptor activator of nuclear factor ␬B ligand axis], fetuin A, vitamin D, and parathyroid hormone [PTH]).2,3 Thus, vascular calcification is now emerging as a systemic disease in which concomitant dysfunction in the bone and kidney might actively contribute to calcium deposition within the arterial wall. In this context, increasing interest now is focusing on the role of the fibroblast growth factor 23 (FGF-23)/ klotho axis. FGF-23 is a phosphaturic hormone produced in bone that controls phosphate excretion and vitamin D biosynthesis in the kidney.4 Elevated FGF-23 plasma levels have been linked in humans to both the presence of vascular damage and risk of Am J Kidney Dis. 2012;59(1):135-144

cardiovascular mortality.5-16 However, the potential contribution of FGF-23 to the vascular, kidney, and bone interplay during kidney function decrease is still unknown.

CASE VIGNETTE A 62-year-old man was hospitalized for fatigue and lower extremity pain with mild exertion and at rest. He had type 2 diabetes mellitus, hypertension, and CKD stage 3. Physical examination showed heart rate of 82 beats/min, blood pressure of 140/90 mmHg, 2/6 systolic heart murmur, and bilateral crackles at the lung bases. A chest radiograph showed peribronchial cuffing, demineralization of thoracic vertebrae, and aortic arch calcification. Blood chemistry tests showed the following values: creatinine, 3.56 mg/dL (313 ␮mol/L); estimated glomerular filtration rate calculated using the MDRD (Modification of Diet in Renal Disease) Study equation, 17 mL/min/1.73 m2 (0.28 mL/s/1.73 m2); urea nitrogen, 62 mg/dL (22.13 mmol/L); calcium, 9.2 mg/dL (2.30 mmol/L); phosphate, 4.8 mg/dL (1.55 mmol/L); PTH, 80.4 pg/mL (80.4 ng/L); 25-hydroxyvitamin D, 20 ng/mL (49.92 nmol/ L); glucose, 140 mg/L (7.77 mmol/L); and hemoglobin A1c, 6.8%.

From the 1Clinica Cardiologica, Dipartimento di Scienze Cardiologiche, Toraciche e Vascolari, and 2Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Padova, Treviso, Italy. Received April 13, 2011. Accepted in revised form July 20, 2011. Originally published online November 10, 2011. Address correspondence to Marcello Rattazzi, MD, PhD, Università degli Studi di Padova, Dipartimento di Medicina Clinica e Sperimentale, Medicina Interna I^, Ospedale Ca’ Foncello, Via Ospedale, 31100 Treviso, Italy. E-mail: [email protected] © 2011 by the National Kidney Foundation, Inc. 0272-6386/$36.00 doi:10.1053/j.ajkd.2011.07.027 135

Zoppellaro et al Doppler studies of the lower limbs showed critical stenoses in the femoral arteries. An echocardiogram showed mild left ventricular hypertrophy, diastolic dysfunction with preserved systolic function, calcification of the mitral annulus, and mild aortic stenosis. Computed tomography angiography of the lower limbs and aortoiliac system showed diffuse atherosclerotic lesions in the abdominal aorta and iliac arteries with multiple critical stenoses in the femoral arteries. Extensive intima/media calcification was observed in all arterial segments. Diastolic heart failure, critical peripheral arterial disease, CKD stage 4 with mineral and bone disorder, and mild aortic stenosis with diffuse vascular calcification were diagnosed. Serum FGF-23 was not measured. Could FGF-23 determination improve medical and therapeutic decisions in this patient? Is FGF-23 only a marker of kidney disease or could it have a role in vascular calcification and bone disease progression?

PATHOGENESIS FGF-23 and Phosphate Metabolism FGF-23 is a phosphaturic hormone synthesized in bone by mineralized tissue matrix-forming cells (mainly osteoblasts and osteocytes),17 which decreases phosphate serum levels independently from PTH and vitamin D activities.4,18 Studies of human genetics, acquired diseases, and recent epidemiology have helped clarify FGF-23 regulatory effects on phosphate homeostasis. FGF-23 gain of function has been identified in several genetic disorders, such as autosomal dominant hypophosphatemic rickets, autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemia, and McCune-Albright syndrome, as well as in acquired disorders such as tumor-induced osteomalacia. These clinical conditions are characterized by elevated FGF-23 levels, hypophosphatemia, inappropriately low 1,25-dihydroxyvitamin D levels, and impaired bone mineralization (rickets or osteomalacia). Conversely, decreased FGF-23 activity was found in tumoral calcinosis, which is characterized by hyperphosphatemia, elevated 1,25-dihydroxyvitamin D levels, bone calcinosis, and extensive ectopic calcification of soft tissues.19 FGF-23 decreases serum phosphate levels through inhibition of sodium-phosphate cotransporter type 2a (NaPi2a) expression in proximal tubules. FGF-23 also has been shown to control vitamin D metabolism by suppressing expression of the vitamin D–activating enzyme (1␣-hydroxylase) and increasing vitamin D degradation through induction of the 24-hydroxylase enzyme. These effects result in a net decrease in circulating 1,25-dihydroxyvitamin D levels, with decreased intestinal phosphate reabsorption.4,20 Therefore, elevated FGF-23 levels promote both increased excretion (by the kidney) and decreased absorption (by the bowel) of phosphate, eventually leading to hypophosphatemia (Fig 1). 136

