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

Abstract

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 stressinduced 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 30-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-20-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 stressinduced miR-200c binds to the KLOTHO mRNA 30-UTR, resulting in reduced KLOTHO expression.