Plasma Klotho Research Articles

The association between klotho and kidney and cardiovascular outcomes: a comprehensive systematic review and meta-analysis.

Chronic kidney disease (CKD) and end-stage renal disease (ESKD) are significant global health challenges associated with progressive kidney dysfunction and numerous complications, including cardiovascular disease and mortality. This study aims to explore the potential association between plasma klotho levels and various prognostic outcomes in CKD and ESKD, including all-cause mortality, cardiovascular events, metabolic syndrome development and adverse renal events necessitating renal replacement therapies. A literature search was conducted through 3 June 2024 using the electronic databases Cochrane Library, Ovid MEDLINE, CINAHL, Web of Science, SCOPUS and PubMed. This systematic review adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Fourteen studies were included. For all-cause mortality, comparing CKD patients with low versus high klotho levels showed a significant association {odds ratio [OR] 1.81 [95% confidence interval (CI) 1.34-2.44], P=.0001}, with substantial heterogeneity (I 2=69%). Excluding one study reduced heterogeneity (I 2=43%) while maintaining significance [OR 1.97 (95% CI 1.45-2.66), P<.0001]. Cardiovascular mortality was higher in patients with low klotho levels [OR 2.11 (95% CI 1.61-2.76), P<.00001], with low heterogeneity (I 2=25%). Excluding one study eliminated heterogeneity (I 2=0%) while maintaining significance [OR 2.39 (95% CI 1.83-3.12), P<.00001]. Composite cardiovascular events did not differ significantly between low and high klotho groups [OR 1.51 (95% CI 0.82-2.77), P=.18], but with high heterogeneity (I 2=72%). Patients with low klotho levels had a higher risk of adverse renal events [OR 2.36 (95% CI 1.37-4.08), P=.002], with moderate heterogeneity (I 2=61%). Sensitivity analysis reduced heterogeneity (I 2=0%) while maintaining significance [OR 3.08 (95% CI 1.96-4.85), P<.00001]. Specifically, for ESKD or kidney replacement therapy risk, low klotho levels were associated with an increased risk [OR 2.30 (95% CI 1.26-4.21), P=.007]. Similarly, CKD progression risk was higher in patients with lower klotho levels [OR 2.48 (95% CI 1.45-4.23), P=.0009]. Lower serum klotho levels serve as a significant predictor of adverse outcomes, including increased risks of all-cause mortality, cardiovascular mortality and progression to end-stage kidney disease among CKD patients.

Cardioprotective effect of the anti-ageing protein Klotho on ischemic heart disease

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII, Ministerio de Universidades, Education and Research Council of Madrid, Biomedicine Network Comunidad de Madrid Background Cardiovascular diseases are the leading cause of death and disability worldwide. Among them, ischemic heart disease, due to myocardial infarction (MI) remains the first cause of cardiovascular death. The anti-ageing protein Klotho is mainly expressed in membrane at renal level. However, Klotho also has a soluble form with different pleiotropic functions that are still undetermined, including a possible cardioprotective role that is currently the target of several studies. Purpose Firstly, we evaluated circulating Klotho levels related to cardiac damage in a cohort of 146 patients after ST-elevation myocardial infarction (STEMI) and in an experimental post-myocardial infarction (PMI) model. Secondly, we investigated soluble Klotho supplementation as a potential novel therapeutic strategy for the prevention of cardiac dysfunction, deleterious remodeling, cardiomyocyte calcium (Ca2+) mishandling and arrhythmias in PMI mice. Methods Study with human samples: we analysed Klotho and NT-proBNP plasma levels in STEMIpatients with preserved renal function. Experimental study: C57BL6 mice subjected to PMI by ligation of the left anterior descending coronary artery were treated with vehicle solution or recombinant Klotho (10 mg/Kg/day) for 15 days after MI. Cardiac function was studied through non-invasive methods including electrocardiogram (ECG) and cardiac magnetic resonance imaging (CMRI). Intracellular Ca2+ handling was analysed using confocal microscopy in isolated ventricular cardiomyocytes. Cardiac remodeling was studied through histological and biomolecular analysis of several markers of cardiac damage and target proteins involved in Ca2+ handling. Results Plasma Klotho levels decrease rapidly after MI in both STEMI patients and PMI mice. Additionally, MI patients in the lowest Klotho tertile showed significantly elevated NT-proBNP levels. Klotho treatment decreased the infarct area in PMI mice accompanied by a decrease in both cardiac hypertrophy and fibrosis. Moreover, reduction in ejection fraction and MI-related ECG alterations were reduced in PMI mice treated with Klotho, including increased QRS, JT, QTc intervals and occurrence of premature ventricular contractions. Klotho also prevented systolic Ca2+ release and cell shortening disturbances in cardiomyocytes from PMI mice without Klotho. Increased diastolic Ca2+ leak was prevented in mice treated with Klotho via CaMKII-dependent pathway by avoiding its phosphorylation and the RyR2 phosphorylation at CaMKII site. Conclusions Klotho supplementation protect against MI by avoiding deleterious cardiac remodeling and ameliorating one of the main complications of MI such as arrhythmias. Thus, recombinant Klotho prevents the intracardiomyocyte Ca2+ mishandling after MI. Our translational findings support the importance of Klotho as a novel biomarker of ventricular damage just after MI and as a novel therapeutic strategy for prevention cardiac events associated to ischemic heart disease.

