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Co-CHAIRS Eduardo Slatopolsky, MD, FACP, Joseph Friedman Professor of Renal Diseases in Medicine, Washington University School of Medicine, St Louis, Missouri, USA Sharon Moe, MD, Professor of Medicine and Vice-Chair for Research, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA PRESENTING FACULTY Roger Bouillon, MD, Laboratory of Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Belgium David Bushinsky, MD, Professor and Associate Chair of Medicine, Nephrology Unit, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA Cyrille Confavreux, MD, Service de Rhumatologie, Université de Lyon-Sud, France Tilman Drueke, MD, INSERM Unité 845 and Service de Néphrologie, Hôpital Necker, Assistance Publique-Hôpitaux de Paris, and Faculté de Médecine René Descartes, Paris, France Keith Hruska, MD, Director of Pediatric Nephrology, Washington University School of Medicine, St Louis, Missouri, USA Harald Jüppner, MD, Associate Professor of Pediatrics, Harvard University Medical School, Boston, Massachusetts, USA Makoto Kuro-o, MD, Associate Professor of Pathology and Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA Martin Pollak, MD, Harvard Institutes of Medicine, Department of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA Mariano Rodriguez, MD, Departamento de Medicina, Hospital Universitaria de Reina Sofia, Cordoba, Spain FACULTY Ezequiel Bellorin-Font, MD, Venezuela Jorge Cannata-Andía, MD, Spain Maria Eugenia Canziani, MD, Brazil

Martin Pollak, MD, Harvard Institutes of Medicine, Department of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA Mariano Rodriguez, MD, Departamento de Medicina, Hospital Universitaria de Reina Sofia, Cordoba, Spain FACULTY Ezequiel Bellorin-Font, MD, Venezuela Jorge Cannata-Andía, MD, Spain Maria Eugenia Canziani, MD, Brazil Aluizio Carvalho, MD, Brazil Ricardo Correa-Rotter, MD, Mexico John Cunningham, MD, UK Masafumi Fukugawa, MD, Japan Vanda Jorgetti, MD, Brazil Tobias Larsson, MD, Sweden Klaus Olgaard, MD, Denmark Anthony Portale, MD, USA Isidro B. Salusky, MD, USA José-Vicente Torregrosa, MD, Spain Myles Wolf, MD, USA INTRODUCTION A Phosphate Centric Forum, supported by Genzyme Corporation, was held on 24–25 June, 2010 at the Sheraton West Park Hotel, Munich, Germany, in which 25 medical and scientific experts in the field of chronic kidney disease-related mineral and bone disorder (CKD-MBD) met to present and discuss the central role of phosphate in the development of CKD-MBD, on which research has now celebrated its 50th anniversary.

the Sheraton West Park Hotel, Munich, Germany, in which 25 medical and scientific experts in the field of chronic kidney disease-related mineral and bone disorder (CKD-MBD) met to present and discuss the central role of phosphate in the development of CKD-MBD, on which research has now celebrated its 50th anniversary. Phosphate plays a central role in the pathophysiology of CKD-MBD and the progression of CKD and contributes to the disproportionate cardiovascular risk faced by patients with CKD. Adaptation of nephrons attempting to preserve phosphate homeostasis requires endocrine tradeoffs that fuel adverse events of hyperphosphatemia in CKD—secondary hyperparathyroidism, calcium and vitamin D derangements, vascular calcification, and metabolic bone disorder. Restricting phosphate absorption by use of phosphate binders as glomerular filtration rate declines reduces the need for nephron adaptation and therefore forestalls physiologic derangements downstream of hyperphosphatemia. A roundtable discussion included the pathophysiology of CKD-MBD, discoveries such as the vitamin D receptor, calcium-sensing receptor, fibroblast growth factor (FGF)-23, and currently available treatment options, as well as future needs regarding research and management of this disorder.

Phosphate plays a central role in the pathophysiology of CKD-MBD and the progression of CKD and contributes to the disproportionate cardiovascular risk faced by patients with CKD. Adaptation of nephrons attempting to preserve phosphate homeostasis requires endocrine tradeoffs that fuel adverse events of hyperphosphatemia in CKD—secondary hyperparathyroidism, calcium and vitamin D derangements, vascular calcification, and metabolic bone disorder. Restricting phosphate absorption by use of phosphate binders as glomerular filtration rate declines reduces the need for nephron adaptation and therefore forestalls physiologic derangements downstream of hyperphosphatemia. A roundtable discussion included the pathophysiology of CKD-MBD, discoveries such as the vitamin D receptor, calcium-sensing receptor, fibroblast growth factor (FGF)-23, and currently available treatment options, as well as future needs regarding research and management of this disorder. Discussants concluded that placebo-controlled trials are needed in CKD stages 2–4 to investigate the effect of phosphate and phosphate binders on mortality. Various other endpoints were suggested, such as bone disease, progression of CKD, and cardiovascular events. Ongoing studies investigating a broad range of cardiovascular surrogates are due for completion in 2011. Monitoring methods using the less expensive option of phosphate and creatinine are preferred over FGF-23 at the present time, and studies investigating patients over 50–55 years of age with high levels of FGF-23 have been suggested to provide the most valuable data. However, it was generally agreed that FGF-23 may be a uremic toxin, which would support development of anti-FGF-23 strategies in the future.

erred over FGF-23 at the present time, and studies investigating patients over 50–55 years of age with high levels of FGF-23 have been suggested to provide the most valuable data. However, it was generally agreed that FGF-23 may be a uremic toxin, which would support development of anti-FGF-23 strategies in the future. Development of other therapeutic targets, including biomarkers that assess function of NaPi2a, b, or c (to decrease the reabsorption of phosphate) or Klotho, is considered to be challenging but should remain a long-term goal. The effects of infusion of soluble Klotho, how the bone senses phosphate, and why high levels of FGF-23 are found in non-renal organs are other areas of interest for future research. This supplement provides a review of five of the topics discussed during the forum: Dr Slatopolsky presents the history of the intact nephron hypothesis and its current implications; Dr Confavreux presents a very new facet of the bone, from a reservoir of minerals to a regulator of energy metabolism; Dr Hruska develops the cardiovascular risk factors in CKD; and Dr Jüppner and Dr Kuro-o revisit the classic physiology in light of the recent discoveries of FGF-23 and Klotho. Publication of this supplement was supported by Genzyme Corporation. Editorial assistance was provided by Kim Coleman Healy, PhD, Envision Scientific Solutions.

This supplement provides a review of five of the topics discussed during the forum: Dr Slatopolsky presents the history of the intact nephron hypothesis and its current implications; Dr Confavreux presents a very new facet of the bone, from a reservoir of minerals to a regulator of energy metabolism; Dr Hruska develops the cardiovascular risk factors in CKD; and Dr Jüppner and Dr Kuro-o revisit the classic physiology in light of the recent discoveries of FGF-23 and Klotho. Publication of this supplement was supported by Genzyme Corporation. Editorial assistance was provided by Kim Coleman Healy, PhD, Envision Scientific Solutions. ES has received consultancy or advisory board fees, lecturer's fees, and research grants from Abbott Laboratories and Genzyme Corporation. ES and Washington University may receive income based on a license-related technology by the University of Wisconsin. SM has received consultancy or advisory board fees from Genzyme Corporation, Amgen Inc, INEOS, Litholink: A LabCorp Company, DiaSorin, Inc, Baxter, and has equity ownership/stock options with Eli Lilly and Company. SM also has received grants from Genzyme Corporation, Amgen Inc, and Shire Plc.

Klotho, named after an ancient Greek goddess of fate, is a putative aging suppressor gene. A defect in Klotho gene expression in mice confers penetrant phenotypes resembling human premature aging syndromes,1 whereas Klotho overexpression confers longevity exceeding the wild type.2 Pathology in Klotho–/– mice includes osteopenia and calcifications (vascular and ectopic) resembling chronic kidney disease-associated mineral and bone disorder (CKD-MBD), in addition to short lifespan and senescent changes in the heart, lungs, thymus, gonads, skin, muscles, hearing, and motor neurons (reviewed by Kuro-o3). The Klotho gene encodes a single-pass transmembrane protein expressed predominantly in the kidney (intensely in the distal convoluted tubule (DCT) and to a lesser extent in the proximal tubule1, 3, 4) and parathyroid gland.5 The phenotypes of Klotho–/– and Fgf23–/– mice are very similar, involving premature aging and abnormal mineral metabolism.1, 6 Both mutants share the senescent phenotypes of short lifespan, growth retardation, hypogonadism, early thymic involution, skin and muscle atrophy, osteoporosis, and emphysema, and deranged mineral metabolism phenotypes including vascular calcification, hyperphosphatemia, hypercalcemia, hypoglycemia, and hypervitaminosis D. These similarities point to the involvement of Klotho and fibroblast growth factor (FGF)-23 in a common physiological pathway. OBJECTIVE This review article will discuss the involvement of Klotho in phosphate metabolism in CKD-MBD and propose the hypothesis that Klotho deficiency is the earliest biomarker of CKD.

The phenotypes of Klotho–/– and Fgf23–/– mice are very similar, involving premature aging and abnormal mineral metabolism.1, 6 Both mutants share the senescent phenotypes of short lifespan, growth retardation, hypogonadism, early thymic involution, skin and muscle atrophy, osteoporosis, and emphysema, and deranged mineral metabolism phenotypes including vascular calcification, hyperphosphatemia, hypercalcemia, hypoglycemia, and hypervitaminosis D. These similarities point to the involvement of Klotho and fibroblast growth factor (FGF)-23 in a common physiological pathway. OBJECTIVE This review article will discuss the involvement of Klotho in phosphate metabolism in CKD-MBD and propose the hypothesis that Klotho deficiency is the earliest biomarker of CKD. THE KLOTHO PROTEIN AS OBLIGATE FGF-23 CO-RECEPTOR Canonical FGF receptors (FGFRs), which require cofactors for specific binding and signal transduction, are expressed in multiple tissues. Most FGFs use heparan sulfate as a cofactor facilitating their binding to FGFRs.3 Endocrine FGFs, however, including FGF-23, use other cofactors (or co-receptors).3 Klotho protein is a co-receptor specific for FGF-23 (refs 7, 8).

cofactors for specific binding and signal transduction, are expressed in multiple tissues. Most FGFs use heparan sulfate as a cofactor facilitating their binding to FGFRs.3 Endocrine FGFs, however, including FGF-23, use other cofactors (or co-receptors).3 Klotho protein is a co-receptor specific for FGF-23 (refs 7, 8). Kidney1 and parathyroid5 Klotho expression identifies these organs as high-affinity FGF-23 endocrine targets. The Klotho/FGFR complex thus mediates FGF-23 participation in the bone–kidney–parathyroid endocrine axis. In the kidney, FGF-23 acting on Klotho/FGFR suppresses phosphate reabsorption and 1,25(OH)2D3 synthesis;9 in the parathyroid, FGF-23 suppresses parathyroid hormone secretion,5, 10, 11 which may also contribute to the ability of FGF-23 to reduce 1,25(OH)2D3 synthesis. Thus, FGF-23 is both a phosphaturic hormone and the counter-regulatory hormone to vitamin D in the bone–kidney–parathyroid endocrine axis, and Klotho is required for the effects of FGF-23 (ref. 12; Figure 1). Both these FGF-23 actions promote negative phosphate balance. Thus, FGF-23 can be identified as ‘phosphatonin', the bone-generated humoral phosphaturic factor postulated more than 10 years ago.13 Secretion of FGF-23 by bone is induced by phosphate and 1,25(OH)2D3(9) and possibly by parathyroid hormone.10, 14 Klotho gene expression is inducible by 1,25(OH)2D3 (ref. 15).

