Klotho as a potential biomarker and therapy for acute kidney injury

Abstract

Klotho is a single-pass transmembrane protein that is highly expressed in the kidney and is known to act as a coreceptor for fibroblast growth factor 23. The extracellular domain can be produced independently or shed from membrane-bound Klotho and functions as an endocrine substance with multiple functions including antioxidation, modulation of ion transport, suppression of fibrosis, and preservation of stem cells. Emerging evidence has revealed that Klotho deficiency is an early event in acute kidney injury (AKI), and a pathogenic factor that exacerbates acute kidney damage and contributes to long-term consequences. Restoration by exogenous supplementation or stimulation of endogenous Klotho might prevent and ameliorate injury, promote recovery, and suppress fibrosis to mitigate development of chronic kidney disease. Although data are still emerging, in this Perspectives article we discuss why this renal-derived protein is a highly promising candidate as both an early biomarker and therapeutic agent for AKI.

Introduction

Acute kidney injury (AKI) is an important clinical problem that has been shown to affect approximately 40% (316/794) of adult patients,¹ and up to 82% (123/150) of pediatric patients² in intensive care units. Acute mortality is high at ≥30% in adult patients³ and, if the patient survives, the long-term outcome of AKI varies from complete recovery, incomplete recovery with progression to chronic kidney disease (CKD), or end-stage renal disease (ESRD).⁴ Even patients with AKI who do not require dialysis are still at risk of death or CKD.⁵ Despite decades of experimental and clinical research, only modest success has been made in translating experimental therapy into clinical practice.

The protein α-Klotho, encoded by KL, was originally identified as the product of an antiaging gene in 1997;⁶ subsequently, a second protein, termed β-Klotho, was identified.⁷ The Klotho proteins are both coreceptors for fibroblast growth factor (FGF) receptors but have distinct functions: α-Klotho regulates mineral homeostasis through FGF23^8,9 whereas β-Klotho transduces FGF21–FGF19 signaling to modulate bile production and energy metabolism.^8,10,11 In this article, we only discuss the role of α-Klotho, referred to as Klotho. KL has alternatively spliced transcripts at exon 3^12,13 resulting in either a secreted soluble form of Klotho (termed soluble Klotho)^14,15 or the full-length single-pass transmembrane protein (termed membrane Klotho), which functions as a coreceptor for FGF23.⁹ Another soluble form of Klotho is generated from the extracellular domain of transmembrane Klotho by secretases. Regardless of whether soluble Klotho is generated by alternative splicing or cleavage, soluble Klotho is secreted into the extracellular space, including blood, urine and cerebrospinal fluid, in which it functions as a hormonal factor that targets multiple remote organs.^16,17 Klotho can exert biological functions via FGF23-dependent^8,9 and FGF23-independent pathways.^15,18 The relative roles of membrane and soluble Klotho remain elusive as overexpression of membrane Klotho also leads to high levels of soluble Klotho.¹⁹

Multiple theories for how each form of Klotho exerts its antiaging activity have been proposed. First, in 2011, intracellular Klotho was shown to have antiaging effects through the suppression of inflammation mediated by probable ATP-dependent RNA helicase DDX58 (also known as retinoic acid- inducible gene 1 protein).²⁰ Second, nuclear Klotho in the brain was also shown to be associated with antiaging, but the molecular mechanism by which this process occurs has not been determined.²¹ Third, soluble Klotho was also found to suppress cell senescence through the inhibition of Wnt signaling.¹⁸

The pleiotropic actions exerted by soluble Klotho include cytoprotection,^22,23 which can be FGF23-dependent or FGF23-independent,^24–26 and is one of the key reasons for administering exogenous soluble Klotho for the treatment of AKI. Indeed, exogenous Klotho protein protects the kidney from ischemia–reperfusion injuries, which clearly supports a role for soluble Klotho in renoprotection. Currently, no solid evidence has confirmed the direct effect of membrane Klotho on renoprotection, because mice that overexpress Klotho (Tg-Kl mice) have high levels of both membrane and soluble Klotho.^19,22

Although the highest expression of Klotho is in the kidney⁶ and it has been in the spotlight in aging research for more than a decade, it has only become a major focus in the renal field in the past 5 years. Indeed, the biology of Klotho is now gaining increasing importance in the fields of mineral metabolism and kidney disease. This Perspectives article discusses recent advances in our understanding of the role of Klotho in AKI and its potential utilization as an early biomarker and therapeutic agent in this disease.

