Evaluation of klotho expression on peripheral blood lymphocytes among hemodialysis patients and its possible contribution to their immuno-compromised status.

Infection is the second most common cause of mortality among end-stage kidney disease (ESKD) patients. Uremic toxins are the main cause of impaired immune response among ESKD patients. Klotho gene, the anti-aging gene, encodes the transmembrane alpha klotho (αKL) protein which acts as an obligate coreceptor for fibroblast growth factor 23 (FGF23). Klotho protein may play a role in immune cell functions, particularly in anti-inflammatory response; however, its role is still incompletely understood. In the present study, we aimed to measure αKL protein expression on peripheral blood lymphocytes (PBLs) among hemodialysis (HD) patients, and we assumed that decreased αKL expression on PBLs may contribute to the impaired immunity among HD patients. This case-control study included 20 ESKD patients on regular hemodialysis for more than 3 months. Their ages ranged from 24 to 69 years. Patients with primary immunodeficiencies, those on systemic immunosuppressive drugs, those with ongoing infections or who had recently recovered from infections, and those with malignancies on active treatment were excluded. A control group of 20 normal subjects of comparable age and gender were also included. We compared αKL protein expression on PBLs by flow cytometry between both groups. Significant reductions in percentages of αKL protein expression on B lymphocytes (CD19), T lymphocytes (CD3), and natural killer cells (CD56) were observed among HD patients compared to controls. We also noticed a significant reduction in the percentages of natural killer cells among HD patients. The present study suggests that decreased αKL expression on PBLs may contribute to the immunocompromised status among HD patients, highlighting the importance of understanding the exact function of αKL protein on immune cells. This may offer a future diagnostic and therapeutic tool to improve the immune response among HD patients.

Klotho gene might antagonize ischemic injury in stroke rats by reducing the expression of AQP4 via P38MAPK pathway.

OBJECTIVES: This study was aimed at exploring whether klotho improved neurologic function in rats with cerebral infarction by inhibiting P38 mitogen-activated protein kinase (MAPK) activation and thus down-regulating aquaporin 4 (AQP4). METHODS: In this study, we induced intracerebral Klotho overexpression in 6-week-old Sprague Dawley rats by injecting lentivirus carrying full-length rat Klotho cDNA into the lateral ventricle of the brain, followed by middle cerebral artery occlusion (MCAO) surgery after three days. Neurologic function was evaluated by neurological deficit scores. Infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The expressions of Klotho, AQP4, and P38 MAPK were detected by Western blot and Immunofluorescence. RESULTS: when rats were subjected to cerebral ischemia, their neurologic function was impaired, the protein expressions of klotho downregulated, the protein expressions of AQP4 and P38 MAPK increased, and the ratios of AQP4 and P-P38-positive area were significantly increased compared with the sham group rats. LV-KL-induced Klotho overexpression greatly improved neurobehavioral deficits and reduced infarct volume in MCAO rats. Klotho overexpression significantly reduced AQP4 and P38 MAPK pathway-related protein expression levels and the ratios of P-P38 and AQP4-positive area in MCAO rats. In addition, SB203580, a P38 MAPK signal pathway inhibitor, improved neurobehavioral deficits, reduced infarct volume, downregulated the expressions levels of AQP4 and P38 MAPK, and reduced the size of P-P38 and AQP4-positive area in MCAO rats. CONCLUSION: Klotho could alleviate the infraction volume and neurological dysfunction in MCAO rats, and its mechanism may involve AQP4 expression downregulation by suppressing P38-MAPK activation.

Klotho promotes AMPK activity and maintains renal vascular integrity by regulating the YAP signaling pathway.

