Urinary Klotho Research Articles

Changes of Kidney and Urinary Klotho Reflect the Involvement of Distal Convoluted Tubules in Lipopolysaccharide (LPS)-Induced AKI in Rats

Changes of Kidney and Urinary Klotho Reflect the Involvement of Distal Convoluted Tubules in Lipopolysaccharide (LPS)-Induced AKI in Rats

Determination of the reference interval for urinary klotho to creatinine ratio of healthy dogs.

For several years, alpha klotho has been considered as a candidate biomarker in chronic kidney disease (CKD), progression of CKD and CKD mineral bone disorders (CKD-MBD). The evidence on the relationship between klotho and kidney function is controversial in some areas. The aim of the study was to identify the influence of age, sex and breed on urinary alpha klotho, values in the early stages of CKD within the studied population and determine a reference interval in a group of healthy dogs. Significantly higher values were measured in older dogs over 6 years old (p = 0.026, p = 0.0007) and in the breed German Shepherd than Belgian Shepherd (p = 0.0401). On the basis of sex and in small breed dogs, no significant differences were noted. In dogs with CKD stage 2, alpha klotho values were significantly lower (p = 0.0135) than in healthy dogs. Within the studied population, a reference interval for urinary klotho to creatinine ratio (UrKl/Cr) was determined in the range of 3.94-23.55 pg/gCr. Since our findings show that alpha klotho is associated with older age, we assume that this may have influenced the results in the group of dogs with CKD stage 1 due to the presence of predominantly old dogs in this group. Future studies would be needed to consider age as a factor affecting urinary alpha klotho in dogs with CKD.

Klotho as an Early Marker of Acute Kidney Injury Following Cardiac Surgery: A Systematic Review.

Acute kidney injury is a common complication following cardiac surgery (CSA-AKI). Serum creatinine levels require a minimum of 24-48 h to indicate renal injury. Nevertheless, early diagnosis remains critical for improving patient outcomes. A PRISMA-compliant systematic review of the PubMed and CENTRAL databases was performed to assess the role of Klotho as a predictive biomarker for CSA-AKI (end-of-search date: 17 February 2024). An evidence quality assessment of the four included studies was performed with the Newcastle-Ottawa scale. Among the 234 patients studied, 119 (50.8%) developed CSA-AKI postoperatively. Serum Klotho levels above 120 U/L immediately postoperatively correlated with an area under the curve (AUC) of 0.806 and 90% sensitivity. Additionally, a postoperative serum creatinine to Klotho ratio above 0.695 showed 94.7% sensitivity and 87.5% specificity, with an AUC of 92.4%, maintaining its prognostic validity for up to three days. Urinary Klotho immunoreactivity was better maintained in samples obtained via direct catheterization rather than indwelling catheter collection bags. Storage at -80 °C was necessary for delayed testing. Optimal timing for both serum and urine Klotho measurements was from the end of cardiopulmonary bypass to the time of the first ICU lab tests. In conclusion, Klotho could be a promising biomarker for the early diagnosis of CSA-AKI. Standardization of measurement protocols and larger studies are needed to validate these findings.

Urinary Excretion of Biomolecules Related to Cell Cycle, Proliferation, and Autophagy in Subjects with Type 2 Diabetes and Chronic Kidney Disease.

