Klotho in Clinical Nephrology

Abstract

αKlotho (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 primarily from the kidney and exerts a myriad of housekeeping functions in just about every organ. The vital role of Klotho is shown by the multiorgan failure with genetic deletion in rodents, with certain features reminiscent of human disease. The most common causes of systemic Klotho deficiency are AKI and CKD. Preclinical data on Klotho biology have advanced considerably and demonstrated its potential diagnostic and therapeutic value; however, multiple knowledge gaps exist in the regulation of Klotho expression, release, and metabolism; its target organs; and mechanisms of action. In the translational and clinical fronts, progress has been more modest. Nonetheless, Klotho has potential clinical applications in the diagnosis of AKI and CKD, in prognosis of progression and extrarenal complications, and finally, as replacement therapy for systemic Klotho deficiency. The overall effect of Klotho in clinical nephrology requires further technical advances and additional large prospective human studies.

Introduction

αKlotho (referred here as Klotho) was serendipitously discovered in 1997 by Kuro-o et al. (1) as a gene linked to aging. This gene was named Klotho after the goddess who spins the thread of life to govern human lifespan, working with two other goddesses of fate, Lachesis who measures the spun thread from Klotho’s spindle and Atropos who ends the life by cutting the thread (2). Kuro-o et al. (1) observed that Klotho hypomorphic mice exhibited a multiorgan failure syndrome resembling premature aging, including short lifespan, disturbed mineral metabolism, and multiple organ degeneration or failure, such as infertility, arteriosclerosis, cardiomyopathy, ectopic calcification, skin atrophy, osteoporosis, and emphysema. Later, both human (3) and rat (4) Klotho genes were cloned with high homology in amino acid sequences with the mouse. Subsequently, two paralogous genes were discovered and termed βKlotho (5) and γKlotho (6) to distinguish them from the first Klotho gene, which was renamed αKlotho. The α- and βKlotho proteins have known biologic roles, but the function of γKlotho is still elusive (7,8). In this manuscript, we only focus on αKlotho (Klotho).

Klotho is mostly expressed in the kidney as a transmembrane protein. In membrane-bound form, Klotho serves as a coreceptor for fibroblast growth factor 23 (FGF23) in conjunction with fibroblast growth factor receptors (FGFRs) (9) (Figure 1). The extracellular domain of transmembrane Klotho is also cleaved by proteases and released from the kidney into circulation, both as a full-length protein or as Kl1 and Kl2 fragments (10,11). These circulating forms of Klotho protein are collectively called “soluble” Klotho (Figure 1). Soluble Klotho protein is also detected in cerebrospinal fluid and urine.

Figure 1.

Overview of Klotho protein. (A) Fibroblast growth factor 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.

Klotho is a pleiotropic protein with multifaceted functions relevant to distinct biology and pathobiology in multiple organs and tissues. Klotho is an inhibitor of apoptosis, fibrosis, and cell senescence and is an inducer of autophagy (13–16). In this review, we discuss its potential clinical application in nephrology, with emphasis on diagnostic and therapeutic utility.

Physiology of Klotho

Klotho Expression and Metabolism

Klotho is highly expressed in the kidney, specifically in the distal tubules and, to a lesser extent, in the proximal tubules (17). Klotho is expressed in other organs, such as the brain, pancreas, and parathyroid glands (1). The Klotho gene encodes a single-pass 130-kD transmembrane protein that consists of two extracellular domains (Kl1 and Kl2), a transmembrane domain, and a short cytoplasmic tail (Figure 1). The extracellular domain of transmembrane Klotho is cleaved at the juxtamembrane region and between Kl1 and Kl2 by proteases (18–20). A secreted form of Klotho from an alternatively spliced transcript has been proposed, but the data are not conclusive (3,21). The spliced Klotho transcript possibly undergoes nonsense-mediated mRNA decay and is not translated to protein (22). The cleaved Klotho is released into the circulation and is known as soluble Klotho (17,22). Soluble Klotho acts as an endocrine or paracrine factor affecting multiple organs, such as the kidney, bone, brain, heart, lungs, and endothelium (23–29). Soluble Klotho suppresses FGF23 production in physiologic concentrations (Figure 2), but it can increase FGF23 expression in the bone (28) or act as a nonenzymatic scaffold protein that enhances FGF23 signaling on the basis of in vitro data in very high, nonphysiologic concentrations (12). Soluble Klotho is not filtered at the glomerulus, but it translocates across the kidney tubules, from the basolateral to the luminal side, and is excreted in the urine (17). Klotho expression is downregulated during acute kidney disease and CKD.

