Soluble Klotho, a Potential Biomarker of Chronic Kidney Disease-Mineral Bone Disorders Involved in Healthy Ageing: Lights and Shadows.

Shortly after the discovery of Klotho, interest grew in its potential role in chronic kidney disease (CKD). There are three isoforms of the Klotho protein: αKlotho, ßKlotho and γKlotho. This review will focus on αKlotho due to its relevance as a biomarker in CKD. αKlotho is synthesized mainly in the kidneys, but it can be released into the bloodstream and urine as soluble Klotho (sKlotho), which undertakes systemic actions, independently or in combination with FGF23. It is usually accepted that sKlotho levels are reduced early in CKD and that lower levels of sKlotho might be associated with the main chronic kidney disease-mineral bone disorders (CKD-MBDs): cardiovascular and bone disease. However, as results are inconsistent, the applicability of sKlotho as a CKD-MBD biomarker is still a matter of controversy. Much of the inconsistency can be explained due to low sample numbers, the low quality of clinical studies, the lack of standardized assays to assess sKlotho and a lack of consensus on sample processing, especially in urine. In recent decades, because of our longer life expectancies, the prevalence of accelerated-ageing diseases, such as CKD, has increased. Exercise, social interaction and caloric restriction are considered key factors for healthy ageing. While exercise and social interaction seem to be related to higher serum sKlotho levels, it is not clear whether serum sKlotho might be influenced by caloric restriction. This review focuses on the possible role of sKlotho as a biomarker in CKD-MBD, highlighting the difference between solid knowledge and areas requiring further research, including the role of sKlotho in healthy ageing.

Klotho inhibits the activation of NLRP3 inflammasome to alleviate lipopolysaccharide-induced inflammatory injury in A549 cells and restore mitochondrial function through SIRT1/Nrf2 signaling pathway.

Acute lung injury is a severe clinical condition constituting a major cause of mortality in intensive care units. This study aimed to investigate the role of klotho in alleviating lipopolysaccharide (LPS)-induced acute lung injury. LPS-induced acute lung injury was used to simulate the acute lung injury caused by severe pneumonia in vitro. The viability and apoptosis of A549 cells were detected by cell counting kit-8 assay and flow cytometry. The inflammatory response, oxidative stress, and mitochondrial function in A549 cells were analyzed by commercial assay kits and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1) staining. The expression of apoptosis-related proteins, Sirtuin 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway-related proteins, and NOD-like receptor family pyrin domain containing 3 (NLRP3) expression in A549 cells was detected by western blot. The mtDNA synthase level in A549 cells was analyzed by reverse transcription-quantitative polymerase chain reaction. The results showed that, klotho had no cytotoxic effect on A549 cells. The viability and mitochondrial function were inhibited and apoptosis, inflammatory response, and oxidative stress were aggravated in LPS-induced A549 cells, which were all reversed by klotho. Klotho activated the SIRT1/Nrf2 signaling pathway to inhibit the LPS-induced NLRP3 inflammasome activation in A549 cells. However, EX527, a SIRT1 inhibitor, attenuated the klotho effect to suppress viability and mitochondrial function and promoted apoptosis, inflammatory response, and oxidative stress of A549 cells. In conclusion, klotho inhibited the activation of NLRP3 inflammasome to alleviate LPS-induced inflammatory injury of A549 cells and restore mitochondrial function through activating the SIRT1/Nrf2 signaling pathway.

Interaction of Klotho and sirtuins.

OBJECTIVE: In this article, we review the articles that have reported the interaction between Klotho and sirtuins. RECENT FINDINGS: Sirtuins are a family of histone deacetylase enzymes that are considered to be the main regulators of biological processes. This family is one of the essential factors for postponing aging and increasing the life span of organisms. Sirtuins play a role in regulating the function of various cellular processes such as cellular metabolism, oxidative stress, apoptosis, and inflammation. It has also been shown that various diseases are related to these enzymes. Klotho is an anti-aging protein that exists as a membrane protein as well as a soluble circulating form. The membrane type of this protein acts as a co-receptor of the FGF endocrine family. It has been shown that the Klotho gene is related to age-related diseases, including osteoporosis, coronary artery, brain diseases, diabetes, etc. At the same time, it is difficult to separate the actions of Klotho and endocrine FGFs. Several studies have shown that Klotho and sirtuins interact with each other at different regulatory levels. However, it is necessary to carry out more in-vivo investigations to create new windows towards the treatment or prevention of various diseases.

