Angiotensin II type 1 receptor blocker attenuates the activation of ERK and NADPH oxidase by mechanical strain in mesangial cells in the absence of angiotensin II

Abstract

It has been reported that mechanical strain activates extracellular signal-regulated protein kinases (ERK) without the involvement of angiotensin II (Ang II) in cardiomyocytes. We examined the effects of mechanical strain on ERK phosphorylation levels in the absence of Ang II using rat mesangial cells. The ratio of phosphorylated ERK (p-ERK) to total ERK expression was increased by cyclic mechanical strain in a time- and elongation strength-dependent manner. With olmesartan [Ang II type 1 receptor (AT1R) antagonist] pretreatment, p-ERK plateau levels decreased in a dose-dependent manner (EC(50) = 1.3 x 10(-8) M, maximal inhibition 50.6 +/- 11.0% at 10(-5) M); a similar effect was observed with RNA interference against Ang II type 1A receptor (AT(1A)R) and Tempol, a superoxide dismutase mimetic. In addition to the inhibition of p-ERK levels, olmesartan blocked the increase in cell surface and phosphorylated p47(phox) induced by mechanical strain and also lowered the mRNA expression levels of NADPH oxidase subunits. These results demonstrate that mechanical strain stimulates AT1R to phosphorylate ERK in mesangial cells in the absence of Ang II. This mechanotransduction mechanism is involved in the oxidative stress caused by NADPH oxidase and is blocked by olmesartan. The inverse agonistic activity of this AT1R blocker may be useful for the prevention of mesangial proliferation and renal damage caused by mechanical strain/oxidative stress regardless of circulating or tissue Ang II levels.

Fig. 1.

Mechanical strain and angiotensin II (Ang II) induce the activation of ERK in a time-dependent and elongation strength (dose)-dependent manner. Phosphorylation (p) levels of ERK were expressed as % change from the control. A: increase in p-ERK by stretch was time dependent; maximal response was obtained at 5 min after the initiation of stretch. B: increase in p-ERK by stretch was elongation strength dependent; maximal response was obtained with 20% elongation. The increase in p-ERK by Ang II was time dependent (C) and concentration dependent (D). Ang II concentration of 10⁻¹⁰ M was used for the time-course study, and an incubation time of 10 min was used for the dose-response study. The maximal response was obtained with 10⁻¹⁰ M of Ang II at an incubation time of 10 min. Means ± SE (n = 4) are given. Ctl, control.

Fig. 2.

Effect of mechanical strain and olmesartan on the phosphorylation levels of ERK. A: concentration-dependent effect of olmesartan (Olm) on mechanical strain-induced activation of ERK. Phosphorylated ERK levels were expressed as % change from the control. *P < 0.01 vs. control. Means ± SE (n = 5) are given. p-ERK levels at the plateau phase (□) were reduced in a concentration-dependent manner (EC₅₀ = 1.3 × 10⁻⁸ M, maximal inhibition 50.6 ± 11.0% at 10⁻⁵ M) with olmesartan treatment. In contrast, p-ERK levels at early phase (•) were not attenuated by pretreatment with olmesartan. B: olmesartan as well as losartan (10⁻⁵ M) did not change basal p-ERK levels but attenuated the elevated levels of p-ERK induced by cyclic mechanical strain at plateau phase. In contrast to olmesartan, losartan did not have any inhibitory effect on the stretch-induced ERK activation at the plateau phase of 60 min. Means ± SE (n = 5) are given. n.s., not significant.

Fig. 3.

Effect of micro RNA (miRNA, miR) against Ang II type 1 receptor (AT1R) on ERK phosphorylation induced by mechanical strain. A: transfection with miRNA significantly decreased the basal expression levels of AT1R (76.8 ± 6.8% reduction). B: miRNA treatment significantly attenuated the stretch-induced elevation of phosphorylation levels of ERK at plateau phase (53.5 ± 9.7% reduction). Means ± SE (n = 3) are given. $P < 0.01 vs. mock control, *P < 0.01 vs. mock + stretch. C: AT1R silencing did not have any inhibitory effect on ERK phosphorylation at initial peak 5 min after stretch application (1,304.38 ± 93.8% increasing of p-ERK in mock group vs. 1,284.98 ± 111.3% in miR group, n = 4). AT_1AR, Ang II type 1A receptor.

Fig. 4.

The effect of Tempol on plateau-phase ERK phosphorylation induced by mechanical strain (Str). Phosphorylation levels of ERK were expressed as % of the control. Similar to the result obtained with olmesartan pretreatment, Tempol dose dependently lowered ERK activity at plateau phase induced by mechanical strain. Maximal inhibition was observed with 10⁻⁵ M of Tempol (Tem). Means ± SE (n = 3) are given. $P < 0.01 from control; *P < 0.01 vs. control.

Fig. 5.

Olmesartan attenuates the membrane translocation and phosphorylation of p47^phox by stretch (Str). Abundance of p47^phox on cell surface membranes (A) and phosphorylation levels of p47^phox (B) are shown. Olmesartan (10⁻⁵ M) significantly decreased membrane translocation and phosphorylation of p47^phox. Means ± SE (n = 4) are given. *P < 0.05 vs. control; $P < 0.05 vs. stretch group. IP, immunoprecipitate.

Fig. 6.

Changes in the mRNA expression levels of NADPH oxidase subunits p47^phox, p22^phox, and p67^phox. Quantification of mRNA was determined on the basis of the C_t value, normalized to β-actin, and expressed as the magnitude of change under mechanical strain application relative to the corresponding control. Means ± SE (n = 3) are given. &P < 0.01 vs. control; *P < 0.01 vs. stretch group.

Fig. 7.

Effect of BAPTA and cytochalasin D on stretch-induced ERK phosphorylation. Phosphorylation levels of ERK were expressed as % change from control. The early-phase (5 min) activation of ERK induced by cyclic mechanical strain was abolished almost completely by preincubation with either 1 μM of BAPTA-TM or 1 μM of cytochalasin D. In contrast, the phosphorylation of ERK at the plateau phase (60 min) by mechanical strain was still observed with BAPTA-TM or cytochalasin D (BAPTA-TM; 273.7 ± 20.5%, cytochalasin D; 208.5 ± 19.8% compared with no stretch control). Means ± SE (n = 4) are given. *P < 0.01 vs. control.