Renal Klotho Expression Research Articles

Сanonical WNT signaling and myocardial remodeling in arterial hypertension and chronic kidney dysfunction

INTRODUCTION . Beta-catenin is a structural protein of adhering junction and intercalated discs of cardiomyocytes as well as the main intracellular messenger of the canonical WNT (cWNT) signaling pathway. The dysregulation of the cWNT signaling and the rearrangement of the cardiomyocyte cytoskeleton accompany the cardiovascular disorders in chronic kidney disease (CKD). THE AIM : to investigate the expression and distribution of β-catenin, calcineurin A, and TGF-β1 in the myocardium of spontaneously hypertensive rats (SHR) with CKD, sham operated SHR and Wistar Kyoto rats of the corresponding age. MATERIAL AND METHODS . Systolic blood pressure (BP), heart rate (HR), myocardial mass index (MMI), creatinine concentration (Cr), myocardial beta-catenin expression and renal Klotho expression, morphological light-optical study of kidney and myocardium tissues was performed in sham operated (SO) Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and SHR with 5/6 nephrectomy (Nx). RESULTS . SHR rats showed higher values of BP, MMI, cardiomyocyte diameter, myocardial fibrosis area, and lower Klotho levels compared to WKY rats. Nx SHR had lower kidney function and renal Klotho expression, higher BP and MMI compared to SHR. An increase in the cardiomyocytes diameter and the area of myocardial fibrosis was accompanied by the overexpression of β-catenin, calcineurin A, and TGF-β1 in the myocardium. CONCLUSION . The upregulation of canonical Wnt signaling and cellular programs of cardiomyocyte hypertrophy mediated by Klotho deficiency can be involved in myocardial remodeling in chronic renal dysfunction.

Klotho ameliorates diabetic nephropathy via LKB1-AMPK-PGC1α-mediated renal mitochondrial protection

Diabetic nephropathy (DN) is associated with renal mitochondrial injury and decreased renal klotho expression. Klotho is known as an aging suppressor, and mitochondrial dysfunction is the hallmark of aging. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is a master regulator of mitochondrial biogenesis, and adenosine monophosphate-activated protein kinase (AMPK) is known as a guardian of mitochondria. Here, we report that recombinant soluble klotho protein (rKL) protects against DN in db/db mice via PGC1α-AMPK-mediated mitochondrial recovery in the kidney. We injected rKL into db/db and db/m mice for 8 weeks and collected the serum and kidney tissue. We treated murine renal tubular cells with rKL in vitro, with and without exposure to 30 mM high glucose (HG). rKL treatment ameliorated major disorders from diabetes, such as obesity, hyperglycemia, and intrarenal reactive oxygen species (ROS) generation, in db/db mice. rKL also diminished albuminuria, recovered renal proximal tubular mitochondria, increased renal p-AMPK and PGC1α, and down-regulated mTOR/TGF-β in db/db mice. In S1 mouse proximal tubular cells, rKL treatment ameliorated HG-mediated cellular and mitochondrial damage and enhanced oxidative phosphorylation, with an increase in PGC1α-AMPK-induced mitochondrial recovery. Our data suggest that klotho exerts a mitochondrial protective effect in diabetic kidney disease by inducing AMPK-PGC1α expression.

Hydrogen sulfide attenuates renal fibrosis by inducing TET-dependent DNA demethylation on Klotho promoter.

Hypoxia is a key pathogenetic characteristic of chronic kidney disease (CKD). Klotho has renoprotective effect and its expression is commonly suppressed in CKD patients. We showed that chronic hypoxia in unilateral ureteral obstruction model mice is associated with renal Klotho promoter methylation and expression silencing. Administration of low-dose of sodium hydrosulfide (NaHS) effectively ameliorated renal tubulointerstitial fibrosis in the mouse model by demethylating Klotho promoter and restoring its expression. Mechanistically, hypoxia microenvironment in CKD reduced cellular oxygen availability and Fe2+ concentration, and led to impaired activity of ten-eleven translocation (TET), which is critical in maintaining Klotho promoter demethylation status. NaHS treatment greatly improved hypoxia condition, restored TET activity, reversed DNA methylation, and thus, increased Klotho expression. Our results strongly suggested that correcting hypoxia condition to restore TET activity could be a promising therapeutic strategy against CKD.

