EXPOSURE OF HUMAN RENAL PROXIMAL TUBULAR CELLS TO GLUCOSE LEADS TO ACCUMULATION OF TYPE-IV COLLAGEN AND FIBRONECTIN BY DECREASED DEGRADATION

Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. In the current st udy we examined the effects of elevated D-glucose concentrations on hu man proximal tubular (HPTC) type IV collagen and fibronectin turnover. Incubation of confluent growth arrested HPTC with 25 mM D-glucose led to accumulation of both type IV collagen and fibronectin. This effect was maximal at 48 hours and represented a sevenfold increase for fibr onectin (N = 4, P = 0.04), and a threefold increase for type IV collag en (N = 3, P = 0.03) over cells exposed to 5 mM D-glucose controls. Th is increase was not dependent on new gene transcription for either pro tein. Tissue inhibitor of metalloproteinases (TIMP 1 + TIMP 2) were in duced following addition of 25 mM D-glucose, but not when cells were e xposed to 5 mM D-glucose. Twenty-four hours after the addition of 25 m M D-glucose there was an eightfold increase in TIMP 1 (P = 0.009, N = 4), and a tenfold increase in TIMP 2 levels (P = 0.003, N = 4), over t he control values for both inhibitors. The increase in both TIMP 1 and TIMP 2 in response to 25 mM D-glucose was abrogated in a dose depende nt manner by the aldose reductase inhibitor sorbinil. Gelatin-substrat e gel zymography showed increased activity of gelatinase A, but not of gelatinase B in response to the addition of 25 mM D-glucose to HPTC. The induction of gelatinase A was accompanied by increased gelatinase A mRNA expression. which was inhibited both by protein kinase C (PKC) depletion using PMA pre-treatment, and by the addition of a PKC inhibi tor. These data demonstrate that the glucose-induced accumulation of t ype IV collagen and fibronectin is unrelated to increased gene transcr iption, but may involve alterations in the degradative pathway of thes e basement membrane constituents. Furthermore, the data demonstrate th at glucose may simultaneously activate two intracellular pathways (the polyol pathway and a PKC dependent activation pathway), which are inv olved in mediating separate, complementary effects on cell function.