Klotho and Clinical Outcomes in CKD: Findings From the Chronic Renal Insufficiency Cohort (CRIC) Study

Rationale & ObjectiveKlotho deficiency may affect clinical outcomes in chronic kidney disease (CKD) through fibroblast growth factor-23 (FGF23)-dependent and independent pathways. However, the association between circulating Klotho and clinical outcomes in CKD remains unresolved and was the focus of this study. Study DesignProspective observational study. Setting & Participants1088 participants of the Chronic Renal Insufficiency Cohort (CRIC) Study with estimated glomerular filtration rate (eGFR) 20-70 ml/min/1.73m2. ExposurePlasma Klotho level at the year-1 study visit. Outcomes5-year risks of all-cause mortality, heart failure hospitalization, atherosclerotic cardiovascular events, and a composite kidney endpoint comprised of a sustained 50% decline in eGFR, dialysis, kidney transplantation, or eGFR <15 ml/min/1.73 m2. Analytical ApproachWe divided Klotho into six groups to account for its non-normal distribution. We used Cox proportional hazards regression and subdistribution hazards models to compare survival and clinical outcomes, respectively, between Klotho groups. We sequentially adjusted for demographics, kidney function, cardiovascular risk factors, sample age, and FGF23. ResultsMean eGFR was 42 ml/min/1.73m2, and median Klotho was 0.31 ng/ml (interquartile range 0.10-3.27 ng/ml). When compared to the lowest Klotho group, survival (hazard ratio [HR] 0.77, 95% confidence interval [CI] 0.32-1.89), heart failure hospitalization (HR 1.10, 95% CI 0.38-3.17), atherosclerotic cardiovascular events (HR 1.19, 95% CI 0.57-2.52), and CKD progression (HR 1.05, 95% CI 0.58-1.91) did not differ in the high Klotho group. In contrast, FGF23 was significantly associated with mortality and heart failure hospitalization independent of Klotho levels. LimitationsDespite adjustments, we cannot exclude potential influence of residual confounding or sample storage on the results. A single measurement of plasma Klotho may not capture Klotho patterns over time. ConclusionsIn a large, diverse, well-characterized CKD cohort, Klotho was not associated with clinical outcomes, and Klotho deficiency did not confound the association of FGF23 with mortality or heart failure hospitalization.

Cardiac Rehabilitation Increases Plasma Klotho Levels.

Background: Mineral metabolism (MM), mainly fibroblast growth factor-23 (FGF-23) and klotho, has been linked to cardiovascular (CV) diseases. Cardiac rehabilitation (CR) has been demonstrated to reduce CV events, although its potential relationship with changes in MM is unknown. Methods: We performed a prospective, observational, case-control study, with acute coronary syndrome (ACS) patients who underwent CR and control patients (matched by age, gender, left ventricular ejection fraction, diabetes, and coronary artery bypass grafting), who did not. The inclusion dates were from August 2013 to November 2017 in CR group and from July 2006 to June 2014 in control group. Clinical, biochemical, and MM biomarkers were collected at discharge and six months later. Our objective was to evaluate differences in the modification pattern of MM in both groups. Results: We included 58 CR patients and 116 controls. The control group showed a higher prevalence of hypertension (50.9% vs. 34.5%), ST-elevated myocardial infarction (59.5% vs. 29.3%), and treatment with angiotensin-converting enzyme inhibitors (100% vs. 69%). P2Y12 inhibitors and beta-blockers were more frequently prescribed in the CR group (83.6% vs. 96.6% and 82.8% vs. 94.8%, respectively). After six months, klotho levels increased in CR patients whereas they were reduced in controls (+63 vs. -49 pg/mL; p < 0.001). FGF-23 was unchanged in the CR group and reduced in controls (+0.2 vs. -17.3 RU/dL; p < 0.003). After multivariate analysis, only the change in klotho levels was significantly different between groups (+124 pg/mL favoring CR group; IC 95% [+44 to +205]; p = 0.003). Conclusions: In our study, CR after ACS increases plasma klotho levels without significant changes in other components of MM. Further studies are needed to clarify whether this effect has a causal role in the clinical benefit of CR.

Associations Between Plasma Klotho with Renal Function and Cerebrospinal Fluid Amyloid-β Levels in Alzheimer's Disease: The Chongqing Ageing & Dementia Study.

The kidney-brain crosstalk has been involved in Alzheimer's disease (AD) with the mechanism remaining unclear. The anti-aging factor Klotho was reported to attenuate both kidney injury and AD pathologies. To investigate whether plasma Klotho participated in kidney-brain crosstalk in AD. We enrolled 33 PiB-PET-positive AD patients and 33 amyloid-β (Aβ)-negative age- and sex-matched cognitively normal (CN) controls from the Chongqing Ageing & Dementia Study (CADS). The levels of plasma Klotho, Aβ, and tau in the cerebrospinal fluid (CSF) were measured by enzyme-linked immunosorbent assay. We found higher plasma Klotho and lower estimated glomerular filtration rate (eGFR) levels in AD patients compared with CN. The eGFR was positively associated with Aβ42, Aβ40 levels in CSF and negatively associated with CSF T-tau levels. Plasma Klotho levels were both negatively correlated with CSF Aβ42 and eGFR. Mediation analysis showed that plasma Klotho mediated 24.96% of the association between eGFR and CSF Aβ42. Renal function impacts brain Aβ metabolism via the kidney-brain crosstalk, in which the plasma Klotho may be involved as a mediator. Targeting Klotho to regulate the kidney-brain crosstalk provides potential therapeutic approaches for AD.

