Evaluation of klotho expression on peripheral blood lymphocytes among hemodialysis patients and its possible contribution to their immuno-compromised status.

Infection is the second most common cause of mortality among end-stage kidney disease (ESKD) patients. Uremic toxins are the main cause of impaired immune response among ESKD patients. Klotho gene, the anti-aging gene, encodes the transmembrane alpha klotho (αKL) protein which acts as an obligate coreceptor for fibroblast growth factor 23 (FGF23). Klotho protein may play a role in immune cell functions, particularly in anti-inflammatory response; however, its role is still incompletely understood. In the present study, we aimed to measure αKL protein expression on peripheral blood lymphocytes (PBLs) among hemodialysis (HD) patients, and we assumed that decreased αKL expression on PBLs may contribute to the impaired immunity among HD patients. This case-control study included 20 ESKD patients on regular hemodialysis for more than 3 months. Their ages ranged from 24 to 69 years. Patients with primary immunodeficiencies, those on systemic immunosuppressive drugs, those with ongoing infections or who had recently recovered from infections, and those with malignancies on active treatment were excluded. A control group of 20 normal subjects of comparable age and gender were also included. We compared αKL protein expression on PBLs by flow cytometry between both groups. Significant reductions in percentages of αKL protein expression on B lymphocytes (CD19), T lymphocytes (CD3), and natural killer cells (CD56) were observed among HD patients compared to controls. We also noticed a significant reduction in the percentages of natural killer cells among HD patients. The present study suggests that decreased αKL expression on PBLs may contribute to the immunocompromised status among HD patients, highlighting the importance of understanding the exact function of αKL protein on immune cells. This may offer a future diagnostic and therapeutic tool to improve the immune response among HD patients.

Klotho gene might antagonize ischemic injury in stroke rats by reducing the expression of AQP4 via P38MAPK pathway.

OBJECTIVES: This study was aimed at exploring whether klotho improved neurologic function in rats with cerebral infarction by inhibiting P38 mitogen-activated protein kinase (MAPK) activation and thus down-regulating aquaporin 4 (AQP4). METHODS: In this study, we induced intracerebral Klotho overexpression in 6-week-old Sprague Dawley rats by injecting lentivirus carrying full-length rat Klotho cDNA into the lateral ventricle of the brain, followed by middle cerebral artery occlusion (MCAO) surgery after three days. Neurologic function was evaluated by neurological deficit scores. Infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The expressions of Klotho, AQP4, and P38 MAPK were detected by Western blot and Immunofluorescence. RESULTS: when rats were subjected to cerebral ischemia, their neurologic function was impaired, the protein expressions of klotho downregulated, the protein expressions of AQP4 and P38 MAPK increased, and the ratios of AQP4 and P-P38-positive area were significantly increased compared with the sham group rats. LV-KL-induced Klotho overexpression greatly improved neurobehavioral deficits and reduced infarct volume in MCAO rats. Klotho overexpression significantly reduced AQP4 and P38 MAPK pathway-related protein expression levels and the ratios of P-P38 and AQP4-positive area in MCAO rats. In addition, SB203580, a P38 MAPK signal pathway inhibitor, improved neurobehavioral deficits, reduced infarct volume, downregulated the expressions levels of AQP4 and P38 MAPK, and reduced the size of P-P38 and AQP4-positive area in MCAO rats. CONCLUSION: Klotho could alleviate the infraction volume and neurological dysfunction in MCAO rats, and its mechanism may involve AQP4 expression downregulation by suppressing P38-MAPK activation.

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.

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.

Evaluation of Klotho gene expression and NGAL levels following acute kidney injury during pregnancy hypertensive disorders.

