Accepted Article

Title page:

The importance of klotho in phosphate metabolism and

kidney disease1

Sven-Jean TAN1,2, Edward R SMITH1, Tim D HEWITSON1,2, Stephen G HOLT1,2 and Nigel D TOUSSAINT1,2 1

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Victoria,

Australia;

2

Department of Medicine (RMH), The University of Melbourne,

Melbourne, Australia

Running head:

Klotho: phosphate metabolism and kidney disease

Word count:

Abstract129

Correspondence:

Dr Sven-Jean Tan

Text3671

Department of Nephrology, The Royal Melbourne Hospital, Grattan Street, Parkville, Victoria 3052, Australia. E-mail: [email protected]

Keywords: acute kidney injury (AKI), cardiovascular disease (CVD), chronic kidney disease (CKD), fibroblast growth factor-23 (FGF23),klotho, phosphate

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nep.12268

This article is protected by copyright. All rights reserved.

1

Accepted Article

Abstract The discovery of fibroblast growth factor23 (FGF23) and its co-receptor α-klotho has broadened our understanding of mineral metabolism and led to a renewed research focus on phosphate homeostatic pathways in kidney disease. Expanding knowledge

of

these

mechanisms,

both

in

normal

physiology

and

in

pathology,identifiestargets for potentialinterventions designed to reduce the

complications of renal disease,particularly the cardiovascular sequelae. FGF23 has emerged as a majorα-klotho-dependent endocrine regulator of mineral metabolism, functioning to activate vitamin D and as aphosphatonin. However, increasingly there is an appreciation that klotho mayact independently as a phosphate regulator, as well as having significant activity in other key biological processes. This review outlines our current understanding of klotho, and its potential contribution to kidney disease

and cardiovascular health.

This article is protected by copyright. All rights reserved.

2

Accepted Article

Introduction Chronic kidney disease (CKD) represents a major and growing public health issue affecting 5-10% of the global population.1 CKD-mineral bone disorder (CKD-MBD)

describes the observationsof disturbances of mineral metabolism (particularly calcium and

phosphate),

bone

remodelling,

andacceleratedvascular

and

soft-tissue

calcification seen in kidney disease.2,3Control of phosphate flux is important in this process as well as being critical to the function of numerous essential biological processes.4 Although a putative phosphate-sensing machinery has been identified in some single cell organisms,5 the homologous sensor in vertebrates remains elusive. Nonetheless, extracellular phosphate levels do appear specifically regulated at the level of absorption through the intestine and excretion via the kidney. Thus in steadystate, the amount of phosphate absorbed fromthe diet is equivalent to the amount excreted in the urine.4A number of hormones act, either directly or indirectly, to

regulate the activity of key phosphate transporters to maintain phosphatehomeostasis in the face of fluctuation in supply (diet) and demand (cellular metabolism and bone mineralisation) (Figure 1).

Klotho, originally identified as the anti-ageing protein, has become an important focus of research in nephrology because of its key role in phosphate homeostasis.6,7The

independent discoveries of fibroblast growth factor23 (FGF23)8,9 and α-klotho,6

have improved our understanding of mineral metabolism and phosphate handling. This review outlines the potential implications and therapeutic potential of this knowledge in kidney and cardiovascular disease.

This article is protected by copyright. All rights reserved.

3

Accepted Article

Klotho

More than 15 years ago, genetic studies identified an ageing gene, klotho whose overexpression extended life span but whosedeletion produced complex phenotypes in animals similar to human ageing.6The klothoknockout mouse is now an established animal model of ageing, allowing further study of well-accepted processes that occur with ageing, such as arteriosclerosis, arterial calcification and osteoporosis, and other less well-studied processes such as angiogenesis.6,10,11The klotho gene encodes a 1012 amino acid long single-pass transmembrane protein,6commonly referred to as αklotho, to differentiate it from two subsequently discovered members of the klotho family; β-klotho and γ-klotho. All three are single-pass transmembrane proteins of

different lengths, which not only share a substantial degree of homology, but function as obligate co-receptors to endocrine fibroblast growth factors (FGFs).12

