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Loss of Klotho, an anti-aging protein, plays a critical role in the pathogenesis of chronic kidney diseases. As Klotho is a large transmembrane protein, it is challenging to harness it as a therapeutic remedy. Here we report the discovery of a Klotho-derived peptide 1 (KP1) protecting kidneys by targeting TGF-β signaling. By screening a series of peptides derived from human Klotho protein, we identified KP1 that repressed fibroblast activation by binding to TGF-β receptor 2 (TβR2) and disrupting the TGF-β/TβR2 engagement. As such, KP1 blocked TGF-β-induced activation of Smad2/3 and mitogen-activated protein kinases. In mouse models of renal fibrosis, intravenous injection of KP1 resulted in its preferential accumulation in injured kidneys. KP1 preserved kidney function, repressed TGF-β signaling, ameliorated renal fibrosis and restored endogenous Klotho expression. Together, our findings suggest that KP1 recapitulates the anti-fibrotic action of Klotho and offers a potential remedy in the fight against fibrotic kidney diseases. Klotho is an anti-ageing protein whose expression is downregulated in chronic kidney disease, but the large size of the protein makes it challenging to deliver therapeutically. Here, the authors develop a Klotho-derived peptide, and show that it recapitulates the anti-fibrotic action of Klotho and prevents kidney fibrosis in mice by targeting TGF-β signalling.

α/βKlotho 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 complex 3 – 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 activities 6 . 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 signalling 2 as therapeutics for human metabolic diseases and cancer. This study reveals how Klotho and heparan sulfate glycosaminoglycan coreceptors enable FGF hormones to induce asymmetric 1:2 FGF–FGFR dimerization mandatory for FGFR kinase activation and hence signalling.

Development of renal fibrosis is a hallmark of renal aging and chronic kidney disease of all etiologies and characterized by extensive renal cell injuries and subsequent myofibroblast transdifferentiations (MTDs), which are significantly influenced by aberrant histone deacetylase (HDAC) activities. However, the key HDAC isoforms and effectors that are causally involved in the processes remain poorly understood. Here, we report that aberrant HDAC3 induction and its inhibition of Klotho, a renal epithelium-enriched aging suppressor, contribute significantly to renal fibrogenesis. HDAC3 was preferentially elevated with concomitant Klotho suppression in fibrotic kidneys incurred by unilateral ureter obstruction (UUO) and aristolochic acid nephropathy (AAN), whereas Hdac3 knockout resisted the fibrotic pathologies. The HDAC3 elevation is substantially blocked by the inhibitors of TGFβ receptor and Smad3 phosphorylation, suggesting that TGFβ/Smad signal activates Hdac3 transcription. Consistently, an HDAC3-selective inhibitor RGFP966 derepressed Klotho and mitigated the renal fibrotic injuries in both UUO and AAN mice. Further, HDAC3 overexpression or inhibition in renal epithelia inversely affected Klotho abundances and HDAC3 was inducibly associated with transcription regulators NCoR and NF-kB and bound to Klotho promoter in fibrotic kidney, supporting that aberrant HDAC3 targets and transcriptionally inhibits Klotho under renal fibrotic conditions. More importantly, the antirenal fibrosis effects of RGFP966 were largely compromised in mice with siRNA-mediated Klotho knockdown. Hence, HDAC3 aberration and the subsequent Klotho suppression constitute an important regulatory loop that promotes MTD and renal fibrosis and uses of HDAC3-selective inhibitors are potentially effective in treating renal fibrotic disorders.

While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the “anti-aging” protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age. While young muscle faithfully regenerates damaged myofibers, aged muscle is impaired. Here the authors show the “anti-aging” protein α-Klotho is upregulated in young muscle after damage via promoter demethylation and this regulation is lost in aging, resulting in mitochondrial damage and an impaired healing response.

