Hunt for the culprit of cardiovascular injury in kidney

Cardiovascular Research Advance Access published October 9, 2015

EDITORIAL

Cardiovascular Research doi:10.1093/cvr/cvv228

Hunt for the culprit of cardiovascular injury in kidney disease Christian Faul 1 and Myles Wolf 2* 1 Division of Nephrology and Hypertension, Department of Medicine, and Department of Cell Biology and Anatomy, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA; and 2Division of Nephrology and Hypertension, Department of Medicine, and Center for Translational Metabolism and Health, Institute for Public Health and Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

This editorial refers to ‘Membrane-bound Klotho is not expressed endogenously in healthy or uraemic human vascular tissue’ by R. Mencke et al., doi:10.1093/cvr/cvv187.

The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.

* Corresponding author: Tel: +1 312 503 8013. E-mail: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected].

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Disordered phosphate homeostasis is thought to contribute mechanistically to the high rates of cardiovascular disease encountered in patients with chronic kidney disease (CKD), but how this occurs is uncertain. As the most recently discovered regulators of phosphate homeostasis, a-klotho, and fibroblast growth factor 23 (FGF23) have seized the investigative spotlight as candidate cardiovascular culprits. a-Klotho is a transmembrane receptor that is most heavily expressed in the kidney tubules,1 whereas FGF23 is an endocrine hormone secreted primarily by osteocytes.2 FGF23 stimulates renal phosphate excretion and lowers circulating levels of calcitriol, which secondarily reduces the efficiency of dietary phosphate absorption in the gastrointestinal tract.3 These effects are mediated by FGF23 binding to and activating heterodimeric complexes of FGF receptors and a-klotho co-receptors in the kidney.4 A circulating soluble form of a-klotho that is derived from cleavage of its extracellular domain may also possess endocrine effects, including FGF23-independent phosphaturia.5 Highlighting their shared roles in regulating total body phosphate economy, deficiency of either a-klotho or FGF23 results in impaired renal phosphate excretion, endogenous calcitriol toxicity, and syndromes of accelerated aging marked by extensive arterial calcification among numerous other manifestations.6,7 Since renal excretion is the only means of phosphate egress from the body, CKD is a prototypical human condition of impaired phosphate excretion. Afflicting millions of people worldwide, it is also the most common. In CKD, renal expression of a-klotho progressively declines and circulating levels of FGF23 progressively increase.3 Enabled by assays that can reliably measure FGF23 in stored blood, prospective studies of large CKD cohorts demonstrated that elevated FGF23 is strongly associated with increased risks of cardiovascular events and death.8 – 10 One possible underlying molecular mechanism is that FGF23 induces pathological cardiac remodelling through a-klotho-independent effects on cardiac myocytes that culminate in left ventricular hypertrophy.11 In

contrast, assays for circulating soluble a-klotho are immature, and there is no current method to quantify renal expression of transmembrane a-klotho in vivo in humans. As a result, human outcome studies of a-klotho deficiency have lagged. Resorting to animal models to fill the gap, studies demonstrated that a-klotho deficiency promotes arterial calcification and left ventricular hypertrophy in rodents, and that these effects can be ameliorated by exogenous administration or transgenic overexpression of a-klotho.12 – 16 How a-klotho deficiency promotes arterial calcification is controversial. Some studies propose loss of direct protective effects of a-klotho that is locally produced and expressed in the vasculature, whereas others propose an indirect effect driven by consequences of reduced a-klotho expression in the kidney, most notably, hyperphosphatemia. At the core of this controversy is the divided literature on whether a-klotho is expressed in the vasculature. In this edition of Cardiovascular Research, Mencke et al. aimed to break this logjam by reporting on their search for a-klotho expression in the human vasculature.17 The investigators studied arterial samples that were harvested from healthy kidney transplant donors and recipients with end-stage renal disease, atherosclerotic plaque specimens that were obtained from patients who underwent carotid endarterectomy, autopsy-derived elastic arterial samples, and primary cultures of human aortic smooth muscle cells. To test for a-klotho protein expression, the authors performed immunohistochemistry analyses using four different anti-a-klotho antibodies, immunofluorescence analyses, and western blot analyses using recombinant human a-klotho as positive control. To test for the presence of a-klotho mRNA, they used quantitative PCR. Since the kidney is known to have the highest a-klotho expression,4 normal human kidneys that were collected at nephrectomy served as positive control for all tissue analyses. Additional controls included HEK293 cells that were transfected with a human a-klotho-expressing transgene. Despite extensive medial calcification in the end-stage renal disease and carotid endarterectomy specimens, and clear-cut evidence of a-klotho staining in the kidney by immunohistochemistry, there was no a-klotho detectable in the vasculature in any patient sample using any antibody. Immunofluorescence of human kidney clearly demonstrated co-localization of a-klotho with other tubular cell markers, but no evidence of its co-localization with smooth muscle cell markers.

