CJASN ePress. Published on July 22, 2020 as doi: 10.2215/CJN.02840320

Klotho in Clinical Nephrology Diagnostic and Therapeutic Implications

Javier A. Neyra,1,2,3 Ming Chang Hu,1,2 and Orson W. Moe1,2,4

Abstract aKlotho (called Klotho here) is a membrane protein that serves as the coreceptor for the circulating hormone fibroblast growth factor 23 (FGF23). Klotho is also cleaved and released as a circulating substance originating 1Charles and Jane Pak primarily from the kidney and exerts a myriad of housekeeping functions in just about every organ. The vital role of Center for Mineral Klotho is shown by the multiorgan failure with genetic deletion in rodents, with certain features reminiscent of Metabolism and Clinical Research, human disease. The most common causes of systemic Klotho deficiency are AKI and CKD. Preclinical data on Klotho Dallas, Texas biology have advanced considerably and demonstrated its potential diagnostic and therapeutic value; however, 2Department of multiple knowledge gaps exist in the regulation of Klotho expression, release, and metabolism; its target organs; and Internal Medicine, mechanisms of action. In the translational and clinical fronts, progress has been more modest. Nonetheless, Klotho University of Texas Southwestern Medical has potential clinical applications in the diagnosis of AKI and CKD, in prognosis of progression and extrarenal Center, Dallas, Texas complications, and finally, as replacement therapy for systemic Klotho deficiency. The overall effect of Klotho in 3Division of clinical nephrology requires further technical advances and additional large prospective human studies. Nephrology, Bone and CJASN 16: ccc–ccc, 2021. doi: https://doi.org/10.2215/CJN.02840320 Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Introduction Soluble Klotho protein is also detected in cerebrospinal Lexington, Kentucky aKlotho (referred here as Klotho) was serendipitously fluid and urine. 4Department of discovered in 1997 by Kuro-o et al. (1) as a linked Klotho is a pleiotropic protein with multifaceted Physiology, University to aging. This gene was named Klotho after the goddess of Texas Southwestern functions relevant to distinct biology and pathobi- Medical Center, who spins the thread of life to govern human lifespan, ology in multiple organs and tissues. Klotho is an Dallas, Texas working with two other goddesses of fate, Lachesis inhibitor of apoptosis, fibrosis, and cell senescence who measures the spun thread from Klotho’s spindle and is an inducer of autophagy (13–16). In this Correspondence: and Atropos who ends the life by cutting the thread (2). review, we discuss its potential clinical application Dr. Javier A. Neyra or Kuro-o et al. (1) observed that Klotho hypomorphic in nephrology, with emphasis on diagnostic and ther- Dr. Ming Chang Hu, mice exhibited a multiorgan failure syndrome re- Charles and Jan Pak apeutic utility. Center for Mineral sembling premature aging, including short lifespan, Metabolism and disturbed mineral metabolism, and multiple organ Clinical Research, degeneration or failure, such as infertility, arterio- Physiology of Klotho University of Texas sclerosis, cardiomyopathy, ectopic calcification, skin Klotho Expression and Metabolism Southwestern Medical fi Center, 5323 Harry atrophy, osteoporosis, and emphysema. Later, both Klotho is highly expressed in the kidney, speci - Hines Boulevard, human (3) and rat (4) Klotho were cloned with cally in the distal tubules and, to a lesser extent, in the Dallas, TX 75390- high homology in amino acid sequences with the proximal tubules (17). Klotho is expressed in other 8856. Email: javier. mouse. Subsequently, two paralogous genes were organs, such as the brain, pancreas, and parathyroid [email protected] or discovered and termed bKlotho (5) and gKlotho (6) to glands (1). The Klotho gene encodes a single-pass 130- ming-chang.hu@ utsouthwestern.edu distinguish them from the first Klotho gene, which was kD that consists of two renamed aKlotho. The a- and bKlotho proteins have extracellular domains (Kl1 and Kl2), a transmembrane known biologic roles, but the function of gKlotho is domain, and a short cytoplasmic tail (Figure 1). The still elusive (7,8). In this manuscript, we only focus on extracellular domain of transmembrane Klotho is aKlotho (Klotho). cleaved at the juxtamembrane region and between Klotho is mostly expressed in the kidney as a trans- Kl1 and Kl2 by (18–20). A secreted form of membrane protein. In membrane-bound form, Klotho Klotho from an alternatively spliced transcript has serves as a coreceptor for fibroblast growth factor 23 been proposed, but the data are not conclusive (3,21). (FGF23) in conjunction with fibroblast growth factor The spliced Klotho transcript possibly undergoes receptors (FGFRs) (9) (Figure 1). The extracellular nonsense-mediated mRNA decay and is not trans- domain of transmembrane Klotho is also cleaved by lated to protein (22). The cleaved Klotho is released proteases and released from the kidney into circula- into the circulation and is known as soluble Klotho tion, both as a full-length protein or as Kl1 and Kl2 (17,22). Soluble Klotho acts as an endocrine or para- fragments (10,11). These circulating forms of Klotho crine factor affecting multiple organs, such as the protein are collectively called “soluble” Klotho (Figure 1). kidney, bone, brain, heart, lungs, and www.cjasn.org Vol 16 January, 2021 Copyright © 2021 by the American Society of Nephrology 1 2 CJASN

Figure 1. | Overview of Klotho protein. (A) 23 (FGF23) engages the fibroblast growth factor receptor (FGFR)-Klotho coreceptor complex that triggers cellular signaling. (B) Transmembrane Klotho with its Kl1 and Kl2 domains and the generation of soluble circulating Klotho by secretases (ADAM10 and ADAM17). (C) Structure of FGFR1c, Klotho, and FGF23 (one molecule each) as determined by Chen et al. (12). RXXR motif, proteolytic cleavage motif. Cont.

(23–29). Soluble Klotho suppresses FGF23 production in Klotho is also expressed in the choroid plexus of the physiologic concentrations (Figure 2), but it can increase brain (1). Klotho-deficient mice display central nervous FGF23 expression in the bone (28) or act as a nonenzymatic system lesions including hypomyelination, synaptic loss, scaffold protein that enhances FGF23 signaling on the basis and behavioral impairments such as dementia and cog- of in vitro data in very high, nonphysiologic concentrations nitive deficits (38). Klotho deficiency in the brain impairs (12). Soluble Klotho is not filtered at the glomerulus, but it blood barrier and promotes immune-mediated central translocates across the kidney tubules, from the basolateral nervous system disorders (39). CKD is associated with to the luminal side, and is excreted in the urine (17). Klotho depression and cognitive impairment (40). Low Klotho in expression is downregulated during acute kidney dis- cerebrospinal fluid was confirmed in Alzheimer disease ease and CKD. (41). Low soluble Klotho in CKD may conceivably contrib- ute to central nervous system dysfunction, although there is no evidence to date of Klotho deficiency in cerebrospinal Klotho and Mineral Metabolism fluid or brain in CKD. Klotho decreases kidney phosphate reabsorption by acting as a coreceptor for FGF23 binding to FGFR1 (30) (Figure 1). Soluble Klotho also directly promotes the Cardiovascular Disease internalization and degradation of the NaPi2a cotrans- A novel risk factor of cardiovascular disease in CKD is fi porter in the kidney proximal tubules contributing to soluble Klotho de ciency (42,43). Hyperphosphatemia phosphate excretion (31). Klotho may also be a suppressor and low soluble Klotho associate with more severe cardiac fi of vitamin D and FGF23 production (32) because Klotho- hypertrophy and brosis. Interestingly, elevated levels of deficient mice exhibit higher CYP27B1 gene (33) and FGF23 FGF23 were associated with pathologic cardiac remodeling fi production (1) (Figure 2). only if Klotho de ciency was also present (34). However, exogenous FGF23, independent of Klotho, induced car- diac hypertrophy through FGFR4-mediated PLC-g sig- naling activation in cardiac myocytes (43,44). Klotho, Klotho in Disease States independent of FGF23, was postulated to protect the CKD heart against cardiac hypertrophy through inhibition Klotho expression in the kidney and soluble Klotho in the of transient receptor potential channel-6 activity in car- blood and urine are decreased in animals and humans with diomyocytes (45). Klotho supplementation also protects CKD from a variety of etiologies, including glomerular and against indoxyl sulfate–mediated cardiac hypertrophy tubulointerstitial diseases (32). Because the kidneys are in mice (46). the primary source of soluble Klotho, circulating soluble Klotho expectedly declines as CKD progresses (Figure 3). Decreased Klotho expression in CKD is not simply due AKI to loss of viable tissue but can be attributable to hyper- AKI is a syndrome with a myriad of inflammatory phosphatemia (34) and hypermethylation or deacetylation such as TNF-a and TNF-like weak inducer of of the Klotho gene promoter by inflammatory cytokines or apoptosis, which downregulate Klotho expression in the uremic toxins, such as indoxyl sulfate (35,36). Similarities kidney through NF-kB activation in murine AKI (47). of clinical features, including short lifespan, cardiac remodel- Similarly, in other inflammatory conditions such as inflam- ing, vascular calcification,bonedisease,musclewasting,and matory bowel disease, TNF-a and IFN-g reduced Klotho hyperphosphatemia, exist in the context of Klotho deficiency expression in murine kidney (48). The reduced kidney Klotho and in CKD (37). is partly mediated by an increase in inducible CJASN 16: ccc–ccc, January, 2021 Klotho in Clinical Nephrology, Neyra et al. 3

