Recombinant Klotho Research Articles

SARS-CoV-2 S Protein Reduces Cytoprotective Defenses and Promotes Human Endothelial Cell Senescence.

Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. The S protein of SARS-CoV2, which acts as the receptor binding protein for the viral infection, is able to induce endothelial cells inflammation and it has been found as an isolated element in the circulation and in human tissues reservoirs months after infection. Here, we investigated whether the S protein is able to directly induce endothelial cell senescence and deciphered some of the mechanisms involved. In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner the cellular content of senescence and DNA damage response markers (senescence-associated-β galactosidase, γH2AX), as well as growth-arrest effectors (p53, p21, p16). In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.

ACTIVATION OF KLOTHO/SIRT1 SIGNALING PATHWAY ATTENUATES MYOCARDIAL ISCHEMIA REPERFUSION INJURY IN DIABETIC RATS.

Diabetes and myocardial ischemia reperfusion (MIR) injury are characterized by oxidative stress, inflammation, autophagy disorders, and cardiac contractile dysfunction. Klotho and SIRT1 regulate the level of oxidative stress to participate in the regulation of many physiological functions such as cell survival, aging, apoptosis, autophagy, mitochondrial biogenesis, and inflammation. We hypothesized that the activation of Klotho/SIRT1 signaling pathway could attenuate MIR in diabetic rats. Type 1 diabetes and MIR injury model were established to examine this hypothesis in vivo . Primary rat cardiomyocytes and H9c2 cells were exposed to high glucose conditions and hypoxia/reoxygenation (H/R) insult in vitro . Hemodynamic parameters of heart function, myocardial infarct size, oxidative stress, markers of MIR injury or cell viability, and the mRNA and protein expression of Klotho and SIRT1 were measured. There was lower expression of Klotho and SIRT1 in diabetic MIR hearts than in nondiabetic rats, as well as significantly increased oxidative stress levels and decreased autophagy levels. Recombinant Klotho (rKlotho) protein and the SIRT1 agonist SRT1720 could significantly attenuate MIR injury in diabetes by activating Klotho/SIRT1 signaling pathway to reduce oxidative stress and restore autophagy levels. These findings suggest that the Klotho/SIRT1 pathway plays an important role in MIR injury in diabetic rats, and rKlotho protein and agonist SRT1720 have therapeutic potential for alleviating diabetic myocardial IR injury by activating Klotho/SIRT1 to reduce oxidative stress and restore autophagy levels.

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.

Recombinant Klotho protein protects pulmonary alveolar epithelial cells against sepsis-induced apoptosis by inhibiting the Bcl-2/Bax/caspase-3 pathway.

Inflammation-induced apoptosis of alveolar type II epithelial cells is a primary contributor to sepsis-induced acute respiratory distress syndrome (ARDS). Klotho is a single-pass transmembrane protein with anti-inflammatory and anti-apoptotic effects. However, the role and mechanism of Klotho in the development of ARDS remains unknown. This study aimed to investigate the effect of Klotho on sepsis-induced apoptosis in human pulmonary alveolar epithelial cells (HPAEpiCs) together with the potential mechanism. Cecal ligation and puncture (CLP) were performed to generate an in vivo sepsis model, and HPAEpiCs were treated with lipopolysaccharide (LPS) to mimic sepsis in vitro. Both models were administered recombinant Klotho protein. The morphology of the lung tissue was observed, and apoptotic cells and cell viability were detected. Interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA), while the expression of Bcl-2, Bax and cleaved caspase-3 was detected with western blotting. Klotho reversed the CLP-induced decrease in mouse survival in vivo (p < 0.001) and increased inflammatory cell infiltration and inflammatory substance exudation in the lung tissue of mice with sepsis (both p < 0.001). Klotho also suppressed apoptosis (p < 0.001) as demonstrated by IL-1β, IL-6 and TNF-α expression (all p < 0.001), and Bcl-2/Bax/caspase-3 pathway activation (p < 0.001). Klotho pretreatment significantly prevented LPS-induced apoptosis in vitro (p < 0.001), as demonstrated by IL-1β, IL-6 and TNF-α upregulation (all p < 0.001); and Bcl-2/Bax/caspase-3 pathway activation in HPAEpiCs (p < 0.001). This study demonstrated that Klotho can ameliorate acute lung injury (ALI) induced by sepsis by inhibiting inflammatory responses and exerting anti-apoptotic effects by suppressing Bcl-2/Bax/caspase-3 pathway activation.