e balance. Thus, FGF-23 can be identified as ‘phosphatonin', the bone-generated humoral phosphaturic factor postulated more than 10 years ago.13 Secretion of FGF-23 by bone is induced by phosphate and 1,25(OH)2D3(9) and possibly by parathyroid hormone.10, 14 Klotho gene expression is inducible by 1,25(OH)2D3 (ref. 15). PATHOGENESIS OF HYPERPHOSPHATEMIA IN KLOTHO–/– MUTANTS Phosphate pathophysiology mediates complex aging-like phenotypes in mice with defects in the Klotho–FGF-23 system. Low-phosphate diet improves the aging-like phenotypes of Klotho mutant mice16 and Fgf23–/– mice.17 Mutant homozygotes consuming 1.03 g phosphorus/100 g diet had typical mutant phenotypes. Male homozygotes consuming 0.4 g phosphorus/100 g diet expressed the Klotho protein in their kidneys and resumed normal spermatogenesis.16 Female homozygotes required zinc supplementation as well as phosphorus restriction for phenotypic rescue.16 Phosphate restriction corrected CKD-MBD-like FGF-23-null phenotypes (hyperphosphatemia, vascular calcifications, and mortality) even though serum calcium and 1,25(OH)2D3 levels remained elevated.17 Several other genetic and dietary interventions that rescue Klotho–/– and/or Fgf23–/– phenotypes18, 19, 20 have lowered serum phosphate as their only common denominator (Table 1). Phosphate retention may thus accelerate aging and/or age-related diseases in mice and humans.21

and 1,25(OH)2D3 levels remained elevated.17 Several other genetic and dietary interventions that rescue Klotho–/– and/or Fgf23–/– phenotypes18, 19, 20 have lowered serum phosphate as their only common denominator (Table 1). Phosphate retention may thus accelerate aging and/or age-related diseases in mice and humans.21 Patients with CKD are far more likely to die of cardiovascular disease than to live to require dialysis.22 CKD-related cardiovascular disease is substantially fueled by hyperphosphatemia and can be seen as phosphate-related accelerated cardiovascular aging.23 A KLOTHO-CENTRIC VIEW OF CKD It may be hypothesized that CKD represents a state of accelerated aging associated with Klotho deficiency and phosphate retention, and that Klotho deficiency is the earliest biomarker of CKD and the initiator of CKD-related mineral dysregulation.

Patients with CKD are far more likely to die of cardiovascular disease than to live to require dialysis.22 CKD-related cardiovascular disease is substantially fueled by hyperphosphatemia and can be seen as phosphate-related accelerated cardiovascular aging.23 A KLOTHO-CENTRIC VIEW OF CKD It may be hypothesized that CKD represents a state of accelerated aging associated with Klotho deficiency and phosphate retention, and that Klotho deficiency is the earliest biomarker of CKD and the initiator of CKD-related mineral dysregulation. Klotho expression declines progressively in CKD as FGF-23 expression increases progressively; high serum phosphate and parathyroid hormone and low 1,25(OH)2D3 accompany these changes (Figure 2). The first measurable decline in urinary secreted Klotho expression (as detected by western blotting of concentrated urine samples, normalized to the same creatinine content) occurs as early as stage 1 CKD24 and is potentially an early clinical marker of nascent acute renal damage. Klotho decline precedes FGF-23 increase as CKD develops in Jck mice, a cystic kidney disease model of early progressive CKD.25 Renal Klotho expression assays (mRNA measurement by RNAse protection, protein measurement by western blotting, and immunohistochemistry) in human kidney specimens from dialysis patients or controls showed that dialysis patients expressed renal membrane Klotho at only 5–15%, most often <5%, of control levels.26 Median Klotho mRNA levels in healthy kidney tissue represented slightly >8% of the level of glyceraldehyde-3-phosphate dehydrogenase, a housekeeping mRNA.26 A sandwich enzyme-linked immunosorbent assay for secreted Klotho in serum also exists and has shown that circulating secreted Klotho in healthy adults ranges from 239 to 1266 pg/ml, decreasing with advancing age and increasing calcemia and increasing with phosphatemia levels.27 More sensitive assays by multiple reaction monitoring using mass spectrometry are currently in progress.

o in serum also exists and has shown that circulating secreted Klotho in healthy adults ranges from 239 to 1266 pg/ml, decreasing with advancing age and increasing calcemia and increasing with phosphatemia levels.27 More sensitive assays by multiple reaction monitoring using mass spectrometry are currently in progress. Reducing serum FGF-23 increases serum 1,25(OH)2D3 and renal Klotho expression. Parathyroidectomy is expected to reduce FGF-23 production,14 which in turn increases 1,25(OH)2D3 synthesis and then renal Klotho expression. Vitamin D administration,15 peroxisome proliferator-activated receptor-γ agonists,28 or angiotensin II inhibitors29, 30 also increase Klotho expression. Large serum FGF-23 increases during CKD progression31 are efforts to maintain FGF-23 signaling as receptor availability decreases. In the normal kidney, Klotho expression is abundant and a small amount of FGF-23 effectively induces phosphaturia. As CKD progresses, ever-increasing serum FGF-23 acts on a resistant kidney with ever-fewer functional nephrons, each of which expresses less Klotho than in a healthy kidney. Serum FGF-23 increases in an attempt to maintain normophosphatemia, but also suppresses 1,25(OH)2D3 synthesis.31 Ultimately, total phosphate excretion can no longer keep pace and serum phosphate increases.

esistant kidney with ever-fewer functional nephrons, each of which expresses less Klotho than in a healthy kidney. Serum FGF-23 increases in an attempt to maintain normophosphatemia, but also suppresses 1,25(OH)2D3 synthesis.31 Ultimately, total phosphate excretion can no longer keep pace and serum phosphate increases. Klotho is a renoprotective factor; when overexpressed it exerts a beneficial effect on mouse glomerulonephritis32 and acute kidney injury33 models. Decline in renal Klotho expression precedes both FGF-23 overexpression and hyperphosphatemia, and may represent the initiating event of CKD. Thus far, we have discussed modulation of the bone–kidney–parathyroid endocrine and phosphaturic axis by renal and parathyroid cell-surface Klotho. However, Klotho also exists as a secreted form produced by clipping the extracellular part of the molecule.34, 35 No form of Klotho protein without FGFR can bind to FGF-23 with high affinity. Secreted Klotho is found in blood, urine, and cerebrospinal fluid,36 and acts as an FGF-23-independent phosphaturic hormone.4 SECRETED KLOTHO, PHOSPHATE EXCRETION, AND RENAL PARACRINE SIGNALING Proximal tubules are where FGF-23 suppresses phosphate reabsorption and 1,25(OH)2D3 synthesis. However, Klotho is expressed most intensely in renal DCT and only weakly in proximal tubules. Two mechanisms for proximal tubule FGF-23 activity are possible:3 either FGF-23 acts directly on the proximal tubules and Klotho's function in the DCT is unexplained, or FGF-23 acts through Klotho on the DCT to induce a paracrine signal to proximal tubules.37

intensely in renal DCT and only weakly in proximal tubules. Two mechanisms for proximal tubule FGF-23 activity are possible:3 either FGF-23 acts directly on the proximal tubules and Klotho's function in the DCT is unexplained, or FGF-23 acts through Klotho on the DCT to induce a paracrine signal to proximal tubules.37 The extracellular portion of Klotho protein is clipped by membrane proteases ADAM10 and ADAM17 (ref. 34) and BACE1 (ref. 35) and secreted into blood, urine, and cerebrospinal fluid.36 Secreted Klotho inhibits the sodium/phosphate transporters NPT2a, NPT2c, and NPT3 (refs 4, 24) and activates ion channels TRPV5 (ref. 38), TRPV6 (ref. 39), and ROMK1 (ref. 40). Secreted Klotho exerts phosphaturic effects independently of FGF-23. Intravenously administered secreted Klotho induces phosphaturia in normal and Fgf23–/– mice.4 NaPi2a mediates proximal tubule phosphate reabsorption (70–80% of total phosphate reabsorption); studies on brush border membrane vesicles from proximal tubule cells show that secreted Klotho inactivates NaPi2a.4 Secreted Klotho conserves serum calcium and reduces calciuria. Some 70% of calcium reabsorption occurs in the proximal tubule and 15% in the DCT (utilizing TRPV5; ref. 41). Whole-cell patch-clamp experiments show that secreted Klotho activates the calcium channel TRPV5 (ref. 38), which is responsible for DCT calcium reabsorption.41

ho conserves serum calcium and reduces calciuria. Some 70% of calcium reabsorption occurs in the proximal tubule and 15% in the DCT (utilizing TRPV5; ref. 41). Whole-cell patch-clamp experiments show that secreted Klotho activates the calcium channel TRPV5 (ref. 38), which is responsible for DCT calcium reabsorption.41 We hypothesize that FGF-23 suppresses renal phosphate reabsorption and promotes calcium reabsorption by promoting the secretion of Klotho from DCT cells. Klotho entering the luminal fluid inhibits NaPi2a in proximal tubules to allow phosphate excretion and activates TRPV5 in distal tubules to reabsorb calcium. Secreted Klotho is present in the luminal fluid of proximal tubules,4 but how it is transported into the proximal tubular lumen is not yet known. CONCLUSIONS Renal and parathyroid Klotho co-receptors make FGFR specific for FGF-23, the humoral phosphatonin secreted by bone. In the kidney, Klotho mediates phosphate excretion and feedback inhibition of 1,25(OH)2D3 synthesis in response to FGF-23. Klotho deficiency causes hyperphosphatemia and accelerated aging phenotypes, which are prevented in animals by resolving phosphate retention. CKD and its complications, including CKD-MBD and vascular calcification, represent accelerated aging triggered by Klotho deficiency. Klotho expression begins declining early in CKD and may precede both hyperphosphatemia and FGF-23 upregulation. Further research is needed to determine whether Klotho decline or increased FGF-23 drives the vicious cycle of phosphate pathology in CKD.

fication, represent accelerated aging triggered by Klotho deficiency. Klotho expression begins declining early in CKD and may precede both hyperphosphatemia and FGF-23 upregulation. Further research is needed to determine whether Klotho decline or increased FGF-23 drives the vicious cycle of phosphate pathology in CKD. Secreted Klotho, an FGF-23-independent phosphaturic hormone, regulates renal sodium/phosphate cotransporters and calcium and potassium ion channels. We hypothesize that FGF-23 induces secretion of Klotho from DCT cells, and secreted Klotho is a paracrine signal to proximal tubule cells to inhibit phosphate reabsorption and stimulate calcium reabsorption. Decreased urinary secreted Klotho may reflect decreased renal Klotho expression and is one of the earliest biomarkers of CKD. It is concluded that phosphate retention induces complex aging-like phenotypes. Thus, maintaining normal phosphate levels with phosphate binders in patients with CKD with declining Klotho expression is expected to reduce mineral and vascular derangements. This article was developed from the author's presentation and discussions at the ‘50 Years of Discovery Following the Intact Nephron Hypothesis' advisory board in Munich, Germany, 24–25 June 2010. The author meets all International Council of Medical Journal Editors criteria and acknowledges the writing assistance of Kim Coleman Healy, PhD, of Envision Scientific Solutions. Publication of this supplement was supported by Genzyme Corporation. TO CITE THIS ARTICLE: Kuro-o M. Phosphate and Klotho. Kidney Int 2011; 79 (Suppl 121):S20–S23.

