Klotho Deficiency Research Articles

MTORC1-Induced Bone Marrow-Derived Mesenchymal Stem Cell Exhaustion Contributes to the Bone Abnormalities in Klotho-Deficient Mice of Premature Aging.

Stem cell exhaustion is a hallmark of aging. Klotho-deficient mice (kl/kl mice) is a murine model that mimics human aging with significant bone abnormalities. The aim of this study is using kl/kl mice to investigate the functional change of bone marrow-derived mesenchymal stem cells (BMSCs) and explore the underlying mechanism. We found that klotho deficiency leads to bone abnormalities. In addition, kl/kl BMSCs manifested hyperactive proliferation but functionally declined both in vivo and in vitro. Mammalian target of rapamycin complex 1 (mTORC1) activity was higher in freshly isolated kl/kl BMSCs, and autophagy in kl/kl BMSCs was significantly decreased, possibly through mTORC1 activation. Conditional medium containing soluble Klotho protein (sKL) rescued hyperproliferation of kl/kl BMSCs by inhibiting mTORC1 activity and restoring autophagy. Finally, intraperitoneal injection of mTORC1 inhibitor rapamycin restored BMSC quiescence, ameliorated bone phenotype, and increased life span of kl/kl mice in vivo. This research highlights a therapeutic strategy to maintain the homeostasis of adult stem cell pool for healthy bone aging.

Partial Genetic Deletion of Klotho Aggravates Cardiac Calcium Mishandling in Acute Kidney Injury.

Acute kidney injury (AKI) is associated with an elevated risk of cardiovascular major events and mortality. The pathophysiological mechanisms underlying the complex cardiorenal network interaction remain unresolved. It is known that the presence of AKI and its evolution are significantly associated with an alteration in the anti-aging factor klotho expression. However, it is unknown whether a klotho deficiency might aggravate cardiac damage after AKI. We examined intracellular calcium (Ca2+) handling in native ventricular isolated cardiomyocytes from wild-type (+/+) and heterozygous hypomorphic mice for the klotho gene (+/kl) in which an overdose of folic acid was administered to induce AKI. Twenty-four hours after AKI induction, cardiomyocyte contraction was decreased in mice with the partial deletion of klotho expression (heterozygous hypomorphic klotho named +/kl). This was accompanied by alterations in Ca2+ transients during systole and an impairment of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) function in +/kl mice after AKI induction. Moreover, Ca2+ spark frequency and the incidence of Ca2+ pro-arrhythmic events were greater in cardiomyocytes from heterozygous hypomorphic klotho compared to wild-type mice after AKI. A decrease in klotho expression plays a role in cardiorenal damage aggravating cardiac Ca2+ mishandling after an AKI, providing the basis for future targeted approaches directed to control klotho expression as novel therapeutic strategies to reduce the cardiac burden that affects AKI patients.

Renal Klotho safeguards platelet lifespan in advanced chronic kidney disease through restraining Bcl‐xL ubiquitination and degradation

BackgroundThrombosis and hemorrhage as two opposite pathologies are prevalent within the chronic kidney disease (CKD) population. Platelet homeostasis, which positions centrally in their pathogenesis, varies among the CKD population, while the underlying mechanism is poorly understood. ObjectiveTo investigate the change character and mechanism of platelet homeostasis in CKD and its association with renal Klotho deficiency. MethodsThe change character of platelet homeostasis and its association with renal Klotho deficiency were determined based on a cohort study as well as CKD mice and Klotho‐deficient mice with CKD. The effects on thrombopoiesis and platelet lifespan were examined by flow cytometry and platelet transfer. The underlying mechanism was explored by proteomics, flow cytometry, western blot, and immunoprecipitation. ResultsWe show that platelet count declines both in patient and mouse models with advanced CKD (Adv‐CKD) and is positively associated with circulating Klotho levels. Mechanistically, we identify that ubiquitin ligase UBE2O governs Bcl‐xL ubiquitination and degradation in platelets, whereas Adv‐CKD–induced oxidative stress in platelets stimulates p38MAPK to promote Bcl‐xL phosphorylation, which facilitates UBE2O binding to Bcl‐xL and subsequent Bcl‐xL degradation. Consequently, platelet lifespan is shortened in Adv‐CKD, culminating in platelet count decline. However, kidney‐secreted soluble Klotho protein restricts oxidative stress in platelets, thereby preserving Bcl‐xL expression and platelet lifespan. ConclusionsOur findings uncover the mechanism of platelet count decline in Adv‐CKD and identify renal Klotho as a long‐range regulator of platelet lifespan, which not only provide a molecular mechanism underlying CKD‐associated thrombocytopenia and hemorrhage but also offer a promising therapy choice.