Figure 2. | Physiologic and pathophysiologic role of Klotho in mineral metabolism with preserved and decreased kidney function. The dashed line indicates putative action on the basis of experimental or clinical data; no evidence supports direct effect yet. PTH, parathyroid hormone. synthase, nitric oxide production, and , but Potential Mechanisms of Klotho-Mediated Kidney and the precise mechanisms of how inflammation downregulates Cardiovascular Protection Klotho in AKI are unknown (48,49). A differential transcrip- Preclinical data strongly suggest a pathogenic role of tional splicing resulting in decay of Klotho mRNA has been Klotho acting through multiple mechanisms in contribut- postulated as a potential mechanism of Klotho downregu- ing to AKI development, AKI-to-CKD transition, progres- lation in settings of acute illness (22). sion of CKD, and development of extrarenal complications Current experimental data support that low Klotho is of CKD (Figure 4, Table 1). not just a biomarker but pathogenic. Klotho-deficient mice have lower kidney and circulatory soluble Klotho, Klotho Renders the Kidney More Resistant to Injury and they have more severe kidney damage and fibrosis and higher risk of AKI-to-CKD transition compared with Inhibition of Cell Senescence. Cell senescence is a complex process in normal aging (52) and in kidney wild-type mice after exposure to distinct kidney insults fi (14,16,23,50,51), indicating that Klotho deficiency ren- disease (53). Klotho de ciency induces and Klotho over- ders the kidney more susceptible to acute insults. expression suppresses cell senescence through increased Wnt signaling activity (54,55). Intracellular Klotho also suppresses cell senescence by inhibiting expression of IL-6 and -8 (56,57). Inhibition of Cell Apoptosis. Klotho deficiency increases apoptosis in cultured cells (58,59) and in the kidney (23,49,50,60). Increasing Klotho decreases apoptotic cell number and improves kidney function and morphology after acute and chronic kidney damage (50,60,61). Antioxidation. The kl/kl mice (low Klotho expression) have higher levels and Tg-Kl mice (Klotho overexpressing) have lower levels of oxidative markers and oxidation- induced cell damage than normal mice (62,63). Klotho exerts its antioxidation effect through stimulating the FOXO family, manganese SOD (55,62), and phosphati- dylinositol 3-kinase/AKT/Nrf2/heme oxygenase- 1 pathways (64). Induction of Autophagy. Autophagy is a conserved process to degrade defective and unwanted cytoplasmic and organelle components and reuse the constituents (65). Either extremely high or low autophagy activity is asso- ciated with kidney damage (16,66). Klotho upregulates Figure 3. | Changes in circulating soluble Klotho according to eGFR autophagy activity, protects kidney against ischemic in- decline and its relationship to other mineral metabolism parameters. jury, and, consequently, mitigates the progression of AKI 1,25(OH)2D3, 1,25-dihydroxyvitamin D. to CKD (16). 4 CJASN

Figure 4. | Role of Klotho in kidney and cardiovascular protection. AKI occurs after exposure to kidney insults. If the insult is strong or long enough, kidney recovery is impaired, and AKI progresses to CKD. With CKD progression, kidney Klotho is decreased followed by increase in circulating FGF23 levels, low 1,25-dihydroxyvitamin D3, high blood phosphate, and increase in PTH. Abnormal mineral hormones individually and synergistically exacerbate each other in the manner of a vortex of downhill spiral (dashed line: putative action) and contribute to end organ complications, including CKD-MBD, uremic cardiomyopathy, and vascular calcification. Klotho replacement provides beneficial effects from protection of kidney against acute injury, promotion of kidney regeneration, retardation of CKD progression, and amelioration of extrarenal complications. Klotho also improves mineral metabolism disturbances and directly or indirectly lessens end organ dysfunction or abnormalities of advanced CKD. CKD-MBD, CKD-mineral and bone disorder; Ca, calcium; 1,25-D3, 1,25-dihydroxyvitamin D; Pi, phosphate.

Klotho Promotes Kidney Recovery injury (69). Klotho exerts proangiogenic actions (70) and When recombinant Klotho protein is injected right after maintains endothelial integrity (71). ischemic injury, mice had similar acute kidney damage but recovered much faster than vehicle injection (16). In- travenous administration of urinary extracellular vesicles Klotho Suppresses Kidney Fibrosis fi carrying Klotho restores kidney Klotho levels in injured Klotho suppresses kidney brosis in several animal b kidney and promotes kidney function recovery after glyc- models (14,72,73) via suppression of TGF- 1 activity, erol injection (67). TGF-receptor II (14,73), and Wnt signaling activity (72). Supplementation of Klotho in rodents inhibits kidney Preservation of Stem Cells. Klotho deficiency is associ- fibrosis and retards CKD progression. ated with depletion (15,68) via augmentation of Wnt signaling and more cell senescence, which were rescued by Klotho protein overexpression (54). Klotho Ameliorates Disordered Mineral Metabolism in CKD CKDisastateofKlothodeficiency, hyperphosphate- Maintenance of Endothelial Function and Angiogene- mia, high FGF23 levels, and low 1,25-dihydroxyvitamin fi sis. Swift and complete recovery of endothelium structure D3 (Figure 3). Klotho de ciency participates in CKD de- and function promotes kidney regeneration after ischemic velopment and progression and contributes to extrarenal

Table 1. Potential mechanisms of beneficial effects of Klotho in kidney disease

AKI AKI to CKD CKD

Protect kidney Promote recovery Inhibit fibrosis Antiapoptosis Preserve stem cells ↓ TGF-b1 signal ↓ Cell senescence ↑ ↓ Wnt signal ↑ Autophagy ↑ Vasculogenesis Mineral metabolism Antioxidation Inhibit fibrosis ↓ Blood phosphate ↓ TGF-b1 signaling Modulate FGF23 activity ↓ Wnt signaling Control 1,25-(OH)2D3 production Control PTH production Control BP

fi FGF23, broblast growth factor 23; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D; PTH, parathyroid hormone. CJASN 16: ccc–ccc, January, 2021 Klotho in Clinical Nephrology, Neyra et al. 5

Table 2. Human studies of soluble Klotho in CKD

Study Clinical Setting Methods Results/Observations

Pavik et al. (106) 87 adults with CKD (stages Serum IBL ELISA Adjustedmean Klotho decreasewas3.2pg/ml 1–5) and 21 controls for each 1-ml/min decrease in eGFR Age and eGFR were independently associated with Klotho Patients with PKD and kidney transplant recipients were excluded Wan et al. (107) 154 children with CKD (stages Plasma IBL ELISA Klotho levels decreased with decreasing eGFR 1–5, 28 on KRT, 44 but no independent association between post-transplant) Klotho and eGFR was found Decreased Klotho was associated with increased FGF23 and PTH levels Kitagawa et al. (97) 114 adults with CKD Serum IBL ELISA Klotho was a significant determinant of arterial stiffness determined by ankle- brachial pulse wave velocity (adjusted OR per 100-pg/ml increase, 0.60; 95% CI, 0.39 to 0.98; P50.008) Positive correlation between Klotho levels and eGFR Patients with established atherosclerotic complications (CAD, CHF, PVOD) or those treated with vitamin D or phosphate binders were excluded Kim et al. (96) 243 adults with CKD Serum IBL ELISA Klotho levels independently predicted the (stages 1–5) composite outcome of doubling SCr, ESKD, or death at median follow-up of 30 mo: adjusted HR per 10-pg/ml increase, 0.96; 95% CI, 0.94 to 0.98; P,0.001 If serum Klotho was #396.3 pg/ml, 35.2% reached the composite outcome versus 15.7% if .396.3 pg/ml (P50.03) Klotho levels were lower at more advanced CKD stages (P value for trend ,0.001) and correlated positively with eGFR and negatively with FGF23 and phosphate levels Klotho was independently associated with eGFR (P,0.001) Exclusion criteria consisted of KRT, organ transplantation, heart failure, cirrhosis, malignancy, pregnancy, acute coronary syndrome or ischemic stroke within 3 mo prior to thestudy, progressiveCKD within 3 mo prior to the study Akimoto et al. (99) 131 adults with CKD Serum and urine IBL ELISA Positive correlation between serum and urine (stages 1–5) Klotho and eGFR 24-h urine Klotho levels were independently associated with eGFR Patients on long-term KRT were excluded Seiler et al. (108) 312 adults with CKD Serum IBL ELISA Klotho levels were significantly associated (stages 2–4) with age but not with eGFR Klotho levels were not associated with the composite outcome of death or KRT initiation at mean follow-up of 2.2 yr Seiler et al. (109) 444 adults with CKD Plasma IBL ELISA Klotho levels (highest versus lowesttertile)did (stages 2–4) not predict atherosclerotic events/death (HR, 0.75; 95% CI, 0.43 to 1.30; P50.30) or ADHF/death (HR, 0.81;95%CI,0.39 to 1.66; P50.56) in adults with CKD at median follow-up of 2.6 yr Events were adjudicated by two independent nephrologists Ozeki et al. (98) 185 adults with CKD Serum IBL ELISA eGFR was an independent predictor of Klotho (stages 1–5) levels, specifically when eGFR was ,60 ml/min Klotho levels were not significantly correlated with serum calcium or phosphate Patients on long-term KRT were excluded 6 CJASN

Table 2. (Continued)

Study Clinical Setting Methods Results/Observations

Cano et al. (110) 31 children on PD and Serum ELISA Baseline Klotho levels were lower in children 45 healthy controls on PD versus controls (132658 versus 3206119 pg/ml; P,0.001) and remained virtually unchanged throughout the observation period of 1 yr Klotho levels did not correlate with FGF23 and phosphate levels Park et al. (111) 24 adults with HTN and CKD Serum IBL ELISA Klotho levels were significantly reduced in (12 with RVH and 12 EH) patients with HTN (mean eGFR 574.7 and and 12 HV controls 48.8 ml/min in EH and RVH, respectively) versus controls (mean eGFR 573 ml/min) after adjustment by eGFR Klotho levels directly correlated with eGFR Exclusion criteria consisted of eGFR,30 ml/ min, uncontrolled BP, diabetes, recent cardiovascular event (within 6 mo), pregnancy, kidney transplant Sawires et al. (112) 40 children with CKD (stages Serum ELISA Serum calcium was an independent predictor 2–5), 44 patients with ESKD of Klotho levels in all groups of on HD, 40 kidney kidney disease transplant recipients, and Serum calcium inversely correlated with 40 HV controls Klotho levels Exclusion criteria consisted of marked hypocalcemia, dysfunctional HD access, combined organ transplantation, surgical parathyroidectomy, and sarcoidosis Oh et al. (100) 78 adults on PD and 30 Serum IBL ELISA Klotho levels were significantly lower in HV controls adults on PD versus HV controls: 329.6 versus 717.8; P,0.001 8-Isoprostane and IL-6 levels were inversely correlated with Klotho levels 8-Isoprostane levels were independently associated with Klotho levels (P50.04) Shroff et al. (113) ESCAPE cohort post hoc Serum IBL ELISA ACEI therapy significantly increased Klotho analysis, 167 children levels without any associated changes in with CKD on ACEI serum calcium or phosphate Klotho levels did not correlate with eGFR at baseline (r50.02; P50.82) or at 8-mo follow- up (r50.06; P50.45) Drew et al. (101) 2496 adults within the Serum IBL ELISA Klotho levels independently associated with Health ABC study 30% kidney function decline, with each (mean eGFR 573 ml/min) doubling of Klotho associated with a 20% decreased odds of significant decline in kidney function over 10 yr This association persisted after adjustment for demographics, cardiovascular disease risk factors, and mineral metabolism measures, including FGF23 Memmos et al. (114) 79 adults on HD Plasma IBL ELISA Lower Klotho levels (#745 versus .745 pg/ ml) independently associated with the composite of death/nonfatal MI or stroke at median follow-up of 5.5 yr (HR, 2.76; 95% CI, 1.22 to 6.22; P50.01) after adjustment for cardiovascular disease risk factors and mineral disease parameters