The development and formation of mammalian blood vessels are closely related to the regulation of signal transduction pathways. Klotho/AMPK and YAP/TAZ signaling pathways are closely related to angiogenesis, but the internal relationship between them is not clear. In this study, we found that Klotho heterozygous deletion mice (Klotho+/- mice) had obvious thickening of the renal vascular wall, obvious enlargement of vascular volume, and significant proliferation and pricking of vascular endothelial cells. Western blot showed that the expression levels of total YAP protein, p-YAP protein (Ser127 and Ser397), p-MOB1, MST1, LATS1, and SAV1 in renal vascular endothelial cells were significantly lower in Klotho+/- mice than in wild-type mice. Knockdown of endogenous Klotho in HUVECs accelerated their ability to divide and form vascular branches in the extracellular matrix. Meanwhile, the results of CO-IP western blot showed that the expression of LATS1 and p-LATS1 interacting with AMPK protein decreased significantly, and the ubiquitination level of YAP protein also decreased significantly in vascular endothelial cells of kidney tissue of Klotho+/- mice. Subsequently, continuous overexpression of exogenous Klotho protein in Klotho heterozygous deficient mice effectively reversed the abnormal renal vascular structure by weakening the expression of the YAP signal transduction pathway. Therefore, we confirmed that Klotho and AMPKα proteins were highly expressed in vascular endothelial cells of adult mouse tissues and organs; this resulted in a phosphorylation modification of YAP protein, closed the activity of the YAP/TAZ signal transduction pathway, and inhibited the growth and proliferation of vascular endothelial cells. When Klotho was absent, the phosphorylation modification of YAP protein by AMPKα was inhibited, resulting in the activation of the YAP/TAZ signal transduction pathway and finally inducing the excessive proliferation of vascular endothelial cells.

Klotho protects chromosomal DNA from radiation-induced damage.

Klotho is an anti-aging, single-pass transmembrane protein found mainly in the kidney. Although aging is likely to be associated with DNA damage, the involvement of Klotho in protecting cells from DNA damage is still unclear. In this study, we examined DNA damage in human kidney cells and mouse kidney tissue after ionizing radiation (IR). The depletion and overexpression of Klotho in human kidney cells reduced and increased the cell survival rates after IR, respectively. The formation of γ-H2AX foci, representing DNA damage, was significantly elevated immediately after IR in cells with Klotho depletion and decreased in cells overexpressing Klotho. These results were confirmed in mouse renal tissues after IR. Quantification of DNA damage by a comet assay revealed that the Klotho knockdown significantly increased the amount of DNA damage immediately after IR, suggesting that Klotho protects chromosomal DNA from the induction of damage, rather than facilitating DNA repair. Consistent with this notion, Klotho was detected in both the nucleus and cytoplasm. In the nucleus, Klotho may serve to protect chromosomal DNA from damage, leading to its anti-aging effects.

VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.

Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

Evaluation of Klotho gene expression and NGAL levels following acute kidney injury during pregnancy hypertensive disorders.

BACKGROUND: This study examined the relationship between hypertensive disorders and acute kidney injury (AKI) during pregnancy by evaluating Klotho (KL) gene expression and Neutrophil gelatinase-associated lipocalin (NGAL) levels in pregnant women. MATERIAL AND METHODS: Pregnant women were divided into 3 groups: (1) Pregnancy related hypertensive disorders with AKI (PR-AKI) (9 cases), (2) hypertensive disorders pregnancy (HDP) (40 cases), and (3) normal pregnancy (30 cases). For each group, Klotho gene transcription levels in the blood, Klotho and NGAL proteins levels, Malondialdehyde (MDA) and superoxide dismutase (SOD) activity levels were measured in serum. Statistical comparisons were made among the three groups. RESULTS: Klotho/ß-actin transcript levels and serum KL protein concentrations were significantly decreased in hypertensive disorder pregnancies with AKI complications. Serum NGAL protein levels were significantly increased in the hypertensive disorder pregnancies with AKI complications. Total serum Klotho protein was negatively correlated with creatinine, while serum NGAL was positively correlated with serum creatinine, urea nitrogen, uric acid, systolic blood pressure, diastolic blood pressure and 24 h urine protein levels. Serum levels of MDA and SOD were significantly increased in the hypertensive disorder pregnancy with AKI and the overall MDA concentration was negatively correlated with Klotho protein concentration. Klotho protein was found to have a direct effect on creatinine, and a mediating effect of MDA was found. CONCLUSION: Decreased expression of Klotho protein in correlation with increased levels of oxidative stress are found during of AKI complications in pregnancy hypertensive disorders.

Investigation of Klotho G395A and C1818T Polymorphisms and Their Association with Serum Glucose Level and Risk of Type 2 Diabetes Mellitus.

