MOLECULAR MECHANISMS OF RENAL INJURY

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Basement membranes are composed of type IV collagen, laminins, proteoglycans and entactin. Type IV collagen comprises a family of six different alpha-chains. Glomerular basement membrane (GBM) collagen, for example, is formed in two phases with alpha-3(IV), and alpha-5(IV) collagens appearing later and eventually dominating protomers initially formed from alpha-1 (IV) and alpha-2(IV) chains. Basement membranes from liver, in contradistinction, do not normally express alpha-5(IV) or alpha-6(IV) collagens after their initial formation. This switch from fetal alpha-1 (IV) and alpha-2(IV) collagens to alpha- 3(IV), alpha-4(IV), and alpha-5(IV) chains in human GBM is a developmental event that advances the fabrication of capillary loops in the glomerulus. We believe this developmental switch improves the structural integrity of the capillary filter by protectively enhancing its resistance to local proteases at the site of glomerular filtration. This switch is defective in most kidneys from patients with x-linked Alport Syndrome (XAS) who have mutations in the alpha-5(IV) gene and go on to renal failure. We propose to examine the mechanism of this developmental switch by studying the supramolecular control of the alpha-5(IV) collagen gene. We hypothesize that switching is gated by the opening of chromatin in selected tissues, and is under the modular control of DNA-binding proteins. Determining coordinates for these locus control regions (LCRs) is the first step in understanding the larger aspects of this regulatory process. Regulatory regions important for the switching of type IV collagen will be studied by mapping unique DNasel hypersensitive sites in kidney chromatin remote to the start of the human alpha-5(IV) gene. We have obtained a BAC clone containing genomic DNA from this region on chromosome Xq22.2, and have developed a strategy for measuring human alpha-5(IV) transcripts in transgenic mice. Candidate hypersensitive sites will be expressed as transgenes and evaluated for functional activity, switching, position-independence and copy number- dependence. Hypersensitive sites identified as LCRs can then e explored at the level of the nucleosome in subsequent, second-order studies.