FGFR-Klotho Complex Within the extensive superfamily of FGFs, only the FGF19 subfamily consisting of FGF19, FGF21 and FGF23 are endocrine FGFs while the other FGFs function as paracrine/autocrine factors.12,13FGF receptors (FGFRs) are detected ubiquitously while klotho expression is limited to certain tissues, thereby determining tissue specificity for the endocrine action of their respective FGFs.14α-klotho is an obligate co-receptor for physiological FGF23 signalling and appears essential for FGF23mediated phosphate regulation in animal models.14-16It is now also evident that klotho proteins play a role in a range of other metabolic processes.6,7,14,17-19β-klotho, that

augments FGF19 and FGF21 signalling, is found in liver, gall bladder, pancreas,

colon and adipose tissue and participates in bile acid metabolic pathways.18,19 γklotho is coupled to FGF19 and is found in the eye, adipose and kidney and its

This article is protected by copyright. All rights reserved.

4

Accepted Article

function remains cryptic.12 The remainder of this review focuses on α-klotho and will henceforth be referred to as klotho.

Transmembrane Klotho and Soluble Klotho Klotho exists in two forms - membrane-bound klotho (mKl) and soluble klotho (sKl). mKlis variably expressed in different tissues includingparathyroid, brain, heart and testis with low-level expression also detected in the aorta.6,20Klothois most abundantly described in the kidney with earlier reports focused on distal convoluted tubule expression,6 though more recently proximal tubule expression of mKl has been reported.21

sKl, on the other hand, is produced in two ways. The first is a result of ectodomain cleavage of mKl (~130 kDa) although factors regulating ectodomain shedding remain poorly characterised. Anumber of proteases have been implicated, most notably Adisintegrin and metalloproteinase (ADAM) 10/17, which is also expressed in the distal convoluted tubule. The second is a product of alternative splicing leading to a shorter form of sKl (~70kDa). Proteomic analysis of various extracellular fluids suggests that the longer form of sKl,generated by cleavage is the major circulating species in humans.22-24

The actions of mKl and sKl differ, with mKl predominantly supporting FGF23 in regulating phosphate.14Circulating sKL, on the other hand, can act as a paracrine or endocrine mediator, with increasing speculation that sKl functions independently of FGF23 signalling pathways.25 sKl displays enzymatic activity that may be important in regulating ion channels such as the sodium-phosphate co-transporter (NaPi-IIa),

This article is protected by copyright. All rights reserved.

5

Accepted Article

renal outer medullary potassium (ROMK) channel and Transient Receptor Potential Vanilloid (TRPV5) ion channel, the latter involved in calcium transport.2628

Furthermore, sKlhasbeen implicated in growth factor signalling as well as

demonstrating anti-insulin, anti-fibrotic and anti-oxidant activities.25,29These actions of klotho can also be dichotomised into either FGF23-dependent or FGF23independent ones (Figure 2).

Some studies have not found a clear relationship between mKl and sKl,30,31but one recent study reported a positive correlation between these levels.32Apotential

endocrine feedback loop has been described whereby sKl stimulates FGF23 expression, which in turn, downregulates kidney mKlabundance.33,34Other reports also raise the possibility that cleaved sKl forms a circulating receptor complex with FGF23, permitting FGFR signalling in tissues where klotho is not expressed or where expression has been lost.33

Klotho Assay The recent development of sandwich enzyme-linked immunoabsorbent assays (ELISA) for the longer form of sKl has provided an opportunity for assessment of circulating

concentrations

in

clinical

studies.35Unfortunately,

the

various

commercially available assays demonstrate poor analytical performance.36 (Table 1) The utility of these assays depends on better comprehension of the relationship between sKl and mKl, as well as improvement in analytical agreement between the available assays, and at present deficiencies in this knowledge greatly compromise our current understanding of klotho.

