RENAL AND EXTRA-RENAL ACTIONS OF KLOTHO

SUMMARY

Klotho is a single-pass transmembrane protein highly expressed in the kidney. Membrane Klotho protein acts as co-receptor for fibroblast growth factor-23. Its extracellular domain is shed from cell surface and functions as an endocrine substance that exerts multiple renal and extra-renal functions. An exhaustive review is beyond the scope and length of this manuscript; thus only effects with pertinence to mineral metabolism and renoprotection will be highlighted here. Klotho participates in mineral homeostasis via interplay with other “calciophosphoregulatory” hormones (PTH, FGF23, and 1,25-(OH)₂ vitamin D₃) in kidney, bone, intestine, and parathyroid gland. Klotho may also be involved in acute and chronic kidney diseases development and progression. Acute kidney injury is a temporary and reversible state of Klotho deficiency and chronic kidney disease is a sustained state of systemic Klotho deficiency. Klotho deficiency renders the kidney more susceptible to acute insults, delays kidney regeneration, and promotes renal fibrosis. In addition to direct renal effects, Klotho deficiency also triggers and aggravates deranged mineral metabolism, secondary hyperparathyroidism, vascular calcification, and cardiac hypertrophy and fibrosis. Although studies examining the therapeutic effect of Klotho replacement were performed in animal models, it is quite conceivable that supplementation of exogenous Klotho and/or up-regulation of endogenous Klotho production may be a viable therapeutic strategy for patients with acute or chronic kidney diseases.

INTRODUCTION

The Klotho gene was identified in 1997 when its disruption in mice caused a phenotype of premature multi-organ failure including shortened life span, growth retardation, accelerated thymic involution, pulmonary emphysema, cognition impairment, skin atrophy, osteopenia, ectopic soft tissue calcification, hyperphosphatemia, and high plasma fibroblast growth factor (FGF)-23.¹ Most of the features observed in Klotho hypomorph or knock-out mice, could be rescued by expressing Klotho,^1,2 indicating that Klotho is anti-aging gene.

The Klotho gene encodes a single-pass transmembrane protein and is expressed in multiple tissues but in particularly high levels in the kidney.^1,3 In the mammalian kidney including mouse, rat and human, Klotho is prominently expressed in distal convoluted tubules (DCT),^1,3,4 but also unequivocally found in the proximal convoluted tubule (PCT)⁴ and also in inner medullary collecting duct-derived cell lines. ^5,6 In addition to membrane-anchored Klotho, a secreted form of Klotho protein is generated from the Klotho gene through alternative splicing. Secreted Klotho is directly released into the extracellular compartment and present in body fluid. Another important form of soluble Klotho in body fluid is ectodomain shedding from membrane Klotho on cell surface by proteases ADAM (acronym for A Desintegrin and Metalloproteinase)10/17 (Figure 1).⁷ Soluble Klotho protein is present in cerebrospinal fluid,⁸ blood,^8,9 and urine of mammals.^4,9

Figure 1. Physiologic roles of Klotho on solute channels and transporters and vitamin D metabolism in the kidney.

Klotho is prominently expressed in distal convoluted tubules (DCT), and less in the proximal convoluted tubules (PCT).⁴ In PCT, membrane Klotho at the basolateral side⁴ functions as coreceptor of FGFRs and drive FGF23 signal transduction to inhibit NaPi cotransporters (NaPi: 2a/c and Pit2) and to suppress cyp27β1 encoding for 1-hydroxylase, and to stimulate cyp24α1 encoding for 24-hydoxylase. The role of Klotho in the cytoplasm of renal tubules is unclear. Whether membrane Klotho at luminal side⁴ inhibits NaPi cotransporters (2a/c and Pit2) in an autocrine mode is not known (dash line). In DCT, membrane Klotho at basolateral side⁴ functions as coreceptor of FGFRs to induce FGF23 signal transduction. What intermediate(s) are released from DCT and how intermediate(s) affect on PCT in paracrine mode is not known. One possible candidate is Klotho release from the DCT to act on the PCT. Whether membrane Klotho at luminal side directly regulates TRPV5 in autocrine mode is unknown. Soluble Klotho in luminal urine derived from either blood or urine exerts regulatory action on NaPi cotransporters in PCT; and on TRPV5 in DCT. Ca: calcium ion; DCT: distal convoluted tubule; FGF23: fibroblast growth factor; FGFR: FGF receptor; NaPi cotransporter: sodium-phosphate dependent cotransporter; PCT: proximal convoluted tubule; Pi: phosphate; TRPV5: transient receptor potential ion channel 5; 1,25 VD₃: 1,25-(OH)₂ vitamin D₃, Dash line: suspected action.

Membrane-anchored and soluble Klotho proteins seem to have distinct functions (Table 1). Membrane Klotho forms a tetrameric complex with FGF receptors (FGFRs) and functions as a co-receptor for FGF23,^10-12 a bone-derived phosphatonin that induces negative phosphate balance through promotion of renal phosphate excretion (Figure 1). Soluble Klotho is a pleiotropic protein functioning as an endocrine factor with a multitude of renal and extra-renal effects (Table 1). Recently, several studies showed that nuclear Klotho¹³ and cytoplasm Klotho¹⁴ are also bioactive molecules to protect cell from senescence and apoptosis (Table 1).

Table 1.