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.

Klotho and Mineral Metabolism

Klotho decreases kidney phosphate reabsorption by acting as a coreceptor for FGF23 binding to FGFR1 (30) (Figure 1). Soluble Klotho also directly promotes the internalization and degradation of the NaPi2a cotransporter in the kidney proximal tubules contributing to phosphate excretion (31). Klotho may also be a suppressor of vitamin D and FGF23 production (32) because Klotho-deficient mice exhibit higher CYP27B1 gene (33) and FGF23 production (1) (Figure 2).

Klotho in Disease States

CKD

Klotho expression in the kidney and soluble Klotho in the blood and urine are decreased in animals and humans with CKD from a variety of etiologies, including glomerular and tubulointerstitial diseases (32). Because the kidneys are 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 to loss of viable tissue but can be attributable to hyperphosphatemia (34) and hypermethylation or deacetylation of the Klotho gene promoter by inflammatory cytokines or uremic toxins, such as indoxyl sulfate (35,36). Similarities of clinical features, including short lifespan, cardiac remodeling, vascular calcification, bone disease, muscle wasting, and hyperphosphatemia, exist in the context of Klotho deficiency and in CKD (37).

Figure 3.

Changes in circulating soluble Klotho according to eGFR decline and its relationship to other mineral metabolism parameters. 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D.

Klotho is also expressed in the choroid plexus of the brain (1). Klotho-deficient mice display central nervous system lesions including hypomyelination, synaptic loss, and behavioral impairments such as dementia and cognitive deficits (38). Klotho deficiency in the brain impairs blood barrier and promotes immune-mediated central nervous system disorders (39). CKD is associated with depression and cognitive impairment (40). Low Klotho in cerebrospinal fluid was confirmed in Alzheimer disease (41). Low soluble Klotho in CKD may conceivably contribute to central nervous system dysfunction, although there is no evidence to date of Klotho deficiency in cerebrospinal fluid or brain in CKD.

Cardiovascular Disease

A novel risk factor of cardiovascular disease in CKD is soluble Klotho deficiency (42,43). Hyperphosphatemia and low soluble Klotho associate with more severe cardiac hypertrophy and fibrosis. Interestingly, elevated levels of FGF23 were associated with pathologic cardiac remodeling only if Klotho deficiency was also present (34). However, exogenous FGF23, independent of Klotho, induced cardiac hypertrophy through FGFR4-mediated PLC-γ signaling activation in cardiac myocytes (43,44). Klotho, independent of FGF23, was postulated to protect the heart against cardiac hypertrophy through inhibition of transient receptor potential channel-6 activity in cardiomyocytes (45). Klotho supplementation also protects against indoxyl sulfate–mediated cardiac hypertrophy in mice (46).

AKI

AKI is a syndrome with a myriad of inflammatory cytokines such as TNF-α and TNF-like weak inducer of apoptosis, which downregulate Klotho expression in the kidney through NF-kB activation in murine AKI (47). Similarly, in other inflammatory conditions such as inflammatory bowel disease, TNF-α and IFN-γ reduced Klotho expression in murine kidney (48). The reduced kidney Klotho is partly mediated by an increase in inducible nitric oxide synthase, nitric oxide production, and oxidative stress, but the precise mechanisms of how inflammation downregulates Klotho in AKI are unknown (48,49). A differential transcriptional splicing resulting in decay of Klotho mRNA has been postulated as a potential mechanism of Klotho downregulation in settings of acute illness (22).