This article is protected by copyright. All rights reserved.

6

Accepted Article

Phosphate regulation by FGF23 and klotho Figure 3represents a conceptualisation of the role of klotho in phosphate control mechanisms. Both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid

hormone (PTH) have established roles in phosphate control. 1,25(OH)2D3 is the major

regulator of active intestinal calcium and phosphate absorption, mainly augmenting jejunal uptake. PTH is predominantly phosphaturic, reducing tubular reabsorption and

increasing urinary excretion, but additionally modulates bone turnover and hence mineral (calcium and phosphate) flux from the skeleton. PTH synthesis by chief cells in the parathyroid glands is directly responsive to extracellular phosphate concentrations via changes in PTH mRNA stability and indirectly via changes in serum calcium concentration conveyed by the calcium-sensing receptor.37,38

Renal handling of phosphate is considered by some the most important mechanism in phosphate homeostasis, with sodium-phosphate (NaPi) co-transporters heralded as the rate-limiting step in phosphate transport.39 Phosphate handling in the kidney and the transporters involved have been reviewed in detail previously.39-42 In brief, between 80-95% of the phosphate is reabsorbed in health, almost exclusively in the proximal tubules facilitated by three different families of solute carrier proteins, also known as NaPi co-transporters.39,42,43 Amongst them are SLC34A1 (NaPi-IIa) or SLC34A1 (NaPi-IIc) from the Type II family. NaPi-IIa is expressed throughout the whole proximal tubule, though in gradually decreasing fashion while NaPi-IIc has only been detected in Segment 1 of the proximal tubule.42Phosphate transport across the apical membrane is dependent on energy created by the electrochemical gradient of sodium ions.40

This article is protected by copyright. All rights reserved.

7

Accepted Article

In order to induce phosphaturia, FGF23 acts on the FGFR-Klotho co-receptor complex to reduce apical expression of NaPi-IIa and NaPi-IIc transporters thereby inhibiting tubular reabsorption of phosphate.44,45sKl candirectly promote phosphaturia via inhibition of NaPi-IIa.46 1,25(OH)2D3-stimulated absorption of phosphate in the

intestine, mediated through the co-transporter NaPi-IIb, is inhibited by FGF23 through inhibition of Cyp271b(1α-hydroxylase) synthesis and inactivation of the active hormone via up-regulation of Cyp24 (24-hydroxylase), thus lowering circulating 1,25(OH)2D3 levels.45 FGF23 also feeds back to suppress PTH synthesis in the parathyroid glands, againin aklotho-dependent manner.47

Although FGF23 has a significant impact on phosphate flux, evidence that phosphate or dietary intake directly regulates FGF23 synthesis is weak. There is little effect of

extracellular phosphate on cultured osteocytes in terms of FGF23 production or FGF23 promoter activity. Intravenous phosphate loading in humans is not associated with a change in circulating FGF23 levels.48,49Studies involving dietary loading

arealso inconsistent, demonstrating a highly variable but modest effect size (if present at all) and sluggish response to intake (over days to weeks).50-52Thus FGF23 appears to be mainly regulated by 1,25(OH)2D3 and locally by changes in bone mineralization that may be secondary to changes in PTH, 1,25(OH)2D3, phosphate or other as yet unidentified bone factors.

The role of klotho in mediating phosphate excretion appears substantial, and has been demonstrated both in vivo and in vitro.15,21 Both klotho knockout mice and FGF23 knockout mice demonstrate similar phenotypes with elevated levels of serum phosphate.6,53 This phenotype results from the inability to manipulate phosphate

This article is protected by copyright. All rights reserved.

