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. 2006 Nov;169(5):1527-40.
doi: 10.2353/ajpath.2006.060169.

Inhibition of p38 mitogen-activated protein kinase and transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis in adriamycin-induced nephropathy

Jinhua Li  1 , Naomi Vittoria Campanale, Rong Jiao Liang, James Antony Deane, John Frederick Bertram, Sharon Denise Ricardo
Affiliations

Inhibition of p38 mitogen-activated protein kinase and transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis in adriamycin-induced nephropathy

Jinhua Li et al. Am J Pathol. 2006 Nov.

Abstract

Inflammation and fibrogenesis are the two determinants of the progression of renal fibrosis, the common pathway leading to end-stage renal disease. The p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta1/Smad signaling pathways play critical roles in inflammation and fibrogenesis, respectively. The present study examined the beneficial renoprotective effect of combination therapy using the p38 MAPK pathway inhibitor (SB203580) and a TGF-beta receptor I (ALK5) inhibitor (ALK5I) in a mouse model of adriamycin (ADR) nephrosis. The p38 MAPK and TGF-beta1/Smad2 signaling pathways were activated in ADR-induced nephropathy in a sequential time course manner. Two weeks after ADR injection, the combined administration of SB203580 (1 mg/kg/24 hours) and ALK5I (1 mg/kg/24 hours) markedly reduced p38 MAPK and Smad2 activities. Moreover, the co-administration of SB203580 and ALK5I to ADR-injected mice resulted in a down-regulation of total and active TGF-beta1 production, reduced myofibroblast accumulation, and decreased expression of collagen type IV and fibronectin. In these mice, retardation in the development of glomerulosclerosis and interstitial fibrosis was observed. In conclusion, although p38 MAPK and TGF-beta1/Smad signaling pathways are distinct they coordinate the progression of renal fibrosis in ADR nephrosis. The co-administration of a p38 MAPK inhibitor and an ALK5 inhibitor may have potential applications in the treatment of renal fibrosis.