Cardiomyocyte TRPC6 overexpression as one of the myocardial hypertrophy mechanisms in chronic kidney dysfunction

BACKGROUND. Klotho is a transmembrane and circulating protein primarily synthesized by the kidney. Klotho deficiency characterizes chronic kidney disease (CKD), as myocardial hypertrophy (GM). The cardioprotective effect of the Klotho protein is due to the negative regulation of a variety of stress signals, leading to the activation of the hypertrophic intracellular signaling pathway calcineurin (CaN) / NFAT in the myocardium. The effect of Klotho may presumably be mediated by the modulation of Ca2 + channels and / or Foxo factors essential for CaN signaling. THE AIM: to study the activity of CaN/ NFAT signaling pathway in the myocardium and to determine the molecular mechanisms of its regulation in conditions of Klotho level decrease in spontaneous hypertensive rats (SHR) with experimental CKD. MATERIAL AND METHODS. The experimental model of CKD was 3/4 or 5/6 nephrectomy (Nx) in SHR. Sham-operated (SO) SHR, and Wistar Kyoto rats (WKY) were used as controls. In all animals were measured systolic blood pressure, myocardial mass index – MMI, creatinine clearance, cardiomyocyte (CM) diameter, Klotho levels in serum (ELISA) and kidney (IHC), myocardial expression of calcineurin (IHC, PCR), transcription factor NFAT (IHC), TRPC6 (IHC), FOXO3A (PCR) and phosphor-Foxo1/3/4 (IHC). The tissue expressions of calcineurin, TRPC6, and Klotho were calculated as the IHC specific product area to the field of view ratio. NFAT expression was evaluated as the positively stained nuclei to the number of nuclei ratio in the field of view. Measurements were performed in 10 fields of view for each histology slide. RESULTS. The model has corresponded to the initial stages of CKD. The increase in MMI (p = 0.005) and CM diameter (p = 0.002) were observed compared in Nx rats to SO. Renal Klotho expression (p < 0.001), and serum Klotho level (p = 0.019) were lower in the Nx. In multiple linear regression analyzes, the values of MMI and CM thickness were independently associated with the level of renal Klotho protein (β = -0.38 ± 0.16, p = 0.026, β = -0.64 ± 0.14, p <0.001, respectively). Nx and systemic hypertension were accompanied by an increase in the expression of the calcineurin gene (p = 0.005) and cytoplasmic calcineurin in CM (p = 0.004), the number of NFAT-positive nuclei (p = 0.007), and an increase in the expression of the FOXO3A gene (p <0.001) in the absence of accumulation of phosphorylated Foxo1/3/4 in CM cytoplasm. SHR rats were characterized by positive IHC staining for TRPC6 compared to WKY (p = 0.004). The expression of calcineurin and TRPC6 varied co-directionally (r = 0.69, p <0.001), and both of these indicators were associated with the Klotho levels (calcineurin vs Klotho in the kidney, r = -0.73, p <0.001; TRPC6 vs Klotho in serum, r = -0.43, p = 0.025). CONCLUSION. The development of Klotho deficiency on early-stage CKD is associated with the expression of transient Ca2+ channels TRPC6 and activation of the calcineurin / NFAT hypertrophic signaling pathway in cardiomyocytes.

MO026TREATMENT WITH ACTIVE VITAMIN D DOES NOT IMPROVE LEFT VENTRICULAR HYPERTROPHY BUT FURTHER INCREASES FGF23 AND ACCELERATES CKD PROGRESSION IN CHILDREN