BACKGROUND: This study examined the relationship between hypertensive disorders and acute kidney injury (AKI) during pregnancy by evaluating Klotho (KL) gene expression and Neutrophil gelatinase-associated lipocalin (NGAL) levels in pregnant women. MATERIAL AND METHODS: Pregnant women were divided into 3 groups: (1) Pregnancy related hypertensive disorders with AKI (PR-AKI) (9 cases), (2) hypertensive disorders pregnancy (HDP) (40 cases), and (3) normal pregnancy (30 cases). For each group, Klotho gene transcription levels in the blood, Klotho and NGAL proteins levels, Malondialdehyde (MDA) and superoxide dismutase (SOD) activity levels were measured in serum. Statistical comparisons were made among the three groups. RESULTS: Klotho/ß-actin transcript levels and serum KL protein concentrations were significantly decreased in hypertensive disorder pregnancies with AKI complications. Serum NGAL protein levels were significantly increased in the hypertensive disorder pregnancies with AKI complications. Total serum Klotho protein was negatively correlated with creatinine, while serum NGAL was positively correlated with serum creatinine, urea nitrogen, uric acid, systolic blood pressure, diastolic blood pressure and 24 h urine protein levels. Serum levels of MDA and SOD were significantly increased in the hypertensive disorder pregnancy with AKI and the overall MDA concentration was negatively correlated with Klotho protein concentration. Klotho protein was found to have a direct effect on creatinine, and a mediating effect of MDA was found. CONCLUSION: Decreased expression of Klotho protein in correlation with increased levels of oxidative stress are found during of AKI complications in pregnancy hypertensive disorders.

α-Klotho released from HK-2 cells inhibits osteogenic differentiation of renal interstitial fibroblasts by inactivating the Wnt-ß-catenin pathway.

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 ß-catenin agonist SKL2001. Mechanistically, s-KL inactivated the Wnt-ß-catenin pathway by directly binding to Wnt2 and upregulating SFRP1. Further investigations identified activation of the Wnt-ß-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-ß-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.

Aristolochic Acid Induces Renal Fibrosis and Senescence in Mice.

The kidney is one of the most susceptible organs to age-related impairments. Generally, renal aging is accompanied by renal fibrosis, which is the final common pathway of chronic kidney diseases. Aristolochic acid (AA), a nephrotoxic agent, causes AA nephropathy (AAN), which is characterized by progressive renal fibrosis and functional decline. Although renal fibrosis is associated with renal aging, whether AA induces renal aging remains unclear. The aim of the present study is to investigate the potential use of AAN as a model of renal aging. Here, we examined senescence-related factors in AAN models by chronically administering AA to C57BL/6 mice. Compared with controls, the AA group demonstrated aging kidney phenotypes, such as renal atrophy, renal functional decline, and tubulointerstitial fibrosis. Additionally, AA promoted cellular senescence specifically in the kidneys, and increased renal p16 mRNA expression and senescence-associated ß-galactosidase activity. Furthermore, AA-treated mice exhibited proximal tubular mitochondrial abnormalities, as well as reactive oxygen species accumulation. Klotho, an antiaging gene, was also significantly decreased in the kidneys of AA-treated mice. Collectively, the results of the present study indicate that AA alters senescence-related factors, and that renal fibrosis is closely related to renal aging.

Clinicopathological and Prognostic Significance of Klotho and Estrogen Receptors Expression in Human Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is male-predominant cancer, but the underlying mechanism remains unclear. This study aimed to investigate the expression of the age-suppressing gene klotho and estrogen receptors (ERs) in HCC patients and analyze their association with clinicopathological variables and their effects on prognosis. METHODS: The expression patterns of klotho, ERα, and ERß were determined by tissue microarray and immunohistochemical technique, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. RESULTS: Klotho expression was significantly lower in HCC than in the adjacent noncancerous tissues (52.7% (49/93) vs. 90.8% (79/87), P = .000), and its protein level in HCC tissue was negatively correlated with clinical staging, histological grade, and stage of the primary tumor (T) (P < .05). Whereas the expression of nuclear ERα and ERß was higher in HCC than their corresponding non-neoplastic tissues (55.9% (52/93) vs. 35.6% (31/87), P = .006; 59.1% (55/93) vs. 43.7% (38/87), P = .038), and the level of nuclear ERα and ERß in HCC tissue was inversely correlated with T stage, tumor size, and clinical staging (P < .05). Correlation analysis showed the expression level of klotho, which is positively correlated with that of nuclear ERα (r = 0.243, P = .019). Patients with klotho-positive tumors had longer survival than those with klotho-negative tumors (P = .002). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (P = .003). CONCLUSION: Klotho, partially regulated by ERα-mediated estrogen pathway, acts as a tumor suppressor and might be a novel biomarker candidate for predicting progression and prognosis in HCC patients.