Current experimental data support that low Klotho is not just a biomarker but pathogenic. Klotho-deficient mice have lower kidney and circulatory soluble Klotho, and they have more severe kidney damage and fibrosis and higher risk of AKI-to-CKD transition compared with wild-type mice after exposure to distinct kidney insults (14,16,23,50,51), indicating that Klotho deficiency renders the kidney more susceptible to acute insults.

Potential Mechanisms of Klotho-Mediated Kidney and Cardiovascular Protection

Preclinical data strongly suggest a pathogenic role of Klotho acting through multiple mechanisms in contributing to AKI development, AKI-to-CKD transition, progression of CKD, and development of extrarenal complications of CKD (Figure 4, Table 1).

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.

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-β1 signal
↓ Cell senescence	↑ Angiogenesis	↓ Wnt signal
↑ Autophagy	↑ Vasculogenesis	Mineral metabolism
Antioxidation	Inhibit fibrosis	↓ Blood phosphate
	↓ TGF-β1 signaling	Modulate FGF23 activity
	↓ Wnt signaling	Control 1,25-(OH)2D3 production
		Control PTH production
		Control BP

FGF23, fibroblast growth factor 23; 1,25-(OH)₂D₃, 1,25-dihydroxyvitamin D; PTH, parathyroid hormone.

Klotho Renders the Kidney More Resistant to Injury

Inhibition of Cell Senescence.

Cell senescence is a complex process in normal aging (52) and in kidney disease (53). Klotho deficiency induces and Klotho overexpression 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 phosphatidylinositol 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 associated with kidney damage (16,66). Klotho upregulates autophagy activity, protects kidney against ischemic injury, and, consequently, mitigates the progression of AKI to CKD (16).

Klotho Promotes Kidney Recovery

When recombinant Klotho protein is injected right after ischemic injury, mice had similar acute kidney damage but recovered much faster than vehicle injection (16). Intravenous administration of urinary extracellular vesicles carrying Klotho restores kidney Klotho levels in injured kidney and promotes kidney function recovery after glycerol injection (67).

Preservation of Stem Cells.

Klotho deficiency is associated with stem cell depletion (15,68) via augmentation of Wnt signaling and more cell senescence, which were rescued by Klotho protein overexpression (54).

Maintenance of Endothelial Function and Angiogenesis.

Swift and complete recovery of endothelium structure and function promotes kidney regeneration after ischemic injury (69). Klotho exerts proangiogenic actions (70) and maintains endothelial integrity (71).

Klotho Suppresses Kidney Fibrosis

Klotho suppresses kidney fibrosis in several animal models (14,72,73) via suppression of TGF-β1 activity, TGF-receptor II (14,73), and Wnt signaling activity (72). Supplementation of Klotho in rodents inhibits kidney fibrosis and retards CKD progression.

Klotho Ameliorates Disordered Mineral Metabolism in CKD

CKD is a state of Klotho deficiency, hyperphosphatemia, high FGF23 levels, and low 1,25-dihydroxyvitamin D₃ (Figure 3). Klotho deficiency participates in CKD development and progression and contributes to extrarenal complications of CKD directly and indirectly through disturbed mineral metabolism (Figures 2 and 4) (29,34,74,–76).

Reduction of Serum Phosphate.

Klotho deficiency impairs phosphaturia (31,77) and, consequently, accelerates phosphate accumulation in CKD. Administration of Klotho protein or enhancement of native Klotho is an effective strategy for the correction of hyperphosphatemia in experimental CKD (75).

Control of Fibroblast Growth Factor 23 Activity.

FGF23 participates in systemic phosphate balance by its triple action on intestine, bone, and kidneys through interplay with Klotho (8), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D₃ (8,12,31,77,–79) (Figures 2 and 4). High serum FGF23 may serve as a diagnostic biomarker and pathogenic intermediate for higher risk of cardiovascular disease and mortality in both patients with AKI (80) and patients with CKD (81). The fact that high FGF23 correlates with cardiac adverse effect only with concomitant low Klotho in animals suggests that Klotho may modulate the toxic effects of FGF23 (34).