Abstract Background and Aims Elevated FGF23, activation of the renin-angiotensin-aldosterone system (RAAS), and vitamin D and Klotho deficiency promote left ventricular hypertrophy (LVH) and LV fibrosis in chronic kidney disease (CKD). Whether treatment with active vitamin D affects uremic cardiomyopathy and/or FGF23/Klotho system in CKD is unclear. Method 5/6 nephrectomized (5/6Nx) rats, as a well-established model of CKD, were dose-dependently treated with calcitriol for 10 weeks and the cardiac phenotype, including local RAAS activation, was investigated compared to untreated 5/6Nx and sham-operated rats. Additionally, 38 children with eGFR ≤ 30 ml/min/1.73 m2 selected from the Cardiovascular Comorbidity in Children with CKD (4C) study were enrolled in a case-control study. Nineteen children were started on active vitamin D treatment with either calcitriol or calcidiol (average dosage 0.4 μg/day), and 19 CKD children with non-active vitamin D treatment matched for age, eGFR, serum calcium, parathyroid hormone, and phosphate served as controls. LV mass index (LVMI), LVH prevalence (LVMI >95th percentile), intact (iFGF23) and C-terminal FGF23 (cFGF23), and soluble Klotho (sKlotho) were assessed at baseline and after nine months. Results In 5/6Nx rats, FGF23 synthesis in bone and heart was enhanced compared to sham-operated rats, and calcitriol further stimulated its expression in bone only. Renal Klotho expression was reduced in 5/6 rats and almost normalized after calcitriol treatment. 5/6Nx induced cardiomyocyte growth and LV fibrosis, and calcitriol treatment significantly ameliorated both. Cardiac RAAS genes, such as angiotensinogen, renin, angiotensin-converting-enzyme, and angiotensin II receptor type I, were significantly upregulated in 5/6Nx rats compared to sham-operated animals, and normalized by calcitriol treatment. In the 4C study cohort, median plasma cFGF23 and iFGF23 were significantly elevated in both groups, whereas sKlotho was not altered compared to healthy children. Forty-seven percent and 42% of active vitamin D-treated patients and controls, respectively, received blood pressure (BP) medications, however diastolic BP was slightly but significantly enhanced in both groups compared to healthy children. Mean LVMI and LVH prevalence of 47% in active vitamin D-treated patients and controls did not differ at baseline. After nine months, median cFGF23 and iFGF23 were significantly increased in active vitamin D-treated patients compared to controls. sKlotho levels were decreased in the active vitamin D-treated group compared to baseline but not in controls. A significant decline in median eGFR was observed in active vitamin D group but not in controls. Likewise, the median eGFR change during the observation period was higher in the active vitamin D group compared to controls. Median LVMI as well as frequency of LVH did not change significantly during vitamin D treatment, nor in controls. Consequently, final median LVMI as well as percentage of patients with LVH did not significantly differ between groups. Conclusion Calcitriol treatment in 5/6Nx rats improves cardiac hypertrophy and fibrosis, reduces uremia-induced cardiac RAAS gene expression, and improves renal Klotho expression. By contrast, treatment with active vitamin D does not decrease the prevalence of LVH in children with advanced CKD compared to matched controls. This may be due to further enhancement of FGF23 levels, faster progression of CKD associated with reduced Klotho in active vitamin D-treated patients and/or concomitant treatment with RAAS inhibitors. The latter may blunt the favorable effects of calcitriol on the activated RAAS.

Klotho supplementation ameliorates blood pressure and renal function in DBA/2-pcy mice, a model of polycystic kidney disease.

Klotho interacts with various membrane proteins such as receptors for transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF). Renal expression of klotho is diminished in polycystic kidney disease (PKD). In the present study, the effects of klotho supplementation on PKD were assessed. Recombinant human klotho protein (10 μg·kg-1·day-1) or a vehicle was administered daily by subcutaneous injection to 6-wk-old mice with PKD (DBA/2-pcy). Blood pressure was measured using tail-cuff methods. After 2 mo, mice were killed, and the kidneys were harvested for analysis. Exogenous klotho protein supplementation reduced kidney weight, cystic area, systolic blood pressure, renal angiotensin II levels, and 8-epi-PGF2α excretion (P < 0.05). Klotho protein supplementation enhanced glomerular filtration rate, renal expression of superoxide dismutase, and klotho itself (P < 0.05). Klotho supplementation attenuated renal expressions of TGF-β and collagen type I and diminished renal abundance of Twist, phosphorylated Akt, and mammalian target of rapamycin (P < 0.05). Pathological examination revealed that klotho decreased the fibrosis index and nuclear staining of Smad in PKD kidneys (P < 0.05). Our data indicate that klotho protein supplementation ameliorates the renin-angiotensin system, reducing blood pressure in PKD mice. Furthermore, the present results implicate klotho supplementation in the suppression of Akt/mammalian target of rapamycin signaling, slowing cystic expansion. Finally, our findings suggest that klotho protein supplementation attenuated fibrosis at least partly by inhibiting epithelial mesenchymal transition in PKD.

Inflammation both increases and causes resistance to FGF23 in normal and uremic rats.

Fibroblast growth factor 23 (FGF23) increases phosphorus excretion and decreases calcitriol (1,25(OH)2D) levels. FGF23 increases from early stages of renal failure. We evaluated whether strict control of phosphorus intake in renal failure prevents the increase in FGF23 and to what extent inflammation impairs regulation of FGF23. The study was performed in 5/6 nephrectomized (Nx) Wistar rats fed diets containing 0.2-1.2% phosphorus for 3 or 15 days. FGF23 levels significantly increased in all Nx groups in the short-term (3-day) experiment. However, at 15 days, FGF23 increased in all Nx rats except in those fed 0.2% phosphorus. In a second experiment, Nx rats fed low phosphorus diets (0.2 and 0.4%) for 15 days received daily intraperitoneal lipopolysaccharide (LPS) injections to induce inflammation. In these rats, FGF23 increased despite the low phosphorus diets. Thus, higher FGF23 levels were needed to maintain phosphaturia and normal serum phosphorus values. Renal Klotho expression was preserved in Nx rats on a 0.2% phosphorus diet, reduced on a 0.4% phosphorus diet, and markedly reduced in Nx rats receiving LPS. In ex vivo experiments, high phosphorus and LPS increased nuclear β-catenin and p65-NFκB and decreased Klotho. Inhibition of inflammation and Wnt signaling activation resulted in decreased FGF23 levels and increased renal Klotho. In conclusion, strict control of phosphorus intake prevented the increase in FGF23 in renal failure, whereas inflammation independently increased FGF23 values. Decreased Klotho may explain the renal resistance to FGF23 in inflammation. These effects are likely mediated by the activation of NFkB and Wnt/β-catenin signaling.