Enhancement of Apo2L/TRAIL signaling pathway receptors by the activation of Klotho gene with CRISPR/Cas9 in Caco-2 colon cancer cells.

Human Klotho gene has many known functions such as anti-aging and anti-tumor, and decreased expression of this gene causes malignant formations in most types of cancer, including colon cancer. Interacting with TRAIL death receptors (DR4 and DR5) induces an apoptotic effect in cancer treatments by reducing the proliferation of cancer cells. The present study aimed to investigate downstream effect of overexpression of Klotho gene, which is known to have an antitumor effect on resistant human colon cancer cells, by examining its action on TRAIL death and decoy (DcR1 and DcR2) receptors for the first time. For this purpose, upregulation of human Klotho gene was achieved with CRISPR/Cas9-mediated system in resistant human colon cancer Caco-2 cells. To determine the effect of upregulation of Klotho gene on cancer cells evaluations with flow cytometry, WST-8, qRT-PCR, ELISA, and immunohistochemical analysis were performed. Then, Klotho gene was knocked out and its apoptotic effect was tested to find out whether it is due to overexpression of Klotho gene or not. Our results indicate that overexpression of Klotho gene in Caco-2 cells via CRISPR/Cas9-sensitized TRAIL death receptor DR4 suppresses the proliferation of cells by leading to apoptosis. Thus, this study conducted on apoptosis-resistant colon cancer cells may bring new insights about the role of Klotho gene in colon cancer.

Klotho ameliorated cognitive deficits in a temporal lobe epilepsy rat model by inhibiting ferroptosis.

Cognitive deficits are among the most common comorbidities of temporal lobe epilepsy (TLE). Ferroptosis associated with the accumulation of iron overload-dependent lipid peroxidation produces significant cognitive deficits in TLE. The anti-aging protein, klotho, has been shown to exert neuroprotective effects while enhancing cognition in neurodegenerative disorders. However, the role of klotho in TLE progression has not been established. In this study, we evaluated the effects and underlying mechanisms of klotho in a rat model of TLE induced by lithium-chloride and pilocarpine (LiCl-Pilo). The expression of klotho was found to be inhibited in the hippocampus following LiCl-Pilo induced TLE in rats. An adeno-virus (AAV), which mediated klotho overexpression (AAV-KL) was injected into the bilateral hippocampus of the rat models. After 3 weeks, rats were treated through intraperitoneal injections of LiCl-Pilo. After 9 weeks, AAV-KL was found to have significantly induced klotho overexpression in the hippocampus, effectively ameliorated cognitive deficits and exerted neuroprotective effects in LiCl-Pilo induced TLE rat models. Klotho significantly prevented ferroptosis and iron overload. Meanwhile, klotho regulated the expressions of divalent metal transporter 1 (DMT 1) and ferroportin (FPN) that were associated with iron accumulation in the hippocampus. Furthermore, klotho significantly elevated glutathione peroxidase-4 (GPX-4) and glutathione (GSH) levels while suppressed reactive oxygen species (ROS) levels. In conclusion, klotho ameliorated cognitive deficits and exerted neuroprotective effects by inhibiting ferroptosis in LiCl-Pilo induced TLE rat models.

Tacrolimus Decreases Cognitive Function by Impairing Hippocampal Synaptic Balance: a Possible Role of Klotho.

