Fig. 7.

HK-2 cells inhibited osteogenic differentiation of human renal interstitial fibroblasts (hRIFs) at least partially via soluble α-Klotho (s-KL) to inactivate the Wnt–β-catenin pathway, and the Wnt–β-catenin pathway was activated in Randall’s plaques (RP). HRIFs were cocultured with recombinant KL (r-KL) or the β-catenin agonist SKL2001 (20 μM). A–C Alizarin Red staining (ARS) of calcium deposits 14 days after osteogenic induction (n = 3). D–F WB was used to determine the relative protein levels of p-β-catenin, β-catenin and osteogenic markers (OCN, MSX2, Runx2) 7 days after osteogenic induction (n = 3). HRIFs were cotransfected with the TOPFlash plasmid and the Renilla luciferase plasmid as an internal control. G The relative luciferase activity was determined in transfected hRIFs cocultured with different concentrations of r-KL under osteogenic conditions for 48 h (n = 3). H The relative luciferase activity was determined in transfected hRIFs cocultured with HK-2 cells, Len-sh-KL-transfected HK-2 cells or Len-KL-transfected HK-2 cells under osteogenic conditions for 48 h (n = 3). I–J Immunohistochemistry (IHC) for transmembrane KL (m-KL), β-catenin and Wnt–β-catenin pathway inhibitors (SFRP4, SFRP1, DKK1, and SOST) in RP (n = 6) and normal renal papillae (NRP; n = 6); the DAB staining density was quantified by the average gray value using the IHC Toolbox plugin in ImageJ. K–M Representative western blots and quantification of m-KL, the ratio of p-β-catenin to β-catenin and Wnt–β-catenin pathway inhibitors in RP (n = 18) and NRP (n = 18) tissues. N–R Correlation analysis between the relative protein expression level of s-KL and the ratio of p-β-catenin to β-catenin N, the expression levels of SFRP1 and Runx2 O, the expression levels of DKK1 and OCN P, the expression levels of s-KL and SFRP1 Q, and the expression levels of s-KL and DKK1 R in RP tissues; n = 18