Analysis of Klotho Expression in the Mouse Kidney Cell Line

Defects in klotho gene expression in the mouse result in a syndrome that resembles human aging. However, the precise properties of klotho protein, the mechanism regulating its expression, etc., have not been clarified. First, using specific antibody we identified klotho expression in mouse kidney as mainly localizing in the distal and collecting duct cells. Based on this information we examined the expression of klotho in a murine inner medullary collecting duct cell line (mIMCD3). We then explored the physiological relevance of regulation of klotho expression under oxidative stress condition. Expression of klotho in mIMCD3 cells was identified by real-time PCR, immunoprecipitation-Western blotting, and immunocytochemical staining. Cells were transfected with recombinant klotho adenovirus and overexpression of klotho gene was induced to confirm the expression of internal klotho. Oxidative stress injury produced by adding hydrogen peroxide to the culture medium dose-dependently reduced klotho expression maximally 24 hours, and it also diminished klotho staining. The results of this study demonstrated that klotho expressed in cultured mIMCD3 cells and that the klotho gene may be involved in the process of oxidative stress injury in these cells