The influence of long-term tacrolimus treatment on cognitive function remains to be elucidated. Using a murine model of chronic tacrolimus neurotoxicity, we evaluated the effects of tacrolimus on cognitive function, synaptic balance, its regulating protein (Klotho), and oxidative stress in the hippocampus. Compared to vehicle-treated mice, tacrolimus-treated mice showed significantly decreased hippocampal-dependent spatial learning and memory function. Furthermore, tacrolimus caused synaptic imbalance, as demonstrated by decreased excitatory synapses and increased inhibitory synapses, and downregulated Klotho in a dose-dependent manner; the downregulation of Klotho was localized to excitatory hippocampal synapses. Moreover, tacrolimus increased oxidative stress and was associated with activation of the PI3K/AKT pathway in the hippocampus. These results indicate that tacrolimus impairs cognitive function via synaptic imbalance, and that these processes are associated with Klotho downregulation at synapses through tacrolimus-induced oxidative stress in the hippocampus.

Inhibitory effect of Astragalus Membranaceus on osteoporosis in SAMP6 mice by regulating vitaminD/FGF23/Klotho signaling pathway.

Spontaneous senile osteoporosis severely threatens the health of the senior population which has emerged as a severe issue for society. A SAMP6 mouse model was utilized to estimate the impact of intragastrically administered Astragalus Membranaceus (AR) on spontaneous senile osteoporosis. Bone mineral density (BMD) and bone microstructure were measured using Micro-CT; contents of calcium and phosphorus were determined with the colorimetric method; and gene and protein expressions of fibroblast growth factor 23 (FGF23), Klotho, Vitamin D receptor (VDR), CYP27B1 and CYP24A1 were detected using qPCR, Western blot and ELISA assays, respectively. The findings indicated that AR could improve the femoral BMD and bone microstructure, elevate the contents of calcium and phosphorus, and increase the expression of Klotho, VDR, and CYP27B1 whereas decreasing the expression of FGF23 and CYP24A1 in SAMP6 mice in a dose independent manner. The present study has demonstrated that AR can promote osteogenesis and alleviate osteoporosis. It is also expected to provide a new insight for the treatment of spontaneous senile osteoporosis and to serve as a research basis for AR application.

Glucagon receptor signaling regulates weight loss via central KLB receptor complexes.

Glucagon regulates glucose and lipid metabolism and promotes weight loss. Thus, therapeutics stimulating glucagon receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating fibroblast growth factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon's weight loss effects. FGF21 signaling requires an obligate coreceptor (ß-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb exhibited a partial reduction in body weight with chronic GCGR agonism (via IUB288) compared with controls, supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist, 1153, also displayed partial weight loss. Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR agonism mediates part of its weight loss properties through central KLB and has implications for future treatments of obesity and metabolic syndrome.

Soluble αKlotho downregulates Orai1-mediated store-operated Ca2+ entry via PI3K-dependent signaling.

αKlotho is a type 1 transmembrane anti-aging protein. αKlotho-deficient mice have premature aging phenotypes and an imbalance of ion homeostasis including Ca2+ and phosphate. Soluble αKlotho is known to regulate multiple ion channels and growth factor-mediated phosphoinositide-3-kinase (PI3K) signaling. Store-operated Ca2+ entry (SOCE) mediated by pore-forming subunit Orai1 and ER Ca2+ sensor STIM1 is a ubiquitous Ca2+ influx mechanism and has been implicated in multiple diseases. However, it is currently unknown whether soluble αKlotho regulates Orai1-mediated SOCE via PI3K-dependent signaling. Among the Klotho family, αKlotho downregulates SOCE while ßKlotho or γKlotho does not affect SOCE. Soluble αKlotho suppresses serum-stimulated SOCE and Ca2+ release-activated Ca2+ (CRAC) channel currents. Serum increases the cell-surface abundance of Orai1 via stimulating vesicular exocytosis of the channel. The serum-stimulated SOCE and cell-surface abundance of Orai1 are inhibited by the preincubation of αKlotho protein or PI3K inhibitors. Moreover, the inhibition of SOCE and cell-surface abundance of Orai1 by pretreatment of brefeldin A or tetanus toxin or PI3K inhibitors prevents further inhibition by αKlotho. Functionally, we further show that soluble αKlotho ameliorates serum-stimulated SOCE and cell migration in breast and lung cancer cells. These results demonstrate that soluble αKlotho downregulates SOCE by inhibiting PI3K-driven vesicular exocytosis of the Orai1 channel and contributes to the suppression of SOCE-mediated tumor cell migration.

