Sex-dependent changes in AMPAR expression and Na, K-ATPase activity in the cerebellum and hippocampus of α-Klotho-Hypomorphic mice.

Aging is characterized by a functional decline in several physiological systems. α-Klotho-hypomorphic mice (Kl-/-) exhibit accelerated aging and cognitive decline. We evaluated whether male and female α-Klotho-hypomorphic mice show changes in the expression of synaptic proteins, N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits, postsynaptic density protein 95 (PSD-95), synaptophysin and synapsin, and the activity of Na+, K+-ATPase (NaK) isoforms in the cerebellum and hippocampus. In this study, we demonstrated that in the cerebellum, Kl-/- male mice have reduced expression of GluA1 (AMPA) compared to wild-type (Kl+/+) males and Kl-/- females. Also, Kl-/- male and female mice show reduced ɑ2/ɑ3-NaK and Mg2+-ATPase activities in the cerebellum, respectively, and sex-based differences in NaK and Mg2+-ATPase activities in both the regions. Our findings suggest that α-Klotho could influence the expression of AMPAR and the activity of NaK isoforms in the cerebellum in a sex-dependent manner, and these changes may contribute, in part, to cognitive decline.

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.

Fibroblast growth factor 23-mediated regulation of osteoporosis: Assessed via Mendelian randomization and in vitro study.

Despite numerous investigations on the influence of fibroblast growth factor 23 (FGF23), α-Klotho and FGF receptor-1 (FGFR1) on osteoporosis (OP), there is no clear consensus. Mendelian randomization (MR) analysis was conducted on genome-wide association studies (GWASs)-based datasets to evaluate the causal relationship between FGF23, α-Klotho, FGFR1 and OP. The primary endpoint was the odds ratio (OR) of the inverse-variance weighted (IVW) approach. Furthermore, we stably transfected FGF23-mimic or siRNA-FGF23 into human bone marrow mesenchymal stem cells (hBMSCs) in culture and determined its cell proliferation and the effects on osteogenic differentiation. Using MR analysis, we demonstrated a strong correlation between serum FGF23 levels and Heel- and femoral neck-BMDs, with subsequent ORs of 0.919 (95% CI: 0.860-0.983, p = 0.014) and 0.751 (95% CI: 0.587-0.962; p = 0.023), respectively. The expression levels of FGF23 were significantly increased in femoral neck of patients with OP than in the control cohort (p < 0.0001). Based on our in vitro investigation, after overexpression of FGF23, compared to the control group, the BMSC's proliferation ability decreased, the expression level of key osteogenic differentiation genes (RUNX2, OCN and OSX) significantly reduced, mineralized nodules and ALP activity significantly decreased. After silencing FGF23, it showed a completely opposite trend. Augmented FGF23 levels are causally associated with increased risk of OP. Similarly, FGF23 overexpression strongly inhibits the osteogenic differentiation of hBMSCs, thereby potentially aggravating the pathological process of OP.

Development and validation of a predictive model based on ß-Klotho for head and neck squamous cell carcinoma.

Head and neck epithelial tissue tumors may be identified as head and neck squamous cell carcinoma (HNSC). Numerous malignancies are encouraged by dysregulation of the FGF19-ß-Klotho (KLB) axis in the tumor microenvironment. Using protein databases and RT-qPCR, we examined KLB expression in HNSC. In HNSC, higher KLB expression was linked to longer survival times and better prognoses. Furthermore, variations in drug susceptibility and immunological infiltration were noted according to KLB expression levels. These results underscore the importance of KLB in the course and management of HNSC by indicating that it may function as a possible prognostic marker and influence immunological and therapeutic responses in these individuals. Further study on HNSC is necessary to investigate KLB's potential as a therapeutic target and prognostic indicator.

Knockout of the longevity gene Klotho perturbs aging and Alzheimer's disease-linked brain microRNAs and tRNA fragments.

Overexpression of the longevity gene Klotho prolongs lifespan, while its knockout shortens lifespan and impairs cognition via perturbation of myelination and synapse formation. However, comprehensive analysis of Klotho knockout effects on mammalian brain transcriptomics is lacking. Here, we report that Klotho knockout alters the levels of aging- and cognition related mRNAs, long non-coding RNAs, microRNAs and tRNA fragments. These include altered neuronal and glial regulators in murine models of aging and Alzheimer's disease and in human Alzheimer's disease post-mortem brains. We further demonstrate interaction of the knockout-elevated tRNA fragments with the spliceosome, possibly affecting RNA processing. Last, we present cell type-specific short RNA-seq datasets from FACS-sorted neurons and microglia of live human brain tissue demonstrating in-depth cell-type association of Klotho knockout-perturbed microRNAs. Together, our findings reveal multiple RNA transcripts in both neurons and glia from murine and human brain that are perturbed in Klotho deficiency and are aging- and neurodegeneration-related.

