Klotho mRNA Levels Research Articles

Pentoxifylline ameliorates subclinical atherosclerosis progression in patients with type 2 diabetes and chronic kidney disease: a randomized pilot trial

BackgroundDiabetic kidney disease (DKD) is associated with a higher risk of cardiovascular disease (CVD). Pentoxifylline (PTF), a nonselective phosphodiesterase inhibitor with anti-inflammatory, antiproliferative, and antifibrotic actions, has demonstrated renal benefits in both clinical trials and meta-analyses. The present work aimed to study the effects of PTF on the progression of subclinical atherosclerosis (SA) in a population of patients with diabetes and moderate to severe chronic kidney disease (CKD).MethodsIn this open-label, randomized controlled, prospective single-center pilot study the evolution of carotid intima-media thickness (CIMT) and ankle-brachial index (ABI) were determined in 102 patients with type 2 diabetes mellitus and CKD assigned to PTF, aspirin or control groups during 18 months. We also determined the variations in the levels of inflammatory markers and Klotho (KL), a protein involved in maintaining cardiovascular health, and their relationship with the progression of SA.ResultsPatients treated with PTF presented a better evolution of CIMT, increased KL mRNA levels in peripheral blood cells (PBCs) and reduced the inflammatory state. The progression of CIMT values was inversely related to variations in KL both in serum and mRNA expression levels in PBCs. Multiple regression analysis demonstrated that PTF treatment and variations in mRNA KL expression in PBCs, together with changes in HDL, were significant determinants for the progression of CIMT (adjusted R2 = 0.24, P < 0.001) independently of traditional risk factors. Moreover, both variables constituted protective factors against a worst progression of CIMT [OR: 0.103 (P = 0.001) and 0.001 (P = 0.005), respectively].ConclusionsPTF reduced SA progression assessed by CIMT variation, a beneficial effect related to KL gene expression in PBCs.Trial registrationThe study protocol code is PTF-AA-TR-2009 and the trial was registered on the European Union Drug Regulating Authorities Clinical Trials (EudraCT #2009–016595–77). The validation date was 2010-03-09.

MicroRNA-200c regulates KLOTHO expression in human kidney cells under oxidative stress.

KLOTHO deficiency is associated with the progression of kidney dysfunction, whereas its overexpression exerts renoprotective effects. Oxidative stress suppresses KLOTHO expression in renal epithelial cells but upregulates microRNA-200c (miR-200c) in human umbilical vein endothelial cells. In this study, we investigated whether oxidative stress-induced miR-200c is implicated in KLOTHO downregulation in human renal tubular epithelium (HK-2) cells. HK-2 cells were stimulated with hydrogen peroxide (H2O2) to examine the effect of oxidative stress. A luciferase reporter containing the KLOTHO 3′-UTR was used to investigate the effect of miR-200c on KLOTHO mRNA metabolism. The expressions of KLOTHO, oxidative stress markers, and miR-200c were determined in human kidney biopsy specimens. H2O2 suppressed KLOTHO expression without a reduction in KLOTHO mRNA levels but upregulated miR-200c expression. Similarly, transfection of a miR-200c mimic reduced KLOTHO levels and luciferase activity without a reduction in KLOTHO mRNA levels. In contrast, transfection of a miR-200c inhibitor maintained KLOTHO expression. Immunofluorescent assay revealed KLOTHO was present in the cytosol and nuclei of HK-2 cells. In human kidney biopsies, KLOTHO expression was inversely correlated with levels of oxidative stress markers (8-hydroxy-2′-deoxyguanosine: ρ = −0.38, P = 0.026; 4-hydroxy-2-hexenal: ρ = −0.35, P = 0.038) and miR-200c (ρ = −0.34, P = 0.043). Oxidative stress-induced miR-200c binds to the KLOTHO mRNA 3′-UTR, resulting in reduced KLOTHO expression.

Klotho suppresses colorectal cancer through modulation of the unfolded protein response.

Klotho is an anti-aging transmembrane protein, which can be shed and function as a hormone. Accumulating data indicate klotho as a tumor suppressor in a wide array of malignancies and indicate the subdomain KL1 as the active region of the protein. We aimed to study the role of klotho as a tumor suppressor in colorectal cancer. Bioinformatics analyses of TCGA datasets indicated reduced klotho mRNA levels in human colorectal cancer, along with negative regulation of klotho expression by hypermethylation of the promoter and 1st exon, and hypomethylation of an area within the gene. Overexpression or treatment with klotho or KL1 inhibited proliferation of colorectal cancer cells in vitro. The in vivo activity of klotho and KL1 was examined using two models recapitulating development of tumors in the normal colonic environment of immune-competent mice. Treatment with klotho inhibited formation of colon polyps induced by the carcinogen azoxymethane, and KL1 treatment slowed growth of orthotopically-implanted colorectal tumors. Gene expression array revealed that klotho and KL1 expression enhanced the unfolded protein response (UPR) and this was further established by increased levels of spliced XBP1, GRP78 and phosphorylated-eIF2α. Furthermore, attenuation of the UPR partially abrogated klotho tumor suppressor activity. In conclusion, this study indicates klotho as a tumor suppressor in colorectal cancer and identifies, for the first time, the UPR as a pathway mediating klotho activities in cancer. These data suggest that administration of exogenous klotho or KL1 may serve as a novel strategy for prevention and treatment of colorectal cancer.

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways.

Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.

Klotho expression in cervical cancer: differential expression in adenocarcinoma and squamous cell carcinoma

AimsKlotho is a trans-membrane protein that serves as a tumour suppressor in a wide array of malignancies. Recent data suggest it as an epigenetically silenced tumour suppressor in cervical cancer....

