Although the early glomerular lesion associated with reduced nephron mass and intracapillary hypertension is focal and segmental, this application tests the hypothesis that all mesangial cells in hemodynamically stressed glomeruli undergo subtle structural and functional changes which precede and contribute to the development of sclerosis. Glomeruli and mesangial cells from rats undergoing 1 5/6 nephrectomy and controls will be examined using three approaches: 1. The biochemical nature of glomerulosclerosis and hyalinosis will be characterized in tissue sections and isolated glomeruli by 1) immunochemical studies using antisera to matrix proteins and plasma constituents; 2) collagen and glycosaminoglycan synthesis studies; 3) collagen subtype analyses; and 4) measurement of gene expression for matrix proteins and proto-ongenes by in situ and in vitro hybridization. 2. Changes in mesangial cell structure and function will be sequentially examined to define their relationship to the development of glomerulosclerosis. Studies will be performed on renal tissue sections and on cultrued mesangial cells. Structural change will be assessed by examining 1) cellular organelle and intracellular filament content using light, immunofluorescence, and electron microscopies; 2) in vitro and in situ hybridization studies for smooth muscle and total actin; and 3) surface receptor changes using antisera to Ia and leukocyte common antigen. Functional change will be assessed by measuring 1) cell contractility; 2, A-II receptor density; 3) gene expression for matrix proteins and proto-oncogenes using in vitro hybridization; 4) collagen and glycosaminoglycan synthesis; 5) collagen subtype analyses; 6) endocytosis; and 7) mesangial macromolecule uptake. 3. Factors which may affect mesangial cell phenotype in vivo will be studied: 1) The effects of heparin on the structure and function of normal cultured mesangial cells; 2) The effect of PDGF on mesangial cell proto-oncogene and extracellular matrix gene expression; and 3) The effect of agents known to inhibit the sclerotic response to intraglomerular hypertension in vivo on the expression of proto-oncogene and extracellular matrix protein genes in glomeruli and cultrued mesangial cells in vitro. This proposal offers unique features likely to enhance our understanding of the events leading to glomerulosclerosis. The application of the highly sensitive tools of molecular biology to the study of glomerular injury should enhance our ability to mesure the subtle, early changes in mesangial cells that may precede the development of sclerosis. The experiments outlined utilize state of the art technologies in molecular biology and extracellular matrix biochemistry to probe the pathogenesis of glomerulosclerosis, a critical issue at the forefront of nephrology.