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Figures

Figure 1-7026
Figure 1-7026
Pathological and functional characterization of ADR-induced nephropathy. A: PAS staining of sections from NS (i) and ADR-injected (ii) mice. Mice with ADR-induced nephropathy exhibited mesangial expansion, well-developed exudative (fibrin-cap) lesions, glomerular sclerosis, tubular collapse, cast formation, and interstitial expansion at day 28. B: Ratio of urinary protein/creatinine. C: Serum creatinine in ADR-injected mice. Data are means ± SD. In B, a: versus NS, P > 0.05; b: versus NS or ADR 3 days, respectively, P < 0.05; c: versus ADR 7 days, P > 0.05; d: versus ADR 14 days, P > 0.05. In C, a: versus NS, P > 0.05; b: versus NS, P < 0.05; c: versus ADR 1 day, P > 0.05; d: versus ADR 3 days, P > 0.05; e: versus ADR 1 day, 3 days, and 14 days, respectively, P < 0.05. Original magnifications, ×400.
Figure 2
Figure 2
Phospho-Smad2 and Phospho-p38 MAPK are up-regulated in ADR nephropathy. A and B: Increase in p-p38 and p-Smad2 in ADR-treated kidneys. Whole kidney lysates from normal mouse kidneys and ADR-treated mouse kidneys 1 day, 3 days, and 14 days after ADR administration were examined for the presence of p-p38 (Ai) and p-Smad2 (Bi) by Western blot analysis. Blots were stripped and probed for β-tubulin as a loading control. Graphs (Aii and Bii) show densitometry analysis (mean ± SD) of the ratio of p-p38 and p-Smad2 to β-tubulin compared with normal saline-treated animals (assigned a normal saline treated group p-p38 or p-Smad2 to β-tubulin ration of 1). Data are means ± SD. In Aii and Bii, a: versus NS, P < 0.05; b: versus ADR 1 day, P > 0.05; c: versus ADR 1 day, P < 0.05; d: versus ADR 3 days, P < 0.05.
Figure 3
Figure 3
p-p38 MAPK and p-Smad2 blockade by SB203580 and ALK5I in ADR nephropathy. Immunohistochemical nuclear staining of p-ATF2 (brown, A–E) and p-Smad2 (brown, F–J) in glomeruli and the tubulointerstitium of NS (A, F), vehicle-treated (B, G), SB203580-treated (C, H), ALK5I-treated (D, I), and after the co-administration of SB203580 and ALK5I (E, J). A and B: In vehicle-treated animals, intrinsic renal and almost all infiltrating inflammatory cells had nuclear staining for p-ATF2 (B) and p-Smad2 (G). Reduced numbers of cells with positive nuclear staining for p-ATF2 and p-Smad2 were observed after treatment with SB203580 (C, H), ALK5I (D, I), and co-administration of SB203580 and ALK5I treatment (E, J). Graphs show the percentage of the numbers of cells with positive nuclear staining for p-ATF2 (K, L) and p-Smad2 (M, N)/total cells. Data are means ± SD. In K–N, a: versus NS, P < 0.05; b: versus ADR + vehicle, P < 0.05; c: versus ADR + SB, P < 0.05; d: versus ADR + ALK5I, P < 0.05. Original magnifications, ×400.
Figure 4
Figure 4
Effects of SB203580 and ALK5I on ADR-induced nephrosis. A: PAS staining on sections from mice: i, NS; ii, ADR + vehicle; iii, ADR + SB203580; iv, ADR + ALK5I; v, ADR + SB203580 + ALK5I. The co-administration of SB203580 and ALK5I to ADR-injected mice ameliorated the progression of renal fibrosis (B, C) and significantly reduced serum creatinine (D) and proteinuria (E), compared with mice administered SB203580 or ALK5I alone (A, D, E). Data are means ± SD. In B–E, a: versus NS, P < 0.05; b: versus ADR + vehicle, P < 0.05; c: versus ADR + SB, P > 0.05; d: versus ADR + SB or ADR + ALK5I, respectively, P < 0.05. Original magnifications, ×400.
Figure 5
Figure 5
Effects of SB203580 and ALK5I on active and total TGF-β1 protein level in kidney tissues in ADR-induced nephrosis. A: Total TGF-β1 protein expression (brown) was detected by immunohistochemistry; i, NS; ii, ADR + vehicle; iii, ADR + SB203580; iv, ADR + ALK5I; v, ADR + SB203580 + ALK5I. B and C: Active form and total TGF-β1 protein levels in kidney tissues were detected by enzyme-linked immunosorbent assay. Data are means ± SD. In B, a: versus NS, P < 0.05; b: versus ADR + vehicle, P < 0.05; c: versus ADR + SB, P > 0.05; d: versus ADR + ALK5I, P < 0.05. In C, a: versus NS, P < 0.05; b: versus ADR + vehicle, P < 0.05; c: versus ADR + ALK5I, P > 0.05; d: versus ADR + SB, P < 0.05. Original magnifications, ×400.
Figure 6
Figure 6
Effects of SB203580 and ALK5I on α-SMA expression in ADR-induced nephrosis. A: Immunohistochemistry for α-SMA expression (brown) in mice with i, NS; ii, ADR + vehicle; iii, ADR + SB203580; iv, ADR + ALK5I; v, ADR + SB203580 + ALK5I. B: Western blot analysis of α-SMA. C: Densitometric analysis showed a significant decrease in α-SMA expression in ADR + SB203580, ADR + ALK5I, ADR + SB203580 + ALK5I groups compared with vehicle control group. Data are means ± SD. In C, a: versus NS, P < 0.05; b: versus NS, P > 0.05; c: versus ADR + vehicle, P < 0.05; d: versus ADR + SB, P > 0.05; e: versus ADR + SB or ADR + ALK5I, respectively, P < 0.05. Original magnifications, ×600.
Figure 7
Figure 7
Western blot analysis of the effects of SB203580 and/or ALK5I on collagen IV (A) and fibronectin (B) deposition in ADR-induced nephrosis. Densitometric analysis of the ratios of collagen type IV/β-tubulin (B) and fibronectin/β-tubulin (D) (assigned a normal saline-treated group collagen IV or fibronectin to β-tubulin ration of 1). Confocal microscopic analysis of the effects of SB203580 and ALK5I on collagen IV deposition in ADR-induced nephrosis: E: NS; F: ADR + vehicle; G: ADR + SB; H: ADR + ALK5I; and I: ADR + SB + ALK5I groups. J: Quantification of the production of collagen IV. Data are means ± SD. In B, D, and J, a: versus NS, P < 0.05; b: versus NS, P > 0.05; c: versus ADR + vehicle, P < 0.05; d: versus ADR + SB, P > 0.05; e: versus ADR + SB or ADR + ALK5I, respectively, P < 0.05.
Figure 8
Figure 8
The effects of treatment of ADR-injected mice with SB203580 and/or ALK5I on interstitial macrophage infiltration. Immunohistochemical staining of F4/80 in NS (i) and ADR-injected mice with vehicle treatment (ii); SB203580-treatment (iii); ALK5I treatment (iv); or SB203580 + ALK5I combined therapy (v). v: Data are means ± SD. In vi, a: versus NS, P < 0.05; b: versus ADR + vehicle, P < 0.05; c: versus ADR + vehicle, P > 0.05; d: versus ADR + SB, P < 0.05; e: versus ADR + SB, P > 0.05; f: versus ADR + ALK5I, P < 0.05. Original magnifications, ×400.
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