Klotho inhibits renal ox-LDL deposition via IGF-1R/RAC1/OLR1 signaling to ameliorate podocyte injury in diabetic kidney disease.

OBJECTIVE: Diabetic kidney disease (DKD) is characterized by the abnormal deposition of oxidized low-density lipoprotein (ox-LDL), which contributes to podocyte damage. Klotho, an aging suppressor that plays a critical role in protecting podocytes in DKD, is mainly expressed in kidney tubular epithelium and secreted in the blood. However, it has not been established whether Klotho can alleviate podocyte injury by inhibiting renal ox-LDL deposition, and the potential molecular mechanisms require further investigation. METHODS: We conducted a comprehensive analysis of serum and kidney biopsy samples obtained from patients diagnosed with DKD. Additionally, to explore the underlying mechanism of Klotho in the deposition of ox-LDL in the kidneys, we employed a mouse model of DKD with the Klotho genotype induced by streptozotocin (STZ). Furthermore, we conducted meticulous in vitro experiments on podocytes to gain further insights into the specific role of Klotho in the deposition of ox-LDL within the kidney. RESULTS: Our groundbreaking study unveiled the remarkable ability of the soluble form of Klotho to effectively inhibit high glucose-induced ox-LDL deposition in podocytes affected by DKD. Subsequent investigations elucidated that Klotho achieved this inhibition by reducing the expression of the insulin/insulin-like growth factor 1 receptor (IGF-1R), consequently leading to a decrease in the expression of Ras-related C3 botulinum toxin substrate 1 (RAC1) and an enhancement of mitochondrial function. Ultimately, this series of events culminated in a significant reduction in the expression of the oxidized low-density lipoprotein receptor (OLR1), thereby resulting in a notable decrease in renal ox-LDL deposition in DKD. CONCLUSION: Our findings suggested that Klotho had the potential to mitigate podocyte injury and reduced high glucose-induced ox-LDL deposition in glomerulus by modulating the IGF-1R/RAC1/OLR1 signaling. These results provided valuable insights that could inform the development of novel strategies for diagnosing and treating DKD.

Structural basis for FGF hormone signalling.

α/ßKlotho coreceptors simultaneously engage fibroblast growth factor (FGF) hormones (FGF19, FGF21 and FGF23)1,2 and their cognate cell-surface FGF receptors (FGFR1-4) thereby stabilizing the endocrine FGF-FGFR complex3-6. However, these hormones still require heparan sulfate (HS) proteoglycan as an additional coreceptor to induce FGFR dimerization/activation and hence elicit their essential metabolic activities6. To reveal the molecular mechanism underpinning the coreceptor role of HS, we solved cryo-electron microscopy structures of three distinct 1:2:1:1 FGF23-FGFR-αKlotho-HS quaternary complexes featuring the 'c' splice isoforms of FGFR1 (FGFR1c), FGFR3 (FGFR3c) or FGFR4 as the receptor component. These structures, supported by cell-based receptor complementation and heterodimerization experiments, reveal that a single HS chain enables FGF23 and its primary FGFR within a 1:1:1 FGF23-FGFR-αKlotho ternary complex to jointly recruit a lone secondary FGFR molecule leading to asymmetric receptor dimerization and activation. However, αKlotho does not directly participate in recruiting the secondary receptor/dimerization. We also show that the asymmetric mode of receptor dimerization is applicable to paracrine FGFs that signal solely in an HS-dependent fashion. Our structural and biochemical data overturn the current symmetric FGFR dimerization paradigm and provide blueprints for rational discovery of modulators of FGF signalling2 as therapeutics for human metabolic diseases and cancer.

Klotho prevents transforming growth factor-ß2-induced senescent-like morphological changes in the retinal pigment epithelium.

