Fig 2: Collagen a1(III) activates DDR1 receptors both in vitro and in vivo. (A) Cells were treated or not with collagen III and after 12 h, stained with antibodies against either total DDR1 or phospho-DDR1 (pDDR1) (bar = 5 µm). (B) Cryosections from 7-week-old wild-type and Alport mice were dual immunostained with antibodies specific for pDDR1 or WT1 (a podocyte nuclear marker) (bar = 15 µm). Results indicate that collagen III activates DDR1 receptors both in vitro and in vivo in glomerular podocytes. Arrowheads denote areas of WT1 and pDDR1 co-localization.

Fig 3: Collagen a1(III) is expressed in the glomerular basement membrane in Alport mice. (A) Dual immunofluorescence analysis was performed on kidney cryosections from 7-week-old wild-type and Alport mice using antibodies for podocin (a slit diaphragm protein) and DDR1 (a collagen receptor). Clear co-localization is apparent, placing DDR1 at the foot processes (bar = 15 µm). (B) Super-resolution structured illumination microscopy (SR-SIM) of dual immunofluorescence staining of a capillary loop from a 7-week-old Alport mouse stained with anti-DDR1 antibodies (in red) and anti-collagen a1(III) antibodies (in green). The adjacent localization (arrowheads) indicates basement membrane localization of collagen a1(III) (bar = 5 µm). (C) RNA-seq results from wild-type and Alport glomeruli show a marked (>20-fold) increase in the expression of Col3a1 mRNA relative to wild-type. These results were confirmed using real-time RT-PCR (data not shown) and microarray analysis [8]. (D) ImageJ analysis of the relative fluorescence for immunostains of wild-type and Alport glomeruli (six independent glomeruli each) shows significant increases of fluorescence intensity in Alport mice. (E) Western blotting shows clear increases in the 139 kDa band corresponding to collagen a1(III). (F) Quantification of the relative band intensity for triplicate blots of wild-type and Alport mouse glomeruli indicates significantly elevated abundance of collagen a1(III) in Alport glomeruli relative to wild-type, consistent with the RNA-seq findings. *p < 0.05, ***p < 0.001.