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

of the earliest renal pathological changes in diabetes is Background. Thickening and reduplication of the tubular basement an increase in tubular basement membrane mass that membrane have been reported as early events in diabetic nephropathy. In this study, we have examined the polar requirements of proximal accompanies the development of renal hypertrophy [2]. tubular cells for the d-glucose‐stimulated accumulation of fibronectin These changes are strongly correlated with glycemic conand the mechanism by which this occurred, with particular emphasis trol and occur independently of the pathological changes on the polyol pathway. Methods. To determine the polarity of fibronectin generation in associated with progressive diabetic nephropathy [3]. It response to glucose, LLC-PK1 cells were grown on porous tissue culture suggests that alterations in renal structure may occur inserts. Monolayer confluence was determined by serial measurement of transepithelial resistance. Confluent cells were growth arrested by that are not specific to nephropathy but reflect a conseserum deprivation, and all experiments were performed under serum- quence of long-standing diabetes/hyperglycemia. We free conditions. and others have demonstrated that in vitro exposure of Results. Application of 25 m md -glucose to either the apical or basolateral aspect of LLC-PK1 cells led to fibronectin accumulation in human renal proximal tubular cells to 25 m md -glucose the basolateral compartment. This reached statistical significance 24 increased generation of type IV collagen and fibronectin hours following apical addition of glucose (2.6-fold increase compared [4‐6]. Our observations suggested that the accumulation with 5 m md -glucose, P 5 0.0025, N 5 6 vs. N 5 4 controls) and 12 hours after the basolateral addition of glucose (2.5-fold increase of these basement membrane constituents was related compared with 5 m md -glucose, P 5 0.03, N 5 6 vs. N 5 4 controls). to alterations in matrix degradation [5]. Exposure of cells to glucose at either their apical or basolateral aspect leads to accumulation of intracellular glucose and polyol pathway acti- In diabetes, proximal tubular cells may be exposed to vation, as assessed by sorbitol accumulation. The increase in fibronectin conditions of elevated glucose either apically as a result concentration in response to glucose was inhibited by the aldose reducof glycosuria or basely as a result of elevated interstitial tase inhibitor sorbinil. At a dose of 100 mm sorbinil, there was a 59% inhibition of fibronectin accumulation in response to apical glucose (P 5 tissue concentrations of glucose. Our studies on the mech0.004, N 5 3 sorbinil vs. N 5 4 controls) and a 66% inhibition in anism of glucose induction of transforming growth factor response to basolateral glucose (P 5 0.008, N 5 3 sorbinil vs. N 5 4 controls) 48 hours after the addition of the inhibitor. Furthermore, b1 (TGF-b1) in proximal tubular cells demonstrated that fibronectin accumulation was also demonstrated following both the induction of TGF-b1 mRNA was a polar phenomenon, apical and basolateral addition of 1 mm sorbitol, but not following the occurring only following the addition of glucose to the addition of 25 mm galactose to either aspect of the cells. Following the addition of sorbitol, there was a 2.8-fold increase in fibronectin 48 basolateral aspect of the cells [7]. hours after apical stimulation (P 5 0.01, N 5 3 treated vs. N 5 4 In this study, we have examined the polar requirements control) and a 2.27-fold increase following basolateral stimulation (P 5 0.04, N 5 3 treated vs. N 5 4 control) at 24 hours. of proximal tubular cells for the d-glucose‐stimulated Conclusions. In summary, these data demonstrate that fibronectin accumulation of fibronectin using the porcine LLC-PK1 generation in response to glucose was nonpolar in terms of application tubular cell line, grown in polarized culture on tissue of glucose but was polar in terms of fibronectin accumulation. The mechanisms of glucose-induced modulation of fibronectin were medi- culture inserts. We also examined the mechanism by ated by polyol pathway activation and were more specifically related which glucose led to the accumulation of fibronectin in to the metabolism of sorbitol to fructose.