8

Accepted Article

reabsorption in the absence of either FGF23 or klotho.53 Nakatani et al. established that serum phosphate levels decreased in FGF23knockout mice following exogenous FGF23 administration, but not in klotho knockout mice identifying the obligate role of klotho.15

Tissue distribution of klotho and FGF-Receptors The site of klotho expression in the nephron and actions related to signal transduction relationships remain controversial. While FGF23 has been found to bind to multiple FGFRs,54signalling by FGF23 was seen only with FGFR-1c, -3c and -4 in cell lines that co-expressklotho.14 In rats, members of the FGFR family are expressed in the

kidney at specific locations. FGFR-3 is expressed in both proximal and distal tubules whilst FGFR-1 and -4 are only seen in distal tubules.55In vitro studies supportFGFR-3 asthe physiologically relevant receptor in FGF23 downstream signallingbecause phosphate transport occurs in the proximal tubules. Interestingly, a study by Liu et al.

concluded that FGFR-3 and FGFR-4 did not mediate renal effects of FGF23 and instead, FGFR-1 was seen to co-localize with klotho in mice in the distal tubules.55 While this study suggests distal tubule distribution, more recent studies have reportedconvincing proximal tubular distribution, which is more physiologically likelysince this is where most phosphate transport occurs.6,20,21,46

Nevertheless, a distal tubule response manifests itself early either directly or indirectly via proximal tubule signalling,56 and exact human mechanisms are yet to be validated.Andrukhova et al. have established that FGF23 directly acts in a murine proximal tubule cell culture model to down-regulate NaPi-IIa through extracellular signal-regulated kinases (ERK)-1/2 and serum/glucocorticoid-regulated kinase-1

This article is protected by copyright. All rights reserved.

9

Accepted Article

(SGK1) signallingand this pathway is dependent on the presence of klotho at

physiological FGF23 levels.21 Through its enzymatic activity, sKl can act directly to

reduce recycling of NaPi-IIa, thereby itself inducing a phosphaturic response.46It is plausible that while mKl is abundant within the distal tubules, proximal tubule distribution of mKl facilitates some degree of phosphate excretion and cleaved sKl through paracrine actionpromotes a response in the proximal tubule despite being cleaved downstream. These exact mechanisms are still unclear.

What happens in CKD? One of the earliest abnormalities that develops as kidney function declines is a sustained reduction in tubular phosphate reabsorption resulting from the phosphaturic actions of FGF23 and PTH.2,57 As the remaining nephrons attempt to preserve

phosphate balance, the amount of phosphate excreted per nephron increases, with increases in single-nephron glomerular filtration and fractional excretion of phosphate (FEPi) measurements.57,58 Ultimately serum phosphate levels are affected when the declining kidney is no longer able to compensate for the reduction in total phosphate excretion.58This is usually observed when the glomerular filtration rate (GFR) falls

below 30ml/min.59

It is widely accepted that circulating FGF23 levels rise early in the course of CKD, perhaps earlier than PTH in most patients,60 yet the trigger for this rise is unclear. Higher FGF23 concentrations have been consistently associated with increased risk of mortality at all stages of CKD, independent of traditional renal and cardiovascular risk factors.61-64 In animal studies FGF23 excess as a result of direct intracardiac administration of a mutant FGF23 (and where klotho is absent) has been shown to

This article is protected by copyright. All rights reserved.

10

Accepted Article

lead to left ventricular hypertrophy and provides a plausible mechanism of direct cardiac injury at the high concentrations observed in advanced disease.65 The significance that these experiments were carried out with mutant FGF23 resistant to furin protease digestion is not known.However,supporting independent links between FGF23 and cardiovascular outcomes and mortality is the integrity of such associations after adjusting for phosphate, PTH and vitamin D levels.61-64 It has yet to be established whether specifically lowering FGF23 or antagonizing its actionwould yield clinical benefit. Indeed, antagonising FGF23 with a specific antibody increased