Butyrate promotes kidney resilience through a coordinated kidney protective response in tubular cells

Acute kidney injury (AKI) is common in hospitalized patients and increases short-term and long-term mortality. Treatment options for AKI are limited. Gut microbiota products such as the short-chain fatty acid butyrate have anti-inflammatory actions that may protect tissues, including the kidney, from injury. However, the molecular mechanisms of tissue protection by butyrate are poorly understood. Treatment with oral butyrate for two weeks prior to folic acid-induced AKI and during AKI improved kidney function and decreased tubular injury and kidney inflammation while stopping butyrate before AKI was not protective. Continuous butyrate preserved the expression of kidney protective factors such as Klotho, PGC-1α and Nlrp6 which were otherwise downregulated. In cultured tubular cells, butyrate blunted the maladaptive tubular cell response to a proinflammatory milieu, preserving the expression of kidney protective factors. Kidney protection afforded by this continuous butyrate schedule was confirmed in a second model of nephrotoxic AKI, cisplatin nephrotoxicity, where the expression of kidney protective factors was also preserved. To assess the contribution of preservation of kidney protective factors to kidney resilience, recombinant Klotho was administered to mice with cisplatin-AKI and shown to preserve the expression of PGC-1α and Nlrp6, decrease kidney inflammation and protect from AKI. In conclusion, butyrate promotes kidney resilience to AKI and decreases inflammation by preventing the downregulation of kidney protective genes such as Klotho. This information may be relevant to optimize antibiotic management during hospitalization.

Therapeutic potential of AAV-FL-Klotho in obesity: Impact on weight loss and lipid metabolism in mice

Klotho, an anti-aging protein, has gained attention for its protective effects against various diseases, including metabolic disorders, through recombinant Klotho administration. However, the potential of Klotho as a target for gene therapy requires further exploration, as it remains relatively understudied in the context of metabolic disorders. In this study, we demonstrate that AAV-full length(FL)-Klotho administration induces weight loss in mice and provides protection against high-fat diet (HFD)-induced obesity and hepatic steatosis, concurrently reducing the weights of white adipose tissue and liver. AAV-FL-Klotho administration also enhanced thermogenic gene expression in brown adipose tissue (BAT) and improved the morphology of interscapular BAT. The weight loss effect of AAV-FL-Klotho was found to be, at least in part, mediated by UCP1-dependent thermogenesis in brown adipocytes, potentially influenced by hepatokines secreted from AAV-FL-Klotho-transduced hepatocytes. These findings suggest that AAV-FL-Klotho is an attractive candidate for gene therapy to combat obesity. Nevertheless, unbiased experiments have also revealed disturbances in lipid metabolism due to AAV-FL-Klotho, as evidenced by the emergence of lipomas and increased expression of hepatic lipogenic proteins.

Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease.