This article was developed from the author's presentation and discussions at the ‘50 Years of Discovery Following the Intact Nephron Hypothesis' advisory board in Munich, Germany, 24–25 June 2010. The author meets all International Council of Medical Journal Editors criteria and acknowledges the writing assistance of Kim Coleman Healy, PhD, of Envision Scientific Solutions. Publication of this supplement was supported by Genzyme Corporation. TO CITE THIS ARTICLE: Kuro-o M. Phosphate and Klotho. Kidney Int 2011; 79 (Suppl 121):S20–S23. MK has received research grant support from Genzyme Corporation, the National Institutes of Health, the Texas Higher Education Coordinating Board, and from Ardelyx. MK has a patent with the Japanese patent filing number H10-529809; Title: Novel polypeptide, novel DNA and novel antibody (Klotho). Figure 1 Endocrine regulation of phosphate metabolism. Circulating 1,25(OH)2D3 turns on the FGF-23 promoter in bone cells. Secreted FGF-23 binds to renal cell Klotho/FGF receptor to turn off the 1α-hydroxylase promoter and turn on the 24-hydroxylase promoter, resulting in net inactivation of conversion of vitamin D to 1,25(OH)2D3. PTH affects these renal promoters in a reverse manner to FGF-23, leading to 1,25(OH)2D3 production. In the parathyroid, FGF-23 binds to Klotho/FGF receptor and shuts off the PTH promoter. FGF-23, fibroblast growth factor-23; PTH, parathyroid hormone.

ulting in net inactivation of conversion of vitamin D to 1,25(OH)2D3. PTH affects these renal promoters in a reverse manner to FGF-23, leading to 1,25(OH)2D3 production. In the parathyroid, FGF-23 binds to Klotho/FGF receptor and shuts off the PTH promoter. FGF-23, fibroblast growth factor-23; PTH, parathyroid hormone. Figure 2 Changes in Klotho protein, FGF-23, PTH, 1,25(OH)2D3, and phosphate as CKD progresses. When Klotho expression first decreases, FGF-23 increases, lowering circulating 1,25(OH)2D3, which depresses Klotho expression further and increases PTH expression. Increased PTH induces further FGF-23 increases, causing large decreases in 1,25(OH)2D3 and large increases in PTH. This cycle results in hyperphosphatemia in late stages of CKD. CKD, chronic kidney disease; FGF-23, fibroblast growth factor-23; PTH, parathyroid hormone. Table 1 Effects on mineral metabolism of interventions rescuing Klotho–/– and Fgf23–/– mouse phenotypes17, 18, 19, 20 Direction of change in serum levels Intervention Phosphate 1,25(OH)2D3 Calcium Low-phosphate diet ↓ ↑ ↑ 1α-Hydroxylase knockout ↓ ↓ ↓ Vitamin D receptor knockout ↓ ↑ ↓ Na–Pi cotransporter IIa knockout ↓ ↑ ↑ Low-vitamin D diet ↓ ↓ ↓

Phosphate is essential for many cellular functions. It is a constituent of DNA, membrane lipids, high-energy phosphates, and second messengers, that is, inositol trisphosphate, cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate, and protein phosphorylation is an essential process, which helps regulate enzyme and receptor activities. Because phosphate is scarce in nature, vertebrate physiology has evolved to conserve phosphate through the efficient use of three sodium-dependent phosphate co-transporters (NaPi-IIa, NaPi-IIb, and NaPi-IIc, also referred to as NPT2a, NPT2b, and NPT2c) that are highly conserved throughout vertebrate taxa, from fish1 to humans.2 Additional molecules contributing to the regulation of phosphate homeostasis include Pit2 and the 1α-hydroxylase, which allows the formation of calcitriol (1,25(OH)2D3). Expression of these proteins in the proximal renal tubules is regulated by serum phosphate concentration and different hormonal systems. For example, low serum phosphate levels induce NPT2b expression in the intestinal tract, thus enhancing absorption of this mineral from the diet. In addition, low serum phosphate levels induce NPT2a and NPT2c expression in the proximal tubules of the kidney, thus maximizing the reabsorption of phosphate and minimizing urinary losses of this mineral. The efficiency of this latter process was strikingly demonstrated in a 1976 study, in which healthy human volunteers on a low-phosphate diet (90 mg/day) reduced their phosphate excretion considerably, thereby avoiding profound hypophosphatemia.2 In contrast to the efficient adaptation to hypophosphatemia, renal excretion of excess phosphate is a more difficult problem for human physiology to solve.

, in which healthy human volunteers on a low-phosphate diet (90 mg/day) reduced their phosphate excretion considerably, thereby avoiding profound hypophosphatemia.2 In contrast to the efficient adaptation to hypophosphatemia, renal excretion of excess phosphate is a more difficult problem for human physiology to solve. The principal hormones that regulate renal phosphate handling are parathyroid hormone (PTH), which is produced by the parathyroid gland, and fibroblast growth factor (FGF)-23, which is produced by osteocytes and osteoblasts in bone. In healthy individuals, increasing serum phosphate concentration induces secretion of PTH and FGF-23. These two phosphaturic hormones reduce expression of NPT2a and NPT2c in the proximal renal tubules, thereby diminishing phosphate reabsorption and increasing urinary phosphate excretion.3, 4 PTH also increases the 1α-hydroxylase in the kidney, thereby increasing 1,25(OH)2D3 production, thus enhancing intestinal calcium and phosphate absorption. Some evidence suggests that PTH induces expression and secretion of FGF-23; FGF-23 in turn decreases 1,25(OH)2D3 production, which is an inhibitor of PTH production.

TH also increases the 1α-hydroxylase in the kidney, thereby increasing 1,25(OH)2D3 production, thus enhancing intestinal calcium and phosphate absorption. Some evidence suggests that PTH induces expression and secretion of FGF-23; FGF-23 in turn decreases 1,25(OH)2D3 production, which is an inhibitor of PTH production. In chronic kidney disease (CKD), increased FGF-23 production enhances the excretion of phosphate per nephron, thereby restoring normophosphatemia. However, it also reduces 1,25(OH)2D3 levels,5 contributing to an increase in PTH secretion, which appears to occur after FGF-23 levels increase. This process disrupts the bone–kidney–parathyroid endocrine axis and eventually fails to prevent the development of hyperphosphatemia as CKD progresses. The resulting changes, constituting CKD-related mineral and bone disorder (CKD-MBD), are a reflection of the trade-offs postulated by Bricker's6 intact nephron hypothesis: ‘If solute intake does not diminish as the number of excretory units diminishes, the adaptive increase in excretion rate per nephron may be accomplished only at the expense of one or more abnormalities of the uremic state.' This review will explore preclinical and clinical studies of the role of FGF-23 in phosphate metabolism in CKD and the contribution of FGF-23 to CKD-MBD.

etory units diminishes, the adaptive increase in excretion rate per nephron may be accomplished only at the expense of one or more abnormalities of the uremic state.' This review will explore preclinical and clinical studies of the role of FGF-23 in phosphate metabolism in CKD and the contribution of FGF-23 to CKD-MBD. RENAL REGULATION OF PHOSPHATE AND CALCIUM HOMEOSTASIS Role of PTH: lessons from familial pseudohypoparathyroidism NPT2a and NPT2c facilitate the efficient reabsorption of phosphate in the proximal tubule; expression of both transporters is regulated by PTH and FGF-23. The acute PTH-dependent regulation of NPT2a and NPT2c expression is mediated predominantly through the cAMP and protein kinase A signaling pathway.4 The PTH-induced increase in urinary phosphate is coupled to cAMP excretion from proximal tubules. Cyclic AMP production requires Gsα, which appears to be derived in this portion of the kidney predominantly, if not exclusively, from the maternal allele, thereby potentially limiting the maximal amount of Gsα protein that can be generated in this tissue. As a consequence of this parent-specific expression of Gsα in the proximal renal tubules, maternally inherited, heterozygous GNAS mutations cause PTH resistance, that is, pseudohyperparathyroidism.7 Inactivating GNAS mutations in those exons that encode Gsα cause pseudohypoparathyroidism type Ia, whereas microdeletions within or upstream of GNAS cause pseudohypoparathyroidism type Ib and are associated with GNAS methylation changes. The mechanism by which cAMP is excreted into the urine remains unknown, but is expected to involve a specific transporter that is expressed in the proximal tubule. In the distal tubule, PTH diminishes calcium excretion through cAMP/protein kinase A- and possibly also through inositol trisphosphate/protein kinase C-dependent actions on the function of calbindin-D and TRPV5. In contrast to the findings in proximal tubular cells, Gsα is expressed in the distal tubular cells from both parental alleles, and maternally inherited GNAS mutations consequently leave the PTH-induced regulation of calcium excretion intact.

/protein kinase C-dependent actions on the function of calbindin-D and TRPV5. In contrast to the findings in proximal tubular cells, Gsα is expressed in the distal tubular cells from both parental alleles, and maternally inherited GNAS mutations consequently leave the PTH-induced regulation of calcium excretion intact. Role of FGF-23: lessons from CKD Similar to the actions of PTH, FGF-23 controls NPT2a and NPT2c expression. FGF-23 is secreted by bone cells in response to 1,25(OH)2D3, which increases the mRNA levels encoding this phosphaturic hormone.8 FGF-23 mediates its action in the kidney through an FGF receptor (FGFR)/Klotho complex to downregulate NPT2a and NPT2c expression in the proximal tubules. However, it is uncertain whether FGF-23 signals initially through receptors in the distal convoluted tubule cells in which ERK phosphorylation occurs in response to FGF-23.9 These cells are adjacent to NPT2a-expressing proximal tubular cells and it has therefore been speculated that a paracrine signal from the distal to the proximal tubules is required for decreasing NPT2a expression. Even small changes in plasma FGF-23 levels are associated with significant changes in urinary phosphate excretion, as shown by the small increase in FGF-23 levels after unilateral nephrectomy in healthy kidney donors, which is associated with increased urinary phosphate excretion.10

red for decreasing NPT2a expression. Even small changes in plasma FGF-23 levels are associated with significant changes in urinary phosphate excretion, as shown by the small increase in FGF-23 levels after unilateral nephrectomy in healthy kidney donors, which is associated with increased urinary phosphate excretion.10 Besides its effect on tubular phosphate handling, FGF-23 reduces PTH secretion, and it inhibits 1α-hydroxylase leading to a decrease in 1,25(OH)2D3 production, which contributes to the development of hypocalcemia and leads to an increase in PTH production.11 The 1,25(OH)2D3 itself increases FGF-23 production.