Modulation of 1,25-Dihydroxyvitamin D₃ Production.

Low 1,25-dihydroxyvitamin D₃ is attributed to bone mineralization defects and secondary hyperparathyroidism in CKD (82). Vitamin D treatment attenuates cardiac FGF23/FGFR4 signaling and hypertrophy (83) and vascular calcification in uremic rats (84) in part through upregulation of circulating Klotho.

Control of Parathyroid Hormone Production.

Secondary hyperparathyroidism is associated with cardiovascular morbidity and mortality in CKD (85,86). In CKD, parathyroid gland loses its response to inhibitory signaling from FGF23 possibly due to lower Klotho and FGFR1 in the parathyroid gland (Figure 2) (87–89). Correction of Klotho to improve mineral homeostasis does not only benefit bone and mineral metabolism but also may attenuate cardiovascular disease and improve the quality of life of patients with CKD (75).

Klotho Reduces Blood Pressure

Hypertension is a complication of CKD and contributes to progression of CKD and cardiovascular disease. Klotho-deficient mice develop salt-sensitive hypertension after high sodium challenge (90) and increased arterial stiffness (91). Klotho single-nucleotide polymorphism (KL rs9536314) associates with salt-sensitive hypertension in adults with newly diagnosed hypertension (92).

Biomarker Candidacy in Human Kidney Disease

Klotho in Human CKD

Kidney Klotho mRNA was significantly lower in patients 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 with eGFR, after adjustment for age and other mineral parameters (95). To date, many studies have shown a positive correlation between Klotho levels (serum and urine) and eGFR in adults with CKD (74,96–99). Soluble Klotho levels correlate with markers of oxidative stress (8-isoprostane) and inflammation (IL-6), although some of these associations dissipated after multivariable analyses (100). In a recent larger study, serum Klotho levels associated with kidney function decline in a cohort of well-functioning older adults aged 70–79 with mean eGFR of 73 ml/min per 1.73 m² (101). With each doubling of serum Klotho, there was an associated 20% decreased odds of significant decline in kidney function over 10 years, which persisted after adjustment for demographics, cardiovascular risk factors, and mineral metabolism parameters, including FGF23 (101). The decline of soluble Klotho levels in adults with mild CKD (e.g., stages ≤2) antedates elevation in FGF23, PTH, and phosphate, and therefore, it may represent an early marker of CKD and CKD-MBD (Figure 3, Table 2) (96,102–104). A recent meta-analysis concluded that there is a significant positive correlation between soluble Klotho and eGFR in patients with CKD (105).

Table 2.

Human studies of soluble Klotho in CKD

Study	Clinical Setting	Methods	Results/Observations
Pavik et al. (106)	87 adults with CKD (stages 1–5) and 21 controls	Serum IBL ELISA	Adjusted mean Klotho decrease was 3.2 pg/ml 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 1–5, 28 on KRT, 44 post-transplant)	Plasma IBL ELISA	Klotho levels decreased with decreasing eGFR but no independent association between 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; P=0.008)
			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 (stages 1–5)	Serum IBL ELISA	Klotho levels independently predicted the 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 (P=0.03)
			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 the study, progressive CKD within 3 mo prior to the study
Akimoto et al. (99)	131 adults with CKD (stages 1–5)	Serum and urine IBL ELISA	Positive correlation between serum and urine 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 (stages 2–4)	Serum IBL ELISA	Klotho levels were significantly associated 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 (stages 2–4)	Plasma IBL ELISA	Klotho levels (highest versus lowest tertile) did not predict atherosclerotic events/death (HR, 0.75; 95% CI, 0.43 to 1.30; P=0.30) or ADHF/death (HR, 0.81; 95% CI, 0.39 to 1.66; P=0.56) 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 (stages 1–5)	Serum IBL ELISA	eGFR was an independent predictor of Klotho 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
Cano et al. (110)	31 children on PD and 45 healthy controls	Serum ELISA	Baseline Klotho levels were lower in children on PD versus controls (132±58 versus 320±119 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 (12 with RVH and 12 EH) and 12 HV controls	Serum IBL ELISA	Klotho levels were significantly reduced in patients with HTN (mean eGFR =74.7 and 48.8 ml/min in EH and RVH, respectively) versus controls (mean eGFR =73 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 2–5), 44 patients with ESKD on HD, 40 kidney transplant recipients, and 40 HV controls	Serum ELISA	Serum calcium was an independent predictor of Klotho levels in all groups of kidney disease
			Serum calcium inversely correlated with 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 HV controls	Serum IBL ELISA	Klotho levels were significantly lower in 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 (P=0.04)
Shroff et al. (113)	ESCAPE cohort post hoc analysis, 167 children with CKD on ACEI	Serum IBL ELISA	ACEI therapy significantly increased Klotho levels without any associated changes in serum calcium or phosphate
			Klotho levels did not correlate with eGFR at baseline (r=0.02; P=0.82) or at 8-mo follow-up (r=0.06; P=0.45)
Drew et al. (101)	2496 adults within the Health ABC study (mean eGFR =73 ml/min)	Serum IBL ELISA	Klotho levels independently associated with 30% kidney function decline, with each 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; P=0.01) 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 and ACE Inhibition on the Progression of CRF in Pediatric Patients; ACEI, angiotensin-converting enzyme inhibitor; ABC, Aging and Body Composition; MI, myocardial infarction.