The Circulating Level of Klotho Is Not Dependent upon Physical Fitness and Age-Associated Methylation Increases at the Promoter Region of the Klotho Gene.

(1) Background: Higher levels of physical fitness are believed to increase the physiological quality of life and impact the aging process with a wide range of adaptive mechanisms, including the regulation of the expression of the age-associated klotho (KL) gene and protein levels. (2) Methods: Here, we tested the relationship between the DNA methylation-based epigenetic biomarkers PhenoAge and GrimAge and methylation of the promoter region of the KL gene, the circulating level of KL, and the stage of physical fitness and grip force in two groups of volunteer subjects, trained (TRND) and sedentary (SED), aged between 37 and 85 years old. (3) Results: The circulating KL level is negatively associated with chronological age in the TRND group (r = -0.19; p = 0.0295) but not in the SED group (r = -0.065; p = 0.5925). The age-associated decrease in circulating KL is partly due to the increased methylation of the KL gene. In addition, higher plasma KL is significantly related to epigenetic age-deceleration in the TRND group, assessed by the biomarker of PhenoAge (r = -0.21; p = 0.0192). (4) Conclusions: The level of physical fitness, on the other hand, does not relate to circulating KL levels, nor to the rate of the methylation of the promoter region of the KL gene, only in males.

Association of Klotho Protein Levels and KL-VS Heterozygosity With Alzheimer Disease and Amyloid and Tau Burden

Identification of proteins and genetic factors that reduce Alzheimer disease (AD) pathology is of importance when searching for novel AD treatments. Heterozygosity of the KL-VS haplotype has been associated with reduced amyloid and tau burden. Whether this association is mediated by the Klotho protein remains unclear. To assess concentrations of Klotho in cerebrospinal fluid (CSF) and plasma among cognitively healthy controls and patients with AD and to correlate these findings with KL-VS heterozygosity status and amyloid and tau burden. This case-control study combined 2 independent case-control AD cohorts consisting of 243 referred patients with AD and volunteer controls recruited from January 1, 2009, to December 31, 2018. Klotho levels were measured in CSF and plasma and correlated with KL-VS heterozygosity status and levels of CSF amyloid-β 42 (Aβ42), total tau, and phosphorylated tau. Statistical analysis was performed from January 1, 2021, to March 1, 2022. Associations of Klotho levels in CSF and plasma with levels of CSF biomarkers were analyzed using linear regression. Association analyses were stratified separately by clinical groups, APOE4 status, and KL-VS heterozygosity. Pearson correlation was used to assess the correlation between CSF and plasma Klotho levels. A total of 243 participants were included: 117 controls (45 men [38.5%]; median age, 65 years [range, 41-84 years]), 102 patients with mild cognitive impairment due to AD (AD-MCI; 59 men [57.8%]; median age, 66 years [range, 46-80 years]), and 24 patients with dementia due to AD (AD-dementia; 12 men [50.0%]; median age, 64.5 years [range, 54-75 years]). Median CSF Klotho levels were higher in controls (1236.4 pg/mL [range, 20.4-1726.3 pg/mL]; β = 0.103; 95% CI, 0.023-0.183; P = .01) and patients with AD-MCI (1188.1 pg/mL [range, 756.3-1810.3 pg/mL]; β = 0.095; 95% CI, 0.018-0.172; P = .02) compared with patients with AD-dementia (1073.3 pg/mL [range, 698.2-1661.4 pg/mL]). Higher levels of CSF Klotho were associated with lower CSF Aβ42 burden (β = 0.519; 95% CI, 0.201-0.836; P < .001) and tau burden (CSF total tau levels: β = -0.884; 95% CI, 0.223 to -0.395; P < .001; CSF phosphorylated tau levels: β = -0.672; 95% CI, -1.022 to -0.321; P < .001) independent of clinical, KL-VS heterozygosity, or APOE4 status. There was a weak correlation between Klotho CSF and plasma levels among the entire cohort (Pearson correlation r = 0.377; P < .001). The findings of this case-control study suggest that Klotho protein levels were associated with clinical stages of AD, cognitive decline, and amyloid and tau burden and that these outcomes were more clearly mediated by the protein directly rather than the KL-VS heterozygosity variant. When selecting individuals at risk for clinical trials, the Klotho protein level and not only the genetic profile should be considered.