Selective inhibition of class IIa histone deacetylases alleviates renal fibrosis.

In this study, we examined the effect of MC1568, a selective class IIa histone deacetylase (HDAC) inhibitor, on the development and progression of renal fibrosis in a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO). All 4 class IIa HDAC isoforms, in particular HDAC4, were up-regulated in renal epithelial cells of the injured kidney. Administration of MC1568 immediately after UUO injury reduced expression of α-smooth muscle actin (α-SMA), fibronectin, and collagen 1. MC1568 treatment or small interfering RNA-mediated silencing of HDAC4 also suppressed expression of those proteins in cultured renal epithelial cells. Mechanistically, MC1568 abrogated UUO-induced phosphorylation of Smad3, NF-κB, and up-regulation of integrin ɑVβ6 in the kidney and inhibited TGF-β1-induced responses in cultured renal epithelial cells. MC1568 also increased renal expression of klotho, bone morphogenetic protein 7, and Smad7. Moreover, delayed administration of MC1568 at 3 d after ureteral obstruction reversed the expression of α-SMA, fibronectin, and collagen 1 and increased expression of matrix metalloproteinase (MMP)-2 and -9. Collectively, these results suggest that selectively targeting class IIa HDAC isoforms (in particular HDAC4) may inhibit development and progression of renal fibrosis by suppressing activation and expression of multiple profibrotic molecules and increasing expression of antifibrotic proteins and MMPs.-Xiong, C., Guan, Y., Zhou, X., Liu, L., Zhuang, M. A., Zhang, W., Zhang, Y., Masucci, M. V., Bayliss, G., Zhao, T. C., Zhuang, S. Selective inhibition of class IIa histone deacetylases alleviates renal fibrosis.

Renal ischemia-reperfusion injury impairs renal calcium, magnesium, and phosphate handling in mice.

Fibroblast growth factor 23 (FGF23) levels are elevated in patients with acute kidney injury (AKI). The consequences on renal Ca2+, Mg2+, and Pi regulatory mechanisms are unknown. We hypothesized that renal ischemia-reperfusion (I/R) injury alters the expression of important renal Ca2+, Mg2+, and Pi transport proteins. I/R injury was induced in male C57BL/6 mice by clamping both renal arteries for 27min. Mice were investigated 18h later. The mRNA and protein levels of renal Ca2+, Mg2+, and Pi transport proteins were measured by RT-qPCR and western blot analysis. I/R injury-induced hyperphosphatemia and hypermagnesemia were paralleled by a decrease in glomerular filtration rate and an increase in the fractional excretion of Ca2+, Mg2+, and Pi. I/R injury affected the fibroblast growth factor 23 (FGF23)-klotho-vitamin D axis by increasing plasma levels of FGF23 and downregulation of renal klotho expression. Plasma levels of PTH and 1,25-dihydroxyvitamin D3 were unchanged. Further, downregulation of key genes for paracellular reabsorption of Ca2+ and Mg2+ (claudin (Cldn)2, Cldn10b, Cldn16, Cldn19) and for active transcellular transport of Ca2+, Mg2+, and Pi (calbindin-D28K, Ncx1, Pmca4, Cnnm2, Trpm7, NaPi-2a, and NaPi-2c) was observed. However, renal expression of Trpv5 and Trpv6 was increased. In vitro studies support a direct effect of proinflammatory cytokines on the mRNA expression of Cldn16, Cldn19, and Trpv6. Our findings indicate that renal I/R injury increases FGF23 blood levels independent of PTH and 1,25-dihydroxyvitamin D3. This increase is associated with hypermagnesemia, hyperphosphatemia, and increased or decreased expression of specific renal Ca2+, Mg2+, and Pi transporters, respectively.

Klotho Ameliorates Medullary Fibrosis and Pressure Natriuresis in Hypertensive Rat Kidneys.