IBL, Immuno-Biologic Laboratories Co., Ltd.; PKD, polycystic kidney disease; FGF23, fibroblast growth factor 23; PTH, parathyroid hormone; OR, odds ratio; 95% CI, 95% confidence interval; CAD, coronary artery disease; CHF, congestive heart failure; PVOD, peripheral vascular occlusive disease; SCr, serum creatinine; HR, hazard ratio; ADHF, acutely decompensated heart failure; PD, peritoneal dialysis; HTN, hypertension; RVH, renovascular hypertension; EH, essential hypertension; HV, healthy volunteer; HD, hemodialysis; ESCAPE, Effect of Strict BP Control andACE Inhibition on theProgression of CRF in Pediatric Patients; ACEI, angiotensin- converting inhibitor; ABC, Aging and Body Composition; MI, myocardial infarction. complications of CKD directly and indirectly through dis- protein or enhancement of native Klotho is an effective turbed mineral metabolism (Figures 2 and 4) (29,34,74–76). strategy for the correction of hyperphosphatemia in exper- Reduction of Serum Phosphate. Klotho deficiency im- imental CKD (75). pairs phosphaturia (31,77) and, consequently, accelerates Control of Fibroblast Growth Factor 23 Activity. FGF23 phosphate accumulation in CKD. Administration of Klotho participates in systemic phosphate balance by its triple JS 16: CJASN ccc – Table 3. Human studies of soluble Klotho in AKI ccc aur,2021 January, , Study Clinical Setting AKI Definition Methods Results/Observations

Hu et al. (50) 17 adults with AKI (inpatient consults) and 14 HV ↑ SCr$50% or Urine immunoblot Mean urine Klotho/Cr levels were significantly lower in AKI $2 mg/dl versus HV (4.8561.69 versus 25.3864.08 fmol/mg of Cr; P50.01) Liu et al. (120) 35 adults undergoing cardiac surgery (19 AKI) AKIN $ stage 1 Serum ELISA Serum Klotho levels were lower in patients with AKI versus (SCr only) patients without AKI immediately after CS: mean 6 SD, 102617 versus 124621 U/L; P50.05. Klotho levels increased as early as day 1 post-CS such that levels were no longer different in patients with AKI versus patients without AKI Torregrosa 60 adults undergoing cardiac surgery or coronary ↑ SCr$50% Urine IBL and Urine samples were collected 12 h post-CS or post-CA (single time et al. (121) angiography (30 AKI) SSBT ELISA point). Urine Klotho/Cr was not different in patients with AKI versus no AKI (mean 6 SD, 2.4560.26 versus 2.0460.20 ng/mg of Cr; P5.0.05 by SSBT and 1.6060.30 versus 1.2460.30; P.0.05 by IBL). No correlation between the two Klotho assays was found Kim et al. (122) 61 adults in the ICU or floor (42 prerenal and 19 AKIN $ stage 1 Serum and urine Patients with prerenal and intrinsic AKI had similar SCr levels. intrinsic AKI) IBL ELISA Urine Klotho/Cr levels were lower in prerenal versus intrinsic AKI (mean 6 SD, 1746292 versus 3816630 ng/g of Cr; P50.001). There was no difference between groups in serum Klotho levels Castellano 30 adults undergoing kidney transplant (15 DGF Serum IBL ELISA Patients with versus without DGF had lower levels of serum et al. (118) developed DGF) Klotho at 2 yr post-transplantation Seibert 46 hospitalized adults (30 AKI; 16 controls) AKIN $ stage 1 Serum IBL ELISA Serum Klotho levels were higher in AKI (assessed at the time of et al. (123) nephrology consultation) versus control patients (mean 6 SD, 567.66294.4 versus 403.56152.5 pg/ml; P50.01). AKI and controls were not matched Neyra 106 adults in the ICU (54 AKI; 52 controls) KDIGO $ stage 2 Urine immunoblot Urine Klotho/Cr levels assessed at a single time point (at time of et al. (116) AKI diagnosis [patients] or within 24 h of ICU admission [controls]) were significantly lower in AKI versus controls: ltoi lnclNprlg,Nyae l 7 al. et Neyra Nephrology, Clinical in Klotho median (IQR), 9.2 (3.0–33.6) versus 25.0 (6.0–92.8) fmol/mg of Cr; P,0.001. Each one-fold higher urine Klotho/Cr level was associated with an 83% (95% CI, 60% to 93%) lower risk of major adverse kidney events at 90 d (composite of death, KRT dependence, or decrease in eGFR$50% from baseline). ICU controls were matched to patients by age, sex, and baseline eGFR

HV, healthy volunteer; SCr, serum creatinine; Cr, creatinine; AKIN, Acute Kidney Injury Network; CS, cardiac surgery; IBL, Immuno-Biologic Laboratories Co., Ltd.; SSBT, Shangai Sunred Biologic Technology Co., Ltd.; CA, coronary angiography; ICU, intensive care unit; DGF, delayed graft function (defined as need for KRT within 7 days post-transplantation); KDIGO, Kidney Disease Improving Global Outcomes; IQR, interquartile range; 95% CI, 95% confidence interval. 8 CJASN

action on intestine, bone, and kidneys through interplay an early marker of CKD and CKD-MBD (Figure 3, Table 2) with Klotho (8), parathyroid hormone (PTH), and 1,25- (96,102–104). A recent meta-analysis concluded that there is – fi dihydroxyvitamin D3 (8,12,31,77 79) (Figures 2 and 4). a signi cant positive correlation between soluble Klotho High serum FGF23 may serve as a diagnostic biomarker and eGFR in patients with CKD (105). and pathogenic intermediate for higher risk of cardiovascular In a cohort study of adult patients with CKD (stages 1–5), disease and mortality in both patients with AKI (80) and serum Klotho levels independently associated with the patients with CKD (81). The fact that high FGF23 correlates composite outcome of doubling serum creatinine, kidney with cardiac adverse effect only with concomitant low Klotho failure, or death, after adjustment for age, diabetes, mean in animals suggests that Klotho may modulate the toxic arterial pressure, eGFR, proteinuria, and PTH (96). Note effects of FGF23 (34). that high-risk patients such as those with acute coronary Modulation of 1,25-Dihydroxyvitamin D3 Production. Low syndrome, ischemic stroke, or progressive CKD within 1,25-dihydroxyvitamin D3 is attributed to bone minerali- 3 months prior to the study enrollment were excluded. zation defects and secondary hyperparathyroidism in CKD (82). Vitamin D treatment attenuates cardiac FGF23/ FGFR4 signaling and hypertrophy (83) and vascular cal- Klotho and Cardiovascular Disease in Human CKD cification in uremic rats (84) in part through upregulation of Serum Klotho is associated with arterial stiffness mea- circulating Klotho. sured by pulse wave velocity in patients with early CKD Control of Parathyroid Hormone Production. Secon- (97). In contrast, a different study in patients with CKD dary hyperparathyroidism is associated with cardiovascu- stages 2–4 showed that plasma Klotho levels were not lar morbidity and mortality in CKD (85,86). In CKD, associated with , acute heart failure, or death parathyroid gland loses its response to inhibitory signaling at 2.6 years (109). Serum Klotho levels were lower in from FGF23 possibly due to lower Klotho and FGFR1 in the patients with primary hypertension compared with healthy parathyroid gland (Figure 2) (87–89). Correction of Klotho volunteers, adjusted for eGFR (111). A post hoc analysis of to improve mineral homeostasis does not only benefitbone the Effect of Strict BP Control and ACE Inhibition on the and mineral metabolism but also may attenuate cardio- Progression of CRF in Pediatric Patients trial in children vascular disease and improve the quality of life of patients with CKD showed that ramipril therapy significantly with CKD (75). increased serum Klotho levels without any changes in serum calcium or phosphate (113). In another study in- Klotho Reduces Blood Pressure cluding adults with acute cerebral infarction, plasma Hypertension is a complication of CKD and contributes Klotho levels were negatively associated with the presence, to progression of CKD and cardiovascular disease. Klotho- burden, and progression of small vessel disease (115). A deficient mice develop salt-sensitive hypertension after recent study showed that low plasma Klotho is associated high sodium challenge (90) and increased arterial stiffness with mortality and cardiovascular events in patients on (91). Klotho single-nucleotide polymorphism (KL hemodialysis (114). The association between Klotho levels rs9536314) associates with salt-sensitive hypertension in and cardiovascular disease has been observed only in small adults with newly diagnosed hypertension (92). studies in humans mostly using surrogate markers of cardiovascular health rather than hard outcomes, such as cardiovascular events or death. Larger prospective studies Biomarker Candidacy in Human Kidney Disease with well defined outcomes are needed to examine if Klotho in Human CKD Klotho is a prognostic marker of cardiovascular dis- Kidney Klotho mRNA was significantly lower in patients ease in CKD. with CKD versus healthy controls and positively correlated with eGFR (93,94). In CKD stages 1–5D with available kidney biopsies, Klotho mRNA levels positively correlated Klotho in AKI with eGFR, after adjustment for age and other mineral In critically ill patients, urinary Klotho levels were lower parameters (95). To date, many studies have shown a in patients with AKI (Kidney Disease Improving Global positive correlation between Klotho levels (serum and Outcomes stage $2; within 48 hours of diagnosis) com- urine) and eGFR in adults with CKD (74,96–99). Soluble pared with well matched intensive care unit controls (by Klotho levels correlate with markers of oxidative stress age, sex, and baseline eGFR; within 48 hours of intensive (8-isoprostane) and inflammation (IL-6), although some care unit admission) (116). In a study of patients with acute of these associations dissipated after multivariable anal- tubular necrosis or acute tubulointerstitial nephritis by yses (100). In a recent larger study, serum Klotho levels kidney biopsy, lower kidney Klotho protein levels associ- associated with kidney function decline in a cohort of well ated with higher peak serum creatinine and more frequent functioning older adults aged 70–79 with mean eGFR of KRT (117). In the kidney transplant population, donor 73 ml/min per 1.73 m2 (101). With each doubling of serum kidney Klotho protein levels decreased following trans- Klotho, there was an associated 20% decreased odds of plantation in those with delayed graft function (118). In significant decline in kidney function over 10 years, which postmortem kidney biopsies of critically ill patients who persisted after adjustment for demographics, cardiovascu- died of sepsis and AKI, there was lower kidney Klotho lar risk factors, and mineral metabolism parameters, in- mRNA and protein in sepsis-associated AKI biopsies cluding FGF23 (101). The decline of soluble Klotho levels in when compared with biopsies from control subjects adults with mild CKD (e.g., stages #2) antedates elevation in undergoing nephrectomy for kidney cancer (119). Not FGF23, PTH, and phosphate, and therefore, it may represent all human studies in AKI have shown an inverse CJASN 16: ccc–ccc, January, 2021 Klotho in Clinical Nephrology, Neyra et al. 9

