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Regular Manuscript

ACE- and chymase-dependent angiotensin II generation in normal and glucose-stimulated human mesangial cells

¹ Federal University of São Paulo

^* To whom correspondence should be addressed. E-mail: mirian{at}nefro.epm.br.

	Abstract

High glucose increases AngII generation in mesangial cells (MC). Chymase, an alternative AngII-generating enzyme, is upregulated in the glomeruli of diabetic kidneys. In this study, we examined AngII synthesis by human MC (HMC) via ACE- and chymase-dependent pathways under normal (NG, 5 mM) and high (HG, 30 mM) glucose conditions. NG cells expressed ACE and chymase mRNA. Under NG the chymase inhibitor chymostatin reduced AngII levels in cell lysates and in the culture medium, and the ACE inhibitor captopril had no effect. HG induced a three-fold increase in chymase mRNA, but not in ACE mRNA. However, HG induced a 10-fold increase in intracellular ACE activity. The increase in AngII generation induced by HG was found in the cell lysate, but not in the culture medium. The rise in intracellular AngII was not prevented by captopril or by chymostatin. Moreover, captopril inhibited the extracellular, but failed to block intracellular ACE activity, suggesting that captopril was unable to reach intracellular ACE. Losartan did not change the intracellular AngII content either in NG or HG conditions, suggesting that the increase in AngII was due to intracellular generation. All together, these results suggest that chymase may be active in HMC and that both enzymes are involved in increased AngII generation during the HG stimulus by different mechanisms including an upregulation of chymase mRNA and a rise in the intracellular ACE activity, favoring the generation and accumulation of intracellular AngII.

Key Words: diabetic nephropathy, angiotensin II, angiotensin converting enzyme, chymase, captopril, chymostatin