In a cohort study of adult patients with CKD (stages 1–5), serum Klotho levels independently associated with the composite outcome of doubling serum creatinine, kidney failure, or death, after adjustment for age, diabetes, mean arterial pressure, eGFR, proteinuria, and PTH (96). Note that high-risk patients such as those with acute coronary syndrome, ischemic stroke, or progressive CKD within 3 months prior to the study enrollment were excluded.

Klotho and Cardiovascular Disease in Human CKD

Serum Klotho is associated with arterial stiffness measured by pulse wave velocity in patients with early CKD (97). In contrast, a different study in patients with CKD stages 2–4 showed that plasma Klotho levels were not associated with atherosclerosis, acute heart failure, or death at 2.6 years (109). Serum Klotho levels were lower in patients with primary hypertension compared with healthy volunteers, adjusted for eGFR (111). A post hoc analysis of the Effect of Strict BP Control and ACE Inhibition on the Progression of CRF in Pediatric Patients trial in children with CKD showed that ramipril therapy significantly increased serum Klotho levels without any changes in serum calcium or phosphate (113). In another study including adults with acute cerebral infarction, plasma Klotho levels were negatively associated with the presence, burden, and progression of small vessel disease (115). A recent study showed that low plasma Klotho is associated with mortality and cardiovascular events in patients on hemodialysis (114). The association between Klotho levels and cardiovascular disease has been observed only in small studies in humans mostly using surrogate markers of cardiovascular health rather than hard outcomes, such as cardiovascular events or death. Larger prospective studies with well-defined outcomes are needed to examine if Klotho is a prognostic marker of cardiovascular disease in CKD.

Klotho in AKI

In critically ill patients, urinary Klotho levels were lower in patients with AKI (Kidney Disease Improving Global Outcomes stage ≥2; within 48 hours of diagnosis) compared with well-matched intensive care unit controls (by age, sex, and baseline eGFR; within 48 hours of intensive care unit admission) (116). In a study of patients with acute tubular necrosis or acute tubulointerstitial nephritis by kidney biopsy, lower kidney Klotho protein levels associated with higher peak serum creatinine and more frequent KRT (117). In the kidney transplant population, donor kidney Klotho protein levels decreased following transplantation in those with delayed graft function (118). In postmortem kidney biopsies of critically ill patients who died of sepsis and AKI, there was lower kidney Klotho mRNA and protein in sepsis-associated AKI biopsies when compared with biopsies from control subjects undergoing nephrectomy for kidney cancer (119). Not all human studies in AKI have shown an inverse relationship between Klotho measurements and kidney function (Table 3).

Table 3.