Table 4. Considerations for soluble Klotho measurements in human samples

Sample Processing Klotho Assay

Sample type and quality Methods of measurement (ELISA versus other) Sample vintage (from sample collection to processing) Reagents Sample vintage (from sample storage to measurement) Specificity of antibodies Methods of collection Additives (e.g., inhibitors) Methods of storage Interassay variability Freeze-thaw cycles Intra-assay variability High-throughput capacity relationship between Klotho measurements and kidney agreement between three different commercial Klotho function (Table 3). ELISAs, suggesting that problems exist beyond just one In a cohort of 35 adults undergoing cardiac surgery, there ELISA. Measurement of soluble Klotho using commercial was no difference in preoperative serum Klotho levels in ELISA was highly unstable in human urine even when those with (n519) versus without (n516) postoperative stored at–80°C (129). An alternative method of Klotho AKI. However, postoperative serum Klotho levels obtained assay using immunoprecipitation-immunoblot uses a syn- immediately and 4 hours following cardiac surgery were thetic anti-Klotho antibody (high affinity for Kl2 domain of both lower in patients with versus without AKI (120). Klotho) for pull down and an anti-Kl1 rat mAb (now Klotho levels increased soon in patients with AKI such that commercially available KM2076; TransGenic Inc., Kobe, they were no longer different between groups by post- Japan) (10) for detection by immunoblot (102). This assay operative day 1 (120). In another study of 60 adults showed progressive decline of serum Klotho levels with undergoing cardiac surgery or coronary angiography increasing stages of CKD in a small single-center study (50% developed postprocedure AKI), urinary Klotho levels using fresh samples (102). were not different in patients with versus without AKI A recent study evaluated two methods of measuring when measured 12 hours after the procedure (121). Neyra serum Klotho in patients with different strata of kidney disease et al. (116) found significantly lower levels of urinary (130). The authors concluded that immunoprecipitation- Klotho in critically ill patients who developed major immunoblot, compared with ELISA, exhibited a stronger adverse kidney events (death, dependence on KRT, or direct correlation with eGFR, better recovery (capture) of decrease in eGFR$50% from baseline) within 90 days of added exogenous Klotho, less susceptibility to variability enrollment than in those who did not. Each one-fold higher from sample additives (protease inhibitors), and much urinary Klotho level was associated with an adjusted 83% better differentiation across different kidney disease lower risk of developing the outcome (Table 3). groups, including AKI and CKD, in reference to healthy Collectively, current clinical data show great promise for volunteers. The immunoprecipitation-immunoblot assay soluble Klotho levels as not merely a diagnostic marker but performance also declined after multiple freeze-thaw as part of novel risk prediction tools of kidney-related cycles, favoring the use of never-thawed samples when outcomes in high-risk patients with CKD or AKI. measuring soluble Klotho. Development of high-throughput Klotho assays and standardized processing and storage conditions will greatly Measurement of Soluble Klotho in Humans benefit the field because suboptimal assays are populating Human studies of measuring circulating soluble Klotho the human database, continuously rendering the unification in serum, plasma, or urine have shown that acute or of data interpretation very difficult. chronic kidney dysfunction is accompanied by lower soluble Klotho (102,103,116), but some studies have re- ported no change or even higher soluble Klotho depending Klotho-Focused Therapies on level of kidney function (16,97,99,106,109,123–125). Counteracting the decrease in Klotho that occurs in These discordant findings may largely be due to problems kidney disease represents a promising strategy to prevent, with performance and reproducibility of current commer- ameliorate, and reverse kidney disease and its extrarenal cially available assays (126–128). An unfortunate fact is that complications (Figure 4). variance in sample type, quality, collection and processing One successful strategy in the laboratory is the use of methods, vintage and conditions of storage, and number of viral delivery of Klotho cDNA in rodent AKI (60). In freeze-thaw cycles can all drastically affect assay perfor- murine models, Klotho cDNA plasmids can be directly mance and yield widely disparate results (Table 4); how- injected or delivered via a viral carrier (nonpathogenic ever, precautions were not usually made to account for adeno-associated virus) to rescue many phenotypes of these confounders. Klotho deficiency (131–134), although its safety in humans Barker et al. (102) showed that a commercial ELISA is not established. Experimental data revealed attenuation yielded different results in fresh versus stored serum of kidney disease in hypertensive rats (131,135), improve- samples and experienced a drop in capture of exogenous ment of kidney function in AKI (60), amelioration of soluble Klotho in patients with advanced CKD as well as angiotensin II–induced kidney injury (132), improvement when samples have undergone freeze-thaw cycles. Heij- of endothelial function (134), and protection from uremic boer et al. (126) showed poor interassay and intra-assay cardiomyopathy in CKD models (45). Another method is by 10 CJASN

Figure 5. | Klotho-centric therapies in kidney disease, including increase in endogenous production or administration of exogenous Klotho protein or cDNA. miRNA, microRNA; PPAR-r, peroxisome proliferator-activated receptor gamma. the minicircle DNA vectors, which allows sustained in vivo and statins (Figure 5) (140–145). Thus far, the efficacy of Klotho gene expression without risk of immunogenicity and these agents has only been demonstrated in preclinical confers kidney protection in models of ischemia reperfusion studies, except for the use of valsartan, which was shown to injury and unilateral ureteral obstruction (136). increase plasma Klotho levels in patients with diabetic Another approach is to increase endogenous Klotho nephropathy following 24 weeks of therapy (143). Off-label production by overcoming the mechanisms that suppress use of already approved drugs to pharmacologically ma- Klotho expression and/or release in kidney disease. In a nipulate endogenous Klotho remains a viable option. It is CKD model, the vitamin D receptor agonist, paricalcitol, important to recognize that pharmacologic interventions increased serum Klotho and reduced serum phosphate and have not been tested in clinical trials of kidney disease as the FGF23. The source of increased soluble Klotho levels was primary outcome. A major hurdle is that the mechanism of not determined to be from the kidneys (84). Experimental how Klotho expression is upregulated needs to be defined. upregulation of Klotho expression by 1,25-dihydroxyvita- Whether these drugs can stimulate endogenous Klotho min D3 was also described in other studies (137,138). One production/release in CKD where the Klotho-generating recent study showed that sevelamer carbonate increased cells are extensively destroyed is unknown. serum Klotho in CKD (139). Other agents that can poten- Another strategy is to target epigenetic modulation of tially increase endogenous Klotho expression are angio- endogenous Klotho expression. Although theoretically tensin II receptor antagonists, PPAR-g agonists, androgen, feasible, modulation of methylation and acetylation of

Figure 6. | Potential diagnostic, prognostic, and therapeutic applications of Klotho in clinical nephrology. Dx, diagnosis. CJASN 16: ccc–ccc, January, 2021 Klotho in Clinical Nephrology, Neyra et al. 11

the Klotho gene promoter needs further confirmation for its studies have provided proof of concept that systemic application in the management of human kidney disease. Klotho protein administration is effective with seemingly Alternatively, administration of exogenous recombi- good safety profiles. Because Klotho deficiency, regardless nant Klotho protein has dual effects: restoring soluble of etiology, could cause or accelerate dysfunction or de- Klotho levels and promoting endogenous production generation in multiple systems or organs, Klotho is a of kidney Klotho. This dual action is particularly im- valuable treatment modality of not just kidney disease but portant in kidney failure, a status in which endoge- also its complications, in particular cardiovascular disease nous capacity is amputated. In a murine model, soluble (Figure 4). The evolution of precision medicine leveraging Klotho injected 30 or 60 minutes after ischemic injury clinical informatics along with genomic, proteomic, and attenuated increase in serum creatinine postinjury and metabolomic data will help the identification of high-risk improved kidney histology (50). Further, repeated ad- patients to be targeted in Klotho-based therapeutic trials. ministration of Klotho starting 1 day after ischemic The ongoing development of Klotho therapies and the injury led to reduced fibrosisandimprovedrecovery evolution of more accurate and precise Klotho assays make from AKI (16). Klotho replacement therapy remains the these clinical trials foreseeable and no longer utopic in the only practical method to control precise levels. Although recombinant Klotho replacement has been successful near future. Clearly, bench and bedside interactive collab- in many animal models, its application in humans still orative research is critically needed to develop and validate faces many hurdles. novel Klotho assays, examine its biomarker potential in diagnosis and prognosis, and identify effective therapeutic windows for Klotho administration in acute kidney disease Summary and Call for Action and CKD. Pragmatic therapeutic trials in selected populations Preclinical, translational, and clinical studies have that are most likely to benefit from Klotho administration or advanced our understanding of Klotho biology, pathobi- modulation should be designed (Figure 6). ology, and potential clinical applications. Despite this accomplishment, the translation to clinical application Disclosures has been slow. Klotho is an evolutionarily conserved All authors have nothing to disclose. housekeeping protein with a myriad of functions on many Funding organs, which can serve as a biomarker for early diagnosis M.C. Hu and O.W. Moe are supported by National Institutes of and/or risk stratification of patients with acute kidney Health grants R01-DK091392 and R01-DK092461, the Charles and disease or CKD. Because of its universal presence and Jane Pak Center Innovative Research Support, and Endowed Pro- panoply of function, specificity remains an issue (e.g., fessor Collaborative Research Support. O.W. Moe is supported by circulating Klotho levels may change in many diseases George O’Brien Kidney Research Center grant P30-DK-07938. J.A. with or without kidney involvement). Neyra is a recipient of National Center for Advancing Translational Despite the emergence of promising human biomarker Sciences Early Career Pilot Grant UL1TR001998. data of Klotho, additional large-scale and multicenter validation is needed. One difficulty to surpass before one can generate large reliable human databases is the stan- References dardization of methodology for soluble Klotho measure- 1. Kuro-o M, MatsumuraY,Aizawa H, Kawaguchi H, Suga T,Utsugi ments. Given the imperfection of assays, one should T,Ohyama Y,Kurabayashi M, Kaname T,Kume E, Iwasaki H, Iida exercise caution in interpretation of multiple small studies A, Shiraki-Iida T,Nishikawa S, Nagai R, Nabeshima YI: Mutation of the mouseklotho geneleadsto a syndrome resemblingageing. where kidney disease occurs under diverse clinical condi- Nature 390: 45–51, 1997 tions and conclusions are made on the basis of variable 2. Ellidag HY,Yilmaz N, Kurtulus F, Aydin O, Eren E, Inci A, Dolu S, sample conditions using different commercial reagents, Ince FDA, Giray O¨ , Yaman A: The three sisters of fate in multiple most of which lack vigorous validation. Further, other sclerosis: Klotho (Clotho), fibroblast growth factor-23 (Lachesis), and vitamin D (Atropos). Ann Neurosci 23: 155–161, 2016 factors known to influence soluble Klotho measurements, fl 3. Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M, such as age, sex, in ammation, high-phosphate diet, FGF23, Nabeshima Y: Identification of the human klotho gene and its vitamin D supplementation, and certain medications, need two transcripts encoding membrane and secreted klotho pro- to be carefully addressed in future prospective studies tein. Biochem Biophys Res Commun 242: 626–630, 1998 testing the biomarker candidacy of Klotho. Finally, there is 4. Ohyama Y, Kurabayashi M, Masuda H, Nakamura T, Aihara Y, Kaname T, Suga T, Arai M, Aizawa H, Matsumura Y, Kuro-o M, a critical need to distinguish and characterize the subtypes Nabeshima Y, Nagail R: Molecular cloning of rat klotho cDNA: of circulating Klotho protein because current assays do not Markedly decreased expression of klotho byacute inflammatory differentiate between full-length soluble Klotho formed stress. Biochem Biophys Res Commun 251: 920–925, 1998 from cleavage of the transmembrane form (18–20), sub- 5. Ito S, Kinoshita S, Shiraishi N, Nakagawa S, Sekine S, Fujimori T, sequent cleaved Kl1 and Kl2 Klotho fragments, and Klotho Nabeshima YI: Molecular cloning and expression analyses of mouse betaklotho, whichencodes a novelKlotho family protein. complexes with other circulating proteins. Therefore, Mech Dev 98: 115–119, 2000 with lack of knowledge as to which form of Klotho is 6. Ito S, Fujimori T, Hayashizaki Y,Nabeshima Y: Identification of a most biologically relevant and/or captured with current novel mouse membrane-bound family 1 glycosidase-like pro- assays, associations drawn between measured soluble tein, which carries an atypical structure. Biochim Klotho levels and clinical outcomes should be circum- Biophys Acta 1576: 341–345, 2002 7. Lee S, Choi J, Mohanty J, Sousa LP, Tome F, Pardon E, Steyaert J, spectly interpreted. Lemmon MA, Lax I, Schlessinger J: Structures of b-klotho reveal a To date, no studies of Klotho protein administration in ‘zip code’-like mechanism for endocrine FGF signalling. Nature humans have been reported. However, ample animal 553: 501–505, 2018 12 CJASN