Klotho as a biomaker for AKI

In the mammalian kidney, Klotho is highly expressed in distal convoluted tubules and is also found in the proximal convoluted tubule, although at lower levels.¹⁵ In contrast to studies in humans and animal models that have shown low levels of Klotho in CKD and ESRD,²⁷ few studies have addressed Klotho levels in AKI (Table 1).

Table 1.

Klotho levels in various rodent models of AKI and in human AKI

Cause of AKI	Renal Klotho protein		Renal KL mRNA		Plasma Klotho protein	Urine Klotho protein	Species	Study
	IB	IHC	RNA blot	qPCR	IP	IB
IRI	ND	ND	ND	ND	ND	↓	Human	Hu et al. (2010)22
IRI	↓	↓	ND	↓	↓	↓	Mouse	Hu et al. (2010)22
IRI	↓	↓	ND	↓	ND	ND	Mouse	Sugiura et al. (2010)23
IRI	↓	↓	ND	↓	↓	↓	Rat	Hu et al. (2010)22
IRI	↓	↓	ND	↓	ND	ND	Rat	Sugiura et al. (2005)33
LPS	ND	ND	↓	ND	ND	ND	Rat	Ohyama et al. (1998)29
Folic acid	↓	ND	ND	↓	↓*	ND	Mouse	Moreno et al. (2011)32
Cisplatin	↓	ND	ND	↓	ND	ND	Mouse	Moreno et al. (2011)32
Cisplatin	↓	↓	ND	↓	ND	ND	Mouse, rat	Panesso et al. (2011)31
Prerenal hypoperfusion	↓	ND	ND	↓	ND	ND	Mouse	Tang et al. (2011)30
Postrenal UUO	ND	ND	ND	↓	ND	ND	Mouse	Doi et al. (2011)28

^*Measured by ELISA.

Abbreviations: AKI, acute kidney injury; IB, immunoblot; IHC, immunohistochemistry; IP, immunoprecipitation; IRI, ischemia–reperfusion injury; LPS, lipopolysaccharide; ND, not determined; qPCR, quantitative PCR; UUO, unilateral ureteral obstruction.

Renal Klotho

In addition to AKI that is induced by ischemia–reperfusion injury, renal Klotho deficiency is clearly found in other types of AKI such as that induced by ureteral obstruction,²⁸ lipopolysaccharide (model of sepsis),²⁹ hypovolemia,³⁰ and nephrotoxins including cisplatin^31,32 and folic acid³² (Table 1). These findings indicate that downregulation of renal Klotho in AKI is a general phenomenon following acute kidney insults such as ischemia, oxidative stress, and exposure to nephrotoxins.

Sugiura et al.³³ monitored the time course of changes in renal Klotho levels in rats after ischemia–reperfusion injury and found that both the protein and mRNA level started to fall in the first day following reperfusion and returned to near baseline levels at days 4 and 3, respectively. A significant reduction in levels of renal Klotho protein was also observed in a similar rat model.²² Furthermore, a time course revealed that changes in kidney morphology were detectable at 5 h following injury: levels of neutrophil gelatinase-associated lipocalin (NGAL) increased between 3 h and 5 h, but renal Klotho levels were sustainably decreased from 3 h, suggesting that renal Klotho protein is one of the earliest biomarkers of kidney injury, at least in a rodent model of AKI induced by ischemia–reperfusion injury.²² In 2011, Moreno et al.³² reported that renal Klotho mRNA levels remained markedly low 7 days after folic acid administration (to induce nephrotoxicity) even when serum creatinine had almost returned to baseline levels; renal Klotho mRNA recovery therefore seems to be delayed when compared with the recovery of renal function. Whether delayed recovery of renal protein Klotho expression is a predictor of progression to CKD in nephrotoxicity induced by folic acid is currently unknown.³²