vascular calcification and mortality in animals with renal impairment.66

The down-regulation of klotho expression in tissues where it is expressed has been linked to enhancement of the klotho-independent effects of FGF23 in other tissues. One explanation is that withless binding to the klotho-FGFR complex, more FGF23 is left in the circulation to bind ‘off-target’ to other FGFRs, where specificity to the receptor is low, yet ligand present in excess so causingactivation of other low specificity FGFRs at non-physiological sites. Aconsistent finding in CKD is the overall decrease in mKl expression in the kidney, parathyroid glands and vasculature.67 Although human studies of mKl have been limited due to difficulty in obtaining tissue to determine expression, there appears to be good evidence of reduced kidney mKl expression in animal CKD models.30,68 A low level of sKl in plasma and urine of mice with CKDhas also been reported.30 Human studies reporting

on associations between circulating sKl and renal function have been capricious even using the same assay (Table 1). Seiler et al. reported no correlation between sKl

levels and renal function69 while other investigators report an increase in sKlwith declining GFR.70-72More than half of the human studies in patients with CKD

This article is protected by copyright. All rights reserved.

11

Accepted Article

however have documented a reduction in sKl levels with reduced GFR.73-78 The aforementioned issues with assay performance may underpin the apparent discordant results, but may also relate to differences in study setting or simply reflect intricacies of klotho metabolism, which as yet we do not understand.

Nonetheless reductions seen in mKl suggest a relative deficiency of klotho in CKD.67This has been supportedby results from a recent study involving CKD patients undergoing renal biopsies, demonstrating not only correlation between mKl and sKl, but a stepwise reduction in both mKl and sKl levels as GFR declines.32Levels of sKl have also been reported to be inversely associated with mortality in an elderly population, approximately one-third of whom had CKD.79This association is consistent withanimal studies where transgenic mice overexpressingklotho conferred a longer lifespan, whilst klotho knockout models age rapidly, highlighting klotho as a potential “protective” factor.6,7,29,79

Arecent reportof 880 adults from the Heart and Soul Study, described an association

between higher urinary phosphate excretion with lower risk of cardiovascular events and a non-significant association with mortality.80 One quarter of the cohort in this study had CKD and analysis of FGF23 levels revealed an association with mortality which was modified by FEPi.81In other words, those with lower FEPi despite higher FGF23 levels had the highest mortality risk implying that an impaired ability to excrete phosphate in responseto FGF23 could be associated with adverse outcomes.This may be the result of relative klotho deficiency.81 Dominguez et al.

further proposed that the concurrent evaluation of plasma FGF23 and FEPi may serve as non-invasive indicators of kidney mKl expression.81

This article is protected by copyright. All rights reserved.

12

Accepted Article

There are a paucity of human tissue studies to validate these hypotheses and early findings, including the concurrent stepwise reduction in mKl and sKl in CKD, as well

as the inverse association of mKl and/or sKl with mortality. Given the abundance of mKl in the kidney and that cleaved sKl is likely to be dependent on overall mKl levels, it is conceivable klotho deficiency in CKD is a result of sustained reduction mKl expression in diseased or damaged kidney. Furthermore, klotho deficiency in CKD may well underpin several of the processes leading to increased morbidity and mortalityobserved in this population, such as mineral metabolism dysregulation and hormonal imbalanceswithin CKD-MBD, as well as possible links with cardiovascular outcomes. Of note, one recent article by Seiler et al. reported no relationship between sKl and cardiovascular outcomes.82However, this study involved a small cohort which had previously been shown to have no correlation between sKl and GFR, and a short follow-up period.69,82Further prospective studies are required to establish consistent findings.

Klotho: a role beyond phosphate metabolism Acute Kidney Injury (AKI) A potential wider role for klotho within the kidney issuggested by a number of other findings. Changes in klotho have been implicated in the course of acute kidney injury (AKI).Despite the heterogeneity of animal models of AKI, studies have consistently shown reduced klotho levels in association with AKI from models including ischaemia reperfusion injury, sepsis, drug-induced, unilateral urinary obstruction (UUO) and others,83-91 althoughthere are differences in the speed and completeness of

This article is protected by copyright. All rights reserved.

