Berberine ameliorates chronic kidney injury caused by atherosclerotic renovascular disease through the suppression of NFκB signaling pathway in rats

Abstract

Background and objectives: Impaired renal function in atherosclerotic renovascular disease (ARD) may be the result of crosstalk between atherosclerotic renovascular stenosis and amplified oxidative stress, inflammation and fibrosis. Berberine (BBR) regulates cholesterol metabolism and exerts antioxidant effects. Accordingly, we hypothesized that BBR treatment may ameliorate ARD-induced kidney injury through its cholesterol-lowering effect and also suppression of the pathways involved in oxidative stress, inflammation and NFκB activation.

Methods: Male rats were subjected to unilateral renal artery stenosis with silver-irritant coil, and then fed with 12-week hypercholesterolemic diet. Rats with renal artery stenosis were randomly assigned to two groups (n = 6 each) - ARD, or ARD+BBR - according to diet alone or in combination with BBR. Similarly, age-matched rats underwent sham operation and were also fed with hypercholesterolemic diet alone or in combination with BBR as two corresponding controls. Single-kidney hemodynamic metrics were measured in vivo with Doppler ultrasound to determine renal artery flow. The metrics reflecting hyperlipidemia, oxidative stress, renal structure and function, inflammation and NFκB activation were measured, respectively.

Results: Compared with control rats, ARD rats had a significant increase in urinary albumin, plasma cholesterol, LDL and thiobarbituric acid reactive substances (TBARS) and a significant decrease in SOD activity. When exposed to 12-week BBR, ARD rats had significantly lower levels in blood pressure, LDL, urinary albumin, and TBARS. In addition, there were significantly lower expression levels of iNOS and TGF-β in the ARD+BBR group than in the ARD group, with attenuated NFκB-DNA binding activity and down-regulated protein levels of subunits p65 and p50 as well as IKKβ.

Conclusions: We conclude that BBR can improve hypercholesterolemia and redox status in the kidney, eventually ameliorating chronic renal injury in rats with ARD, and that BBR can act against proinflammatory and profibrotic responses through suppression of the NFκB signaling pathway.

Figure 1. Doppler ultrasound velocity at the left renal artery was detected at 300 mm/sec in CTL rats (A); renal blood flow acceleration was detected at 600 mm/sec in ARD rats (B).

Figure 2. Effects of BBR on renal tissue architecture and fibrosis.

A: Representative light microscopic features of HE-stained kidney sections. a) Normal histological structure. b) Almost normal histological structure after BBR treatment in CTL+BBR rats. c) Tubulointerstitial inflammation and cells infiltration were evident in ARD rats. d) The renal histological morphology is almost normal after BBR treatment in ARD+BBR rats. B: Assessment of kidney tissue architecture, and interstitial and perivascular fibrosis by Masson's trichrome staining. a) Normal histological structure. b) Almost normal histological structure after BBR treatment in CTL+BBR rats. c) Compared with CTL rats, ARD rats had more tubular degeneration and ectasia, more interstitial, periglomerular, and periarterial fibrosis (light blue staining areas). d) The renal histological morphology was almost normal after BBR treatment in ARD+BBR rats. C: Quantification of Masson's trichrome for tubulointerstitial injury score. Data expressed as means ± SEM, n = 6 per group. *P<0.05 vs. CTL group. ^# P<0.05 vs. ARD group. ^$ P<0.05 vs. CTL+BBR

Figure 3. Effects of BBR on glomerular ultrastructural structure.

A: a) EM analysis of a normal glomerulus revealed normal ultrastructure of the mesangial area. b) Almost normal ultrastructure of the mesangial area in CTL+BBR rats. c) Proliferation of mesangial cell and matrix were evident in ARD rats, with mesangial matrix expansion indicated by an asterisk. d) The mesangial area morphology was almost normal after BBR treatment in ARD+BBR rats. Original magnification ×6000. B: a) EM analysis of a normal glomerulus showing normal ultrastructure of the foot processes. b) Foot process morphology was normal in CTL+BBR rats. c) Focal areas of foot process effacement were evident in ARD rats. d) Foot process morphology was normal after BBR treatment in ARD+BBR rats. Original magnification ×15000. EM, electron micrograph; Mes, mesangial cell; CP, capillary lumen.

Figure 4. Effect of BBR on expression of pro-inflammatory and fibrotic molecules in rat kidney.

A and B: Representative Immunohistochemical staining for iNOS and TGF-β, respectively. a) CTL rats had little staining for iNOS or TGF-β. b) Faint staining for iNOS or TGF-β in CTL+BBR kidneys. c) ARD caused stronger staining for iNOS or TGF-β in the renal endothelium and tubulointerstitium. d) BBR treatment caused a significant reduction in the expression of iNOS or TGF-β in ARD+BBR group kidney. e) Negative control. C) Expression of pro-inflammatory and fibrotic molecules in the kidney was detected by western blots; D–E: Each molecule densitometry analysis of western blots from 3 independent experiments. Magnification, ×200. Data expressed as mean ± SEM, n = 6 per group. ^* P<0.05 vs. normal group. ^# P<0.05 vs. ARD group. ^$ P<0.05 vs. CTL+BBR

Figure 5. Effect of BBR on NFκB signaling pathway in rat kidney.

A: Specificity and effect of BBR on NFκB-DNA binding activities in rat kidney. Lane 1: negative extract control; Lane 2: positive extract control; Lane 3: CTL group rats; Lane 4: CTL+BBR group rats; Lane 5: ARD group; Lane 6: BBR-treated rats. B: Densitometric analysis of NFκB-DNA binding activities. Densitometry was performed on autographs. Values were median relative density unit (RDU) of each group. C: Expression of phosphorylated-IKKβ, NFκB subunit p65/p50 in kidneys was detected by western blots.D–E: Each molecule densitometry analysis of western blots from 3 independent experiments. Data expressed as mean ± SEM, n = 6 per group. ^* P<0.05 versus CTL. ^# P<0.05 vs. ARD. n = 6 per group. *P<0.05 versus CTL group. ^# P<0.05 vs. ARD group. ^$ P<0.05 vs. CTL+BBR