Chronic kidney disease (CKD) is linked to greater prevalence and rapid progression of calcific aortic valve disease (CAVD) characterized by valvular leaflet fibrosis and calcification. Fibroblast growth factor 23 (FGF23) level is elevated, and anti-aging protein Klotho is reduced in CKD patients. However, the roles of FGF23 and Klotho in the mechanism of aortic valve fibrosis and calcification remain unclear. We hypothesized that FGF23 mediates CKD-induced CAVD by enhancing aortic valve interstitial cell (AVIC) fibrosis and calcification, while soluble Klotho inhibits FGF23 effect. Methods and Results: In an old mouse model of CKD, kidney damages were accompanied by aortic valve thickening and calcification. FGF23 levels in plasma and aortic valve were increased, while Klotho levels were decreased. Recombinant FGF23 elevated the inflammatory, fibrogenic, and osteogenic activities in AVICs. Neutralizing antibody or shRNA targeting FGF23 suppressed the pathobiological activities in AVICs from valves affected by CAVD. FGF23 exerts its effects on AVICs via FGF receptor (FGFR)/Yes-associated protein (YAP) signaling, and inhibition of FGFR/YAP reduced FGF23's potency in AVICs. Recombinant Klotho downregulated the pathobiological activities in AVICs exposed to FGF23. Incubation of FGF23 with Klotho formed complexes and decreased FGF23's potency. Further, treatment of CKD mice with recombinant Klotho attenuated aortic valve lesions. Conclusion: This study demonstrates that CKD induces FGF23 accumulation, Klotho insufficiency and aortic valve lesions in old mice. FGF23 upregulates the inflammatory, fibrogenic and osteogenic activities in AVICs via the FGFR/YAP signaling pathway. Soluble Klotho suppresses FGF23 effect through molecular interaction and is capable of mitigating CKD-induced CAVD.

Recombinant Klotho alleviates vancomycin-induced acute kidney injury by upregulating anti-oxidative capacity via JAK2/STAT3/GPx3 axis

Emerging studies support that Klotho protects against different kidney diseases. However, the role of Klotho in vancomycin induced acute kidney injury (Van-AKI) is largely unclear. Hence this study aimed to explore the regulatory mechanism of Klotho in Van-AKI. The mRNA expression of Klotho and the JAK2/STAT3/GPx3 in renal tissue were assessed by RNA sequence analysis after 600 mg/kg Van daily for seven days; Small interfering RNA and recombinant protein are applied to examine the mechanism action of Klotho in vitro and in vivo respectively. Flow cytometry and spectrophotometry detected the expression of reactive oxygen species and antioxidant enzymes. Transmission electron microscopy scanned the structural damage of mitochondria. Western blotting, qPCR, and immunofluorescence were employed to explore the JAK2/STAT3/GPx3 expression. RNA sequence analysis found that Van challenging reduced Klotho and GPx3 expression but increased JAK2/STAT3 in renal tissue. In HK-2 cells, Klotho were decreased by Van in a dose-dependent manner. Klotho siRNA enhanced the production of reactive oxygen species and the cell apoptosis ratio by regulating the JAK2/STAT3, and JAK2/STAT3 inhibitors prevented the decrease of GPx3. Meanwhile, 1 μg/ml recombinant human Klotho showed the opposite function to 120 pmol Klotho siRNA. In Van-AKI BALB/c mice, 20 μg/kg recombinant mouse Klotho once every two days improved the anti-oxidative enzyme expression, mitochondria structure, renal dysfunction, and histological damage. In conclusion, Klotho enhances antioxidant capacity through the JAK2/STAT3/GPx3 axis, which in turn improves Van-AKI.

Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells.