13

Accepted Article

klotho recovery in the different models.83-85In a recent review, Hu and Moe speculated that a delayed response may predict unfavourable long-term prognosis.86In

support of this,both sKl and mKl were reduced 3 hours post reperfusion83and the administration of exogenous klotho reduced renal injury especially when given within 60 minutes of reperfusion.83 Further transgenic overexpression of klotho conferred more resistance to ischaemia reperfusion injury compared to wild-type.83Therefore klotho deficiency as an early event in AKI and its potential role as apathogenic factor that exacerbates acutekidney damage may makethis renal-derived protein a highly promising candidate for both an early biomarker and therapeutic agent for AKI.

Klotho, Fibrosis and Inflammation Progression from AKI to CKD or end-stage kidney disease inevitably follows a common pathway, characterized by progressive interstitial fibrosis.92 Transforming growth factor-β1 (TGF-β1) is a key player in mesenchymal transition and has an important role in fibrosis.90 In the UUO model TGF-β1 is elevated and correlates with the severity of fibrosis following injury.91 Administration of recombinant klotho was observed to inhibit TGF-β1 signalling by directly binding to its receptor, thereby

inhibiting the binding of TGF-β1 and ultimately alleviating renal fibrosis.90 In a murine model of folic acid nephropathy and with cell culture, Moreno et al.demonstratedklotho down-regulation by inflammation through thetumour necrosis

factor (TNF) family of cytokines in a nuclear factor-kappa B (NFκB)-dependent manner.85 This reduced gene expression was demonstrated to be a result of histone deacetylation, with inhibition of this mechanism resulting in reversal of the effects of TNFα,85arguing again for a possible therapeutic role using sKl, not only as a novel

AKI biomarker but as potential therapy in kidney injury. This article is protected by copyright. All rights reserved.

14

Accepted Article

Klotho and Angiotensin II Angiotensin-II (AngII) is a well-recognised potent pro-inflammatory, pro-oxidant and pro-fibrotic agent traditionally considered exclusively involved in blood pressure and electrolyte control that is up-regulated in a variety of renal pathology.93,94AngII blockade using angiotensin converting enzyme inhibitors (ACE-i) and angiotensin (type-1) receptor blockers (ARB) have not only demonstrated the pleiotropic effects of AngIIbut blockade confers cardio-renal protection beyond that of blood pressure control.94-96In examiningthese mechanisms, Zhou et al. studied rat renal tubular epithelial cells (NRK-52E) treated with AngII, ACE-i and ARB, alone and in combination.97 The authors determined that several markers of fibrosis and inflammation including TGF-β1, were up-regulated as a result of treatment with AngII and down-regulated when treated in combination with ACE-i and/or ARB.

Concurrently, klotho mRNA and protein levels in the cells showed relative inverse regulation, suggesting potential mechanistic pathways of AngII-induced kidney

damage and klotho protection.97Others have reported similar findings independent of blood pressure in animal studies, where continuous AngII administration resulted in down-regulation of both klotho gene and protein levels in the kidney, andAngII blockade, either with an ARB and/or ACE-i conferred protection by up-regulation of klotho mRNA levels.98-102 Furthermore, Mitani et al. established klotho gene transfer as a potential rescue therapy in mice with AngII-induced renal damage, exhibiting improved functional and morphological kidney status,98 further supporting a potential role of klotho in therapy for kidney injury. Two post-hoc human studies have assessed

sKl levels and the effects of ARB treatment. Both studies reported significant increases in sKL levels followingadministration of ARB in diabetic patients with

This article is protected by copyright. All rights reserved.

15

Accepted Article

relatively preserved GFR,103,104 providing some in vivo data on the link between AngII and klotho.