The Role of Klotho G395A Gene Polymorphism in Atherosclerotic Cardiovascular Disease and Mortality Risk Scores in Non-dialysis Chronic Kidney Disease

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Klotho expression was reduced in patients with CKD, leading to vascular calcification, endothelial dysfunction, and atherosclerosis. We investigated the role of the klotho G395A gene polymorphism and plasma klotho level in the ten-year risk of atherosclerotic cardiovascular disease (ASCVD) and CVD mortality in CKD patients.METHODS: We used the PCR-CTPP assay method to genotype klotho G395A single nucleotide polymorphism (SNP) in 72 non-dialysis CKD patients. The klotho level was determined using the enzyme-linked immunoassay (ELISA) method. Path analysis was used to determine the relationship between the klotho G395A SNP, plasma klotho level, ASCVD risk score, and CVD mortality risk score.RESULTS: Our results showed that the GA genotype had lower plasma klotho levels than the GG genotype (path coefficient=-0.185, p=0.000). There was a significant negative correlation between plasma klotho level and the ASCVD risk score (r=-0.243, p=0.040), but no significant correlation was found between plasma klotho level and the CVD mortality risk score (r=-0.145, p=0.225). Path analysis showed that plasma klotho level had a significant negative direct effect on ASCVD risk score (path coefficient=-0.272, p=0.000) and an indirect effect on CVD mortality risk score (path coefficient=0.187, p=0.005).CONCLUSION: Klotho G395A SNP might reduce lower plasma klotho levels, which increased ASCVD and CVD mortality risk scores in non-dialysis CKD patients. However, other risk factors such as age, CKD stages, hypertension, and smoking should be taken into consideration. Therefore, large-scale genetic association studies with adjusted variables could be conducted in various ethnic groups for a more robust result.KEYWORDS: klotho, single nucleotide polymorphism, cardiovascular disease, chronic kidney disease

FC022: IPI3K/AKT/MTOR Pathway Regulates Renal Expression of Klotho

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) represents an important and growing burden on health worldwide, mainly driven by the increase in the incidence of associated risk factors like diabetes. As kidney function declines, changes in mineral metabolism occur, which contribute to the increased risk of cardiovascular disease in CKD patients. In this respect, Klotho was described as an anti-ageing protein, mainly expressed in the kidney, where it acts as an obligatory co-receptor of the Fibroblast Growth Factor (FGF23), which is involved in urinary phosphate excretion. The earliest mineral metabolism change in CKD is the fall in renal and plasma Klotho levels, followed by an increase in FGF23. Klotho loss is related to inflammatory and fibrotic processes contributing not only to the progression of CKD but also to the increase in cardiovascular risk. Clinically, there is a growing interest in maintaining renal Klotho expression in patients, but the mechanisms in Klotho decline remain unknown. PTEN is the main negative regulator of PI3K-AKT-mTOR pathway. PTEN degrades PIP3 to PIP2 decreasing the activation of the canonical insulin pathway. The involvement of PTEN in cancer has been broadly investigated. Furthermore, the PI3K/PTEN signalling pathways are involved in a wide variety of diseases including cardiac hypertrophy, heart failure, hypertension and acute kidney injury. However, its potential role in CKD has not been properly assessed. The objective of this study was to investigate the role of PTEN and the PI3K/AKT/mTOR pathways in kidney Klotho levels and the modulation of phosphorus metabolism. METHOD We generated a proximal tubule conditional PTEN knockout mouse model (PT-PTEN-cKO), in which a high phosphorus diet (HPD) and rapamycin treatment were administered in order to analyse how PI3K/AKT/mTOR pathway participates in phosphorus metabolism and kidney Klotho levels. We used human proximal tubular cell (PTC) line HK-2 to knock down PTEN by means of transfection with shPTEN lentiviral vector, to study the direct effect of PTEN elimination in Klotho levels in vitro. RESULTS PT-PTEN-cKO mice present a specific activation of the PI3K/AKT/mTOR pathway in the proximal tubule. PTEN mRNA decrease correlates with a drop in kidney Klotho levels, accompanied by an increment of inflammatory and fibrotic markers. In addition, our model showed an increase in both, plasma phosphate and FGF23, with a decrease in the fractional excretion of phosphate (% FEPi). This fact can be explained by the inability of kidney cells to respond to FGF23, because of Klotho loss. Moreover, parathyroid hormone (PTH) and bone resorption were increased. We observe that PT-PTEN-KO mice in an HPD needed higher FGF23 levels in order to excrete phosphate than control mice. Rapamycin administration restored Klotho levels. In vitro, PTEN silencing in PTC, leads to an increase in PI3K/AKT/mTOR activity and also to a reduction in Klotho expression. Finally, the inhibition of mTOR phosphorylation by rapamycin reestablished Klotho expression in vitro. CONCLUSION The overactivation of PI3K/AKT/mTOR pathway, in PTC, decreases Klotho kidney levels. Our findings constitute an important breakthrough in the research of new therapeutic targets in order to maintain renal Klotho levels and it may be useful in the treatment of kidney disease patients.

Implications of Klotho Protein for Managing Kidney Disease - an Emerging Role in Therapeutics and Molecular Medicine.

