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. 2021 Mar 26;296:100590. doi: 10.1016/j.jbc.2021.100590

Figure 4.

Figure 4

Zurich variant knock-in homozygous mouse (Col4a3z/zmouse) phenocopies features of Alport syndrome in GBM. Immunofluorescence staining of renal sections demonstrates deposition of genetically modified α3 chain along with native α4 and α5 chains within collagen IV α345 scaffold in a Col4a3z/z mouse (top left). This scaffold did not form in the kidney of Col4a3 KO mouse lacking α3 chain (top left). The Zurich variant also did not interfere with hexamer assembly and formation of sulfilimine cross-links as shown by western blot using collagen IV chain-specific antibodies (top left, D indicates dimer and M indicates monomer). The shift in the position of mutant α3 bands relative to the control is due to the difference in sequence length. Samples were normalized on kidney weight and tubulin (bottom of the western blot). Histological analysis of kidney sections revealed varying degree of glomerular sclerosis in 20% to 70% of the glomeruli in the Col4a3z/z mice (periodic acid–Schiff (PAS)-stained representative glomeruli are shown, magnification 600×) and occasional formation of crescents (bottom left, H&E staining). Representative transmission electron microscopy (TEM) images of the glomerular capillary loops from two different Col4a3z/z mice and a wild-type control mouse are shown at the top right. In Col4a3z/z mice exhibiting high albuminuria (see corresponding urine albumin-to-creatinine ratio (ACR) values and albumin SDS-PAGE bands above the TEM images), the glomerular basement membrane (GBM) was irregularly thinned and thickened, lamellated, and occasionally split with foot process effacement. Animals with moderate albuminuria demonstrated mainly irregular thickening of the GBM (arrows and higher magnification for GBM structure). GBM thickness (measured from two glomeruli with five images each, from each mouse) was increased in WT (n = 2) versus Col4a3z/z (n = 3) mice (bottom middle) (p < 0.001, Student’s t test). Col4a3z/z mice (n = 6) had significantly elevated ACR compared with WT mice (n = 5) (bottom right). Both experimental groups included male and female mice.