Dysregulation of cell cycle, proliferation, and autophagy plays a pivotal role in diabetic kidney disease. In this study, we assessed urinary excretion of molecular regulators of these processes that mediate their effects via the PI3K/AKT/mTOR pathway in subjects with long-term type 2 diabetes (T2D) and different patterns of chronic kidney disease (CKD). We included 140 patients with T2D and 20 non-diabetic individuals in a cross-sectional study. Urinary PTEN, Beclin-1, sirtuin 1 (SIRT1), Klotho, fibroblast growth factor 21 (FGF21), and connective tissue growth factor (CTGF) were assessed using ELISA. Patients with T2D, when compared to control, demonstrated increased excretion of PTEN, Beclin-1, SIRT1, FGF21, CTGF, and decreased urinary Klotho (all p < 0.05). In the diabetic group, PTEN, FGF21, and CTGF were significantly higher in patients with declined renal function, while Klotho was lower in those with elevated albuminuria. FGF21 and PTEN correlated inversely with the estimated glomerular filtration rate. There was a negative correlation between Klotho and urinary albumin-to-creatinine ratio. In multivariate models, Klotho and PTEN were associated with albuminuric CKD independently. The results provide further support for the role of PTEN, BECN1, FGF21, Klotho, and CTGF in development albuminuric and non-albuminuric CKD in diabetes.

Urinary Klotho Excretion: A Key Regulator of Sodium Homeostasis in Chronic Kidney Disease Stage 2-4.

BACKGROUND Soluble alpha-klotho (klotho) is considered an important regulator of mineral homeostasis in patients with chronic kidney disease (CKD). Since the mineral transport proteins are located on the apical membrane of renal tubular cells, we hypothesized that urine klotho may also be involved in their homeostasis. We aimed to investigate the associations between serum and urine klotho and their impacts on mineral homeostasis in patients with stage 2 to 4 CKD. MATERIAL AND METHODS Serum, spot urine, and 24-h urine of klotho were measured by using enzyme-linked immunosorbent assay. Fractional excretion of sodium, potassium, calcium, phosphate, magnesium, and klotho were calculated. RESULTS A total of 53 patients with CKD stages 2 to 4 were enrolled in this cross-sectional study. The mean age was 71.1±10.5 years, and 68% were men. Linear regression analysis showed that serum log-transformed klotho was negatively associated with log-transformed fractional excretion of klotho (log-FEKlotho) (ß=-0.085, P=0.02), showing that urinary klotho excretion could negatively regulate serum klotho levels. Moreover, our multivariate stepwise regression showed log-fractional excretion of sodium was positively associated with log-FEKlotho (ß=0.138, P=0.032). This implied urinary klotho excretion positively regulated urinary sodium excretion. CONCLUSIONS Our study showed that urine klotho excretion resulted in decreased serum klotho levels and enhanced urinary sodium excretion in patients with CKD stages 2 to 4. In addition to serum klotho, we found, for the first time, that urine klotho also played a significant role in sodium homeostasis.

Optimization of fluorescent substrates for ADAM17 and their utility in the detection of diabetes

ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase that releases various types of membrane-associated proteins, including adhesive molecules, cytokines and their receptors, and inflammatory mediators. Evidence suggests that the enzyme is involved in the proteolytic cleavage of antiaging transmembrane protein Klotho (KL). What is more, reduced serum and urinary KL levels are observed in the early stages of chronic kidney disease. This study aimed to optimise the ADAM17 specific and selective fluorescent substrates. Then, the obtained substrate was used to detect the enzyme in urine samples of patients diagnosed with diabetes. It turned out that in all cases we were able to detect proteolytic activity, which was the opposite of the healthy samples.