Human studies of soluble Klotho in AKI

Study	Clinical Setting	AKI Definition	Methods	Results/Observations
Hu et al. (50)	17 adults with AKI (inpatient consults) and 14 HV	↑ SCr≥50% or ≥2 mg/dl	Urine immunoblot	Mean urine Klotho/Cr levels were significantly lower in AKI versus HV (4.85±1.69 versus 25.38±4.08 fmol/mg of Cr; P=0.01)
Liu et al. (120)	35 adults undergoing cardiac surgery (19 AKI)	AKIN ≥ stage 1 (SCr only)	Serum ELISA	Serum Klotho levels were lower in patients with AKI versus patients without AKI immediately after CS: mean ± SD, 102±17 versus 124±21 U/L; P=0.05. 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 et al. (121)	60 adults undergoing cardiac surgery or coronary angiography (30 AKI)	↑ SCr≥50%	Urine IBL and SSBT ELISA	Urine samples were collected 12 h post-CS or post-CA (single time point). Urine Klotho/Cr was not different in patients with AKI versus no AKI (mean ± SD, 2.45±0.26 versus 2.04±0.20 ng/mg of Cr; P=>0.05 by SSBT and 1.60±0.30 versus 1.24±0.30; 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 intrinsic AKI)	AKIN ≥ stage 1	Serum and urine IBL ELISA	Patients with prerenal and intrinsic AKI had similar SCr levels. Urine Klotho/Cr levels were lower in prerenal versus intrinsic AKI (mean ± SD, 174±292 versus 381±630 ng/g of Cr; P=0.001). There was no difference between groups in serum Klotho levels
Castellano et al. (118)	30 adults undergoing kidney transplant (15 developed DGF)	DGF	Serum IBL ELISA	Patients with versus without DGF had lower levels of serum Klotho at 2 yr post-transplantation
Seibert et al. (123)	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 nephrology consultation) versus control patients (mean ± SD, 567.6±294.4 versus 403.5±152.5 pg/ml; P=0.01). AKI and controls were not matched
Neyra et al. (116)	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 AKI diagnosis [patients] or within 24 h of ICU admission [controls]) were significantly lower in AKI versus controls: 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.

In a cohort of 35 adults undergoing cardiac surgery, there was no difference in preoperative serum Klotho levels in those with (n=19) versus without (n=16) postoperative AKI. However, postoperative serum Klotho levels obtained immediately and 4 hours following cardiac surgery were both lower in patients with versus without AKI (120). Klotho levels increased soon in patients with AKI such that they were no longer different between groups by postoperative day 1 (120). In another study of 60 adults undergoing cardiac surgery or coronary angiography (50% developed postprocedure AKI), urinary Klotho levels were not different in patients with versus without AKI when measured 12 hours after the procedure (121). Neyra et al. (116) found significantly lower levels of urinary Klotho in critically ill patients who developed major adverse kidney events (death, dependence on KRT, or decrease in eGFR≥50% from baseline) within 90 days of enrollment than in those who did not. Each one-fold higher urinary Klotho level was associated with an adjusted 83% lower risk of developing the outcome (Table 3).

Collectively, current clinical data show great promise for soluble Klotho levels as not merely a diagnostic marker but as part of novel risk prediction tools of kidney-related outcomes in high-risk patients with CKD or AKI.

Measurement of Soluble Klotho in Humans

Human studies of measuring circulating soluble Klotho in serum, plasma, or urine have shown that acute or chronic kidney dysfunction is accompanied by lower soluble Klotho (102,103,116), but some studies have reported no change or even higher soluble Klotho depending on level of kidney function (16,97,99,106,109,123–125). These discordant findings may largely be due to problems with performance and reproducibility of current commercially available assays (126–128). An unfortunate fact is that variance in sample type, quality, collection and processing methods, vintage and conditions of storage, and number of freeze-thaw cycles can all drastically affect assay performance and yield widely disparate results (Table 4); however, precautions were not usually made to account for these confounders.