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Editorial

Likewise, there was no a-klotho detectable in cultured human vascular smooth muscle cells by immunofluorescence or in human aortic smooth muscle cells by immunoblottting, whereas HEK293 cells that were transfected with the human a-klotho cDNA clearly expressed the protein. Western blot analyses confirmed the absence of a-klotho protein from all human vascular specimens but high expression levels in kidney. The authors did detect extremely low levels of a-klotho mRNA in human arteries, but appropriately conclude that this is probably insufficient to be physiologically relevant given the absence of protein expression. Conclusively confirming a null hypothesis, in this case, that a-klotho is not present in the vasculature, is much more challenging than rejecting the null hypothesis and concluding presence of a-klotho. There are numerous technical reasons why investigators could commit a type II error in which they fail to detect a-klotho that is truly present in a tissue or cellular sample. Mencke et al. should be commended for their exhaustive search that used multiple different antibodies, multiple mutually confirmatory screening approaches, and multiple positive controls. Their results are convincing. Type II error is exceedingly unlikely.

Whereas the positive controls were critical to the experiments by Mencke et al. problems with the negative controls in other studies likely underlie how these could have committed the type I error of incorrectly concluding that the vasculature does express a-klotho. The gold standard experiment for validating the specificity of an antibody is to demonstrate strong expression by western blot analysis in tissue from wild-type mice but absolutely no protein detection in the same tissue from a knock-out mouse in which expression does not occur. Any bands detected in the knock-out specimens represent non-specific antibody cross-reactivity. To our knowledge, the KM2076 antibody is the only commercially available anti-a-klotho antibody that has undergone this level of published scrutiny. Although the expected 130 kDa signal of a-klotho was missing from kidney extracts from a-klotho-null mice, KM2076 detected other bands of lower-molecular weight in extracts from both wild-type and a-klotho-null mice.18 Using KM2076 or other similarly non-specific antibodies for tissue immunostaining will promote false-positive staining for a-klotho. Antibody cross-reactivity is probably also a major factor that undermines the accuracy of commercially available ELISA tests of soluble a-klotho levels. It is absolutely essential that


Figure 1 Hypotheses on how a-klotho deficiency promotes arterial calcification in chronic kidney disease. Under Hypothesis 1, vascular expression of the transmembrane form of a-klotho, which is most highly expressed in the kidney, protects vessels from calcification such that deficiency of vascular a-klotho in chronic kidney disease induces calcification directly. The report by Mencke et al. contradicts this hypothesis. Under Hypothesis 2, reduced circulating levels of the soluble form of a-klotho, which is released into circulation from ectodomain shedding of renal transmembrane a-klotho, render vessels susceptible to calcification. This hypothesis was not tested by Mencke et al. Based on an extensive body of prior in vitro, animal and human data, hyperphosphatemia is likely an independent causal factor in the pathogenesis of arterial calcification in chronic kidney disease (central panel). Under this model, concomitant deficiencies of the transmembrane and soluble forms of a-klotho in chronic kidney disease are indirectly associated with calcification.

Editorial

Conflict of interest: M.W. has received research support, honoraria, or consultant fees from Amgen, Astra Zeneca, DiaSorin, Keryx, Luitpold, Opko, Pfizer, Sanofi, Shire, and Vifor.

Funding C.F. is supported by grant R01HL128714 from the National Institutes of Health, and he has received research support from U3 Pharma GmbH, Germany. M.W. is supported by grants R01DK076116, R01DK081374, R01DK094796, K24DK093723, R21DK100754, and U01DK099930 from the National Institutes of Health, and a Strategically Focused Research Network Center Grant from the American Heart Association.