#2598 KLOTHO DEFICIENCY INDUCES REGULATORY T CELLS IN MURINE LUPUS NEPHRITIS

Abstract Background and Aims Recent studies demonstrate that klotho deficiency participate in various chronic kidney disease. However, it has not been fully assessed the influence of klotho in autoimmune kidney diseases. On the one hand, klotho is known to binds transforming growth factor β (TGFβ) receptor to antagonize its pathophysiological actions including renal fibrosis. On the other hand, TGFβ is required to generate and maintain regulatory T cells with inducing FOXP3, an important cell population for immunological tolerance. Method NZBWF1 mice were used as a model of lupus nephritis. NZBWF1 mice were housed separately in metabolic cage, and divided into two groups (n=10 for each): one group was treated with daily subcutaneous injection of klotho protein (20 µg/kg/day), and the other received vehicle alone. Systolic blood pressure was measured by tail-cuff method. Glomerular filtration rate was assessed using FITC-inulin. Four weeks later, the animals were killed to harvest the spleen and kidneys for analyses. Results Klotho supplementation suppressed blood pressure, 8-epi-prostaglandin F2a excretion and renal angiotensin II levels (p&amp;lt;0.05 for all) without changes in albuminuria and glomerular filtration rate in NZBWF1 mice. Exogenous klotho protein supplementation increased serum klotho levels, urine klotho excretion, and endogenous renal expression of klotho in NZBWF1 mice (p&amp;lt;0.05 for all). Surprisingly, anti-double strand DNA antibody was slightly elevated in klotho-treated NZBWF1 mice (p&amp;lt;0.05). Glomerular pathology and interstitial cell infiltration were similar between 2 groups. The spleen tended to be greater in klotho-treated group, but statistical significance was not attained. In consistent, CD8+FOXP3+ T cells were unaltered between 2 groups. However, klotho supplementation reduced CD4+FOXP3+ T cells in spleen of NZBWF1 mice (p&amp;lt;0.05). Conclusion The present data indicated that klotho protein supplementation suppressed renal renin-angiotensin system, ameliorating blood pressure and oxidative stress. Our results suggest that klotho supplementation worsened auto-antibody, possibly by inhibiting TGFβ with resultant deterioration in regulatory T cells. The present findings implicate that although klotho may not suite for the management of autoimmune kidney diseases, such as lupus and ANCA-related nephritis, klotho supplementation for dialysis patients could mitigate against the defect in cellular immunity and susceptibility to infections including tuberculosis and COVID-19.

Noninvasive Assessment of Renal Fibrosis of Chronic Kidney Disease in Rats by [68Ga]Ga-FAPI-04 Small Animal PET/CT and Biomarkers

Renal fibrosis is the most common pathological feature and common pathway of progression in chronic kidney disease (CKD). We evaluated [68Ga]Ga-FAPI-04 small animal positron emission tomography/computed tomography (PET/CT) and biomarkers as noninvasive assessments of renal fibrosis (RF) in CKD rats to generate new ideas for clinical diagnosis. A rat model of renal fibrosis was administered adenine by gavage (n = 28), and the control group was given 0.9% NaCl by gavage (n = 20). At different time points (weeks 1, 2, 4, and 6), five rats were randomly selected from the two groups for [68Ga]Ga-FAPI-04 small animal PET/CT imaging. At the same time, the expression of Fibroblast activation protein (FAP) in renal tissue and the expression levels of type III procollagen N-terminal peptide (PIIINP), transforming growth factor (TGF-β1), Klotho, and sex-determining region Y-box protein 9 (SOX9) in blood and urine were determined. FAP was highly expressed in the renal tissue of rats in the CKD group and expression increased with the progression of renal fibrosis. [68Ga]Ga-FAPI-04 small animal PET/CT examination showed that the uptake of radioactive tracers in the CKD group was higher than that in the control group, and SUVmax (r = 0.9405) and target-to-background ratio (TBR) (r = 0.9392) were positively correlated with renal fibrosis. The serum levels of PIIINP, TGF-β1, and SOX9 in CKD rats were significantly higher than those in the control group and were positively correlated with RF (r = 0.8234, r = 0.7733, and r = 0.7135, respectively) and SUVmax (r = 0.8412, r = 0.7763, and r = 0.6814, respectively). Compared with the control group, the level of serum Klotho decreased and was negatively correlated with RF (r = -0.6925) and SUVmax (r = -0.6322). Compared with the control group, the levels of PIIINP and TGF-β1 in urine were positively correlated with RF (r = 0.8127 and r = 0.8077, respectively) and SUVmax (r = 0.8400 and r = 0.8177, respectively). Urine Klotho decreased compared with the control group and was negatively correlated with RF (r = -0.5919) and SUVmax (r = -0.5995). The change in urine SOX9 was not statistically significant. In conclusion, compared with renal biopsy, [68Ga]Ga-FAPI-04 small animal PET/CT shows renal fibrosis quickly and noninvasively. PIIINP, TGF-β1, and Klotho in serum and urine may be used as biomarkers of RF, and serum SOX9 is expected to become a new diagnostic biomarker of RF.