Degenerative changes of the retinal pigment epithelium (RPE) triggered by transforming growth factor-ß2 (TGF-ß2) and oxidative stress play a critical role in the progression of age-related macular degeneration (AMD). The expression of α-klotho, an antiaging protein, declines with age, increasing the risk factors for age-related diseases. Here, we investigated the protective effects of soluble α-klotho on TGF-ß2-induced RPE degeneration. The morphological changes induced by TGF-ß2, including epithelial-mesenchymal transition (EMT), were attenuated in the mouse RPE by the intravitreal injection (IVT) of α-klotho. In ARPE19 cells, EMT and morphological alterations by TGF-ß2 were attenuated by co-incubation with α-klotho. TGF-ß2 decreased miR-200a accompanied by zinc finger e-box binding homeobox1 (ZEB1) upregulation and EMT, all of which were prevented by α-klotho co-treatment. Inhibitor of miR-200a mimicked TGF-ß2-induced morphological changes, which were recovered by ZEP1 silencing, but not by α-klotho, implying the upstream regulation of α-klotho on miR-200a-ZEP1-EMT axis. α-Klotho inhibited receptor binding of TGF-ß2, Smad2/3 phosphorylation, extracellular signal-regulated protein kinase 1/2 (ERK1/2)-a mechanistic target of rapamycin (mTOR) activation and oxidative stress via NADPH oxidase 4 (NOX4) upregulation. Furthermore, α-klotho recovered the TGF-ß2-induced mitochondrial activation and superoxide generation. Interestingly, TGF-ß2 upregulated α-klotho expression in the RPE cells, and genetic suppression of endogenous α-klotho aggravated TGF-ß2-induced oxidative stress and EMT. Lastly, α-klotho abrogated senescence-associated signaling molecules and phenotypes induced by long-term incubation with TGF-ß2. Hence, our findings indicate that the antiaging α-klotho plays a protective role against EMT and degeneration of the RPE, demonstrating the therapeutic potential for age-related retinal diseases, including the dry type of AMD.

Pathophysiologic Implications and Therapeutic Approach of Klotho in Chronic Kidney Disease: A Systematic Review.

The Klotho protein, known as an antiaging protein, is expressed mainly in the kidney, and kidney disorders may contribute to the disrupted expression of renal Klotho. The purpose of this systematic review was to determine if there are biological and nutraceutical therapies that increase the expression of Klotho and can help prevent complications associated with chronic kidney disease. A systematic literature review was carried out through the consultation of PubMed, Scopus, and Web of Science. Records between the years 2012 and 2022 in Spanish and English were selected. Cross-sectional or prevalence and analytical studies were included that evaluated the effects of Klotho therapy. A total of 22 studies were identified after the critical reading of these selected studies: 3 investigated the association between Klotho and growth factors, 2 evaluated the relationship between the concentration of Klotho and the type of fibrosis, 3 focused on the relationship between vascular calcifications and vitamin D, 2 assessed the relationship between Klotho and bicarbonate, 2 investigated the relationship between proteinuria and Klotho, 1 demonstrated the applicability of synthetic antibodies as a support for Klotho deficiency, 1 investigated Klotho hypermethylation as a renal biomarker, 2 investigated the relationship between proteinuria and Klotho, 4 linked Klotho as an early marker of chronic kidney disease, and 1 investigated Klotho levels in patients with autosomal dominant polycystic kidney disease. In conclusion, no study has addressed the comparison of these therapies in the context of their use with nutraceutical agents that raise the expression of Klotho.

Anti-Inflammatory Klotho Protein Serum Concentration Correlates with Interferon Gamma Expression Related to the Cellular Activity of Both NKT-like and T Cells in the Process of Human Aging.