Acute Kidney Injury (AKI) and Chronic Kidney Disease (CKD) are a growing public health problem. There is a paucity of sensitive biomarkers to detect AKI, early CKD, and ameliorate extra-renal complications. Klotho protein, detected mainly in the kidneys, regulates renal health and functions as a co-receptor for fibroblast growth factor 23 (FGF-23) signaling. It is now coming to be known for its extreme pleiotropic actions. These include cytoprotection via anti-oxidation, anti-senescence, anti-apoptosis, renoprotective effects, promotion of angiogenesis and vascularisation, inhibition of fibrogenesis, and stem cell preservation. Emerging clinical studies suggest kidney damage to be a perpetual state of renal Klotho deficiency. In AKI, Klotho levels in plasma and/or urine possibly will serve as an initial biomarker for kidney parenchymal injury. In CKD, Klotho levels may also be an indicator of early disease as well as predict the rate of progression. Earlier studies using ELISA as a technique reveal a correlation between plasma Klotho, eGFR, serum creatine, and Blood Urea Nitrogen (BUN) levels. Thereby preventing the decline of Klotho levels by various mechanisms can retard CKD advancement and improve renal function. Substantial data indicate Klotho can be therapeutically included as an individualized regimen for managing CKD patients. Considerable research is required in investigating the role of soluble Klotho as a biomarker in patients with different types and severity of kidney diseases, which will be highlighted in our review.

Lumbar spine abnormalities in patients with obstructive sleep apnoea

Previous studies suggested cervical spondylosis as a risk factor for development of obstructive sleep apnoea (OSA). We aimed to assess lumbar disc degeneration in patients with OSA and correlate the findings with symptoms and disease severity. Twenty-seven patients with OSA and 29 non-OSA controls underwent sleep studies and lumbar magnetic resonance imaging (MRI), and completed the Epworth Sleepiness Scale and the 24-item Roland‐Morris Disability Questionnaire (RMDQ) questionnaires. Plasma klotho was determined with enzyme-linked immunosorbent assay. Patients with OSA had higher number of disc bulges (4.6 ± 3.7 vs. 1.7 ± 2.5, p < 0.01) and anterior spondylophytes (2.7 ± 4.2 vs. 0.8 ± 2.1, p < 0.01), increased disc degeneration (total Pfirrmann score 16.7 ± 4.7 vs. 13.2 ± 4.1, p < 0.01) and vertebral fatty degeneration (7.8 ± 4.7 vs. 3.8 ± 3.7, p < 0.01). There was no difference in the RMDQ score (0/0–3.5/ vs. 0/0–1/, p > 0.05). Markers of OSA severity, including the oxygen desaturation index and percentage of total sleep time spent with saturation < 90% as well as plasma levels of klotho were correlated with the number of disc bulges and anterior spondylophytes (all p < 0.05). OSA is associated with lumbar spondylosis. Our study highlights the importance of lumbar imaging in patients with OSA reporting lower back pain.

The Long-Term Study of Urinary Biomarkers of Renal Injury in Spontaneously Hypertensive Rats

Background: The age-related increase in blood pressure in spontaneously hypertensive rats (SHRs) is associated to cardiac hypertrophy, heart failure, and renal injury. Here, we investigated for the first time the urinary enzymatic activities of glutamil aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), and Klotho urinary levels, proteins that are strongly expressed in the kidney, as early biomarkers of renal injury in SHRs. Methods: Male SHR and Wistar Kyoto (WKY) rats were studied from 2 to 8 months old. Systolic blood pressure (SBP), the heart rate (HR), metabolic variables, and urinary markers were measured monthly. At the end of the study, a histopathological evaluation of the kidney was performed. Results: Kidneys of SHR did not develop signs of relevant histopathological changes, but showed increased glomerular area and cellularity. Plasma creatinine was decreased, and creatinine clearance was augmented in SHR at the end of the study. Urinary excretion of Klotho was higher in SHR at 5 and 8 months old, whereas plasma Klotho levels were similar to WKY. GluAp, AlaAp, and DPP4 urinary activities were increased in SHR throughout the time-course study. A positive correlation between glomerular area and cellularity with creatinine clearance was observed. Urinary GluAp, AlaAp, DPP4, and Klotho showed positive correlations with SBP. Conclusions: GluAp, AlaAp, DPP4, and Klotho in the urine are useful tools for the evaluation of renal damage at early stages, before the whole histopathological and biochemical manifestations of renal disease are established. Moreover, these observations may represent a novel and noninvasive diagnostic approach to assess the evolution of kidney function in hypertension and other chronic diseases.

Increased Plasma Level of Longevity Protein Klotho as a Potential Indicator of Cognitive Function Preservation in Patients With Schizophrenia

Cognitive impairments are a core feature of schizophrenia. Klotho is an anti-aging protein with demonstrated cognitive-enhancing effects on the brain. The purpose of this study was to investigate the differences in levels of plasma klotho between patients with schizophrenia and healthy controls, as well as the relationship between klotho level and cognitive function in patients. Forty patients with schizophrenia and 40 gender- and age-matched healthy individuals were recruited. Positive and Negative Syndrome Scale (PANSS) was used to assess the psychopathology of patients. A neuropsychological battery was performed to evaluate the cognitive function of participants. Plasma klotho was measured using enzyme-linked immunosorbent assay. We show that patients with schizophrenia performed worse in the neurocognitive tests than the healthy controls. The levels of plasma klotho were significantly higher in schizophrenia patients than in healthy controls (p < 0.001). In patients, plasma klotho levels were positively correlated with cognitive function with regard to attention (p = 0.010), working memory (p < 0.001), verbal memory (p = 0.044), executive function (p < 0.001), and composite cognitive score (p < 0.001). Stepwise linear regression analysis shows that executive function had the highest correlation with plasma klotho levels (β = 0.896, t = 8.290, p < 0.001). Collectively, these results indicate that anti-aging protein klotho may be implicated in the pathogenesis of schizophrenia, and increased klotho may act as a compensatory factor for the preservation of cognitive function in schizophrenia. Further studies are needed to investigate the dynamic changes of klotho and the mechanisms by which klotho modulates cognition in schizophrenia.