Klotho and Vascular Disease Studies that have examined associations of sKl in populations without kidney disease (Table 1) collectively suggesting that klotho may play a protective role in biological processes. One cohort study reported reduced longevity associated with a prevalent functional klotho gene variant (when in homozygosity).105 Furthermore, this allele has been reported to be independently associated with early-onset occult coronary artery disease supporting a possible protective role for klotho in the cardiovascular system.106Treatment with statins in klotho-mutant mice, where angiogenesis and vasculogenesis are impaired subsequent to unilateral hindlimb ischaemia, improved blood flow and limb salvage through enhanced angiogenesis and vasculogenesis, independent of lipid lowering effects.11Studies in cell lines and in animal modelssupport findings that statins upregulate mKl in a dose dependent manner.10,107,108Furthermore, klotho gene delivery into rat aortic smooth muscle cells demonstrated decreased oxidative stressand reduced apoptosis109 and adenovirus-

delivered-klotho in fatty rats increased nitric oxide production, and restored

endothelial function.110 Taken together, this body of evidence strengthens the rationale that klotho deficiency has far-reaching implications beyond phosphate control, providing plausible pathophysiological pathways linking klotho, CKD and detrimental outcomes.

Conclusion

This article is protected by copyright. All rights reserved.

16

Accepted Article

Both FGF23 andklotho have been established as key players in bone and mineral metabolism butthere are still many unanswered questions. Whilst mKl is abundant in distal tubules, reported proximal tubule expression provides a credible explanation of klotho-dependent FGF23 phosphate regulation within the proximal tubules.

Althoughthe degree of correlation between mKl and sKl needs to be further validated, differences between them are becoming evident, where sKl may have a much wider biological role than previously described.The availability of sKl assays will likely expand our comprehension of phosphate homeostasis as well as the intricacies of klotho regulation. Consequently, this may providea greater understanding of the roles of klotho and FGF23 in the pathogenesis of CKD-MBD andbe helpful in examination of potential therapeutic targets of injury and disease.

Acknowledgements SJT is a current recipient of a National Health and Medical Research Council (NHMRC) Postgraduate Research Scholarship. The contents of this review article are solely the views of the individual authors and do not reflect the views of NHMRC.

This article is protected by copyright. All rights reserved.

17

Accepted Article

Figures and Tables Figure 1.Pathways of phosphate flux in humans. Representative daily fluxes (mmol/24h) in a healthy 70 kg adult. Extracellular phosphate concentration is chiefly regulated by modulation of intestinal absorption and renal tubular reabsorption. Major positive (green) and negative (red) hormonal regulators and their transporter/enzyme targets (purple) are indicated. Abbreviations: Cyp24, 24-hydroxylase; Cyp27b1, 1αhydroxylase; FGF-23, fibroblast growth factor-23; mKL, membrane klotho; sKL, soluble klotho; NaPi, sodium-dependent phosphate transporter; PTH, parathyroid hormone. Reproduced with modification from Cundy et al.111

Figure 2. Mechanism of klotho action. A) Transmembrane klotho (orange) functions as co-receptor for FGF23 (blue). Activation of the FGFR:klotho complex reduces reabsorption of phosphate and inhibits synthesis of 1,25(OH)2D3 in the renal tubules and suppresses PTH synthesis in the parathyroid glands. Ectodomain shedding of transmembrane klotho is mediated by ADAM10/17 or other unidentified secretases and releases soluble klotho (red) into the extracellular fluid. B) Soluble klotho (red) may bind FGF23 in circulation and form mobile receptor complex capable of activation FGFR on cells where transmembrane klotho expression is low or absent. C)

Modification

of

ion

channel

(green)

glycosylation

by

β-

glucuronidase/neuraminidase activity of soluble klotho alters channel occupancy in the plasma membrane either resulting in internalisation or retention via binding of galectin-1.

This article is protected by copyright. All rights reserved.

18

Accepted Article

D) Soluble klotho has a number of cytoprotective effects by unknown mechanisms.