Objective: The objective was to study the association of Klotho gene G395A and C1818T single nucleotide polymorphisms with glycemia, serum, glycosylated hemoglobin (HbA1c) level and the risk of type 2 diabetes mellitus (T2DM) in the Pashtun population of Pakistan. Methods: In this study, 195 normal individuals and 217 T2DM patients were enrolled. All subjects were divided into three groups, namely overall subjects (control + T2DM patients), control individuals and T2DM patients, and their fasting glucose, HbA1c level, lipid profile and C1818T and G395A polymorphisms were determined. Results: The allele frequencies of G395A in overall subjects were 0.568 for A and 0.432 for G. Similarly, allele frequencies for G395A in overall subjects were 0.597 and 0.403 for C and T alleles, respectively. The AA genotype of G395A was observed to be a risk factor for T2DM. In normal individuals, no significant (p > 0.05) association was observed between klotho C1818T and G395A polymorphisms and hyperglycemia. In overall subjects, the C1818T polymorphism was associated (p < 0.05) with high fasting glucose and HbA1c levels in female subjects only. In T2DM patients, both C1818T and G395A polymorphisms were found to be significantly (p < 0.05) associated with high fasting glucose and HbA1c levels both in males and females. Conclusion: The G395A polymorphism was observed to increase the risk of T2DM. Both C1818T and G395 were associated with high fasting glucose and HbA1c levels in T2DM patients.

Klotho in diabetic kidney disease: more than dust in the Wnt.

Activation of the Wnt/ß-catenin pathway represents a hallmark in the development of kidney fibrosis. Herein, Chen et al. report that Klotho-derived peptide 6, a peptide mimicking the function of the protein Klotho, directly binds to endogenous Wnt ligands and, thereby, serves as a small-molecule inhibitor of canonical Wnt/ß-catenin signaling. In diabetic kidney disease, Klotho-derived peptide 6 reduces glomerular injury and preserves kidney function, highlighting Klotho-derived peptide 6 as a novel therapeutic agent.

The anti-aging protein Klotho affects early postnatal myogenesis by downregulating Jmjd3 and the canonical Wnt pathway.

Modulating the number of muscle stems cells, called satellite cells, during early postnatal development produces long-term effects on muscle growth. We tested the hypothesis that high expression levels of the anti-aging protein Klotho in early postnatal myogenesis increase satellite cell numbers by influencing the epigenetic regulation of genes that regulate myogenesis. Our findings show that elevated klotho expression caused a transient increase in satellite cell numbers and slowed muscle fiber growth, followed by a period of accelerated muscle growth that leads to larger fibers. Klotho also transcriptionally downregulated the H3K27 demethylase Jmjd3, leading to increased H3K27 methylation and decreased expression of genes in the canonical Wnt pathway, which was associated with a delay in muscle differentiation. In addition, Klotho stimulation and Jmjd3 downregulation produced similar but not additive reductions in the expression of Wnt4, Wnt9a, and Wnt10a in myogenic cells, indicating that inhibition occurred through a common pathway. Together, our results identify a novel pathway through which Klotho influences myogenesis by reducing the expression of Jmjd3, leading to reductions in the expression of Wnt genes and inhibition of canonical Wnt signaling.

FGF23-Klotho Axis and Fractures in Patients Without and With Early CKD: A Case-Cohort Analysis of CARTaGENE.

CONTEXT: Whether fibroblast growth factor-23 (FGF23) and α-Klotho are associated with fractures, especially in chronic kidney disease (CKD), remains controversial. OBJECTIVE: We evaluated how FGF23, α-Klotho, and traditional mineral parameters predict fractures in individuals with and without early CKD. METHODS: We conducted a stratified case-cohort analysis using CARTaGENE, a population-based survey from Quebec, Canada. Individuals aged 40 to 69 years were selected according to outcome and CKD status (non-CKD: eGFR > 60 mL/min/1.73 m2; CKD stage 3: eGFR 30-60 mL/min/1.73 m2]). Baseline levels of c-terminal FGF23 (cFGF23), α-Klotho, parathyroid hormone (PTH), phosphate, and calcium were analyzed for associations with osteoporotic fracture incidence from recruitment (2009-2010) through March 2016. Adjusted Cox models were used, and predictors were treated linearly or flexibly using splines. RESULTS: A total of 312 patients (159 non-CKD; 153 CKD) were included; 98 had ≥ 1 fracture at any site during a median follow up of 70 months. Compared with non-CKD, CKD patients had increased levels of cFGF23 but similar levels of α-Klotho. cFGF23 was linearly associated with increased fracture incidence (adjusted HR = 1.81 [1.71, 1.93] per doubling for all participants). The association of α-Klotho with fracture followed a U-curve (overall P = 0.019) but was attenuated by adjustment for potential mediators (bone mineral density, phosphate, PTH). PTH and phosphate also had U-shaped associations with fracture. Associations were mostly similar between non-CKD and CKD. Adjustment for cFGF23 strongly attenuated the association between CKD status and fractures. CONCLUSION: cFGF23 is associated linearly with fracture incidence while α-Klotho, PTH, and phosphate levels have a U-shaped association.