P1573EFFECT OF INFLAMMATION ON MUSCLE-BONE INTERACTIONS IN HEMODIALYSIS PATIENTS

Abstract Background and Aims Chronic hemodialysis patients show decreased muscle mass, low physical activity and chronic weakness. Hemodialysis treatments results in increased risk of inflammation, which impairs physical activity. The calcium-phosphate and bone metabolism, as well as muscle mass, are regulated by a range of factors like FGF-23 and its co-factor Klotho protein, myostatin, follistatin and sclerostin. Complex interactions between these cytokines and inflammation have been described. The aim of the study was to assess the influence of inflammation on plasma concentrations of factors that regulate muscle and bone-mineral metabolism Method 55 hemodialysis patients were divided into study group (32 patients) and the reference group (23 patients). The former were the patients who had inflammation confirmed by plasma CRP &amp;gt;50 mg /L. Blood was collected before and after hemodialysis during the inflammation, and before hemodialysis after the resolution of inflammation Results Mean CRP in study group was 105.9±73.9 mg/l. The mean period between blood sampling during inflammation and resolution of inflammation was 15±7.9 days. Statistically significant increase of plasma cFGF-23 concentration was observed after hemodialysis. Its median concentration increased from 1992.8 RU/ml; IQR = [1350; 4320 RU/ml] before hemodialysis to 2421.3 RU/ml IQR = [1351; 7348 RU/ml] after HD (p = 0.001). Significant increase of sclerostin was observed after hemodialysis. Serum sclerostin increased from 1346.2 pg/ml IQR = [1034; 2243 pg/ml] before HD to 1583.8 pg/ml IQR = [1198; 2559 pg/ml] after HD (p = 0.002). Follistatin concentration increased from 2669 pg/ml IQR = [2129; 5702 pg/ml] before HD to 4220.5 pg/ml IQR = [2583; 6727 pg/ml] (p = 0.009). Hemodialysis had no significant effect on plasma Klotho protein, iFGF-23, FGF-21, and myostatin. After the resolution of inflammation there was statistically significant decrease in FGF-21 from 3591.4 pg/ml. IQR = [1174.3; 6,537.3 pg/ml] to 2454 pg/ml IQR = [1221.1; 3448.4 pg/ml] p = 0.006). There were significant changes of the concentration of Klotho protein, cFGF-23, iFGF-23, sclerostin, myostatin, follistatin after the resolution of inflammation vs. baseline. Conclusion Hemodialysis modulates the concentration of factors involved in regulation of calcium-phosphate metabolism, bone turnover and muscle growth and strength. The resolution of inflammation in hemodialysis patients leads to the decrease of plasma FGF-21 concentration.

P0541KLOTHO IS INVOLVED IN EARLY AND LONG-TERM PROTECTION AGAINST RHABDOMYOLYSIS ASSOCIATED AKI

Abstract Background and Aims Rhabdomyolysis is characterized by the breakdown of the skeletal muscle and the subsequent myoglobin (Mb) release into the bloodstream. A common complication of this syndrome is acute renal injury (AKI). Once filtered by the kidney, Mb causes oxidative stress, inflammation and tubular cell death. There is no specific treatment for rhabdomyolysis-AKI, so it is crucial a better understanding of this syndrome to identify new therapeutic targets. Klotho is an anti-aging protein mostly expressed by the kidney. In addition to its functions in the regulation of mineral metabolism, Klotho protects from AKI-harmful effects. However, no previous studies analyzed the role of Klotho in rhabdomyolysis. Method We performed a pre-clinical model of rhabdomyolysis in C57BL/6J mice (male, 12 weeks old, n=30) by intramuscular injection of 10 ml/kg of 50% glycerol (≥99.5% m/v). Mice were sacrificed 3 and 6 hours or 1, 3, 7 and 30 days after glycerol administration. To evaluate to beneficial effect of Klotho in rhabdomyolysis, C57BL/6J mice were injected intraperitoneally with 0.1 mg/kg recombinant mouse Klotho (1819-KL, R&amp;D Systems), or vehicle (PBS) 30 minutes before and 1, 3 and 5 days after glycerol injection. Blood, urine and renal samples were collected to analyze renal function, Klotho/FGF23 levels, oxidative stress, inflammation, fibrosis and cell death, all of them pathological processes affecting Klotho expression. In addition, we carried out studies in murine tubular cells (MCTs) to study the molecular mechanisms involved in Klotho regulation. Results Our results indicate that rhabdomyolysis induces an early decrease in Klotho renal mRNA and protein expression as well as Klotho serum levels. Klotho levels decreased in line with augmentation of creatinine concentration, kidney inflammation (CCL2 and IL-6 mRNA expression) and tubular injury marker NGAL. Moreover, patients with rhabdomyolysis-AKI also showed lower plasma Klotho levels and increased FGF23 plasma concentration than age-matched healthy individuals. Renal klotho protein expression remained reduced one month after rhabdomyolysis-induction, in line with long term renal fibrosis and pro-inflammatory macrophage accumulation (F4/80+ cells). Exogenous recombinant Klotho administration ameliorated renal function and reduced rhabdomyolysis-mediated tubular cell death oxidative stress (4-HNE staining) and tubular injury 24h after glycerol injection. In the same line, Klotho administration during AKI development reduced long term renal fibrosis and macrophage infiltration one month later. Antioxidant therapies with N-acetylcysteine (NAC) and sulforaphane, a potent Nuclear factor erythroid-2-related factor 2 (Nrf2) inducer, reduced Mb-mediated Klotho decrease in cultured tubular cells. Inhibition of TNF-α and IL-6 with infliximab and tocilizumab, respectively, also reverted Mb-mediated Klotho decrease. Inhibition of the inflammatory NFkB and p38 pathways also prevented Mb-mediated Klotho reduction. Conclusion Our findings are the first to demonstrate decreased renal and soluble Klotho levels not only in the early phases of rhabdomyolysis-induced AKI, but also when renal function was recovered, indicating that long-term consequences of AKI, such as inflammation and fibrosis, are also involved in Klotho downregulation. In addition, our results also indicate that Klotho administration may be a potential strategy to decrease rhabdomyolysis- long term negative effects.