FGF-23 is not the only factor involved in urinary phosphate excretion. Studies performed in human hypophosphatemic syndromes showed a group of mediators, collectively known as “phosphatonins,” that appear to be involved in phosphate homeostasis.21 This group includes PHEX (phosphate-regulating gene with homology to endopeptidasis on the X chromosome), MEPE (matrix extracellular phosphoglycoprotein), DMP-1 (dentin matrix protein-1), and sFRP4 (secreted frizzled related protein 4). FGF-23 exerts its action through interaction with FGF receptors (mainly FGFR-1) that are ubiquitous in the body. Concerted interaction between FGFR-1 and klotho (a single pass membrane protein) confers the tissue specificity for FGF-23 action.22,23 In the absence of klotho, the affinity of FGF-23 for its receptor is significantly decreased, and FGF-23 alone cannot properly regulate systemic phosphate balance.24,25 How the bone “senses” changes in phosphate balance and adjusts FGF-23 secretion is not yet clear. After 5 days of oral phosphate supplementation, healthy humans significantly increase FGF-23 serum levels and phosphate urinary excretion.26-31 However, an acute change in phosphate levels through nondietary intervention such as infusion of dibasic potassium phosphate solution did not modify FGF-23 levels.32 These observations suggest the existence of an unknown intestinal mediator that might regulate FGF-23 secretion, possibly vitamin D itself.18,33 Regulation of FGF-23 serum levels also is linked to activities of other well-known modulators of bone homeostasis and calcium-phosphate balance, including vitamin D and PTH. Vitamin D is a potent stimulus for FGF-23 secretion, especially in the presence of high phosphate levels,34,35 thus suggesting the existence of a feedback control between the 2 hormones. Also, PTH, for which the phosphaturic activity has been known for decades, appears to have a stimulatory effect on FGF-23. It has been shown that (1) mice overexpressing PTH exhibited high FGF-23 serum levels,36 (2) total parathyroidectomy in animals is accompanied by a decrease in circulating FGF-23 levels,37 and (3) infusion of (1-34) PTH in both healthy persons and patients with CKD induced a significant increase in FGF-23 serum levels.38 Moreover, this stimulatory effect appears to be independent of increases in vitamin D levels.39 However, both in vitro and in vivo studies showed that FGF-23 inhibits PTH secretion in a klotho-dependent way, highlighting the existence of a negative feedback control between FGF-23 and PTH.40-42 It should be emphasized that high FGF-23 levels in advanced CKD are unable to prevent the secondary PTH increase. This effect could be exAm J Kidney Dis. 2012;59(1):135-144

FGF-23 and the Bone-Vascular Axis

Figure 1. Fibroblast growth factor 23 (FGF-23) and phosphate homeostasis in normal and decreased kidney function. (A) In normal conditions, low phosphate levels are accompanied by decreased FGF-23 and parathyroid hormone (PTH) circulating levels. As a consequence, phosphate tubular excretion in the kidneys is decreased and active vitamin D is available to promote phosphate bowel absorption. (B) In response to increased phosphate levels, bone and parathyroid glands release FGF-23 and PTH, which are responsible for augmented urinary phosphate excretion and decreased vitamin D production. Phosphate bowel absorption is decreased, but not completely prevented, by the vitamin D lowering. The result is that phosphate levels are normalized. (C) Even in the setting of chronic kidney disease, hyperphosphatemia promotes PTH and FGF-23 release. However, due to decreased kidney function, the increased level of these phosphaturic hormones is not followed by adequate phosphate excretion. At this point, the persistence of high phosphate levels further promotes the release of FGF-23 and PTH. The presence of secondary hyperparathyroidism coupled with decreased vitamin D activity contributes to renal osteodystrophy. Persistent hyperphosphatemia is a major contributor to ectopic calcification in the vasculature. Abbreviations: ESRD, end-stage renal disease; Pi, phosphate serum levels.

plained by klotho downregulation in the parathyroid glands of patients with CKD, a condition that creates resistance to FGF-23 action.43 All these biological aspects and their clinical implications are now subjects of intense investigation.44 Am J Kidney Dis. 2012;59(1):135-144

Clinical Evidence on the Role of FGF-23 A series of recent epidemiologic studies clearly show elevated FGF-23 levels in patients with CKD. The degree of FGF-23 elevation directly correlates 137

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with the decrease in kidney function and precedes the increase in circulating phosphate levels.45,46 Studies conducted in dialysis patients show that FGF-23 levels are associated in a linear and dose-dependent way with all-cause mortality.5,7,8 This strong correlation is still present after adjustment for traditional risk factors (such as age, sex, and blood pressure) and confounders (such as causes of kidney disease and vitamin D, calcium, phosphate, and PTH levels). The Heart and Soul study extended these findings to a cohort of patients with heart disease, with no significant alteration in calcium-phosphate balance and level of kidney function. Also in this study, FGF-23 levels were associated directly with increased risk of allcause mortality and cardiovascular events, independent of vascular risk factors and calcium-phosphate levels.9 A similar finding was seen by the investigators of the CRIC (Chronic Renal Insufficiency Cohort) study, which showed elevated FGF-23 levels as independent predictors of progression toward endstage renal disease in patients with relatively preserved kidney function.10 Nevertheless, studies conducted in non-CKD/noncoronary patients were not able to show an association between circulating FGF23, the extent of coronary calcification,47 and risk of cardiovascular mortality.48 Thus, at the present time, the predictive value of FGF-23 should be restricted to patients with CKD and/or coronary disease and should not be transferred to patients with normal kidney function. Other clinical studies have focused on the association between FGF-23 levels and the presence of cardiovascular damage. In this context, Gutierrez et al11 showed in patients with advanced CKD the existence of a direct correlation between FGF-23 level and the presence of left ventricular hypertrophy. In a similar group of patients, other investigators described a direct association between FGF-23, degree of carotid intima-media thickening,12 and endothelial dysfunction.13 Similar findings were observed in the elderly patients without end-stage renal disease in the PIVUS (Prospective Investigation of the Vasculature in Uppsala Seniors) study, in which FGF-23 serum levels directly correlated with the presence of left ventricular hypertrophy,14 arterial stiffness/endothelial dysfunction,6 and severity of total-body atherosclerosis.15 On the whole, these clinical findings emphasize the potential of FGF-23 as a novel biomarker of mortality and cardiovascular organ damage in patients with different CKD stages and normal phosphate levels. However, the clinical evidence collected to date is not sufficient to establish whether FGF-23 is directly involved in the pathogenesis of the vascular/kidney/ bone damage observed in patients with CKD or could 138

represent a counteracting mechanism aimed at limiting the detrimental consequences of hyperphosphatemia and kidney function decrease. In the last few years, genetically modified mice have been generated to investigate the phenotypical consequences of artificially decreased/increased FGF-23 expression. A closer look at the findings of these studies may furnish pathophysiologic details useful to delineate the actual role of FGF-23 in the pathologic processes observed in the bone, arteries, and kidney (Table 1).