Abstract 14399: Novel Mechanisms of Diastolic Dysfunction in Cardiac Aging

Aging is associated with a progressive decline in cardiac diastolic function. Membranous α-klotho, predominantly produced by the kidney, can be cleaved and form circulating α-klotho, which acts as an anti-aging hormone. Echocardiography showed klotho deficiency in 20-24-month-old heterozygous klotho (KL +/- ) mice aggravates left ventricular hypertrophy (LVH) and diastolic dysfunction (DD) compared with old wild type (OWT) littermates. Supplementation with α-Klotho (0.1mg/kg/day. i.p) for 10 weeks significantly reduced LV mass and ameliorated DD in both OWT and OKL. The improvement in DD was confirmed by Millar catheter measurement of -dP/dt. To gain mechanistic insights, we performed unbiased acetylomics using Acetyl-lysine antibody enrichment, followed by Tandem mass tag proteomics analysis. We identified 1215 lysine acetylation sites (K-Ac) on >1000 cardiac proteins, of which 220 K-Ac sites were significantly hyperacetylated in OWT hearts. There were 63 Ac-K sites >2-fold hyperacetylated in aged hearts (OWT vs. YWT). The comparison of OKL to YWT augmented the number of Ac-K sites to 82. These sites include redox proteins, several metabolic and mitochondrial proteins involved in fatty acid and amino acid metabolism, and the TCA cycle. Several isoforms of myosin heavy chains (MHC) were also hyperacetylated at multiple sites. Some hyperacetylated MHC (e.g. K941) are predicted to alter the molecular structure and relaxation kinetics by Molecular Dynamic Simulation. In addition to delayed Ca 2+ reuptake by myocardial sarcoplasmic reticulum ATPase (SERCA2a), decreased SERCA2a expression and increased acetylated SERCA2 in aged hearts, which was all corrected upon klotho supplementation, we found potential novel mechanisms of DD with hyperacetylation of MHC, concomitant with a decrease in Sirt1 deacetylase, in OWT mice that was aggravated in OKL, which was replenished upon klotho supplementation. Collectively, we demonstrated klotho deficiency aggravates cardiac aging phenotypes, associated with hyperacetylation of several cardiac proteins due to diminished Sirt1. Klotho supplementation rejuvenates several cardiac aging phenotypes, including regression of LVH and improvement of DD, in parallel with reversal of protein acetylation.

Klotho Deficiency Induces Regulatory T Cells in Mice With Lupus Nephritis

Klotho Deficiency Induces Regulatory T Cells in Mice With Lupus Nephritis

Klotho Ameliorates Vascular Calcification via Promoting Autophagy.

Vascular calcification (VC) is regarded as a common feature of vascular aging. Klotho deficiency reportedly contributes to VC, which can be ameliorated by restoration of Klotho expression. However, the specific mechanisms involved remain unclear. Here, we investigated the role of autophagy in the process of Klotho-inhibiting VC. The clinical study results indicated that, based on Agatston score, serum Klotho level was negatively associated with aortic calcification. Then, Klotho-deficient mice exhibited aortic VC, which could be alleviated with the supplementation of Klotho protein. Moreover, autophagy increased in the aorta of Klotho-deficient mice and protected against VC. Finally, we found that Klotho ameliorated calcification by promoting autophagy both in the aorta of Klotho-deficient mice and in mouse vascular smooth muscle cells (MOVAS) under calcifying conditions. These findings indicate that Klotho deficiency induces increased autophagy to protect against VC and that Klotho expression further enhances autophagy to ameliorate calcification. This study is beneficial to exploring the underlying mechanisms of Klotho regulating VC, which has important guiding significance for future clinical studies in the treatment of VC.