Effect of vitamin D supplementation on klotho protein, antioxidant status and nitric oxide in the elderly: A randomized, double-blinded, placebo-controlled clinical trial

IntroductionVitamin D has anti-aging/wellness properties because of its ability to induce klotho, a bona fide anti-senescence protein, and suggesting that optimum levels of vitamin D directly affect anti-aging. Plasma klotho and vitamin D both decrease with age. Higher levels of klotho are associated with lower mortality through lowering the risk of chronic disease, so vitamin D supplementation might have favorable effects on the amount of klotho, longevity and could promote healthy aging. This study was conducted to examine the effect of vitamin D supplementation on plasma klotho, antioxidant status and nitric oxide in the elderly. MethodsIn this double-blinded, randomized placebo-controlled clinical trial, 90 participants aged over 60 years, with vitamin D deficiency, were randomly assigned either to a vitamin D 50,000 IU/week provided as cholecalciferol weekly, for 12 weeks or to a placebo. ResultsAfter 12 weeks, mean plasma 25(OH) D,TAC, CAT and NO concentrations significantly increased in the vitamin D group (P < 0.001,P < 0.001,P < 0.001& P = 0.03 respectively) but mean plasma klotho and SOD concentration increased non significantly (P = 0.164 and P = 0.397, respectively). Changes in plasma klotho, TAC and vitamin D were statistically significant different (P < 0.001, P < 0.001 & P < 0.001 respectively) but CAT and NO had no significant change (P = 0.142& P = 0.202, respectively) between the two groups at the end of the study. ConclusionThe findings revealed that the intake of 25OHD increased the plasma TAC, CAT, NO and 25OHD and prevented the reduction in plasma klotho so it may provide a potentially beneficial role in healthy aging and the elderly.

Study of plasma klotho and plasma copeptin level in adolescents with type 1 diabetes mellitus and relation to microvascular complications

Background Type 1 diabetes, previously known as juvenile diabetes or insulin-dependent diabetes, is a form of diabetes characterized by the body’s inability to produce insulin owing to the autoimmune destruction of the insulin-producing beta cells in the pancreas. Klotho (KL), known as an anti-aging protein, was discovered recently to have a multitude of biological effects. Major attention has been paid to the role of α-klotho (α-KL) in diabetes and its relation with diabetic nephropathy. The circulating form of α-KL, named as soluble KL, functions as an endocrine substance that exerts heterogeneous actions, including the modulation of renal function upon hyperglycemia, regulation of cell compensation, downregulation of inflammation and anti-oxidation effects. Aim The aim of this work is to study plasma KL and plasma copeptin in adolescents with type 1 diabetes and their relation to microvascular complications. Patients and methods This study was conducted in diabetes outpatient clinic in national institute of diabetes and endocrinology. The sample of 117 patients was divided into three groups: diabetes group, with 39 patients; diabetes with complication group, with 39 patients; and healthy controls, with 39 participants. Results Our study shows significant elevation of plasma copeptin level in type 1 diabetic adolescents, which was also significantly higher in the presence of microvascular complications particularly diabetic kidney disease (P=0.000). Although plasma KL is found to be significantly higher in type 1 diabetes without complications group, its level is dropped in the diabetes with complications group (P=0.005). Conclusion Plasma KL and plasma copeptin can be used as novel biomarkers for predicting early kidney injury in type 1 diabetic adolescents.

The Association Between Circulating Klotho and Dipeptidyl Peptidase-4 Activity and Inflammatory Cytokines in Elderly Patients With Alzheimer Disease.