Interferon gamma (IFNγ) is a cytokine implicated in the pathogenesis of autoimmune diseases. SAM and HD domain-containing protein 1 (SAMHD1) is an IFNγ-inducible protein that modulates cellular dNTP levels. Mutations in the human SAMHD1 gene cause Aicardi-Goutières (AG) syndrome, an autoimmune disease sharing similar clinical features with systemic lupus erythematosus (SLE). Klotho is an anti-inflammatory protein which suppresses aging through multiple mechanisms. Implication of Klotho in autoimmune response is identified in rheumatologic diseases such as SLE. Little information exists regarding the effect of Klotho in lupus nephritis, one of the prevalent symptoms of SLE. The present study verified the effect of IFNγ on SAMHD1 and Klotho expression in MES-13 glomerular mesangial cells, a special cell type in glomerulus that is critically involved in lupus nephritis. IFNγ upregulated SAMHD1 expression in MES-13 cells through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) and the nuclear factor kappa B (NFκB) signaling pathways. IFNγ decreased Klotho protein expression in MES-13 cells. Treatment of MES-13 cells with recombinant Klotho protein inhibited SAMHD1 expression by blocking IFNγ-induced NFκB nuclear translocation, but showed no effect on JAK-STAT1 signaling. Collectively, our findings support the protective role of Klotho in attenuating lupus nephritis through the inhibition of IFNγ-induced SAMHD1 expression and IFNγ downstream signaling in MES-13 cells.

The transcription factor Fosl1 preserves Klotho expression and protects from acute kidney injury

Increased expression of AP-1 transcription factor components has been reported in acute kidney injury (AKI). However, the role of specific components, such as Fosl1, in tubular cells or AKI is unknown. Upstream regulator analysis of murine nephrotoxic AKI transcriptomics identified AP-1 as highly upregulated. Among AP-1 canonical components, Fosl1 was found to be upregulated in two transcriptomics datasets from nephrotoxic murine AKI induced by folic acid or cisplatin and from proximal tubular cells exposed to TWEAK, a cytokine mediator of AKI. Fosl1 was minimally expressed in the kidneys of control uninjured mice. Increased Fosl1 protein was localized to proximal tubular cell nuclei in AKI. In human AKI, FOSL1 was found present in proximal tubular cells in kidney sections and in urine along with increased urinary FOSL1 mRNA. Selective Fosl1 deficiency in proximal tubular cells (Fosl1Δtub) increased the severity of murine cisplatin- or folate-induced AKI as characterized by lower kidney function, more severe kidney inflammation and Klotho downregulation. Indeed, elevated AP-1 activity was observed after cisplatin-induced AKI in Fosl1Δtub mice compared to wild-type mice. More severe Klotho downregulation preceded more severe kidney dysfunction. The Klotho promoter was enriched in Fosl1 binding sites and Fosl1 bound to the Klotho promoter in cisplatin-AKI. In cultured proximal tubular cells, Fosl1 targeting increased the proinflammatory response and downregulated Klotho. Invivo, recombinant Klotho administration protected Fosl1Δtub mice from cisplatin-AKI. Thus, increased proximal tubular Fosl1 expression during AKI is an adaptive response, preserves Klotho, and limits the severity of tubular cell injury and AKI.

Klotho Protein Decreases MMP-Mediated Degradation of Contractile Proteins during Ischaemia/Reperfusion Injury to the Cardiomyocytes

Ischaemia, followed by reperfusion, causes the generation of reactive oxygen species, overproduction of peroxynitrite, activation of matrix metalloproteinases (MMPs), and subsequently the degradation of heart contractile proteins in the cardiomyocytes. Klotho is a membrane-bound or soluble protein that regulates mineral metabolism and has antioxidative activity. This study aimed to examine the influence of Klotho protein on the MMP-mediated degradation of contractile proteins during ischaemia/reperfusion injury (IRI) to the cardiomyocytes. Human cardiac myocytes (HCM) underwent in vitro chemical IRI (with sodium cyanide and deoxyglucose), with or without the administration of recombinant Klotho protein. The expression of MMP genes, the expression and activity of MMP proteins, as well as the level of contractile proteins such as myosin light chain 1 (MLC1) and troponin I (TnI) in HCM were measured. Administration of Klotho protein resulted in a decreased activity of MMP-2 and reduced the release of MLC1 and TnI that followed in cells subjected to IRI. Thus, Klotho protein contributes to the inhibition of MMP-dependent degradation of contractile proteins and prevents injury to the cardiomyocytes during IRI.

