Figure 7.

Myofibroblast-specific Wnt4 knockout does not impact renal fibrosis. (A) Breeding strategy results in 25% control mice and 25% KO mice. lacZ recombination is evident in interstitial pericytes, mesangial cells, and VSMCs in both Wnt4^flox/+ and Wnt4^del/flox mice. As expected, there is evidence of expansion of lacZ+ interstitial cells, whereas labeling of mesangial cells and VSMCs remains unchanged. (B) Ten days after UUO, Wnt4 transcript is increased approximately 30-fold in control mice over CLK. Wnt4 transcript levels are significantly decreased in UUO samples from Wnt4^del/flox (knockouts) compared with control mice. (C–E) Transcript levels of fibrotic marker genes αSMA, collagen 1α1, and fibronectin are all increased 10 days after UUO in control and KO mice compared with CLK, however, are not different from each other. (F–H) Western blots comparing expression of Wnt4 and αSMA within genotypes and across genotypes in the 10 day UUO and CLK samples. (I) Quantification of integrated densities of Wnt4 protein bands indicates a significant increase in Wnt4 protein in control mice after UUO. Comparison of relative Wnt4 protein levels between control and KO samples after UUO shows an approximately 93%–94% decrease of Wnt4 in the KO kidneys. Wnt4 protein levels in UUO kidneys of KO mice are similar to Wnt4 protein levels in CLK kidneys. Data analyzed by two-way ANOVA comparing between genotypes in CLK and UUO conditions. In F–H, blots are analyzed by comparing integrated optical densities of bands and normalizing with GAPDH bands followed by two-way ANOVA (Bonferroni’s post test). ***P<0.001. Scale bars, 25 µM in A. KO, knockout; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.