Introduction:Klotho and Dipeptidyl Peptidase-4 (DPP4) are two proteins that modulate inflammatory pathways. We investigated the association between circulating klotho and DPP4 activity and their relationship with inflammatory cytokines, miR-29a, and miR-195 in Alzheimer Disease (AD).Methods:This study was conducted on 16 AD patients and 16 healthy age-matched controls. Plasma levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β, interleukin-6 (IL-6), klotho, and DPP4 were measured by enzyme-linked immunosorbent assay. Plasma expression of miR-29a and miR-195 were also measured and compared by a real-time polymerase chain reaction.Results:There was a significant increase in TNF-α (p=0.006), IL-1β (p=0.012), and IL-6 (p=0.012) levels in the AD subjects compared with controls. Also, we found a decrease in plasma levels of klotho and an increase in plasma levels of DPP4 in the AD group that was not significant compared with the controls. Lower expression of miR-29a (P=0.009) and higher expression of miR-195 (P=0.003) were observed in the AD group that was significant than controls. Further analysis showed a negative correlation between klotho and plasma levels of IL-6 (r=−0.58, p=0.01). Also, there was a positive correlation between plasma DPP4 activity and TNF-α levels (r=0.50, P=0.04) and IL-1β (r=0.62, P=0.01). Likewise, plasma klotho concentration showed a negative correlation with the age of AD subjects (r=−0.56, P=0.02).Conclusion:TNF-α, IL-1β, and IL-6 are involved in AD pathophysiology, and dysregulation of DPP4 and klotho may be associated with the inflammatory response of AD. Down-regulation of miR-29a and up-regulation of miR-195 indicated the role of miRNAs in the AD process.

Correlation of Plasma Klotho Levels and Carotid Intima- Media Thickness in CKD Patients on Regular Hemodialysis

Background: The incidence of heart death is 5-10 times more in patients with chronic kidney disease (CKD) with regular hemodialysis compared to the general population. Vascular calcification is a risk factor. In CKD patients there is a decrease in the distal renal cell cells which are the main producer of Klotho in the body. The functions of Klotho are maintaining integrity, endothelial permeability and endogenous inhibitors of vascular calcification. Previous studies reported an association between vascular calcification and Klotho plasma levels. Vascular calcification for heart disease can be determined by the thickness Intima Media (TIM) Carotid Artery. Objective: To determine the correlation of plasma Klotho levels with the Intima Media thickness of the Carotid Artery in CKD patients with regular hemodialysis. Methode: This cross-sectional study was conducted at the Rasyida Kidney General Hospital in Medan from April to July 2018. Patients with CKD who have undergone routine hemodialysis are examined for plasma levels of Klotho and real time ultrasound examinations to determine the thickness of arterial intima media carotid. Data are analyzed with Mann Whitney test, binominal logistic regression and Pearson correlation. Results: Seventy patients with CKD showed an average plasma Klotho level of 281.43 + 298.63 pg / ml with an average TIM of the carotid artery of 0.22 + 0.09 cm. Patients were divided into 2 groups, namely the calcification group (n = 39) and the noncalcified group (n = 31). Mann-Whitney test was performed with the results that there were differences in plasma Klotho levels between the calcification group and the noncalcified carotid artery group which were statistically significant (p = 0.001). The binominal logistic regression analysis test was performed on risk factors associated with the incidence of vascular calcification and the results of Klotho serum levels, Diabetes Mellitus and Hypertension were found to be significant risk factors for calcification (p &lt;0.05). With the Pearson correlation test, the negative correlation of plasma Klotho levels with the thickening of Intima Carotid Arterial Media was statistically significant with moderate correlation strength (p = 0.002; r = -0.368). Conclusion: Patients with chronic kidney disease with low plasma Klotho levels have thickening of the intima media of carotid artery.

Plasma Klotho concentration is associated with the presence, burden and progression of cerebral small vessel disease in patients with acute ischaemic stroke.

Klotho is a soluble or membrane-bound anti-aging protein, whose protective actions are important for a prudential function of many organs. Because Klotho and cerebral small vessel disease (SVD) are associated with ageing process and endothelial dysfunction, it is possible that Klotho has an association with cerebral SVD. We aimed to investigate the association of plasma Klotho concentration with the presence, burden and progression of cerebral SVD. We prospectively enrolled 262 patients with first-ever acute cerebral infarction, performed brain MRI and collected their blood samples within 24 hours of admission. An enzyme-linked immunosorbent assay was used for evaluating plasma Klotho concentration. We estimated the total SVD score of each patient after determining the presence and burden of high-grade white matter hyperintensities (HWMHs), cerebral microbleeds (CMBs), high-grade perivascular spaces (HPVSs) and asymptomatic lacunar infarctions (ALIs). Univariate and multivariate analyses were conducted to investigate association of Klotho with cerebral SVD and the total SVD score. Of the 262 patients, 152 (58.0%) were men. The mean age of these patients was 64.7 years. The mean ± standard deviation of plasma Klotho concentration was 329.8 ± 194.1 pg/mL. In multivariate analysis, plasma Klotho concentration was negatively associated with the presence of HWMHs [Odds ratio (OR): 0.13, p = 0.047], HPVSs (OR: 0.22, p = 0.024) and ALIs (OR: 0.53, p = 0.021) but not associated with the presence of CMBs (OR: 0.39, p = 0.404). Plasma Klotho concentration was also negatively related to the total SVD score (unstandardized coefficients beta: −0.895, standard error = 0.317, p = 0.005, R2 = 0.239). Furthermore, plasma Klotho concentration was negatively related to the presence (OR: 0.75, 95% CI: 0.59–0.96, p = 0.025) and severity of cerebral SVD progression (OR: 0.72, 95% CI: 0.56–0.92, p = 0.009). In conclusion, plasma Klotho concentration was negatively associated with the presence, burden and progression of cerebral SVD.