A Human-Mouse Chimera of the α3α4α5(IV) Collagen Protomer Rescues the Renal Phenotype in Col4a3−/− Alport Mice

Abstract

Collagen IV is a major structural component of basement membranes. In the glomerular basement membrane (GBM) of the kidney, the alpha3, alpha4, and alpha5(IV) collagen chains form a distinct network that is essential for the long-term stability of the glomerular filtration barrier, and is absent in most patients affected with Alport syndrome, a progressive inherited nephropathy associated with mutation in COL4A3, COL4A4, or COL4A5 genes. To investigate, in vivo, the regulation of the expression, assembly, and function of the alpha3alpha4alpha5(IV) protomer, we have generated a yeast artificial chromosome transgenic line of mice carrying the human COL4A3-COL4A4 locus. Transgenic mice expressed the human alpha3 and alpha4(IV) chains in a tissue-specific manner. In the kidney, when expressed onto a Col4a3(-/-) background, the human alpha3(IV) chain restored the expression of and co-assembled with the mouse alpha4 and alpha5(IV) chains specifically at sites where the human alpha3(IV) was expressed, demonstrating that the expression of all three chains is required for network assembly. The co-assembly of the human and mouse chains into a hybrid network in the GBM restores a functional GBM and rescues the Alport phenotype, providing further evidence that defective assembly of the alpha3-alpha4-alpha5(IV) protomer, caused by mutations in any of the three chains, is the pathogenic mechanism responsible for the disease. This line of mice, humanized for the alpha3(IV) collagen chain, will also provide a valuable model for studying the pathogenesis of Goodpasture syndrome, an autoimmune disease caused by antibodies against this chain.

Loss of α3/α4(IV) Collagen from the Glomerular Basement Membrane Induces a Strain-Dependent Isoform Switch to α5α6(IV) Collagen Associated with Longer Renal Survival in Col4a3 −/− Alport Mice

“…Upon analysis of NC1 hexamers from B6 Col4a3 ϩ/Ϫ mouse kidneys, the ␣3, ␣4, and ␣5NC1 domains were bound quantitatively to immobilized mAb against ␣3NC1 and ␣5NC1 domains, along with small amounts of ␣1 and ␣2NC1 domains, whereas the unbound fraction contained only ␣1 and ␣2NC1 domains. This indicates the presence of ␣3␣4␣5(IV) protomers, assembled with each other or with ␣1␣2(IV) protomers, as previously shown in wild-type mice (17). In contrast, upon analysis of NC1 hexamers from Col4a3 Ϫ/Ϫ mouse kidneys, no material was bound by the ␣3NC1 mAb, whereas the anti-␣5NC1 mAb co-precipitated quantitatively the ␣5 and ␣6(IV) NC1 domains, accompanied by some ␣1 and ␣2(IV) NC1 domains.…”

“…Rat mAb RH42 (anti-␣4NC1 [17]), b14 (anti-␣5NC1 [18]), and B66 (anti-␣6NC1 [4]) were used for immunofluorescence staining, and affinity-purified rabbit polyclonal antibodies were used for detection of ␣1/2(IV) collagen. Rat mAb M54 and M69 (19) were used for specific detection of murine ␣5 and ␣6 NC1 domains in Western blots; other chain-specific mAb were as described (17). mAb 8D1 and b14 coupled to Affigel-10 were used for separation of NC1 hexamers that contained ␣3 and ␣5 subunits, respectively.…”

Goodpasture Autoantibodies Unmask Cryptic Epitopes by Selectively Dissociating Autoantigen Complexes Lacking Structural Reinforcement

“…The absence of ␣3NC1 monomers (implying an absence of GP-reactive M-␣3␣4␣5 hexamers) in the lungs of some patients may explain the phenotype of anti-GBM disease without pulmonary hemorrhage. Finally, the well recognized difficulty in inducing anti-GBM disease in mice may be related to the relative impenetrability of their GBM hexamers, which in turn is determined by the higher content of ␣3NC1 dimers in the murine GBM compared with that found in the human kidneys (34).…”

Cellular Origins of Type IV Collagen Networks in Developing Glomeruli

“…Moreover, the monoclonal antibody used in these immunoelectron microscopic localization experiments reacts specifically with the hexamer of NC1 domains of collagen ␣3␣4␣5(IV), 20 indicating that collagen IV network polymerization was beginning to occur intracellularly. Examination of sections from several animals and multiple glomeruli at various stages of development failed to show anti-collagen ␣3␣4␣5(IV) labeling of biosynthetic organelles within glomerular endothelium or mesangial cells.…”