Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.

Abstract

A mouse model for the autosomal form of Alport syndrome was produced. These mice develop a progressive glomerulonephritis with microhematuria and proteinuria, consistent with the human disease. End-stage renal disease develops at -14 weeks of age. TEM analysis of the glomerular basement membranes (GBM) during development of renal pathology revealed focal multilaminated thickening and thinning beginning in the external capillary loops at 4 weeks and spreading throughout the GBM by 8 weeks. By 14 weeks, half of the glomeruli were fibrotic with collapsed capillaries. Immunofluorescence analysis of the GBM showed the absence of type IV collagen ~-3, e~-4, and c~-5 chains and a persistence of ~-1 and ~-2 chains (these chains normally localize to the mesangial matrix). Northern blot analysis using probes specific for the collagen chains illustrate the absence of COL4A3 in the knockout, whereas mRNAs for the remaining chains are unchanged. An accumulation of fibronectin, heparan sulfate proteoglycan, laminin-1, and entactin was observed in the GBM of the affected animals. The temporal and spatial pattern of accumulation was consistent with that for thickening of the GBM as observed by TEM. Thus, expression of these basement membrane-associated proteins may be involved in the progression of Alport renal disease pathogenesis. The levels of mRNAs encoding the basement membrane-associated proteins at 7 weeks were unchanged.

Glomerular Expression of Type IV Collagen Chains in Normal and X-Linked Alport Syndrome Kidneys

“…Two of these, affected with a frameshift mutation and a large deletion, respectively, had no detectable COL4A5 transcript, a result similar to that reported in the Samoyed dog 55 and suggesting that such mutations are responsible for destabilization of the mutated message and its rapid degradation. 35,36 In the third patient, affected with a 3Ј splice mutation, a COL4A5 transcript was clearly expressed. Splicing of such RNA would be predicted to give rise either to a transcript with an in-frame deletion (arising from exon skipping), and/or to an out-of-frame transcript and a putative truncated protein.…”

“…39 The same holds true in Samoyed dogs affected with an X-linked nephritis closely resembling the human AS, and because of a stop mutation in COL4A5 exon 35 55 and in mouse or dog models for autosomal recessive AS. [35][36][37] In these situations the ␣3(IV) to ␣5(IV) network is replaced, throughout the width of AS GBM, by an ␣1(IV) to ␣2(IV) network which is normally confined to the subendothelial region of the GBM. The precise mechanisms resulting in the lack of all three chains when only one gene is mutated and in their replacement by ubiquitous ␣1(IV) and ␣2(IV) chains remains speculative.…”

“…28 -31 The presence of cysteine-rich ␣3(IV) and ␣4(IV) chains, forming with ␣5(IV) a network containing loops and supercoiled triple helices stabilized by disulfide bonds between the chains, seems to be important with regards to the longterm stability of the GBM and its role as a filter. 26,32 Despite the increasing number of AS mutations reported in the literature [12][13][14][15][16][17][18][19] and the existence of AS animal models, [33][34][35][36][37] several questions regarding the consequences of AS mutations on the collagen organization within the GBM and the mechanisms responsible for the progressive development of AS nephropathy remain unanswered. A striking feature observed in the majority of AS is the absence of all three ␣3(IV), ␣4(IV), and ␣5(IV) chains within the GBM although only one of these chains is actually mutated.…”

Irradiation Prolongs Survival of Alport Mice

“…20 The mice were bred in specific pathogen-free areas, and all of these experiments were approved by Institutional Animal Care and Use Committee regulations. Typing was performed using the PCR with tail DNA.…”

“…The predicted band sizes of Col4a3 were 361 bp in WT mice and 1458 bp in KO mice because a 1097-bp neo cassette was inserted into the exon 48 to generate the KO mice. 20 …”