Aging-suppressor Klotho: Prospects in diagnostics and therapeutics.

The protein Klotho (KL) was first discovered in KL-deficient mice, which developed a syndrome similar to premature aging in humans. Since then, KL has been implicated in multiple molecular signaling pathways and diseases. KL has been shown to have anti-aging, healthspan and lifespan extending, cognitive enhancing, anti-oxidative, anti-inflammatory, and anti-tumor properties. KL levels decrease with age and in many diseases. Therefore, it has been of great interest to develop a KL-boosting or restoring drug, or to supplement endogenous Klotho with exogenous Klotho genetic material or recombinant Klotho protein, and to use KL levels in the body as a marker for the efficacy of such drugs and as a biomarker for the diagnosis and management of diseases. The goal of this study was to provide a comprehensive review of KL levels across age groups in individuals who are healthy or have certain health conditions, using four sources: blood, cerebrospinal fluid, urine, and whole biopsy/necropsy tissue. By doing so, baseline KL levels can be identified across the lifespan, in the absence or presence of disease. In turn, these findings can be used to guide the development of future KL-based therapeutics and biomarkers, which will heavily rely on an individual's baseline KL range to be efficacious. A total of 65 studies were collected primarily using the PubMed database. Research articles that were published up to April 2022 were included. Statistical analysis was conducted using RStudio. Mean and median blood KL levels in healthy individuals, mean blood KL levels in individuals with renal conditions, and mean blood KL levels in individuals with metabolic or endocrine conditions were shown to decrease with age. Similarly, CSF KL levels in patients with AD also declined compared with age-matched controls. The present study confirms the trend that KL levels in blood decrease with age in humans, among those who are healthy, and even further among those with renal and endocrine/metabolic illnesses. Further, by drawing this trend from multiple published works, we were able to provide a general idea of baseline KL ranges, specifically in blood in these populations. These data add to the current knowledge on normal KL levels in the body and how they change with time and in disease, and can potentially support efforts to create KL-based treatments and screening tools to better manage aging, renal, and metabolic/endocrine diseases.

Abstract 13265: Soluble Klotho Captures FGF23 to Mitigate Aortic Valve Inflammocalcification Associated With Chronic Kidney Disease

Calcific aortic valve disease (CAVD) is prevalent in the elderly people, particularly those with chronic kidney disease (CKD). Klotho (KL), an anti-aging protein, is expressed mainly in the kidney and the cardiovascular system as soluble and membrane forms. CKD patients have reduced levels of KL and increased levels of fibroblast growth factor 23 (FGF23), a pro-inflammatory mediator that may interact with soluble KL. We have reported that KL reduces the osteogenic response to high phosphate levels, a condition seen in CKD, in human aortic valve interstitial cells (AVICs). This study tested the hypothesis that soluble KL captures FGF23 to prevent aortic valve inflammocalcification induced by CKD. Methods and Results: We developed a novel CKD-CAVD model in old C57 mice (20 months) on AIN-76A diet and treated with adenine and calcitriol. Histology and immunostaining revealed tubular damage and monocyte/macrophage accumulation in kidney tissue of CKD mice. Aortic valve lesions, thickening and calcification, were evident in CKD mice. Serum FGF23 levels were increased while serum KL levels decreased. In addition, KL expression in the aortic valve was reduced. Recombinant FGF23 (0.04 mg/ml) elevated the inflammosteogenic activity in human AVICs. Treatment with recombinant KL (0.5 μg/ml) markedly decreased FGF23-induced inflammosteogenic activation in AVICs. Systemic analysis revealed that FGF23 upregulates YAP/SMAD3 signaling, and inhibition of YAP and SMAD3 reduced the effect of FGF23 on the inflammosteogenic activity in AVICs. In addition, recombinant KL suppressed the activation of this signaling mechanism in AVICs, and treatment of CKD mice with recombinant KL attenuated aortic valve lesions. However, denaturation of KL protein resulted in a loss of its effect on FGF23-induced AVIC inflammosteogenic activation. Further, incubation of FGF23 with recombinant KL not only resulted in the formation of complexes of these two proteins, but also markedly decreased the pro-inflammosteogenic effect of FGF23 in AVICs. Conclusion: This study demonstrates that CKD induces KL insufficiency and FGF23 accumulation that promote aortic valve lesions. Recombinant KL is capable of binding to FGF23 to suppress its pro-inflammosteogenic effect in the aortic valve.

