Glomerular development is marked by a sequence of type IV collagen isotype replacements in the GBM. Specifically, collagen IV a1a2a1 network, present in the earliest glomerular basement membrane (GBM) of immature nephrons, is replaced in mature GBM by collagen IV a3a4a5 network. Our central hypothesis is: GBM collagen IV network switching is critical for development and maintenance of both endothelial cells and podocytes. We speculate that: cells survey their evolving type IV collagen networks through integrins;synthesis of type IV collagen and its cognate integrin receptors are linked;and when receptors engage abnormal collagen networks, or if receptor expression is incorrect, altered signals are conveyed. This abnormal signaling then thwarts normal basement membrane protein synthesis, leading to further defects in glomerular structure and function.
Our aims are:
Aim 1) To examine possible defects in GBM-integrin signaling in Col4a3 mutant mice, a model of Alport disease. We will (a) define the GBM composition from Col4a3 mutants;(b) characterize integrin expression during progression of Alport disease;and (c) inventory changes in glomerular cell proteins between normal and Alport mice using a proteomics approach.
Aim 2) To characterize glomeruli of Col4a1 heterozygous mice, which are microalbuminuric with thin GBM. We will (a) define the GBM composition from Col4a1 mutants;(b) characterize their integrins and (c) compare their glomerular proteomes with wild type.
Aim 3) To determine the contribution of integrins a1 ?1 and a2?1 on collagen IV network formation. Integrin a1?1is anti-fibrotic whereas a2?1 is pro-fibrotic. We will (a) cross integrin a1-null mice onto Col4a3 and Col4a1 backgrounds expecting increased fibrosis;and (b) cross integrin a2-null mice onto the same, expecting amelioration. We will also test for GBM assembly perturbations through in vivo administration of heterotrimeric triple helical collagen peptides containing integrin binding sites and by injection of antibodies against the type IV collagen integrin binding sites.
Aim 4) Determine whether GBM protein synthesis and integrin expression are linked. This will be tested in glomerular cell cultures by qRT-PCR, Western blotting, siRNA knockdowns, and confocal immunofluorescence and immunoelectron microscopy. Together, these studies will provide fundamentally new information on the dynamic reciprocity between glomerular cells and collagen IV networks and how integrins mediate this critically important interrelationship.
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