Biological functions of Klotho protein

Form of Klotho	Nuclear	Intracellular	Membrane	Extracellullar
Locale	Nucleus	Cytoplasm	Cell surface	Blood, urine, cerebrospinal fluid
Domain	N/A	Kl1 or full length	Full length	Ectodomain containing Kl1 and Kl2
Biologic function	Anti-age	↓ cytokine production Anti-senescence Anti-age Modulation of Na/K-ATPase PTH release Ca homeostasis	FGF23 dependent:(FGF23 co-receptor for FGF23 signal) Anti-age Anti-IGF Calciophosphoregulatory hormone Suppress PTH, 1,25 VD3 FGF23 independent: release soluble Klotho	FGF23 dependent: same as transmembrane but in endocrine or paracrine mode FGF23 independent: Anti-oxidation Modulation of renal ion channels Anti-Wnt signal Anti-apoptosis Anti-senescence Anti-RAA

CSF: cerebrospinal fluid; IGF: Insulin-like growth factor; N/A: no information; RAA: rennin-angiotensin- aldosterone

PHYSIOLOGICAL ROLE OF KLOTHO IN THE KIDNEY

The kidney is not a mere excretory organ but also a hormonal source producing several active molecules such as 1,25-(OH)₂-Vitamin D₃ (1,25 VD₃), renin, erythropoietin, and Klotho. Klotho exerts multiple actions on the kidney but only selected functions will be highlighted in this article. This includes regulation of 1,25 VD₃ production and modulation of urinary phosphate (Pi), Ca and K excretion.

Interaction with Renal 1,25-(OH)₂-Vitamin D₃ Production

Klotho and the vitamin D system reciprocally regulate each other. In homozygous Klotho-deficient (Kl^−/−) mice, extremely high plasma 1,25 VD₃ was noted as well as up-regulation of 1α-hydroxylase and down-regulation of 24-hydrolase,¹⁵ which provides in vivo genetic, but indirect, evidence that the high circulating 1,25 VD₃ is due to over production and low degradation of 1,25 VD₃ (Figure 1).¹⁵ In addition, Yoshida et al. found that normal genetic responses to vitamin D supplementation including down-regulation of 1α-hydroxylase transcripts, and up-regulation of 24-hydroxylase and vitamin D receptor (VDR) transcripts were impaired in Kl^−/− mice¹⁵ suggesting that normal expression of renal Klotho is required for normal vitamin D homeostasis. On the other hand, the dysregulation of the vitamin D system may be associated with high mortality in both Klotho-deficient and FGF23-deficient mice as diminution of vitamin D activity by a vitamin D-deficient diet¹⁶ or genetically deleting the 1α-hydroxylase gene¹⁷ successfully normalizes plasma 1,25 VD₃ levels and rescues a significant fraction of the phenotypes including renal Pi retention, vascular calcification and short life span in Kl^−/− mice. But the mechanism whereby inactivation of vitamin D activity rescues most phenotypes in Kl^−/− mice is unknown. Recently, Ohnishi and colleagues proposed the role phosphate toxicity in mammalian aging, because reducing blood Pi levels by genetic deletion of Na-Pi cotransporter-2a (NaPi-2a) rescue most phenotypes in Kl^−/− mice.¹⁸ These beneficial effects disappear and premature aging-like features reappear when hyperphosphatemia is induced in mice with double deletion of NaPi-2a and Klotho by feeding with a high-phosphate diet.¹⁸

1,25 VD₃ administration up-regulates renal Klotho expression in normal animals.¹⁶ In vitro study performed by Foster and colleagues showed that there are vitamin D responsive elements in the vicinity of Klotho gene promoter, and that 1,25 VD₃ induces Klotho transcripts in cultured mouse and human kidney cell lines.⁵ Thus 1,25 VD₃ and Klotho forms a negative feedback control loop similar to that of parathyroid hormone (PTH) and vitamin D. A primary increase in 1,25 VD₃ up-regulates Klotho expression which in turn suppresses 1,25 VD₃ production and likewise an increase in Klotho will suppress 1,25 VD₃ to remove a major stimulator of Klotho production (Figure 2 left panel). Novel insights into Klotho-vitamin D interaction will be valuable in understanding 1,25 VD₃ therapy in CKD patients.

Figure 2. Proposed physiological role of Klotho in mineral metabolism and pathophysiologic consequences of Klotho deficiency in CKD.

Left panel: In the setting of normal kidney function with normal Klotho levels, Klotho may suppress FGF23 production and release from the bone. But there is no data to date to prove direct effect of Klotho on FGF23 production in the bone. Klotho functions as coreceptor of FGFR to allow FGF23 to suppress PTH production and release from parathyroid. PTH stimulates and increases plasma levels of FGF23 and 1,25 VD₃. Increased 1,25 VD₃ further stimulates FGF23, and directly and indirectly suppresses PTH levels. Increased 1,25 VD₃ also stimulates Klotho production in the kidney. Taken together, through several negative or positive feedback loops, Klotho functions as both a phosphate and calcium regulatory hormone to directly or indirectly suppress PTH, 1,25 VD₃ and FGF23 production and release. The final outcome of Klotho’s action on the kidney is to prevent renal Pi retention and to prevent renal Ca loss. Right panel: In CDK and ESRD, the network is deranged (red arrows). Renal Klotho is decreased followed by decrease in plasma Klotho. The down-regulation of Klotho increases FGF23 production via unknown mechanism, which in turn suppresses 1,25 VD₃ production in the kidney. Whether low plasma Klotho renders parathyroid gland resistant to the suppressive effect of FGF23 on PTH production is not proven. However, decreased FGFR1/3 and Klotho expression in the uremic parathyroid gland could make the gland resistant to FGF23, and triggers or/and promotes secondary hyperparathyroidism. Low plasma Ca also participates in SHPT development. Hyperphosphatemia amplifies the high FGF23 and PTH, and low Klotho in the blood. The high plasma PTH, Pi and FGF23, and low plasma 1,25 VD₃ and Klotho in concert contribute to development of complications such as metabolic bone disease, secondary hyperparathyroidism, cardiomyopathy, and vascular calcification. Dash line: unproven putative roles of Klotho. Ca: ion calcium; CKD: chronic kidney disease; ESRD: End-stage renal disease; FGFR: FGF receptor; PTH: parathyroid hormone; Pi: phosphate; 1,25 VD₃: 1,25-(OH)₂ vitamin D₃

