Modified Huangqi Chifeng decoction alleviates podocyte injury on rat with experimental membranous nephropathy

Abstract

Objective: This study aims to investigate the therapeutic effects of modified Huangqi Chifeng decoction (MHCD) on proteinuria in membranous nephropathy (MN) and its potential protective effects on podocytes. Furthermore, we explored whether these effects are associated with the inhibition of the nuclear factor kappa-B (NF-κB) pathway.

Methods: Passive Heymann nephritis (PHN) rat model was applied with a single tail vein injection of sheep anti-rat Fx1A serum (0.4 ml/100g). All rats were divided into four groups: normal group, PHN group, benazepril group (10 mg/kg), and MHCD group (12.5 g/kg), and were treated for 6 weeks. 24-hour urine protein levels and serum biochemical parameters were measured. Optical microscopy and transmission electron microscopy were performed to assess pathological changes in renal tissues. Additionally, the expression levels of IgG, C5b-9, nephrin, podocin, Wilms' tumor gene 1 (WT-1), and NF-κB p65 were evaluated.

Results: PHN rats exhibited progressive proteinuria over time. However, MHCD treatment significantly reduced levels of proteinuria and triglyceride, while increased levels of albumin. Moreover, MHCD alleviated pathological damage in renal tissues, and reduced the expression of IgG and membrane attack complex (C5b-9). Immunohistochemistry analysis revealed that MHCD increased the expression of nephrin, podocin, and WT-1. Western blot analysis showed that MHCD increased the expression of nephrin and podocin while inhibiting the activation of NF-κB p65.

Conclusions: Our findings indicate that MHCD exert reno-protective effects in the experimental rat model of MN by alleviating podocyte damage and inhibiting the NF-κB pathway.

Keywords: Membranous nephropathy; NF-κB pathway; modified Huangqi Chifeng decoction; passive Heymann nephritis; podocyte injury.

Figure 1.

MHCD significantly reduced proteinuria in PHN rats. The 24-h proteinuria of all groups before and after drug administration. Each bar represents the mean ± SD of 9 rats for each group. One-way ANOVA followed by tukey’s test. *p < 0.05, **p < 0.01 versus the normal group, ^#p < 0.05, ^#p < 0.05 versus the model group.

Figure 2.

Effects of MHCD on serum ALB, TC, TG, BUN and Scr in PHN rats. ALB was significantly decreased in model group as compared to the normal group, whlie MHCD treatment could improve it. TC, TG and BUN were increased in the PHN rats, MHCD and benazepril have the potential tendency to lower TG. (A) ALB; (B) TC; (C) TG; (D) BUN; (E) Scr; each bar represents the mean ± SD of 9 rats for each group. One-way ANOVA followed by tukey’s test. **p < 0.01 versus the normal group, ^#p < 0.05, ^#p < 0.05 versus the model group.

Figure 3.

MHCD improved renal pathology in PHN rats. (A) Representative images of pathologic changes by PAS (Scale bar: 100 μm); black arrows: infiltration of interstitial inflammatory cells. (B) Representative images of pathologic changes by Masson staining. Green arrows: thickened GBM. (Scale bar: 100 μm). (C) Quantitative analysis of Masson-positive area in four groups. Each bar represents the mean ± SD of 9 rats for each group. One-way ANOVA followed by tukey’s test. **p < 0.01 versus the normal group, ^##p < 0.01 versus the model group.

Figure 4.

MHCD decreased IgG and C5b-9 deposition in PHN rats. (A) Representative images of IgG immunofluorescence deposition. Scale bar: 100 μm. (B) Representative images of C5b-9 immunofluorescence deposition. Scale bar: 50 μm.

Figure 5.

Representative transmission electron microgarphs showing the ultrastructure changes of kidneys in different groups. Green arrows: diffuse fusion of podocyte foot process, thickening of the glomerular basement membrane (GBM). Scale bar: 2 µm (upper panels) and 1 µm (lower panels).

Figure 6.

MHCD improved expression of proteins related to podocyte in PHN rats. (A) Immunohistochemistry analysis of nephrin and podocin. Scale bar: 100 µm. (B) Immunohistochemistry analysis of WT-1. Scale bar: 100 µm. (C-D) Representative Western blotting bands and analysis of nephrin and podocin. Each bar represents the mean ± SD of 3 rats for each group. One-way ANOVA followed by tukey’s test. **p < 0.01 versus the normal group. ^#p < 0.05, ^##p < 0.01 versus the model group. ^&&p < 0.01 versus the benazepril group.

Figure 7.

MHCD inhibited NF-κB p65 activation in kidneys of PHN rats. Representative Western blotting bands and analysis of pNF-κB p65 and NF-κB p65. Each bar represents the mean ± SD of 3 rats for each group. One-way ANOVA followed by tukey’s test. **p < 0.01 versus the normal group. ^##p < 0.01 versus the model group. ^&&p < 0.01 versus the benazepril group.