Abstract 13254: Klotho Suppresses Endothelial-to-Mesenchymal Transition in the Aorta of Mice With Chronic Kidney Disease by Inhibition of Endothelial Senescent Activity

Background: Klotho is an anti-aging protein expressed primarily in the kidney and vasculature. Chronic kidney disease (CKD) results in hyperphosphatemia. We reported that high levels of phosphate downregulate Klotho expression in vascular cells. Endothelial-to-mesenchymal transition (EndMT) in arteries promotes the development of atherosclerosis, and CKD is a significant risk factor for atherosclerosis. However, the impact of CKD on EndMT in arteries is unclear and whether Klotho insufficiency plays a role in the mechanism underlying vascular EndMT remains unknown. This study tested the hypothesis that Klotho insufficiency associated with CKD induces senescence in arterial endothelial cells to upregulate EndMT. Methods and Results: We developed a CKD model in C57/BL6 mice (5-6 months old) on AIN-76A diet and treated with adenine and calcitriol. CKD was confirmed by markedly elevated blood levels of creatinine and phosphate, as well as by renal tubular damage. En face immunofluorescence staining of aorta and image quantification revealed that the levels of biomarkers of senescence (P21) and EndMT (vimentin) were greater, while Klotho levels were lower, in the aortic arch endothelial cells of CKD mice compared to sham control mice. P21 and vimentin were co-localized in a greater number of endothelial cells in the aortic arch of CKD mice. High phosphate treatment upregulated the levels of P21 and vimentin and reduced the levels of Klotho in both primary human and mouse aortic endothelial cells, and knockdown P21 attenuated EndMT induced by high phosphate. Recombinant Klotho suppressed both senescence and EndMT in primary human aortic endothelial cells. Further, prolonged treatment of CKD mice with recombinant Klotho attenuated endothelial senescence and EndMT in the aortic arch. Conclusion: Klotho insufficiency associated with CKD promotes endothelial senescence and EndMT in aorta. High phosphate is capable of inducing endothelial senescent change that provokes EndMT. Administration of recombinant Klotho or upregulation of Klotho expression may have therapeutic potential for suppressing pro-atherosclerosis activity in large arteries by prevention of endothelial senescence and resultant EndMT.

Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death

Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle-like "LAM" cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genesTSC1and/orTSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show that treatment of these cells with the AGTR1 inhibitor losartan or silencing of theAgtr1gene leads to increased cell deathinvitroand attenuates tumor progressioninvivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increased expression of Klotho. In TSC2-deficient cells, we showed overexpression of Klotho or treatment with recombinant (soluble) Klotho mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1 inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the RAS in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.

Klotho relieves inflammation and exerts a cardioprotective effect during renal ischemia/reperfusion-induced cardiorenal syndrome