Plasma Klotho

When the level of soluble Klotho in the blood was examined in a rat model by immunoprecipitation, it was found to be dramatically and sustainably decreased at 3 h following ischemia–reperfusion injury.²² This time profile is almost identical to that of renal Klotho, suggesting that the changes in levels of plasma and renal Klotho in response to injury occur in parallel.²² Levels of plasma Klotho protein started to decrease as early as 3 h after ischemia–reperfusion injury, after a brief increase at 1 h; protein levels started to increase approximately 48 h after injury, reaching baseline levels after approximately 7 days.²² Furthermore, decreased levels of plasma Klotho were also observed in mouse models of nephrotoxic AKI induced by both folic acid³² and cisplatin^31,32 (Table 1). Thus, AKI is not only a state of renal Klotho deficiency, but also one of endocrine Klotho deficiency. Data published in the literature for human plasma Klotho includes many healthy individuals but only a limited number of patients with CKD.^34–38 Data of plasma Klotho levels in patients with CKD are inconsistent.^37,39 To date, no study has examined plasma Klotho in patients with AKI. One study showed a negative linear relationship of plasma Klotho levels when compared with serum creatinine concentration and age in healthy children (n = 39) and adults (n = 142);⁴⁰ further studies are, however, required to establish a normal range for plasma Klotho levels.

Urinary Klotho

Soluble Klotho has an enzymatic function in the urinary lumen where it modulates the function of transporters and channels;⁴¹ urinary Klotho might, therefore, provide a viable window to monitor the level of renal and blood Klotho. To date, few studies have examined urinary Klotho in either patients with AKI or animal models of AKI. Assaying human plasma Klotho levels by either immunoblot or ELISA has not been fully validated and access to kidney biopsy samples is limited. Measurement of Klotho levels in urine as a surrogate marker of renal Klotho might therefore be feasible as urinary and plasma Klotho seemed to vary in para llel in a rodent model of AKI.²² Urinary Klotho decreased in the first day following ischemia– reperfusion injury; levels began to rise by day 2 and were at normal levels by day 7.²² Furthermore, patients with AKI had undetectable or notably lower levels of Klotho protein in the urine than did healthy volunteers²² (Table 1). In our opinion, a large prospective, cohort study is required to validate the specificity and sensitivity of urinary Klotho as a marker of renal Klotho levels.

Animal studies are now showing that downregulation of Klotho in AKI is a common and general phenomenon of kidney damage regardless of etiology.^{22,28–31,33} The prognostic value of plasma and urine Klotho to predict the outcome of AKI needs to be explored. Interestingly, removing 10–15% of total blood volume did not markedly decrease Klotho mRNA or protein levels in the kidney.²⁸ At present, however, it is unclear whether Klotho levels decrease gradually during the trans ition from a prerenal to intrinsic renal state or whether there are any distinct cut-offs that mark tubular necrosis that are of diagnostic value to clinicians. Nonetheless, Klotho measurement should be explored as a potential biomarker for distinguishing prerenal from intrinsic AKI.

Urinalysis, urinary electrolytes, urine osmolarity and volume, and serum creatinine levels are traditional parameters that are used for AKI diagnosis. Owing to the poor sensitivity, delayed response, and lack of specificity of these parameters, one must exercise caution in interpreting them.⁴² Novel biomarkers perform better for early diagnosis and prediction of outcome of AKI than do traditional parameters (Box 1). The multicenter Translational Research Investigating Biomarker Endpoints in Acute Kidney Injury (TRIBE-AKI) cohort study clearly indicated that levels of urine IL-18 and urine NGAL were associated with subsequent AKI and poor outcomes after cardiac surgery; plasma NGAL enabled a risk prediction to be made in an adult, but not a pediatric, population.^43,44 Kidney injury molecule 1 (KIM-1) is expressed at low levels in healthy adult kidney.^45,46 Elevated levels of KIM-1 in urine is a more specific, but less sensitive and a later marker than NGAL in AKI induced by ischemia– reperfusion injury.⁴⁷ A combination of biomarkers might improve early diagnosis of AKI onset following exposure to renal insults.⁴⁸ In AKI, levels of NGAL, KIM-1 and IL-18 are elevated⁴⁷ whereas Klotho is suppressed.^{22,28–31,33} In our opinion, combined measurement of two biomarkers that change in an opposite direction following renal injury, for instance NGAL–Klotho or KIM-1–Klotho, would markedly amplify the sensitivity for early detection of AKI, and should, therefore, be explored.

Box 1. Biomarkers and therapies for AKI.