Modulation of renal ion channel and transporters

Klotho protein was considered as one of the novel phosphatonin soon after its discovery because the Klotho-deficient mouse has severe hyperphosphatemia and transgenic mouse overexpressing Klotho has hypophosphatemia. Subsequent animal and cell culture studies revealed that Klotho does not only control phosphate homeostasis through modulating Na-dependent phosphate cotransporters (NaPi-2a and 2c),^4,19,20 but also regulate calcium homeostasis by modulating renal calcium channel, transient receptor potential ion channel (TRPV5);^21,22 and potassium homeostasis by regulation of the renal outer medullary K channel 1 (ROMK1).²³ One unique aspect is that as a regulator of ion transport, Klotho functions as an enzyme. The two Kl1 and Kl2 repeats in the extracellular domain of membrane Klotho share 20-40% amino acid sequence homology to family 1 glycosidase.^1,24 But two important and highly preserved glutamate residues critical for the glycosidase activity are replaced by an asparagine within Kl1 domain and an alanine or a serine within Kl2 domain respectively rendering the glycosidase activity null.^1,24 However, in vitro studies showed that Klotho possesses β-glucuronidase^4,22,24 or sialidase activity^21,23 through which Klotho exerts regulation of several renal ion channels and transporters. Therefore, Klotho is emerging as a principal “calciophosphoregulatory” hormone.

Inhibition of NaPi-2a activity

Three NaPi cotransporters, NaPi-2a, NaPi-2c, and Pit-2 with different expression in the kidney proximal tubules participate in renal regulation of phosphate homeostasis with different isoforms possessing different substrate specificities, pH sensitivities, and kinetics of response in regulation.²⁵

Two lines of independent studies showed that hyperphosphatemic Kl^−/− mice display increase in activity of NaPi cotransport in the kidney,⁴ and in NaPi-2a and NaPi-2c protein expression compared with wild-type (WT) mice,^4,19 suggesting that hyperphosphatemia at least in part is of renal origin. Most importantly, phosphate restriction^26,27 or induction of renal phosphate leak by deletion of NaPi-2a,¹⁸ successfully rescues the multi-organ failure in the Klotho-deficient mice indicating that phosphotoxicity is a principal pathogenic factor in these animals. Transgenic Klotho-overexpressing mice (Tg-Kl) have lower plasma Pi, while renal fractional excretion of Pi (FE_phos) is increased,⁴ indicating a renal leak of Pi. Soluble Klotho increases FE_phos and decreases plasma Pi in the normal rat and in FGF23 knock-out mice,⁴ indicating that the Klotho-induced phosphaturia is FGF23-independent as well as FGF23 independent.

The direct suppression of NaPi-2a by soluble Klotho protein in a FGF23-independent fashion is mediated by direct inhibition of NaPi cotransport activity without change in protein abundance on cell surface which is a novel mode of regulation for this class of transporters.⁴ This can be mimicked by recombinant β-glucuronidase and blocked by glucuronidase inhibitor, but not affected by sialidase.⁴ The model of direct effect of Klotho on NaPi-2a protein is proposed as follows: glucuronate on a yet unknown substrate is removed by Klotho leading to suppression of NaPi cotransport activity which subsequently renders NaPi-2a protein more susceptible to proteases residing in BBM. While protease inhibitors abolish the proteolysis, they do not reverse the Klotho-induced inhibition of transport,⁴ supporting that Klotho-induced deglycosylation is sufficient to suppress NaPi cotransport activity and that subsequent proteolysis is not required to suppress NaPi transport at acute phase (Figure 1).

Regulation of TRPV5

In addition to being a potent phosphaturic hormone, Klotho also maintains calcium homeostasis.¹⁵. Through its suppression of PTH²⁸ and 1,25 VD₃,¹⁵ Klotho indirectly decreases intestinal calcium absorption. Suppression of PTH is also expected to promote calciuria but the potent action of Klotho on distal calcium reabsorption partially counteracts the reduction in gut absorption. The net effect at the whole organism level is maintenance of balance but with lower turnover. This is supported by the fact that Klotho gene deletion or overexpression has rather mild effects on plasma calcium as opposed to the severe hyper- and hypophosphatemia in Klotho-deficient and Klotho-excess animals respectively.^1,4 We will focus on the most novel aspect, which is the direct effect of Klotho on renal calcium reabsorption (Figure 1).

Chang et al. first showed that soluble Klotho stimulates TRPV5, one of key regulator for urinary calcium excretion by stabilizing TRPV5 on the cell surface and proposed that Klotho function as a glucuronidase.²² Glucuronic acid will be a very uncommon moiety of N-glycan chains on a membrane channel such as TRPV5. A second study by Cha et al. supported an alternative mode of Klotho effect on TRPV5.²¹ Klotho functions as a sialidase which removes α2,6-sialic acids from the N-glycan chains on TRPV5 and exposes underlying N-acetyl-D-lactosamine for binding to galectin-1.²¹ This association prevents TRPV5 from internalization via a dynamin-dependent process, leading to stabilization of more TRPV5 on cell surface. These results clearly support the concept that Klotho is a calciotropic protein to prevent renal calcium loss.