red for decreasing NPT2a expression. Even small changes in plasma FGF-23 levels are associated with significant changes in urinary phosphate excretion, as shown by the small increase in FGF-23 levels after unilateral nephrectomy in healthy kidney donors, which is associated with increased urinary phosphate excretion.10 Besides its effect on tubular phosphate handling, FGF-23 reduces PTH secretion, and it inhibits 1α-hydroxylase leading to a decrease in 1,25(OH)2D3 production, which contributes to the development of hypocalcemia and leads to an increase in PTH production.11 The 1,25(OH)2D3 itself increases FGF-23 production. FGF-23 AND PHOSPHATE IN CKD FGF-23 appears to be an important biomarker for an abnormal regulation of phosphate homeostasis in CKD and is likely to be involved in CKD-MBD pathophysiology. As CKD progresses, plasma FGF-23 levels increase. This increase occurs earlier and to a greater extent than observed for serum phosphate; in late CKD, plasma FGF-23 levels can be elevated by several orders of magnitude.5, 12 Findings in experimental renal disease suggest that the FGF-23 increase also precedes the increase in PTH levels.11 In fact, bone biopsies of patients with CKD have shown increased expression of FGF-23 already by CKD stage 2, along with a marked increase in DMP1 protein (a negative regulator of FGF-23), which may be improperly processed and could thus be inactive.13 Furthermore, in a prospective study of patients with mild-to-moderate CKD,14 higher plasma FGF-23 levels were shown to predict a more rapid progression toward end-stage renal disease (ESRD); similarly elevated FGF-23 levels predict CKD progression in patients with diabetic nephropathy.15 In addition, elevated FGF-23 levels are independently associated with left ventricular hypertrophy in patients with CKD,16 an important finding because CKD-MBD, and particularly hyperphosphatemia, worsen the cardiovascular prognosis in CKD. Aortic calcification, a major reason for cardiovascular morbidity, in hemodialysis recipients is independently predicted by plasma FGF-23.17

ted with left ventricular hypertrophy in patients with CKD,16 an important finding because CKD-MBD, and particularly hyperphosphatemia, worsen the cardiovascular prognosis in CKD. Aortic calcification, a major reason for cardiovascular morbidity, in hemodialysis recipients is independently predicted by plasma FGF-23.17 Phosphate is a major regulator of FGF-23 expression.18 Dietary phosphate loading increases FGF-23 expression, whereas phosphate depletion with binders decreases the circulating levels of this hormone.19 In mild CKD, FGF-23 seems to function as a protective factor, as it triggers adaptive changes that maintain normophosphatemia. For example, in animal models, FGF-23 protects against hyperphosphatemia by increasing urinary phosphate excretion and reducing 1,25(OH)2D3 production; diminished 1,25(OH)2D3 levels lead to hypocalcemia and thus to an increase in PTH levels, which further enhances renal phosphate excretion.11 The importance of FGF-23 in mediating these effects is shown by the effects of anti-FGF-23 antibodies in rats with experimental CKD.11 Animals with early CKD that were treated with anti-FGF-23 antibodies showed an increase in 1,25(OH)2D3 levels, a normalization of serum calcium levels, and a decrease in PTH levels. Furthermore, fractional phosphate excretion decreased because of inactivation of FGF-23 resulting in hyperphosphatemia.

xperimental CKD.11 Animals with early CKD that were treated with anti-FGF-23 antibodies showed an increase in 1,25(OH)2D3 levels, a normalization of serum calcium levels, and a decrease in PTH levels. Furthermore, fractional phosphate excretion decreased because of inactivation of FGF-23 resulting in hyperphosphatemia. As CKD progresses, elevated FGF-23 levels are no longer able to enhance urinary phosphate excretion, thus leading to the development of hyperphosphatemia. This may be partly related to declining Klotho expression and a reduction in functional nephrons. When patients require treatment by dialysis, the levels of immunoreactive FGF-23 can be markedly elevated (Figure 1).20 However, unlike the appearance of large amounts of C-terminal PTH fragments, most of which are biologically inactive, practically all of the circulating FGF-23 present in patients with ESRD is intact and biologically active. This conclusion is based on observations with a reporter-cell assay (human embryonic kidney cells expressing Klotho and a luciferase reporter expressed under the control of the EGR-1 promoter) designed to quantify biologically active FGF-23, which demonstrated excellent correlation with immunoreactive measurement of FGF-23. The immunometric assays used for these measurements quantify the intact FGF-23 molecule alone (Kainos assay) or the intact protein along with a C-terminal fragment (Immutopics assay).21, 22 Consistent with the excellent correlation between bioactive and immunoreactive FGF23, Western blot analyses of plasma FGF-23 from dialysis patients showed that most of the circulating FGF-23 is intact.21 Thus, with CKD progression, the excess of biologically active FGF-23, which is thought to occur in response to hyperphosphatemia, ceases to be protective and may lead to pathological off-target effects that potentially contribute to the increase in mortality as FGF-23 increases in patients with ESRD23, 24 (Figure 2).

1 Thus, with CKD progression, the excess of biologically active FGF-23, which is thought to occur in response to hyperphosphatemia, ceases to be protective and may lead to pathological off-target effects that potentially contribute to the increase in mortality as FGF-23 increases in patients with ESRD23, 24 (Figure 2). Interventions that lower serum phosphate, such as oral phosphate binders to prevent intestinal absorption or possibly long-acting PTH analogs that reduce NPT2a or NPT2c expression, may help prevent CKD-related FGF-23 increases (Figure 3). For example, two recently reported, long-acting PTH analogs (M-PTH(1-28) and Trp1-M-PTH(1-28)) were found to efficiently reduce NPT2a expression in proximal tubules, leading to sustained hypophosphatemia in wild-type mice.25 Targeting the proximal tubules with long-acting PTH agents in early CKD may add to the effects of phosphate binders in normalizing serum phosphate levels and may help avoid an increase in FGF-23, unless PTH has a role in the synthesis and/or secretion of FGF-23. Proactive interventions to avoid the development of transient hyperphosphatemia in CKD may thus prevent an increase in FGF-23 and the hormone's putative off-target effects in CKD-MBD. In support of this conclusion, a recent post hoc analysis of a randomized clinical trial has shown that treatment with phosphate binders can markedly decrease FGF-23 levels in patients undergoing hemodialysis.26

emia in CKD may thus prevent an increase in FGF-23 and the hormone's putative off-target effects in CKD-MBD. In support of this conclusion, a recent post hoc analysis of a randomized clinical trial has shown that treatment with phosphate binders can markedly decrease FGF-23 levels in patients undergoing hemodialysis.26 CONCLUSIONS FGF-23 is a bone-generated major regulator of phosphate homeostasis, which appears to be an important biomarker of phosphate homeostasis in patients with CKD. Plasma FGF-23 concentrations begin to increase early in CKD, with increases by orders of magnitude by the time patients reach ESRD. Increasing FGF-23 levels are independently associated with left ventricular hypertrophy, CKD progression, and mortality, possibly through off-target actions. Lowering serum phosphate levels through the use of phosphate binders may lower FGF-23 levels. Further research is needed to show whether lowering FGF-23 levels improves outcomes in patients with CKD. This article was developed from the author's presentation and discussions at the ‘50 Years of Discovery Following the Intact Nephron Hypothesis' symposium in Munich, Germany, 24–25 June 2010. The author meets all International Council of Medical Journal Editors criteria and acknowledges the writing assistance of Kim Coleman Healy, PhD, of Envision Scientific Solutions. Publication of this supplement was supported by Genzyme Corporation. TO CITE THIS ARTICLE: Jüppner H. Phosphate and FGF-23. Kidney Int 2011; 79 (Suppl 121):S24–S27.

This article was developed from the author's presentation and discussions at the ‘50 Years of Discovery Following the Intact Nephron Hypothesis' symposium in Munich, Germany, 24–25 June 2010. The author meets all International Council of Medical Journal Editors criteria and acknowledges the writing assistance of Kim Coleman Healy, PhD, of Envision Scientific Solutions. Publication of this supplement was supported by Genzyme Corporation. TO CITE THIS ARTICLE: Jüppner H. Phosphate and FGF-23. Kidney Int 2011; 79 (Suppl 121):S24–S27. HJ has served as a paid consultant to Genzyme and Roche, and is a named inventor on the patent outlining the development of immunometric assays for the detection of FGF-23. Figure 1 Spectrum of serum fibroblast growth factor (FGF)-23 levels in early chronic kidney disease and end-stage renal disease (ESRD) as compared with the normal condition and with different disorders affecting FGF-23. ADHR, autosomal dominant hypophosphatemic rickets; ARHP, autosomal recessive hypophosphatemia; TIO, tumor-induced osteomalacia; XLH, X-linked hypophosphatemia. Adapted with permission from Isakova et al.20 Figure 2 Mortality in patients with end-stage renal disease (ESRD) receiving hemodialysis in relationship to quartiles of serum fibroblast growth factor (FGF)-23 concentration at initiation of dialysis. R, reference. Reproduced with permission from Gutiérrez et al.23 Copyright The Massachusetts Medical Society.

Figure 1 Spectrum of serum fibroblast growth factor (FGF)-23 levels in early chronic kidney disease and end-stage renal disease (ESRD) as compared with the normal condition and with different disorders affecting FGF-23. ADHR, autosomal dominant hypophosphatemic rickets; ARHP, autosomal recessive hypophosphatemia; TIO, tumor-induced osteomalacia; XLH, X-linked hypophosphatemia. Adapted with permission from Isakova et al.20 Figure 2 Mortality in patients with end-stage renal disease (ESRD) receiving hemodialysis in relationship to quartiles of serum fibroblast growth factor (FGF)-23 concentration at initiation of dialysis. R, reference. Reproduced with permission from Gutiérrez et al.23 Copyright The Massachusetts Medical Society. Figure 3 ‘Trade-off hypothesis' revisited. Involvement of fibroblast growth factor (FGF)-23 in end-stage renal disease (ESRD) pathology, reflecting potentially adverse effects of increased secretion of this phosphaturic hormone, which helps normalize phosphate homeostasis but contributes to the development of secondary hyperparathyroidism. PTH, parathyroid hormone.