Novel biomarkers

• Cystatin C

• γ-Glutathione-S-transferase

• Urine N-acetyl-β-D-glucosaminidase

• Liver-type fatty-acid-binding protein

• KIM-1

• IL-18

• NGAL

• MicroRNAs

• Klotho

Novel therapies

• Antioxidants (e.g. oxygen free radical scavengers, N-acetylcysteine)

• Modulation of renal adenosine receptors

• Inducible nitric oxide synthase inhibitors

• Atrial natriuretic peptide

• Erythropoietin

• Modulators of hypoxia-inducible factors

• Stem cells

• Klotho

Abbreviations: AKI, acute kidney injury; KIM-1; kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin.

Mechanisms of Klotho regulation

The mechanism of how Klotho is reduced in AKI remains elusive. Clearly, renal destruction would decrease production of Klotho, but the pathophysiological mechanism underlying Klotho downregulation in AKI is much more complex than loss of renal tissue alone. The immediate but transient response of Klotho following ischemia–reperfusion injury is in fact upregulation followed by reduction.²² The temporary rise in Klotho protein levels in kidney and blood may represent a defensive response to acute insults.²²

Klotho downregulation occurs early after kidney injury and prior to changes in other markers of kidney damage, indicating that renal tissue destruction is unlikely to be the only mechanism responsible for the decrease in Klotho mRNA and protein levels. Oxidative stress can decrease Klotho mRNA and protein prior to onset of cell death.²⁴ Experiments in cultured kidney cell lines (Table 2) were not as consistent as the data from animal models, which suggests that changes of single factors is insufficient to fully explain the mechanism.

Table 2.

Klotho protein or transcript levels in cultured kidney cell lines

Cell line	Kidney insults	Klotho protein		KL mRNA		Study
		IB	ICC	RNA Blot	qPCR
Human cell lines
HEK 293	TNF	ND	ND	↓	↓	Ohyama et al. (1998)29
HEK 293	IL-1β	ND	ND	↔	↔	Ohyama et al. (1998)29
HEK 293	LPS	ND	ND	↔	↔	Ohyama et al. (1998)29
RPTEC	LPS + IL-1β + TNF-β	ND	ND	↔	↔	Thurston et al. (2010)50
Mouse cell lines
mpkDCT4	IFN-β or IL-1β or TNF-β	ND	ND	ND	↓	Thurston et al. (2010)50
mpkDCT4	IL-6	ND	ND	ND	↔	Thurston et al. (2010)50
mpkDCT4	SNAP	ND	ND	ND	↓	Thurston et al. (2010)50
mIMCD3	TNF-β	ND	ND	ND	↓	Thurston et al. (2010)50
mIMCD3	TNF-β + IFN-γ	ND	ND	ND	↓	Thurston et al. (2010)50
MCT	TNF-β or TNFSF12	ND	ND	ND	↓	Moreno et al. (2011)32
mIMCD3	H2O2	↓	↓	ND	↓	Mitobe et al. (2005)24

Abbreviations: HEK 293, human embryonic kidney cell; IB, immunoblot; ICC, immunocytochemistry; LPS, lipopolysaccharide; MCT, mouse kidney cell; mIMCD3, mouse inner medullary collecting duct; mpkDCT4, mouse distal convoluted tubule cell; ND, not determined; qPCR, quantitative PCR; RPTEC, human renal proximal tubular epithelial cells; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; TNF, tumor necrosis factor; TNFSF12, TNF ligand superfamily member 12.

A paucity of data are available on levels of Klotho protein and mRNA in vitro (Table 2). Proinflammatory cytokines or oxidative stress decrease Klotho expression in part through epigenetic modulation of Klotho promoter methylation⁴⁹ or deacetylation.^32,49 Tumor necrosis factor (TNF) and IFN-γ reduced Klotho mRNA and protein levels in cultured renal epithelial cells.⁵⁰ Exogenous administration of TNF ligand super family member 12 (TNFSF12, also known as TWEAK) decreased renal expression of Klotho; moreover, the blockade or absence of TNFSF12 abrogated the decrease in renal and plasma Klotho in AKI induced by folic acid.³² Furthermore, inhibition of Klotho by TNFSF12 and TNF was associated with activation of nuclear factor κB and promotion of histone deacetylation of the Klotho promoter.³² Inhibition of histone deacetylase reversed TNFSF12-induced Klotho downregulation, presenting a novel therapeutic approach to upregulate renal Klotho expression.