Regulation of ROMK1

ROMK1 is one of the major mediators of urinary K reabsorption. Cha et al. found that acute infusion of Klotho leads to anti-kaliuresis mediated by increased apical membrane abundance of ROMK1.²³ They proposed a mechanism of Klotho action similar to that of TRPV5.²³ Klotho removes terminal sialic acids from N-glycan of ROMK1, and exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galectin-1. Binding to galectin-1 at cell surface prevents clathrin-mediated endocytosis and causes accumulation of functional ROMK1 on the plasma membrane.²³ At the moment, the role of Klotho as a potassium homeostatic hormone is unclear as there does not appear to be disturbances in plasma potassium concentrations in either the Klotho-deficient or over-expressing mice. If Klotho has significant antikaliuretic effects, one should not anticipate undesirable effects in the Klotho-deficient states in AKI and CKD but may theoretically be more concerned with therapeutic administration of Klotho leading to hyperkalemia. These theoretical effects remain to be explored.

PHYSIOLOGICAL ROLE OF KLOTHO IN EXTRA-RENAL ORGANS

Membrane Klotho and soluble Klotho protein exert distinct but possibly overlapping actions. FGFRs are ubiquitously expressed, but FGF23 signal transduction is controlled by co-expression with membrane Klotho.¹² The restricted expression of Klotho protein in very few organs (kidney, heart, brain and parathyroid gland) in concert with multi-functions in multiple tissues (Table 1) suggested that soluble Klotho may function independently of FGFRs as an endocrine hormones and an enzyme to directly modulate target proteins.

Modulation of PTH production

Klotho modulates PTH directly and indirectly. Klotho indirectly regulates PTH production through modulation of plasma levels of 1,25 VD₃, Pi and FGF23. In addition, Klotho may exert a direct effect on PTH production and release (Figure 2 left panel).

In genetically-manipulated mouse whose Klotho gene is replaced by a LacZ reporter, β-X-gal staining is clearly positive in the kidney, the sinoatrial node region of the heart, choroid plexus of brain, and the parathyroid gland,²⁹ which is compatible with earlier findings by RT-PCR.¹ Immunohistochemistry further confirmed the localization of Klotho in the parathyroid and but not the surrounding thyroid tissue,²⁸ thus excluding the possibility that the positive RT-PCR results are due to contamination. Ben-Dov.et al found FGFR1 and FGFR3 in the parathyroid tissue,²⁸ suggesting that FGFRs may form a complex with Klotho and FGF23 in parathyroid gland. FGF23 binds to Klotho/FGFR and activates MAPK cascade signal pathway in cultured cells transfected with Klotho,¹² parathyroid cells,²⁸ as well as in the kidney,^4,10; indicating that parathyroid gland is a likely target organ of FGF23, and Klotho is prerequisite for FGF23 to modulate PTH production.

In vivo animal and in vitro cell culture experiments performed in independent laboratories revealed that FGF23 decreases PTH production, increases expression of both the parathyroid calcium-sensing receptor and the vitamin D receptor (both of which contributes to suppression of PTH), and decreases cell proliferation,^28,30 when normal Klotho and FGFR is expressed in parathyroid glands. Interestingly, FGF23 seems to increase Klotho in parathyroid gland,²⁸ which may positively facilitate FGF23’s action on suppression of PTH production.

The expression of Pit-1, one of NaPi-3 isoforms was found in parathyroid gland and was up-regulated by 1,25 VD₃ and low Pi diet; and down-regulated by vitamin D deficient diet.³¹ Hu et al. showed that Klotho suppresses the activity and expression of Pit-1 in rat vascular smooth muscle cell line in vitro,⁹ but to date, there is no evidence for Klotho effect on Pit-1 in parathyroid.

Ionized calcium activity is a key modulator of PTH synthesis and release from parathyroid. An intriguing model was proposed as an alternative mode of Klotho action on the parathyroid cells.³² In this model, intracellular Klotho binds to Na/K-ATPase to form a complex in response to low intracellular [Ca²⁺] and bring Na/K-ATPase to the cell surface.³² The resultant change in electrochemical gradient was proposed to trigger the release of PTH through unidentified signal pathway.³² Thus Klotho expression in the parathyroid may suppress PTH production by FGF23 signal pathway and increase PTH production by Na/K-ATPase signal pathway stimulated by low blood Ca. However, how the complex of Na/K-ATPase with Klotho is formed in response to low Ca²⁺, how Na/K-ATPase activity is stimulated, and what intracellular signal is required to couple Na/K-ATPase to PTH release remain to be illustrated.

Inhibition of intestinal phosphate absorption

In addition to increased renal reabsorption of Pi, increased absorption of dietary Pi may exacerbate hyperphosphatemia in Kl^−/− mice,¹⁹ because expression of the intestinal phosphate transporter NaPi-2b is significantly higher in Kl^−/− mice than in WT mice.¹⁹ In vitro studies revealed that Klotho directly decreases Pi-induced current in NaPi-2b expressing oocytes,²⁰ which supports the model that Klotho inhibits Pi absorption in the intestine.

In addition, recent study showed that another Na-coupled phosphate transporter Pit-1 protein is also present in the apical membrane of enterocytes of rat duodenum and jejunum; but not in the ileum.³³ Unlike NaPi-2b, PiT-1 protein in the brush border membrane and Pit-1 mRNA expression are not changed in the duodenum or jejunum as a function of dietary Pi.³³ Therefore, the relative contribution of PiT-1 to intestinal Pi absorption remains to be determined; and Klotho’s effect on intestinal Pit-1 remains to be addressed.

Taken together, Klotho is a calciophosphoregulatory protein. The direct effect of Klotho on the kidney is to promote phosphaturia and to prevent renal calcium loss. Klotho regulates Pi and Ca homeostasis directly and by interplay with other calcium and phosphate regulatory hormones: FGF23, PTH, and 1,25 VD₃ (Figure 1 and Figure 2 left panel).