Klotho is a beta-glucuronidase that reveals both anti-inflammatory and anti-oxidative properties that have been associated with mechanisms of aging. The study aimed to analyze the relationships between the serum concentration of soluble α-Klotho and cellular activity of two populations of lymphocytes; T and NKT-like cells corresponding to the level of cytokine secretion; i.e., IFN-γ, TNF-α, and IL-6. The studied population comprised three age groups: young individuals ('young'), seniors aged under 85 ('old'), and seniors aged over 85 ('oldest'). Both NKT-like and T cells were either non-cultured or cultured for 48 h and stimulated appropriately with IL-2, LPS or PMA with ionomycin to compare with unstimulated control cells. In all studied age groups non-cultured or cultured NKT-like cells revealed higher expressions of TNF-α, IL-6, and IFN-γ than T cells. α-Klotho concentration in serum decreased significantly in the process of aging. Intriguingly, only IFN-γ expression revealed a positive correlation with α-Klotho protein serum concentration in both non-cultured and cultured T and NKT-like cells. Since IFN-γ is engaged in the maintenance of immune homeostasis, the observed relationships may indicate the involvement of α-Klotho and cellular IFN-γ expression in the network of adaptive mechanisms developed during the process of human aging.

Parishin alleviates vascular ageing in mice by upregulation of Klotho.

Senescence of vascular endothelial cells is the major risk of vascular dysfunction and disease among elderly people. Parishin, which is a phenolic glucoside derived from Gastrodia elata, significantly prolonged yeast lifespan. However, the action of parishin in vascular ageing remains poorly understood. Here, we treated human coronary artery endothelial cells (HCAEC) and naturally aged mice by parishin. Parishin alleviated HCAEC senescence and general age-related features in vascular tissue in naturally aged mice. Network pharmacology approach was applied to determine the compound-target networks of parishin. Our analysis indicated that parishin had a strong binding affinity for Klotho. Expression of Klotho, a protein of age-related declines, was upregulated by parishin in serum and vascular tissue in naturally aged mice. Furthermore, FoxO1, on Klotho/FoxO1 signalling pathway, was increased in the parishin-intervened group, accompanied by the downregulated phosphorylated FoxO1. Taken together, parishin can increase Klotho expression to alleviate vascular endothelial cell senescence and vascular ageing.

The role of Klotho and FGF23 in cardiovascular outcomes of diabetic patients with chronic limb threatening ischemia: a prospective study.

Cardiovascular complications after lower extremity revascularization (LER) are common in diabetic patients with peripheral arterial disease (PAD) and chronic limb threatening ischemia (CLTI). The Klotho-fibroblast growth factor 23 (FGF23) axis is associated with endothelial injury and cardiovascular risk. We aimed to analyze the relationship between Klotho and FGF23 serum levels and the incidence of major adverse cardiovascular events (MACE) and major adverse limb events (MALE) after LER in diabetic patients with PAD and CLTI. Baseline levels of Klotho and FGF23, and their association with subsequent incidence of MACE and MALE were analyzed in a prospective, non-randomized study in a population of diabetic patients with PAD and CLTI requiring LER. A total of 220 patients were followed for 12 months after LER. Sixty-three MACE and 122 MALE were recorded during follow-up period. Baseline lower Klotho serum levels (295.3 ± 151.3 pg/mL vs. 446.4 ± 171.7 pg/mL, p < 0.01), whereas increased serum levels FGF23 (75.0 ± 11.8 pg/mL vs. 53.2 ± 15.4 pg/mL, p < 0.01) were significantly associated with the development of MACE. Receiver operating characteristic (ROC) analysis confirmed the predictive power of Klotho and FGF23 baseline levels. Furthermore, decreased Klotho levels were associated with the occurrence of MALE after LER (329.1 ± 136.8 pg/mL vs 495.4 ± 183.9 pg/mL, p < 0.01). We found that Klotho and FGF23 baseline levels are a potential biomarker for increased cardiovascular risk after LER in diabetic patients with PAD and CLTI.

Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure.