Lipopolysaccharide decreased renal Klotho expression in a rodent model (Table 1),²⁹ but not in cultured kidney cell lines (Table 2),^29,50 suggesting that lipopolysaccharide does not directly decrease renal Klotho. The effect of interleukins, including IL-6⁵⁰ and IL-1β,^29,50 on downregulation of Klotho expression in cultured kidney cell lines was also inconsistent (Table 2).

The rapid decrease in Klotho protein level was associated with a modest decrease in Kl mRNA in a rat model of AKI,²² suggesting that Klotho reduction cannot be solely attributed to low levels of Kl mRNA, but rather attributable to Klotho protein destabilization and/or arrest of translation. Further support of this model came from the observation that renal Klotho over-expressed from a transgene is also reduced in this rat model of AKI induced by ischemia–reperfusion injury.²²

MicroRNAs (miRNAs) are a class of small, noncoding RNAs capable of post-transcriptional regulation of gene expression and act as post-transcriptional repressors by binding 3′ untranslated regions of target genes. Deregulation of miRNAs during AKI is involved in AKI development and progression to CKD.^51,52 KL mRNA contains a long 3′ untranslated region,¹³ suggesting that hypothetically, Klotho is a target for miRNAs.

Therapeutic role of Klotho in AKI

Although an early and sensitive marker for renal injury is urgently required, an equal necessity is a specific therapy to alter the course of AKI. In addition to its potential as a biomarker for AKI, the fall in Klotho level may have pathophysiological importance. In a rat model of ischemia–reperfusion injury, adenovirus-mediated Kl gene delivery resulted in considerable improvement in serum creatinine concentration, appreciable amelioration of renal histological changes, and diminution of apoptotic cells in the kidneys.³³ It is important to note that the transferred Kl gene was only expressed in the liver, not in the kidney, indicating that Klotho functions as a circulating substance to exert reno protection. Moreover, as Kl gene delivery preceded induction of ischemia–reperfusion injury, Klotho might serve as a prophylactic agent against AKI.

For pragmatic reasons, it would be wise to examine if Klotho protein is as effective as KL gene delivery for patients with AKI. When a single dose of Klotho protein was intraperitoneally injected into rats with AKI 30 min after ischemia–reperfusion injury, it considerably improved renal function, ameliorated kidney histology, preserved renal Klotho levels, and suppressed an increase in renal NGAL levels.²² By contrast, Klotho was substantially less effective when it was given 60 min after reperfusion, indicating that the earlier Klotho is administered the better the renal outcome.²² Tg-Kl mice that express 1.5–2-fold greater levels of plasma Klotho¹⁹ than do wild-type mice were more resistant to ischemic kidney injury.²² If these results are translated into a clinical setting, then Klotho would be promising as a prophylactic agent for patients with high risk of AKI such as those with hypovolemia, shock, and patients undergoing cardio pulmonary bypass, abdominal aortic aneurysm or kidney transplantation.

Nephrotoxicity is one of the serious adverse effects of cisplatin—a widely used antitumor agent,⁵³ but the mechanism of cisplatin-induced nephrotoxicity is still incompletely understood. Cisplatin-induced AKI was shown to be exaggerated in haplo-insufficient Kl^−/+ mice and ameliorated in Tg-Kl mice. NGAL expression in the kidney was markedly higher in Kl^−/+ mice and lower in Tg-Kl than in wild-type mice.³¹ Klotho is, therefore, a potentially useful agent to prevent cisplatin-induced AKI.

Renoprotection by Klotho

AKI is a complex pathological process involving tissue injury followed by tissue repair. Even though marked progress has been made in our understanding of the pathophysiology of AKI and in exploring potential novel therapies, few effective therapies have been translated from the bench to bedside (Box 1). The multifaceted function of Klotho suggests that Klotho might protect the kidney from ischemic injury by multiple mechanisms, including cytoprotection, cell regeneration and antifibrosis. One can broadly categorize the beneficial effects of any substance, whether endogenous or exo genous, in the setting of AKI into three broad classes: direct cytoprotection to prevent and ameliorate damage; promotion of regenerative repair; and avoidance of fibrotic reaction (Figure 1).