PATHOPHYSIOLOGICAL ROLE OF KLOTHO DEFICIENCY IN KIDNEY DISEASE

Klotho deficiency renders the kidney more susceptible to injury

Liu et al. have shown increased senescence in progenitor cells in many tissues of Kl^−/− mice.³⁴ Knock-down of endogenous Klotho promotes augmentation of senescence in cultured cells.³⁴ Administration of exogenous Klotho significantly decreases senescence in endothelial cells³⁵ and fibroblasts.³⁶ Furthermore, Klotho depletion-induced cell senescence may be associated with up-regulation of Wnt signaling activity because administration of exogenous Wnt accelerates cell senescence in vivo and in vitro, and Wnt signaling is significantly increased in Kl^−/− mice, and suppressed by genetic Klotho overexpression.³⁴ Soluble Klotho can bind to various Wnt family members and inhibit their biological activity.³⁴ Recently, intracellular Klotho is shown to be able to suppress cell senescence by inhibiting retinoic-acid-inducible gene-I-induced expression of IL-6 and IL-8 both in vitro and in vivo.¹⁴ Klotho deficiency from kidney diseases enhances cell senescence induced by oxidative stress.^37-39 Excessive senescence or resultant apoptosis and stem cell deletion may decrease the kidney’s ability to defend against renal insults and impair regeneration.

Klotho is an anti-apoptotic protein. Klotho deficiency significantly increases apoptosis in cultured kidney and endothelial cells;^35,40 and in the kidney.³⁹ Oxidative stress down-regulates Klotho expression and in turn increases cell damage when exposure to H₂O₂ ⁶. Elevation of Klotho by genetic manipulation or viral delivery decreases the number of apoptotic cells and improves kidney function and renal morphology after acute⁴⁰ and chronic kidney damage.³⁹ These results form the basis for potential clinical application of Klotho in the treatment of AKI and CKD.

Klotho deficiency delays kidney recovery

Two independent studies showed that Kl^−/− mice have stem cell depletion in several organs³⁴ and progenitor cell senescence in the kidney,³⁹ which may delay tissue recovery after kidney injury. In addition, Kl^−/− mice have severe abnormal endothelial function⁴¹ and integrity,⁴² and impairment of angiogenesis and vasculogenesis after ischemic limbs⁴³. If those results were translated into ischemic kidney damage, kidney regeneration may be delayed after kidney injury. Increased Klotho by genetic manipulation was shown to accelerate angiogenesis and vasculogenesis, and decrease limb loss, and promote limb recovery after ischemic injury in Kl^−/− mice,⁴⁴ which suggest that Klotho replacement may promote kidney recovery.

Klotho deficiency promote renal fibrosis

Renal fibrosis is not only a histological characteristic in CKD, but also is pathogenic for chronic progression. Transforming growth factor (TGF)-βis considered to contribute to renal fibrosis.⁴⁵ Kl^−/− mice have more glomerular and tubulointerstitial fibrin deposition and higher active plasminogen activator inhibitor-1 (PAI-1) antigen in plasma and PAI-1 mRNA expression in the kidney;⁴⁶ and Kl^−/+ mice have more fibrosis than WT mice do at baseline and after unilateral ureteral ligation to induce unilateral ureteral obstruction (UUO).⁴⁷ Kl^−/+ mice with UUO also have higher TGF-β, and lower Klotho mRNA and protein than WT mice.⁴⁷ The obstructed kidneys from Kl^−/+ mice express significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and TGF-β than those from WT mice.⁴⁷ In cultured rat kidney cell lines, Klotho alleviates TGF-β-induced epithelial-mesenchymal transition or phenotype, suppresses TGF-β-induced target genes activation, and reduces TGF-β1-induced Smad2 phosphorylation,⁴⁸ suggesting that Klotho protein inhibits renal fibrosis primarily through inhibiting TGF-β1 signaling. More importantly and interestingly, Doi et al. recently showed that intraperitoneal injection of soluble Klotho protein suppresses renal fibrosis induced by UUO,⁴⁸ suggesting that soluble Klotho protein may be a novel therapeutic agent for renal fibrosis. In addition to the effect of Klotho on TGF-β1, suppressive effects of Klotho on Insulin-like growth factor pathway² and on Wnt signal pathway³⁴ may be also associated with Klotho’s inhibitory action on renal fibrosis, but so far there is no direct evidence to confirm this concept.

We propose that Klotho deficiency renders the kidney more susceptible to injury, accelerates renal fibrogenesis, retards renal tissue regeneration, and eventually promotes chronic progression. Supplementation of Klotho may provide beneficial impact in preventing and slowing down CKD progression.

PATHOPHYSIOLOGICAL ROLE OF KLOTHO IN THE METABOLIC SYNDROME

The metabolic syndrome (MS) is characterized by obesity, serum lipid profile alterations, hypertension, and fasting hyperglycemia, and is a risk factor for the development of diabetes, cardiovascular disease.^49,50 Recent studies indicate that the MS is also independently associated with an increased risk for incident CKD in non-diabetic adults,⁵¹ as MS patients are at significantly higher risk for microalbuminuria and/or CKD, and the level of risk is related to the number of components of the syndrome. Any component of MS may independently favor the development of renal abnormalities and is potentially considered a modifiable risk factor for CKD. Thus, correct of any component should be a rationale for intervention of MS and can effectively prevent the development and progression of renal damage.

One cross-sectional study examining genetic variants of the Klotho gene polymorphism with MS showed association of the KL-VS variant (F352V and/or C370S within exon 2, simply termed VS alleles) to high blood glucose, high blood pressure, insulin resistance, and trend towards its association with hypertriglyceridemia in Indians.⁵² Several animal models such as diabetes induced by streptozotocin,⁵³ diabetes from leptin deficiency,⁵⁴ spontaneous non-insulin-dependent diabetes (Otsuka Long-Evans Tokushima Fatty, OLETF),^55,56 and hypertension (spontaneous hypertension and volume-dependent hypertension)^55,57 have disclosed dramatic reduction of renal Klotho mRNA or/and protein expression, suggesting that decrease in Klotho may be part of the MS. At present, one does not know whether Klotho is a cause of result of MS, or a parallel phenomenon.

