Figure 2.
Ilk KO in PCs causes kidney fibrosis and activates the TGF-β/Smad signaling pathway. (A) Immunofluorescence staining revealed the increased expression of α-SMA (red and arrows, top panels), ECM collagen type 1 (Col I; red and arrows, middle panels), and fibronectin (FN; red and arrows, bottom panels) in the cortex of 4-week-old KO mice. CD PCs were highlighted by immunostaining with anti-AQP2 antibody (green). Nuclei were stained blue with DAPI. Scale bar, 10 μm. (B) Deposition of collagen fibrils in 4-week-old wild-type (WT) and 4- and 8-week-old KO kidney was examined by picrosirius red staining. Collagen type 1 (red) and type 3 fibrils (green) were observed under polarized light (bottom three panels). Top three panels are images obtained under the bright light. Scale bar, 50 μm. (C) Masson trichrome staining revealed the excessive deposition of collagen fibers in 4- and 8-week-old KO kidney compared with the control. Scale bar, 50 μm. (D) Representative immunoblotting revealed increased expression of NGAL, FN, α-SMA, phosphorylated Smad3 (Ser423/425, p-Smad3, arrows), and total Smad2/3 (T-Smad2/3) in 4-week-old Ilk KO kidney (left). Individual protein signal intensity was quantified by densitometry and plotted over the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ratio of p-Smad3 over T-Smad2/3 is also shown in the graph (right). **P<0.01, ***P<0.001 versus wild-type mice. (E–G) Real-time PCR confirmed the augmented mRNA levels of (E) FN, (F) Col I, and (G) TGF-βi in Ilk KO kidney. Expression of individual genes was normalized to β-actin. ***P<0.001 versus 4-week-old wild-type mice; #P<0.05, ##P<0.01 versus 8-week-old wild-type mice. Data presented as mean±SEM, n=6–7 for (D–G). Statistics were performed using the t test. MW, molecular weight.