Renoprotective Effects Of Isoliquiritin Against Cationic Bovine Serum Albumin-Induced Membranous Glomerulonephritis In Experimental Rat Model Through Its Anti-Oxidative And Anti-Inflammatory Properties

Abstract

Background: Membranous glomerulonephritis (MGN) is a nephrotic syndrome which shows the symptoms of heavy proteinuria and immune complex deposition in glomerular sub-epithelial space and finally leads to chronic kidney disease. Isoliquiritin (ILQ) is a flavonoid with a wide range of pharmacological properties, including antioxidant and anti-inflammatory activity. The present study was undertaken to investigate the possible mechanisms by which ILQ ameliorates cationic bovine serum albumin (C-BSA) induced MGN in rat model.

Methods: The MGN condition was confirmed by the 24 hr proteinuria and ILQ (10 mg/kg/bw/day) or TPCA-1 (10 mg/kg/bw/day; IKKβ inhibitor) was administered to successfully induce rats for 4 weeks.

Results: The present study revealed that MGN rats treated with ILQ showed significantly ameliorated kidney dysfunction and histopathological changes in kidneys. ILQ treated MGN rats alleviated the oxidative stress and were presented with increased anti-oxidative status in kidneys. Furthermore, ILQ treatment to MGN rats showed anti-oxidative effects through the prominent stimulation of Nrf2 signaling pathway and inhibition of Keap1, which consequently increases the Nrf2 nuclear translocation and thereby induces expression of NQO1 and HO-1. In addition, ILQ-treated MGN rats demonstrated anti-inflammatory effects by inhibiting NF-κB signaling pathway through decreased mRNA and protein expressions of NF-κB p65, IKKβ, COX-2, iNOS, p38-MAPK, p-p38-MAPK, TNF-α, IL-1β, IL-8, ICAM-1, E-selectin and VCAM-1 and reduced the nuclear translocation of NF-κB p65.

Conclusion: The protective effect of ILQ on MGN can be explained by its anti-oxidative and anti-inflammatory activities, which in turn due to the activation of Nrf2 and downregulation of NF-κB pathway.

Keywords: inflammation; isoliquiritin; membranous glomerulonephritis; oxidative stress.

Figure 1

Effects of ILQ on (A) body weight, (B) kidney weights and (C) kidney somatic index (KSI) on C-BSA induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group.

Figure 2

Effects of ILQ on (A) urinary protein, (B) serum creatinine, (C) BUN, (D) adiponectin, (E) total cholesterol and (F) triglycerides on C-BSA induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group.

Figure 3

Effects of ILQ on histopathological changes on C-BSA-induced MGN rats. (A) Lower magnification (200× magnification; scale bar represents 100 μm). Hematoxylin and eosin (H&E). (B) Higher magnification (600× magnification; scale bar represents 10 μm) of Hematoxylin and eosin (H&E). n=6. Abbreviations: BC, bowman capsule; DT, distal-convoluted tubules; G, glomerulus; GA, glomerular atrophy; P, podocytes; PT, proximal-convoluted tubules.

Figure 4

Effects of ILQ on mRNA levels of (A) Nrf2, (B) Keap-1, (C) HO-1 and (D) NQO1 on C-BSA-induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group.

Figure 5

Effects of ILQ on immunohistochemistry and quantitative analysis of (A, E) Nrf2, (B, F) Keap1, (C, G) HO-1, and (D, H) NQO1 on C-BSA-induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group. Scale bar represents 50 μm (400× magnification). Abbreviations: BC, bowman capsule; DT, distal-convoluted tubules; G, glomerulus; GA, glomerular atrophy; PT, proximal-convoluted tubules.

Figure 6

Representative Western blots images. Qualification of fold-change of (A) nuclear Nrf2, (B) cytosol Nrf2 and (C) Keap1. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group.

Figure 7

Effects of ILQ on (A) MDA, (B) NO, (C) SOD, (D) CAT, (E) GPx and (F) GSH levels on C-BSA-induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group.

Figure 8

Effects of ILQ on (A) nuclear NF-κβ p65, (B) IKKβ mRNA, (C) ELISA-TNF-α levels, (D) ELISA-IL-1β levels, (E) IL-8 mRNA, (F) ICAM-1 mRNA, (G) VCAM-1 mRNA, (H) MCP-1 mRNA and (I) E-selectin mRNA levels on C-BSA-induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group.

Figure 9

Effects of ILQ on immunohistochemistry and quantitative analysis of (A, E) NF-κβ p65, (B, F) IKKβ (C, G) and COX2 (D, H) iNOS on C-BSA-induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group. Scale bar represents 50 μm (400× magnification). Abbreviations: BC, bowman capsule; DT, distal-convoluted tubules; G, glomerulus; GA, glomerular atrophy; PT, proximal-convoluted tubules.

Figure 10

Effects of ILQ on Western blots images and qualification of fold-change of (A) nuclear NF-κβ p65, (B) cytosol NF-κβ p65 and (C) p-IKKβ on C-BSA-induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group.

Figure 11

Effects of ILQ on immunohistochemistry and quantitative analysis of (A, C) p38 MAPK, (B, D) p-p38 MAPK and Western blots images and qualification of fold-change of (E) p38 MAPK, (F) p-p38 MAPK on C-BSA-induced MGN rats. Data were expressed as mean ± S.D. for 6 rats per group. ^#p < 0.05 vs MGN group and *p < 0.05 vs ILQ or TPCA1 treatment group. Scale bar represents 50 μm (400× magnification). Abbreviations: BC, bowman capsule; DT, distal-convoluted tubules; G, glomerulus; GA, glomerular atrophy; PT, proximal-convoluted tubules.