Abstract 234: AGEs Enhance the Metabolism of Angiotensin-(1-7) and Lower Intracellular Peptide Expression

Abstract

Advancedglycated end products (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may be important mediators of diabetic pathologies. An accompanying abstract described AGE-dependent cellular hypertrophy and myofibroblast transition (MT) in NRK-52E renal epithelial cells that was abolished by treatment with the alternative angiotensin (Ang) product Ang-(1-7); the inhibitory effect of Ang-(1-7) was likely mediated by attenuation of ERK and TGF-β stimulated pathways. AGEs evoke multiple signaling events, but the impact on the Ang-(1-7) system is unknown. The present study addressed whether AGEs suppress the Ang-(1-7) axis, and thus facilitate their deleterious actions. MGA exposure for 48 hrs significantly reduced the cellular expression of Ang-(1-7) [396 ± 59 vs. 219 ± 54 fmol/mg protein; p<0.05] in the NRK-52E cells; however, the cellular content of Ang II and Ang I was not changed. We then assessed the processing pathways of Ang I to Ang-(1-7) in the 100,000 xg soluble fraction, as well as the activity secreted extracellularly in the media. Conversion of Ang I to Ang-(1-7) was also not changed following MGA exposure in the cellular supernatant [56 ± 7 vs. 66 ± 8 fmol/min/mg protein]. In contrast, the metabolism of Ang-(1-7) to Ang-(1-4) was significantly enhanced in the soluble fraction of the MGA exposed cells [175 ± 9 vs. 115 ± 11 fmol/min/mg protein, p<0.05]. The Ang-(1-7) endopeptidase activity was abolished by the thiol inhibitor PCMB, but was insensitive to the metallopeptidase inhibitor JMV390. AGE slightly but significantly induced the metabolism of Ang-(1-7) to Ang-(1-4) in the cell media [1.36 ± 0.01 vs. 1.42 ± 0.01 fmol/min/ml, p<0.05]. In this case, the secreted endopeptidase exhibited marked sensitivity to JMV390 [IC50 = 7.6 nM]. Finally, chronic treatment with the ERK1/2 inhibitor PD98059 abrogated the increase in Ang-(1-4), and suggests that downregulation of the peptidase is linked to AGE-induced MT. We conclude that the cellular actions of AGE may reflect a loss of Ang-(1-7) tone through enhanced metabolism of the peptide. Moreover, we identify two distinct endopeptidase activities that metabolize Ang-(1-7) to Ang-(1-4) and both are increased following AGE exposure.