Reduced expression and growth inhibitory activity of the aging suppressor klotho in epithelial ovarian cancer

Klotho is an anti-aging transmembrane protein, which can be shed and function as a hormone. Accumulating data indicate klotho as a tumor suppressor in a wide array of malignancies, and we identified klotho as an inhibitor of the insulin-like growth factor (IGF-1) pathway in cancer cells. As this pathway is significant in the development of epithelial ovarian cancer (EOC) we studied klotho expression and activity in this tumor. Klotho mRNA levels were reduced in 16 of 19 EOC cell lines and immunohistochemistry analysis revealed high expression in normal ovaries, and reduced expression in 100 of 241 high grade papillary-serous adenocarcinoma of the ovaries, fallopian tubes and peritoneum. Reduced expression was associated with wild-type BRCA status. Klotho reduced EOC cell viability, enhanced cisplatin sensitivity, and reduced expression of mesenchymal markers. Finally, klotho inhibited IGF-1 pathway activation and inhibited transcriptional activity of the estrogen receptor. In conclusion, klotho is silenced in a substantial subset of the tumors and restoring its expression slows growth of EOC cells and inhibits major signaling pathways. As klotho is a hormone, treatment with klotho may serve as a novel treatment for EOC.

25-Hydroxyvitamin D3 1-Alpha-Hydroxylase-Dependent Stimulation of Renal Klotho Expression by Spironolactone

Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)₂D₃-generating 25-hydroxyvitamin D₃ 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)₂D₃ stimulates, by activating the vitamin D₃ receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)₂D₃ and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D₃ receptor (VDR) or of mineralocorticoid receptor (NR3C2). Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D₃ 1-alpha-hydroxylase with subsequent activation of the vitamin D₃ receptor by 1,25(OH)₂D₃, an effect possibly independent from the mineralocorticoid receptor.

Epigenetic silencing of the tumor suppressor klotho in human breast cancer

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.

Expression of FGF23/KLOTHO system in human vascular tissue

BackgroundFibroblast growth factor (FGF)-23 levels have been associated with impaired vasoreactivity, increased arterial stiffness, and cardiovascular morbi-mortality, whereas a protective function of KLOTHO against endothelial dysfunction has been reported. Since expression of the FGF23–KLOTHO system in human vascular tissue remains unproved, we aimed to study the expression of FGF23, FGF receptors (FGFR) and KLOTHO in human aorta. In addition, we analyzed the FGF23–KLOTHO expression in occlusive coronary thrombi. MethodsThoracic aorta specimens from 44 patients underwent elective cardiac surgery, and thrombus material from 2 patients with acute coronary syndrome (ACS), were tested for FGF23–KLOTHO system expression. ResultsExpression of KLOTHO (mean expression level 4.85±5.43, arbitrary units) and two of the three cognate FGFR (FGFR-1 and -3) were detected and confirmed by RT-PCR, sequencing and qRT-PCR. KLOTHO expression was confirmed within occlusive coronary thrombi from patients with ACS. However, expression of FGF23 and FGFR4 was not observed. We also detected the aortic expression of membrane-anchored A Desintegrin and Metalloproteinases (ADAM)-17, the enzyme responsible for the shedding of KLOTHO from the cell surface, and the anti-inflammatory cytokine interleukin (IL)-10. Interestingly, in aortic samples there was a direct association between KLOTHO mRNA levels and those of ADAM-17 and IL-10 (r=0.54, P<0.001; r=0.51, P<0.01, respectively). ConclusionsHuman vascular tissue expresses members of the FGF23–KLOTHO system, indicating that it can be a direct target organ for FGF23. In addition, KLOTHO expression is also detected in occlusive coronary thrombi. These findings suggest a putative role of FGF23–KLOTHO axis in human vascular pathophysiology and cardiovascular disease.

Type I Membrane Klotho Expression Is Decreased and Inversely Correlated to Serum Calcium in Primary Hyperparathyroidism

The type I membrane protein Klotho was recently shown to mediate PTH secretion in parathyroid cells in response to low extracellular calcium. In contrast, Klotho inhibits PTH secretion indirectly through the action of fibroblast growth factor-23. Abnormal Klotho expression in parathyroid disorders remains to be elucidated. The aim of the study was to determine: 1) Klotho expression in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT) compared to normal tissue; and 2) its relation to the serum calcium and PTH levels. Surgically removed parathyroid glands (n = 40) and four normal parathyroid tissue specimens were analyzed for Klotho mRNA and protein levels by quantitative real-time PCR and immunohistochemistry. In vitro effects of calcium on Klotho mRNA expression were studied in bovine parathyroid cells. Klotho mRNA levels were significantly decreased (n = 23) or undetectable (n = 17) in parathyroid adenomas compared to normal tissues (P < 0.001). Reduced Klotho protein expression was confirmed by immunohistochemistry. Klotho mRNA levels were inversely correlated to serum calcium (r = -0.97; P < 0.0001), and calcium dose-dependently decreased Klotho mRNA expression in normal parathyroid cells in vitro (P < 0.01). Serum calcium was the only significant marker of Klotho expression in multivariate analysis with calcium, phosphate, PTH, and adenoma weight as independent variables. Parathyroid Klotho expression is decreased or undetectable in pHPT. We provide evidence that 1) serum calcium is strongly associated with parathyroid Klotho expression in pHPT; and 2) abnormal PTH secretion in hypercalcemic pHPT subjects is mediated by Klotho-independent mechanisms.