In advancing chronic kidney disease (CKD), there is a multitude of biochemical, physiological, and clinical alterations. In the nineteenth century, Bright1 recognized the relationship between intrinsic kidney disease and the many abnormalities seen in the uremic state. The exact nature of events leading from the initial destruction of nephrons to the pathophysiological changes of end-stage renal function was not fully understood then. However, there is now significant evidence demonstrating that the many forms of CKD could give rise to the same pattern of derangements that relate principally to the extent of nephron destruction. The physiopathology of ‘chronic Bright's disease', an exposition of the ‘intact nephron hypothesis' published in Bricker et al.,2 provided a logical framework for understanding the changes in renal function that occur in animal models and in patients with CKD. Bricker et al. observed adaptive changes in renal function and in homeostasis that result from progressive CKD.

n exposition of the ‘intact nephron hypothesis' published in Bricker et al.,2 provided a logical framework for understanding the changes in renal function that occur in animal models and in patients with CKD. Bricker et al. observed adaptive changes in renal function and in homeostasis that result from progressive CKD. Bricker3 emphasized that, as the number of functioning nephrons decreases, each remaining nephron must perform a greater fraction of total renal excretion. Bricker clearly explained the differences between substances such as creatinine, which are mainly handled by glomerular filtration, and sodium and phosphate, which are mainly handled by renal tubular reabsorption. For substances handled by glomerular filtration, a decrease in glomerular filtration induces a progressive increase in their plasma concentration early in the course of CKD. On the other hand, with substances such as sodium and phosphate that are mainly handled by tubular reabsorption, a decrease in renal tubular reabsorption of these substances will prevent a significant change in their blood concentrations, and their levels will remain normal in serum until advanced deterioration of renal function. Similarly, with substances such as potassium, which are mainly handled by renal tubular secretion, adaptive processes that will significantly increase the potassium secretion per nephron will maintain the levels of serum potassium within the normal range despite the fact that the patient may have severe renal impairment.

milarly, with substances such as potassium, which are mainly handled by renal tubular secretion, adaptive processes that will significantly increase the potassium secretion per nephron will maintain the levels of serum potassium within the normal range despite the fact that the patient may have severe renal impairment. In the 1950s, if there was a consensus, ‘it was that the processes of disease transformed the afflicted kidney into a disorganized and heterogeneous population of nephrons marred by a spectrum of functional disturbances which varied from one kidney to another, from one disease to another, from one patient to another.'3 Bricker3 elaborated in great detail and challenged the idea that anatomical abnormalities cannot be followed by functional adaptations. He emphasized that ‘the functional capacity of the residual nephrons of the diseased kidney is largely independent of the specific form of renal disease. The decrease in the number of nephrons is clearly responsible for many of the abnormalities that develop in the patient; the persistent nephrons permit the patient to survive.' One of the best examples is related to sodium excretion. A normal person with a glomerular filtration rate (GFR) of 120 ml/min will filter 17,000 to 18,000 mEq/day of Na. This person will excrete ∼1% of the filtered load of sodium provided the patient has a normal salt intake (8–12 g/day).

rmit the patient to survive.' One of the best examples is related to sodium excretion. A normal person with a glomerular filtration rate (GFR) of 120 ml/min will filter 17,000 to 18,000 mEq/day of Na. This person will excrete ∼1% of the filtered load of sodium provided the patient has a normal salt intake (8–12 g/day). If the patient with CKD continues with the same diet but loses 50% of his/her renal function, the renal tubule must adapt and excrete twice the amount of Na per nephron, or roughly 2%. As the disease progresses, every time the patient loses 50% of his renal function, the fractional excretion of sodium should double. This hypothesis is depicted in Figure 1. Studies by Slatopolsky et al.4 validated this hypothesis, comparing patients with normal GFRs (120 ml/min) with those with GFRs as low as 2.0 ml/min (Figure 2). When patients with inulin clearances from 25 to 2.6 ml/min were fed diets containing 60 or 120 mEq/day of Na, all patients were in perfect balance, clearly demonstrating that the lower the GFR and consequently the lower the number of nephrons, the greater the fractional excretion of Na. In other words, as GFR decreases, the greater the amount of Na excreted per remaining nephron. This indicates that patients with very low GFRs (<5 ml/min) ingesting 120 mEq/day of Na must excrete >30% of the filtered load of Na to maintain sodium balance.

number of nephrons, the greater the fractional excretion of Na. In other words, as GFR decreases, the greater the amount of Na excreted per remaining nephron. This indicates that patients with very low GFRs (<5 ml/min) ingesting 120 mEq/day of Na must excrete >30% of the filtered load of Na to maintain sodium balance. To further correlate the relationship between Na excretion and bicarbonate reabsorption, Slatopolsky et al.5 performed bicarbonate titrations in patients with CKD stages 3–5. Normal subjects and patients with CKD with GFRs >30 ml/min fell into one group. When extracellular fluid volume expansion was minimized by salt deprivation and diuretics in advance of the titration study, after the infusion of sodium bicarbonate, no definitive maximal tubular reabsorption for bicarbonate was observed. In the same group of patients, exaggerated expansion of the extracellular fluid led to an alteration in the patterns of bicarbonate reabsorption. Results from patients with GFRs of ⩽20 ml/min resembled increased expansion, although extracellular fluid volume expansion was not exaggerated. However, those patients had a significant increase in Na excretion per nephron. On the other hand, patients with severe CKD stage 5 and nephrotic syndrome characterized by a low excretion of Na per nephron had a maximal tubular reabsorption for bicarbonate similar to normal subjects. There was a significant correlation between fractional excretion of sodium and bicarbonate reabsorption (P<0.0001; ref. 5).

ther hand, patients with severe CKD stage 5 and nephrotic syndrome characterized by a low excretion of Na per nephron had a maximal tubular reabsorption for bicarbonate similar to normal subjects. There was a significant correlation between fractional excretion of sodium and bicarbonate reabsorption (P<0.0001; ref. 5). THE INTACT NEPHRON AND CKD-RELATED MINERAL AND BONE DISORDER CKD is characterized by several alterations in mineral homeostasis. Secondary hyperparathyroidism is present even in the early stages of CKD and leads to the development of high bone turnover, pathological fractures, vascular calcification, and other systemic alterations. The maintenance of phosphate balance requires the excretion into the urine each day of the same amount of phosphorus that enters the extracellular fluid.3 Several decades ago we demonstrated that, in dogs with experimental renal disease and in patients with different degrees of CKD, tubular reabsorption of phosphate decreases in proportion to the severity of CKD. At a GFR of 120 ml/min, ∼10% of the filtered phosphate is excreted (that is, tubular reabsorption of phosphate is 90%). As GFR diminishes, the degree of phosphate excretion per nephron increases, and at very low filtration rate levels tubular reabsorption of phosphate is 10–20%, or 80–90% of the filtered load of phosphate is excreted into the urine6 (Figure 3).

d phosphate is excreted (that is, tubular reabsorption of phosphate is 90%). As GFR diminishes, the degree of phosphate excretion per nephron increases, and at very low filtration rate levels tubular reabsorption of phosphate is 10–20%, or 80–90% of the filtered load of phosphate is excreted into the urine6 (Figure 3). In our initial studies in dogs with 5/6 nephrectomy,7 we demonstrated that if we greatly restricted phosphate intake from 1200 to 100 mg/day there was no need for adaptation, and the surviving nephrons could reabsorb 90% of the filtered load of phosphate. Moreover, in the same studies we demonstrated that, if phosphate is restricted from the beginning of nephron reduction, secondary hyperparathyroidism can be prevented, suggesting a critical role for phosphate retention in the elevation of parathyroid hormone (PTH). Subsequently, we developed the concept of ‘proportional reduction'. In other words, if GFR is reduced in a step-wise manner over a period of weeks or months by gradual ligation of the branches of the renal artery, and at the same time phosphate intake is reduced in proportion to the decrease in GFR, there is no need for adaptation. We clearly demonstrated this hypothesis in a paper published in 1972 (ref. 8). In a group of dogs, GFR was gradually reduced from 56 to 40 to 19, and finally to 12 ml/min. At the same time, phosphate intake was gradually and proportionally reduced from 1200 to 234 mg/day. There were no changes in serum phosphorus, calcium, magnesium, tubular reabsorption of phosphate, or PTH, clearly indicating that we could prevent the development of secondary hyperparathyroidism by preventing the adaptation of phosphate excretion.

ntake was gradually and proportionally reduced from 1200 to 234 mg/day. There were no changes in serum phosphorus, calcium, magnesium, tubular reabsorption of phosphate, or PTH, clearly indicating that we could prevent the development of secondary hyperparathyroidism by preventing the adaptation of phosphate excretion. Subsequently, we learned the critical role of 1,25(OH)2D3 (calcitriol) in the transcriptional repression of the PTH gene.9, 10 Because in advanced CKD the reduced renal mass may limit the production of calcitriol, studies were conducted11 to clarify whether phosphate restriction improves secondary hyperparathyroidism in advanced CKD directly or indirectly. Dogs with experimental CKD were fed a low-calcium, low-phosphate diet for several weeks (Figure 4). It is well known that a low-phosphate diet can increase the levels of calcitriol, presumably by decreasing the levels of fibroblast growth factor (FGF)-23, which reduces the activity of 1α-hydroxylase. The results confirm a marked effect of phosphate restriction in secondary hyperparathyroidism. In humans and rats, calcium and phosphorus are well-known modulators of renal 1α-hydroxylase. However, in contrast to previous findings in patients with early CKD,12 in the dogs with advanced CKD, significant reduction of phosphate intake did not increase the levels of calcitriol. In these studies, we observed a significant decrease in serum-ionized Ca and phosphate conditions known to stimulate 1α-hydroxylase, but plasma calcitriol did not increase. Finally, in 1996 (ref. 13) we demonstrated in vitro that phosphate per se, independent of ionized calcium or calcitriol, has a direct effect on PTH secretion. Parathyroid glands obtained from normal rats were incubated in vitro with culture media containing 0.2 or 2.8 mmol/l phosphorus. Again, the concentrations of ionized calcium and calcitriol were the same in both situations. The 2.8 mmol/l phosphorus concentration in the media greatly increased the secretion of PTH in comparison with the results obtained with 0.2 mmol/l (Figure 5). In the same year, two other laboratories14, 15 clearly demonstrated a direct effect of phosphate on PTH secretion. The effect of phosphate on the secretion of PTH is posttranscriptional.16, 17 Figure 6 summarizes the mechanisms involved in the pathogenesis of secondary hyperparathyroidism.

d with 0.2 mmol/l (Figure 5). In the same year, two other laboratories14, 15 clearly demonstrated a direct effect of phosphate on PTH secretion. The effect of phosphate on the secretion of PTH is posttranscriptional.16, 17 Figure 6 summarizes the mechanisms involved in the pathogenesis of secondary hyperparathyroidism. I believe it is important to emphasize that the initial hypothesis regarding the mechanisms by which phosphate controls the regulation of PTH was made 50 years ago, and since then many factors have been discovered that also participate in the regulation of PTH. Regardless of these new developments, the initial idea that phosphate per se controls PTH is correct. PHOSPHATE–PARATHYROID–INTESTINAL–RENAL AXIS Several studies in experimental animal models have shown that a high phosphate intake can accelerate uremia-induced parathyroid hyperplasia and secondary hyperparathyroidism, whereas phosphate restriction prevents these abnormalities. The effect of phosphate is independent of serum calcium and 1,25(OH)2D3 (refs 13, 18).

everal studies in experimental animal models have shown that a high phosphate intake can accelerate uremia-induced parathyroid hyperplasia and secondary hyperparathyroidism, whereas phosphate restriction prevents these abnormalities. The effect of phosphate is independent of serum calcium and 1,25(OH)2D3 (refs 13, 18). We conducted further studies in uremic rats with established parathyroid hyperplasia and secondary hyperparathyroidism to determine whether the change to a low-phosphate diet could modify the above abnormalities.19 After 1 week of a low-phosphate diet, serum PTH returned to normal despite the persistence of parathyroid hyperplasia. Our studies did not show a change in the synthesis of PTH, but did show a suppression of PTH release. We also demonstrated an increase in intracellular PTH in the parathyroid gland in animals that were fed a high-phosphate diet and subsequently treated for 1 week with a low-phosphate diet. No apoptosis was observed in these glands. Because of our findings, we called our studies ‘Hyperplasia of the parathyroid glands without secondary hyperparathyroidism.'19 Studies by Ritter et al.20 in our laboratory demonstrated that after 2 weeks of CKD and a high-phosphate diet there was a significant loss of the calcium-sensing receptor. However, when the diet was switched to one that was lower in phosphate, serum PTH returned to normal in only 1 day but it took ∼2 weeks for the calcium-sensing receptor to return to normal.

ory demonstrated that after 2 weeks of CKD and a high-phosphate diet there was a significant loss of the calcium-sensing receptor. However, when the diet was switched to one that was lower in phosphate, serum PTH returned to normal in only 1 day but it took ∼2 weeks for the calcium-sensing receptor to return to normal. Further studies by Martin et al.21 in our laboratory demonstrated that in rats with established secondary hyperparathyroidism there was a 60–80% decrease in serum phosphorus and PTH, with no change in serum calcium only 2 h after feeding the uremic rats a low-phosphate diet. Moreover, when a low-phosphate diet was given by gavage, serum PTH and phosphorus decreased significantly after only 15 min. On the other hand, when serum phosphate was acutely decreased by an infusion of glucose, no change in serum PTH was observed. All these results raise the question, ‘Is there an acute regulation of PTH by dietary phosphate, and if so, is it mediated by a hormone possibly derived from the gastrointestinal tract?'