Klotho in Kidney Transplantation: A New and Important Target?

Klotho in Kidney Transplantation: A New and Important Target?

The controversy of klotho as a potential biomarker in chronic kidney disease.

Klotho is an identified longevity gene with beneficial pleiotropic effects on the kidney. Evidence shows that a decline in serum Klotho level occurs in early chronic kidney disease (CKD) and continues as CKD progresses. Klotho deficiency is associated with poor clinical outcomes and CKD mineral bone disorders (CKD-MBD). Klotho has been postulated as a candidate biomarker in the evaluation of CKD. However, the evidence for the clinical significance of the relationship between Klotho and kidney function, CKD stage, adverse kidney and/or non-kidney outcomes, and CKD-MBD remains inconsistent and in some areas, contradictory. Therefore, there is uncertainty as to whether Klotho is a potential biomarker in CKD; a general consensus regarding the clinical significance of Klotho in CKD has not been reached, and there is limited evidence synthesis in this area. To address this, we have systematically assessed the areas of controversy, focusing on the inconsistencies in the evidence base. We used a PICOM strategy to search for relevant studies and the Newcastle–Ottawa Scale scoring to evaluate included publications. We reviewed the inconsistent clinical findings based on the relationship of Klotho with CKD stage, kidney and/or non-kidney adverse outcomes, and CKD-MBD in human studies. Subsequently, we assessed the underlying sources of the controversies and highlighted future directions to resolve these inconsistencies and clarify whether Klotho has a role as a biomarker in clinical practice in CKD.

Klotho restoration via ACE2 activation: A potential therapeutic strategy against acute kidney injury-diabetes comorbidity

Acute kidney injury (AKI) is a collection of clinical syndromes with persistent increases in morbidity and mortality rates. Hyperglycemia is a risk factor for AKI development. Renin-angiotensin-aldosterone system (RAS) disequilibrium and Klotho downregulation also play a pivotal role in the pathogenesis of AKI. Moreover, the relationship between Klotho and ACE2 (a component of non-conventional RAS) regulation in AKI remains an unexplored area of research. Hence, in this study, we investigated ACE2 and Klotho regulation in AKI using ischemic Wistar rats and NRK52E cells under normal and hyperglycemic conditions. Our findings suggested that hyperglycemia exacerbates renal ischemia-reperfusion injury (IRI)/hypoxia-reperfusion injury (HRI) induced AKI. Systemic and renal Klotho deficiency is a novel hallmark of AKI. Additionally, ACE2 is a protective component of the RAS, and its inhibition/deficiency leads to inflammation, apoptosis, Klotho downregulation, and thus AKI development. However, ACE2 activation resulted in the amelioration of AKI. Importantly, ACE2 plays an important role in Klotho upregulation, which might act as an intermediate for ACE2-mediated reno-protection. In conclusion, ACE2 activator i.e. DIZE restored endogenous ACE2-Ang-(1-7)-Klotho level, inhibited apoptosis and inflammation, and ameliorates IRI/HRI induced AKI under diabetic and non-diabetic conditions. Hence, in future, targeting ACE2-Ang-(1-7)-Klotho axis may prove a novel therapeutic strategy against AKI, where further preclinical and clinical investigations are required to verify the clinical potential of this finding.

Aging impairs arterial compliance via Klotho-mediated downregulation of B-cell population and IgG levels.

