N-Acetylcysteine Attenuates the Development of Renal Fibrosis in Transgenic Mice with Dilated Cardiomyopathy

Abstract

Mechanisms underlying the renal pathology in cardiorenal syndrome (CRS) type 2 remain elusive. We hypothesised that renal glutathione deficiency is central to the development of CRS type 2. Glutathione precursor, N-acetylcysteine (NAC;40 mg/kg/day; 8 weeks) or saline were administered to transgenic mice with dilated cardiomyopathy (DCM) and wild-type (WT) controls. Cardiac structure, function and glutathione levels were assessed at the end of this protocol. Renal fibrosis, glutathione content, expression of inflammatory and fibrotic markers, and function were also evaluated. In both genotypes, NAC had minimal effect on cardiac glutathione, structure and function (P ≥ 0.20). In NAC treated DCM mice, loss of glomerular filtration rate (GFR), tubulointerstitial and glomerular fibrosis and renal oxidised glutathione levels were attenuated by 38%, 99%, 70% and 52% respectively, compared to saline treated DCM mice (P ≤ 0.01). Renal expression of PAI-1 was greater in saline treated DCM mice than in WT mice (P < 0.05). Renal PAI-1 expression was less in NAC treated DCM mice than in vehicle treated DCM mice (P = 0.03). Renal IL-10 expression was greater in the former cohort compared to the latter (P < 0.01). These data indicate that normalisation of renal oxidized glutathione levels attenuates PAI-1 expression and renal inflammation preventing loss of GFR in experimental DCM.

Figure 1

(a,b) Representative images of tubulointerstitial fibrosis at baseline. Scale bars are 16 µm. Blue staining indicates fibrosis. (c) Percentage of tubulointerstitial fibrosis at baseline (n = 5). (d–g) Representative images of tubulointerstitial fibrosis after 8 weeks of NAC or saline treatments. (h) Percentage of tubulointerstitial fibrosis after 8 weeks of NAC or saline treatments (n = 4–6). Data are mean ± SEM. *P < 0.05, ***P < 0.001 vs age matched saline treated WT mice. ⁺⁺⁺ P < 0.001 vs age matched vehicle treated DCM mice. ^# P < 0.05 vs DCM mice at baseline. P values were derived from a one-way ANOVA followed by Tukey post-hoc test. An unpaired t-test was used to compare baseline fibrosis vs fibrosis at the end of NAC treatment in DCM mice. WT = wild type mice, DCM = transgenic mice with dilated cardiomyopathy, VEH = vehicle, NAC = N-acetylcysteine.

Figure 2

(a,b) Representative images of glomerular fibrosis at baseline. Scale bars are 16 µm. Blue staining indicates fibrosis. (c) Percentage of glomerular fibrosis at baseline (n = 5). (d–g) Representative images of glomerular fibrosis after 8 weeks of NAC or saline treatments. (h) Percentage of glomerular fibrosis after 8 weeks of NAC or saline treatments (n = 4–6). Data are mean ± SEM. **P < 0.01, ***P < 0.001 vs age matched saline treated WT mice. ⁺⁺⁺ P < 0.001 vs age matched vehicle treated DCM mice. ^# P < 0.05 vs DCM mice at baseline. P values were derived from a one-way ANOVA followed by Tukey post-hoc test. An unpaired t-test was used to compare baseline fibrosis vs fibrosis at the end of NAC treatment in DCM mice. Abbreviations are as for Fig. 1.

Figure 3

(a) Glomerular filtration rate (as measured by creatinine clearance) and (b) albuminuria in WT and DCM mice administered NAC or saline (n = 7–9). Data are mean ± SEM. **P < 0.01 vs saline treated WT mice. ⁺⁺ P < 0.01 vs saline treated DCM mice. P values were derived from a one-way ANOVA followed by Tukey post-hoc test. GFR = glomerular filtration rate. Other abbreviations are as for Fig. 1.

Figure 4

Levels of glutathione (a–d) at baseline (n = 4–6) and (e–h) after 8 weeks of NAC or saline treatments (n = 6). Data are mean ± SEM. *P ≤ 0.05 vs age matched WT mice. ⁺⁺ P < 0.01 vs age matched saline treated DCM mice. P values were derived from a one-way ANOVA followed by Tukey post-hoc test. Total GSH = total glutathione content, GSSG = oxidised glutathione content, GSH = reduced glutathione content, GSH:GSSG = reduced glutathione: oxidised glutathione ratio. Other abbreviations are as for Fig. 1.

Figure 5

(a–d) Expression of total collagen, collagen types I, III and IV in renal cortical tissues of WT and DCM mice administered NAC or saline (n = 4–8). Data are mean ± SEM. *P < 0.05, **P < 0.01 vs saline treated WT mice. P values were derived from a one-way ANOVA followed by Tukey post-hoc test. Col1a1 = collagen type I, Col3a1 = collagen type III, Col4a1 = collagen type IV. Other abbreviations are as for Fig. 1.

Figure 6

(a–e) Markers associated with inflammation induced renal fibrosis. mRNA expression of Il1a, Il1b, Il6, Tnfα and Il10 in renal cortical tissues of WT and DCM mice administered NAC or saline (n = 6–8). Data are mean ± SEM. *P < 0.05, **P < 0.01 vs saline treated WT mice. ⁺⁺⁺ P < 0.001 vs saline treated DCM mice. Il1a = interleukin 1 alpha, Il1b = interleukin 1 beta, Il6 = interleukin 6, Tnfα = tumor necrosis factor alpha, Il10 = interleukin 10. Other abbreviations are as for Fig. 1.

Figure 7

Representative images of renal expression of (a–d) IL-10 and (f–i) PAI-1 after 8 weeks of NAC or saline treatments. Magnified section of each representative image (outlined with dotted lines) has been included to provide clear representation of positive cells. Here, black arrows were used to indicate positive cells. Scale bars are 100 µm. Quantification of the mean immunoexpression of (e) IL-10 and (j) PAI-1 in each group after 8 weeks of NAC or saline treatments (n = 3–4). Data are mean ± SEM. *P < 0.05 vs saline treated WT mice. ⁺ P < 0.05, ⁺⁺ P < 0.01 vs vehicle treated DCM mice. P values were derived from a one-way ANOVA followed by Tukey post-hoc test. IL-10 = interleukin 10, PAI-1 = plasminogen activator inhibitor-1. Other abbreviations are as for Fig. 1.

Figure 8

(a–e) Markers associated with TGF pathway induced renal fibrosis. mRNA expression of Pai1, Tgfβ, αSMA, Mmp2 and Timp2 in renal cortical tissues of WT and DCM mice administered NAC or saline (n = 5–8). Data are mean ± SEM. *P < 0.05 vs saline treated WT mice. P values were derived from a one-way ANOVA followed by Tukey post-hoc test. Pai1 = plasminogen activator inhibitor-1, Tgfβ = transforming growth factor beta, αSMA = alpha smooth muscle actin, Mmp2 = matrix metalloproteinase-2, Timp2 = tissue inhibitor of metalloproteinase 2. Other abbreviations are as for Fig. 1.