The aging-protective gene α-Klotho (KL) produces two main transcripts. The full-length mRNA generates a transmembrane protein that after proteolytic ectodomain shedding can be detected in serum as processed Klotho (p-KL), and a shorter transcript which codes for a putatively secreted protein (s-KL). Both isoforms exhibit potent pleiotropic beneficial properties, although previous reports showed negative side effects on mineral homeostasis after increasing p-KL concentration exogenously. Here, we expressed independently both isoforms using gene transfer vectors, to assess s-KL effects on mineral metabolism. While mice treated with p-KL presented altered expression of several kidney ion channels, as well as altered levels of Pi and Ca2+ in blood, s-KL treated mice had levels comparable to Null-treated control mice. Besides, bone gene expression of Fgf23 showed a fourfold increase after p-KL treatment, effects not observed with the s-KL isoform. Similarly, bone microstructure parameters of p-KL-treated mice were significantly worse than in control animals, while this was not observed for s-KL, which showed an unexpected increase in trabecular thickness and cortical mineral density. As a conclusion, s-KL (but not p-KL) is a safe therapeutic strategy to exploit KL anti-aging protective effects, presenting no apparent negative effects over mineral metabolism and bone microstructure.

Klotho protects against aged myocardial cells by attenuating ferroptosis.

Klotho (KL) is a renal protein with aging-suppression properties that mediates its regulatory effect during cardiac fibroblast aging. However, to determine whether KL can protect aged myocardial cells by attenuating ferroptosis, this study aimed to investigate the protective effect of KL on aged cells and to explore its potential mechanism. Cell injury of H9C2 cells was induced with D-galactose (D-gal) and treated with KL in vitro. This study demonstrated that D-gal induces aging in H9C2 cells. D-gal treatment increased ß-GAL(ß-galactosidase) activity, decreased cell viability, enhanced oxidative stress, reduced mitochondrial cristae, and decreased the expression of solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase-4 (GPx4), and P53, which are primary regulators of ferroptosis. The results showed that KL can eliminate D-gal-induced aging in H9C2 cells, likely due to its ability to increase the expression of the ferroptosis-associated proteins SLC7A11 and GPx4. Moreover, pifithrin-α, a P53-specific inhibitor, enhanced the expression of SLC7A11 and GPx4. These results suggest that KL may be involved in D-gal-induced H9C2 cellular aging during ferroptosis, mainly through the P53/SLC7A11/GPx4 signaling pathway.

Inhibition of EZH2 mitigates peritoneal fibrosis and lipid precipitation in peritoneal mesothelial cells mediated by klotho.

BACKGROUND: Peritoneal fibrosis caused by long-term peritoneal dialysis (PD) is the main reason why patients withdraw from PD treatment. Lipid accumulation in the peritoneum was shown to participate in fibrosis, and klotho is a molecule involved in lipid metabolism. GSK343 (enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibitor) has been verified to inhibit epithelial mesenchymal transdifferentiation (EMT) and peritoneal fibrosis, but its related mechanism remains unclear. This study aimed to investigate whether lipid accumulation was involved in the effect of GSK343 and its related mechanism. MATERIALS AND METHODS: First, the expression of EZH2, klotho and EMT indices in human peritoneal mesothelial cells (HMrSV5) incubated with high glucose (HG) levels was detected. After EZH2 was inhibited by GSK343, Western blot (WB), wound healing and Transwell assays were used to explore the effect of GSK343. EZH2 and klotho expression was also detected. Oil red O and Nile red staining and triglyceride (TG) detection kits were used to detect lipid accumulation. A rescue experiment with small interfering RNA specific for klotho (si-klotho) on the basis of GSK343 was also conducted to verify that GSK343 exerted its effect via klotho. In in vivo experiments, rats were administered GSK343, and the related index was assessed. RESULTS: In our study, we revealed that the expression of EZH2 was significantly upregulated and klotho was significantly downregulated in HMrSV5 cells induced by high glucose. With the aid of GSK343, we found that lipid deposition caused by HG was significantly decreased. In addition, EMT and fibrosis were also significantly alleviated. Moreover, GSK343 could also restore the downregulation of klotho. To further verify whether klotho mediated the effect of EZH2, a rescue experiment with si-klotho was also conducted. The results showed that si-klotho could counteract the protective effect of GSK343 on high glucose-induced lipid accumulation and fibrosis. In vivo experiments also revealed that GSK343 could relieve peritoneal fibrosis, lipid deposition and EMT by mitigating EZH2 and restoring klotho expression. CONCLUSIONS: Combining these findings, we found that EZH2 regulated lipid deposition, peritoneal fibrosis, and EMT mediated by klotho. To our knowledge, this is the first study to demonstrate the effect of the EZH2-klotho interaction on peritoneal fibrosis. Hence, EZH2 and klotho could act as potential targets for the treatment of peritoneal fibrosis.

