Changes of ZO-1 Expression in Diabetic Rat Glomeruli and Cultured Mouse Podocyte Under High Glucose Conditions and the Effect of Angiotensin II Type 1 Receptor Blocker

Kim, Beom Seok; Xu, Zhong Gao; Park, Hyeong Cheon; Kang, Shin Wook; Choi, Kyu Hun; Ha, Seung kyu; Lee, Ho Yung; Han, Dae suk

Abstract

BACKGROUND
Diabetic nephropathy (DN) is clinically characterized by persistent proteinuria. The underlying pathologic changes responsible for the nephropathy are the loss of size selective and/or charge selective properties of the glomerular filtration barrier. Size selectivity is maintained primarily by the slit diaphragm and ZO-1 is one of the basic components of it. However, the precise role of the ZO-1 in the pathogenesis of the glomerular diseases is not fully understood. We investigated the changes of ZO-1 expression in diabetic glomeruli in vivo, and by high glucose in cultured podocyte in vitro. We also evaluated the effect of angiotensin II type 1 receptor blocker (ARB) on the ZO-1 changes induced by diabetes or high glucose. METHODS: To determine the effect of ARB on podocytes ZO-1 protein and mRNA expression, immortalized mouse podocytes were incubated with RPMI medium containing normal glucose (NG, 5.6 mM) or high glucose (HG, 30 mM) with or without ARB (10-6 M, L-158, 809). For animal studies, rats were injected with diluent (Control, C, n=18) or streptozotocin. The latter were left untreated (DM, n=18) or treated with 1 mg/kg/day ARB (DM+ARB, n=18). Six rats from each group were sacrificed monthly, and Western blot and RT?PCR were performed for ZO-1 with sieved glomeruli. Renal sections were stained for ZO-1 by immunohistochemistry. RESULTS: The ZO-1 mRNA and protein expressions in podocytes exposed to HG conditions were significantly higher than those in podocytes exposed to NG media (p<0.05). ARB treatment inhibited the HG induced increase in ZO-1 mRNA and protein expression by 73% and 64%, respectively (p<0.05). Compared to the C rats (19.8+/-3.2 mg/day), 24 hour urinary protein excretion at 3 month was significantly higher in the DM rats (90.6+/-11.3 mg/day, p< 0.05), and ARB treatment partly reversed the increase in proteinuria in DM rats (51.6+/-6.6 mg/day, p<0.05). Glomerular ZO-1 mRNA and protein expressions were also significantly increased in DM than corresponding C at all duration (p<0.05). ARB treatment for 3 months in DM rats inhibited the increase in ZO-1 mRNA and protein expression by 57.5% and 70.6%, respectively (p<0.05). ARB treatment for 3 months significantly ameliorated increased glomerular ZO-1 expression in DM rats as assessed by immunohistochemistry. CONCLUSION: In conclusion, ZO-1 mRNA and protein expressions were increased in podocytes exposed to HG and in DM glomeruli, and this increment in ZO-1 expression was ameliorated with ARB. Taken together, these data suggest that change of ZO-1 expression in podocytes is implicated in the early changes of diabetic nephropathy and may contribute to the development of proteinuria.