BackgroundRenal ischemia and reperfusion injury (IRI) is the main cause of acute kidney injury (AKI). AKI induces the development of cardiac hypertrophy (CH) during cardiorenal syndrome (CRS), and cardiomyocyte calcium mishandling though systemic inflammation after 8 days of renal IRI. Klotho has recently been described as an anti-inflammatory component. Given this, Klotho treatment could prevent or attenuate the inflammation, thereby also preventing electrical cardiac outcomes incurred by CRS. The aim of this study was to investigate the therapeutic role of Klotho in CRS after unilateral renal IRI through its anti-inflammatory action. MethodsWe examined renal tissue structure and function, intracellular Ca2+ dynamics in adult ventricular cardiomyocytes and serum cytokine levels from C57BL/6 mice that suffered unilateral renal IRI by occluding the left pedicle for 60 min and reperfusion for 8 days. The animals were treated with recombinant Klotho protein starting from the day of the surgery, then daily for 8 days. ResultsAfter Klotho treatment for 8 days, the left renal tissue remained damaged, however the renal function was restored due to the right kidney tissue preservation. In parallel, Klotho also prevented an increase in serum interleukin (IL-) 6, IL-1β, and tumor necrosis factor alpha (TNF-α) levels. CH and low cell contraction were also prevented, as well as a decrease in systolic Ca2+ transients and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity measured as Ca2+ transient decay, an increase in spontaneous Ca2+ release and the incidence of pro-arrhythmic events. ConclusionsThe Klotho treatment showed promise, playing an important role in the pathophysiology of CRS. We were unable to observe a total renoprotective role of the compound in the model; in turn, a cardioprotective role of Klotho was demonstrated through the prevention of hypertrophy and normalization of the Ca2+ cycle dysfunction of cardiomyocytes. We propose that Klotho acts in the cardiorenal syndrome by systematically preventing inflammation and increased FGF23, alleviating cardiac outcomes.

Recombinant Klotho Protein Ameliorates Myocardial Ischemia/Reperfusion Injury by Attenuating Sterile Inflammation

Currently, no effective therapy and potential target have been elucidated for preventing myocardial ischemia and reperfusion injury (I/R). We hypothesized that the administration of recombinant klotho (rKL) protein could attenuate the sterile inflammation in peri-infarct regions by inhibiting the extracellular release of high mobility group box-1 (HMGB1). This hypothesis was examined using a rat coronary artery ligation model. Rats were divided into sham, sham+ rKL, I/R, and I/R+ rKL groups (n = 5/group). Administration of rKL protein reduced infarct volume and attenuated extracellular release of HMGB1 from peri-infarct tissue after myocardial I/R injury. The administration of rKL protein inhibited the expression of pro-inflammatory cytokines in the peri-infarct regions and significantly attenuated apoptosis and production of intracellular reactive oxygen species by myocardial I/R injury. Klotho treatment significantly reduced the increase in the levels of circulating HMGB1 in blood at 4 h after myocardial ischemia. rKL regulated the levels of inflammation-related proteins. This is the first study to suggest that exogenous administration of rKL exerts myocardial protection effects after I/R injury and provides new mechanistic insights into rKL that can provide the theoretical basis for clinical application of new adjunctive modality for critical care of acute myocardial infarction.

The anti-aging factor Klotho protects against acquired long QT syndrome induced by uremia and promoted by fibroblast growth factor 23

BackgroundChronic kidney disease (CKD) is associated with increased propensity for arrhythmias. In this context, ventricular repolarization alterations have been shown to predispose to fatal arrhythmias and sudden cardiac death. Between mineral bone disturbances in CKD patients, increased fibroblast growth factor (FGF) 23 and decreased Klotho are emerging as important effectors of cardiovascular disease. However, the relationship between imbalanced FGF23-Klotho axis and the development of cardiac arrhythmias in CKD remains unknown.MethodsWe carried out a translational approach to study the relationship between the FGF23–Klotho signaling axis and acquired long QT syndrome in CKD-associated uremia. FGF23 levels and cardiac repolarization dynamics were analyzed in patients with dialysis-dependent CKD and in uremic mouse models of 5/6 nephrectomy (Nfx) and Klotho deficiency (hypomorphism), which show very high systemic FGF23 levels.ResultsPatients in the top quartile of FGF23 levels had a higher occurrence of very long QT intervals (> 490 ms) than peers in the lowest quartile. Experimentally, FGF23 induced QT prolongation in healthy mice. Similarly, alterations in cardiac repolarization and QT prolongation were observed in Nfx mice and in Klotho hypomorphic mice. QT prolongation in Nfx mice was explained by a significant decrease in the fast transient outward potassium (K+) current (Itof), caused by the downregulation of K+ channel 4.2 subunit (Kv4.2) expression. Kv4.2 expression was also significantly reduced in ventricular cardiomyocytes exposed to FGF23. Enhancing Klotho availability prevented both long QT prolongation and reduced Itof current. Likewise, administration of recombinant Klotho blocked the downregulation of Kv4.2 expression in Nfx mice and in FGF23-exposed cardiomyocytes.ConclusionThe FGF23–Klotho axis emerges as a new therapeutic target to prevent acquired long QT syndrome in uremia by minimizing the predisposition to potentially fatal ventricular arrhythmias and sudden cardiac death in patients with CKD.Graphical abstract

