Prolyl-3 Hydroxylation of Type IV Collagen in Kidney Development

Baylor College Of Medicine, Houston TX

Investigators

Abstract

[unreadable] DESCRIPTION (provided by applicant): Prolyl-3 hydroxylases are a recently identified family of enzymes involved in post-translational modification of collagens. Fibrillar collagens have a single residue that undergoes prolyl-3 hydroxylation, whereas type IV basement membrane collagen has 12-15 modified residues. The kidney is composed of basement membranes with unique collagen hetero-trimers contributing to their specific functions, including the glomerular basement membrane, mesangial matrix, and tubular basement membranes. Preliminary in- situ hybridization data shows prolyl-3 hydroxylases 2 & 3 (P3H2 and P3H3) are expressed in distinct distributions within the kidney. P3H2 is specific to the glomeruli, while P3H3 localizes to a central mesangial distribution, tubules, and collecting system. We propose to study the biologic function of P3H2 and P3H3 in kidney development by generating targeted knock-out mice for each gene. The targeting strategy will be deletion of interior exons via homologous recombination, leading to disruption of the open reading frame. Study of these mice will include histology, electron microscopy, urine protein and serum creatinine measurements, and frozen tissue collection for evaluation of gene expression. The kidney phenotype will be studied at multiple post-natal and embryonic time points. Novel P3H3 sequence variations have been identified in the genes of patients with hereditary nephritis. These sequence variants localize to the functional dioxygenase domain, and are conserved throughout vertebrates. We plan to study the function of these variants compared to wild-type recombinant human protein produced in insect cells. The in-vivo assay will use recombinant human collagen as a substrate, measuring succinate production coupled to NADH oxidation. If these variants are deleterious, we will proceed to analyze a larger population with chronic kidney disease to evaluate what role abnormal prolyl-3 hydroxylation plays in this group. Relevance: In the kidney, specialized collagens in the membranes surrounding cells aid in the kidney's function of filtering between the blood and urinary spaces in the body. This study focuses on prolyl-3hydroxylases, proteins that make changes to collagen that affects its form and function. Understanding the role of these proteins in the kidney may lead to improved screening methods, earlier detection, and new treatments for patients with chronic kidney disease. [unreadable] [unreadable] [unreadable]