Resveratrol ameliorates renal damage, increases expression of heme oxygenase-1, and has anti-complement, anti-oxidative, and anti-apoptotic effects in a murine model of membranous nephropathy

Abstract

Background: Idiopathic membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis and a common cause of nephrotic syndrome in adults. There are limited available treatments for MN. We assessed the efficacy of resveratrol (RSV) therapy for treatment of MN in a murine model of this disease.

Methods: Murine MN was experimentally induced by daily subcutaneous administration of cationic bovine serum albumin, with phosphate-buffered saline used in control mice. MN mice were untreated or given RSV. Disease severity and pathogenesis was assessed by determination of metabolic and histopathology profiles, lymphocyte subsets, immunoglobulin production, oxidative stress, apoptosis, and production of heme oxygenase-1 (HO1).

Results: MN mice given RSV had significantly reduced proteinuria and a marked amelioration of glomerular lesions. RSV also significantly attenuated immunofluorescent staining of C3, although there were no changes of serum immunoglobulin levels or immunocomplex deposition in the kidneys. RSV treatment of MN mice also reduced the production of reactive oxygen species (ROS), reduced cell apoptosis, and upregulated heme oxygenase 1 (HO1). Inhibition of HO1 with tin protoporphyrin IX partially reversed the renoprotective effects of RSV. The HO1 induced by RSV maybe via Nrf2 signaling.

Conclusion: Our results show that RSV increased the expression of HO1 and ameliorated the effects of membranous nephropathy in a mouse model due to its anti-complement, anti-oxidative, and anti-apoptotic effects. RSV appears to have potential as a treatment for MN.

Fig 1. Effect of resveratrol on laboratory indicators of membranous nephropathy.

(A) proteinuria, (B) serum albumin, (C) serum cholesterol, (D) serum creatinine. Abbreviations: NC, normal control; NC-RSV, NC with resveratrol; MN, membranous nephropathy; MN-RSV, MN with RSV; *, p < 0.05.

Fig 2. Effect of resveratrol on renal histology.

Representative kidney sections from mice in the NC group (A, D, G, J, M), MN group (B, E, H, K, N), and MN-RSV group (C, F, I, L, O) following H&E staining (A–C) and immunofluorescence staining of IgG (D–F), C3 (G–I), C4 (J–L), and C1q (M–O). The quantifications of H&E staining (P) and immunofluorescence staining of IgG (Q), C3 (R), C4 (S), and C1q (T) were listed. All images are ×400. Abbreviations as in Fig 1. *, p < 0.05.

Fig 3. Effect of resveratrol on distribution of lymphocyte subsets and production of immunoglobulins.

(A) Percentages of CD4⁺, CD8⁺, and CD19⁺ immune cells and (B) serum anti-cBSA immunoglobulins IgG1 and IgG2a. Each bar shows the mean ± standard deviation of 5 mice. Abbreviations as in Fig 1. *, p < 0.05.

Fig 4. Effect of resveratrol on production of superoxide anion and kidney cell apoptosis.

(A-C, G) Fluorescence micrographs of dihydroethidium (DHE)-positive cells in the kidneys of mice from the NC, MN, and MN-RSV groups (respectively) and their quantification. (D-F, H) Fluorescence micrographs of TUNEL-positive cells in the kidneys of mice from the NC, MN, and MN-RSV groups (respectively), and their quantification. All images are ×400. Abbreviations as in Fig 1. *, p < 0.05.

Fig 5. Effect of resveratrol on renal expression of heme oxygenase-1.

(A) Expression of HO1 mRNA of mice in the NC, MN, and MN-RSV groups, (B-D, E) immunohistochemical staining of HO1 protein of mice in the NC, MN, and MN-RSV groups and their quantification. All images are ×400. Abbreviations as in Fig 1. *, p < 0.05.

Fig 6. Effect of heme oxygenase-1 inhibition on the renoprotective effects of resveratrol.

(A) Proteinuria in controls (MN and MN-RSV groups) and in mice given an HO1 competitive inhibitor (NC-SnPP and MN-RSV-SnPP groups). (B-F) Representative kidney sections of mice in the MN-RSV-SnPP group were subjected to H&E staining, IgG staining, C3 staining, DHE staining, and TUNNEL staining, respectively. All images are ×400. Abbreviations as in Fig 1. *, p < 0.05.

Fig 7. RSV activates HO1 expression via stimulating Nrf2 binding activity in E11 podocytes.

(A and B) E11 podocyte cells were treated with or without RSV as indicated. RNA expression levels from RSV-untreated or RSV-treated E11 podocytes were determined by RT-qPCR. Data are presented as the mean ± SD from three independent experiments. **P<0.01. (C) Lysates from RSV-untreated or RSV-treated E11 podocytes were immunoblotted with antibodies against the indicated target. (D) Western blot analysis of immunoprecipitated Nrf2 antibody or an IgG control antibody by anti-Nrf2 antibody from RSV-untreated or RSV-treated E11 podocytes. Arrowhead represents the Nrf2 protein. (E) Chromatin immunoprecipitation (ChIP) assays were performed with antibodies against Nrf2 or an IgG control. Subsequent qPCR analysis was carried out using primers specific for 4 promoter regions of HO1. Input represents 1% of the chromatin used for immunoprecipitation. Data are presented as the mean ± SD from three mice. **P<0.01. (F) Real-time qPCR analyses of E11 podocytes transfected with indicated siRNA then treated with 20 um RSV for 6h. Data are presented as the mean ± SD from three independent experiments. **P<0.01.