Aging is associated with compromised immune function and arterial remodeling and stiffness. The purpose of this study is to investigate whether in vivo AAV-based delivery of secreted Klotho (SKL) gene (AAV-SKL) improves aging- and senescence-associated immune dysfunction and arterial stiffness. Senescence-accelerated mice prone strain 1 (SAMP1, 10months) and old mice (20months) were used. Serum SKL levels, B-cell population and serum IgG levels were markedly decreased in SAMP1 and old mice. Rescue of downregulation of serum SKL levels by in vivo AAV2-based delivery of SKL gene (AAV-SKL) increased B-cell population and serum IgG levels and attenuated arterial stiffness in SAMP1 and old mice. Thus, Klotho deficiency may play a role in senescence- and aging-associated humoral immune dysfunction and arterial stiffness. Vascular infiltration of inflammatory cells and expression of TGFβ1, collagen 1, scleraxis, MMP-2 and MMP-9 were increased while the elastin level was decreased in aortas of SAMP1 and old mice which can be rescued by AAV-SKL. Interestingly, treatment with IgG effectively rescued arterial inflammation and remodeling and attenuated arterial stiffness and hypertension in aging mice. In cultured B-lymphoblast cells, we further showed that SKL regulates B-cell proliferation and maturation partly via the NFkB pathway. Aging-associated arterial stiffening may be largely attributed to downregulation of B-cell population and serum IgG levels. AAV-SKL attenuates arterial stiffness in aging mice partly via restoring B-cell population and serum IgG levels which attenuates aging-associated vascular inflammation and arterial remodeling.

Impact of cytotoxic agents or apoptosis stimulants on αklotho in MDCK, NRK-52E and HK2 kidney cells.

αKlotho is a transmembrane protein acting as a co-receptor for FGF23, a bone hormone regulating renal phosphate and vitamin D metabolism. αKlotho expression is controlled by PPARγ. Soluble αklotho (sKL) regulates cellular signaling impacting stress resistance and death. αKlotho deficiency causes early onset of aging-associated diseases while its overexpression markedly increases lifespan. Cellular stress due to cytotoxic therapeutics or apoptosis induction through caspase activation or serum deficiency may result in cell death. Owing to αklotho’s role in cellular stress and aging, this study explored the effect of cytotoxic agents or apoptosis stimulants on cellular αklotho expression. Experiments were performed in renal MDCK, NRK-52E and HK-2 cells. Gene expression was determined by qRT-PCR, sKL by ELISA, apoptosis and necrosis by annexin V binding and a fluorescent DNA dye, and cell viability by MTT assay. Cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis induction with caspase 3 activator PAC-1 and serum deprivation induced αklotho and PPARG gene expression while decreasing viability and proliferation and inducing apoptosis of MDCK and NRK-52E cells to a variable extent. PPARγ antagonism attenuated up-regulation of αklotho in MDCK cells. In HK-2 cells, αklotho gene expression and sKL protein were down-regulated by chemotherapeutics. SKL serum levels in patients following chemotherapy were not significantly changed. In summary, potentially fatal stress results in up-regulation of αKlotho gene expression in MDCK and NRK-52E cells and down-regulation in HK-2 cells. These results indicate that different renal cell lines may exhibit completely different regulation of αklotho.

Phosphate Metabolic Inhibition Contributes to Irradiation-Induced Myelosuppression through Dampening Hematopoietic Stem Cell Survival.

Myelosuppression is a common and intractable side effect of cancer therapies including radiotherapy and chemotherapy, while the underlying mechanism remains incompletely understood. Here, using a mouse model of radiotherapy-induced myelosuppression, we show that inorganic phosphate (Pi) metabolism is acutely inhibited in hematopoietic stem cells (HSCs) during irradiation-induced myelosuppression, and closely correlated with the severity and prognosis of myelosuppression. Mechanistically, the acute Pi metabolic inhibition in HSCs results from extrinsic Pi loss in the bone marrow niche and the intrinsic transcriptional suppression of soluble carrier family 20 member 1 (SLC20A1)-mediated Pi uptake by p53. Meanwhile, Pi metabolic inhibition blunts irradiation-induced Akt hyperactivation in HSCs, thereby weakening its ability to counteract p53-mediated Pi metabolic inhibition and the apoptosis of HSCs and consequently contributing to myelosuppression progression. Conversely, the modulation of the Pi metabolism in HSCs via a high Pi diet or renal Klotho deficiency protects against irradiation-induced myelosuppression. These findings reveal that Pi metabolism and HSC survival are causally linked by the Akt/p53–SLC20A1 axis during myelosuppression and provide valuable insights into the pathogenesis and management of myelosuppression.