The administration of thiazolidinedione, an agonist of peroxisome proliferator-activated receptor-gammas (PPAR-γ) increases renal Klotho mRNA expression, attenuates abnormal lipid and glucose metabolism, and reduces systolic blood pressure in OLETF rats.⁵⁶ More interestingly, adenovirus-mediated Klotho gene delivery can repeat thiazolidinedione’s action,⁵⁸ indicating the therapeutic potential of Klotho gene delivery in MS.

In rats with streptozotocin-induced diabetes,⁵⁹ Klotho protein in the kidney is notably decreased along with kidney destruction.⁵⁹ Both insulin and phloridzin corrects hyperglycemia, reverses the reduced renal Klotho expression, and improves kidney function and histology of diabetic rats. Klotho protein in Madin-Darby canine kidney cell is reduced by incubation in high glucose medium.⁷ Insulin has been shown to stimulate shedding of extracellular domain of membrane Klotho protien,⁷ which may increase the blood Klotho concentrations.

Similarly, in rats with spontaneous hypertension, Klotho gene delivery via adeno-associated virus carrying mouse Klotho full length cDNA reverses reduced Klotho expression in the kidney, controls blood pressure, improves kidney function, and attenuates renal fibrosis.⁵⁷

Taken together, MS appears to be a state of Klotho deficiency prior to development of kidney injury. Klotho deficiency may render the kidneys more susceptible to acute and chronic renal insults and kidney damage further exacerbates Klotho deficiency. Improvement of glucose metabolism or control of blood pressure could considerably increase Klotho expression in the kidney. On the other hand, Klotho possesses anti-insulin activity and induce insulin resistance.² Unger in 2006 proposed that insulin resistance induced by Klotho may decrease insulin-stimulated intracellular glucose availability and may prevent intracellular caloric and lipid overload and toxicity.⁶⁰ A frequently used medication, PPAR-γ agonist for type II diabetes mellitus has been confirmed to increase Klotho.⁶¹ Thus, modulation of Klotho expression in the kidney is potentially a future treatment option for MS.

PATHOPHYSIOLOGIC ROLE OF KLOTHO DEFICIENCY IN COMPLICATIONS OF CHRONIC KIDNEY DISEASE

Secondary hyperparathyroidism

Secondary hyperparathyroidism (SHPT) is a common and severe complication in CKD.⁶² contributing to renal metabolic bone disease, cardiac disease, and anemia. In addition, hyperphosphatemia, hypovitaminosis D and low expression of VDR are proposed to contribute to maintaining elevation of PTH levels in CKD patients. But if one analyzes the profile of changes of those parameters, it is more likely that these abnormalities are involved in the acceleration of SHPT in later stage rather than triggering them early.⁶² Epidemiologic observation⁶³ and animal studies⁶⁴ show that plasma FGF23 elevation is a very early event. Plasma Klotho decline may even be an earlier event,⁶⁵ which in turn can stimulate FGF23 overproduction. Thus, the conceptual mode of development of SHPT is revised to include aberrant FGF23/Klotho activity to play a more fundamental role in SHPT development (Figure 2 right panel).

CKD in both humans and animals is a state of high plasma level of FGF23⁶⁶ and low FGFR1 and Klotho expression in parathyroid gland;^67-69 which renders FGF23 to lose its inhibitory actions on PTH production, and to fail to increase calcium-sensing receptor and vitamin D receptor (VDR).³⁰ Indeed, Galitzer and colleagues have confirmed that FGF23 fails to decrease plasma PTH, suppress cell proliferation, and activate the MAPK pathway in parathyroid glands of rats with advanced CKD,⁷⁰ indicating that the gland is resistant to FGF23, possibly from low FGFR1 and Klotho (Figure 2 right panel). In addition, aberrant Klotho-Na/K-ATPase axis, a FGF23-independent mode, may be also involved in SHPT development in CKD. Hofman-Bang et al. reported an unexpected finding of an increase in Klotho, FGFR and Na/K ATPase in the parathyroid glands of early CKD rats,⁷¹ which is seemingly opposite to findings by other laboratories,^30,68-70 but may be due to different stages of CKD model and different levels of plasma calcium concentrations. These authors further reasoned that when plasma calcium levels are in normal range, the elevated expression of Klotho in parathyroid glands will be reduced,⁷¹ because Imura and colleagues proposed that low plasma calcium increases intracellular Klotho.³² Thus far, a model can be constructed that PTH production in CKD may be controlled by two Klotho dependent signal pathways. In early CKD, Klotho-Na/K-ATPase signaling is over-active due to higher expression of Klotho and FGFR in the parathyroid glands and possibly regulated by plasma calcium concentration. Hypocalcemia promotes formation of Klotho/Na/K-ATPase complex and induce PTH synthesis in the parathyroid. The elevation of plasma FGF23 with high expression of Klotho and FGFR in the parathyroid glands should decrease PTH secretion to maintain normal mineral metabolism. However in advanced CKD, low expression of Klotho and FGFR in the parathyroid glands blunts suppressive activity of FGF23/Klotho signaling. The non-unanimous blood levels of PTH in CKD patients would be interpreted by the complicated mechanisms of PTH modulation. Thus, Klotho restoration in the parathyroid with normalization of plasma Ca may be a novel approach to block SHPT development.

