Figure 4.

Klotho promoter hypermethylation and suppressed protein expression following UUO are abolished in Ybx1-deficient kidneys following UUO. (a) Diagram illustrating that Klotho promoter hypermethylation subsequently repressed expression, the WNT/β-catenin signaling pathway, and fibrosis. (b) Representative methylation-specific PCR of the Klotho promoter from contralateral and UUO mouse kidneys. (c) Expression of Klotho, αSMA, fibronectin, and DNMT1 in wild-type and Ybx1^ΔRosaERT+TX mouse kidneys (3 samples from each group are shown). (d) Transcript levels of DNMT1, 3a, and 3b assayed by RT-PCR. (e) Representative immunohistochemistry of Klotho in healthy and diseased kidney tissues of wild-type and Ybx1^ΔRosaERT+TX animals. (f) A comparison of Pai1, Kl (Klotho), Snail1, CTGF, and αSMA with known YB-1-binding motifs (red box) of matrix metalloproteinase2 (Mmp2), Col1a1, DNA polymerase-a (DPA), IL10, and Sglt2 genes reveals homologies and stem-loop motifs (indicated by arrows). (g) Schematic view of the Klotho promoter, including the YB-1 binding motif, CpG islands, and transcriptional start (ATG). (h) Under healthy conditions, the YB-1-DNMT1 complex is located in the cytosol. Following pro-fibrogenic stress, the complex translocates to the nucleus, where it binds to promoter regions such as Klotho, leading to promoter hypermethylation and the reduced expression of Klotho. The expression of the pro-fibrogenic genes Col1a1, αSMA, CTGF, and TGFβ is induced. A different scenario takes place in the kidney tissue of Ybx1^ΔRosaERT+TX animals. No methylation of the aforementioned genes takes place due to the lack of DNMT1 [29,38,48,54,55,56,57], (*** p < 0.001).