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., protease inhibitors)
Methods of storage	Interassay variability
Freeze-thaw cycles	Intra-assay variability
	High-throughput capacity

Barker et al. (102) showed that a commercial ELISA yielded different results in fresh versus stored serum samples and experienced a drop in capture of exogenous soluble Klotho in patients with advanced CKD as well as when samples have undergone freeze-thaw cycles. Heijboer et al. (126) showed poor interassay and intra-assay agreement between three different commercial Klotho ELISAs, suggesting that problems exist beyond just one ELISA. Measurement of soluble Klotho using commercial ELISA was highly unstable in human urine even when stored at –80°C (129). An alternative method of Klotho assay using immunoprecipitation-immunoblot uses a synthetic anti-Klotho antibody (high affinity for Kl2 domain of Klotho) for pull down and an anti-Kl1 rat mAb (now commercially available KM2076; TransGenic Inc., Kobe, Japan) (10) for detection by immunoblot (102). This assay showed progressive decline of serum Klotho levels with increasing stages of CKD in a small single-center study using fresh samples (102).

A recent study evaluated two methods of measuring serum Klotho in patients with different strata of kidney disease (130). The authors concluded that immunoprecipitation-immunoblot, compared with ELISA, exhibited a stronger direct correlation with eGFR, better recovery (capture) of added exogenous Klotho, less susceptibility to variability from sample additives (protease inhibitors), and much better differentiation across different kidney disease groups, including AKI and CKD, in reference to healthy volunteers. The immunoprecipitation-immunoblot assay performance also declined after multiple freeze-thaw cycles, favoring the use of never-thawed samples when measuring soluble Klotho.

Development of high-throughput Klotho assays and standardized processing and storage conditions will greatly benefit the field because suboptimal assays are populating the human database, continuously rendering the unification of data interpretation very difficult.

Klotho-Focused Therapies

Counteracting the decrease in Klotho that occurs in kidney disease represents a promising strategy to prevent, ameliorate, and reverse kidney disease and its extrarenal complications (Figure 4).

One successful strategy in the laboratory is the use of viral delivery of Klotho cDNA in rodent AKI (60). In murine models, Klotho cDNA plasmids can be directly injected or delivered via a viral carrier (nonpathogenic adeno-associated virus) to rescue many phenotypes of Klotho deficiency (131–134), although its safety in humans is not established. Experimental data revealed attenuation of kidney disease in hypertensive rats (131,135), improvement of kidney function in AKI (60), amelioration of angiotensin II–induced kidney injury (132), improvement of endothelial function (134), and protection from uremic cardiomyopathy in CKD models (45). Another method is by the minicircle DNA vectors, which allows sustained in vivo Klotho gene expression without risk of immunogenicity and confers kidney protection in models of ischemia reperfusion injury and unilateral ureteral obstruction (136).

Another approach is to increase endogenous Klotho production by overcoming the mechanisms that suppress Klotho expression and/or release in kidney disease. In a CKD model, the vitamin D receptor agonist, paricalcitol, increased serum Klotho and reduced serum phosphate and FGF23. The source of increased soluble Klotho levels was not determined to be from the kidneys (84). Experimental upregulation of Klotho expression by 1,25-dihydroxyvitamin D₃ was also described in other studies (137,138). One recent study showed that sevelamer carbonate increased serum Klotho in CKD (139). Other agents that can potentially increase endogenous Klotho expression are angiotensin II receptor antagonists, PPAR-γ agonists, androgen, and statins (Figure 5) (140–145). Thus far, the efficacy of these agents has only been demonstrated in preclinical studies, except for the use of valsartan, which was shown to increase plasma Klotho levels in patients with diabetic nephropathy following 24 weeks of therapy (143). Off-label use of already approved drugs to pharmacologically manipulate endogenous Klotho remains a viable option. It is important to recognize that pharmacologic interventions have not been tested in clinical trials of kidney disease as the primary outcome. A major hurdle is that the mechanism of how Klotho expression is upregulated needs to be defined. Whether these drugs can stimulate endogenous Klotho production/release in CKD where the Klotho-generating cells are extensively destroyed is unknown.

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.

Another strategy is to target epigenetic modulation of endogenous Klotho expression. Although theoretically feasible, modulation of methylation and acetylation of the Klotho gene promoter needs further confirmation for its application in the management of human kidney disease.

