p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

Abstract

Activation of p38 mitogen-activated protein kinase (MAPK) is associated with tissue fibrosis, and inhibition of p38 MAPK can attenuate the progression of fibrosis. We aimed to investigate whether p38 MAPK activity in kidney tissue confirmed by immunohistochemical staining is associated with renal tubulointerstitial fibrosis in chronic kidney disease patients with IgA nephropathy. We collected kidney biopsy specimens from 341 IgA nephropathy patients and 15 control patients to identify the clinical and histopathological factors associated with kidney tubulointerstitial fibrosis and to find an association between kidney phosphorylated p38 immunoactivity and pathological grading. In addition, we aimed to investigate whether the anti-fibrotic effect of p38 MAPK inhibition can be identified by assessing the immunostaining intensity of phosphorylated p38 in kidney tissue. A renal tubulointerstitial fibrosis model was introduced using 7-week-old C57BL/6 mice subjected to unilateral ureteral obstruction (UUO). The p38 MAPK inhibitor SB-731445 was injected intraperitoneally every day for 7 days, and changes in renal fibrosis-associated markers were investigated. Assessment of kidney biopsy specimens from IgA nephropathy patients revealed that the degree of interstitial fibrosis was significantly associated with the tissue immunoactivity of phosphorylated p38. High-grade interstitial fibrosis was associated with a low glomerular filtration rate, high proteinuria, and high-grade histopathological changes, including tubular atrophy, interstitial inflammation, and glomerular sclerosis. In a mouse UUO model, renal protein expression of COL1 and phosphorylated p38 were significantly increased, and the protein expression of COL1 and phosphorylated p38 decreased in mice administered 10 mg/kg/day p38 MAPK inhibitor. We found that kidney interstitial fibrosis is associated with increased immunoactivity of phosphorylated p38 in a UUO mouse model and in human IgA nephropathy patients and that the anti-fibrotic effect of p38 MAPK inhibition can be confirmed using immunohistochemical staining for phosphorylated p38 in kidney tissue.

Fig 1. Immunoactivity of phosphorylated p38 in IgA nephropathy patients according to the grade of interstitial fibrosis (original magnification x100).

Control panel shows the phosphorylated p38 kidney tissue activity among patients who have no pathologic changes in kidney biopsy specimens. (Fig 1-A) The immunoactivity of phosphorylated p38 in tissues from IgA nephropathy patients with moderate interstitial fibrosis (Fig 1-D) was higher than that in tissues from patients with no interstitial fibrosis (Fig 1-B) or mild interstitial fibrosis (Fig 1-C). The immunoactivity of phosphorylated p38 in patients with a high degree of interstitial fibrosis was higher than immunoactivity in those with a low degree of kidney interstitial fibrosis (Fig 1-E; overall P < 0.001; control vs. none, P = 0.588; none vs. moderate to severe, P = 0.001; mild vs. moderate to severe, P = 0.021).

Fig 2. Histologic Masson's trichrome stain findings of renal fibrosis after UUO and administration of a p38 MAPK inhibitor.

Scale bar = 100 μm (original magnification x100). Mice from the sham-operated control group (A) show sparse tubulointerstitial fibrosis. UUO-operated group with administered 20% DMSO vehicle (B) shows prominent tubulointerstitial fibrosis. UUO-operated group administered p38 MAPK inhibitor at 5 mg/kg/day (C). UUO-operated group administered p38 MAPK inhibitor at 10 mg/kg/day (D). The extent of tubulointerstitial fibrosis in the groups administered p38 MAPK inhibitor was lower than that in the vehicle group (E). *, P < 0.05; UUO, unilateral ureteral obstruction. (n = 5 per group for each experiment, and n = 2 for sham-operated control group).

Fig 3. Fibrosis-related mRNA expression as assessed with RT-PCR.

(A) Expression of αSMA was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (B) Expression of COL1 was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (C) Expression of fibronectin was decreased in the 5 and 10 mg/kg/day p38 MAPK inhibitor groups, with a dose-dependent pattern. (D) Expression of p38 MAPK was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (E) Expression of MCP-1 was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (F) The decrease in periostin expression was not statistically significant. αSMA, α-smooth muscle actin; COL1, type 1 collagen; MCP-1, monocyte chemoattractant protein-1; UUO, unilateral ureteral obstruction. *, P < 0.01; ^†, P < 0.05. (n = 5 per group for each experiment, and n = 2 for sham-operated control group).

Fig 4. Fibrosis-related renal protein expression as assessed with Western blot.

(A) Expression of αSMA did not decrease after p38 MAPK inhibitor administration. (B) Expression of COL1 decreased significantly after administration of 10 mg/kg/day p38 MAPK inhibitor. (C) Expression of phosphorylated p38 MAPK decreased significantly after administration of 10 mg/kg/day p38 MAPK inhibitor. αSMA, α-smooth muscle actin; COL1, type 1 collagen. ^†, P < 0.05. (n = 4 per group for each experiment, and n = 2 for sham-operated control group).

Fig 5. Immunohistochemical staining showing the expression of phosphorylated p38 in the mouse renal tubulointerstitial area after UUO and administration of p38 MAPK inhibitor.

Scale bar = 50 μm (original magnification x300). (A) Sham-operated group. (B) UUO-operated group administered 20% DMSO vehicle. (C) UUO-operated group administered 5 mg/kg/day p38 MAPK inhibitor. (D) UUO-operated group administered 10 mg/kg/day p38 MAPK inhibitor. Tubulointerstitial expression of phosphorylated p38 decreased significantly in the groups treated with p38 MAPK inhibitor. UUO, unilateral ureteral obstruction.

Fig 6. Microscopic morphological changes in hTECs after treatment with p38 MAPK inhibitor.

(A) Control hTECs show a normal ellipsoid shape and clear contour. (B) Exposure to TGF-β (2 ng/ml) induced bursting and speckled morphological changes in hTECs. (C) Exposure to TGF-β (2 ng/ml) in the presence of p38 MAPK inhibitor (2 μM). (D) Exposure to TGF-β (2 ng/ml) in the presence of p38 MAPK inhibitor (4 μM).

Fig 7. Cellular mRNA expression of fibrosis-related markers after exposure to TGF-β in the presence of p38 MAPK inhibitor.

(A) COL1 expression was increased after exposure to TGF-β (2 ng/ml). Treatment with the p38 MAPK inhibitor significantly reduced the cellular expression of COL1. (B) The decrease in the expression of fibronectin was not significant after concomitant exposure to TGF-β and p38 MAPK inhibitor. (C) Expression of periostin was increased after exposure to TGF-β (2 ng/ml). Treatment with p38 MAPK inhibitor reduced TGF-β-induced periostin expression. (n = 6 per group for each experiment, and in vitro experiments were repeated 3 times to confirm reproducibility) *, P < 0.01.