Triiodothyronine enhances various forms of kidney-specific Klotho protein and suppresses the Wnt/ß-catenin pathway: Insights from in-vitro, in-vivo and in-silico investigations.

Age-related diseases are intricately linked to the molecular processes underlying aging, with the decline of the antiaging protein Klotho being a key factor. Investigating these processes is crucial for developing therapeutic strategies. The age-associated reduction in Klotho expression, coupled with a decline in the endocrine hormone triiodothyronine (T3), prompted a detailed exploration of their potential interplay. Our research, conducted through both in-vitro and in-vivo studies on BALB/c mice, unveiled a significant capacity of T3 to upregulate various forms of Klotho via ATF-3/p-c-Jun transcription factor. This effect was particularly noteworthy in aged individuals, where Klotho expression had waned compared to their younger counterparts. Importantly, T3 demonstrated a promising therapeutic impact in rejuvenating Klotho expression in this context. Further investigations elucidated the molecular mechanisms underlying T3's impact on aging-related pathways. In-vitro and in-vivo experiments established T3's ability to downregulate the Wnt/ß-Catenin pathway by enhancing Klotho expression. In-silico analyses provided insights into Klotho's intricate role, showing its capacity to inhibit Wnt ligands such as Wnt3 and Wnt8a, consequently disrupting their interaction with the Wnt receptor. Additionally, T3 was found to downregulate kidney-specific GSK-3ß expression through the augmentation of Klotho expression. The study also highlighted T3's role in maintaining calcium and phosphate homeostasis via Klotho. This comprehensive investigation not only sheds light on the intricate mechanisms governing aging processes but also presents promising avenues for therapeutic interventions targeting the Wnt/ß-Catenin pathway implicated in various age-associated diseases.

Klotho is highly expressed in the chief sites of regulated potassium secretion, and it is stimulated by potassium intake.

Klotho regulates many pathways in the aging process, but it remains unclear how it is physiologically regulated. Because Klotho is synthesized, cleaved, and released from the kidney; activates the chief urinary K+ secretion channel (ROMK) and stimulates urinary K+ secretion, we explored if Klotho protein is regulated by dietary K+ and the potassium-regulatory hormone, Aldosterone. Klotho protein along the nephron was evaluated in humans and in wild-type (WT) mice; and in mice lacking components of Aldosterone signaling, including the Aldosterone-Synthase KO (AS-KO) and the Mineralocorticoid-Receptor KO (MR-KO) mice. We found the specific cells of the distal nephron in humans and mice that are chief sites of regulated K+ secretion have the highest Klotho protein expression along the nephron. WT mice fed K+-rich diets increased Klotho expression in these cells. AS-KO mice exhibit normal Klotho under basal conditions but could not upregulate Klotho in response to high-K+ intake in the K+-secreting cells. Similarly, MR-KO mice exhibit decreased Klotho protein expression. Together, i) Klotho is highly expressed in the key sites of regulated K+ secretion in humans and mice, ii) In mice, K+-rich diets increase Klotho expression specifically in the potassium secretory cells of the distal nephron, iii) Aldosterone signaling is required for Klotho response to high K+ intake.

ABI3BP promotes renal aging through Klotho-mediated ferroptosis.

The aging process of the kidneys is accompanied with several structural diseases. Abnormal fiber formation disrupts the balance of kidney structure and function, causing to end-stage renal disease and subsequent renal failure. Despite this, the precise mechanism underlying renal damage in aging remains elusive. In this study, ABI3BP gene knockout mice were used to investigate the role of ABI3BP in renal aging induced by irradiation. The results revealed a significant increase in ABI3BP expression in HK2 cells and kidney tissue of aging mice, with ABI3BP gene knockout demonstrating a mitigating effect on radiation-induced cell aging. Furthermore, the study observed a marked decrease in Klotho levels and an increase in ferroptosis in renal tissue and HK2 cells following irradiation. Notably, ABI3BP gene knockout not only elevated Klotho expression but also reduced ferroptosis levels. A significant negative correlation between ABI3BP and Klotho was established. Further experiments demonstrated that Klotho knockdown alleviated the aging inhibition caused by ABI3BP downregulation. This study identifies the upregulation of ABI3BP in aged renal tubular epithelial cells, indicating a role in promoting ferroptosis and inducing renal aging by inhibiting Klotho expression.