RECENT ADVANCES FGF-23 and Bone As mentioned, impairment of FGF-23 activity is accompanied by modification in bone morphology as FGF-23 gain of function (ie, X-linked hypophosphatemia and tumor-induced osteomalacia) is characterized by the presence of hypophosphatemic rickets with widened growth plates and osteoidosis, and FGF-23 loss of function causes unregulated mineral deposition with impaired cortical bone production (ie, tumoral calcinosis).19 Although a detailed understanding of the mechanisms controlled by the FGF-23/ klotho axis during bone remodeling are lacking, findings obtained to date emphasize the importance of FGF-23’s indirect effect in the generation of these bone phenotypes (ie, through modulation of vitamin D and phosphate homeostasis). Mouse models of either FGF-23 overexpression or inactivation are characterized by the presence of bone architecture derangement, which in several ways resembles changes seen in human diseases. In particular, FGF-23 overexpression42,54,55 induces a bone phenotype similar to human osteomalacia/rickets, whereas FGF-23 deficiency49,50,52 promotes an osteoporosis-like phenotype. Interestingly, FGF-23 inactivation also is accompanied by extensive ectopic calcification, with bone involvement documented by the increase in bone mineral content (Table 1). Of note, FGF-23⫺/⫺ and Klotho⫺/⫺ mice show strikingly similar bone morphology, suggesting that the integrity of the FGF-23/klotho axis might have a crucial role in controlling bone homeostasis.25,49,61 Moreover, the observation that klotho-deficient mice exhibit a bone phenotype similar to FGF-23⫺/⫺, even in a model of FGF-23 overexpression,56 suggests the existence of a klotho-mediated FGF-23 effect in bone metabolism. However, even if some in vitro studies suggest a direct effect of FGF-23 on bone,62,63 to date, any effort to show klotho expression in bone tissue has been in vain.23 Alternatively, we can speculate that the bone phenotype is not directly related to the effects of local and/or systemic FGF-23 levels and its interaction with klotho, but instead should be attributed to modification in the Am J Kidney Dis. 2012;59(1):135-144

FGF-23 and the Bone-Vascular Axis Table 1. Vascular, Bone, and Kidney Modification in Animal Studies Investigating the FGF-23/Klotho Axis Mouse Model

Pi

FGF-23

1,25(OH)2D

PTH

Bone

Kidney

Vessels

FGF-23⫺/⫺ (described in49-53)

1

22

11

⫽/1

Osteoporosis-like phenotype (low bone turnover, 2BMD, 2chondrocytes) with narrowed growth plates; excessive unregulated mineral deposition (11BMC)

Marked vascular calcification; 1expression of 1␣(OH)ase and NaPi2a

Extensive vascular and soft tissue calcification

tFGF-23 (described in20,42,54-56)

2

11

⫽/2

1/2

Osteomalacia/rickets-like phenotype (2BMD, 1chondrocytes, 1ALP, disorganized structure and reduced thickness) with widening of growth plates; no ectopic calcification (2BMC)

No calcification; 2 expression of NaPi2a and 1␣(OH)ase, 1expression of 24(OH)ase

No calcification described

Klotho⫺/⫺ (described in60,61,64)

1

11

1/⫽

⫽/2

Osteoporosis-like phenotype (low bone turnover, hypomineralized osteoid, 2osteoblast, 2BMD 1OPG, 2osteoclast differentiation) with narrowed growth plates

Marked calcification; 1 expression of NaPi2a and 1␣(OH)ase

Extensive vascular and soft tissue calcification

FGF-23⫺/⫺ Klotho⫺/⫺ (Nakatani et al25)

1

22

1

2

Not described

Marked calcification; 1 NaPi2a and 1␣(OH)ase expression

Extensive vascular and soft tissue calcification

tFGF-23 Klotho⫺/⫺ (Bai et al56)

1

11

1

2

Osteoporosis-like phenotype (low bone turnover, hypomineralized osteoid, 2BMD) with narrowed growth plates

Renal calcification; 1expression of 1␣(OH)ase

Extensive vascular and soft tissue calcification

FGF-23⫺/⫺ 1␣(OH)ase⫺/⫺ (described in52,57)

2

22

22

11

Osteomalacia/rickets-like phenotype (22BMD, 1chondrocytes, disorganized structure) with widening of growth plates; no ectopic calcification (2BMC)

2 NaPi2a expression (secondary to hyperparathyroidism)

No soft tissue calcification

Klotho⫺/⫺ 1␣(OH)ase⫺/⫺ (Ohnishi et al58)

2

2

22

11

Not described

No calcification; 22 NaPi2a expression

No soft tissue calcification

FGF-23⫺/⫺ VDR ⌬/⌬ (Hesse et al51)

⫽

22

11

11

Normal bone architecture with slightly reduced BMD and BMC (secondary to hyperparathyroidism)

No calcification

No soft tissue calcification

FGF-23⫺/⫺ NaPi2a⫺/⫺ (Sitara et al59)