Circular RNA ITCH suppresses metastasis of gastric cancer via regulating miR-199a-5p/Klotho axis.

Circular RNAs (circRNAs) are considered as a new regulatory factor in growth, metastasis and therapeutic resistance of human cancers. But the clinical significance and underlying mechanism of circular RNA ITCH (circ-ITCH) in gastric cancer (GC) remain unknown. In the present study, we found that circ-ITCH was down-regulated in GC cell lines, GC tissues and their serum-derived exosomes. The level of circ-ITCH was related to invasion depth. Functional assays showed that circ-ITCH overexpression inhibited the proliferation, migration, invasion and epithelial mesenchymal transition (EMT) of GC cells, whereas circ-ITCH knockdown appeared an opposite effect. Bioinformatic analysis and luciferase reporter assay confirmed that circ-ITCH acted as miR-199a-5p sponge and increased the level of Klotho. The expression level of miR-199-5p was up-regulated in GC tissues and negatively correlated with that of circ-ITCH. MiR-199a-5p mimics reversed the effects on inhibiting metastasis induced by circ-ITCH overexpression and decreased the level of Klotho in GC cells. Our findings indicate that circ-ITCH suppresses metastasis of GC by acting as the sponge of miR-199a-5p and increasing Klotho expression, which serves as a potential biomarker and targets for the diagnosis and therapy of GC.Abbreviations: CircRNAs: circular RNAs; GC: gastric cancer; circ-ITCH: circular RNA Itchy E3 ubiquitin protein ligase; ceRNA: competitive endogenous RNA; EMT: Epithelial-mesenchymal transition; siRNA: Small interfering RNA; TEM: transmission electron microscope; NTA: nanoparticle tracking analysis.

Wnt-induced, TRP53-mediated Cell Cycle Arrest of Precursors Underlies Interstitial Cell of Cajal Depletion During Aging.

BACKGROUND & AIMS: Gastric dysfunction in the elderly may cause reduced food intake, frailty, and increased mortality. The pacemaker and neuromodulator cells interstitial cells of Cajal (ICC) decline with age in humans, and their loss contributes to gastric dysfunction in progeric klotho mice hypomorphic for the anti-aging Klotho protein. The mechanisms of ICC depletion remain unclear. Klotho attenuates Wnt (wingless-type MMTV integration site) signaling. Here, we examined whether unopposed Wnt signaling could underlie aging-associated ICC loss by up-regulating transformation related protein TRP53 in ICC stem cells (ICC-SC). METHODS: Mice aged 1-107 weeks, klotho mice, APCΔ468 mice with overactive Wnt signaling, mouse ICC-SC, and human gastric smooth muscles were studied by RNA sequencing, reverse transcription-polymerase chain reaction, immunoblots, immunofluorescence, histochemistry, flow cytometry, and methyltetrazolium, ethynyl/bromodeoxyuridine incorporation, and ex-vivo gastric compliance assays. Cells were manipulated pharmacologically and by gene overexpression and RNA interference. RESULTS: The klotho and aged mice showed similar ICC loss and impaired gastric compliance. ICC-SC decline preceded ICC depletion. Canonical Wnt signaling and TRP53 increased in gastric muscles of klotho and aged mice and middle-aged humans. Overstimulated canonical Wnt signaling increased DNA damage response and TRP53 and reduced ICC-SC self-renewal and gastric ICC. TRP53 induction persistently inhibited G1/S and G2/M cell cycle phase transitions without activating apoptosis, autophagy, cellular quiescence, or canonical markers/mediators of senescence. G1/S block reflected increased cyclin-dependent kinase inhibitor 1B and reduced cyclin D1 from reduced extracellular signal-regulated kinase activity. CONCLUSIONS: Increased Wnt signaling causes age-related ICC loss by up-regulating TRP53, which induces persistent ICC-SC cell cycle arrest without up-regulating canonical senescence markers.