ASK 1 inhibitor treatment suppresses p38/ JNK signalling with reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis

Figure 1. Glomerular injury and activation of p38 and c‐Jun amino terminal kinase (JNK) in day 1 nephrotoxic serum nephritis (NTN). Disease was induced in groups of 6 SD rats which were treated with GS‐444217 (drug; NTN‐D) or vehicle (NTN‐V) alone. A group of 6 normal rats served as control. A, urine total protein:creatinine ratio, (B) serum creatinine, (C) percentage of glomeruli exhibiting thrombosis in periodic acid Schiff (PAS) stained sections. D‐F, PAS stained sections: D, normal rat kidney; E, vehicle‐treated disease exhibiting capillary thrombosis (*); F, drug treated disease. G‐I, immunostaining for phosphorylated p38 (p‐p38): G, weak staining for p‐p38 in some glomerular cells in normal rat kidney; H, vehicle‐treated day 1 NTN (NTN‐V) shows many p‐p38+ cells in the glomerular tuft; I, GS‐444217 (drug; NTN‐D) treatment abolishes p‐p38 staining in day 1 NTN. J, Quantification of p‐p38+ cells within the glomerular tuft. K‐M, immunostaining for phosphorylated c‐JunSer63 (p‐c‐Jun): K, No staining in normal rat kidney; (L) p‐c‐Jun+ cells are seen in the glomerular tuft and Bowman's capsule in vehicle‐treated day 1 NTN; M, GS‐444217 (drug) treatment reduces the number of p‐c‐Jun+ cells…

Figure 2. Glomerular leucocyte recruitment and inflammation in day 1 nephrotoxic serum nephritis (NTN). Disease was induced in groups of 6 SD rats which were treated with GS‐444217 (drug; NTN‐D) or vehicle (NTN‐V) alone. A group of 6 normal rats served as control. A, Glomerular neutrophils. B, Glomerular macrophages. C, Glomerular T cells. Glomeruli were isolated for RT‐PCR analysis of: D, TNF‐α, and (E) NOS2. Data are mean ± SD. Analysis by one‐way ANOVA with Tukey's multiple comparison test

Figure 3. Kidney injury in day 14 nephrotoxic serum nephritis (NTN). Disease was induced in groups of 8 WKY rats which were treated with GS‐444217 (drug; NTN‐D) or vehicle (NTN‐V) alone. A group of 6 normal rats served as control. A, Urine total protein:creatinine ratio. B, Serum creatinine. C‐G, periodic acid Schiff stained sections: C, normal rat kidney; D, vehicle‐treated disease exhibiting hypercellularity, crescent formation (*) and thrombosis; E, drug‐treated disease showing hypercellularity; F, vehicle‐treated disease showing extensive tubulointerstitial damage with cast formation, tubular dilation and inflammatory cell infiltration: G, drug‐treated disease showing mild tubulointerstitial damage. H, Percentage of glomeruli exhibiting crescent formation. I, RT‐PCR analysis of mRNA levels for the tubular damage marker, KIM‐1. Original magnification, ×400 (C‐G), ×200 (F,G). Data are mean ± SD. A, analysed by repeated measures two‐way ANOVA; B,H,I, analysed by one‐way ANOVA with Tukey's multiple comparison test

Figure 4. Glomerular fibrin deposition and kidney p38 and c‐Jun amino terminal kinase activation in day 14 nephrotoxic serum nephritis (NTN). Disease was induced in groups of 8 WKY rats which were treated with GS‐444217 (drug; NTN‐D) or vehicle (NTN‐V) alone. A‐C, Immunostaining for fibrinogen: A, little staining is seen in normal rat kidney; B, pronounced fibrin deposition is seen in the glomerular tuft and in crescents in vehicle‐treated disease, (C) drug treatment partially reduces fibrin deposition in day 14 NTN. D, Quantification of the glomerular fibrin staining score (0‐3+). E, Western blot analysis of whole kidney lysates for p‐p38 and p‐c‐Jun with tubulin reprobe. Graphs show quantification for p‐p38 (F) and p‐c‐Jun (G). Data are mean ± SD. D, Analysis by nonparametric one‐way ANOVA with Dunn's multiple comparison test; F,G, analysis by one‐way ANOVA with Tukey's multiple comparison test

Figure 5. Macrophage recruitment and inflammation in day 14 nephrotoxic serum nephritis (NTN). Disease was induced in groups of 8 WKY rats which were treated with GS‐444217 (drug; NTN‐D) or vehicle (NTN‐V) alone. A‐C, Immunostaining for ED1+ macrophages: A, normal rat kidney; B, vehicle‐treated disease shows prominent macrophage accumulation in glomeruli and through the interstitium; C, drug treatment also has substantial glomerular and interstitial macrophage accumulation. Quantification of the number of ED1+ macrophages in: D, glomeruli, and (E) the interstitium. RT‐PCR analysis of mRNA levels for: F, CD68; G, CCL2; H, NOS2; I, MMP‐12; J, MMP‐9, and; K, CD206. Data are mean ± SD. Analysis by one‐way ANOVA with Tukey's multiple comparison test

Figure 6. T‐cell infiltration in day 14 nephrotoxic serum nephritis (NTN). Disease was induced in groups of 8 WKY rats which were treated with GS‐444217 (drug; NTN‐D) or vehicle (NTN‐V) alone. A‐C, Immunostaining for R73+ T cells: A, normal rat kidney; B, vehicle treated disease shows a substantial infiltrate of T cells in glomeruli and the interstitium; C, drug treatment shows a marked reduction in T‐cell infiltration in both compartments. Quantification of the number of R73+ T cells in: D, glomeruli, and (E) the interstitium. RT‐PCR analysis of mRNA levels for: F, CD3; G, CCL5; H, IL‐2, and; I, FoxP3. Data are mean ± SD. Analysis by one‐way ANOVA with Tukey's multiple comparison test

Figure 7. Renal fibrosis in day 14 nephrotoxic serum nephritis (NTN). Disease was induced in groups of 8 WKY rats which were treated with GS‐444217 (drug; NTN‐D) or vehicle (NTN‐V) alone. A‐C, Immunostaining for collagen IV: A, normal rat kidney shows collagen IV in the glomerular and tubular basement membranes; B, vehicle‐treated disease shows increased deposition of collagen IV in both glomeruli and the interstitium; C, drug treatment reduces shows a mild increase in collagen IV staining compared to normal. D‐F, Immunostaining for α‐SMA: D, normal rat kidney shows α‐SMA staining in vessel walls; E, vehicle‐treated disease shows α‐SMA+ myofibroblasts within glomeruli, in the periglomerular area and in the interstitium, and; F, drug‐treated disease shows only small numbers of α‐SMA+ cells in glomeruli and the interstitium. RT‐PCR analysis of mRNA levels for: G, collagen I; H, α‐SMA; and; I, TGF‐β1. Data are mean ± SD. Analysis by one‐way ANOVA with Tukey's multiple comparison test