8. Hu MC, Shiizaki K, Kuro-o M, Moe OW: Fibroblast growth factor 27. Wang Y,Chen L, Huang G, He D, He J, Xu W, Zou C, Zong F, Li Y, 23 and Klotho: Physiology and pathophysiology of an endocrine Chen B, Wu S, Zhao W, Wu J: Klotho sensitizes human lung network of mineral metabolism. Annu Rev Physiol 75: 503–533, cancer cell line to cisplatin via PI3k/Akt pathway. PLoS One 8: 2013 e57391, 2013 9. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, 28. Smith RC, O’Bryan LM, Farrow EG, Summers LJ, Clinkenbeard Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o EL, Roberts JL, Cass TA, Saha J, Broderick C, Ma YL, Zeng QQ, M: Regulation of fibroblast growth factor-23 signaling by klotho. Kharitonenkov A, Wilson JM, Guo Q, Sun H, Allen MR, Burr DB, JBiolChem281: 6120–6123, 2006 Breyer MD, White KE: Circulating aKlotho influences phosphate 10. Kato Y, Arakawa E, Kinoshita S, Shirai A, Furuya A, Yamano K, handling by controlling FGF23 production. J Clin Invest 122: Nakamura K, Iida A, Anazawa H, Koh N, Iwano A, Imura A, 4710–4715, 2012 Fujimori T, Kuro-o M, Hanai N, Takeshige K, Nabeshima Y: 29. Hu MC, Shi M, Gillings N, Flores B, Takahashi M, Kuro-O M, Establishment of the anti-Klotho monoclonal antibodies and Moe OW: Recombinant a-Klotho may be prophylactic and detection of Klotho protein in kidneys. Biochem Biophys Res therapeutic for acute to chronic kidney disease progression and Commun 267: 597–602, 2000 uremic cardiomyopathy. Kidney Int 91: 1104–1114, 2017 11. Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, 30. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Fujimori T, Nabeshima Y: Secreted Klotho protein in sera and Okawa K, Fujita T, Fukumoto S, Yamashita T: Klotho converts CSF: Implication for post-translational cleavage in release of canonical FGF receptor into a specific receptor for FGF23. Klotho protein from cell membrane. FEBS Lett 565: 143–147, Nature 444: 770–774, 2006 2004 31. Hu MC, Shi M, Zhang J, Pastor J, Nakatani T, Lanske B, Razzaque 12. Chen G, Liu Y, Goetz R, Fu L, Jayaraman S, Hu MC, Moe OW, MS, Rosenblatt KP, Baum MG, Kuro-o M, Moe OW: Klotho: A Liang G, Li X, Mohammadi M: a-Klotho is a non-enzymatic novel phosphaturic substance acting as an autocrine enzyme in molecular scaffold for FGF23 hormone signalling. Nature 553: the renal proximal tubule. FASEB J 24: 3438–3450, 2010 461–466, 2018 32. Neyra JA, Hu MC: aKlotho and chronic kidney disease. Vitam 13. Sugiura H, Yoshida T, Shiohira S, Kohei J, Mitobe M, Kurosu H, Horm 101: 257–310, 2016 Kuro-o M, Nitta K, Tsuchiya K: Reduced Klotho expression level 33. Ohnishi M, Nakatani T, Lanske B, Razzaque MS: Reversal of in kidney aggravates renal interstitial fibrosis. Am J Physiol Renal mineral ion homeostasis and soft-tissue calcification of klotho Physiol 302: F1252–F1264, 2012 knockout mice by deletion of vitamin D 1alpha-hydroxylase. 14. Doi S, Zou Y, Togao O, Pastor JV, John GB, Wang L, Shiizaki K, Kidney Int 75: 1166–1172, 2009 Gotschall R, Schiavi S, Yorioka N, Takahashi M, Boothman DA, 34. Hu MC, Shi M, Cho HJ, Adams-Huet B, Paek J, Hill K, Shelton J, Kuro-o M: Klotho inhibits transforming growth factor-beta1 Amaral AP,Faul C, Taniguchi M, Wolf M, Brand M, Takahashi M, (TGF-beta1) signaling and suppresses renal fibrosis and cancer Kuro-O M, Hill JA, Moe OW: Klotho and phosphate are mod- metastasis in mice. JBiolChem286: 8655–8665, 2011 ulators of pathologic uremic cardiac remodeling. JAmSoc 15. Bian A, Neyra JA, Zhan M, Hu MC: Klotho, stem cells, and aging. Nephrol 26: 1290–1302, 2015 Clin Interv Aging 10: 1233–1243, 2015 35. Young GH, Wu VC: KLOTHO methylation is linked to uremic 16. Shi M, Flores B, Gillings N, Bian A, Cho HJ, Yan S, Liu Y,Levine B, toxinsand chronickidney disease.KidneyInt 81: 611–612, 2012 Moe OW, Hu MC: aKlotho mitigates progression of AKI to CKD 36. Sun CY,Chang SC, Wu MS: Suppression of Klotho expression by through activation of autophagy. J Am Soc Nephrol 27: protein-bound uremic toxins is associated with increased DNA 2331–2345, 2016 methyltransferase expression and DNA hypermethylation. 17. Hu MC, Shi M, Zhang J, Addo T, Cho HJ, Barker SL, Ravikumar P, Kidney Int 81: 640–650, 2012 Gillings N, Bian A, Sidhu SS, Kuro-o M, Moe OW: Renal pro- 37. Kooman JP, Kotanko P, Schols AM, Shiels PG, Stenvinkel P: duction, uptake, and handling of circulating aKlotho. JAmSoc Chronic kidney disease and premature . Nat Rev Nephrol Nephrol 27: 79–90, 2016 10: 732–742, 2014 18. Bloch L, Sineshchekova O, Reichenbach D, Reiss K, Saftig P, 38. Chen CD, Sloane JA, Li H, Aytan N, Giannaris EL, Zeldich E, Kuro-o M, Kaether C: Klotho is a substrate for alpha-, beta- and Hinman JD, Dedeoglu A, Rosene DL, Bansal R, Luebke JI, Kuro-o gamma-secretase. FEBS Lett 583: 3221–3224, 2009 19. Chen CD, Podvin S, Gillespie E, Leeman SE, Abraham CR: M, Abraham CR: The antiaging protein Klotho enhances oli- stimulates the cleavage and release of the extracellular domain godendrocyte maturation and myelination of the CNS. of Klotho by ADAM10 and ADAM17. Proc Natl Acad Sci U S A J Neurosci 33: 1927–1939, 2013 104: 19796–19801, 2007 39. Zhu L, Stein LR, Kim D, Ho K, YuGQ, Zhan L, Larsson TE, Mucke 20. Chen CD, Tung TY, Liang J, Zeldich E, Tucker Zhou TB, Turk BE, L: Klotho controls the brain-immune system interface in the Abraham CR: Identification of cleavage sites leading to the shed choroid plexus. Proc Natl Acad Sci U S A 115: E11388–E11396, form of the anti-aging protein klotho. Biochemistry 53: 2018 5579–5587, 2014 40. Tanaka S, Okusa MD: Crosstalk between the nervous system and 21. Shiraki-Iida T, Aizawa H, Matsumura Y, Sekine S, Iida A, the kidney. Kidney Int 97: 466–476, 2020 Anazawa H, Nagai R, Kuro-o M, Nabeshima Y: Structure of the 41. Semba RD, Moghekar AR, Hu J, Sun K, Turner R, Ferrucci L, mouse klotho gene and its two transcripts encoding membrane O’Brien R: Klotho in the cerebrospinal fluid of adults with and and secreted protein. FEBS Lett 424: 6–10, 1998 without Alzheimer’s disease. Neurosci Lett 558: 37–40, 2014 22. Mencke R, Harms G, Moser J, van Meurs M, Diepstra A, 42. Faul C, Amaral AP, Oskouei B, Hu MC, Sloan A, Isakova T, Leuvenink HG,Hillebrands JL: HumanalternativeKlothomRNA Gutie´rrez OM, Aguillon-Prada R, Lincoln J, Hare JM, Mundel P, is a nonsense-mediated mRNA decay target inefficiently spliced Morales A, Scialla J, Fischer M, Soliman EZ, Chen J, Go AS, Rosas in renal disease. JCI Insight 2: e94375, 2017 SE, Nessel L, Townsend RR, Feldman HI, St John Sutton M, Ojo A, 23. Panesso MC, Shi M, Cho HJ, Paek J, YeJ, Moe OW,Hu MC: Klotho Gadegbeku C, Di Marco GS, Reuter S, Kentrup D, Tiemann K, has dual protective effects on cisplatin-induced acute kidney Brand M, Hill JA, Moe OW, Kuro-O M, Kusek JW, Keane MG, injury. Kidney Int 85: 855–870, 2014 Wolf M: FGF23 induces left ventricular hypertrophy. J Clin Invest 24. Cheng MF, Chen LJ, Niu HS, Yang TT, Lin KC, Cheng JT: Signals 121: 4393–4408, 2011 mediating Klotho-induced neuroprotection in hippocampal 43. Grabner A, Amaral AP, Schramm K, Singh S, Sloan A, Yanucil C, neuronal cells. Acta Neurobiol Exp (Warsz) 75: 60–71, 2015 Li J, Shehadeh LA, Hare JM, David V, Martin A, Fornoni A, Di 25. Sun S, Cheng B, Sun PG, Wu XH, Wu QQ, He P: RTEF-1 protects Marco GS, Kentrup D, Reuter S, Mayer AB, Pavensta¨dt H, against oxidative damage induced by H2O2 in human umbilical Stypmann J, Kuhn C, Hille S, Frey N, Leifheit-Nestler M, Richter vein endothelial cells through Klotho activation. Exp Biol Med B, Haffner D, Abraham R, Bange J, Sperl B, Ullrich A, Brand M, (Maywood) 240: 1606–1613, 2015 Wolf M, Faul C: Activation of cardiac fibroblast growth factor 26. Ravikumar P,YeJ, Zhang J, Pinch SN, Hu MC, Kuro-o M, Hsia CC, receptor 4 causes left ventricular hypertrophy. Cell Metab 22: Moe OW: a-Klotho protects against oxidative damage in pul- 1020–1032, 2015 monary epithelia. Am J Physiol Lung Cell Mol Physiol 307: 44. Han X,CaiC, Xiao Z,QuarlesLD: FGF23 inducedleftventricular L566–L575, 2014 hypertrophy mediated by FGFR4 signaling in the myocardium is CJASN 16: ccc–ccc, January, 2021 Klotho in Clinical Nephrology, Neyra et al. 13