Renal expression of klotho is reduced in hypertension. Experiments were performed to examine whether exogenous klotho protein supplementation ameliorates pressure natriuresis in early phase of hypertension, using stroke-prone spontaneously hypertensive rats (sp-SHR). The interactions between klotho protein and renal renin-Ang (angiotensin) system were examined with immunoprecipitation and cell culture methods. Uninephrectomy was performed in sp-SHRs to induce nephrosclerosis, and they were treated with exogenous klotho protein or vehicle. Exogenous klotho protein supplementation to sp-SHR decreased blood pressure, renal Ang II levels, AGT (angiotensinogen) expression, HIF (hypoxia-inducible factor)-1α abundance, and medullary fibronectin levels, with increased renal klotho expression and serum and urine klotho levels. Klotho supplementation also reduced kidney weight, renal phosphorylated Akt, and mTOR (mammalian target of rapamycin) abundance. Furthermore, klotho supplementation restored renal autoregulation of glomerular filtration rate and enhanced pressure-induced natriuresis in sp-SHR. Klotho protein bound to AT1R (Ang II type-1 receptor) and decreased the presence of AT1R on HK-2 (human proximal tubular) cells, attenuating inositol triphosphate generation. Klotho protein suppressed Ang II-induced increments of AGT expression in HK-2 cells. Collectively, the present data demonstrate that klotho binds with the AT1R to suppress Ang signal transduction, participating in inactivating renal renin-Ang system. Our results also suggest that exogenous klotho supplementation represses Akt-mTOR signaling to reduce renal hypertrophy and restore the autoregulatory ability of glomerular filtration rate in uninephrectomized sp-SHRs. Finally, the present findings implicate that klotho supplementation inhibits HIF-1α pathway and medullary fibrosis, contributing to enhancements of pressure natriuresis and reduction in blood pressure.

Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes.

Klotho interacts with various membrane proteins, such as receptors for transforming growth factor (TGF)-β and insulin-like growth factor (IGF), to alter their function. Renal expression of klotho is diminished in diabetes. The present study examined whether exogenous klotho protein supplementation ameliorates kidney injury and renin-angiotensin system (RAS) in db/db mice. We investigated the effects of klotho supplementation on diabetic kidney injury and RAS. Recombinant human klotho protein (10μg/kg/d) was administered to db/db mice daily. Klotho protein supplementation reduced kidney weight, systolic blood pressure (SBP), albuminuria, glomerular filtration rate, and 8-epi-prostaglandin F2α excretion without affecting body weight. Although klotho supplementation did not alter glycated albumin, it reduced renal angiotensin II levels associated with reduced renal expression of angiotensinogen. Klotho supplementation improved renal expression of superoxide dismutase (SOD), and endogenous renal expression of klotho. Klotho supplementation reduced the levels of hypoxia-inducible factor, phosphorylated Akt, and phosphorylated mTOR and decreased the renal expression of TGF-β, tumour necrosis factor (TNF), and fibronectin. These data indicate that klotho supplementation reduces blood pressure and albuminuria along with ameliorating renal RAS activation in db/db mice. Furthermore, these results suggest that klotho inhibits IGF signalling, induces SOD expression to reduce oxidative stress, and suppresses Akt-mTOR signalling to inhibit abnormal kidney growth. Collectively, the results suggest that klotho inhibits TGF-β and TNF signalling, resulting in a decline in renal fibrosis.

Endotoxaemia differentially regulates the expression of renal Ca2+ transport proteins in mice.

Alterations in parathyroid hormone (PTH) and/or vitamin D signalling are frequently reported in patients with sepsis. The consequences on renal and intestinal Ca2+ and Pi regulatory mechanisms are still unclear. We hypothesized that endotoxaemia alters the expression of important renal and intestinal Ca2+ and Pi transport proteins. Male C57BL/6 mice were treated with lipopolysaccharide (LPS; 3mg/kg; i.p.). The mRNA and protein levels of renal and intestinal Ca2+ and Pi transport proteins were measured by RT-qPCR, immunohistochemistry and western blot analysis. Lipopolysaccharide-induced hypocalcaemia and hyperphosphataemia was paralleled by a decrease in glomerular filtration rate and urinary excretion of Ca2+ and Pi . Endotoxaemia augmented plasma levels of PTH and affected the fibroblast growth factor 23 (FGF23)-klotho-vitamin D axis by increasing plasma levels of FGF23 and downregulation of renal klotho expression. Renal expression of CYP27b1 and plasma levels of 1,25-dihydroxyvitamin D3 were increased in response to LPS. Endotoxaemia augmented the renal expression of TRPV5, TRPV6 and PiT1, whereas the renal expression of calbindin-D28K , NCX1, NaPi -2a and NaPi -2c were decreased. Incubation of primary distal tubule cells with LPS increased TRPV6 mRNA levels. Furthermore, LPS decreased the intestinal expression of TRPV6, calbindin-D9K and of NaPi -2b. Our findings indicate that endotoxaemia is associated with hypocalcaemia and hyperphosphataemia and a disturbed FGF23-klotho-vitamin D signaling. Further, LPS-induced acute kidney injury was accompanied by an increased or decreased expression of specific renal and intestinal Ca2+ and Pi transporters respectively. It seems unlikely that LPS-induced hypocalcaemia is due to renal loss of Ca2+ .

