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Abstract: This study investigates whether angiotensin converting enzyme (ACE) inhibitors can block high glucose-induced increase of angiotensin II (Ang II) levels in cultured rat mesangial cells. Incubation of cells with high glucose for 5 days increased Ang II in the cell media (extracellular) and cell lysates (intracellular) by ∼1.5-fold and ∼2.2-fold, respectively. Captopril blocked high glucose-induced increase in extracellular Ang II levels but not in intracellular Ang II levels. Since the inability of captopril to inhibit intracellular Ang II in intact cells could be explained by failure of the drug to enter the cells, further studies were performed using lysates from cells pre-treated with high glucose for 5 days. Addition of captopril to cell lysates was indeed able to inhibit Ang I conversion to Ang II but only partially. These findings suggest the involvement of non-ACE mechanisms in Ang II formation from Ang I in mesangial cells. [Copyright &y& Elsevier]

Accumulation of mesangial matrix in diabetic nephropathy is caused by increased synthesis and decreased degradation. We have previously demonstrated that incubation in high-glucose medium decreases mesangial cell collagenase activity (Diabetes 44:929-935, 1995). Because angiotensin II (AII) is involved in the pathogenesis of diabetic nephropathy, the present studies were performed to determine if AII mediates glucose-induced 1) inhibition of mesangial collagenase activity, 2) mesangial matrix accumulation, and 3) in-crease in transforming growth factor (TGF)-beta1 secretion in mesangial cells. The direct effect of high glucose on AII generation in mesangial cells was also determined. Primary mesangial cells from normal Sprague-Dawley rats were used in all studies. Collagenase activity in cell medium was determined using three methods: 1) zymography; 2) quantitative assay using fluoresceinated gelatin as substrate; and 3) a new enzyme-linked immunosorbent assay (ELISA) that specifically measures 72-kDa collagenase (MMP-2), the principal collagenase synthesized by mesangial cells. Matrix accumulation was estimated by immunoperoxidase assay on cell layers using anti-glomerular basement membrane (GBM) antibodies. TGF-beta1 and AII levels were determined by ELISA. Exposure of mesangial cells to 30 mmol/l glucose (high glucose) vs. 5 mmol/glucose (normal glucose) for 5 days resulted in a significant decrease in collagenase activity (25%) that was normalized by 10(-4) mol/l losartan, a type 1 angiotensin II (AT1) receptor antagonist. High glucose increased anti-GBM binding compared with normal glucose; this effect of glucose was reversed by losartan. Incubation of cells with 30 mmol/l glucose increased total TGF-beta1 secretion, which was also normalized by losartan. Addition of AII (10(-6) mol/l) for 24 h to the culture medium inhibited collagenase activity by 33%; losartan (10(-4) mol/l) blocked this inhibition of enzyme activity. Also, AII decreased collagenase (MMP-2) levels but stimulated TGF-beta1 secretion in mesangial cells. Finally, glucose increased mesangial AII generation in a concentration-dependent manner, with incubation in 30 mmol/l glucose increasing AII by 25% compared with 5 mmol/l glucose. We conclude that glucose increases AII production by mesangial cells, which results in stimulation of TGF-beta1 secretion, decreased matrix degradation, and increased matrix accumulation. These effects of AII are mediated by the AT1 receptor. [ABSTRACT FROM AUTHOR]

Background/Aims: Reactive oxygen species, and especially superoxide (O2·–),have been implicated in diabetic nephropathy. O2·– accumulation in cells is dependent on O2·– production (by NADH/NADPH oxidase) as well as scavenging by superoxide dismutase (SOD) activity. This study was designed to investigate the effects of high glucose (HG) on O2·– accumulation and SOD activity in human mesangial cells (HMC) and to determine if these effects are mediated by angiotensin II (Ang II). Methods: HMC were incubated in media containing 10 mMglucose (control, C), 30 mM glucose (HG), 10 mM glucose + either 20 mM 2-deoxy-D-glucose (2-DG) or 20 mM mannitol (high mannitol, HM) (osmotic controls), or Ang II (10–5M). Ang II action was antagonized by employing 10–4M of Ang II receptor antagonists (losartan or irbesartan) or 10–4M of NADH/NADPH oxidase inhibitors [diphenyleneiodonium chloride (DPI) or apocynin]. Superoxide and total SOD activity were assayed using chemiluminescence of lucigenin. Results: Incubation of HMC in HG resulted in a 1.6-fold increase in Ang I (p < 0.05) and a 1.4-fold increase in Ang II levels (p < 0.05) in cell lysates. These changes were accompanied by a >2-fold increase in O2·– accumulation (p < 0.01), which was inhibited by losartan and irbesartan. Exogenous Ang II increased net O2·– accumulation by 2.7-fold (p < 0.01), which was normalized by losartan and irbesartan. DPI and apocynin blocked the HG and Ang II-induced increases in O2·– (p < 0.01). HG but not exogenous Ang II inhibited total SOD activity by 30%, which was not affected by losartan. Conclusion: High glucose increases O2–· accumulation in HMC primarily by increasing its production via the Ang II-NADH/NADPH oxidase system. Copyright © 2005 S. Karger AG. Basel. [ABSTRACT FROM AUTHOR]

Role of angiotensin II in diabetic nephropathy. Considerable evidence suggests that the intrarenal renin-angiotensin system plays an important role in diabetic nephropathy. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II (Ang II) receptor blockers (ARBs) can attenuate progressive glomerulosclerosis in disease models and can slow disease progression in humans. Because agents that interfere with Ang II action may decrease glomerular injury without altering glomerular pressures, it has been suggested that Ang II has direct effects on glomerular cells to induce sclerosis independent of its hemodynamic actions. To study nonhemodynamic effects of Ang II on matrix metabolism, many investigators have used cell culture systems. Glucose and Ang II have been shown to produce similar effects on renal cells in culture. For instance, incubation of mesangial cells in high-glucose media or in the presence of Ang II stimulates matrix protein synthesis and inhibits degradative enzyme (e.g., collagenase, plasmin) activity. Glucose and Ang II also can inhibit proximal tubule proteinases. Glucose increases expression of the angiotensinogen gene in proximal tubule cells and Ang II production in primary mesangial cell culture, which indicates that high glucose itself can activate the renin-angiotensin system. The effects of glucose and Ang II on mesangial matrix metabolism may be mediated by transforming growth factor-β (TGF-β). Exposure of mesangial cells to glucose or Ang II increases TGF-β expression and secretion. Their effects on matrix metabolism can be blocked by anti-TGF-β antibody or ARBs such as losartan, which also prevents the glucose-induced increment in TGF-β secretion. Taken together, these findings support the hypothesis that the high-glucose milieu of diabetes increases Ang II production by renal, and especially, mesangial cells, which results in stimulation of TGF-β1 secretion, leading to increased synthesis and decreased degradation of matrix proteins, thus producing matrix accumulation. This may be an important mechanism linking hyperglycemia and Ang II in the pathogenesis of diabetic nephropathy. [ABSTRACT FROM AUTHOR]