The association of soluble Klotho levels with anemia and hemoglobin variability in hemodialysis patients.

The anti-aging protein Klotho levels are decreased, and Klotho deficiency is associated with cardiovascular diseases in patients with chronic kidney disease. There are recent studies about the relation between soluble Klotho levels and anemia. We aimed to investigate the correlation of anemia and hemoglobin variability with soluble Klotho levels in hemodialysis patients. Ninety-one hemodialysis patients were included in this study. The mean hemoglobin value, hemoglobin variability, and coefficient of variation of hemoglobin for each patient were calculated. According to mean hemoglobin levels, two groups were defined as under 11 and ≥11 g/dl. Soluble Klotho levels of each patient were studied. Mean hemoglobin levels, hemoglobin variability, and coefficient of variation of hemoglobin were not significantly correlated with soluble Klotho levels. According to mean hemoglobin levels under 11 and ≥ 11 g/dl, there was no statistically significant correlation between anemia and soluble Klotho levels. Soluble Klotho levels were not associated with anemia and hemoglobin variability in hemodialysis patients. Further studies are needed to reveal the complicated relation between anemia and soluble Klotho levels.

MO1012: Variability of Klotho and FGF-23 Levels After Kidney Transplantation Depending on the Glomerular Filtration Rate

Abstract BACKGROUND AND AIMS Klotho is a transmembrane protein expressed mainly in distal tubular epithelial cells of the kidneys. Its gene expression and soluble form levels play a crucial role in the interaction of the aging-inflammation binomial and act as a co-receptor for the phosphaturic fibroblast growth factor 23 (FGF-23). Klotho deficiency, which is the norm in chronic kidney disease (CKD), has been reported as an independent risk factor for cardiovascular disease and subclinical atherosclerosis [1, 2]. This study investigates the evolution of Klotho and FGF-23 levels during the first 2 years after kidney transplantation and their variability depending on the glomerular filtration rate (GFR). METHOD Serum levels of Klotho and FGF-23 were determined in 42 patients right before kidney transplantation (KT) and at 3rd, 12th and 24th month after KT. The measurements were made by ELISA. The GFR of the kidney graft was performed by indirect methods (formula: MDRa and CKD-EPI) at each study visit. Variables were reported as mean ± SD, and a value of P < .05 was considered to be statistically significant for each test. RESULTS KT recipients were divided into two groups arbitrary: the first group with a GFR > 40 mL/min (n = 26) and the other one with a GFR ≤ 40 mL/min (n = 16). At the starting point, there was not found any statistical difference in Klotho and FGF-23 levels between groups (GFR > 40 mL/min: 462.65 ± 264.99 pg/mL and 786.84 ± 521.67 pg/mL, respectively; versus GFR ≤ 40 mL/min: 436.5 ± 189.33 and 779.02 ± 589.86). Those with better kidney function after KT significantly increased Klotho levels on 12th and 24th month of follow-up (baseline 462.65 ± 264.99 versus 12th month 561.96 ± 314.68 and 24th month 581.15 ± 350.10; P = .001), whereas KT recipients with a GFR ≤ 40 mL/min only proved an increase in Klotho levels at 12th month after a non-significant initial decrease at 3rd month (462 ± 204.91 versus 356.96 ± 177.07, respectively; P = .028). This trend was not confirmed at the 24th month (397.12 ± 182.05). Klotho levels were found significantly different at the 24th month between groups (P = .031). See Fig. 1. Figure 2 shows the changes in FGF-23 levels. There are significant differences in both groups from baseline to month 24. (P < .001 group GFR > 40 mL/min and P < .001 group GFR ≤ 40 mL/min). FGF-23 dropped significantly in both groups at the 3rd month (P = .018) and 12th months (P = .003). CONCLUSION Vascular disease and mineral metabolism malfunction are relevant abnormalities in patients with CKD as well as in KT recipients. This study shows that Klotho is increasingly produced after KT in those patients whose allografts achieve a better function. We failed to prove the same effect on those with a GFR < 40 mL/min. Nonetheless, FGF-23 production declines satisfactorily independently of GRF. These results suggest the promotion of cardiovascular health after KT, particularly in those with higher GFR. The clinical and prognostic value of these changes are yet to be determined.

