Proximal tubule angiotensin AT2 receptors mediate an anti-inflammatory response via interleukin-10: role in renoprotection in obese rats

Abstract

The angiotensin type 2 receptor (AT2R) has been shown to lower inflammation in the kidney. However, the role of the anti-inflammatory cytokine interleukin (IL)-10 in AT2R-mediated attenuation of inflammation has not been elucidated. We hypothesized that AT2R activation is renoprotective by directly increasing the levels of anti-inflammatory cytokine IL-10 in the kidney via nitric oxide (NO) signaling. For in vitro studies, the human proximal tubule epithelial cell-line (human kidney-2 [HK-2]) was activated with lipopolysaccharide (10 μg/mL) and AT2R agonist C21 (1 μmol/L) for 24 hours, and media cytokine levels were assessed. Lipopolysaccharide modestly downregulated AT2R expression. Treatment with C21 lowered lipopolysaccharide-induced levels of both tumor necrosis factor-α and IL-6, but increased IL-10 levels. Treatment with neutralizing IL-10 antibody (1 μg/mL) or NO synthase inhibitor L-NAME (1 mmol/L) abolished this effect. For in vivo studies, prehypertensive obese Zucker rats and age-matched lean Zucker rats were treated for 2 weeks with C21 (300 μg/kg per day, IP) and AT2R antagonist (PD123319; 50 μg/kg per minute, SC infusion). Compared with lean Zucker rats, obese Zucker rats had higher levels of renal AT2R expression, tumor necrosis factor-α, and IL-6. C21 treatment decreased levels of tumor necrosis factor-α by 75% and IL-6 by 60%. Conversely, PD treatment lowered the renal IL-10 levels in obese Zucker rats by ≈60%. Renal morphometry revealed increased mesangial matrix expansion and glomerular macrophage infiltration, which was improved by C21 treatment in obese Zucker rats. Our findings suggest that proximal tubule AT2R activation is anti-inflammatory by increasing IL-10 production, which is largely NO dependent and thus offers renoprotection by preventing early inflammation-induced renal injury in obesity.

Keywords: AT2 Receptor; C21; anti-inflammatory; interleukin-10; nitric oxide; renoprotection.

Fig. 1

Concentration of (A) tumor necrosis factor-α (TNF- α), (B) interleukin-6 (IL-6) and (C) interleukin-10 (IL-10) in the media collected from HK-2 proximal tubule epithelial cells after activation with lipopolysaccharide (LPS, 10μg/ml) and/or AT₂R agonist (C21, 1μmol/L) for 24 hours. Cytokine concentrations in media were measured by ELISA. Data are represented as mean ± SEM. * indicates p<0.05 vs control, # indicates p<0.05 vs LPS treated and $ indicates p<0.05 vs C21 treated HK-2 cells (n=7).

Fig. 2

Effect of increasing concentrations of neutralizing interleukin-10 (IL-10) antibody (0.25, 0.5, 1, 2.5 μg/ml) on the concentration of (A) tumor necrosis factor-α (TNF- α) and (B) interleukin-6 (IL-6) in the media collected from HK-2 proximal tubule epithelial cells after activation with lipopolysaccharide (LPS, 10μg/ml) and/or AT₂R agonist (C21, 1μmol/L) for 24 hours. Cytokine concentrations in media were measured by ELISA. Data are represented as mean ± SEM. * indicates p<0.05 vs control, # indicates p<0.05 vs LPS treated and $ indicates p<0.05 vs LPS+C21 treated HK-2 cells (n=7).

Fig. 3

Effect of nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on the concentration of (A) interleukin-6 (IL-6) and (B) interleukin-10 (IL-10) in the media collected from HK-2 proximal tubule epithelial cells after activation with lipopolysaccharide (LPS, 10μg/ml) and/or AT₂R agonist (C21, 1μmol/L) for 24 hours. Cytokine concentrations in media were measured by ELISA. Data are represented as mean ± SEM. * indicates p<0.05 vs respective control (A), # indicates p<0.05 vs LPS+C21 (B) treated HK-2 cells (n=5). ND indicates not detected.

Fig.4

Expression of angiotensin (A) AT₁ (AT₁R) and (B) AT₂ receptors (AT₂R) in the kidney cortex of lean and prehypertensive obese Zucker rats after 2 weeks treatment with vehicle (Veh), AT₂R antagonist PD123319 (PD), AT₂R agonist (C21) and both PD123319+C21 (PD+C21). The upper panels show representative western blots for AT₁R, AT₂R and β-actin. Lower panels represent quantitative results normalized to β-actin. Data are mean ± SEM. * indicates p<0.05 vs lean vehicle treated rats (n=5-6).

Fig. 5

Pro-inflammatory cytokine expression in the plasma (A,B) and concentration in the kidney cortex (C,D) of lean and pre-hypertensive obese Zucker rats after 2 weeks treatment with vehicle (Veh), AT₂R antagonist PD123319 (PD), AT₂R agonist (C21) and both PD123319+C21 (PD+C21). (A) Tumor necrosis factor- α (TNF- α) and (B) Interleukin-6 (IL-6) expression in the plasma normalized with total plasma protein stained with amido black. Top panels show representative western blots for TNF- α and IL-6 (C) TNF-α and (D) IL-6 levels in the kidney normalized to total protein. Data are mean ± SEM. * indicates p<0.05 vs lean vehicle treated, # indicates p<0.05 vs obese vehicle treated and $ indicates p<0.05 vs obese C21 treated rats (n=6).

Fig. 6

Anti-inflammatory cytokine interleukin-10 (IL-10) expression in the plasma (A) and concentration in the kidney cortex (B) of lean and pre-hypertensive obese Zucker rats after 2 weeks treatment with vehicle (Veh), AT₂R antagonist PD123319 (PD), AT₂R agonist (C21) and both PD123319+C21 (PD+C21). (A) IL-10 expression in the plasma was normalized with total plasma protein stained with amido black. Top panels show representative western blots for IL-10. (B) IL-10 levels in the kidney normalized to total protein. Data are mean ± SEM. * indicates p<0.05 vs lean vehicle treated, # indicates p<0.05 vs obese vehicle treated and $ indicates p<0.05 vs obese C21 treated rats (n=6).

Fig. 7

Renal morphometry (A) Periodic Acid Schiff (PAS) stained sections from the kidney cortex of lean (L, upper panel) and obese (O, lower panel) Zucker rats after 2 weeks treatment with vehicle (V), AT₂R antagonist PD123319 (PD), AT₂R agonist (C21) and both PD123319+C21 (PD+C21). (B) Semi-quantitative mesangial matrix expression (MME) score from PAS stained sections. An average MME score was obtained from analysis of 30 glomeruli per rat. Data are mean ± SEM (n=6). * indicates p<0.05 vs lean vehicle treated, # indicates p<0.05 vs obese vehicle treated rats (400x magnification).

Fig. 8

Monocyte/macrophage infiltration in glomeruli (A) CD68 immunostained sections from the kidney cortex of lean (L, upper panel) and obese (O, lower panel) Zucker rats after 2 weeks treatment with vehicle (V), AT₂R antagonist PD123319 (PD), AT₂R agonist (C21) and both PD123319+C21 (PD+C21). (B) Quantitative measurement of CD68+ monocyte/macrophages from immunostained sections. An average number of macrophage infiltration was obtained from analysis of 30 glomeruli per rat. Data are mean ± SEM (n=6). * indicates p<0.05 vs lean vehicle treated, # indicates p<0.05 vs obese vehicle treated rats (400x magnification).