Cardiovascular calcification

Cardiovascular disease is the leading cause of mortality of CKD and cardiovascular calcification is prevalent in CKD. In addition to the classical traditional factors, FGF23 and Klotho are novel contributors to ectopic calcification in soft tissues including aorta (Figure 2B).^1,9,72,73 Thus far, there are no effective targeted therapies for cardiovascular disease in CKD other than manipulation of the classical risk factors.

Tangri et al. examined association of functional Kl-VS variant of Klotho with valvular and vascular calcification on 1389 cases and 2139 controls from the Framingham Heart Study Offspring Cohort⁷⁴ and did not observe any association of the Kl-VS variant of Klotho valvular or vascular calcification.⁷⁴ However, experimental animal studies documented that CKD is a state of systemic Klotho deficiency (Table 2). Kl^−/− mice have extensive ectopic calcification in soft tissues akin to that observed in CKD subjects suggesting a pathogenetic association between Klotho deficiency and calcification. Increase in Klotho by genetic manipulation inhibits vascular calcification in CKD animals.⁹ The suppressive influence of Klotho on vascular calcification in CKD is multi-factorial including (1) decreasing plasma Pi by promoting negative Pi balance,^4,72,73 (2) inhibiting Pi-induced Pit-1 and Pit-2 activation in the vasculature,⁹ (3) suppressing cell senescence, apoptosis, and death in vascular endothelial cells and smooth muscle cells induced by a variety of insults including Pi,^36,38,75 (4) serving as an anti-inflammatory modulator.^14,54

Table 2.

Comparison of phenotypes Klotho deficiency and CKD

	Klotho deficiency	Chronic kidney disease
Blood chemistry
Phosphate	↑ ↑ ↑ ↑	↑ or ↑↑↑*
Calcium	↑	↔ or ↓↓
Creatinine	↑	↑ ↑ ↑
1,25 VD3	↑ ↑ ↑	↓ ↓ ↓
PTH	↔ or ↓	↑ ↑
FGF23	↑ ↑ ↑	↑ ↑
Klotho	↓↓↓ or disappear	↓↓ at ESRD**
Gross phenotypes
Body weight	↓ ↓ ↓	↓ ↓
Growth retardation	↓ ↓ ↓ ↓	↓↓ in children
Physical activity	↓ ↓ ↓	↓
Fertility	↓ ↓ ↓ ↓	↓ ↓
Life span	↓ ↓ ↓ ↓	↓ ↓
Cardiovascular disease
Cardiac hypertrophy	↑ ↑	↑ ↑ ↑
Cardiac fibrosis	↑ ↑	↑ ↑ ↑
Vascular calcification	↑ ↑ ↑ ↑	↑ ↑
Arthrosclerosis	↑ ↑	↑ ↑ ↑
Blood pressure	↑	↑ ↑ ↑ ↑
Hematocrit levels	↓	↓ ↓ ↓ ↓
Bone disease	↓ ↓ ↓	↓ ↓ ↓

^*at early CKD, blood Pi is in normal range;

^**blood Klotho may be increased in early CKD⁸⁹; ESRD: end-stage renal disease

Data from independent laboratories showed that Klotho and FGFR1/3 protein or/and mRNA, but not FGF23 and FGFR4, are expressed in human vasculature.^76,77 and human aorta derived-smooth muscle cells.⁷⁷ Interestingly, both Klotho and FGFR1/3 expression are down-regulated in human arteries from CKD patients who have vascular calcification.⁷⁷ When human aorta derived-smooth muscle cells are incubated with pooled uremic blood, high Pi, and Ca; both FGFR1/3 and Klotho expression is down-regulated and vascular smooth muscle cells undergoing “osteogenic-chondrogenic” trans-differentiation are triggered.⁷⁷_ENREF_87 Vascular Klotho deficiency by Klotho knockdown potentiates the development of calcification and decrease the ability of vascular tissue to respond to FGF23.⁷⁷_ENREF_87 They also showed that vascular Klotho deficiency driven by pro-calcific stressors could be restored by vitamin D receptor (VDR) activators in vitro cell culture model and ex vivo model by using human arterial organ cultures from CKD patients.⁷⁷ Furthermore, VDR activators exert its anti-calcific effects by restoration of Klotho and regain of FGF-23 responsiveness.⁷⁷ This study provides some novel insights into vascular calcification in uremia: both chronic metabolic and mechanistic stress could induce vascular calcification by dysregulation of Klotho/FGF-23 signaling. Administration of VDR activators can restore Klotho expression and unmask FGF-23 anti-calcific effect.⁷⁷ This concept is similar to the mode of the effect of defect in FGF23/Klotho signaling in SHPT in CKD (Figure 2 right panel).

Recently, one study examining the role of stanniocalcin (STC) 1 and 2, which are up-regulated in the kidney of Kl^−/− mice,⁷⁸ in ectopic calcification. STC2 protein is focally localized with the calcified lesions of renal arterioles, renal tubular cells, heart and aorta in Kl^−/− mice. High Pi medium increases STC2 mRNA levels as well as that of osteocalcin, osteopontin, and PiT-1 in rat aortic vascular smooth muscle. Interestingly, knockdown with a small interfering RNA or the overexpression of STC2 showed acceleration and inhibition of Pi-induced calcification respectively in this cell line.⁷⁹ These results suggest that STC2 might represent a novel target for ectopic calcification in the condition of Klotho deficiency.

The prospect of Klotho restoration affecting the course of CKD in terms of prevention, forestalling, slowing, or reversal of vascular calcification is a high priority question in terms of therapeutics and requires investigation.

