No abstract

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC ␤ isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC ␣ and ␤ 1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC ␤ selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC ␤ 1 isoform without decreasing PKC ␣ isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E 2 production, and inhibition of Na ϩ -K ϩ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-␤ 1 and extracellular matrix components such as fibronectin and ␣ 1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the ␤ isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na

Cell Culture. Bovine retinal pericytes were obtained from fresh eyes as described (12) and cultured in Dulbecco's modified Eagle's medium (DMEM; 5.5 mM glucose) with 20% (vol/vol) fetal bovine serum. Prior to RNA isolation, the pericytes were grown to confluence and shifted into DMEM plus 1% (vol/vol) calf serum with glucose levels of either 5.5 mM, 22 mM, or 5.5 mM with 16.5 mM mannitol. Media were changed daily for 7 days, and the cells were stimulated with DMEM plus 10% fetal bovine serum for 8 hr (30 hr for Western blots). Pericyte growth is inhibited under similar conditions (13). Bovine aortic endothelial and smooth muscle cells were obtained from calf aorta as described (14) and