Figure 3. A framework depicting the various actions and feedback loops of the multiple hormones and proteins in phosphate control. * Phosphaturic actions of FGF23 and suppression of PTH by FGF23 are klotho dependent. The relationship between FGF23 and Klotho are unclear with some in vivo data suggesting the negative feedback mechanism (dashed lines). The association between dietary phosphate loading and FGF23 levels is inconsistent (dash/dotted line, see text).

This article is protected by copyright. All rights reserved.

19

Accepted Article

Table 1: Summary of all reported human studies examining soluble klotho Study

Year

Population Studied

Sample Size, n

Assay

Association with soluble Klotho (sKl)

Studies involving patients with kidney disease

2013

CKD Stage 2-4

321

IBL

114

2013

Kidney Donors, Kidney Recipients

20

IBL

115

9. Hryszko et al. 10. Karalliedde et al.103

2013 2013

22 76

IBL IBL

11. Lim et al.104

2013

Haemodialysis (cholecalciferol) Diabetic kidney disease (RCT, valsartan) Diabetic kidney disease (RCT, losartan, quinapril)

sKl correlated with mKl mKl with progressive CKD sKl -ve correlation with residual diuresis sKl weak +ve correlation with phosphate clearance sKl  with progressive CKD sKl  with progressive CKD sKl  with progressive CKD sKl +ve correlation with 1,25(OH)2D3 sKl -ve correlation with PTH and FEPi sKl independently associated with arterial stiffness sKl did not relate to kidney function sKl with donor nephrectomy No appreciable change with transplantation sKlotho  with cholecalciferol sKl  with valsartan

33

IBL

sKl  with losartan

12. Pavik et al.116

2012

ADPcKD, CKD, XLH, Healthy controls

163

IBL

13. Komaba et al.31

2012

Dialysis (cinacalcet)

51

IBL

14. Devaraj et al.71

2012

Diabetes + no CKD, CKD (SCr > 2mg/dL), Healthy controls

200

Cusabio

15. Kacso et al.72

2012

Diabetic Nephropathy, Healthy Controls

189

USCN

2012 2012 2012 2012 2011

Peritoneal dialysis Dialysis, Healthy controls CKD Stage 1-5 CKD Stage 1-5 CKD, Healthy controls

36 73 292 131 40

IBL IBL IBL IBL KHK^

sKl  in ADPcKD sKl -ve correlation with cyst volume/kidney growth sKl  initially before returning to baseline with cinacalcet sKl  in diabetics sKl  in CKD sKl  in early CKD sKl  in late CKD Urinary sKl +ve correlation with residual function sKl  in dialysis sKl  in early CKD sKl  in late CKD (serum and urine) sKl  in CKD

77

IBL

No difference noted between groups

34

IBL

sKl  in underweight and overweight groups

1. Sakan et al.32

2014

CKD Stage 1-5

236

KHK^

2. Golembiewska et al.112

2013

Incident peritoneal dialysis

35

Demeditec

3. Pavik et al. 4. Wan et al.77 5. Kim et al.78

2013 2013 2013

CKD Stage 1-5, Healthy controls Children with CKD CKD Stage 1-5

98 154 243

IBL IBL IBL

6. Kitagawa et al.113

2013

CKD Stage 2-4

114

IBL

7. Seiler et al.69

76

8. Akimoto et al.

16. 17. 18. 19. 20.

Akimoto et al.117 Yokoyama et al.73 Shimamura et al.74 Akimoto et al.75 Sugiura et al.70

Studies not involving patients with kidney disease Diabetes + MVD, Non-diabetes + MVD, 21. van Ark et al.118 2013 Healthy Controls (eGFR

The importance of klotho in phosphate metabolism and kidney disease.

The discovery of fibroblast growth factor-23 (FGF23) and its co-receptor α-klotho has broadened our understanding of mineral metabolism and led to a r...
736KB Sizes 0 Downloads 3 Views