Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model

Abstract

Renal artery stenosis (RAS) is an important cause of chronic renal dysfunction. Recent studies have underscored a critical role for CCL2 (MCP-1)-mediated inflammation in the progression of chronic renal damage in RAS and other chronic renal diseases. In vitro studies have implicated p38 MAPK as a critical intermediate for the production of CCL2. However, a potential role of p38 signaling in the development and progression of chronic renal disease in RAS has not been previously defined. We sought to test the hypothesis that inhibition of p38 MAPK ameliorates chronic renal injury in mice with RAS. We established a murine RAS model by placing a cuff on the right renal artery and treated mice with the p38 inhibitor SB203580 or vehicle for 2 wk. In mice treated with vehicle, the cuffed kidney developed interstitial fibrosis, tubular atrophy, and interstitial inflammation. In mice treated with SB203580, the RAS-induced renal atrophy was reduced (70% vs. 39%, P < 0.05). SB203580 also reduced interstitial inflammation and extracellular matrix deposition but had no effect on the development of hypertension. SB203580 partially blocked the induction of CCL2, CCL7 (MCP-3), CC chemokine receptor 2 (CCR2), and collagen 4 mRNA expression in the cuffed kidneys. In vitro, blockade of p38 hindered both TNF-α and TGF-β-induced CCL2 upregulation. Based on these observations, we conclude that p38 MAPK plays a critical role in the induction of CCL2/CCL7/CCR2 system and the development of interstitial inflammation in RAS.

Fig. 1.

Treatment of SB203580 inhibits renal artery stenosis (RAS)-induced p38 activity. A: Western blot analysis from renal homogenates from sham or stenotic kidneys with or without SB203580 treatment for 2 wk were probed with and antibodies as indicated. Images are blots of representative experiments. Phospho-MK-2 level is an indicator for p38 MAPK activity. Tubulin was used as loading control. B: phospho-MK-2 (p-MK-2) and total MK-2 density were quantitated from Western blot analysis above and relative ratios of p-MK-2 /total MK-2 are illustrated. Values are means ± SE, *P < 0.05.

Fig. 2.

Inhibition of p38 does not affect blood pressure and plasma activity. A: mice systolic blood pressure was measured 1 day before and 1 and 2 wk after the surgery in different groups as indicated. Values are means ± SE. Blood pressure (BP) was significantly increased in mice at 1 and 2 wk after RAS surgery with or without SB203580 treatment. *P < 0.01. B: 2 wk after sham or RAS surgery, renin activity in EDTA plasma was assessed via production of ANG I from exogenously added angiotensinogen substrate using GammaCoat Renin Activity ¹²⁵I RIA Kit (Diasorin, Stillwater, MN). Values are means ± SE, *P < 0.05, NS, not statistically significant.

Fig. 3.

Inhibition of p38 attenuates renal atrophy in RAS mice. Histological sections obtained from sham and RAS mice with or without 2 wk SB203580 treatment were stained with hematoxylin and eosin. Representative images from each group are shown (magnification, ×400). A: sham operated, vehicle treated; B: sham operated, SB203580 treated; C: RAS, vehicle treated; D: RAS, SB203580 treated. E: atrophy rate (atrophy/total) from each group was quantitated via MetaVue image analysis system and visualized. Values are means ± SE, *P < 0.01, **P < 0.05.

Fig. 4.

Inhibition of p38 partially blocks renal fibrosis in RAS mice. Histological sections were prepared as Fig. 3 but stained with trichrome. Representative images from each group are shown (magnification, ×400). A: sham operated, vehicle treated; B: sham operated, SB203580 treated; C: RAS, vehicle treated; D: RAS, SB203580 treated. E: %collagen staining (blue) from each group was quantitated via MetaVue image analysis system and illustrated. Values are means ± SE. *P < 0.01; **P < 0.05.

Fig. 5.

Inhibition of p38 reduces interstitial collagen 4 deposition in stenotic kidneys. Histological sections were prepared as Fig. 3 but immunostained with collagen 4. Representative images from each group are shown (magnification, ×200). A: sham operated, vehicle treated; B: sham operated, SB203580 treated; C: RAS, vehicle treated; D: RAS, SB203580 treated. E: %collagen 4 staining (brown) from each group was quantitated via MetaVue image analysis system and illustrated. Values are means ± SE. *P < 0.01; **P < 0.05.

Fig. 6.

Inhibition of p38 reduces T cells infiltration in stenotic kidneys. Histological sections were prepared as Fig. 3 but immunostained with CD3. Representative images from each group are shown (magnification, ×400). A: sham operated, vehicle treated; B: sham operated, SB203580 treated; C: RAS, vehicle treated; D: RAS, SB203580 treated. E: %CD3 staining (brown) from each group was quantitated via MetaVue Image Analysis System and illustrated. Values are means ± SE, *P < 0.01, **P < 0.05.

Fig. 7.

Inhibition of p38 reduces the upregulation of CCL2 in RAS mice. A: immunoblot analysis of CCL2 expression. Western blot analysis from renal homogenates from sham or stenotic kidneys with or without SB203580 treatment for 2 wk were probed with antibodies as indicated. Images are blots of representative experiments. B: densitometric quantitation of above blots. CCL2 levels were normalized to tubulin and relative density was illustrated Values are means ± SE, *P < 0.01, **P < 0.05.

Fig. 8.

p38 blockade affects CCL2/CCL7/CCR2 expressions in RAS mice. Gene expression was measured by absolute quantitation real-time PCR and the gene copy numbers in 1 μg total RNA were double-normalized to GAPDH and 18S rRNA. A: CCL2; B: CCR2; C: CCL7; D: Col4a1. CCR2, CC chemokine receptor 2. Values are means ± SE, *P < 0.01, **P < 0.05, NS: not statistically significant.

Fig. 9.

Inhibition of p38 blocks TGF-β-induced upregulation of CCL2 and col4 expression in rat glomerular mesangial cells (MC). MC were pretreated with vehicle (0.1% DMSO) or p38 inhibitor SB202190 for 1 h, followed by 10 ng/ml TGF-β1 for 6 h. Total RNA was isolated and the copy number of each gene in 1 μg total RNA was measured by absolute quantitation real-time PCR and normalized by 18S rRNA. CCL2 (A); Col4a (B); CCR2 (C). Values are means ± SE, *P < 0.01; **P < 0.05.

Fig. 10.

Inhibition of p38 reduces TNF-α-stimulated CCL2 expression in rat MC. MC were pretreated with vehicle or p38 inhibitor SB202190 for 1 h, followed by 10 ng/ml TNF-α for 6 h. The concentration of CCL2 secreted into the culture medium was quantified by using a rat-specific CCL2 ELISA kit. Values are means ± SE, *P < 0.01.