Klotho ameliorates angiotension-II-induced endothelial senescence via restoration of autophagy by inhibiting Wnt3a/GSK-3ß/mTOR signaling: A potential mechanism involved in prognostic performance of Klotho in coronary atherosclerotic disease.

OBJECTIVE: We aimed to evaluate the prognostic performance of circulating Klotho in coronary atherosclerotic disease (CAD), and to further explore the effect of Klotho on stress-mediated endothelial senescence and underlying mechanism. METHODS: A cohort of 295 patients had a 12-month follow-up for major adverse cardiovascular events (MACE). Serum Klotho was detected by enzyme linked immunosorbent assay. Cell viability, SA-ß-Gal staining, the expression of P53 and P16 were analyzed for endothelial senescence. Oxidative stress was evaluated by measurement of reactive oxygen species, superoxide dismutase and malondialdehyde. LC3, P62, Wnt3a, GSK-3ß and mTOR were analyzed by western blotting. Autophagosome formation was detected by adenovirus transfection. RESULTS: In epidemiological analysis, low Klotho (≤295.9 pg/ml) was significantly associated with MACE risk (HR=2.266, 95 %CI 1.229-4.176). In experimental analysis, Klotho alleviated endothelial senescence and oxidative stress caused by Ang-II exposure; Klotho restored impaired autophagic flux to ameliorate Ang-II induced endothelial senescence; Ang-II activated Wnt3a/GSK-3ß/mTOR signaling to inhibit autophagy, whereas Klotho restored autophagy through blockade of Wnt3a/GSK-3ß/mTOR signaling; Klotho ameliorated endothelial senescence by suppressing Wnt3a/GSK-3ß/mTOR pathway under Ang-II exposure. CONCLUSIONS: Prognostic significance of Klotho in CAD is potentially ascribed to its anti-endothelial senescence effect via autophagic flux restoration by inhibiting Wnt3a/ GSK-3ß/mTOR signaling.

Klotho protects chromosomal DNA from radiation-induced damage.

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

Androgen increases klotho expression via the androgen receptor-mediated pathway to induce GCs apoptosis.

BACKGROUND: Many epidemiological studies have shown that anovulatory polycystic ovary syndrome (PCOS) is accompanied by hyperandrogenism. However, the exact mechanism of hyperandrogen-induced anovulation remains to be elucidated. In this study, we aimed to investigate the potential mechanism of anovulation in PCOS. To investigate the role of klotho as a key factor in the androgen receptor (AR)-mediated development of PCOS, we investigated the effects of testosterone on ovarian klotho expression in vivo and in vitro. RESULTS: Testosterone propionate (TP)-induced rats showed cycle irregularity, hyperandrogenism, polycystic ovarian changes, dyslipidemia. However, inhibition of AR expression could relieve PCOS traits. We also found that AR and klotho showed relatively high expression in PCOS rat ovarian tissue and in TP-induced granulosa cells (GCs), which was inhibited by the addition of flutamide. TP-induced GCs apoptosis was suppressed by AR antagonist, as well as silencing klotho expression in human GCs. Chromatin immunoprecipitation assay demonstrated that AR indirectly binds to the klotho promoter. CONCLUSIONS: Our results demonstrated TP mediates the expression of klotho via androgen receptor and klotho alterations could be a reason for ovarian dysfunction in PCOS.

Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity.