NRF2 pathway activation attenuates ageing-related renal phenotypes due to α-klotho deficiency.

Oxidative stress is one of the major causes of the age-related functional decline in cells and tissues. The KEAP1-NRF2 system plays a central role in the regulation of redox balance, and NRF2 activation exerts antiageing effects by controlling oxidative stress in aged tissues. α-Klotho was identified as an ageing suppressor protein based on the premature ageing phenotypes of its mutant mice, and its expression is known to gradually decrease during ageing. Because α-klotho has been shown to possess antioxidant function, ageing-related phenotypes of α-klotho mutant mice seem to be attributable to increased oxidative stress at least in part. To examine whether NRF2 activation antagonizes ageing-related phenotypes caused by α-klotho deficiency, we crossed α-klotho-deficient (Kl-/-) mice with a Keap1-knockdown background, in which the NRF2 pathway is constitutively activated in the whole body. NRF2 pathway activation in Kl-/- mice extended the lifespan and dramatically improved ageing-related renal phenotypes. With elevated expression of antioxidant genes accompanied by an oxidative stress decrease, the antioxidant effects of NRF2 seem to make a major contribution to the attenuation of ageing-related renal phenotypes of Kl-/- mice. Thus, NRF2 is expected to exert an antiageing function by partly compensating for the functional decline of α-Klotho during physiological ageing.

Cardiovascular remodeling as a result of fibroblast growth factor-23 (FGF-23)/Klotho imbalance in patients with CKD.

BACKGROUND: In chronic kidney disease (CKD) cardiovascular remodeling (CVR) is very frequent compared with general population and, as suppose, may be associated with «new¼ renal risk factors. The aim of study was to estimate association of new serum biomarkers (FGF-23, Klotho) and traditional biomarker of cardiac damage-serum Troponin I (sTr-I) with signs of CVR. METHODS: One hundred thirty CKD G1-5D patients without cardiovascular disease (CVD) clinical manifestation were included. We measured serum FGF-23, Klotho and sTr-I. The instrumental methods were: echocardiography, SphygmoCor test [Pulse Wave Velocity (PWV), Central (aortic) Blood Pressure (CBP), Subendocardial Blood Supply (SBS)]. RESULTS: FGF-23 level correlated with: sTr-I (r = 0.512; p < 0.01), eccentric left ventricular hypertrophy, LVH (r = 0.543; p < 0.01), SBS (r = - 0.499; p < 0.05). There were no differences of FGF-23 level in patients with normal and high CBP. Klotho correlated with concentric LVH (r = - 0.451; p < 0.01), PWV (r = - 0.667; p < 0.001), Cardiac Calcification Score, CCS (r = - 0.581; p < 0.01). Multivariate analysis revealed positive independent association of FGF-23 with eccentric LVH (OR = 1.036, 95% CI (1.004-1.068); p = 0.038). Klotho was a negative determinant for concentric LVH (OR = 0.990, 95% CI 0.987-0.994; p < 0.001), increased PWV (OR = 0.984, 95% CI (0.977-0.991); p < 0.001) and CCS (OR = 0.991, 95% CI (0.988-0.995); p < 0.001). In addition, multivariate analysis revealed a relationship between serum Klotho (OR = 0.980, 95% CI (0.964-0.996); p = 0.016), FGF-23 (OR = 3.145, 95% CI (1.020-9.695); p = 0.046) and troponin I level. CONCLUSION: In CKD patients without CVD clinical manifestation increased serum FGF-23 level and decreased Klotho are associated with CVR: FGF-23 with eccentric LVH (independently of CBP), Klotho determinate concentric LVH, PWV and CCS. Moderately elevated sTr-I levels may be a manifestation of FGF-23/Klotho imbalance in CKD.