Empagliflozin, SGLT2 inhibitor, attenuates renal fibrosis in rats exposed to unilateral ureteric obstruction: potential role of klotho expression.

Chronic kidney disease (CKD) is a global healthcare problem; however until now, there is no effective treatment that can stop its progression. In this study, we aimed to investigate the effect of empagliflozin, a sodium-glucose linked transporter-2 inhibitor (SGLT2I) in a model of unilateral ureteric obstruction (UUO) in rats, as a model of progressive renal interstitial fibrosis in vivo and the possibility of inclusion of klotho protein. Rats were randomly divided into five groups: group 1: control group, group 2: UUO untreated group, group 3: prophylactic SGLT2I treatment before UUO, group 4: immediate SGLT2I treatment after UUO, and group 5: delayed SGLT2I treatment (this group received distilled water 1week after UUO then empagliflozin for 2weeks). At the end of the experiment period, animals were sacrificed, and kidney fibrotic and inflammatory parameters were measured. Also kidney sections were examined histopathologically for CTGF expression. UUO resulted in renal dysfunction and fibrosis through upregulating inflammatory cascade (NF-κB-TLR4) as well as many fibrotic pathways (as TGF-β1, αSMA, Wnt, CTGF, and fibronectin) with significant reduction in the klotho protein expression. We hypothesized that both prophylactic and immediate treatment with empagliflozin after UUO in rats exert more renoprotective effect in comparison with delayed treatment via enhancement of renal klotho expression and activity, for further investigations.

Contribution of volume overload to progression of cardiovascular disease in a rat model of chronic kidney disease.

Volume overload is a common phenomenon in patients with chronic kidney disease that is associated with cardiovascular risk factors. However, its contribution to the development of adverse cardiovascular outcomes in those patients is not fully understood. Thus, the present work investigated the effect of salt-induced volume overload on cardiac functions and geometry in a rat model of chronic kidney disease. Thirty adult male Sprague-Dawley rats were randomly divided. One set of animals received a sham operation, while another set of animals underwent uninephrectomy. Rats were then fed either a normal-salt (0.4%) or high-salt (8.0%) diet for 6 weeks. The salt-loaded, uninephrectomized rats were treated with indapamide (3 mg·kg-1·day-1, orally) for 6 weeks. We found that uninephrectomized rats subjected to a high-salt diet (8.0%) for 6 weeks presented with hypertension, proteinuria, decreased renal Klotho expression, and deterioration in cardiac hemodynamics and histology. Echocardiography to assess cardiac function showed that ejection fraction and fractional shortening were positively correlated with relative renal Klotho expression. In conclusion, salt-induced volume overload in a rat model of chronic kidney disease has an adverse cardiovascular outcome and is associated with inflammatory activation and decrease in renal Klotho expression.

Impact of Altered Mineral Metabolism on Pathological Cardiac Remodeling in Elevated Fibroblast Growth Factor 23.