Sodium-glucose co-transporter-2 inhibitors increase Klotho in patients with diabetic kidney disease: A clinical and experimental study

Sodium-glucose co-transporter-2 inhibitors (SGLT2i) provide cardiorenal protection. However, the molecular mechanisms remain poorly understood. We explored the impact of SGLT2i on Klotho, a kidney-derived protein with antiaging, renal-protective and heart-protective properties. A real world prospective observational study addressed the impact of initiating SGLT2i (canagliflozin, dapagliflozin, empagliflozin) or dipeptidyl peptidase-4 inhibitors (DPP4i) in patients with early diabetic kidney disease (DKD). Serum and urinary soluble Klotho, albuminuria and serum and urinary tumor necrosis factor-alpha (TNFa) were measured. The effect of SGLT2i on Klotho mRNA and protein was explored in vitro in kidney proximal tubular cells stressed with high glucose concentrations to simulate the diabetic milieu, albumin to simulate albuminuria, and the inflammatory cytokine TWEAK to simulate the inflammatory environment in DKD. Baseline urinary Klotho was negatively associated with albuminuria (r − 0.45, P < 0.001) and urinary TNFa (r − 0.40, P < 0.01). Both DPP4i and SGLT2i reduced HbA1c similarly, but only SGLT2i decreased eGFR, albuminuria and urinary TNFa and increased (P < 0.001) serum (5.2 %) and urinary Klotho (38.9 %). Changes in urinary TNFa (β − 0.53, P = 0.001) and albuminuria (β − 0.31, P < 0.05) were independently associated with changes in urinary Klotho (adjusted R2 = 0.54, P < 0.001). Studies in renal tubular cells demonstrated that high glucose, albumin and TWEAK decreased Klotho mRNA expression and protein levels, an effect similarly prevented by SGLT2i. SGLT2i increase Klotho availability in type 2 diabetic patients with poorly controlled diabetes and early DKD, as well as in stressed tubular cells. This effect on Klotho may contribute to the kidney and heart protection afforded by SGLT2i.

Urinary Klotho Abnormalities in Children with Sickle Cell Disease

Background: Klotho is a trans membrane protein expressed in the renal tubules and serves as an obligatory co-receptor for fibroblast growth factor 23(FGF23) to aid in phosphorus excretion. Prior studies have shown FGF23 resistance in sickle cell disease (SCD). The purpose of the study is to investigate urinary klotho/creatinine (Ur Kl/Cr) in pediatric SCD with normal traditional markers of renal function (eGFR &gt; 90 ml/min and no micro albuminuria) and to compare it with the healthy control population. Methods: Cross-sectional observational study to compare Ur Kl/Cr in pediatric SCD and controls. To do a subgroup analysis among the study population to assess the effect of hydroxyl urea (HU) on Ur Kl/Cr. Results: 20 controls and 22 pediatric SCD were enrolled. In the SCD group, 13 were on treatment with HU. The baseline characteristics of the study and control group were the same. Wilcoxon rank-sum test was used to compare the levels of Ur Kl/Cr ratio between SCD and control. For P value of 0.05, the levels of Ur Kl/Cr were statistically significantly higher in the sickle cell group (752.7 ± 1101.0) over the control group (216.8 ± 225.3). Subgroup analysis in the SCD group showed high Urinary Kl excretion in the non-HU group (1346.7 ± 1523.4) vs. non HU group (341.4 ± 355.3) but not statistically significant. Conclusion: Children with SCD tend to have increased secretion of Ur Kl/Cr compared to control likely due to tubular receptor resistance. HU may reverse this phenomenon by its effect on preventing tubular damage.