2

22

1

2

Osteoporosis-like phenotype (2BMD, 2 chondrocytes, unmineralized osteoidosis); unregulated ectopic calcification (1BMC)

1 1␣(OH)ase

No soft tissue calcification

Klotho⫺/⫺ NaPi2a⫺/⫺ (Ohnishi et al60)

⫽

11

11

2

Not described

Not described

Reduced vascular and soft tissue calcification compared with Klotho⫺/⫺

Abbreviations: 1-2, significant increase-decrease in respect to the control mouse; 11-22, relevant increase-decrease (ie, more than doubling the control mouse); 1␣(OH)ase, vitamin D 1␣-hydroxylase; 1,25(OH)2D, 1,25 dihydroxyvitamin D; 24(OH)ase, vitamin D 24-hydroxylase; ALP, alkaline phosphatase activity; BMC, bone mineral content; BMD, bone mineral density; FGF-23, fibroblast growth factor 23; NaPi2a, sodium-phosphate cotransporter type 2a; OPG, osteoprotegerin; Pi, phosphate serum levels; PTH, parathyroid hormone; tFGF-23, FGF-23 transgenic mouse; VDR ⌬/⌬, double mutant vitamin D receptor.

activity of other bone modulators that accompany the changes in FGF-23 expression. As listed in Table 1, it is apparent that the FGF-23⫺/⫺ and Klotho⫺/⫺ mice, with a similar bone phenotype, also show identical Am J Kidney Dis. 2012;59(1):135-144

levels of phosphate and vitamin D. These parameters instead are reversed in transgenic FGF-23 mice, which show a bone phenotype characterized by osteomalacialike architecture. Imbalance in phosphate levels ap139

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pears to have a limited effect on bone metabolism, as suggested by a study of mice double deficient for FGF-23 and NaPi2a, in which the development of hypophosphatemia did not reverse the FGF-23⫺/⫺ bone phenotype.59 On the contrary, vitamin D seems to be more relevant as shown by the analysis of bone tissues from the mouse double deficient for FGF-23 and 1␣-hydroxylase,52,57 which showed skeletal features similar to the 1␣-hydroxylase single knockout mouse. This finding suggests that the bone phenotype found in the single FGF-23–deficient mouse is mediated at least in part by the high vitamin D levels observed in this model. FGF-23 expression and activity during bone remodeling also could be regulated by other factors involved in bone homeostasis, such as the OPG/RANK/ RANKL triad. Interestingly, increased expression of OPG, a well-known inhibitor of osteoclast maturation, has been documented in bone of the Klotho⫺/⫺ mouse,64 an animal model characterized by features of impaired osteoclastogenesis. Of note, the mouse double deficient for OPG⫺/⫺ and Klotho⫺/⫺ rescued the bone phenotype of the Klotho⫺/⫺ single knockout mouse.65 These data suggest that modifications observed in the Klotho⫺/⫺ mouse are caused at least in part by increased OPG levels, which would be responsible for the decreased osteoclast activity observed in the bone. On the whole, it appears that the actual role played in vivo by FGF-23 during physiologic/pathologic bone remodeling is complicated. As listed in Table 1, the bone turnover seen in mice models of FGF-23 overexpression/downregulation are observed in the context of a complex interplay that includes changes in vitamin D, phosphate, and PTH levels. Thus, even if the evidence collected to date does not fully exclude a direct pathogenetic effect of FGF-23 on bone derangement, most changes described in the animal models appear as changes in the activity of other bone modulators, mainly vitamin D. Additional in vivo and in vitro studies are needed to confirm these findings in the context of decreased kidney function. FGF-23 and the Kidney As mentioned, several epidemiologic studies distinctly showed a significant increase in FGF-23 serum levels in patients with CKD.5,7,8,46,66 Nevertheless, this clinical/biohumoral correlation does not provide a complete explanation of the role of FGF-23 during kidney disease progression. FGF-23 is augmented in patients with early stages of CKD long before hyperphosphatemia is detectable. FGF-23 acts by promoting phosphate urinary excretion, thus representing first-line protection from detrimental consequences of increased phosphate levels. 140

However, despite high levels of intact and biologically active FGF-23,67 patients with advanced CKD show a significant decrease in urinary phosphate excretion, which likely promotes a further secondary increase in FGF-23 levels (Fig 1). Impairment of the FGF-23 phosphaturic effect in the damaged kidney could be explained by the decrease in nephron mass and/or the development of resistance to the hormone activity. In line with the latter hypothesis, downregulation of klotho expression has been documented in renal tubules of dialysis patients.68 Unfortunately, a detailed description of histologic changes occurring in kidneys of mouse models overexpressing or deficient for FGF-23 is available in only a few cases (Table 1). In these studies, a relevant role for the onset/progression of the pathologic processes in renal tissue seems to be had by ectopic calcification, which is located mainly in the vasculature.52 Of note, independent of FGF-23 serum levels, the histologic kidney damage in the animal models may be caused solely by increased circulating phosphate levels (Table 1). As expected, most studies based on FGF-23– deficient or transgenic mice explore changes in NaPi2a expression on the brush border of proximal tubules, together with modification of the enzymes involved in vitamin D metabolism. On the whole, data collected to date are concordant about the inhibitory effects of FGF-23 on NaPi2a expression and 1,25-dihydroxyvitamin D production. FGF-23 and the Vasculature Even if only a few studies have been specifically designed to investigate the relationship between FGF-23 and vascular homeostasis, the evidence available to date seems to exclude the presence of a direct pathogenetic effect. This again emphasizes the negative impact of hyperphosphatemia. Human studies have shown that syndromes characterized by low FGF-23 serum levels are accompanied by extensive ectopic calcification.69 In addition, animal studies have shown that mice overexpressing FGF-23 display a normal vascular phenotype, whereas FGF-23 knockout models are characterized by extensive arterial calcification (Table 1). Based on these observations, it is tempting to speculate that FGF-23 may have a beneficial role in preserving vessel integrity. However, patients with CKD, despite elevated circulating FGF-23 levels, are prone to ectopic calcification, arterial stiffening, coronary artery disease, and endothelial dysfunction, a series of clinical findings that are associated with increased cardiovascular mortality.5,6,9 How should the augmented FGF-23 levels be interpreted in respect to vascular disease progression? The association between FGF-23 levels and the Am J Kidney Dis. 2012;59(1):135-144