Cardiac remodeling

Cardiac “remodeling” which is often used as a general term of changes in cardiac structure and function with implied negative connotations is used here to describe the cardiac hypertrophy and fibrosis in CKD; sometimes referred to as “uremic cardiomyopathy”, which distinguishes it pathogenically from the highly prevalent hypertensive and ischemic cardiomyopathy. In addition to traditional cardiovascular risk factors, novel risk factors such as vitamin D deficiency,⁸⁰ high plasma FGF23,^81,82 and low plasma Klotho⁸¹ can all contribute to cardiac hypertrophy (Figure 2 right panel).

In the heart, Klotho is expressed solely at the sinoatrial node.²⁹ Kl^−/− mice have sinoatrial conduction defects²⁹ which may be a cause of sudden death under restraint stress. The absence of degenerative structural change in sinoatrial node of Kl^−/− mice²⁹ suggests that haplo-sufficient levels of Klotho might be sufficient to maintain the function of sinoatrial node as a pacemaker.

One recent study showed that Klotho deficiency is associated with cardiac hypertrophy.⁸¹ Klotho-deficient mice have left ventricular hypertrophy which is not hypertension-dependent, but may be FGF23-dependent because (1) either intramyocardial and intravenous injection of FGF23 result in cardiac hypertrophy in WT mice, (2) an FGFR blocker attenuates cardiac hypertrophy in 5/6 nephrectomized CKD, but does not change blood pressure.⁸¹ One interpretation is the importance of FGF23 but the effect of low Klotho is equally likely due to possible interplay between Klotho and FGF23 in the pathogenesis of cardiac remodeling in CKD. It is clear that uremic cardiomyopathy is a complex metabolic disease that is way beyond the classical cardiac risk factors. In addition to FGF23 and Klotho, aberrant vitamin D and phosphate metabolism and cardiac renin-angiotension-aldosterone activation may also participate in cardiac hypertrophy in CKD (Figure 2 right panel).

KLOTHO AS POTENTIAL THERAPEUTIC AGENT

Although there is no published clinical study showing therapeutic efficacy of Klotho administration in either acute or chronic kidney disease, animal experiments thus far have shown unequivocal therapeutic effects of Klotho gene delivery or direct administration of Klotho protein on several models. Restoration of endogenous Klotho or administration of exogenous Klotho potentially provides novel treatment strategies for CKD patients. We will highlight recent advances of Klotho administration in animal models of kidney disease.

Administration of exogenous Klotho

Animal studies have shown that delivery of Klotho gene via viral carrier efficiently protects kidney from acute injury induced by ischemia reperfusion;⁸³ and also affect the course of disease progression of CKD.^57,58,84 Compared with Klotho gene delivery to animal subjects, administration of recombinant Klotho protein might be a safer and easier approach to correct endocrine Klotho deficiency.

In vitro studies have shown that soluble Klotho, a full length of the extracellular domain, is active in inhibition of IGF signal transduction,² suppression of Wnt signal,³⁴ modulation of several ion channel or transporter^4,21,23 or control of FGF23 signal transduction.¹² In vivo studies further revealed its therapeutic potential in several kidney disease models. Klotho administration has been proven successful in protection of kidney function in AKI animals induced by IRI.⁸⁵ More recently, same Klotho preparation was also documented to be efficient in inhibition of renal fibrosis and in better preservation of renal function in UUO model.⁴⁸ Thus, exogenous Klotho protein supplementation may be a potentially feasible way of replacement therapy in Klotho-deficient states.

Up-regulation of endogenous Klotho protein

Transgenic overexpression of Klotho to plasma levels about twice normal² is effective in preventing kidney injury acutely from ischemia-reperfusion,⁸⁵ immune-mediated glomerulonephritis,³⁹ and in a renal ablation model of CKD.⁹ This provides the basis for developing ways to increase endogenous Klotho expression via stimulation or removal of suppression of Klotho when residual kidney function is somewhat preserved and endogenous Klotho-producing cells in the kidney are not destroyed but simply suppressed. Furthermore, strategies to increase Klotho production in extra-renal tissues might be of particular importance for ESRD patients whose functional kidney tissue is totally lost.

It has been shown that peroxisome proliferator-activated receptor-γ agonist,⁸⁶ and anti-oxidants⁶ increase renal Klotho expression in AKI animals. Some animal experiments and cell culture studies showed that restricting Pi intake,²⁶ active vitamin D,¹⁶ inhibition of 3-hydroxy-3-methylglutaryl CoA reductase,⁸⁷, and inhibition of Ang II,⁸⁸ elevate Klotho expression in the kidney or/and in cultured cell lines. Therefore, these conventional maneuvers potentially offer additional therapeutic mechanisms for treating Klotho deficiency and their clinical utility needs to be explored.

CONCLUSION AND PERSPECTIVES

In concert, animal data and the limited clinical observations to date are overwhelmingly strong to suggest that Klotho is a pleiotropic protein and plays multiple physiological roles in modulation of kidney function and pathophysiological roles in acute kidney damage, progression of CKD, and extra-renal complications in CKD. Klotho is not merely an early biomarker for kidney damage, but also has a pathogenic role for kidney disease. The understanding of the renal and extra-renal actions of Klotho will advance novel strategies for both diagnosis and treatment of AKI or/and CKD.

The potential utility of Klotho in clinical practice is anticipated to be at least two-fold. First, Klotho may serve as an early and sensitive biomarker to kidney diseases. But its specificity and its prognostic value and differential diagnostic value in humans remain to be examined. Second, Klotho exogenous supplementation or/and up-regulation of endogenous Klotho production may provide novel therapy for AKI patients to retard or block its progression to CKD and for CKD by arresting or slowing progression as well as preventing and reversing complications. As one is moving to use Klotho as novel diagnostic, prognostic, and therapeutic strategy for CKD patients, an identification of proper indications including CKD stage, composition of abnormal mineral metabolism, and state of complications is of the highest priority.