Serum and urinary soluble α-Klotho levels in patients with chronic kidney disease

Background: Chronic kidney disease is an epidemic global health problem that leads to death. To prevent any disease progression and treatment, the diagnosis must be made in the early stage by studying renal damage. Klotho is a protein found in many organs of the human body, but it is mainly abundant in the kidney. Objectives: This study is aimed to evaluate klotho’s clinical significance as an additional biomarker for diagnosing chronic kidney disease in its early stages. Methods: One hundred subjects were included in this study to measure their serum and urinary klotho. Forty patients with chronic kidney disease (pre-dialysis) and sixty normal subjects were enrolled in this study. Serum and urinary klotho were determined using the ELISA technique in addition to other renal function tests. Results: Serum and urinary alpha klotho were decreased in CKD patients when compared with control subjects. A positive correlation was found between serum creatinine and urinary alpha klotho in the patients’ group. Conclusion: Serum and urinary alpha klotho levels were decreased significantly in patients with chronic kidney disease compared with healthy controls.

Investigation on urinary and serum alpha klotho in dogs with chronic kidney disease

BackgroundAs a co-receptor for fibroblast growth factor 23, klotho plays a pivotal role in phosphate metabolism. The kidney is known to be the main source of soluble alpha-klotho and the principal regulator of its concentration. Previous studies in human participants showed that the concentration of soluble alpha-klotho in serum and urine decreased in chronic kidney disease (CKD) patients. However, no previous study has assessed soluble alpha-klotho levels in dogs. This study aimed to measure serum and urinary alpha-klotho levels in CKD dogs and identify their associations with International Renal Interest Society (IRIS) CKD stages and other parameters known to be associated with CKD.ResultsSerum and urinary alpha klotho concentrations were measured by a commercially available canine-specific sandwich enzyme-linked immunosorbent assay kit and compared between groups by a nonparametric Kruskal–Wallis test. Spearman’s correlation coefficient was used to evaluate the relationships between variables. A stepwise multiple regression analysis was performed to estimate the effects of independent predictors on klotho concentrations. The urine klotho-to-creatinine ratio (UrKl/Cr) was significantly lower in stage 3 dogs than the control group and was significantly lower in dogs with stage 3 and 4 CKD than in those with stage 1 and 2 disease. UrKl/Cr was negatively correlated with serum symmetric dimethylarginine (sSDMA), blood urea nitrogen (BUN), creatinine, and phosphorus concentration. Serum alpha-klotho concentration in dogs with stages 2 and 3 CKD was significantly lower than those in the control group. There was no significant correlation between serum alpha-klotho and BUN, creatinine, and phosphorus concentrations. No statistically significant differences were observed in UrKl/Cr and serum alpha-klotho concentration between groups based on sex, age, urine protein-to-creatinine ratio (UPC), or blood pressure.ConclusionsUrKl/Cr decreased in dogs with advanced CKD, and it was negatively correlated with sSDMA, BUN, creatinine, and phosphorus concentrations. Thus, klotho is associated with CKD and its clinical consequences, including CKD-mineral bone disorder, in dogs. Although serum klotho concentration was negatively correlated with sSDMA levels, it was not apparently related to IRIS CKD stage or other parameters known to be associated with CKD.

Urine klotho is a potential early biomarker for acute kidney injury and associated with poor renal outcome after cardiac surgery