Protective association of Klotho rs495392 gene polymorphism against hepatic steatosis in non-alcoholic fatty liver disease patients.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease (NAFLD) is closely associated with metabolic dysfunction. Among the multiple factors, genetic variation acts as important modifiers. Klotho, an enzyme encoded by the klotho (KL) gene in human, has been implicated in the pathogenesis of metabolic dysfunctions. However, the impact of variants in KL on NAFLD risk remains poorly understood. The aim of this study was to investigate the impact of KL rs495392 C>A polymorphism on the histological severity of NAFLD. METHODS: We evaluated the impact of the KL rs495392 polymorphism on liver histology in 531 Chinese with NAFLD and replicated that in the population-based Rotterdam Study cohort. The interactions between the rs495392, vitamin D, and patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 polymorphism were also analyzed. RESULTS: Carriage of the rs495392 A allele had a protective effect on steatosis severity (odds ratio [OR], 0.61; 95% confidence interval [CI], 0.42-0.89; P=0.010) in Chinese patients. After adjustment for potential confounders, the A allele remained significant with a protective effect (OR, 0.66; 95% CI, 0.45-0.98; P=0.040). The effect on hepatic steatosis was confirmed in the Rotterdam Study cohort. Additional analysis showed the association between serum vitamin D levels and NAFLD specifically in rs495392 A allele carriers, but not in non-carriers. Moreover, we found that the rs495392 A allele attenuated the detrimental impact of PNPLA3 rs738409 G allele on the risk of severe hepatic steatosis. CONCLUSION: The KL rs495392 polymorphism has a protective effect against hepatic steatosis in patients with NAFLD.

Epigenetic restoration of endogenous Klotho expression alleviates acute kidney injury-diabetes comorbidity.

AIMS: The present study aimed at exploring the mechanisms behind Klotho regulation in hyperglycemia augmented AKI. In addition, epigenetic ways to restore the Klotho expression in AKI-diabetes comorbidity have been evaluated. MAIN METHODS: Bilateral ischemia-reperfusion injury (IRI) and chemical hypoxia-reperfusion injury (HRI) were developed in diabetic rats and, NRK52E cells under high glucose conditions respectively, to mimic the AKI condition. Plasma, urine, tubular lysate of the kidney and NRK52E cell lysate were used for biochemical, ELISA, histology, immunoblotting, RT-PCR and RNA interference studies. KEY FINDINGS: Hyperglycemia significantly aggravated IRI/HRI induced AKI as evidenced by biochemical and histological results. We also observed a significant increase in expressions of kidney specific histone deacetylases (HDACs), apoptotic and inflammatory proteins, and decrease in levels of endogenous Klotho, H3K9Ac and H3K27Ac proteins in hyperglycemic IRI/HRI groups. SIGNIFICANCE: Diabetes comorbidity exaggerates AKI, where endogenous Klotho loss could be a potential connecting link. However, kidney-specific HDACs inhibition showed reno-protection via restoring the endogenous Klotho loss and thus prevention of inflammation and apoptosis, which could prove to be a potential therapeutic strategy against diabetes-AKI comorbidity.

Clinicopathological and Prognostic Significance of Klotho and Estrogen Receptors Expression in Human Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is male-predominant cancer, but the underlying mechanism remains unclear. This study aimed to investigate the expression of the age-suppressing gene klotho and estrogen receptors (ERs) in HCC patients and analyze their association with clinicopathological variables and their effects on prognosis. METHODS: The expression patterns of klotho, ERα, and ERß were determined by tissue microarray and immunohistochemical technique, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. RESULTS: Klotho expression was significantly lower in HCC than in the adjacent noncancerous tissues (52.7% (49/93) vs. 90.8% (79/87), P = .000), and its protein level in HCC tissue was negatively correlated with clinical staging, histological grade, and stage of the primary tumor (T) (P < .05). Whereas the expression of nuclear ERα and ERß was higher in HCC than their corresponding non-neoplastic tissues (55.9% (52/93) vs. 35.6% (31/87), P = .006; 59.1% (55/93) vs. 43.7% (38/87), P = .038), and the level of nuclear ERα and ERß in HCC tissue was inversely correlated with T stage, tumor size, and clinical staging (P < .05). Correlation analysis showed the expression level of klotho, which is positively correlated with that of nuclear ERα (r = 0.243, P = .019). Patients with klotho-positive tumors had longer survival than those with klotho-negative tumors (P = .002). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (P = .003). CONCLUSION: Klotho, partially regulated by ERα-mediated estrogen pathway, acts as a tumor suppressor and might be a novel biomarker candidate for predicting progression and prognosis in HCC patients.