Figure 1.

Proposed mechanisms by which Klotho exerts its beneficial effects on renal epithelia in acute ischemia–reperfusion injury. Ischemia reperfusion downregulates Klotho production in the kidney and decreases blood Klotho levels, which may be an early biomarker of AKI and also a pathological intermediate for exacerbation of kidney damage. Administration of exogenous Klotho protein can effectively prevent and limit AKI induced by ischemia–reperfusion injury. In addition, Klotho is shown to promote normal healing and prevent fibrosis. Abbreviation: AKI, acute kidney injury.

Direct cytoprotection

Inhibition of cell senescence

Cell senescence is a complex process present not only in normal aging but also in many types of kidney disease.⁵⁴ Senescent cells may alter secretion of growth factors, exhibit increased susceptibility to apoptosis and delayed repair, and impair regeneration after exposure to insults. Kl overexpression ameliorates renal injury that is associated with reduction in senescent cells, decreased oxidant stress, and reduced apoptosis in the kidneys of mice with spontaneous chronic glomerular disease with mutation in Tenc1.⁵⁴ Klotho depletion directly promoted senescence of renal epithelial cells, and Klotho supplementation blocked senescence induced by oxidative stress.⁵⁵

Inhibition of cell apoptosis

Apoptosis in AKI is associated with loss of renal epithelial cells, and is perhaps one way to remove damaged cells. Excessive apoptosis, however, considerably decreases the total pool of cells and consequently might alter kidney morphology and impair regeneration.⁵⁶ An in vitro study showed that Klotho attenuates apoptosis in kidney cell lines treated with H₂O₂.²⁴ An in vivo study found that Kl gene transfer decreases apoptotic cells and suppresses apoptotic protein expression in ischemic kidneys of rats.^23,24,33

Antioxidation

Kl^−/− mice have high levels and Tg-Kl mice have lower levels of oxidative markers and oxidation-induced cell damage than do healthy mice.⁵⁷ In vitro, Klotho rendered cultured medullary collecting duct cells resistant to H₂O₂, and decreased the number of apoptotic cells following treatment with H₂O₂.²⁴ In addition, a paraquatinduced increase in lipid peroxidation was suppressed by Klotho in HeLa cells.⁵⁷ Klotho prevents oxidative injury and apoptosis through activation of the FOXO family of transcription factors and stimulation of manganese superoxide dismutase.⁵⁷

Inhibition of fibrosis

The risk of CKD is increased 13 times in all patients with AKI, but in patients with AKI at RIFLE failure stage, the risk of CKD is 41 times greater than in individuals without AKI.⁵⁸ Although the causes of AKI are multi factorial, progression of AKI to CKD and ESRD tends to follow a single route irrespective of the cause of AKI characterized largely by renal fibrosis. Indeed, ischemic renal injury in animals consistently results in long-term morphological alteration and renal dysfunction.^59,60 6 months after right nephrectomy and left renal ischemia, rats developed CKD with renal hypertrophy.⁵⁹ Indeed, immunohistochemistry showed the accumulation of inflammatory cells, fibroblast cells and collagen IV at 6 months.⁵⁹ Rats developed proteinuria 16 weeks after ischemia–reperfusion injury, and severe renal fibrosis with elevation of transforming growth factor (TGF)-β1 by 40 weeks.⁶⁰ Therefore, severe ischemic injury results in renal fibrosis in rodents.⁶⁰

Yang et al.⁶¹ compared the incidence and severity of renal fibrosis among four types of models of AKI: moderate and severe bilateral ischemia–reperfusion injury, unilateral ischemia–reperfusion injury, aristolochic acid toxic nephropathy, and unilateral ureteral obstruction (UUO).⁶¹ All mice except moderate bilateral ischemia–reperfusion injury had severe azotemia within 1 week of ischemia–reperfusion injury.⁶¹ Although this azotemia later improved, the mice still had high serum creatinine levels 3 weeks after injury.⁶¹ Renal fibrosis was present in all models of AKI.⁶¹ Therefore, despite restoration of renal function after acute insults, alteration of kidney histology with deleterious loss of renal function occurs in the long term.