FGF-23 and the Bone-Vascular Axis

presence of vascular calcification has been investigated recently by Giachelli’s group70 in a mouse model of mildly decreased GFR. In particular, these authors observed a direct correlation between FGF-23 circulating levels and the extent of aortic calcium deposition in mice fed a high-phosphate diet. Similar to this finding, human studies showed an association between FGF-23 plasma levels and calcium accumulation in the aorta and coronaries of dialysis patients.16,71 If we assume a protective effect of circulating FGF-23 on arterial wall integrity, it could be hypothesized that decreased kidney function is accompanied by the development of vascular tissue resistance to FGF-23 actions. This effect may be due to downregulation of klotho expression in the vascular cells, similar to that observed in kidneys of patients with CKD.24,25 Unfortunately, at the present time, no clear evidence is available about the existence of FGF-23/klotho interaction/effects within the arterial wall, even if a recent study showed an inhibitory effect of soluble klotho on phosphate-induced calcification in vascular smooth muscle cells.72 Expression and accumulation of FGF-23 within the affected arterial wall to date have been poorly investigated. A recent histopathologic study of carotid plaques showed the presence of FGF-23 within calcified areas of the atherosclerotic lesions, although the origin of the cell types involved in FGF-23 production and deposition is still unknown.73 If we revisit data obtained from animal studies of mice overexpressing or deficient for FGF-23, we observe that the presence/absence of vascular calcification appears to be dependent mainly on circulating phosphate levels (Table 1). Ectopic vascular calcification can be documented in only animal models with hyperphosphatemia, regardless of changes observed in PTH, vitamin D, and FGF-23 levels.53,60 Therefore, it is reasonable to conclude that the calcific degeneration of vessels observed in mouse models deficient for klotho and FGF-23 is related mainly to the accompanying increase in phosphate levels. The absence of a direct FGF-23 pathogenetic effect during arterial calcific degeneration also is suggested by the lack of significant changes in the vascular phenotype of mice with increased/decreased FGF-23 levels and normal/ decreased phosphate levels (such as mice double deficient for FGF-23/NaPi2a and overexpressing FGF23).58,59 Thus, phosphate levels, more than FGF-23 level fluctuations, appear to be the major determinant of vascular calcification observed in these animal models. Nevertheless, further clarification of the expression/activation of the FGF-23/klotho axis in vascular cells is necessary before considering the association between FGF-23 and vascular disease a process entirely mediated by phosphate circulating levels. Am J Kidney Dis. 2012;59(1):135-144

SUMMARY Based on the evidence described, we begin to construct the role of FGF-23 in bone-kidney-vascular axis dysregulation. In particular, most FGF-23 effects in the skeleton appear to be mediated by changes in other bone homeostatic modulators, such as vitamin D and OPG. No clear evidence emerges from animal studies about a significant direct role of circulating FGF-23 in kidney disease progression. The pathologic changes observed in the kidneys of some animal models primarily could be a consequence of the vascular damage provoked by ectopic calcium deposition. In addition, no evidence is available yet about the existence of a direct FGF-23 contribution in modulating vascular integrity. Arterial calcification observed in mice with high/low FGF-23 circulating levels can be documented only in cases of a concomitant increase in phosphate serum levels. A major limitation for the application of these findings to decreased kidney function is that none of the animal studies has been conducted using a CKD model. Thus, it cannot be excluded that other factors specifically associated with kidney disease progression might interfere with FGF-23 activities and alter the pathophysiologic mechanisms observed in animal models with normal kidney function. Moreover, the proposed scenario and interpretation of the data need better elucidation of the actual role of klotho in all involved tissues, both as a membrane-associated protein and a humoral mediator. Comparison of the data obtained from animal models with an impaired FGF-23/klotho axis highlights the participation of phosphate serum levels in the pathologic processes observed in bone, kidney, and vascular tissue. In particular, hyperphosphatemia is associated with the presence of kidney damage, calcium deposition in the vasculature, and dysregulated bone mineralization. Thus, increased FGF-23 levels may be considered an attempt to protect these tissues from the detrimental consequences of hyperphosphatemia.74 In the last few years, phosphate has been widely recognized as a strong inducer of both in vivo and in vitro vascular calcification. Mechanisms driven by hyperphosphatemia include phenotypic transdifferentiation of valve cells/smooth muscle cells toward a procalcific profile75,76 and initiation of crystal nucleation by overwhelming calcification inhibitors.77 However, from a clinical perspective, phosphate serum values emerged in only a few epidemiologic studies as independent markers of cardiovascular risk and vascular damage. This weak association could be explained by the wide intraindividual variability observed among repeated phosphate serum determinations.78 Therefore, a single phosphate measurement might not be representative of systemic phosphate homeostasis, 141

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especially in a long time frame. On the contrary, FGF-23 determination might result in the near future as a representative indicator of the averaged phosphate balance over time. Future studies should be aimed at better clarifying the significance of FGF-23 measurement in patients with a mild-moderate decrease in kidney function or with CKD and extensive vascular disease, such as the patient described in the vignette. If the capacity of FGF-23 to predict cardiovascular risk and vascular disease progression can be confirmed, intervention studies could be designed to establish whether initiation of phosphate-lowering pharmacologic therapy might be based on a threshold of FGF-23 values independent from serum phosphate levels. In conclusion, findings obtained from animal models suggest that the increase in FGF-23 circulating levels commonly observed during kidney function decrease is not involved directly in the initiation or progression of the associated vascular complications. Instead, a central role seems to be played by high phosphate levels, which invariably are associated with the presence of arterial calcification regardless of FGF-23 levels. Additional basic and clinical science studies are needed to confirm this hypothesis and establish the clinical/therapeutic relevance of FGF-23 serum determination in the different stages of CKD.