BackgroundCurrent paradigms of detecting acute kidney injury (AKI) are insensitive and non-specific. Klotho is a pleiotropic protein that is predominantly expressed in renal tubules. In this study, we evaluated the diagnostic and prognostic roles of urine Klotho for AKI following cardiac surgery.MethodsWe conducted a prospective study involving 91 patients undergoing cardiac surgery. AKI was defined according to the AKIN definition. The renal outcomes within 7 days after operation were evaluated. Perioperative levels of urine Klotho and urine neutrophil gelatinase-associated lipocalin (NGAL) were measured by using ELISA.ResultsOf 91 participants, 33 patients (36.26%) developed AKI. Of these AKI patients, 21 (63.64%), 8 (24.24%), and 4 (12.12%) were staged 1, 2, and 3, respectively. Serum creatinine in AKI patients began to slightly increase at first postoperative time and reached the AKI diagnostic value 1 day after operation.Postoperative urine Klotho peaked at the first postoperative time (0 h after admission to the intensive care unit (ICU)) in patients with AKI, and was higher than that in non-AKI patients up to day 3. The AUC of detecting AKI for urine Klotho was higher than urine NGAL at the first postoperative time and 4 h after admission to the ICU. In a multivariate model, increased first postoperative urine Klotho may be an independent predictor for AKI occurrence following cardiac surgery. The concentrations of first postoperative urine Klotho were higher in AKI stage 2 and 3 than those in stage 1 (p < 0.05), and were higher in patients with incomplete recovery of renal function than those with complete recovery (p < 0.05).ConclusionsUrine Klotho may serve as an early biomarker for AKI and subsequent poor short-term renal outcome in patients undergoing cardiac surgery.

Urine Klotho Is Lower in Critically Ill Patients With Versus Without Acute Kidney Injury and Associates With Major Adverse Kidney Events.

Klotho and Fibroblast Growth Factor (FGF)-23 were recently postulated as candidate biomarkers and/or therapeutic targets in acute kidney injury (AKI). We examined whether urine Klotho and serum intact FGF23 levels were differentially and independently associated with major adverse kidney events (MAKE) in critically ill patients with and without AKI. Single-center, prospective, case-control study. ICU in a tertiary medical center. 54 AKI patients and 52 controls without AKI admitted to the ICU. None. AKI was defined by KDIGO criteria and included only AKI stage ≥2. Controls were matched by age, gender, and baseline eGFR. Paired serum and urine samples were obtained 24-48h after AKI diagnosis (cases) or ICU admission (controls). The primary outcome was 90-day MAKE, which was the composite of all-cause death, dependence on renal replacement therapy or a 50% or higher decrease in eGFR from baseline. Forty-four (41.5%) patients developed MAKE-90. Patients who developed MAKE-90 had more comorbidity, higher acuity of illness scores and more prevalent AKI. Levels of urine Klotho adjusted by creatinine (Cr) were lower and serum intact FGF23 levels were higher in AKI patients vs. ICU controls. In adjusted models, the highest vs. lowest tertile of urine Klotho/Cr was independently associated with an overall 95% lower risk of MAKE-90 (81% lower risk in patients with AKI). The highest vs. lowest tertile of serum intact FGF23 was associated with >300% higher risk of MAKE-90. Urine Klotho/Cr levels were significantly lower and serum intact FGF23 levels significantly higher in critically ill patients with AKI vs. matched-controls without AKI. When measured in the first 48h of ICU admission or AKI diagnosis, urine Klotho/Cr independently associated with major adverse kidney events, particularly in patients with AKI. These results show promise for testing these biomarkers -individually or in combination- as part of novel risk-prediction models of renal outcomes in the ICU.

Bicarbonate Supplement Restores Urinary Klotho Excretion in Chronic Kidney Disease: A Pilot Study