Clinical and experimental studies indicate a possible link between high serum levels of fibroblast growth factor 23 (FGF23), phosphate, and parathyroid hormone (PTH), deficiency of active vitamin D (1,25D) and klotho with the development of pathological cardiac remodeling, i.e., left ventricular hypertrophy and myocardial fibrosis, but a causal link has not been established so far. Here, we investigated the cardiac phenotype in klotho hypomorphic (kl/kl) mice and Hyp mice, two mouse models of elevated FGF23 levels and klotho deficiency, but differing in parameters of mineral metabolism, by using histology, quantitative real-time PCR, immunoblot analysis, and serum and urine biochemistry. Additionally, the specific impact of calcium, phosphate, PTH, and 1,25D on hypertrophic growth of isolated neonatal rat cardiac myocytes was investigated in vitro. Kl/kl mice displayed high serum Fgf23 levels, increased relative heart weight, enhanced cross-sectional area of individual cardiac myocytes, activated cardiac Fgf23/Fgf receptor (Fgfr) 4/calcineurin/nuclear factor of activated T cell (NFAT) signaling, and induction of pro-hypertrophic NFAT target genes including Rcan1, bMHC, brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) as compared to corresponding wild-type (WT) mice. Investigation of fibrosis-related molecules characteristic for pathological cardiac remodeling processes demonstrated ERK1/2 activation and enhanced expression of Tgf-β1, collagen I, and Mmp2 in kl/kl mice than in WT mice. In contrast, despite significantly elevation of serum and cardiac Fgf23, and reduced renal klotho expression, Hyp mice showed no signs of pathological cardiac remodeling. Kl/kl mice showed enhanced serum calcium and phosphate levels, while Hyp mice showed unchanged serum calcium levels, lower serum phosphate, and elevated serum iPTH concentrations compared to corresponding WT mice. In cultured cardiac myocytes, treatment with both calcium or phosphate significantly upregulated endogenous Fgf23 mRNA expression and stimulated hypertrophic cell growth and expression of pro-hypertrophic genes. The treatment with PTH induced hypertrophic cell growth only, and stimulation with 1,25D had no significant effects. In conclusion, our data indicate that Hyp mice, in contrast to kl/kl mice appear to be protected from pathological cardiac remodeling during conditions of high FGF23 levels and klotho deficiency, which may be due, at least in part, to differences in mineral metabolism alterations, i.e., hypophosphatemia and lack of hypercalcemia.

Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy.

BackgroundThe role of Klotho (KL) in sepsis-induced acute kidney injury (AKI) and the potential relationship between KL and autophagy in septic AKI were investigated.Materials and methodsA murine model of sepsis-induced AKI was established by cecal ligation and puncture (CLP). Mice undergoing CLP and immortalized proximal tubular epithelial human HK-2 cells that were exposed to lipopolysaccharide (LPS) were treated with recombinant KL, autophagy stimulator rapamycin (Rap), and autophagy suppressor 3-methyladenine (3-MA).ResultsAutophagy activation and KL reduction reached maximum levels in mice 24 hours after CLP. Recombinant KL and/or Rap significantly attenuated CLP-induced renal dysfunction (P<0.05) and partially restored endogenous renal KL expression (P<0.05). Recombinant KL had no impact on CLP-induced autophagy and apoptosis, whereas Rap significantly stimulated autophagy and reduced apoptosis in mice. 3-MA significantly exacerbated renal dysfunction, increased apoptosis, and inhibited autophagy in mice with CLP-induced AKI (all P<0.05). In LPS-treated HK-2 cells, Rap significantly enhanced autophagy and reduced apoptosis (all P<0.05), whereas recombinant KL had no impact, and 3-MA inhibited autophagy and significantly increased apoptosis (P<0.05).ConclusionRecombinant KL alleviates renal dysfunction and restores renal KL expression in mice with sepsis-induced AKI, but the underlying mechanism may not be related to autophagy induction.

Chronic nicotine exposure reduces klotho expression and triggers different renal and hemodynamic responses in klotho-haploinsufficient mice.

The klotho gene, which encodes a single-pass transmembrane protein and a secreted protein, is expressed predominantly by the distal renal tubules and is related to calcium phosphorus metabolism, ion channel regulation, intracellular signaling pathways, and longevity. Klotho deficiency aggravates acute kidney injury and renal fibrosis. Exposure to nicotine also worsens kidney injury. Here, we investigated renal Klotho protein expression in a mouse model of chronic (28-day) nicotine exposure, in which mice received nicotine or vehicle (saccharine) in drinking water, comparing wild-type (WT) mice, klotho-haploinsufficient ( kl/+) mice, and their respective controls, in terms of the effects of that exposure. Nicotine exposure was associated with a significant decline in renal Klotho expression in WT and kl/+ mice as well as a reduction in the glomerular filtration rate in WT mice. Although plasma electrolytes were similar among the groups, fractional excretion of sodium was reduced in both nicotine-exposed groups. The nicotine-WT mice presented augmented baroreflex sensitivity to nitroprusside and augmented sympathetic cardiac modulation. However, nicotine- kl/+ mice presented higher plasma levels of urea and aldosterone together with a higher α-index (spontaneous baroreflex) and higher peripheral sympathetic modulation, as evaluated by spectral analysis. We can conclude that nicotine downregulates Klotho expression as well as that renal and autonomic responses to nicotine exposure are modified in kl/+ mice.

Klotho and Postmenopausal Hormone Replacement Therapy in Women with Chronic Kidney Disease.