Aristolochic Acid Induces Renal Fibrosis and Senescence in Mice.

The kidney is one of the most susceptible organs to age-related impairments. Generally, renal aging is accompanied by renal fibrosis, which is the final common pathway of chronic kidney diseases. Aristolochic acid (AA), a nephrotoxic agent, causes AA nephropathy (AAN), which is characterized by progressive renal fibrosis and functional decline. Although renal fibrosis is associated with renal aging, whether AA induces renal aging remains unclear. The aim of the present study is to investigate the potential use of AAN as a model of renal aging. Here, we examined senescence-related factors in AAN models by chronically administering AA to C57BL/6 mice. Compared with controls, the AA group demonstrated aging kidney phenotypes, such as renal atrophy, renal functional decline, and tubulointerstitial fibrosis. Additionally, AA promoted cellular senescence specifically in the kidneys, and increased renal p16 mRNA expression and senescence-associated ß-galactosidase activity. Furthermore, AA-treated mice exhibited proximal tubular mitochondrial abnormalities, as well as reactive oxygen species accumulation. Klotho, an antiaging gene, was also significantly decreased in the kidneys of AA-treated mice. Collectively, the results of the present study indicate that AA alters senescence-related factors, and that renal fibrosis is closely related to renal aging.

α-Klotho released from HK-2 cells inhibits osteogenic differentiation of renal interstitial fibroblasts by inactivating the Wnt-ß-catenin pathway.

Randall's plaques (RP) are well established as precursor lesions of idiopathic calcium oxalate (CaOx) stones, and the process of biomineralization driven by osteogenic-like cells has been highlighted in RP formation, but the mechanism is poorly understood. Given the inhibitory role of α-Klotho (KL), an aging suppressor protein with high expression in kidneys, in ectopic calcification and the close association between KL gene polymorphisms and urolithiasis susceptibility, we determined the potential role of KL in RP formation. This study found that both soluble KL (s-KL) and transmembrane KL (m-KL) were downregulated, and that s-KL but not m-KL was inversely correlated with upregulation of osteogenic markers in RP tissues. Additionally, s-KL expression was markedly suppressed in human renal interstitial fibroblasts (hRIFs) and slightly suppressed in HK-2 cells after osteogenic induction, intriguingly, which was echoed to the greater osteogenic capability of hRIFs than HK-2 cells. Further investigations showed the inhibitory effect of s-KL on hRIF osteogenic differentiation in vitro and in vivo. Moreover, coculture with recombinant human KL (r-KL) or HK-2 cells suppressed osteogenic differentiation of hRIFs, and this effect was abolished by coculture with KL-silenced HK-2 cells or the ß-catenin agonist SKL2001. Mechanistically, s-KL inactivated the Wnt-ß-catenin pathway by directly binding to Wnt2 and upregulating SFRP1. Further investigations identified activation of the Wnt-ß-catenin pathway and downregulation of SFRP1 and DKK1 in RP tissues. In summary, this study identified s-KL deficiency as a pathological feature of RP and revealed that s-KL released from HK-2 cells inhibited osteogenic differentiation of hRIFs by inactivating the Wnt-ß-catenin pathway, not only providing in-depth insight into the role of s-KL in renal interstitial biomineralization but also shedding new light on the interaction of renal tubular epithelial cells with interstitial cells to clarify RP formation.

Enhancement of Apo2L/TRAIL signaling pathway receptors by the activation of Klotho gene with CRISPR/Cas9 in Caco-2 colon cancer cells.