TGF-β1 is thought to have a key role in renal epithelial–mesenchymal transition.²⁸ Kl^−/− mice have high levels of renal tubulointerstitial fibrosis,⁶² which is associated with increased TGF-β1 levels in the kidneys. The renal fibrosis in UUO is accompanied by upregulation of TGF-β1 and fibronectin, and downregulation of Klotho mRNA and protein.⁶² These alterations were exaggerated in Kl^+/− mice with UUO.⁶² Klotho supplementation alleviated renal fibrosis, and suppressed the expression of fibrosis markers and TGF-β1 target genes such as Snai1 and Twist1, but did not reduce TGF-β1 expression in the kidneys of mice with UUO.²⁸

TGF-β1 stimulates plasminogen activator inhibitor 1 (PAI-1) expression in the kidney through the Smad–p53–Usf2 pathway in the UUO mouse model.⁶³ Abnormal elevation of PAI-1 activity is associated with activation of profibrotic signals and kidney fibrosis.⁶³ TNF release in AKI synergistically increased hypoxia-induced PAI-1 expression.⁶⁴ PAI1 mRNA and PAI-1 protein activity were elevated in multiple tissues in Kl^−/− mice compared with healthy mice.⁶⁵ We predict that Klotho supplementation might decrease PAI-1 and in turn suppress renal fibrosis. Indeed, exogenous Klotho suppresses the increase in fibrogenic markers in cultured kidney cell lines induced by TGF-β1.⁶⁶

Acceleration of kidney regeneration

Preservation of stem cells

Klotho deficiency is associated with stem cell depletion, which is part of normal aging.¹⁸ The extracellular domain of Klotho blocks Wnt binding to its membrane receptor and inhibits biological activity.¹⁸ Substantial augmentation of Wnt signaling, high levels of cell senescence and stem cell depletion were found in renal tissues of Kl^−/− mice and these alterations were reversed by Klotho protein overexpression.¹⁸ Furthermore, accelerated cell senescence and stem cell depletion as a result of Wnt was inhibited by Klotho protein, suggesting that Klotho is a secreted Wnt antagonist.

Endothelial function and angiogenesis

Abnormal endothelial function and impairment of angiogenesis and vasculogenesis can delay kidney regeneration, whereas quick and complete recovery of endothelium morphology and function promotes kidney regeneration after ischemia– reperfusion injury in rodent models.^67,68 Statins attenuated kidney morphological alteration and improved kidney function induced by ischemia– reperfusion injury⁶⁹ and restored renal angiogenesis and renal microcirculation.⁷⁰ In addition, statins upregulated renal Klotho mRNA and protein in cultured kidney cells.⁷¹ Whether this increase in Klotho following exposure to statins results in acceleration of angiogenesis and vasculogenesis is yet to be determined.

Conclusions

Ample data from animal studies show decreased Klotho levels in the kidney and plasma following ischemia–reperfusion injury and during AKI. These findings suggest that AKI is a transient renal and endocrine Klotho-deficient state and this deficiency might have a role in kidney dysfunction, recovery, and AKI progression to CKD. The field now needs to move from a preclinical phase to clinical testing. It is important to note that we still do not understand how Klotho is decreased nor do we understand the mechanisms of Klotho action so the clinical data may be somewhat of an empirical nature but it will nonetheless be of the utmost importance for several reasons. First, Klotho is a potential early biomarker for making the diagnosis of AKI²² and in distinguishing intrinsic renal failure from prerenal hypoperfusion; the sensitivity and specificity of Klotho as a biomarker, however, remains to be established. Second, Klotho levels may have prognostic value in prediction of AKI recovery or progression to CKD. Third, early administration of exogenous Klotho protein,²² delivery of KL,³³ or enhancement of endogenous Klotho by novel small molecules,⁷² peroxisome proliferator-activated receptor-γ agonist,⁷³ or statin⁷¹ may prevent AKI. Fourth, administration of Klotho protein and upregulation of endogenous Klotho expression after AKI may accelerate recovery. Fifth, even if Klotho does not change the course of AKI, treating Klotho deficiency could decrease the risk of progression to CKD by inhibiting renal fibrosis. Elucidating the molecular mechanisms of how Klotho functions as a cytoprotective protein and as an inhibitor of fibrogenesis will be important in understanding how Klotho protects the kidney from ischemia-reperfusion and prevents CKD development after an acute episode of ischemic injury.