Therapeutic potential of Ganoderma lucidum polysaccharide peptide in Doxorubicin-induced nephropathy: modulation of renin-angiotensin system and proteinuria

Figures

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  FIGURE 1. Graphical workflow of the experimental design. Abbreviations: i.p., intraperitoneal injection; GL-PP, Ganoderma lucidum polysaccharide peptide; DOX, Doxorubicin.

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  FIGURE 2. GL-PP alleviates DOX-induced proteinuria and renal dysfunction in mice. (A) Temporal progression of proteinuria. (B) Change in body weight over the course of the experiment (expressed as a percentage of initial body weight). Body weight % was computed using the formula (actual body weight/initial body weight) x 100. (C) Blood urea nitrogen. (D) Blood creatinine. (E) Plasma triglycerides. (F) Representative image of renal PAS staining (×200), scale bar = 20 μm. (G) Semi-quantitative scoring of glomerular injury. Calculated as the average score of 20 fields per mouse in each group. (H) Semi-quantitative scoring of tubulointerstitial injury, calculated as the average score of 10 fields per mouse in each group. Data are presented as mean ± SEM, N = 5-6 per group. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. CTL (control); # p < 0.05; ### p < 0.001 vs. DOX (Doxorubicin). For statistical analysis, (A, B) were analyzed using a two-way ANOVA with Tukey’s multiple comparison test. (C, D, F) underwent a one-way ANOVA with Tukey’s multiple comparison test. (G ,H) were subjected to the Kruskal-Wallis test with Dunn’s multiple comparison test.

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  FIGURE 3. DOX-induced kidney fibrosis and inflammation are suppressed by GL-PP. (A) The amounts of COX-2 and iNOS proteins were assessed using Western blotting. (B) Quantification of the protein levels of COX-2 and iNOS adjusted to actin. (C) TGF-β1 and COL-1 protein levels were assessed by Western blotting. (D) TGF-β1 and COL-1 protein levels were measured and adjusted to β-actin. (E) Illustrations of Masson’s trichrome staining that are typical (×200), scale bar = 100 µm. (F) Masson-stained kidney slice morphometric analysis. N = 3-6 per group. ** p < 0.01; *** p < 0.001 vs. CTL (control); ## p < 0.01; ### p < 0.001 vs. DOX (Doxorubicin). For statistical analysis, (B, D, F) underwent a one-way ANOVA with Tukey’s multiple comparison test.

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  FIGURE 4. In mice, GL-PP reduces the glomerular damage brought on by DOX. (A) Nephrin and podocin protein levels were assessed using Western blots. (B) Nephrin and podocin protein levels were quantified and adjusted to β-actin in. (C) Nephrin immunofluorescence staining (×200), scale bar: 20 μm. (D) Podocin immunofluorescence staining (×200), scale bar: 20 μm. N = 3-6. ** p < 0.01; *** p < 0.001 vs. CTL (control); # p < 0.05; ### p < 0.001 vs. DOX (Doxorubicin). For statistical analysis, Figures 4B underwent a one-way ANOVA with Tukey’s multiple comparison test.

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  FIGURE 5. DOX-induced kidney cell death is inhibited by GL-PP. (A) Bax, Bcl-2, and Caspase-3 protein levels were assessed using Western blots. (B) Measuring the amounts of the proteins Bax, Bcl-2, Bax/Bcl-2, and Caspase-3, normalized to β-actin. (C) Green fluorescence TUNEL staining (×200), scale bar = 100 µm. (D) The TUNEL Relative Fluorescence Ratio. N = 3-6. ** p < 0.01; *** p < 0.001 vs. CTL (control); # p < 0.05; ## p < 0.01; ### p < 0.001 vs. DOX (Doxorubicin). For statistical analysis, (B, D) underwent a one-way ANOVA with Tukey’s multiple comparison test.

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  FIGURE 6. In mice, GL-PP reduces oxidative damage brought on by DOX. (A) Amounts of TBARS in renal tissues. (B) The amount of SOD in renal tissues. (C) GSH-Px concentrations in renal tissues. (D) H2O2 concentrations in renal tissues. (E) A Western blot was used to examine NOX4 protein levels. NOX4 protein levels were measured and adjusted to -actin in (F). N = 3-6 per group. ** p < 0.01; *** p < 0.001 vs. CTL (control); # p < 0.05; ## p < 0.01 vs. DOX (Doxorubicin). For statistical analysis, (A, B, C, D, F) underwent a one-way ANOVA with Tukey’s multiple comparison test.

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  FIGURE 7. In mice with renal damage brought on by DOX, GL-PP prevents PRR-RAS activation. (A) The protein levels of fPRR and sPRR in the renal cortex were assessed using a Western blot. (B) Analysis of the protein levels of fPRR and sPRR adjusted to β-actin. (C) Levels of sPRR in urine. (D) Renin activity in the urine. (E) Ang Ⅱ excretion in the urine. N = 3–6 per group. *** p < 0.001 vs. CTL (control); ### p < 0.001 vs. DOX (Doxorubicin). For statistical analysis, (B, C, D, E) underwent a one-way ANOVA with Tukey’s multiple comparison test.

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  FIGURE 8. Graphical abstract illustrating the schematic diagram of the signaling pathways. Abbreviations: GL-PP, Ganoderma lucidum polysaccharide peptide; PRR, the (pro)renin receptor; RAS, the renin-angiotensin system; NOX4, nicotinamide adenine nucleotide phosphate oxidase 4; H2O2, hydrogen peroxide; Bax, BCL2-Associated X; Bcl-2: B-cell lymphoma-2; Caspase-3: cysteinyl aspartate specific proteinase-3; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; TGF-β1, transforming growth factor-β1; TBARS, thiobarbituric acid reactive substances; GSH-Px, glutathione peroxidase; SOD, Superoxide D.ismutase.