Berberine improves kidney function in diabetic mice via AMPK activation

Abstract

Diabetic nephropathy is a major cause of morbidity and mortality in diabetic patients. Effective therapies to prevent the development of this disease are required. Berberine (BBR) has several preventive effects on diabetes and its complications. However, the molecular mechanism of BBR on kidney function in diabetes is not well defined. Here, we reported that activation of AMP-activated protein kinase (AMPK) is required for BBR-induced improvement of kidney function in vivo. AMPK phosphorylation and activity, productions of reactive oxygen species (ROS), kidney function including serum blood urea nitrogen (BUN), creatinine clearance (Ccr), and urinary protein excretion, morphology of glomerulus were determined in vitro or in vivo. Exposure of cultured human glomerulus mesangial cells (HGMCs) to BBR time- or dose-dependently activates AMPK by increasing the thr172 phosphorylation and its activities. Inhibition of LKB1 by siRNA or mutant abolished BBR-induced AMPK activation. Incubation of cells with high glucose (HG, 30 mM) markedly induced the oxidative stress of HGMCs, which were abolished by 5-aminoimidazole-4-carboxamide ribonucleoside, AMPK gene overexpression or BBR. Importantly, the effects induced by BBR were bypassed by AMPK siRNA transfection in HG-treated HGMCs. In animal studies, streptozotocin-induced hyperglycemia dramatically promoted glomerulosclerosis and impaired kidney function by increasing serum BUN, urinary protein excretion, and decreasing Ccr, as well as increased oxidative stress. Administration of BBR remarkably improved kidney function in wildtype mice but not in AMPKα2-deficient mice. We conclude that AMPK activation is required for BBR to improve kidney function in diabetic mice.

Figure 1. BBR activates AMPK in cultured HGMCs.

(A) Cultured primary HGMCs were incubated with BBR (100 µM) for indicated time after starvation overnight. AMPK thr172 phosphorylation in total cell lysate was detected by western blot. The blot is a representative of three blots from three independent experiments. *P<0.05 VS control. (B) Dose-dependent effects of BBR on AMPK-Thr172 phosphorylation in HGMECs. N is 3 in each group. *P<0.05 VS control. (C) Confluent HGMCs were treated with BBR (100 µM) for 2 hours. AMPK activity was assayed using the SAMS peptide as a substrate. N is 5 in each group. *P<0.05 VS control.

Figure 2. BBR-induced AMPK activation in cultured HGMCs is LKB1-dependent.

(A) HGMCs were transfected with control siRNA or LKB1 siRNA for 48 h. Then cells were treated with BBR (100 µM) for 2 hours. AMPK thr172 phosphorylation in total cell lysate was detected by western blot. The blot is a representative of three blots from three independent experiments. *P<0.05 VS control. NS indicates no significance. (B) HGMCs were infected with adenovirus containing LKB1-WT, LKB1-S428A or LKB1-S307A for 48 h. The infected cells were then treated with BBR (100 µM) for 2 hours. AMPK-Thr172 phosphorylation was detected by Western blot. The blot is a representative blot from three independent experiments. *P<0.05 VS ad-GFP. NS indicates no significance.

Figure 3. AMPK upregulations by AICAR or gene overexpression attenuate HG-induced oxidative stress in cultured HGMCs.

(A) HGMCs were incubated with D-glucose (30 mM) in presence or absence of AICAR (2 mM) for 12 hours. ROS productions were detected by DHE fluorescence. N is 5 in each group. ^* P<0.05 vs. control, ^# P<0.05 vs HG alone. (B) HGMCs infected with Ad-GFP or Ad-AMPK-CA for 48 hours were incubated with HG for 12 hours. ROS productions were detected by DHE fluorescence. The picture is a representative from 3 independent experiments. ^* P<0.05 vs. control GFP. NS indicates no significance.

Figure 4. BBR via AMPK activation reverses HG-induced oxidative stress in HGMCs.

HGMCs were transfected with AMPKα1/2 siRNA for 48 hours. Then cells were pre-incubated with BBR (100 µM) for 30 minutes followed by treatment with D-glucose (30 mM) for 12 hours. (A) AMPKα protein expression in total cell lysates was assayed by Western blot. (B) ROS productions were detected by DHE fluorescence. (C) Quantitative data of ROS productions. N is 5 in each group. ^* P<0.05 vs. control, ^# P<0.05 vs HG plus control siRNA. NS indicates no significance.

Figure 5. BBR prevents hyperglycemia-induced renal dysfunction in WT but not in AMPKα2^-/- mice.

Permanent hyperglycemia in WT and AMPKα2^-/- mice was induced by a low-dose STZ induction. All mice were received with or without BBR administration (200 mg/kg body weight daily) for 8 weeks after the stable diabetic model was established. At the end of experiments, mice were sacrificed. (A) Blood glucose in all mice. 5–10 mice in each group. ^* P<0.05 vs. WT alone, ^# P<0.05 vs STZ plus WT. NS indicates no significance. (B) Morphological and quantitative analysis of glomerulus by HE staining. a, WT; b, WT + STZ; c, WT + STZ + BBR; d, AMPKα2^-/- + STZ; e, AMPKα2^-/- + STZ + BBR. (C) Serum BUN, (D) creatine clearance rate (Ccr), and (E) urinary protein excretion were assayed. 5–10 mice in each group. ^* P<0.05 vs. control, ^# P<0.05 vs DM in WT. NS indicates no significance.

Figure 6. BBR via AMPK prevents hyperglycemia-induced oxidative stress in mice.

Permanent hyperglycemia in WT and AMPKα2^-/- mice was induced by a low-dose STZ induction as described in method section. All mice were received with or without BBR administration (200 mg/kg body weight daily) for 8 weeks after the stable diabetic model was established. At the end of experiments, mice were sacrificed to detect MDA and 3-NT by IHC. (A) Representative pictures of MDA and 3-NT. (B) Quantitative analysis of 3-NT. (C) Quantitative analysis of MDA. 5–10 mice in each group. ^* P<0.05 vs. control, ^# P<0.05 vs DM in WT. NS indicates no significance.