Fig 2: Blockade of CXCR4 represses ß-catenin signalling in UIRI mice. A, Representative micrographs show renal expression of p-GSK3ß (Ser9) and ß-catenin in three groups. Paraffin sections were immunostained with antibodies against p-GSK3ß (Ser9) and ß-catenin. Yellow arrows indicate positive staining. Scale bar, 50 µm. B-D, Representative (B) Western blots and graphical representations of (C) p-GSK3ß (Ser9) and (D) ß-catenin in three groups. Numbers 1-3 indicate each individual animal in a given group. *P < .05 versus sham control (n = 5-6); †P < .05 versus UIRI alone (n = 5-6). E-I, Representative (E) Western blots and graphical representations of (F) PAI-1, (G) Snail 1, (H) MMP-7 and (I) E-cadherin in three different groups. Numbers 1-3 indicate each individual animal in a given group. *P < .05 versus sham controls (n = 5-6); †P < .05 versus UIRI alone (n = 5-6)

Fig 3: In vivo expression of exogenous Wnt1 before IR downregulates renal Wnt/ß-catenin target genes in mice after AKI-CKD progression. (A,B) mRNA expression of PAI-1 and MMP-7 in different groups as indicated. (C–E) Representative Western blot analyses of PAI-1 and Klotho protein levels. (F) mRNA expression of Klotho. *P < 0.05; **P < 0.01. n = 5.

Fig 4: Renal Wnt/ß-catenin signaling is time-dependently induced in kidneys during aging. (a–c) Western blot analyses show renal expression of Wnt1 and Wnt10b. Graphical representations of (b) Wnt1 and (c) Wnt10b protein expression levels in different groups are presented. *p < .05 versus 7-month-old mice (n = 5–6). (d) Representative micrographs show renal expression of Wnt1 and ß-catenin. Arrows indicate positive staining. Scale bar, 50 µm. (e–i) Western blot analyses show renal expression of the active form and total ß-catenin, as well as its targets PAI-1 and Snail1. Graphical representations of (f) active ß-catenin, (g) ß-catenin, (h) PAI-1, and (i) Snail1 protein expression levels in different groups are presented. *p < .05 versus 7-month-old mice (n = 5–6). (j) Graphical representation of the relative abundance of MMP-7 mRNA. *p < .05 versus 7-month-old mice (n = 5–6). (k) Colocalization of active ß-catenin and various segment-specific tubular markers in aging kidneys. Frozen kidney sections from 24-month-old mice were immunostained for various segment-specific tubular markers (green) and active ß-catenin (red). Segment-specific tubular markers were used as follows: proximal tubule, Lotus tetragonolobus lectin (LTL); distal tubule, thiazide-sensitive NaCl cotransporter (NCC); and collecting duct, aquaporin-3 (AQP3). Sections incubated with secondary antibodies alone (omitting primary antibody) were used as negative controls (right panel). Arrows indicate positive tubules with colocalization of active ß-catenin and specific tubular makers. Scale bar, 75 µm. (l) Colocalization of ß-catenin and multiple cell type markers in kidneys from 24-month-old mice. Frozen kidney sections were co-stained with various cell type-specific markers (red) and ß-catenin (green). Cell markers were used as follows: macrophage, mannose R; fibroblasts, PDGFRß; and endothelial cell, EMCN. Sections incubated with secondary antibodies alone (omitting primary antibody) were used as negative controls (right panel). Arrowheads indicate colocalization of ß-catenin and specific cell makers. Scale bar, 75 µm

Fig 5: Vardenafil inhibited matrix-associated protein generation by fibroblasts. Quiescent AKR-2B fibroblasts in 0.1% FBS/DMEM were pretreated for 60 min with the indicated vardenafil concentration (PDE5 inhibitors). Vehicle (-) or TGF-ß (+) was directly added to a final concentration of 10 ng/mL, and following 24 h of incubation, lysates were prepared and western blotted for Collagen I, PAI-1, CTGF, a-SMA, and fibronectin. GAPDH was used as a loading control. Data are representative of three separate experiments.