Diabetic glomerulosclerosis occurs in a setting of attenuated responsiveness of glomerular aterioles and mesangila cells to angiotensin II. Persistent arteriolar vasodilitation in this setting predisposes the glomerulus to increased plasma flow and hydrostatic pressure with the attendant development of proteinuria and glomerular sclerosis in the insulin treated streptozotocin rat. Glomerular mesangial cells are contiguous with the smooth muscle cells surrounding the afferent and efferent arteriole and are capable of contraction in response to angiotensin II in vitro only in the presence of insulin. It has been demonstrated that rat glomeruli bear insulin receptors. It is therefore likely that glomerular mesangial cells bear insulin or insulin-like growth factor receptors as well. It is postulated that an insulin resistant condition may be induced in these cells, in part, by current insulin vehicles. Insulin resistance may predispose glomerular mesangial cells to become hyporesponsive to vasoconstrictors. The insulin resistant state would also predispose the kidney to ongoing unopposed gluconeogenesis. Gluconegenic provision of substrate for aldose reductase would in turn favor the production of sorbitol an abnormal mesangial matrix constituent found in diabetic rat glomeruli. To assess the insulin resistant state, rat glomerular mesangila cells will be examined for insulin or insulin-like growth factor I and II binding. Should these receptors be found, insulin will be used to down regulate receptors and desensitize these cells. Following this maneuver, the cells will be tested for angiotensin II mediated contraction and increases in cytosolic free calcium. Should contraction be absent or attenuated in this setting, it would suggest similar phenomona occur in vivo. Pyruvate carboxylase is a mitochondrial biotin dependent enzyme which mediates the first rate limiting step of gluconeogenesis whose activity is inhibitable by insulin. Phosphoenolpyruvate carboxykinase (PEPCK) is a cytosolic enzyme which mediates the second rate limiting step of gluconeogenesis. PEPCK activity is also inhibitable by insulin. To assess distal post-receptor attenuation of the insulin signal, which would be expected in the insulin resistant state, measurement of pyruvate carboxylase and PEPCK RNA transcription and RNA content will be performed in explanted glomeruli and tubules of insulin treated diabetic rats utilizing antisense transcripts from plasmid clones containing the cDNA for rat liver pyruvate carboxylase and PEPCK. Should the insulin resistant state be demonstrated in these experiments, it would suggest insulin resistance is a permissive metabolic condition which predisposes the diabetic to hemodynamically triggered events causing glomerular structural damage and proteinuria seen in diabetic glomerulosclerosis.
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