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

Renal fibrosis is an unavoidable end result of all forms of progressive chronic kidney diseases (CKD). Discovery of efficacious drugs against renal fibrosis is in crucial need. In a preliminary study we found that a derivative of artemisinin, dihydroartemisinin (DHA), exerted strong renoprotection, and reversed renal fibrosis in adenine-induced CKD mouse model. In this study we investigated the anti-fibrotic mechanisms of DHA, particularly its specific target in renal cells. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) or oral administration of adenine (80 mg · kg −1 ), the mice received DHA (30 mg · kg −1  · d −1 , i.g.) for 14 or 21 days, respectively. We showed that DHA administration markedly attenuated the inflammation and fibrotic responses in the kidneys and significantly improved the renal function in both the renal fibrosis mouse models. In adenine-treated mice, DHA was more effective than 5-azacytidine against renal fibrosis. The anti-fibrotic effects of DHA were also observed in TGF-β1-treated HK-2 cells. In order to determine the target protein of DHA, we conducted pull-down technology coupled with shotgun proteomics using a small-molecule probe based on the structure of DHA (biotin-DHA). As a results, DNA methyltransferase 1 (DNMT1) was identified as the anti-fibrotic target of DHA in 3 different types of renal cell lines (HK-2, HEK293 and 3T3). We demonstrated that DHA directly bound to Asn 1529 and Thr 1528 of DNMT1 with a Kd value of 8.18 μM. In primary mouse renal tubular cells, we showed that DHA (10 μM) promoted DNMT1 degradation via the ubiquitin–proteasome pathway. DHA-reduced DNMT1 expression effectively reversed Klotho promoter hypermethylation, which led to the reversal of Klotho protein loss in the kidney of UUO mice. This subsequently resulted in inhibition of the Wnt/β-catenin and TGF-β/Smad signaling pathways and consequently conferred renoprotection in the animals. Knockdown of Klotho abolished the renoprotective effect of DHA in UUO mice. Our study reveals a novel pharmacological activity for DHA, i.e., renoprotection. DHA exhibits this effect by targeting DNMT1 to reverse Klotho repression. This study provides an evidence for the possible clinical application of DHA in the treatment of renal fibrosis.

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. Matrix stiffening is a quintessential feature of aged tissues. Authors show that an aged (stiff) matrix epigenetically represses the gene encoding the longevity factor, α-Klotho, resulting in chondrocyte dysfunction, a leading cause of osteoarthritis.

Aging is associated with a high prevalence of hypertension due to elevated susceptibility of BP to dietary salt, but its mechanism is unknown. Serum levels of Klotho, an anti-aging factor, decline with age. We found that high salt (HS) increased BP in aged mice and young heterozygous Klotho-knockout mice and was associated with increased vascular expression of Wnt5a and p-MYPT1, which indicate RhoA activity. Not only the Wnt inhibitor LGK974 and the Wnt5a antagonist Box5 but Klotho supplementation inhibits HS-induced BP elevation, similarly to the Rho kinase inhibitor fasudil, associated with reduced p-MYPT1 expression in both groups of mice. In cultured vascular smooth muscle cells, Wnt5a and angiotensin II (Ang II) increased p-MYPT1 expression but knockdown of Wnt5a with siRNA abolished Ang II-induced upregulation of p-MYPT1, indicating that Wnt5a is indispensable for Ang II-induced Rho/ROCK activation. Notably, Klotho inhibited Wnt5a- and Ang II-induced upregulation of p-MYPT1. Consistently, Klotho supplementation ameliorated HS-induced augmentation of reduced renal blood flow (RBF) response to intra-arterial infusion of Ang II and the thromboxane A2 analog U46619, which activated RhoA in both groups of mice and were associated with the inhibition of BP elevation, suggesting that abnormal response of RBF to Ang II contributes to HS-induced BP elevation. Thus, Klotho deficiency underlies aging-associated salt-sensitive hypertension through vascular non-canonical Wnt5a/RhoA activation.

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

Purpose Parkinson’s Disease (PD), a neurodegenerative disorder, is associated with substantial morbidity. α-Klotho, an anti-aging protein known for its neuroprotective properties, has gained attention. This study aims to assess serum levels of α-Klotho in PD patients. Methods This study is a cross-sectional case-control study. PD was diagnosed according to UK Parkinson Disease Society Brain Bank criteria. Serum α-Klotho level was measured using a commercially available enzyme-linked immunosorbent assay. Results Of the 314 participants in the study, 157 were patients with PD and 157 were controls. Lower levels of α-Klotho were observed in PD (0.85 nmol/L) in comparison to the controls (1.47 nmol/L, p  < 0.001). α-Klotho levels were also significantly lower among PD patients with dementia compared to PD patients without dementia. In logistic regression analysis, α-Klotho (OR: 0.04, p  < 0.001) demonstrated a significant relationship between PD. A significant correlation was identified between α-Klotho levels and Mini-Mental State Examination scores in PD patients. The sensitivity and the specifity of α-Klotho were 90% and 65% for predicting PD. Conclusions Our findings suggest that α-klotho could potentially serve as a biomarker. However additional studies are needed to confirm our findings. Key points Aim This study aimed to investigate the potential association between α-Klotho, a protein implicated in longevity, and Parkinson’s Disease, the most prevalent motor system disorder. Findings Our findings revealed both an association between serum α-Klotho protein and Parkinson’s Disease and its potential value for diagnostic applications. Message This study demonstrates an association between α-Klotho protein levels and Parkinson’s Disease, suggesting its potential as a biomarker for Parkinson’s Disease diagnosis.