Human Klotho gene has many known functions such as anti-aging and anti-tumor, and decreased expression of this gene causes malignant formations in most types of cancer, including colon cancer. Interacting with TRAIL death receptors (DR4 and DR5) induces an apoptotic effect in cancer treatments by reducing the proliferation of cancer cells. The present study aimed to investigate downstream effect of overexpression of Klotho gene, which is known to have an antitumor effect on resistant human colon cancer cells, by examining its action on TRAIL death and decoy (DcR1 and DcR2) receptors for the first time. For this purpose, upregulation of human Klotho gene was achieved with CRISPR/Cas9-mediated system in resistant human colon cancer Caco-2 cells. To determine the effect of upregulation of Klotho gene on cancer cells evaluations with flow cytometry, WST-8, qRT-PCR, ELISA, and immunohistochemical analysis were performed. Then, Klotho gene was knocked out and its apoptotic effect was tested to find out whether it is due to overexpression of Klotho gene or not. Our results indicate that overexpression of Klotho gene in Caco-2 cells via CRISPR/Cas9-sensitized TRAIL death receptor DR4 suppresses the proliferation of cells by leading to apoptosis. Thus, this study conducted on apoptosis-resistant colon cancer cells may bring new insights about the role of Klotho gene in colon cancer.

FGFR4 and Klotho Polymorphisms Are Not Associated with Cardiovascular Outcomes in Chronic Kidney Disease.

INTRODUCTION: High plasma fibroblast growth factor 23 (FGF-23) predicts cardiovascular events in chronic kidney disease (CKD) patients. Experimental evidence suggests FGF receptor 4 (FGFR4) activation by FGF-23, and deficiency of the soluble form of its co-receptor Klotho promotes left-ventricular hypertrophy (LVH). To evaluate the clinical relevance of these findings, a Mendelian randomization study analyzed the association of genetic variants of FGFR4 and Klotho with echocardiographic parameters and cardiac events in CKD patients. METHODS: The prospective Cardiovascular and Renal Outcome in CKD 2-4 Patients-The Fourth Homburg Evaluation study recruited CKD G2-G4 patients, of whom 519 consented to SNP genotyping (FGFR4: rs351855; Klotho: rs9536314). Echocardiographic examinations at baseline and 5 years later assessed prevalence of LVH by measurement of left-ventricular mass index (LVMI). Patients were followed for 5.1 ± 2.1 years for the primary endpoints of cardiac decompensation and atherosclerotic cardiovascular disease (ASCVD). RESULTS: Carriers of the different alleles did neither differ in baseline LVMI (rs351855: p = 0.861; rs9536314: p = 0.379) nor in LVMI changes between baseline and follow-up (rs351855: p = 0.181; rs9536314: p = 0.995). Hundred and four patients suffered cardiac decompensation, and 144 patients had ASCVD. Time to cardiac decompensation (rs351855: p = 0.316; rs9536314: p = 0.765) and ASCVD (p = 0.508 and p = 0.800, respectively) did not differ between carriers of different alleles. DISCUSSION/CONCLUSION: rs351855 and rs9536314 were not associated with LVMI or cardiac events. These findings do not provide evidence for a relevant clinical role of either FGFR4 stimulation or soluble form of Klotho deficiency in LVH development.

METTL14 promotes glomerular endothelial cell injury and diabetic nephropathy via m6A modification of α-klotho.

BACKGROUND: N6-Methyladenosine (m6A) modification has been implicated in many bioprocesses. However, its functions in diabetic nephropathy (DN) have not been determined. Here, we investigated the role of METTL14, a key component of the m6A methyltransferase complex, in DN. METHODS: The expression of METTL14 was detected in DN patients and human renal glomerular endothelial cells (HRGECs). In vitro and in vivo experiments were performed to explore the functions of METTL14 on high glocse-induced HRGECs and renal injury of DN mice. We also investigated whether METTL14 works by regulating α-klotho expression through m6A modification. RESULTS: METTL14 were highly expressed in kidneys of DN patients and high glocse-induced HRGECs both at the mRNA and protein level. Overexpression of METTL14 increased ROS, TNF-α and IL-6 levels and apoptosis in HRGECs. Conversely, METTL14 silence decreased the levels of ROS, TNF-α and IL-6 and cell apoptosis. We confirmed that METTL14 down-regulated α-klotho expression in an m6A-dependent manner. In addition, we also found that METTL14 aggravated renal injury and inflammation of db/db mice, which could partially rescued by α-klotho. CONCLUSION: Our data revealed that METTL14 plays a vital role in high glucose-induced glomerular endothelial cells and diabetic nephropathy through m6A modification of α-klotho.