Renal Klotho and inorganic phosphate are extrinsic factors that antagonistically regulate hematopoietic stem cell maintenance.

The composition and origin of extrinsic cues required for hematopoietic stem cell (HSC) maintenance are incompletely understood. Here we identify renal Klotho and inorganic phosphate (Pi) as extrinsic factors that antagonistically regulate HSC maintenance in the bone marrow (BM). Disruption of the Klotho-Pi axis by renal Klotho deficiency or Pi excess causes Pi overload in the BM niche and Pi retention in HSCs, leading to alteration of HSC maintenance. Mechanistically, Pi retention is mediated by soluble carrier family 20 member 1 (SLC20A1) and sensed by diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) to enhance Akt activation, which then upregulates SLC20A1 to aggravate Pi retention and augments GATA2 activity to drive the expansion and megakaryocyte/myeloid-biased differentiation of HSCs. However, kidney-secreted soluble Klotho directly maintains HSC pool size and differentiation by restraining SLC20A1-mediated Pi absorption of HSCs. These findings uncover a regulatory role of the Klotho-Pi axis orchestrated by the kidneys in BM HSC maintenance.

In Vivo Cardiac-specific Expression of Adenylyl Cyclase 4 Gene Protects against Klotho Deficiency-induced Heart Failure

Klotho is an aging-suppressor gene. Klotho gene deficiency causes heart failure in Klotho-hypomorphic mutant (KL (-/-)) mice. RNA-seq and western blot analysis showed that adenylyl cyclase type IV (AC4) mRNA and protein expression was largely decreased in cardiomyocytes of KL (-/-) mice. The objective of this study was to investigate whether in vivo cardiac-specific expression of AC4 gene protects against Klotho deficiency-induced heart failure. Interestingly, in vivo AAV-based cardiac-specific AC4 gene expression increased left ventricular fractional shortening, ejection fraction, stroke volume, and left ventricular end-diastolic volume in KL (-/-) mice, suggesting that cardiac-specific AC4 gene expression improves Klotho deficiency-induced heart dysfunction. Cardiac-specific AC4 gene expression also decreased Klotho deficiency-induced cardiac hypertrophy. Cardiac-specific AC4 gene expression alleviated Klotho deficiency-induced cardiac fibrosis and calcification. Furthermore, cardiac-specific AC4 gene expression attenuated mitochondrial dysfunction, superoxide accumulation and cardiomyocyte apoptotic cell death. Thus, downregulation of AC4 may contribute to Klotho deficiency-induced heart failure. Mechanistically, AAV2/9-αMHC-AC4 increased cardiomyocytic cAMP levels and thus regulated the PKA-PLN-SERCA2 signal pathway, which is critical in modulating calcium flux and mitochondrial function. In conclusion, cardiac-specific AC4 gene expression protects against Klotho deficiency-induced heart failure through increasing cardiomyocytic cAMP levels, which alleviates cAMP-dependent mitochondrial dysfunction, superoxide accumulation and apoptotic cell death. AC4 regulates superoxide levels via the cAMP-PKA pathway. AC4 could be a potential therapeutic target for heart failure associated with Klotho deficiency. Heart failure is the major cause of mortality in patients with chronic kidney disease (CKD). A decrease in Klotho levels is linked to CKD.

