Angiotensin-(1–7) abolishes AGE-induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2

Abstract

Angiotensin-(1-7) (Ang-(1-7)/AT7-Mas receptor axis is an alternative pathway within the renin angiotensin system (RAS) that generally opposes the actions of Ang II/AT1 receptor pathway. Advanced glycated end product (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may contribute to the development and progression of diabetic nephropathy in part through activation of the Ang II/AT1 receptor system; however, the influence of AGE on the Ang-(1-7) arm of the RAS within the kidney is unclear. The present study assessed the impact of AGE on the Ang-(1-7) axis in NRK-52E renal epithelial cells. MGA exposure for 48 hours significantly reduced the intracellular levels of Ang-(1-7) approximately 50%; however, Ang I or Ang II expression was not altered. The reduced cellular content of Ang-(1-7) was associated with increased metabolism of the peptide to the inactive metabolite Ang-(1-4) [MGA: 175 ± 9 vs. Control: 115 ± 11 fmol/min/mg protein, p<0.05, n=3] but no change in the processing of Ang I to Ang-(1-7). Treatment with Ang-(1-7) reversed MGA-induced cellular hypertrophy and myofibroblast transition evidenced by reduced immunostaining and protein expression of α-smooth muscle actin (α-SMA) [0.4±0.1 vs. 1.0±0.1, respectively, n=3, p<0.05]. Ang-(1-7) abolished AGE-induced activation of the MAP kinase ERK1/2 to a similar extent as the TGF-β receptor kinase inhibitor SB58059; however, Ang-(1-7) did not attenuate the MGA-stimulated release of TGF-β. The AT7-Mas receptor antagonist D-Ala7-Ang-(1-7) abolished the inhibitory actions of Ang-(1-7). In contrast, AT1 receptor antagonist losartan did not attenuate the MGA-induced effects. We conclude that Ang-(1-7) may provide an additional therapeutic approach to the conventional RAS blockade regimen to attenuate AGE-dependent renal injury.

Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute?

“…ANG II content was determined by a direct Peninsula ELISA, and the relative peptide expression in control and high glucoseexposed cardiomyocytes was confirmed by antibody capture and HPLC-MS (132). Finally, we assessed the effect of advanced glycation end products on angiotensin expression in renal NRK-52E cells (9). Basal cellular content was quantified by direct RIAs for ANG I (85 fmol/mg protein; Peninsula), ANG II (150 fmol/mg protein; ALPCO), and ANG-(1-7) (400 fmol/mg protein; custom RIA) (9).…”

“…Danser and colleagues (113,139) suggest that urinary renin may be an alternative measure of an active RAS within the kidney. Quenched fluorescent substrates based on the ANG- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) sequence are available to measure renin. Although this approach does not rely on the detection of ANG I or an exogenous source of angiotensinogen, the sensitivity and specificity of the peptide substrates are not comparable with angiotensinogen.…”

“…Finally, we assessed the effect of advanced glycation end products on angiotensin expression in renal NRK-52E cells (9). Basal cellular content was quantified by direct RIAs for ANG I (85 fmol/mg protein; Peninsula), ANG II (150 fmol/mg protein; ALPCO), and ANG-(1-7) (400 fmol/mg protein; custom RIA) (9). Advanced glycation end product exposure significantly reduced ANG-(1-7) and increased metabolism of the peptide that was associated with myofibroblast transition but did not impact ANG II or ANG I levels (9).…”

“…In lieu of these approaches, the NH 2 -terminally intact forms of ANG II, ANG-(1-7), and ANG I are the predominant peptides identified in plasma and various tissues. Quantitation of the cell and urinary data reflects direct RIA-or ELISA-based methods (9,34,38,45,50,70,77,115,131,133,139,141,148).…”

“…Basal cellular content was quantified by direct RIAs for ANG I (85 fmol/mg protein; Peninsula), ANG II (150 fmol/mg protein; ALPCO), and ANG-(1-7) (400 fmol/mg protein; custom RIA) (9). Advanced glycation end product exposure significantly reduced ANG-(1-7) and increased metabolism of the peptide that was associated with myofibroblast transition but did not impact ANG II or ANG I levels (9). As these cells express an intracellular RAS, nuclear content of ANG II and ANG-(1-7) was quantified at ϳ60 fmol/mg protein consistent with nuclear immunostaining for both peptides (10).…”