ADAM10 as a major activator of reactive oxygen species production and klotho shedding in podocytes under diabetic conditions.

Early stages of diabetes are characterized by elevations of insulin and glucose concentrations. Both factors stimulate reactive oxygen species (ROS) production, leading to impairments in podocyte function and disruption of the glomerular filtration barrier. Podocytes were recently shown to be an important source of αKlotho (αKL) expression. Low blood Klotho concentrations are also associated with an increase in albuminuria, especially in patients with diabetes. We investigated whether ADAM10, which is known to cleave αKL, is activated in glomeruli and podocytes under diabetic conditions and the potential mechanisms by which ADAM10 mediates ROS production and disturbances of the glomerular filtration barrier. In cultured human podocytes, high glucose increased ADAM10 expression, shedding, and activity, NADPH oxidase activity, ROS production, and albumin permeability. These effects of glucose were inhibited when cells were pretreated with an ADAM10 inhibitor or transfected with short-hairpin ADAM10 (shADAM10) or after the addition soluble Klotho. We also observed increases in ADAM10 activity, NOX4 expression, NADPH oxidase activity, and ROS production in αKL-depleted podocytes. This was accompanied by an increase in albumin permeability in shKL-expressing podocytes. The protein expression and activity of ADAM10 also increased in isolated glomeruli and urine samples from diabetic rats. Altogether, these results reveal a new mechanism by which hyperglycemia in diabetes increases albumin permeability through ADAM10 activation and an increase in oxidative stress via NOX4 enzyme activation. Moreover, αKlotho downregulates ADAM10 activity and supports redox balance, consequently protecting the slit diaphragm of podocyteσ under hyperglycemic conditions.

The Effect of Propofol on the Hippocampus in Chronic Cerebral Hypoxia in a Rat Model Through Klotho Regulation.

BACKGROUND/AIM: Chronic cerebral hypoxia often leads to brain damage and inflammation. Propofol is suggested to have neuroprotective effects under anaesthesia. MATERIALS AND METHODS: This study used rat models with carotid artery coarctation or closure. Four groups of rats were compared: a control group, a propofol-treated group, a group with bilateral common carotid artery blockage (BCAO), and a BCAO group treated with propofol post-surgery. RESULTS: The Morris water maze test indicated cognitive impairment in BCAO rats, which also showed hippocampal structure changes, oxidative stress markers alteration, and reduced Klotho expression. Propofol treatment post-BCAO surgery improved these outcomes, suggesting its potential in mitigating chronic cerebral hypoxia effects. CONCLUSION: Propofol may increase klotho levels and reduce apoptosis and inflammation linked to oxidative stress in cognitively impaired mice.

Cognitive Impairment Related to Chronic Kidney Disease Is Associated with a Decreased Abundance of Membrane-Bound Klotho in the Cerebral Cortex.

Cognitive impairment (CI) is a complication of chronic kidney disease (CKD) that is frequently observed among patients. The aim of this study was to evaluate the potential crosstalk between changes in cognitive function and the levels of Klotho in the brain cortex in an experimental model of CKD. To induce renal damage, Wistar rats received a diet containing 0.25% adenine for six weeks, while the control group was fed a standard diet. The animals underwent different tests for the assessment of cognitive function. At sacrifice, changes in the parameters of mineral metabolism and the expression of Klotho in the kidney and frontal cortex were evaluated. The animals with CKD exhibited impaired behavior in the cognitive tests in comparison with the rats with normal renal function. At sacrifice, CKD-associated mineral disorder was confirmed by the presence of the expected disturbances in the plasma phosphorus, PTH, and both intact and c-terminal FGF23, along with a reduced abundance of renal Klotho. Interestingly, a marked and significant decrease in Klotho was observed in the cerebral cortex of the animals with renal dysfunction. In sum, the loss in cerebral Klotho observed in experimental CKD may contribute to the cognitive dysfunction frequently observed among patients. Although further studies are required, Klotho might have a relevant role in the development of CKD-associated CI and represent a potential target in the management of this complication.

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.

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.

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.

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.

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.

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.

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.