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 h 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-Ala(7)-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.

Keywords: Advanced glycation endproducts; Angiotensin; EMT; ERK1/2; TGF-B.

Figure 1. MGA exposure reduces intracellular levels of Ang-(1-7) in the in the NRK-52E cells

Panel A: Ang I peptide levels are not different between control and MGA treated cells. Panel B: There are no difference in Ang II peptide levels between MGA treated cells and control cells. Panel C: Ang-(1-7) peptide levels were significantly lower in the MGA treated cells than control cells. Intracellular peptide content was quantified by separate RIAs to Ang I, Ang II and Ang-(1-7). Data are the mean ± SEM from 3 different cell passages. *P< 0.05.

Figure 2. MGA exposure increases Ang-(1-7) metabolism in the NRK-52E cells

Peptide metabolism was assessed in the 100,000 × g supernatant fraction from control and MGA-treated (48 hours) NRK-52E cells for 30 minutes at 37°C. Peptide products were determined by HPLC separation with isocratic elution conditions (38). Panel A: Ang-(1-7) conversion to Ang-(1-4) in control cells. Panel B: Ang-(1-7) conversion to Ang-(1-4) in MGA-treated cells. Panel C: Quantification of Ang-(1-4) generation from Ang-(1-7) revealed a higher rate of Ang-(1-7) conversion with MGA exposure. Data are the mean ± SEM of 3 different cell passages. *P< 0.05 vs. control.

Figure 3. MGA exposure does not influence Ang I to Ang-(1-7) formation in the NRK-52E cells

Peptide metabolism was assessed in the 100,000 × g supernatant fraction from control and MGA-treated (48 hours) NRK-52E cells for 30 minutes at 37°C. Peptide products were determined by HPLC separation under gradient conditions (1). Panel A: Ang I conversion to Ang-(1-7) in control cells. Panel B: Ang I conversion to Ang-(1-7) in MGA treated cells. Panel C: Quantification of Ang-(1-7) generation from Ang I revealed no differences between control and MGA treated cells. Data are the mean ± SEM of 3 different cell passages.

Figure 4. Ang-(1-7) attenuates MGA-induced cell hypertrophy in the NRK-52E cells

Left: MGA treatment for 48 hours significantly increased ³H-leucine incorporation approximately 180% of control. Ang-(1-7) (A7, 100 nM) significantly reduced the extent of hypertrophy induced by MGA. The Ang-(1-7) receptor antagonist D-Ala⁷-Ang-(1-7) (DAL, 10 μM), reversed the inhibitory effects of Ang-(1-7) on MGA-induced hypertrophy. The ERK 1/2 inhibitor PD98059 (PD, 1 μM), abolished the cellular hypertrophy induced by MGA. Ang-(1-7) and PD98059 exhibit no additive effects on hypertrophy. The DAL antagonist alone or the AT1 antagonist losartan (LOS) alone did not influence the MGA-dependent hypertrophy. ³H-leucine incorporation is expressed as percentage (%) from control cells for each experiment. Data are the mean ± SEM from 5 different cell passages. Inset: MGA treatment for 48 hours increased cell area as measured in immunofluorescent images. Ang-(1-7) (A7, 100 nM) and PD98059 (PD, 1 μM), reversed the increase in cell area induced by MGA.*P < 0.05 vs. control, #P < 0.05 vs. MGA-treated cells.

Figure 5. Ang-(1-7) appears to inhibit MGA-induced fluorescent staining for de novo expression of α-SMA in NRK 52-E cells

Cells were incubated with serum free media (A and C) or MGA (100 μM, B, D-I) or TGF-β (5 ng, J) for 72 hrs. MGA-treated cells were treated with either Ang-(1-7) (100 nM, E), Ang-(1-7) and DAL (10 μM, F), ERK1/2 inhibitor PD98059 (1 μM, G), TGF-β receptor kinase inhibitor SB525334 (1 μM, H) or AT1 receptor antagonist losartan (1 μM, I). The immunofluorescent images are representative of 3 different cell passages.

Figure 6. Ang-(1-7) inhibits MGA-induced protein expression of expression of α-SMA in NRK 52-E cells

Western blot analysis of MGA (100 μM) induced α-SMA expression after 48 hours. Ang-(1-7) (A7, 100 nM) attenuated the increase in α-SMA, which was blocked by the Mas receptor antagonist D-Ala⁷-Ang-(1-7) (DAL, 10 μM). Both the ERK1/2 inhibitor PD98059 (PD, 1 μM) and TGF-β receptor kinase inhibitor SB525334 (SB, 1 μM) abolished the increase in α-SMA expression. The AT₁ antagonist losartan (LOS, 1 μM) did not influence the α-SMA expression. Data are the mean ± SEM from 3-4 different cell passages. *P < 0.05 vs. control, #P < 0.05 vs. MGA.

Figure 7. Ang-(1-7) does not influence MGA-induced release of TGF-β in NRK-52E cells

Cells were exposed to MGA for 48 hours and TGF-β in the cell media determined by ELISA. MGA exposure increased TGF-β release approximately 3-fold. There were no significant effects on MGA-induced TGF-β release by Ang-(1-7) (A7, 100 nM), D-Ala⁷-Ang-(1-7) (DAL, 10 μM), or the AT₁ receptor antagonist losartan (LOS, 1 μM). TGF-β release was not significantly different between the ERK1/2 inhibitor PD98059 (PD, 1 μM) or combined Ang-(1-7) and PD (A7/PD) treatment to control. Data are the mean ± SEM from 4-5 different cell passages. *P < 0.05 vs. control.

Figure 8. Ang-(1-7) inhibits MGA-induced phosphorylation of ERK 1/2 in NRK 52-E cells

Western blot analysis of ERK1/2 with MGA treatment for 48 hours. MGA increased the phosphorylation of ERK1/2. Ang-(1-7) attenuated that MGA-induced phosphorylation which was blocked by D-Ala⁷-Ang-(1-7) (DAL, 10 μM). Inhibitors to TGF-β₁ receptor kinase (SB, 1 μM) and ERK1/2 (PD, 1 μM) abolished ERK1/2 phosphorylation. The AT₁ receptor antagonist losartan (LOS, 1 μM) did not inhibit the MGA-induced ERK1/2 activation. Data are the mean ± SEM from 3 different cell passages. *P < 0.05 vs. control, #P < 0.05 vs. MGA.

Figure 9. Ang-(1-7) inhibits TGF-β induced phosphorylation of ERK 1/2 in NRK-52E cells

Western blot analysis of TGF-β-induced ERK1/2 phosphorylation. TGF-β (5 ng/ml) stimulated ERK1/2 phosphorylation after a 15 min incubation. Ang-(1-7) (A7, 100 nM) pretreatment inhibited TGF-β induced ERK1/2 phosphorylation. The inhibitory effect of Ang-(1-7) was reversed by the antagonist D-Ala⁷-Ang-(1-7) (DAL, 10 μM). Pretreatment with TGF-β receptor kinase inhibitor SB525334 (SB, 1 μM) abolished ERK1/2 phosphorylation and there was no additive effect of Ang-(1-7) and SB. Data are the mean ± SEM from 3 different cell passages. *P < 0.05 vs. control, #P < 0.05 vs. MGA treated cells.