Early diabetic nephropathy is characterized by an elevation in the glomerular filtration rate that preceeds clinically apparent renal dysfunction by decades. This suggests that chronic derangements in glomerular hemodynamics may lead to the progressive glomerular sclerosis of end-stage diabetic nephropathy. Enhanced prostaglandin synthesis has been demonstrated to occur in diabetic nephropathy and is likely to account for the derangements in glomerular hemodynamics. Release of arachidonic acid, the rate-limiting step in prostanoid synthesis, has been shown to be under hormonal control in a variety of cell types. Phospholipase A2 (PLA2) occupies a central role in the release of arachidonic acid and ultimately in the regulation of prostanoid production. It is proposed, therefore, that abnormalities in the hormonal regulation of renal PLA2 leads to increases in prostaglandin production accounting for the derangements in glomerular hemodynamics seen in early diabetic nephropathy. The long-term objective of these studies will be to elucidate the mechanisms of PLA2 regulation and identify abnormalities in diabetic nephropathy. To accomplish this, the hormone-induced release of arachidonic acid and synthesis of prostaglandins will be characterized in glomerular epithelial and mesangial cells from normal and diabetic rats. Assays will be developed to directly demonstrate PLA2 regulation by hormones resulting in arachidonic acid release. The role of guanine nucleotide binding proteins as modulators of PLA2 activity will be investigated. To obtain a better understanding of the molecular regulation of this enzyme, the membrane-associated PLA2 will be purified to homogeneity. Polyclonal and monoclonal antibodies raised against the enzyme will be utilized to study the mechanisms of regulation in cultured cells and tissue from normal and diabetic animals. Finally, the cDNA for the enzyme will be isolated from rat renal cDNA libraries. This cDNA clone will be useful in future studies to explore differences in the expression and regulation of PLA2 in normal and diabetic states. A detailed understanding of the regulation of PLA2 will provide insights into disease states associated with abnormalities in prostanoid synthesis such as diabetic nephropathy.
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