Yi Shen An, a Chinese traditional prescription, ameliorates membranous glomerulonephritis induced by cationic bovine serum albumin in rats

Abstract

Context: Yi Shen An (YSA) is an investigational composite of traditional Chinese medicine (Reference: 2010L000974) for the treatment of renal disease.

Objective: To investigate the protective effects of YSA against membranous glomerulonephritis (MGN).

Materials and methods: Male Sprague-Dawley rats were injected with cationic bovine serum albumin (C-BSA) to create a model of MGN. Then, rats were orally treated with YSA at doses of 0.25, 0.5, 1 and 2 g/kg for 35 successive days; prednisone (5 mg/kg) was used as a positive control. At the end of the experimental period, we performed a series of tests, including 24 h urinary protein, and biochemical, immunological, antioxidative, coagulation indices, and histopathological examination.

Results: YSA-1 g/kg significantly lowered urinary protein from 68.37 to 30.74 mg (p < 0.01). Meantime, total protein (TP) and albumin (ALB) recovered from 66.26 and 20.51 g/L to 76.08 and 35.64 g/L (p < 0.01), respectively. YSA removed the deposition of immunoglobulin G (IgG) and complement 3c (C3c), prevented inter-capillary cell hyperplasia on the glomerular basement membrane (GBM), and reduced electron-dense deposits and fusion of podocytes. In addition, serum IgG and superoxide dismutase were significantly elevated. In contrast, malondialdehyde, total cholesterol, triglyceride, circulating immune complex (CIC), and immunoglobulin M decreased in the YSA-treated group. Moreover, the blood coagulation dysfunction was adjusted.

Discussion and conclusions: These findings indicate YSA may exert a therapeutic effect against MGN through the inhibition of CIC formation, and the removal of IgG and C3c deposition from the GBM, thus supporting the development of further clinical trials.

Keywords: Nephritis; circulating immune complex; podocytes; proteinuria.

Figure 1.

Experimental procedure. s.c., subcutaneous injection; i.v., intravenous injection; C-BSA, cationic bovine serum albumin.

Figure 2.

HPLC chromatograms of quality control components. R1, Panax notoginseng saponins R1; Rg1, ginseng saponin Rg1; Rb1, ginsenoside Rb1. (A) Reference substance for R1, Rg1, Rb1; (B) R1, Rg1, Rb1 in Yi-shen-An (YSA); (C) Reference substance for tanshinone IIA; (D) Tanshinone IIA in YSA; (E) Reference substance for emodin; (F) Reference substance for aloe-emodin; (G) Emodin and aloe-emodin in YSA; (H) Reference substance for chlorogenic acid; I, Chlorogenic acid in YSA.

Figure 3.

Bar graphs representing levels of 24 h urinary protein. Data are shown as means ± standard error of the mean. Statistical differences are represented as ^##p < 0.01 vs. Control, **p < 0.01 vs. Model group. YSA, Yi Shen An. PRE, prednisone acetate positive control.

Figure 4.

Bar graphs representing the levels of CIC, IgG, IgA, and IgM. Statistical differences are represented as ^#/##p < 0.05/0.01 vs. Control, ^*/**p < 0.05/0.01 vs. Model group. CIC, circulating immune complex; IgG, immunoglobulin G; IgA, immunoglobulin A; IgM, immunoglobulin M; YSA, Yi Shen An; PRE, prednisone acetate positive control.

Figure 5.

Bar graphs representing the levels of SOD and MDA. Statistical differences are represented as ^##p < 0.01 vs. Control, *p < 0.05 vs. Model group. SOD, superoxide dismutase; MDA, malondialdehyde; YSA, Yi Shen An; PRE, prednisone acetate positive control.

Figure 6.

Renal immunofluorescence staining and scanning electron microscope analysis of CBSA-induced MGN rats. YSA, Yi Shen An; PRE, prednisone acetate positive control. Statistical differences are represented as ^##p < 0.01 vs. Control, *p < 0.05 vs. Model group. (A) Immunohistochemical staining of immunoglobulin G (IgG, original magnification ×20); (B) Immunohistochemical staining of complement 3c (C3c, original magnification ×20); (C, D) Quantitative analysis of IgG (C) and C3c deposits (D); (E) Transmission electron micrographs of renal tissue. (Original magnification ×30,000). ‘→’ represents the presence of glomerular podocyte fusion, the proliferation of mesangial cells and matrix, and electron-dense deposits in the GBM and epithelia.

Figure 7.

The effects of YSA on histopathological changes in the kidney tissues of MGN rats. At the end of the study, rat kidneys were harvested and fixed using 4% neutralized formalin. Paraffin sections were then prepared and stained with haematoxylin & eosin, periodic acid-Schiff, and Masson’s trichrome staining. A double-blinded method was used in the pathological analysis, the results were qualitatively identical, and representative results are shown. YSA, Yi Shen An; PRE, prednisone acetate positive control. (A) Representative haematoxylin-eosin staining of kidney samples (original magnification ×40). ‘→’ represents hyperplasia of mesangial cells and the matrix, as well as the luminal stenosis of renal capsule and capillaries. (B) Representative periodic acid-Schiff staining of kidney sections (original magnification ×40). ‘→’ represents glomerular mesangial cell proliferation and basement membrane thickness. (C) Representative Masson’s trichrome staining of kidney sections (original magnification ×100). ‘→’ represents the deposition of polytrophin in the mesangial area. (D–F) The scores for glomerular injury (D), glomerular diameter (E) and tubular atrophy (F). Statistical differences are represented as ^##p < 0.01 vs. Control, **p < 0.01 vs. Model group.