ACKNOWLEDGEMENTS Support: None. Financial Disclosure: The authors declare that they have no relevant financial interests.

REFERENCES 1. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med. 2004;351(13):12961305. 2. Moe SM, Chen NX. Mechanisms of vascular calcification in chronic kidney disease. J Am Soc Nephrol. 2008;19(2):213-216. 3. Demer LL, Tintut Y. Vascular calcification: pathobiology of a multifaceted disease. Circulation. 2008;117(22):2938-2948. 4. Shimada T, Hasegawa H, Yamazaki Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res. 2004;19(3):429-435. 5. Gutierrez OM, Mannstadt M, Isakova T, et al. Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. N Engl J Med. 2008;359(6):584-592. 6. Mirza MA, Larsson A, Lind L, Larsson TE. Circulating fibroblast growth factor-23 is associated with vascular dysfunction in the community. Atherosclerosis. 2009;205(2):385-390. 7. Jean G, Terrat JC, Vanel T, et al. High levels of serum fibroblast growth factor (FGF)-23 are associated with increased mortality in long haemodialysis patients. Nephrol Dial Transplant. 2009;24(9):2792-2796. 8. Seiler S, Reichart B, Roth D, Seibert E, Fliser D, Heine GH. FGF-23 and future cardiovascular events in patients with chronic kidney disease before initiation of dialysis treatment. Nephrol Dial Transplant. 2010;25(12):3983-3989. 142

9. Parker BD, Schurgers LJ, Brandenburg VM, et al. The associations of fibroblast growth factor 23 and uncarboxylated matrix Gla protein with mortality in coronary artery disease: the Heart and Soul Study. Ann Intern Med. 2010;152(10):640-648. 10. Isakova T, Xie H, Yang W, et al. Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease. JAMA. 2011;305(23):2432-2439. 11. Gutierrez OM, Januzzi JL, Isakova T, et al. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. Circulation. 2009;119(19):2545-2552. 12. Balci M, Kirkpantur A, Gulbay M, Gurbuz OA. Plasma fibroblast growth factor-23 levels are independently associated with carotid artery atherosclerosis in maintenance hemodialysis patients. Hemodial Int. 2010;14(4):425-432. 13. Yilmaz MI, Sonmez A, Saglam M, et al. FGF-23 and vascular dysfunction in patients with stage 3 and 4 chronic kidney disease. Kidney Int. 2010;78(7):679-685. 14. Mirza MA, Larsson A, Melhus H, Lind L, Larsson TE. Serum intact FGF23 associates with left ventricular mass, hypertrophy and geometry in an elderly population. Atherosclerosis. 2009; 207(2):546-551. 15. Mirza MA, Hansen T, Johansson L, et al. Relationship between circulating FGF23 and total body atherosclerosis in the community. Nephrol Dial Transplant. 2009;24(10):3125-3131. 16. Srivaths PR, Goldstein SL, Silverstein DM, Krishnamurthy R, Brewer ED. Elevated FGF 23 and phosphorus are associated with coronary calcification in hemodialysis patients. Pediatr Nephrol. 2011;26(6):945-951. 17. Yoshiko Y, Wang H, Minamizaki T, et al. Mineralized tissue cells are a principal source of FGF23. Bone. 2007;40(6):15651573. 18. Shimada T, Yamazaki Y, Takahashi M, et al. Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism. Am J Physiol Renal Physiol. 2005;289(5): F1088-F1095. 19. Fukumoto S. Physiological regulation and disorders of phosphate metabolism—pivotal role of fibroblast growth factor 23. Intern Med. 2008;47(5):337-343. 20. Saito H, Kusano K, Kinosaki M, et al. Human fibroblast growth factor-23 mutants suppress Na⫹-dependent phosphate co-transport activity and 1alpha,25-dihydroxyvitamin D3 production. J Biol Chem. 2003;278(4):2206-2211. 21. Berndt TJ, Schiavi S, Kumar R. “Phosphatonins” and the regulation of phosphorus homeostasis. Am J Physiol Renal Physiol. 2005;289(6):F1170-F1182. 22. Urakawa I, Yamazaki Y, Shimada T, et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006;444(7120):770-774. 23. Li SA, Watanabe M, Yamada H, Nagai A, Kinuta M, Takei K. Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice. Cell Struct Funct. 2004; 29(4):91-99. 24. Nakatani T, Ohnishi M, Razzaque MS. Inactivation of klotho function induces hyperphosphatemia even in presence of high serum fibroblast growth factor 23 levels in a genetically engineered hypophosphatemic (Hyp) mouse model. FASEB J. 2009;23(11):3702-3711. 25. Nakatani T, Sarraj B, Ohnishi M, et al. In vivo genetic evidence for klotho-dependent, fibroblast growth factor 23 (Fgf23) -mediated regulation of systemic phosphate homeostasis. FASEB J. 2009;23(2):433-441. 26. Ferrari SL, Bonjour JP, Rizzoli R. Fibroblast growth factor-23 relationship to dietary phosphate and renal phosphate handling in healthy young men. J Clin Endocrinol Metab. 2005;90(3): 1519-1524. Am J Kidney Dis. 2012;59(1):135-144

