Renal proximal tubular cell fibronectin accumulation in response to glucose is polyol pathway dependent

Background Thickening and reduplication of the tubular basement membrane ha ve been reported as early events in diabetic nephropathy. In this study, we have examined the polar requirements of proximal tubular cells for the D-g lucose-stimulated accumulation of fibronectin and the mechanism by which th is occurred, with particular emphasis on the polyol pathway. Methods. To determine the polarity of fibronectin generation in response to glucose, LLC-PK1 cells were grown on porous tissue culture inserts. Monola yer confluence was determined by serial measurement of transepithelial resi stance. Confluent cells were growth arrested by serum deprivation, and all experiments were performed under serum-free conditions. Results. Application of 25 mM D-glucose to either the apical or basolateral aspect of LLC-PK1 cells led to fibronectin accumulation in the basolateral compartment. This reached statistical significance 24 hours following apic al addition of glucose (2.6-fold increase compared with 5 mM D-glucose, P = 0.0025, N = 6 vs. N = 4 controls) and 12 hours after the basolateral addit ion of glucose (2.5-fold increase compared with 5 mM D-glucose, P = 0.03, N = 6 vs. N = 4 controls). Exposure of cells to glucose at either their apic al or basolateral aspect leads to accumulation of intracellular glucose and polyol pathway activation, as assessed by sorbitol accumulation. The incre ase in fibronectin concentration in response to glucose was inhibited by th e aldose reductase inhibitor sorbinil. At a dose of 100 mu M sorbinil, ther e was a 59% inhibition of fibronectin accumulation in response to apical gl ucose (P = 0.004, N = 3 sorbinil vs. N = 4 controls) and a 66% inhibition i n response to basolateral glucose (P = 0.008, N = 3 sorbinil vs. N = 4 cont rols) 48 hours after the addition of the inhibitor. Furthermore, fibronecti n accumulation was also demonstrated following both the apical and basolate ral addition of 1 mM sorbitol, but not following the addition of 25 mM gala ctose to either aspect of the cells. Following the addition of sorbitol, th ere was a 2.8-fold increase in fibronectin 48 hours after apical stimulatio n (P = 0.01, N = 3 treated vs. N = 4 control) and a 2.27-fold increase foll owing basolateral stimulation (P = 0.04, N = 3 treated vs. N = 4 control) a t 24 hours. Conclusions. In summary, these data demonstrate that fibronectin generation in response to glucose was nonpolar in terms of application of glucose but was polar in terms of fibronectin accumulation. The mechanisms of glucose- induced modulation of fibronectin were mediated by polyol pathway activatio n and were more specifically related to the metabolism of sorbitol to fruct ose.