15 min. On the other hand, when serum phosphate was acutely decreased by an infusion of glucose, no change in serum PTH was observed. All these results raise the question, ‘Is there an acute regulation of PTH by dietary phosphate, and if so, is it mediated by a hormone possibly derived from the gastrointestinal tract?' Recently, Berndt et al.22 demonstrated that the instillation of phosphate in the duodenum of normal rats rapidly increases the renal excretion of phosphate. This was also seen in thyroparathyroidectomized rats, and there were no changes in the filtered load of phosphate or serum levels of FGF-23 or secreted frizzled-related protein. Moreover, these observations were also seen when the kidney was denervated, and the phosphaturia was not elicited when phosphate was instilled in other parts of the gastrointestinal tract, such as the stomach. Furthermore, when homogenates of duodenal mucosa were infused intravenously there was a rapid increase in renal phosphate excretion. Berndt et al.22 believe that there is a direct existence of an intestinal–renal axis specific for phosphate that is mediated by an unknown factor ‘intestinal phosphatonin'. Moreover, they also propose the existence of an intestinal phosphate sensor.23 Although this sensor has not yet been demonstrated, it is possible that the parathyroid gland also has a ‘phosphate sensor'. Again, to date there is no scientific proof for the existence of such a sensor. This author, however, strongly believes in the existence of a ‘phosphate sensor' in parathyroid glands. To date, all the important factors that control the release of PTH have a sensor in the parathyroid gland (that is, vitamin D receptor, calcium sensor receptor, Klotho–FGFR-1 receptor complex), and as phosphate also has a crucial role in the development of secondary hyperparathyroidism the existence of a phosphate sensor seems to be a reasonable possibility.

control the release of PTH have a sensor in the parathyroid gland (that is, vitamin D receptor, calcium sensor receptor, Klotho–FGFR-1 receptor complex), and as phosphate also has a crucial role in the development of secondary hyperparathyroidism the existence of a phosphate sensor seems to be a reasonable possibility. PHOSPHATE MODULATION OF PARATHYROID HYPERPLASIA Parathyroid cell proliferation rather than hypertrophy is the main determinant of parathyroid enlargement and secondary hyperparathyroidism. The lack of an appropriate parathyroid cell line has impeded the identification of the mechanisms underlying the switch from a quiescent parathyroid cell to one that divides rapidly in response to hypocalcemia, hyperphosphatemia, or a decrease in 1,25(OH)2D3 as part of the progression of CKD. Studies conducted by Dusso et al.24 centered on phosphate control of parathyroid cell proliferation, as no change in the number of cells undergoing apoptosis has been observed in experimental conditions affecting parathyroid cells.19 Dusso et al.24 focused on dietary phosphate regulation of parathyroid expression of the cyclin/cyclin-dependent kinase inhibitor p21. Her studies demonstrate that, in early CKD, phosphate restriction may arrest uremia-induced parathyroid hyperplasia through a specific induction of the mRNA and protein levels of the cyclin-dependent kinase inhibitor p21. In addition, high phosphate intake induces transforming growth factor (TGF)-α, which seems to function as an autocrine signal to stimulate cell growth, thus worsening the parathyroid hyperplasia and the resultant secondary hyperparathyroidism. Growth signals from enhanced parathyroid TGF-α require activation of the epidermal growth factor receptor (EGFR): a 170-kDa membrane glycoprotein with intrinsic tyrosine kinase activity.25 TGF-α-activated EGFR translocates to the nucleus, where it functions as a transcription factor. TGF-α/EGFR-driven growth involves TGF-α binding to the EGFR, which in turn induces EGFR dimerization and tyrosine phosphorylation of the EGFR by an intrinsic EGFR tyrosine kinase. This step is critical for the onset of downstream growth signal. Studies conducted by Cozzolino et al.26 clearly demonstrate that a high phosphate dietary intake increases not only TGF-α but also the EGFR in the parathyroid gland of rats with CKD. The TGF-α–EGFR complex produces growth acceleration of benign and malignant tissue.

s critical for the onset of downstream growth signal. Studies conducted by Cozzolino et al.26 clearly demonstrate that a high phosphate dietary intake increases not only TGF-α but also the EGFR in the parathyroid gland of rats with CKD. The TGF-α–EGFR complex produces growth acceleration of benign and malignant tissue. Moreover, in the same studies,26 the use of erlotinib, a tyrosine kinase inhibitor, counteracted the potent mitogenic signals triggered by kidney disease and phosphate retention. Low-calcium diets also increase the TGFα–EGFR complex. This study26 demonstrates that the enhancement of parathyroid TGF-α and EGFR expression and growth signal occurs early after the onset of renal disease and is aggravated by a high-phosphate or a low-calcium diet.

triggered by kidney disease and phosphate retention. Low-calcium diets also increase the TGFα–EGFR complex. This study26 demonstrates that the enhancement of parathyroid TGF-α and EGFR expression and growth signal occurs early after the onset of renal disease and is aggravated by a high-phosphate or a low-calcium diet. Further studies by Dusso et al.27 identify a tumor necrosis factor-α-converting enzyme (TACE) as the cause of the initial increase in parathyroid TGF-α, and this starts the vicious cycle for progression of parathyroid hyperplasia. TACE, also known as ADAM17, is a metalloproteinase essential for TGF-α signaling and several other EGFR ligands. The location of TACE at the cell membrane is mandatory for its function. Preliminary results27 in uremic rats have shown that high phosphate intake also enhances parathyroid TACE content in early and established secondary hyperparathyroidism. However, high dietary phosphate may not affect parathyroid TACE expression directly, but may induce its translocation to the cell surface, causing the release of TGF-α required to initiate EGFR activity during parathyroid cell growth and TACE stabilization.

ontent in early and established secondary hyperparathyroidism. However, high dietary phosphate may not affect parathyroid TACE expression directly, but may induce its translocation to the cell surface, causing the release of TGF-α required to initiate EGFR activity during parathyroid cell growth and TACE stabilization. CONCLUSIONS The intact nephron hypothesis has fueled 50 years of research and discovery in CKD. The adverse events of hyperphosphatemia in CKD, secondary hyperparathyroidism, calcium and vitamin D derangements, vascular calcification, and metabolic bone disorder are the results of the endocrine trade-offs required by the adaptation of nephrons attempting to preserve phosphate homeostasis. Restricting phosphate intake or, when that is not possible, restricting phosphate absorption by use of phosphate binders as GFR declines reduces the need for nephron adaptation and forestalls endocrine sequelae such as hyperparathyroidism. PTH is regulated at the levels of mRNA stability and protein exocytosis by extracellular phosphate concentration. Rapid (15-min) regulation of PTH by phosphate may require the release of an intestinal hormone. The increase in fractional excretion of phosphate after the instillation of phosphate in the intestine is rapid (15–20 min) and does not require PTH or FGF-23. Potentially, an ‘intestinal phosphatonin' may be the mediator of this action. Finally, in the parathyroid gland, enhanced TGF-α and EGFR coexpression drives parathyroid hyperplasia in early CKD. Parathyroid TACE activity, itself controlled by phosphate and vitamin D, is crucial in transducing these growth signals and will be an important therapeutic target. Clearly, phosphate has direct effects at multiple organ sites suggesting a phosphate sensor. However, further studies are necessary to scientifically prove the existence of a phosphate sensor in the parathyroid glands and gastrointestinal tract.

nsducing these growth signals and will be an important therapeutic target. Clearly, phosphate has direct effects at multiple organ sites suggesting a phosphate sensor. However, further studies are necessary to scientifically prove the existence of a phosphate sensor in the parathyroid glands and gastrointestinal tract. This research was supported in part by NIDDK grants DK-09976, DK-30178, DK-07126, Research in Renal Diseases, Washington University School of Medicine grant and WUCKDR O'Brien Center (P30DK079333). The author expresses his appreciation to Jane Finch for valuable suggestions and critical review of the manuscript. Publication of this supplement was supported by Genzyme Corporation. TO CITE THIS ARTICLE: Slatopolsky E. The intact nephron hypothesis: the concept and its implications for phosphate management in CKD-related mineral and bone disorder. Kidney Int 2011; 79 (Suppl 121): S3–S8. ES has received consultancy or advisory board fees, lecturer's fees, and research grants from Abbott Laboratories and Genzyme Corporation. ES and Washington University may receive income on the basis of a license-related technology by the University of Wisconsin. Figure 1 Hypothetical treatment of sodium excretion in chronic kidney disease (see text for details). GFR, glomerular filtration rate; FENa, fractional excretion of sodium; FLNa, filtered load of sodium. Figure newly constructed from data in Bricker et al.2 and Bricker.3

ES has received consultancy or advisory board fees, lecturer's fees, and research grants from Abbott Laboratories and Genzyme Corporation. ES and Washington University may receive income on the basis of a license-related technology by the University of Wisconsin. Figure 1 Hypothetical treatment of sodium excretion in chronic kidney disease (see text for details). GFR, glomerular filtration rate; FENa, fractional excretion of sodium; FLNa, filtered load of sodium. Figure newly constructed from data in Bricker et al.2 and Bricker.3 Figure 2 Urinary sodium excretion in patients with advanced chronic kidney disease (stages 4–5). All patients with glomerular filtration rates ranging from 25 to 2.6 ml/min maintain perfect Na balance after ingestion of 60 or 120 mEq of Na/day. Reproduced by permission from Slatopolsky et al.4 Copyright 1968 by the American Society for Clinical Investigation. Reproduced with permission from the American Society for Clinical Investigation in the format Journal via Copyright Clearance Center. Figure 3 Comparison of tubular reabsorption of phosphate (TRP) and glomerular filtration rate (GFR) in a group of normal subjects and 24 patients with different degrees of chronic kidney disease. The lower the GFR, the lower the TRP or the greater the excretion of phosphate per nephron. Reproduced by permission from Slatopolsky et al.6 Copyright 1968 by the American Society for Clinical Investigation. Reproduced with permission from the American Society for Clinical Investigation in the format Journal via Copyright Clearance Center.