Alternatively, administration of exogenous recombinant Klotho protein has dual effects: restoring soluble Klotho levels and promoting endogenous production of kidney Klotho. This dual action is particularly important in kidney failure, a status in which endogenous capacity is amputated. In a murine model, soluble Klotho injected 30 or 60 minutes after ischemic injury attenuated increase in serum creatinine postinjury and improved kidney histology (50). Further, repeated administration of Klotho starting 1 day after ischemic injury led to reduced fibrosis and improved recovery from AKI (16). Klotho replacement therapy remains the only practical method to control precise levels. Although recombinant Klotho replacement has been successful in many animal models, its application in humans still faces many hurdles.

Summary and Call for Action

Preclinical, translational, and clinical studies have advanced our understanding of Klotho biology, pathobiology, and potential clinical applications. Despite this accomplishment, the translation to clinical application has been slow. Klotho is an evolutionarily conserved housekeeping protein with a myriad of functions on many organs, which can serve as a biomarker for early diagnosis and/or risk stratification of patients with acute kidney disease or CKD. Because of its universal presence and panoply of function, specificity remains an issue (e.g., circulating Klotho levels may change in many diseases with or without kidney involvement).

Despite the emergence of promising human biomarker 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 standardization of methodology for soluble Klotho measurements. Given the imperfection of assays, one should exercise caution in interpretation of multiple small studies where kidney disease occurs under diverse clinical conditions and conclusions are made on the basis of variable sample conditions using different commercial reagents, most of which lack vigorous validation. Further, other factors known to influence soluble Klotho measurements, such as age, sex, inflammation, high-phosphate diet, FGF23, vitamin D supplementation, and certain medications, need to be carefully addressed in future prospective studies testing the biomarker candidacy of Klotho. Finally, there is a critical need to distinguish and characterize the subtypes of circulating Klotho protein because current assays do not differentiate between full-length soluble Klotho formed from cleavage of the transmembrane form (18–20), subsequent cleaved Kl1 and Kl2 Klotho fragments, and Klotho complexes with other circulating proteins. Therefore, with lack of knowledge as to which form of Klotho is most biologically relevant and/or captured with current assays, associations drawn between measured soluble Klotho levels and clinical outcomes should be circumspectly interpreted.

To date, no studies of Klotho protein administration in humans have been reported. However, ample animal studies have provided proof of concept that systemic Klotho protein administration is effective with seemingly good safety profiles. Because Klotho deficiency, regardless of etiology, could cause or accelerate dysfunction or degeneration in multiple systems or organs, Klotho is a valuable treatment modality of not just kidney disease but also its complications, in particular cardiovascular disease (Figure 4). The evolution of precision medicine leveraging clinical informatics along with genomic, proteomic, and metabolomic data will help the identification of high-risk patients to be targeted in Klotho-based therapeutic trials. The ongoing development of Klotho therapies and the evolution of more accurate and precise Klotho assays make these clinical trials foreseeable and no longer utopic in the near future. Clearly, bench and bedside interactive collaborative research is critically needed to develop and validate novel Klotho assays, examine its biomarker potential in diagnosis and prognosis, and identify effective therapeutic windows for Klotho administration in acute kidney disease and CKD. Pragmatic therapeutic trials in selected populations that are most likely to benefit from Klotho administration or modulation should be designed (Figure 6).

Figure 6.

Potential diagnostic, prognostic, and therapeutic applications of Klotho in clinical nephrology. Dx, diagnosis.

Disclosures

All authors have nothing to disclose.

Funding

M.C. Hu and O.W. Moe are supported by National Institutes of Health grants R01-DK091392 and R01-DK092461, the Charles and Jane Pak Center Innovative Research Support, and Endowed Professor Collaborative Research Support. O.W. Moe is supported by George O’Brien Kidney Research Center grant P30-DK-07938. J.A. Neyra is a recipient of National Center for Advancing Translational Sciences Early Career Pilot Grant UL1TR001998.