Molecular biological study of mechanisms leading towards glomerulosclerosis. study of reactive oxygen intermediates and antioxidans enzymes in normal and pathological renal cortex, in isolated glomeruli and in cultured cells.

Our aim is to investigate the involvement of reactive oxygen intermediates (ROI) and antioxidant enzymes (AOE) in the synthesis of extracellular matrix proteins in animal models of chronic renal failure with glomerulosclerosis as well as in cultured mesangial cells with an increased extracellular matrix production. The role of and the relationship between angiotensin II, ROI and TGF-B will be investigated. We will study time-related changes of different antioxidant enzymes in 3 different animal models of chronic renal failure. This research implies the study of the expression, synthesis and activity of each antioxidant enzyme in homogenates of kidney cortex and isolated glomeruli as well as the integrins a1B1 and a5B1. We will identify the ROI involved in the induction of TGF-B in angiotensin II-stimulated mesangial cells using specific scavengers. The role of the renin-angiotensin-aldosteron axis upon changes of AOE will be clarified. Using specific cDNA- and/or RNA-probes the changes in expression of antioxidant enzyme genes, of the TGF-B gene and genes encoding for extracellular matrix proteins such as laminin will be monitored and using the same probes we will identify the cells that express the genes for antioxidant anzymes, TGF-B and extracellular martix proteins (in situ hybridisation). Using specific antibodies we will perform a semi-quantitative determination of changes in synthesis of antioxidant enzymes, TGF-B, matrix proteins and integrins in the kidney. Enzyme cytochemistry with the use of specific substrates will permit to measure the activity of each antioxidant enzyme (enzyme assays), and localise their activity in kidney sections.