Pharmacological Blockade of NLRP3 Inflammasome/IL-1β-Positive Loop Mitigates Endothelial Cell Senescence and Dysfunction.

The clinical relevance of IL-1β in chronic inflammation underlying atherosclerosis has been reinforced by recent evidence associating pharmacological inhibition of the cytokine with lower cardiovascular risk. Previously, we have demonstrated a direct involvement of IL-1β in endothelial senescence. Therefore, this can be a key mechanism contributing to the sterile inflammatory milieu associated with aging, termed inflammaging. In the present study, we have evaluated whether a positive feedback of IL-1β in the NLRP3 inflammasome via NF-κB could promote human endothelial senescence in vitro and murine endothelial dysfunction in vivo. Our results indicate that the NLRP3 inflammasome is pivotal in mediating the detrimental effects of IL-1β, showing that auto-activation is a crucial feature boosting endothelial cell senescence in vitro, which is paralleled by vascular dysfunction in vivo. Hence, the inhibitor of NLRP3 inflammasome assembly, MCC 950, was able to disrupt the aforementioned positive loop, thus alleviating inflammation, cell senescence and vascular dysfunction. Besides, we explored alternative NLRP3 inflammasome inhibitory agents such as the RAS heptapeptide Ang-(1-7) and the anti-aging protein klotho, both of which demonstrated protective effects in vitro and in vivo. Altogether, our results highlight a fundamental role for the hereby described NLRP3 inflammasome/IL-1β positive feedback loop in stress-induced inflammaging and the associated vascular dysfunction, additionally providing evidence of a potential therapeutic use of MCC 950, Ang-(1-7) and recombinant klotho to block this loop and its deleterious effects.

Retinoic acid induces hyaluronic acid production through the klotho-mediated EGFR signaling pathway in human epidermal keratinocytes

All-trans retinoic acid (RA) is an effective anti-aging chemical substance widely used in skin-care products. RA compromises epidermal differentiation and induces keratinocyte proliferation, causing hyaluronic acid production through mechanisms that are not completely understood. Klotho protein causes the differentiation of human epidermal keratinocytes. Klotho gene expression is mediated by epidermal growth factor (EGF), which inhibits cell apoptosis in aging-related diseases. The klotho gene causes human aging syndrome, including short lifespan, skin atrophy, and osteoporosis. We investigated the relationship between RA and klotho in epidermal keratinocytes for the first time. In human epidermal keratinocytes, RA induced klotho gene expression. Treatment with both RA and recombinant klotho induced hyaluronic acid production in human epidermal keratinocytes. However, in klotho small interfering RNA (siRNA)-transfected keratinocytes, RA produced less hyaluronic acid than in the control group, indicating that RA may partially regulate hyaluronic acid production through a klotho-dependent pathway. Knockdown of klotho gene expression inactivated the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway, which is involved in hyaluronic acid production. We concluded that the effect of RA on hyaluronic acid production is partly regulated through the klotho-mediated EGFR signaling pathway in human epidermal keratinocytes.