Functional roles of klotho in the process of renal fibrosis assessed in the cultured renal epithelial and fibroblast cells

Introduction and Aims: Klotho is a protein that has anti-aging properties and is known to function as an obligate co-receptor of FGF23 in the regulation of phosphate homeostasis and also to be involved in insulin/IGF1 and WNT signaling, suggesting that Klotho has several biological properties in physiological and pathophysiological conditions. It has been reported that the renal expression of the Klotho gene is markedly suppressed in chronic kidney disease (CKD). Acceleration of renal fibrosis is a basic pathophysiology for progression of CKD, and previously, we showed that the renal interstitial fibrosis was severer in the kl +/mice than those in the wild-type mice by UUO treatment. Thus, in the present study we investigated the biological role of Klotho in the process of fibrosis in cultured cells. Methods:We explored the relationship between Klotho and fibrosis-related factors in cultured renal epithelial cells (mIMCD and HK2) and fibroblast cells (NRK49F) under TGF-β stimulation, which were quantified by using CL-Quant software to analyze time-lapse images in a Nikon Biostation CT in combination with a high-throughput cell migration assay, and the relationship between Klotho and cell polarity by measuring the expression of Na/K ATPase/PKC-ζ, a marker of cell signaling (β-catenine) in 3D culture with collagen mixture. The expression levels of fibrotic marker, such as α-SMA, fibronectin and TGF-β1 was immunohistochemically stained and mRNA expression levels were assessed by quantitative RT-PCR. Internal expression of klotho was modified by siRNA transfection. Results: TGF-β1 reduced Klotho expression in IMCD and HK2 cells. In addition, in NRK49F cell in which internal Klotho expression was negligible, expression levels of α-SMA and PAI-1 mRNAwere significantly suppressed in by addition of recombinant Klotho protein to the medium. Migration of NRK49F cells was accelerated and expression of fibrotic markers was up-regulated by addition of TGF-β1, in contrast, Klotho addition suppressed the effect. SiRNA reduced the expression of klotho in epithelial cells, resulting in accelerating cell migration. Klotho affected the localization of membrane protein, namely, addition of Klotho protein promoted to translocate Na/ K-ATPase and PKC-ζ to the cell membrane area and attenuate migration/proliferation, especially, Klotho exerts its effects in proliferating or (re-) differentiating cell condition. Conclusions: Taken together, it is likely that klotho protects against renal fibrotic process accompanied by down-regulation of fibrotic markers, counteracting to TGF-β, and one of possible mechanism mediating translocation of Na/K ATPase, resulting in Ca channel stabilization/alternation of Ca ion concentration. Klotho should be involved in the accentuation of the progression of renal fibrosis in CKD.