FGF-23 and the Bone-Vascular Axis 27. Burnett SM, Gunawardene SC, Bringhurst FR, Juppner H, Lee H, Finkelstein JS. Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women. J Bone Miner Res. 2006;21(8):1187-1196. 28. Antoniucci DM, Yamashita T, Portale AA. Dietary phosphorus regulates serum fibroblast growth factor-23 concentrations in healthy men. J Clin Endocrinol Metab. 2006;91(8):3144-3149. 29. Perwad F, Azam N, Zhang MY, Yamashita T, Tenenhouse HS, Portale AA. Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25-dihydroxyvitamin D metabolism in mice. Endocrinology. 2005;146(12):5358-5364. 30. Vervloet MG, van Ittersum FJ, Buttler RM, Heijboer AC, Blankenstein MA, Ter Wee PM. Effects of dietary phosphate and calcium intake on fibroblast growth factor-23. Clin J Am Soc Nephrol. 2011;6(2):383-389. 31. Nishida Y, Taketani Y, Yamanaka-Okumura H, et al. Acute effect of oral phosphate loading on serum fibroblast growth factor 23 levels in healthy men. Kidney Int. 2006;70(12):2141-2147. 32. Ito N, Fukumoto S, Takeuchi Y, et al. Effect of acute changes of serum phosphate on fibroblast growth factor (FGF)23 levels in humans. J Bone Miner Metab. 2007;25(6):419-422. 33. Masuyama R, Stockmans I, Torrekens S, et al. Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts. J Clin Invest. 2006;116(12): 3150-3159. 34. Yamamoto R, Minamizaki T, Yoshiko Y, et al. 1alpha,25dihydroxyvitamin D3 acts predominately in mature osteoblasts under conditions of high extracellular phosphate to increase fibroblast growth factor 23 production in vitro. J Endocrinol. 2010; 206(3):279-286. 35. Saito H, Maeda A, Ohtomo S, et al. Circulating FGF-23 is regulated by 1alpha,25-dihydroxyvitamin D3 and phosphorus in vivo. J Biol Chem. 2005;280(4):2543-2549. 36. Kawata T, Imanishi Y, Kobayashi K, et al. Parathyroid hormone regulates fibroblast growth factor-23 in a mouse model of primary hyperparathyroidism. J Am Soc Nephrol. 2007;18(10): 2683-2688. 37. Lavi-Moshayoff V, Wasserman G, Meir T, Silver J, NavehMany T. PTH increases FGF23 gene expression and mediates the high-FGF23 levels of experimental kidney failure: a bone parathyroid feedback loop. Am J Physiol Renal Physiol. 2010;299(4):F882F889. 38. Wesseling-Perry K, Harkins GC, Wang HJ, et al. The calcemic response to continuous parathyroid hormone (PTH)(134) infusion in end-stage kidney disease varies according to bone turnover: a potential role for PTH(7-84). J Clin Endocrinol Metab. 2010;95(6):2772-2780. 39. Lopez I, Rodriguez-Ortiz ME, Almaden Y, et al. Direct and indirect effects of parathyroid hormone on circulating levels of fibroblast growth factor 23 in vivo. Kidney Int. 2011;80(5):475482. 40. Krajisnik T, Bjorklund P, Marsell R, et al. Fibroblast growth factor-23 regulates parathyroid hormone and 1alpha-hydroxylase expression in cultured bovine parathyroid cells. J Endocrinol. 2007;195(1):125-131. 41. Ben-Dov IZ, Galitzer H, Lavi-Moshayoff V, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest. 2007;117(12):4003-4008. 42. Shimada T, Urakawa I, Yamazaki Y, et al. FGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa. Biochem Biophys Res Commun. 2004;314(2):409-414. 43. Galitzer H, Ben-Dov IZ, Silver J, Naveh-Many T. Parathyroid cell resistance to fibroblast growth factor 23 in secondary hyperparathyroidism of chronic kidney disease. Kidney Int. 2011; 77(3):211-218. Am J Kidney Dis. 2012;59(1):135-144

44. Isakova T, Wolf MS. FGF23 or PTH: which comes first in CKD? Kidney Int. 2010;78(10):947-949. 45. Fliser D, Kollerits B, Neyer U, et al. Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the Mild to Moderate Kidney Disease (MMKD) Study. J Am Soc Nephrol. 2007;18(9):2600-2608. 46. Larsson T, Nisbeth U, Ljunggren O, Juppner H, Jonsson KB. Circulating concentration of FGF-23 increases as renal function declines in patients with chronic kidney disease, but does not change in response to variation in phosphate intake in healthy volunteers. Kidney Int. 2003;64(6):2272-2279. 47. Roos M, Lutz J, Salmhofer H, et al. Relation between plasma fibroblast growth factor-23, serum fetuin-A levels and coronary artery calcification evaluated by multislice computed tomography in patients with normal kidney function. Clin Endocrinol (Oxf). 2008;68(4):660-665. 48. Taylor EN, Rimm EB, Stampfer MJ, Curhan GC. Plasma fibroblast growth factor 23, parathyroid hormone, phosphorus, and risk of coronary heart disease. Am Heart J. 2011;161(5):956962. 49. Sitara D, Razzaque MS, Hesse M, et al. Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol. 2004;23(7):421432. 50. Shimada T, Kakitani M, Yamazaki Y, et al. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest. 2004;113(4):561-568. 51. Hesse M, Frohlich LF, Zeitz U, Lanske B, Erben RG. Ablation of vitamin D signaling rescues bone, mineral, and glucose homeostasis in Fgf-23 deficient mice. Matrix Biol. 2007;26(2): 75-84. 52. Razzaque MS, Sitara D, Taguchi T, St-Arnaud R, Lanske B. Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. FASEB J. 2006;20(6):720722. 53. Stubbs JR, Liu S, Tang W, et al. Role of hyperphosphatemia and 1,25-dihydroxyvitamin D in vascular calcification and mortality in fibroblastic growth factor 23 null mice. J Am Soc Nephrol. 2007;18(7):2116-2124. 54. Larsson T, Marsell R, Schipani E, et al. Transgenic mice expressing fibroblast growth factor 23 under the control of the alpha1(I) collagen promoter exhibit growth retardation, osteomalacia, and disturbed phosphate homeostasis. Endocrinology. 2004; 145(7):3087-3094. 55. Bai X, Miao D, Li J, Goltzman D, Karaplis AC. Transgenic mice overexpressing human fibroblast growth factor 23 (R176Q) delineate a putative role for parathyroid hormone in renal phosphate wasting disorders. Endocrinology. 2004;145(11): 5269-5279. 56. Bai X, Dinghong Q, Miao D, Goltzman D, Karaplis AC. Klotho ablation converts the biochemical and skeletal alterations in FGF23 (R176Q) transgenic mice to a Klotho-deficient phenotype. Am J Physiol Endocrinol Metab. 2009;296(1):E79E88. 57. Sitara D, Razzaque MS, St-Arnaud R, et al. Genetic ablation of vitamin D activation pathway reverses biochemical and skeletal anomalies in Fgf-23-null animals. Am J Pathol. 2006; 169(6):2161-2170. 58. Ohnishi M, Nakatani T, Lanske B, Razzaque MS. Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1alpha-hydroxylase. Kidney Int. 2009;75(11):1166-1172. 143