lower the TRP or the greater the excretion of phosphate per nephron. Reproduced by permission from Slatopolsky et al.6 Copyright 1968 by the American Society for Clinical Investigation. Reproduced with permission from the American Society for Clinical Investigation in the format Journal via Copyright Clearance Center. Figure 4 Effects of a low-phosphate, low-calcium diet on serum calcium, calcitriol, phosphorus, and parathyroid hormone (PTH) in dogs with chronic kidney disease and established secondary hyperparathyroidism. *p<0.05. Figure newly constructed from data in Lopez-Hilker et al.11 Figure 5 The effects of phosphorus in the culture media on the secretion of parathyroid hormone (PTH) in a normal rat parathyroid gland in vitro. In the two experimental conditions, the concentrations of ionized calcium and 1,25(OH)2D3 were identical. Error bars denote standard error. Adapted with permission from Slatopolsky et al.13

fects of phosphorus in the culture media on the secretion of parathyroid hormone (PTH) in a normal rat parathyroid gland in vitro. In the two experimental conditions, the concentrations of ionized calcium and 1,25(OH)2D3 were identical. Error bars denote standard error. Adapted with permission from Slatopolsky et al.13 Figure 6 Summary of the factors involved in the pathogenesis of secondary hyperparathyroidism. A decrease in ionizing calcium (ICa) is crucial in the development of secondary hyperparathyroidism. This change in ICa is secondary to phosphate retention and to low levels of 1,25(OH)2D3. Phosphate retention increases fibroblast growth factor (FGF)-23, which, in conjunction with its cofactor, the Klotho protein, decreases the activity of the 1α-hydroxylase and increases the 24α-hydroxylase, thus decreasing the levels of circulating 1,25(OH)2D3. In addition, phosphate retention, independent of change in ICa, posttranscriptionally increases the synthesis of parathyroid hormone (PTH). The 1,25(OH)2D3, independent of calcium, suppresses the transcription of the PTH gene. Decreases in the vitamin D receptor, calcium sensor receptor, and Klotho–FGFR1 receptor complex in the parathyroid gland also aggravate the development of secondary hyperparathyroidism.

Among the 25 million American patients with stage 2–5 chronic kidney disease (CKD), cardiovascular disease causes a disproportionately high mortality risk. Patients with CKD are more likely to die (often of cardiovascular causes) than to progress to dialysis.1 The risk of death is especially high in late-stage kidney disease; a 30-year-old patient with end-stage renal disease faces an equivalent risk of death to a 90-year-old without CKD.2 The cardiovascular risk factors generally considered do not explain the heightened cardiovascular risk in CKD.3 Vascular calcification4, 5 and hyperphosphatemia6 drive cardiovascular risk in CKD, and they are related. Risk factors are rigorously defined by four types of evidence: (1) observational studies; (2) definitive prospective translational or clinical studies; (3) mechanism of action studies; and (4) outcome studies showing risk reduction when the putative factor is corrected. Serum phosphorus, at the current state of our knowledge, is associated with the first three of these four lines of evidence. One early observational outcome study has also shown risk reduction benefits with correction of phosphate levels in patients with CKD. This review will summarize current evidence for phosphate as a cardiovascular risk factor in the general population and among patients with CKD.

t three of these four lines of evidence. One early observational outcome study has also shown risk reduction benefits with correction of phosphate levels in patients with CKD. This review will summarize current evidence for phosphate as a cardiovascular risk factor in the general population and among patients with CKD. OBSERVATIONAL STUDIES OF PHOSPHATE AND CARDIOVASCULAR RISK Serum phosphorus and mortality risk have been analyzed in multiple observational studies. In patients with end-stage renal disease on dialysis (40,538 hemodialysis patients), those with serum phosphorus >6.0 to 7.0 mg/dl had a relative risk of death 1.25 times that of those with serum phosphorus ⩽5 mg/dl. The lowest serum phosphate category (<3 mg/dl) showed slightly increased risk (1.2 versus 4.5 mg/dl).7 In a retrospective cohort study of 6730 US veterans with CKD not receiving dialysis (those transplanted or without phosphorus measurements were excluded), the adjusted hazard ratio for death rose to 1.90 in patients with serum phosphorus >5.0 mg/dl (compared with patients with serum phosphorus <2.5 mg/dl).8

.5 mg/dl).7 In a retrospective cohort study of 6730 US veterans with CKD not receiving dialysis (those transplanted or without phosphorus measurements were excluded), the adjusted hazard ratio for death rose to 1.90 in patients with serum phosphorus >5.0 mg/dl (compared with patients with serum phosphorus <2.5 mg/dl).8 In the general population, serum phosphorus has been associated with the risk of cardiovascular disease (Figure 1). The Framingham Offspring Study,9 deleted of participants with histories of CKD, revealed that increasing serum phosphorus was associated with a continuous increasing risk of cardiovascular disease (heart attack, stroke, angina, peripheral vascular disease, or heart failure). In a post hoc analysis of the Cholesterol and Recurrent Events study10 (Figure 1), serum phosphate showed a graded independent relationship to risk of death and new cardiovascular events in patients who had suffered heart attacks previously and had normal kidney function. In both the Cholesterol and Recurrent Events study and the US Third Health and Nutrition Examination Survey populations, elevation of serum alkaline phosphatase and serum phosphate together conferred greater risk than either parameter alone.11

ts who had suffered heart attacks previously and had normal kidney function. In both the Cholesterol and Recurrent Events study and the US Third Health and Nutrition Examination Survey populations, elevation of serum alkaline phosphatase and serum phosphate together conferred greater risk than either parameter alone.11 How might serum phosphorus affect atherosclerosis? In CKD, we associate hyperphosphatemia causally with vascular calcification, which stiffens arteries and leads to cardiovascular events. In young and middle-aged adults without CKD, serum phosphorus levels were associated with vascular stiffness and coronary artery calcium levels in the Multi-Ethnic Study of Atherosclerosis. In middle-aged participants with mild-to-moderate CKD,12 the ankle–brachial index increased in parallel with serum phosphorus levels within the normal range. In the Coronary Artery Risk Development in Young Adults study, which was a 15-year prospective observational study,13 10% of the participants with 15-year data experienced significant coronary calcification during follow-up, related to the serum phosphorus level at the beginning of the follow-up period.

e normal range. In the Coronary Artery Risk Development in Young Adults study, which was a 15-year prospective observational study,13 10% of the participants with 15-year data experienced significant coronary calcification during follow-up, related to the serum phosphorus level at the beginning of the follow-up period. DEFINITIVE PROSPECTIVE TRANSLATIONAL OR CLINICAL STUDIES: HOW DOES PHOSPHORUS FUNCTION AS A RISK FACTOR FOR CARDIOVASCULAR MORTALITY? The complexity of human disease causing cardiovascular risk requires a translational model to dissect the role of phosphorus in vascular calcification and coronary artery disease development in CKD. There are two types of translational studies that address the issue of phosphorus as a cardiovascular risk. In the first, genetic engineering was used to produce deficiency of the fibroblast growth factor-23 skeletal hormone, which is responsible for regulating renal phosphate excretion. Mice with fibroblast growth factor-23 deficiency develop hyperphosphatemia, excess calcitriol and vascular calcification, and have shortened life spans. Feeding these mice low-phosphate diets corrects hyperphosphatemia, eliminates vascular calcification, and lengthens their life span.

ulating renal phosphate excretion. Mice with fibroblast growth factor-23 deficiency develop hyperphosphatemia, excess calcitriol and vascular calcification, and have shortened life spans. Feeding these mice low-phosphate diets corrects hyperphosphatemia, eliminates vascular calcification, and lengthens their life span. The second type of study that demonstrates the function of inorganic phosphate as a cardiovascular risk factor comprises studies in animal models of atherosclerosis in which hyperphosphatemia is induced by CKD. We have used the low-density lipoprotein receptor-deficient mouse (ldlr–/–) fed a high-fat diet with ablative CKD. LDL–/– mice fed high-fat diets develop hypercholesterolemia, metabolic syndrome, and vascular calcification. CKD added to this model induces hyperphosphatemia (beginning at stage 3 CKD) and increases vascular, especially aortic, calcification.14 Administration of phosphate binders and bone morphogenetic protein (BMP)-7 in this model prevented hyperphosphatemia by reducing phosphate absorption and sending serum phosphate into the skeleton.15, 16 In mice with established vascular calcification (atherogenic diet begun at 10 weeks, CKD induced by 14 weeks, and phosphate binder or BMP-7 administered from 22 to 28 weeks), control of phosphate for weeks 22–28 with sevelamer carbonate or BMP-7 diminished vascular calcification and prevented cardiac hypertrophy.15, 16 The study with BMP-7 originally addressed concerns that BMP-7 might stimulate vascular calcification, but in fact showed BMP-7 to be therapeutic, decreasing aortic calcification below control levels and stimulating bone formation. The stimulation of bone formation was the mechanism of BMP-7-induced correction of hyperphosphatemia, and a component of the action against vascular calcification.

ate vascular calcification, but in fact showed BMP-7 to be therapeutic, decreasing aortic calcification below control levels and stimulating bone formation. The stimulation of bone formation was the mechanism of BMP-7-induced correction of hyperphosphatemia, and a component of the action against vascular calcification. MECHANISM OF ACTION STUDIES A scientific consensus more advanced than mere plausibility has developed with regard to the mechanism by which high serum phosphate leads to vascular calcification and cardiovascular risk. Pathologically, there are two types of large artery calcification stimulated by CKD, calcification of the smooth muscle tunica media and atherosclerotic calcification of smooth muscle cells in plaque neointima (Figure 2). Atherosclerotic calcification is more important in cardiovascular mortality risk, and tunica media calcification is more important in vascular stiffness.17 In atherosclerotic calcification, neointimal cells express an osteoblastic phenotype, either by migration and differentiation of pericytes or by dedifferentiation, migration, and redifferentiation of smooth muscle cells. Initial renal injury induces dedifferentiation of vascular smooth muscle cells, which makes them migratory and vulnerable to osteoblastic induction by BMP-2. Additional studies have shown phosphorus to be a molecule capable of stimulating signal transduction.18, 19 We have shown inorganic phosphate to stimulate expression of osterix both in vivo in our ldlr–/– high-fat fed CKD mice and in vitro in human aortic smooth muscle cells derived from atherosclerotic donors with early CKD20 (Figure 3). Osterix is an osteoblast transcription factor required for cellular stimulation of matrix mineralization.21 Primary mouse or human smooth muscle cells in vitro transitioned from normal to high-phosphate culture medium (1 to 2 mmol/l) will mineralize their extracellular matrix in 2–3 weeks.20 Blocking of osterix expression in the presence of high phosphorus prevents mineralization.