attenuated by soluble Klotho in mice. J Mol Cell Cardiol 138: effect of Klotho on endothelial cells through cAMP activation. 66–74, 2020 Endocrine 31: 82–87, 2007 45. Xie J, Yoon J, An SW, Kuro-o M, Huang CL: Soluble klotho 64. Cui W, Leng B, Wang G: Klotho protein inhibits H2O2-induced protects against uremic cardiomyopathy independently of fi- oxidative injury in endothelial cells via regulation of PI3K/AKT/ broblast growth factor 23 and phosphate. J Am Soc Nephrol 26: Nrf2/HO-1 pathways. Can J Physiol Pharmacol 97: 370–376, 1150–1160, 2015 2019 46. Yang K, Wang C, Nie L, Zhao X, Gu J, Guan X, Wang S, Xiao T,Xu 65. Feng Y, He D, Yao Z, Klionsky DJ: The machinery of macro- X, He T, Xia X, Wang J, Zhao J: Klotho protects against indoxyl autophagy. Cell Res 24: 24–41, 2014 sulphate-induced myocardial hypertrophy. J Am Soc Nephrol 66. Li P,Shi M, Maique J, Shaffer J, YanS, Moe OW, Hu MC: Beclin 1/ 26: 2434–2446, 2015 Bcl-2 complex-dependent autophagy activity modulates renal 47. Moreno JA, Izquierdo MC, Sanchez-Nino~ MD, Sua´rez-Alvarez susceptibility to ischemia-reperfusion injury and mediates re- B, Lopez-Larrea C, Jakubowski A, Blanco J, Ramirez R, Selgas R, noprotection by Klotho. Am J Physiol Renal Physiol 318: Ruiz-Ortega M, Egido J, Ortiz A, Sanz AB: The inflammatory F772–F792, 2020 cytokines TWEAK and TNFa reduce renal klotho expression 67. Grange C, Papadimitriou E, Dimuccio V, Pastorino C, Molina J, through NFkB. J Am Soc Nephrol 22: 1315–1325, 2011 O’Kelly R, Niedernhofer LJ, Robbins PD, Camussi G, Bussolati B: 48. Thurston RD, Larmonier CB, Majewski PM, Ramalingam R, Urinary extracellular vesicles carrying Klotho improve the re- Midura-Kiela M, Laubitz D, Vandewalle A, Besselsen DG, covery of renal function in an Acute Tubular Injury model. Mol Muhlbauer M, Jobin C, Kiela PR, Ghishan FK: Tumor necrosis Ther 28: 490–502, 2019 factor and interferon-gamma down-regulate Klotho in mice with 68. Sato S, Kawamata Y, Takahashi A, Imai Y, Hanyu A, Okuma A, colitis. Gastroenterology 138: 1384–1394, 2010 Takasugi M, Yamakoshi K, Sorimachi H, Kanda H, Ishikawa Y, 49. Mitobe M, Yoshida T, Sugiura H, Shirota S, Tsuchiya K, Nihei H: Sone S,Nishioka Y,OhtaniN, Hara E:Ablation of the p16(INK4a) Oxidative stress decreases klotho expression in a mouse kidney tumour suppressor reverses ageing phenotypes of klotho mice. cell line. Nephron, Exp Nephrol 101: e67–e74, 2005 Nat Commun 6: 7035, 2015 50. Hu MC, Shi M, ZhangJ, Quinones~ H, Kuro-o M, Moe OW:Klotho 69. Polichnowski AJ: Microvascular rarefaction and hypertension in deficiency is an early biomarker of renal ischemia-reperfusion the impaired recovery and progression of kidney disease fol- injury and its replacement is protective. Kidney Int 78: lowing AKI in preexisting CKD states. Am J Physiol Renal Physiol 1240–1251, 2010 315: F1513–F1518, 2018 51. Jorge LB, Coelho FO,Sanches TR, Malheiros DMAC, Ezaquiel de 70. Mazzotta C, Manetti M, Rosa I, Romano E, Blagojevic J, SouzaL,DosSantos F,deSa´ LimaL,ScavoneC, IrigoyenM,Kuro- Bellando-Randone S, Bruni C, Lepri G, Guiducci S, Ibba- O M, Andrade L: Klotho deficiency aggravates sepsis-related Manneschi L, Matucci-Cerinic M: Proangiogenic effects of multiple organ dysfunction. Am J Physiol Renal Physiol 316: soluble a-Klotho on systemic sclerosis dermal microvascular F438–F448, 2019 endothelial cells. Arthritis Res Ther 19: 27, 2017 52. Ferrucci L, Gonzalez-Freire M, Fabbri E, Simonsick E, Tanaka T, 71. Kusaba T,Okigaki M, Matui A, Murakami M, Ishikawa K, Kimura Moore Z, Salimi S, Sierra F, de Cabo R: Measuring biological T,Sonomura K,Adachi Y,ShibuyaM,Shirayama T,TandaS, Hatta aging in humans: A quest. Aging Cell 19: e13080, 2020 T,Sasaki S, Mori Y,Matsubara H: Klotho is associated with VEGF 53. Knoppert SN, Valentijn FA, Nguyen TQ, Goldschmeding R, receptor-2 and the transient receptor potential canonical-1 Falke LL: Cellular senescence and the kidney: Potential thera- Ca21 channel to maintain endothelial integrity. Proc Natl Acad peutic targets and tools. Front Pharmacol 10: 770, 2019 Sci U S A 107: 19308–19313, 2010 54. Liu H, Fergusson MM, Castilho RM, Liu J, Cao L, Chen J, Malide 72. Zhou L, Li Y, Zhou D, Tan RJ, Liu Y: Loss of Klotho contributes to D, Rovira II, Schimel D, Kuo CJ, Gutkind JS, Hwang PM, Finkel T: kidney injury by derepression of Wnt/b-catenin signaling. JAm Augmented Wnt signaling in a mammalian model of accelerated Soc Nephrol 24: 771–785, 2013 aging. Science 317: 803–806, 2007 73. Li S, Yu L, He A, Liu Q: Klotho inhibits unilateral ureteral 55. Kuro-o M: Klotho as a regulator of oxidative stress and senes- obstruction-induced endothelial-to-mesenchymal transition via cence. Biol Chem 389: 233–241, 2008 TGF-b1/Smad2/Snail1 signaling in mice. Front Pharmacol 10: 56. Liu F, Wu S, Ren H, Gu J: Klotho suppresses RIG-I-mediated 348, 2019 senescence-associated inflammation. Nat Cell Biol 13: 74. Hu MC, Shi M, Zhang J, Quinones~ H, Griffith C, Kuro-o M, Moe 254–262, 2011 OW: Klotho deficiency causes vascular calcification in chronic 57. Zeng Y, Wang PH, Zhang M, Du JR: Aging-related renal injury kidney disease. J Am Soc Nephrol 22: 124–136, 2011 and inflammation are associated with downregulation of Klotho 75. Neyra JA, Hu MC: Potential application of klotho in human and induction of RIG-I/NF-kB signaling pathway in senescence- chronic kidney disease. Bone 100: 41–49, 2017 accelerated mice. Aging Clin Exp Res 28: 69–76, 2016 76. Lu X, Hu MC: Klotho/FGF23 axis in chronic kidney disease and 58. Ikushima M, Rakugi H, Ishikawa K, Maekawa Y, Yamamoto K, cardiovascular disease. Kidney Dis 3: 15–23, 2017 Ohta J, Chihara Y, Kida I, Ogihara T: Anti-apoptotic and anti- 77. Hu MC, Shi M, Moe OW: Role of aKlotho and FGF23 in regu- senescence effects of Klotho on vascular endothelial cells. Bi- lation of type II Na-dependent phosphate co-transporters. ochem Biophys Res Commun 339: 827–832, 2006 Pflugers Arch 471: 99–108, 2019 59. Maekawa Y, Ohishi M, Ikushima M, Yamamoto K, Yasuda O, 78. Goetz R, Nakada Y, Hu MC, Kurosu H, Wang L, Nakatani T, Shi Oguro R, Yamamoto-Hanasaki H, Tatara Y, Takeya Y, Rakugi H: M, Eliseenkova AV, Razzaque MS, Moe OW, Kuro-o M, Klothoproteindiminishesendothelialapoptosis andsenescence Mohammadi M: Isolated C-terminal tail of FGF23 alleviates via a mitogen-activated kinase pathway. Geriatr Gerontol Int 11: hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex 510–516, 2011 formation. Proc Natl Acad Sci U S A 107: 407–412, 2010 60. Sugiura H, Yoshida T, Tsuchiya K, Mitobe M, Nishimura S, 79. Ide N, YeR, Courbebaisse M, Olauson H, Densmore MJ, Larsson Shirota S, Akiba T, Nihei H: Klotho reduces apoptosis in ex- TE,HanaiJI, Lanske B:Invivoevidence foraninterplayof FGF23/ perimental ischaemic acute renal failure. Nephrol Dial Trans- Klotho/PTH axis on the phosphate handling in renal proximal plant 20: 2636–2645, 2005 tubules. Am J Physiol Renal Physiol 315: F1261–F1270, 2018 61. Haruna Y,Kashihara N, Satoh M, Tomita N, Namikoshi T, Sasaki 80. Leaf DE, Siew ED, Eisenga MF, Singh K, Mc Causland FR, T,Fujimori T,Xie P,Kanwar YS: Amelioration of progressive renal Srivastava A, Ikizler TA, Ware LB, Ginde AA, Kellum JA, Palevsky injury by genetic manipulation of Klotho gene. Proc Natl Acad PM, Wolf M, Waikar SS: Fibroblast growth factor 23 associates Sci U S A 104: 2331–2336, 2007 with death in critically ill patients. Clin J Am Soc Nephrol 13: 62. Yamamoto M, Clark JD, Pastor JV,Gurnani P,Nandi A, Kurosu H, 531–541, 2018 Miyoshi M, Ogawa Y, Castrillon DH, Rosenblatt KP, Kuro-o M: 81. Rodelo-Haad C, Santamaria R, Munoz-Casta~ neda~ JR, Regulation of oxidative stress by the anti-aging hormone klotho. Pendo´n-Ruiz de Mier MV,Martin-Malo A, Rodriguez M: FGF23, J Biol Chem 280: 38029–38034, 2005 biomarker or target? Toxins (Basel) 11: 175, 2019 63. Rakugi H, Matsukawa N, Ishikawa K, Yang J, Imai M, Ikushima 82. Gutie´rrez OM: Fibroblast growth factor 23 and disordered vi- M, Maekawa Y, Kida I, Miyazaki J, Ogihara T: Anti-oxidative tamin D metabolism in chronic kidney disease: Updating the 14 CJASN