References 1. Hu MC, Shiizaki K, Kuro-o M, Moe OW. Fibroblast growth factor 23 and Klotho: physiology and pathophysiology of an endocrine network of mineral metabolism. Annu Rev Physiol 2013;75:503–533. 2. Quarles LD. Endocrine functions of bone in mineral metabolism regulation. J Clin Invest 2008;118:3820 –3828. 3. Wolf M. Update on fibroblast growth factor 23 in chronic kidney disease. Kidney Int 2012;82:737 –747. 4. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 2006;444:770 – 774. 5. Hu MC, Shi M, Zhang J, Pastor J, Nakatani T, Lanske B, Razzaque MS, Rosenblatt KP, Baum MG, Kuro-o M, Moe OW. Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. FASEB J 2010;24:3438 –3450.

6. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45 –51. 7. Shimada T, Kakitani M, Yamazaki Y, Hasegawa H, Takeuchi Y, Fujita T, Fukumoto S, Tomizuka K, Yamashita T. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest 2004;113: 561 –568. 8. Gutie´rrez OM, Januzzi JL, Isakova T, Laliberte K, Smith K, Collerone G, Sarwar A, Hoffmann U, Coglianese E, Christenson R, Wang TJ, deFilippi C, Wolf M. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. Circulation 2009;119:2545 –2552. 9. Isakova T, Xie H, Yang W, Xie D, Anderson AH, Scialla J, Wahl P, Gutie´rrez OM, Steigerwalt S, He J, Schwartz S, Lo J, Ojo A, Sondheimer J, Hsu CY, Lash J, Leonard M, Kusek JW, Feldman HI, Wolf M; Chronic Renal Insufficiency Cohort (CRIC) Study Group. Fibroblast growth factor 23 and risks of mortality and endstage renal disease in patients with chronic kidney disease. JAMA 2011;305: 2432 – 2439. 10. Scialla JJ, Xie H, Rahman M, Anderson AH, Isakova T, Ojo A, Zhang X, Nessel L, Hamano T, Grunwald JE, Raj DS, Yang W, He J, Lash JP, Go AS, Kusek JW, Feldman H, Wolf M; Chronic Renal Insufficiency Cohort (CRIC) Study Investigators. Fibroblast growth factor-23 and cardiovascular events in CKD. J Am Soc Nephrol 2014;25:349 –360. 11. Faul C, Amaral AP, Oskouei B, Hu MC, Sloan A, Isakova T, Gutie´rrez OM, Aguillon-Prada R, Lincoln J, Hare JM, Mundel P, Morales A, Scialla J, Fischer M, Soliman EZ, Chen J, Go AS, Rosas SE, Nessel L, Townsend RR, Feldman HI, St John Sutton M, Ojo A, Gadegbeku C, Di Marco GS, Reuter S, Kentrup D, Tiemann K, Brand M, Hill JA, Moe OW, Kuro-O M, Kusek JW, Keane MG, Wolf M. FGF23 induces left ventricular hypertrophy. J Clin Invest 2011;121:4393 –4408. 12. Hu MC, Shi M, Zhang J, Quin˜ones H, Griffith C, Kuro-o M, Moe OW. Klotho deficiency causes vascular calcification in chronic kidney disease. J Am Soc Nephrol 2011;22: 124 –136. 13. Chen TH, Kuro-O M, Chen CH, Sue YM, Chen YC, Wu HH, Cheng CY. The secreted Klotho protein restores phosphate retention and suppresses accelerated aging in Klotho mutant mice. Eur J Pharmacol 2013;698:67 –73. 14. Hu MC, Shi M, Cho HJ, Adams-Huet B, Paek J, Hill K, Shelton J, Amaral AP, Faul C, Taniguchi M, Wolf M, Brand M, Takahashi M, Kuro-O M, Hill JA, Moe OW. Klotho and phosphate are modulators of pathologic uremic cardiac remodeling. J Am Soc Nephrol 2014;26:1290 –1302. 15. Xie J, Yoon J, An SW, Kuro-o M, Huang CL. Soluble klotho protects against uremic cardiomyopathy independently of fibroblast growth factor 23 and phosphate. J Am Soc Nephrol 2014;26:1150 –1160. 16. Yang K, Wang C, Nie L, Zhao X, Gu J, Guan X, Wang S, Xiao T, Xu X, He T, Xia X, Wang J, Zhao J. Klotho protects against indoxyl sulphate-induced myocardial hypertrophy. J Am Soc Nephrol 2015;26:2434 –2446. 17. Mencke R, Harms G, Mirkovic´ K, Struik J, Van Ark J, Van Loon E, Verkaik M, De Borst MH, Zeebregts CJ, Hoenderop JG, Vervloet MG, Hillebrands JL; NIGRAM Consortium; NIGRAM Consortium. Membrane-bound Klotho is not expressed endogenously in healthy or uraemic human vascular tissue. Cardiovasc Res 2015;108: 220 –231. 18. Barker SL, Pastor J, Carranza D, Quin˜ones H, Griffith C, Goetz R, Mohammadi M, Ye J, Zhang J, Hu MC, Kuro-o M, Moe OW, Sidhu SS. The demonstration of alphaKlotho deficiency in human chronic kidney disease with a novel synthetic antibody. Nephrol Dial Transplant 2015;30:223 – 233. ¨ stman Wernerson A, Lanske B, 19. Lindberg K, Amin R, Moe OW, Hu MC, Erben RG, O Olauson H, Larsson TE. The kidney is the principal organ mediating klotho effects. J Am Soc Nephrol 2014;25:2169 – 2175. 20. Scialla JJ, Lau WL, Reilly MP, Isakova T, Yang HY, Crouthamel MH, Chavkin NW, Rahman M, Wahl P, Amaral AP, Hamano T, Master SR, Nessel L, Chai B, Xie D, Kallem RR, Chen J, Lash JP, Kusek JW, Budoff MJ, Giachelli CM, Wolf M; Chronic Renal Insufficiency Cohort Study Investigators. Fibroblast growth factor 23 is not associated with and does not induce arterial calcification. Kidney Int 2013;83: 1159 – 1168. 21. Shalhoub V, Shatzen EM, Ward SC, Davis J, Stevens J, Bi V, Renshaw L, Hawkins N, Wang W, Chen C, Tsai MM, Cattley RC, Wronski TJ, Xia X, Li X, Henley C, Eschenberg M, Richards WG. FGF23 neutralization improves chronic kidney disease-associated hyperparathyroidism yet increases mortality. J Clin Invest 2012; 122:2543 – 2553.