ular stimulation of matrix mineralization.21 Primary mouse or human smooth muscle cells in vitro transitioned from normal to high-phosphate culture medium (1 to 2 mmol/l) will mineralize their extracellular matrix in 2–3 weeks.20 Blocking of osterix expression in the presence of high phosphorus prevents mineralization. Reducing serum phosphorus (for example, with phosphate binders) reverses osteoblastic differentiation of vascular cells and reverses vascular calcification.20 Osteoblastic transition and calcification of smooth muscle cells (akin to bone formation) in the atherosclerotic plaque is an active and reversible process. Osteoclasts and large multinucleated macrophages are present in plaques and can actually reabsorb calcification when serum phosphorus is decreased.

alcification.20 Osteoblastic transition and calcification of smooth muscle cells (akin to bone formation) in the atherosclerotic plaque is an active and reversible process. Osteoclasts and large multinucleated macrophages are present in plaques and can actually reabsorb calcification when serum phosphorus is decreased. EFFECTS OF CKD ON PHOSPHATE HOMEOSTASIS The phosphate balance diagram is shown in Figure 4. In health, bone formation and resorption balance each other, and there is capacity in the skeletal mineralization fronts to absorb a transient positive phosphate balance, which would be converted into bone. This permits the skeleton to function physiologically as a phosphate and calcium reservoir. In CKD-related mineral and bone disorder, renal osteodystrophy causes excess bone resorption over bone formation in both high- and low-turnover forms of osteodystrophy. This blocks the normal reservoir function of the skeleton. In CKD, increasing fractional excretion of phosphorus (via adaptation of intact nephrons) maintains phosphorus balance in the early stages, but eventually reduction in the tubular reabsorption of phosphate cannot keep pace and phosphate balance becomes positive in stage 3 CKD before actual hyperphosphatemia occurs. The increased bone resorption contributes to the exchangeable phosphate pool, and to hyperphosphatemia. When extra phosphorus is present from a positive balance, in this situation it exits from the exchangeable pool into heterotopic deposition sites. Therefore, the skeleton contributes to heterotopic calcification especially in the vasculature in CKD.

tributes to the exchangeable phosphate pool, and to hyperphosphatemia. When extra phosphorus is present from a positive balance, in this situation it exits from the exchangeable pool into heterotopic deposition sites. Therefore, the skeleton contributes to heterotopic calcification especially in the vasculature in CKD. The pathophysiology of phosphorus balance in CKD is dependent on a recently discovered skeletal hormone, fibroblast growth factor-23, and its relevant co-receptor, Klotho, which are required for renal phosphate excretion. Their deficiency causes hyperphosphatemia and heterotopic mineralization,22 which can be rescued by a low-phosphate diet. In this translational model, vascular calcification appears to be the mechanism by which high serum phosphorus increases cardiovascular and mortality risk. Low-phosphate diets improved survival along with preventing vascular calcification.

and heterotopic mineralization,22 which can be rescued by a low-phosphate diet. In this translational model, vascular calcification appears to be the mechanism by which high serum phosphorus increases cardiovascular and mortality risk. Low-phosphate diets improved survival along with preventing vascular calcification. OUTCOME STUDIES OF PHOSPHORUS CORRECTION Human studies demonstrating that reducing serum phosphorus concentrations decreases cardiovascular risk have not been conducted. However, some studies have been conducted that are supportive of the conclusion that phosphorus is a cardiovascular risk factor. The Accelerated Mortality on Renal Replacement study23 (Figure 5) was a prospectively observed cohort study of 10,044 incident hemodialysis patients, comparing those who did with those who did not receive any phosphate-binder treatment during the first 90 days of dialysis. Phosphate binder use was associated with significantly reduced mortality risk on multivariate analysis (on an intent-to-treat or as-treated basis), as well as in a propensity score-matched comparison. Benefits were independent of baseline serum phosphate levels, and thus may reflect lowered levels of the phosphaturic factor fibroblast growth factor-23, which is induced up to 100-fold in untreated hyperphosphatemic dialysis patients.

-to-treat or as-treated basis), as well as in a propensity score-matched comparison. Benefits were independent of baseline serum phosphate levels, and thus may reflect lowered levels of the phosphaturic factor fibroblast growth factor-23, which is induced up to 100-fold in untreated hyperphosphatemic dialysis patients. The Renagel in New Dialysis study,24 an 18-month randomized, controlled trial of sevelamer HCl versus calcium-containing phosphate binder in 109 new hemodialysis patients, showed that among patients with baseline coronary artery calcification, calcium binder use was associated with more rapid and severe progression of calcification than sevelamer HCl use. A pre-specified secondary end-point analysis of the Renagel in New Dialysis study25 revealed that, in 127 new hemodialysis patients randomized to sevelamer versus calcium-containing phosphate binders and followed up for 44 months, baseline coronary artery calcification score was a predictor of all-cause mortality. Sevelamer was associated with a significant survival benefit versus calcium-containing binders in this study.

new hemodialysis patients randomized to sevelamer versus calcium-containing phosphate binders and followed up for 44 months, baseline coronary artery calcification score was a predictor of all-cause mortality. Sevelamer was associated with a significant survival benefit versus calcium-containing binders in this study. CONCLUSIONS From the discussion above, the following conclusions are drawn. First, observational studies suggest that serum phosphate is a cardiovascular risk factor in patients with CKD and in the general population. Second, translational studies demonstrate that hyperphosphatemia stimulates atherosclerotic vascular calcification by inducing osteoblastic gene expression in the aorta, and that correction of hyperphosphatemia decreases vascular calcification. Third, studies in vitro demonstrate that medium inorganic phosphate is an active signaling molecule, the receptor may be Pit1, and phosphate directly stimulates osteoblastic transcription factors. Fourth, human studies are consistent in showing the effects of phosphate and its correction on vascular calcification and cardiovascular mortality. However, these studies are not formal outcome studies and are insufficient evidence without the translational data. Fifth, the strong epidemiologic and mechanistic evidence suggesting that serum phosphate is a cardiovascular risk factor needs to be confirmed by human prospective and outcome studies to satisfy the rigorous definition of phosphorus as a cardiovascular risk factor. Finally, in the meantime, before formal outcome studies are completed, interventions to maintain serum phosphate normal in patients with CKD before end-stage renal disease are clearly warranted.

ed by human prospective and outcome studies to satisfy the rigorous definition of phosphorus as a cardiovascular risk factor. Finally, in the meantime, before formal outcome studies are completed, interventions to maintain serum phosphate normal in patients with CKD before end-stage renal disease are clearly warranted. KH, assisted by his co-authors, wrote this article from his presentation and discussions at the ‘50 Years of Discovery Following the Intact Nephron Hypothesis' symposium in Munich, Germany, 24–25 June 2010. All authors meet the International Council of Medical Journal Editors criteria and acknowledge editorial assistance (initial outline preparation from KH's presentation materials; formatting of authors' final text for journal submission) by Kim Coleman Healy, PhD, of Envision Scientific Solutions. Publication of this supplement was supported by Genzyme Corporation. TO CITE THIS ARTICLE: Hruska K, Mathew S, Lund R, Fang Y and Sugatani T. Cardiovascular risk factors in chronic kidney disease: does phosphate qualify? Kidney Int 2011; 79 (Suppl 121): S9–S13. KH has received consultancy and advisory board fees from Genzyme Corporation and Shire Pharmaceuticals, and his laboratory receives research grants from Fresenius Medical Care, Shire Pharmaceuticals, National Institutes of Health/Biolink, and National Institutes of Health/National Institute of Digestive Diseases, Diabetes, and Kidney Disease.

ncy and advisory board fees from Genzyme Corporation and Shire Pharmaceuticals, and his laboratory receives research grants from Fresenius Medical Care, Shire Pharmaceuticals, National Institutes of Health/Biolink, and National Institutes of Health/National Institute of Digestive Diseases, Diabetes, and Kidney Disease. Figure 1 Observational studies from the general population, which suggest that phosphorus is a cardiovascular risk factor. The left panel assembles data from Dhingra et al.9 Cardiovascular disease (CVD) was scored as fatal/non-fatal MI, angina, cerebrovascular events, peripheral vascular disease, or heart failure. Hazard ratios are adjusted for age, sex, body mass index, diabetes, blood pressure, treatment of hypertension, smoking, alcohol consumption, total cholesterol/high-density cholesterol ratio, hemoglobin, serum albumin, estimated glomerular filtration rate, proteinuria, and high-sensitivity C-reactive protein. The right panel is adapted with permission from Tonelli et al.10 Hazard ratio values are adjusted for baseline age, sex, race, smoking status, diabetes, waist-to-hip circumference, fasting glucose, glomerular filtration rate, hemoglobin, serum albumin, aspirin use, and left ventricular ejection fraction. N=4127. CKD, chronic kidney disease; MI, myocardial infarction. Figure 2 The two major types of large artery calcification stimulated by chronic kidney disease (CKD) are atherosclerotic neointimal calcification and medial calcification. Reproduced with permission from Dr Gerard London.

Figure 1 Observational studies from the general population, which suggest that phosphorus is a cardiovascular risk factor. The left panel assembles data from Dhingra et al.9 Cardiovascular disease (CVD) was scored as fatal/non-fatal MI, angina, cerebrovascular events, peripheral vascular disease, or heart failure. Hazard ratios are adjusted for age, sex, body mass index, diabetes, blood pressure, treatment of hypertension, smoking, alcohol consumption, total cholesterol/high-density cholesterol ratio, hemoglobin, serum albumin, estimated glomerular filtration rate, proteinuria, and high-sensitivity C-reactive protein. The right panel is adapted with permission from Tonelli et al.10 Hazard ratio values are adjusted for baseline age, sex, race, smoking status, diabetes, waist-to-hip circumference, fasting glucose, glomerular filtration rate, hemoglobin, serum albumin, aspirin use, and left ventricular ejection fraction. N=4127. CKD, chronic kidney disease; MI, myocardial infarction. Figure 2 The two major types of large artery calcification stimulated by chronic kidney disease (CKD) are atherosclerotic neointimal calcification and medial calcification. Reproduced with permission from Dr Gerard London. Figure 3 Hyperphosphatemia stimulates expression of osterix in the aortas of low-density lipoprotein receptor-deficient mouse (ldlr–/–) with chronic kidney disease (CKD) fed high-fat diets. LaCO3, a non-calcium-containing phosphate binder, reverses osterix expression. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2 The two major types of large artery calcification stimulated by chronic kidney disease (CKD) are atherosclerotic neointimal calcification and medial calcification. Reproduced with permission from Dr Gerard London. Figure 3 Hyperphosphatemia stimulates expression of osterix in the aortas of low-density lipoprotein receptor-deficient mouse (ldlr–/–) with chronic kidney disease (CKD) fed high-fat diets. LaCO3, a non-calcium-containing phosphate binder, reverses osterix expression. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Figure 4 The phosphate balance diagram in chronic kidney disease (CKD) showing positive balance and a skeletal contribution to hyperphosphatemia, which blocks skeletal reservoir function. Reproduced with permission from Mathew et al.20 Figure 5 Effects of serum phosphate and phosphate binder use on survival in the Accelerated Mortality in Renal Replacement study.23 Survival of treated and untreated patients in the overall propensity score-matched cohort (a) and according to quartiles of baseline serum phosphate (b–e). Reproduced by permission from Isakova et al.23