Kidney function is highly susceptible to age-related changes, with chronic kidney disease (CKD) serving as an important cause of morbidity and mortality in older patients. The prevalence of CKD in Korea is higher among the elderly, relative to the general population, with the most significant increases seen following the onset of menopause. Under normal conditions, estrogen attenuates renal superoxide production and protects the kidney from oxidative damage. As estrogen levels are known to decrease by as much as 80% during menopause, this represents a significant risk for older women. Postmenopausal hormone replacement therapy (HRT) modulates the renin-angiotensin system, thereby reducing the progressive deterioration of renal function. Use of estrogen-based HRT has been shown to ameliorate renal function in postmenopausal women, and delay CKD progression. Renal expression of klotho, an important suppressor of aging, is markedly decreased in CKD patients, making it a promising candidate for use as a prognostic biomarker in CKD. Here, we review the key links between renal function, sex, age, and estrogen levels during menopause, and discuss the use of postmenopausal HRT in CKD attenuation.

The mechanism of attenuation of epithelial-mesenchymal transition by a phosphodiesterase 5 inhibitor via renal klotho expression.

SummaryPhosphodiesterase‐5 (PDE‐5) inhibitors induces vasodilation in several organs by blocking cyclic GMP (guanosine monophosphate) degradation. However, the existence of alternative mechanism of action in case of an impaired nitric oxide (NO) system remains controversial. Previous studies suggested that decreased NO bioavailability may result in the downregulation of klotho expression, but the relationship between klotho and NO remains obscure. Therefore, we investigated whether a PDE‐5 inhibitor could preserve epithelial–mesenchymal transition (EMT) and relationship exists between the NO and renal klotho expression. Ten‐week‐old SD rats (N = 24, 200 g, male) were divided (N = 6) into four groups, which received: A LSD, L‐NAME 1 mg/mL in drinking water, Udenafil 5 mg/kg subcutaneously and both for 4 weeks. Urine nitrate/nitrite, NGAL (Neutrophil gelatinase‐associated lipocalin), and cGMP were measured using ELISA. Kidney was subjected to evaluate PCNA (proliferative cell nuclear antigen), α‐SMA (smooth muscle cell antigen), E‐cadherin, and klotho expression. Urine cGMP decreased after treatment of PDE‐5 inhibitor compared with control due to blocking degradation of cGMP (P < .05, control vs Udenafil and L‐NAME with Udenafil groups). Urine NGAL increased after treating of L‐NAME and attenuated after using PDE‐5 inhibitor (P < .05, control vs L‐NAME and L‐NAME with Udenafil). PCNA, α‐SMA, and E‐cadherin (EMT markers) increased after L‐NAME treatment and normalized after using PDE‐5 inhibitor. Klotho expression showed trend to increase in the L‐NAME with PDE‐5 inhibitor group compared with the L‐NAME group, however, eNOS expression did not change after treatment of L‐NAME or PDE‐5 inhibitor compared with control. PDE‐5 inhibitor alleviates EMT in the kidney via klotho modulation independent of the NO system.

Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.

In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)-23. Abnormal regulation of the FGFR1-Klotho receptor complex may cause a resistance to the phosphaturic action of FGF23. The purpose of the present study was to investigate the regulation of renal Klotho and FGF receptor (FEFR)-1 in healthy and uremic rats induced by 5/6 nephrectomy. In normal rats, the infusion of rat recombinant FGF23 enhanced phosphaturia and increased renal FGFR1 expression; however, Klotho expression was reduced. Uremic rats on a high-phosphate (HP) diet presented hyperphosphatemia with marked elevation of FGF23 and an increased fractional excretion of phosphate (P) that was associated with a marked reduction of Klotho expression and an increase in FGFR1. After neutralization of FGF23 by anti-FGF23 administration, phosphaturia was still abundant, Klotho expression remained low, and the FGFR1 level was reduced. These results suggest that the expression of renal Klotho is modulated by phosphaturia, whereas the FGFR1 expression is regulated by FGF23. Calcitriol (CTR) administration prevented a decrease in renal Klotho expression. In HEK293 cells HP produced nuclear translocation of β-catenin, together with a reduction in Klotho. Wnt/β-catenin inhibition with Dkk-1 prevented the P-induced down-regulation of Klotho. The addition of CTR to HP medium was able to recover Klotho expression. In summary, high FGF23 levels increase FGFR1, whereas phosphaturia decreases Klotho expression through the activation of Wnt/β-catenin pathway.-Muñoz-Castañeda, J. R., Herencia, C., Pendón-Ruiz de Mier, M. V., Rodriguez-Ortiz, M. E., Diaz-Tocados, J. M., Vergara, N., Martínez-Moreno, J. M., Salmerón, M. D., Richards, W. G., Felsenfeld, A., Kuro-O, M., Almadén, Y., Rodríguez, M. Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.