Fatty kidney: A possible future for chronic kidney disease research.

Metabolic syndrome is a growing twenty-first century pandemic associated with multiple clinical comorbidities ranging from cardiovascular diseases, non-alcoholic fatty liver disease and polycystic ovary syndrome to kidney dysfunction. A novel area of research investigates the concept of fatty kidney in the pathogenesis of chronic kidney disease, especially in patients with diabetes mellitus or metabolic syndrome. To review the most updated literature on fatty kidney and provide future research, diagnostic and therapeutic perspectives on a disease increasingly affecting the contemporary world. We performed an extensive literature search through three databases including Embase (Elsevier) and the Cochrane Central Register of Controlled Trials (Wiley) and PubMed/Medline Web of Science in November 2021 by using the following terms and their combinations: 'fatty kidney', 'ectopic fat', 'chronic kidney disease', 'cardiovascular event', 'cardio-metabolic risk', 'albuminuria' and 'metabolic syndrome'. Each study has been individually assessed by the authors. Oxidative stress and inflammation, Klotho deficiency, endoplasmic reticulum stress, mitochondrial dysfunction and disruption of cellular energy balance appear to be the main pathophysiological mechanisms leading to tissue damage following fat accumulation. Despite the lack of large-scale comprehensive studies in this novel field of research, current clinical trials demonstrate fatty kidney as an independent risk factor for the development of chronic kidney disease and cardiovascular events. The requirement for future studies investigating the pathophysiology, clinical outcomes and therapeutics of fatty kidney is clear.

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

Klotho: An Emerging Factor With Ergogenic Potential.

Sarcopenia and impaired cardiorespiratory fitness are commonly observed in older individuals and patients with chronic kidney disease (CKD). Declines in skeletal muscle function and aerobic capacity can progress into impaired physical function and inability to perform activities of daily living. Physical function is highly associated with important clinical outcomes such as hospitalization, functional independence, quality of life, and mortality. While lifestyle modifications such as exercise and dietary interventions have been shown to prevent and reverse declines in physical function, the utility of these treatment strategies is limited by poor widespread adoption and adherence due to a wide variety of both perceived and actual barriers to exercise. Therefore, identifying novel treatment targets to manage physical function decline is critically important. Klotho, a remarkable protein with powerful anti-aging properties has recently been investigated for its role in musculoskeletal health and physical function. Klotho is involved in several key processes that regulate skeletal muscle function, such as muscle regeneration, mitochondrial biogenesis, endothelial function, oxidative stress, and inflammation. This is particularly important for older adults and patients with CKD, which are known states of Klotho deficiency. Emerging data support the existence of Klotho-related benefits to exercise and for potential Klotho-based therapeutic interventions for the treatment of sarcopenia and its progression to physical disability. However, significant gaps in our understanding of Klotho must first be overcome before we can consider its potential ergogenic benefits. These advances will be critical to establish the optimal approach to future Klotho-based interventional trials and to determine if Klotho can regulate physical dysfunction.

Abnormally decreased renal Klotho is linked to endoplasmic reticulum-associated degradation in mice.

Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded proteins retained in the ER, is implicated in various diseases including chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial fibrosis (RIF).Methods: Following establishment of a mouse RIF model by unilateral ureteral obstruction (UUO), a specific ERAD inhibitor, Eeyarestatin I (EerI), was administered to experimental animals by intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by enzyme-linked immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1, glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and protein disulfide isomerase (PDI). HK-2 cells were exposed to transforming growth factor (TGF)-β1 with or without EerI, and expressions of related proteins including Klotho, Derlin-1, GRP78, ATF4 and PDI were determined by western blotting analyses.Results: UUO induced severe kidney injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-β1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded proteins, subsequently contributing to the additional production of mature Klotho protein.Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.