Extracellular matrix stiffening is a quintessential feature of cartilage aging, a leading cause of knee osteoarthritis. Yet, the downstream molecular and cellular consequences of age-related biophysical alterations are poorly understood. Here, we show that epigenetic regulation of α-Klotho represents a novel mechanosensitive mechanism by which the aged extracellular matrix influences chondrocyte physiology. Using mass spectrometry proteomics followed by a series of genetic and pharmacological manipulations, we discovered that increased matrix stiffness drove Klotho promoter methylation, downregulated Klotho gene expression, and accelerated chondrocyte senescence in vitro. In contrast, exposing aged chondrocytes to a soft matrix restored a more youthful phenotype in vitro and enhanced cartilage integrity in vivo. Our findings demonstrate that age-related alterations in extracellular matrix biophysical properties initiate pathogenic mechanotransductive signaling that promotes Klotho promoter methylation and compromises cellular health. These findings are likely to have broad implications even beyond cartilage for the field of aging research.

Klotho in kidney diseases: a crosstalk between the renin-angiotensin system and endoplasmic reticulum stress.

Klotho is a transmembrane anti-ageing protein that exists in three forms, i.e. α-Klotho, ß-Klotho and γ-Klotho, with distinct organ-specific expression and functions in the body. Here we focus on α-Klotho (hereafter Klotho), abundantly expressed by the distal and proximal convoluted tubules of the kidney. A significant decline in systemic and renal Klotho levels is a new hallmark for kidney disease progression. Emerging research portrays Klotho as a promising diagnostic and therapeutic target for diabetic and non-diabetic kidney disease. Even so, the underlying mechanisms of Klotho regulation and the strategies to restore its systemic and renal levels are still lacking. Angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers are the current standard of care for kidney diseases, but the molecular mechanisms for their nephroprotective action are still ambiguous. Moreover, endoplasmic reticulum (ER) stress also plays a crucial role in kidney disease progression. Few studies have claimed that the renin-angiotensin-aldosterone system (RAAS) has a direct relation with ER stress generation and vice versa in kidney disease. Interestingly, RAAS and ER stress modulation are associated with Klotho regulation in kidney disease. Here we focus on how the RAAS and ER stress connect with Klotho regulation in kidney disease. We also discuss Klotho and ER stress in an alliance with the concept of haemodynamic and metabolic overload in kidney disease. In addition, we highlight novel approaches to implement Klotho as a therapeutic target via RAAS and ER stress modulation for the treatment of diabetic and non-diabetic kidney diseases.

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

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

Fibroblast growth factor 23-Klotho and mineral metabolism in the first year after pediatric kidney transplantation: A single-center prospective study.

BACKGROUND: The role of fibroblast growth factor 23 (FGF23) levels in mineral metabolism before and after kidney transplantation in pediatric patients is poorly understood. METHODS: We prospectively evaluated 24 patients under 18 years of age (4.5 [3.3-9.8] years) who underwent living kidney transplantation between July 2016 and March 2018, and measured intact FGF23 and serum αKlotho levels, and other parameters of mineral metabolism before and after transplantation (Day 7, 1 and 4 months, and 1 year). Relationships between parameters were examined by linear analysis. RESULTS: FGF23 level was 440.8 [63.4-5916.3] pg/ml pre-transplant and decreased significantly to 37.1 [16.0-71.5] pg/ml at Day 7 post-transplant (-91.6%, p < .001). Thereafter, it remained at normal levels until 1 year. αKlotho level was 785 [568-1292] pg/ml pre-transplant and remained low at Day 7 and 1 month post-transplant, with an increasing trend at 4 months. Post-transplant phosphorus levels were significantly decreased compared with pre-transplant, with a lowest level of 1.7 [1.3-2.9] mg/dl, -5.7 [-6.8, -3.8] SD at Day 4, followed by gradual recovery. Phosphorus levels and the ratio of tubular maximum phosphate reabsorption were significantly and negatively associated with pre-transplant FGF23 until 4 months of post-transplant. Pre-transplant αKlotho was negatively associated with pre-transplant FGF23 but not FGF23 or other parameters after transplantation. CONCLUSION: FGF23 in pediatric kidney transplant patients decreased rapidly after transplantation and associated with post-transplant hypophosphatemia and increased phosphorus excretion. Post-transplant αKlotho was low early post-transplant but tended to increase subsequently. Post-transplant αKlotho was unaffected by pre-transplant FGF23 or other factors, suggesting pre-transplant chronic kidney disease status has no effect.