We tested the hypothesis that correcting acidosis may improve urinary Klotho excretion and serum α-Klotho. This is a prospective, interventional, nonrandomized, open-label trial study. In this study setting, metabolic acidosis is commonly observed during chronic kidney disease (CKD). We reported a positive relationship between serum bicarbonate (Sbicar) and serum α-Klotho in these patients. The study involved 20 patients with a known kidney disease referred for renal checkup. Inclusion criteria were age ≥18years, CKD stage 3-5 non dialysis, Sbicar < 22mmol/L, and not receiving bicarbonate supplementation. Patients were then prescribed 1 g of oral sodium bicarbonate 3 times per day for 4weeks. Patients were evaluated at two and 4weeks by blood and urine measurements. Mean serum Klotho was 615±287 pg/mL, and mean serum Sbicar was 19.3±1.7mmol/L at baseline. Sbicar increased from baseline at two (23.9±2.9mmol/L, P<.001) and 4weeks (23.4±1.9mmol/L, P<.001). There was no change in serum Klotho at two (630±333mmol/L) and 4weeks (632±285mmol/L). By contrast, urine Klotho/creatinine ratio, which was very low at baseline (34.6±31.6 pg/mmoL), increased by 320% at two weeks (P<.005) and by 280% at 4weeks (P<.01). Correcting acidosis by oral administration of sodium bicarbonate rapidly increases the urine excretion of soluble α-Klotho in CKD patients. However, a 4-week bicarbonate treatment was not able to increase serum α-Klotho. A longer study may confirm this pilot observation and increase serum Klotho, which has been shown to exert a protective cardiovascular effect during CKD.

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.

Effects of Pentoxifylline on Soluble Klotho Concentrations and Renal Tubular Cell Expression in Diabetic Kidney Disease.

The effect of pentoxifylline on Klotho levels in patients with type 2 diabetes mellitus with chronic kidney disease (CKD) was assessed in a post hoc analysis of the Pentoxifylline for Renoprotection in Diabetic Nephropathy (PREDIAN) trial. Circulating and urinary tumor necrosis factor-α (TNF-α) and Klotho were measured before and after 1 year of pentoxifylline. The effect on Klotho expression was assessed in cultured renal tubular cells. Pentoxifylline administration resulted in decreased serum and urinary TNF-α, whereas serum and urinary Klotho increased significantly. Changes in urinary Klotho, urinary TNF-α, and phosphorus were associated with changes in serum Klotho; changes in estimated glomerular filtration rate, urinary TNF-α, and albuminuria were related to urinary Klotho variation. In renal tubular cells, pentoxifylline prevented the decrease in Klotho expression induced by inflammatory cytokines or albumin. Pentoxifylline increased Klotho levels in patients with diabetes with stage 3-4 CKD and prevented reduced Klotho expression in vitro. This beneficial effect may be related to anti-inflammatory and antialbuminuric activity.

Soluble klotho as a marker of renal fibrosis and podocyte injuries in human kidneys.

Klotho deficiency is relevant to renal fibrosis and podocyte injury in vivo and in vitro. We examined whether histological findings of renal biopsy specimens were associated with the levels of soluble klotho in humans. We investigated renal biopsy specimens of 67 patients and detailed microscopic findings were reviewed. Soluble serum/urinary klotho and urinary angiotensinogen were assessed by enzyme-linked immunosorbent assays, and tissue klotho expression was assessed by immunohistochemical staining. The median age of the study participants was 35.6 years. High serum klotho levels (≥14 pg/mL) were associated with decreased odds ratios (ORs) of interstitial fibrosis (OR = 0.019, P = 0.003) and segmental sclerosis (OR = 0.190, P = 0.022) in multivariable logistic regression analysis. Patients with a lower urinary klotho-to-creatinine ratio (UKCR) were significantly more likely to have diffuse foot process effacement (OR = 0.450, P = 0.010). The area under the receiver-operating characteristic curve (AUC) of serum klotho for predicting interstitial fibrosis was 0.920 (95% CI, 0.844–0.996), and the best cut-off value of serum klotho was 138.1 pg/mL. The AUC of UKCR for predicting diffuse foot process effacement was 0.754 (95% CI, 0.636–0.872), and the best cut-off value of UKCR was 96.7 pg/mgCr. Urinary angiotensinogen-to-creatinine ratio was not associated with serum klotho, UKCR, or any pathological finding. Our data suggested that soluble serum and urinary klotho levels represent a potential biomarker to predict renal fibrosis and podocyte injury in humans.