Zoppellaro et al 59. Sitara D, Kim S, Razzaque MS, et al. Genetic evidence of serum phosphate-independent functions of FGF-23 on bone. PLoS Genet. 2008;4(8):e1000154. 60. Ohnishi M, Nakatani T, Lanske B, Razzaque MS. In vivo genetic evidence for suppressing vascular and soft-tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin d levels. Circ Cardiovasc Genet. 2009;2(6):583-590. 61. Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse Klotho gene leads to a syndrome resembling ageing. Nature. 1997;390(6655):45-51. 62. Wang H, Yoshiko Y, Yamamoto R, et al. Overexpression of fibroblast growth factor 23 suppresses osteoblast differentiation and matrix mineralization in vitro. J Bone Miner Res. 2008;23(6): 939-948. 63. Shalhoub V, Ward SC, Sun B, et al. Fibroblast growth factor 23 (FGF23) and alpha-klotho stimulate osteoblastic MC3T3.E1 cell proliferation and inhibit mineralization. Calcif Tissue Int. 2011;89(2):140-150. 64. Kawaguchi H, Manabe N, Miyaura C, Chikuda H, Nakamura K, Kuro-o M. Independent impairment of osteoblast and osteoclast differentiation in Klotho mouse exhibiting low-turnover osteopenia. J Clin Invest. 1999;104(3):229-237. 65. Yamashita T, Okada S, Higashio K, Nabeshima Y, Noda M. Double mutations in klotho and osteoprotegerin gene loci rescued osteopetrotic phenotype. Endocrinology. 2002;143(12):4711-4717. 66. Imanishi Y, Inaba M, Nakatsuka K, et al. FGF-23 in patients with end-stage renal disease on hemodialysis. Kidney Int. 2004; 65(5):1943-1946. 67. Shimada T, Urakawa I, Isakova T, et al. Circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by peritoneal dialysis is intact and biologically active. J Clin Endocrinol Metab. 2010;95(2):578-585. 68. Koh N, Fujimori T, Nishiguchi S, et al. Severely reduced production of klotho in human chronic renal failure kidney. Biochem Biophys Res Commun. 2001;280(4):1015-1020.

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69. Benet-Pages A, Orlik P, Strom TM, Lorenz-Depiereux B. An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet. 2005;14(3):385390. 70. El-Abbadi MM, Pai AS, Leaf EM, et al. Phosphate feeding induces arterial medial calcification in uremic mice: role of serum phosphorus, fibroblast growth factor-23, and osteopontin. Kidney Int. 2009;75(12):1297-1307. 71. Nasrallah MM, El-Shehaby AR, Salem MM, Osman NA, El Sheikh E, Sharaf El Din UA. Fibroblast growth factor-23 (FGF-23) is independently correlated to aortic calcification in haemodialysis patients. Nephrol Dial Transplant. 2010;25(8): 2679-2685. 72. Hu MC, Shi M, Zhang J, et al. Klotho deficiency causes vascular calcification in chronic kidney disease. J Am Soc Nephrol. 2011;22(1):124-136. 73. Voigt M, Fischer DC, Rimpau M, Schareck W, Haffner D. Fibroblast growth factor (FGF)-23 and fetuin-A in calcified carotid atheroma. Histopathology. 2010;56(6):775-788. 74. Razzaque MS. Phosphate toxicity: new insights into an old problem. Clin Sci (Lond). 2010;120(3):91-97. 75. Jono S, McKee MD, Murry CE, et al. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000;87(7): E10-E17. 76. Rattazzi M, Iop L, Faggin E, et al. Clones of interstitial cells from bovine aortic valve exhibit different calcifying potential when exposed to endotoxin and phosphate. Arterioscler Thromb Vasc Biol. 2008;28(12):2165-2172. 77. Sage AP, Lu J, Tintut Y, Demer LL. Hyperphosphatemiainduced nanocrystals upregulate the expression of bone morphogenetic protein-2 and osteopontin genes in mouse smooth muscle cells in vitro. Kidney Int. 2011;79(4):414-422. 78. Chonchol M, Dale R, Schrier RW, Estacio R. Serum phosphorus and cardiovascular mortality in type 2 diabetes. Am J Med. 2009;122(4):380-386.

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