future investigations of the tissue distribution of a-klotho protein expression validate their antibodies’ specificity in a-klotho-null mice. The broader biological conclusion suggested by the current results is that a-klotho deficiency must injure the vasculature indirectly (Figure 1). This is supported by findings from transgenic mice in which deletion of a-klotho specifically from the kidney was sufficient to completely recapitulate the phenotype of global a-klotho ablation, including hyperphosphatemia, elevated FGF23 levels, and arterial calcification, at least in the kidney.19 Based on evidence from epidemiological and laboratory studies, hyperphosphatemia rather than FGF23 excess most likely drives calcification in states of a-klotho deficiency in rodents and humans with CKD.20,21 What remains less clear is how mice that are prone to calcification are protected by exogenous a-klotho administration or its transgenic overexpression. Soluble a-klotho might be indirectly protective by reversing hyperphosphatemia,12 or it could have direct beneficial effects on the vasculature. Direct end-organ actions have been also invoked to explain the salutary effects of soluble a-klotho on left ventricular hypertrophy,14 – 16 but underlying cellular mechanisms have not been demonstrated in either vessel or heart. Are hormonal effects of soluble a-klotho accountable? If so, what receptors and signalling pathways respond to soluble a-klotho stimulation? Are proposed enzymatic actions of soluble a-klotho on transport channels in the kidney5 also operative in vessel and heart? If so, what is the substrate in these targets? Could the beneficial effects of soluble a-klotho be mediated by altering FGF23 levels or acting as a decoy receptor that prevents FGF23 from activating its a-klotho-independent effects in the heart? Given these open questions, the extent to which coexisting a-klotho deficiency vs. FGF23 excess vs. hyperphosphatemia are cardiovascular disease culprits in CKD remains controversial. The hunt goes on.

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