Albumin downregulates Klotho in tubular cells.

Kidney tubular cells are the main sources of Klotho, a protein with phosphaturic action. Genetic Klotho deficiency causes premature cardiovascular aging in mice. Human chronic kidney disease (CKD) is characterized by acquired Klotho deficiency. Despite the lack of uremic toxin accumulation, Category G1 CKD [(normal glomerular filtration rate (GFR)] is already associated with decreased Klotho and with premature cardiovascular aging. We have explored whether albuminuria, a criterion to diagnose CKD when GFR is normal, may directly decrease Klotho expression in human CKD, preclinical models and cultured tubular cells. In a CKD cohort, albuminuria correlated with serum phosphate after adjustment for GFR, age and sex. In this regard, urinary Klotho was decreased in patients with pathological albuminuria but preserved GFR. Proteinuria induced in rats by puromycin aminonucleoside and in mice by albumin overload was associated with interstitial inflammation and reduced total kidney Klotho messenger ribonucleic acid (mRNA) expression. Western blot disclosed reduced kidney Klotho protein in proteinuric rats and mice and immunohistochemistry localized the reduced kidney Klotho expression to tubular cells in proteinuric animals. In cultured murine and human tubular cells, albumin directly decreased Klotho mRNA and protein expression. This was inhibited by trichostatin A, an inhibitor of histone deacetylases, but unlike cytokine-induced Klotho downregulation, not by inhibitors of nuclear factor kappa-light-chain-enhancer of activated B cells. In conclusion, albumin directly decreases Klotho expression in cultured tubular cells. This may explain, or at least contribute to, the decrease in Klotho and promote fibroblast growth factor 23 resistance in early CKD categories, as observed in preclinical and clinical proteinuric kidney disease.

Abstract P419: Klotho Ameliorate Medullary Fibrosis and Pressure Natriuresis in Hypertensive Rat Kidneys

Recent data indicate that klotho interacts with the renin-angiotensin system (RAS). However, the effects of klotho protein supplementation on hypertensive renal injury have not been examined yet. First, the in vivo experiments were performed using spontaneously hypertensive rats (SHR). To elucidate in vivo observations, the in vitro studies were carried out to examine the interactions between klotho protein and angiotensin type-1 (AT1) receptor with immunoprecipitation and cell culture (HK-2 cell) methods. Uninephrectomized SHRs were treated with exogenous klotho protein or vehicle. Exogenous klotho protein supplementation to uninephrectomized SHRs decreased blood pressure, renal angiotensin II levels, angiotensinogen expression, HIF-1α abundance, and medullary fibronectin with increased renal klotho expression and serum and urine klotho levels. Klotho supplementation also reduced kidney weight, renal phosphorylated Akt and mTOR abundance. Furthermore, klotho supplementation restored renal autoregulation of glomerular filtration rate and renal plasma flow, and improved pressure-induced natriuresis in SHR. Klotho protein bound to AT1 receptors without affecting the bindings of angiotensin II to AT1 receptor. Klotho decreased the presence of AT1 receptors on HK-2 cells, attenuating inositol triphosphate generation. Angiotensin II elevated angiotensinogen expression in HK-2 cells, and klotho protein suppressed angiotensin II-induced increases in angiotensinogen expression. Of note, although angiotensin II improves glomerular autoregulatory ability especially at lower pressure range, angiotensin II impairs pressure-induced natriuresis. Collectively, the present data demonstrate that klotho binds with the AT1 receptors to suppress angiotensin signal transduction and inactivate the renal RAS. In addition, our results suggest that exogenous klotho supplementation suppresses Akt-mTOR signaling to decline renal hypertrophy and restores renal autoregulatory ability in uninephrectomized SHRs. Finally, the present findings implicate that klotho supplementation inhibits HIF-1α pathway to suppress medullary fibrosis, presumably involved in improvements of pressure-natriuresis and reduction in blood pressure.