“trade-off” hypothesis. Clin J Am Soc Nephrol 5: 1710–1716, with glomerular filtration rate and proteinuria among cardiac 2010 patients. BMC Nephrol 15: 147, 2014 83. Leifheit-Nestler M, Grabner A, Hermann L, Richter B, Schmitz K, 99. Akimoto T, Yoshizawa H, Watanabe Y, Numata A, Yamazaki T, Fischer DC, Yanucil C, Faul C, Haffner D: Vitamin D treatment Takeshima E, Iwazu K, Komada T, Otani N, Morishita Y, Ito C, attenuates cardiac FGF23/FGFR4 signaling and hypertrophy in Shiizaki K, Ando Y, Muto S, Kuro-o M, Kusano E: Characteristics uremic rats. Nephrol Dial Transplant 32: 1493–1503, 2017 of urinary and serum soluble Klotho protein in patients with 84. Lau WL, Leaf EM, Hu MC, Takeno MM, Kuro-o M, Moe OW, different degrees of chronic kidney disease. BMC Nephrol 13: Giachelli CM: Vitamin D receptor agonists increase klotho and 155, 2012 osteopontin while decreasing aortic calcification in mice with 100. Oh HJ, Nam BY,Lee MJ, Kim CH, Koo HM, Doh FM, Han JH, Kim chronic kidney disease fed a high phosphate diet. Kidney Int 82: EJ, Han JS, Park JT, Yoo TH, Kang SW, Han DS, Han SH: De- 1261–1270, 2012 creased circulating klotho levels in patients undergoing dialysis 85. Carrillo-Lo´pez N, Panizo S, Alonso-Montes C, Martı´nez-Arias L, and relationship to oxidative stress and inflammation. Perit Dial Avello N, Sosa P, Dusso AS, Cannata-Andı´a JB, Naves-Dı´az M: Int 35: 43–51, 2015 High-serum phosphate and parathyroid hormone distinctly 101. Drew DA, Katz R, Kritchevsky S, Ix J, Shlipak M, Gutie´rrez OM, regulate bone loss and vascular calcification in experimental Newman A, Hoofnagle A, Fried L, Semba RD, Sarnak M: As- chronic kidney disease. Nephrol Dial Transplant 34: 934–941, sociation between soluble klotho and change in kidney func- 2019 tion: The health aging and body composition study. JAmSoc 86. Isakova T, Cai X, Lee J, Mehta R, Zhang X, Yang W, Nessel L, Nephrol 28: 1859–1866, 2017 Anderson AH, Lo J, Porter A, Nunes JW, Negrea L, Hamm L, 102. Barker SL, Pastor J, Carranza D, Quinones~ H, Griffith C, Goetz R, Horwitz E, Chen J, Scialla JJ, de Boer IH, Leonard MB, Feldman MohammadiM, YeJ, ZhangJ,HuMC, Kuro-oM, Moe OW,Sidhu HI, Wolf M; CRIC Study Investigators: Longitudinal evolution of SS: The demonstration of aKlotho deficiency in human chronic markers of mineral metabolism in patients with CKD: The kidney disease with a novel synthetic antibody. Nephrol Dial chronic renal insufficiency cohort (CRIC) study. Am J Kidney Dis Transplant 30: 223–233, 2015 75: 235–244, 2020 103. Shimamura Y,Hamada K, Inoue K, Ogata K, Ishihara M, Kagawa 87. Galitzer H, Ben-Dov IZ, Silver J, Naveh-Many T: Parathyroid cell T,Inoue M, Fujimoto S, Ikebe M, Yuasa K, Yamanaka S, Sugiura T, resistance to fibroblast growth factor 23 in secondary hyper- Terada Y: Serum levels of soluble secreted a-Klotho are de- parathyroidism of chronic kidney disease. Kidney Int 77: creased in the early stages of chronic kidney disease, making it a 211–218, 2010 probable novel biomarker for early diagnosis. Clin Exp Nephrol 88. Kuro-O M: Klotho and endocrine fibroblast growth factors: 16: 722–729, 2012 Markers of chronic kidney disease progression and cardiovas- 104. Rotondi S, Pasquali M, Tartaglione L, Muci ML, Mandanici G, cular complications? Nephrol Dial Transplant 34: 15–21, 2019 Leonangeli C, Sales S, Farcomeni A, Mazzaferro S: Soluble a 89. Patel S, Barron JL, Mirzazedeh M, Gallagher H, Hyer S, Cantor T, -klotho serum levels in chronic kidney disease. Int J Endocrinol Fraser WD: Changes in bone mineral parameters, vitamin D 2015: 872193, 2015 metabolites, and PTH measurements with varying chronic 105. Wang Q, Su W, Shen Z, Wang R: Correlation between soluble kidney disease stages. J Bone Miner Metab 29: 71–79, 2011 a-Klotho and renal function in patients with chronic kidney 90. Zhou X, Chen K, Lei H, Sun Z: Klotho gene deficiencycauses salt- disease: A review and meta-analysis. BioMed Res Int 2018: sensitive hypertension via monocyte chemotactic protein-1/CC 9481475, 2018 chemokine receptor 2-mediated inflammation. JAmSoc 106. Pavik I, Jaeger P, Ebner L, Wagner CA, Petzold K, Spichtig D, Nephrol 26: 121–132, 2015 Poster D, Wu¨thrich RP, Russmann S, Serra AL: Secreted klotho 91. Gao D, Zuo Z, Tian J, Ali Q, Lin Y, Lei H, Sun Z: Activation of and FGF23 in chronic kidney disease stage 1 to 5: A sequence SIRT1 attenuates klotho deficiency-induced arterial stiffness and suggested from a cross-sectional study. Nephrol Dial Transplant hypertension by enhancing AMP-activated protein kinase ac- 28: 352–359, 2013 tivity. Hypertension 68: 1191–1199, 2016 107. Wan M, Smith C, Shah V, Gullet A, Wells D, Rees L, Shroff R: 92. Citterio L, Delli Carpini S, Lupoli S, Brioni E, Simonini M, Fibroblast growth factor 23 and soluble klotho in children with Fontana S, Zagato L, Messaggio E, Barlassina C, Cusi D, Manunta chronic kidney disease. Nephrol Dial Transplant 28: 153–161, P, Lanzani C: Klotho gene in human salt-sensitive hypertension. 2013 Clin J Am Soc Nephrol 15: 375–383, 2020 108. Seiler S, Wen M, Roth HJ, Fehrenz M, Flu¨gge F, Herath E, 93. Asai O, Nakatani K, Tanaka T, Sakan H, Imura A, Yoshimoto S, Weihrauch A, Fliser D, Heine GH: Plasma Klotho is not related to Samejima K, Yamaguchi Y, Matsui M, Akai Y, Konishi N, Iwano kidney function and does not predict adverse outcome in pa- M, Nabeshima Y, Saito Y: Decreased renal a-Klotho expression tientswith chronickidneydisease.KidneyInt83:121–128,2013 in early diabetic nephropathy in humans and mice and its 109. Seiler S, Rogacev KS, Roth HJ, Shafein P, Emrich I, Neuhaus S, possible role in urinary calcium excretion. Kidney Int 81: Floege J, Fliser D,HeineGH:Associationsof FGF-23and sKlotho 539–547, 2012 with cardiovascular outcomes among patients with CKD stages 94. Koh N, Fujimori T,Nishiguchi S, Tamori A, Shiomi S, Nakatani T, 2-4. Clin J Am Soc Nephrol 9: 1049–1058, 2014 Sugimura K, Kishimoto T, Kinoshita S, Kuroki T, Nabeshima Y: 110. Cano FJ, Freundlich M, Ceballos ML, Rojo AP, Azocar MA, Severely reduced production of klotho in human chronic renal Delgado IO, Ibacache MJ, Delucchi MA, Lillo AM, Irarra´zabal failure kidney. Biochem Biophys Res Commun 280: 1015–1020, CE, Ugarte MF: Longitudinal FGF23 and Klotho axis charac- 2001 terization in children treated with chronic peritoneal dialysis. 95. Sakan H, Nakatani K, Asai O, Imura A, Tanaka T, Yoshimoto S, Clin Kidney J 7: 457–463, 2014 Iwamoto N, Kurumatani N, Iwano M, Nabeshima Y, Konishi N, 111. Park MY, Herrmann SM, Saad A, Eirin A, Tang H, Lerman A, Saito Y: Reduced renal a-Klotho expression in CKD patients and Textor SC, Lerman LO: Biomarkers of kidney injury and klotho in its effect on renal phosphate handling and vitamin D metabo- patients with atherosclerotic renovascular disease. Clin J Am Soc lism. PLoS One 9: e86301, 2014 Nephrol 10: 443–451, 2015 96. Kim HR, Nam BY,Kim DW,Kang MW, Han JH, Lee MJ, Shin DH, 112. Sawires HK, Essam RM, Morgan MF, Mahmoud RA: Serum Doh FM, Koo HM, Ko KI, Kim CH, Oh HJ, YooTH, Kang SW, Han klotho: Relation to fibroblast growth factor-23 and other regu- DS, Han SH: Circulating a-klotho levels in CKD and relationship lators of phosphate metabolism in children with chronic kidney to progression. Am J Kidney Dis 61: 899–909, 2013 disease. Nephron 129: 293–299, 2015 97. Kitagawa M, Sugiyama H, Morinaga H, Inoue T, Takiue K, 113. Shroff R, Aitkenhead H, Costa N, Trivelli A, Litwin M, Picca S, Ogawa A, Yamanari T, Kikumoto Y, Uchida HA, Kitamura S, Anarat A, Sallay P, Ozaltin F, Zurowska A, Jankauskiene A, Maeshima Y,Nakamura K, Ito H, Makino H: A decreased level of Montini G, Charbit M, Schaefer F, Wu¨hl E; ESCAPE Trial Group: serum soluble Klotho is an independent biomarker associated Normal 25-hydroxyvitamin D levels are associated with less with arterial stiffness in patients with chronic kidney disease. proteinuria and attenuate renal failure progression in children PLoS One 8: e56695, 2013 with CKD. J Am Soc Nephrol 27: 314–322, 2016 98. Ozeki M, Fujita S, Kizawa S, Morita H, Sohmiya K, Hoshiga M, 114. Memmos E, Sarafidis P, Pateinakis P, Tsiantoulas A, Faitatzidou Ishizaka N: Association of serum levels of FGF23 and a-Klotho D, Giamalis P, Vasilikos V, Papagianni A: Soluble Klotho is CJASN 16: ccc–ccc, January, 2021 Klotho in Clinical Nephrology, Neyra et al. 15

