(A) Representative micrographs show that Shh did not significantly affect podocyte integrity and ultrastructure ex vivo. Isolated glomeruli from normal rats were incubated in the absence or presence of Shh (50 ng/ml) for 24 hours. Transmission electron microscopy (TEM) demonstrates that podocyte foot processes and slit diaphragm were largely preserved after Shh incubation. Arrows indicate slit diaphragm. Dotted lines indicate the boundary of glomerular basement membrane. Scale bar: 25 μm (top); 500 nm (bottom). (B and C) Western blots show that Shh did not affect the abundance of podocyte-specific proteins, such as nephrin, podocalyxin, α-actinin-4, and WT1, ex vivo. Representative Western blot (B) and quantitative data (C) are presented (n = 3, t test). (D and E) Western blots show that Shh induced mesangial cell activation in glomerular miniorgan culture. Isolated glomeruli were incubated with Shh (50 ng/ml) for 24 hours, and glomerular lysates were immunoblotted with antibodies against PDGFR-β, fibronectin, α-SMA, β-catenin, and α-tubulin, respectively. Representative Western blot (D) and quantitative data (E) are presented. *P < 0.05 versus controls (n = 3, t test). (F and G) Immunofluorescence staining shows that Shh did not change the expression and localization of podocyte-specific proteins ex vivo. Isolated glomeruli were incubated without or with Shh for 24 hours, and glomerular sections were immunostained for nephrin, podocalyxin, and α-actinin-4 proteins. Representative micrographs (F) and quantitation of immunofluorescence (IF) intensity (arbitrary unit) (G) are shown. Data were obtained from 3 glomeruli per experiment; 3 independent experiments per group. Arrows indicate positive staining. Scale bar: 20 μm. (H and I) Double staining shows that Shh selectively promoted mesangial cell activation. Isolated glomeruli were incubated without or with Shh for 24 hours and subjected to double immunostaining for podocyte marker nestin (green) and mesangial cell marker PDGFR-β (red). Representative micrographs (H) and quantitation of IF intensity (arbitrary unit) (I) are shown. *P < 0.05 versus controls, t test. Data were obtained from 3 glomeruli per experiment; 3 independent experiments per group. White arrows indicate PDGFR-β⁺ mesangium, whereas yellow arrows denote nestin⁺ podocytes. Scale bar: 20 μm.