associated with mortality and cardiovascular events in hemo- 130. Neyra JA, Moe OW, Pastor J, Gianella F, Sidhu SS, Sarnak MJ, Ix dialysis. BMC Nephrol 20: 217, 2019 JH, Drew DA: Performance of soluble Klotho assays in clinical 115. Woo HG, Chang Y, Ryu DR, Song TJ: Plasma Klotho concen- samples of kidney disease. Clin Kidney J 13: 235–244, 2019 tration is associated with the presence, burden and progression 131. Wang Y, Sun Z: Klotho gene delivery prevents the progression of of cerebral small vessel disease in patients with acute ischaemic spontaneous hypertension and renal damage. Hypertension 54: stroke. PLoS One 14: e0220796, 2019 810–817, 2009 116. NeyraJA, LiX, Mescia F,Ortiz-SorianoV,Adams-HuetB, Pastor J, 132. Mitani H, Ishizaka N, Aizawa T, Ohno M, Usui S, Suzuki T, HuM-C, Toto RD, Moe OW: Urine klotho is lower in critically ill Amaki T, Mori I, Nakamura Y, Sato M, Nangaku M, Hirata Y, patients with versus without acute kidney injury and associates Nagai R: In vivo klotho gene transfer ameliorates angiotensin II- with major adverse kidney events. Crit Care Explor 1: e0016, induced renal damage. Hypertension 39: 838–843, 2002 2019 133. Shiraki-Iida T, Iida A, Nabeshima Y, Anazawa H, Nishikawa S, 117. Seo MY, Yang J, Lee JY, Kim K, Kim SC, Chang H, Won NH, Kim Noda M, Kuro-o M, Nabeshima Y: Improvement of multiple MG, Jo SK, Cho W, Kim HK: Renal Klotho expression in patients pathophysiological phenotypes of klotho (kl/kl) mice by with acute kidney injury is associated with the severity of the adenovirus-mediated expression of the klotho gene. J Gene Med injury. Korean J Intern Med (Korean Assoc Intern Med) 30: 2: 233–242, 2000 489–495, 2015 134. Saito Y, Nakamura T, Ohyama Y, Suzuki T, Iida A, Shiraki-Iida T, 118. Castellano G, Intini A, Stasi A, Divella C, Gigante M, Pontrelli P, Kuro-o M, Nabeshima Y,Kurabayashi M, Nagai R: In vivo klotho Franzin R, Accetturo M, Zito A, Fiorentino M, Montinaro V, gene delivery protects against in mul- Lucarelli G, Ditonno P, Battaglia M, Crovace A, Staffieri F, tiple risk factor syndrome. Biochem Biophys Res Commun 276: Oortwijn B, van Amersfoort E, Pertosa G, Grandaliano G, 767–772, 2000 Gesualdo L: Complement modulation of anti-aging factor klotho 135. Wang Y, Sun Z: Antiaging gene Klotho regulates endothelin-1 in ischemia/reperfusion injury and delayed graft function. Am levels and endothelin receptor subtype B expression in kidneys J Transplant 16: 325–333, 2016 of spontaneously hypertensive rats. J Hypertens 32: 1629–1636, 119. Jou-Valencia D, Molema G, Popa E, Aslan A, van Dijk F, Mencke 2014 R, Hillebrands JL, Heeringa P, Hoenderop JG, Zijlstra JG, van 136. Shin YJ, Luo K, Quan Y, Ko EJ, Chung BH, Lim SW, Yang CW: Meurs M, Moser J: Renal klotho is reduced in septic patients and Therapeutic challenge of minicircle vector encoding klotho in animal model. Am J Nephrol 49: 413–424, 2019 pretreatment with recombinant klotho attenuates organ injury in ~ ~ lipopolysaccharide-challenged mice. Crit Care Med 46: 137. Munoz-Castaneda JR, Herencia C, Pendo´n-Ruiz de Mier MV, e1196–e1203, 2018 Rodriguez-Ortiz ME, Diaz-Tocados JM, Vergara N, Martı´nez- 120. Liu YJ, Sun HD, Chen J, Chen MY,Ouyang B, Guan XD: Klotho: A Moreno JM, Salmero´n MD, Richards WG, Felsenfeld A, Kuro-O M, Almade´n Y, Rodrı´guez M: Differential regulation of renal novel and early biomarker of acute kidney injury after cardiac Klotho and FGFR1 in normal and uremic rats. FASEB J 31: valve replacement surgery in adults. Int J Clin Exp Med 8: 3858–3867, 2017 7351–7358, 2015 138. Takenaka T, Inoue T, Ohno Y, Miyazaki T, Nishiyama A, Ishii N, 121. Torregrosa I, Montoliu C, Urios A, Gime´nez-Garzo´ C, Toma´sP, Suzuki H: Calcitriol supplementation improves endothelium- Solı´s MA, Ramos C, Juan I, Puchades MJ, Saez G, Blasco ML, dependent in rat hypertensive renal injury. Kidney Miguel A: Urinary Klotho measured by ELISA as an early bio- Blood Press Res 39: 17–27, 2014 marker of acute kidney injury in patients after cardiac surgery or 139. Liabeuf S, Ryckelynck JP, El Esper N, Urena~ P, Combe C, Dussol coronary angiography. Nefrologia 35: 172–178, 2015 B, Fouque D, Vanhille P,Frimat L, Thervet E, Mentaverri R, Prie´ D, 122. Kim AJ, Ro H, Kim H, Chang JH, Lee HH, Chung W, Jung JY: Choukroun G; FRENCH Study collaborators: Randomized Klotho and S100a8/A9 as discriminative markers between pre- clinical trial of sevelamer carbonate on serum klotho and fi- renal and intrinsic acute kidney injury. PLoS One 11: e0147255, broblast growth factor 23 in CKD. Clin J Am Soc Nephrol 12: 2016 1930–1940, 2017 123. Seibert E, Radler D, Ulrich C, Hanika S, Fiedler R, Girndt M: 140. Zhang H, Li Y, Fan Y, Wu J, Zhao B, Guan Y, Chien S, Wang N: Serum klotho levels in acute kidney injury. Clin Nephrol 87: Klotho is a target gene of PPAR-gamma. Kidney Int 74: 732–739, 173–179, 2017 2008 124. Devaraj S, Syed B, Chien A, Jialal I: Validation of an immuno- 141. Yang HC, Deleuze S, Zuo Y, Potthoff SA, Ma LJ, Fogo AB: The assay for soluble klotho protein: Decreased levels in diabetes PPARgamma agonist pioglitazone ameliorates aging-related and increased levels in chronic kidney disease. Am J Clin Pathol progressive renal injury. J Am Soc Nephrol 20: 2380–2388, 137: 479–485, 2012 2009 125. Kim Y, Kim JH, Nam YJ, Kong M, Kim YJ, Yu KH, Lee BC, Lee C: 142. Narumiya H, Sasaki S, Kuwahara N, Irie H, Kusaba T,Kameyama Klotho is a genetic risk factor for ischemic stroke caused by H, Tamagaki K, Hatta T, Takeda K, Matsubara H: HMG-CoA cardioembolism in Korean females. Neurosci Lett 407: reductase inhibitors up-regulate anti-aging klotho mRNA via 189–194, 2006 RhoA inactivation in IMCD3 cells. Cardiovasc Res 64: 331–336, 126. Heijboer AC, Blankenstein MA, Hoenderop J, de Borst MH, 2004 Vervloet MG; NIGRAM consortium: Laboratory aspects of cir- 143. Karalliedde J, Maltese G, Hill B, Viberti G, Gnudi L: Effect of culating a-Klotho. Nephrol Dial Transplant 28: 2283–2287, renin-angiotensin system blockade on soluble Klotho in patients 2013 with type 2 diabetes, systolic hypertension, and albuminuria. 127. Pedersen L, Pedersen SM, Brasen CL, Rasmussen LM: Soluble Clin J Am Soc Nephrol 8: 1899–1905, 2013 serum Klotho levels in healthy subjects. Comparison of two 144. Yoon HE, Ghee JY, Piao S, Song JH, Han DH, Kim S, Ohashi N, different immunoassays. Clin Biochem 46: 1079–1083, 2013 Kobori H, Kuro-o M, Yang CW: Angiotensin II blockade upre- 128. Yamazaki Y,Imura A, UrakawaI, Shimada T,Murakami J, Aono Y, gulates the expression of Klotho, the anti-ageing gene, in an Hasegawa H, Yamashita T, Nakatani K, Saito Y, Okamoto N, experimental model of chronic cyclosporine nephropathy. Kurumatani N, NambaN, Kitaoka T,Ozono K, Sakai T,Hataya H, Nephrol Dial Transplant 26: 800–813, 2011 Ichikawa S, Imel EA, Econs MJ, Nabeshima Y: Establishment of 145. Hsu SC, Huang SM, Lin SH, Ka SM, Chen A, Shih MF, Hsu YJ: sandwich ELISA for soluble alpha-Klotho measurement: Age- Testosterone increases renal anti-aging klotho gene expression dependent change of soluble alpha-Klotho levels in healthy via the androgen receptor-mediated pathway. Biochem J 464: subjects. Biochem Biophys Res Commun 398: 513–518, 2010 221–229, 2014 129. Adema AY, Vervloet MG, Blankenstein MA, Heijboer AC: a-Klotho is unstable in human urine. Kidney Int 88: 1442–1444, Published online ahead of print. Publication date available at 2015 www.cjasn.org.