Berberine Reduces Lipid Accumulation by Promoting Fatty Acid Oxidation in Renal Tubular Epithelial Cells of the Diabetic Kidney

Abstract

Abnormal lipid metabolism in renal tubular epithelial cells contributes to renal lipid accumulation and disturbed mitochondrial bioenergetics which are important in diabetic kidney disease. Berberine, the major active constituent of Rhizoma coptidis and Cortex phellodendri, is involved in regulating glucose and lipid metabolism. The present study aimed to investigate the protective effects of berberine on lipid accumulation in tubular epithelial cells of diabetic kidney disease. We treated type 2 diabetic db/db mice with berberine (300 mg/kg) for 12 weeks. Berberine treatment improved the physical and biochemical parameters of the db/db mice compared with db/m mice. In addition, berberine decreased intracellular lipid accumulation and increased the expression of fatty acid oxidation enzymes CPT1, ACOX1 and PPAR-α in tubular epithelial cells of db/db mice. The mitochondrial morphology, mitochondrial membrane potential, cytochrome c oxidase activity, mitochondrial reactive oxygen species, and mitochondrial ATP production in db/db mice kidneys were significantly improved by berberine. Berberine intervention activated the AMPK pathway and increased the level of PGC-1α. In vitro berberine suppressed high glucose-induced lipid accumulation and reversed high glucose-induced reduction of fatty acid oxidation enzymes in HK-2 cells. Importantly, in HK-2 cells, berberine treatment blocked the change in metabolism from fatty acid oxidation to glycolysis under high glucose condition. Moreover, berberine restored high glucose-induced dysfunctional mitochondria. These data suggested that berberine alleviates diabetic renal tubulointerstitial injury through improving high glucose-induced reduction of fatty acid oxidation, alleviates lipid deposition, and protect mitochondria in tubular epithelial cells.

Keywords: berberine; diabetic kidney disease; fatty acid oxidation; mitochondria; renal tubular epithelial cells.

FIGURE 1

Effects of BBR on histopathological alterations of diabetic mice. (A) Representative photomicrographs of PAS staining and immunohistochemistry images of collagen IV, fibronectin (magnification 400×). (B) Semiquantitative analyses of the glomerular volume. (C) Semiquantitative analyses of the glomerular injury index. (D) Semiquantitative analyses of the mesangial matrix fraction. (E) Semiquantitative expression levels of collagen IV and fibronectin. db/m: normal mice + vehicle (0.9% saline of the same volume); db/m + BBR: db/m + BBR (300 mg/kg/d); db/db: diabetic mice + vehicle (0.9% saline of the same volume); db/db + BBR: db/db + BBR (300 mg/kg/d); (n = 8 mice/group). Data are expressed as means ± SD. **p < 0.01 versus the db/m group; #p < 0.05, compared with the db/db group by ANOVA.

FIGURE 2

Effects of BBR on tubulointerstitial fibrosis of diabetic mice. (A) Representative photomicrographs of PAS staining and immunohistochemistry images of KIM1, α-SMA, E-cadherin, collagen I and TGFB1 (magnification: ×400). (B) Semiquantitative analyses of proximal tubular area according to the outer diameter. (C) Semiquantitative analyses of the tubulointerstitial damage index. (D) Representative western blotting images of KIM1, α -SMA, E-cadherin, collagen I and TGFB1. (E) Quantification of protein expression of KIM1, α -SMA, E-cadherin, collagen I and TGFB1. db/m: normal mice + vehicle (0.9% saline of the same volume); db/m + BBR: db/m + BBR (300 mg/kg/d); db/db: diabetic mice + vehicle (0.9% saline of the same volume); db/db + BBR: db/db + BBR (300 mg/kg/d); (n = 8 mice/group). Data are expressed as means ± SD. **p < 0.01 versus the db/m group; #p < 0.05, compared with the db/db group by ANOVA.

FIGURE 3

BBR decreased intracellular lipid accumulation and increased the expression of fatty acid oxidation enzymes in proximal tubules of diabetic kidney. (A) Lipid droplets accumulation was detected by Oil Red O staining (magnification: ×400). (B) The triglyceride levels of renal cortical tissues. (C) The expression level of CPT1, ACOX1 and PPAR-α in the renal cortex tissues was analyzed by western blotting. (D) Representative immunohistochemistry staining analysis of PPAR-α expression in the renal tissues of mice (magnification: ×400). db/m: normal mice + vehicle (0.9% saline of the same volume); db/m + BBR: db/m + BBR (300 mg/kg/d); db/db: diabetic mice + vehicle (0.9% saline of the same volume); db/db + BBR: db/db + BBR (300 mg/kg/d); (n = 8 mice/group). Data are expressed as means ± SD. **p < 0.01 versus the db/m group; #p < 0.05, compared with the db/db group by ANOVA.

FIGURE 4

BBR restored mitochondrial morphology, mitochondrial function and increased mitochondrial biogenesis in db/db mice. (A) Mitochondrial morphology were examined by transmission electron microscopy (magnification: ×20,000). (B) mitochondrial membrane potentia (ΔΨ) was measured using a fluorescent probe JC-1. The ratio of red/green fluorescence represented ΔΨ m. (C) Subcellular extracts of CYCS was evaluated by western blotting analysis. (D) ATP levels were measured using an ATP bioluminescent assay kit. (E) Mitochondrial ROS was detected using MitoSOX by flow cytometry. (F) The expression levels of PGC-1α and p-AMPK were analyzed by western blotting and the relative intensity of PGC-1α and p-AMPK was normalized to the β-actin and total AMPK, respectively. db/m: normal mice + vehicle (0.9% saline of the same volume); db/m + BBR: db/m + BBR (300 mg/kg/d); db/db: diabetic mice + vehicle (0.9% saline of the same volume); db/db + BBR: db/db + BBR (300 mg/kg/d); (n = 8 mice/group). Data are expressed as means ± SD. **p < 0.01 versus the db/m group; #p < 0.05, compared with the db/db group by ANOVA.

FIGURE 5

BBR decreased intracellular lipid accumulation and increases the expression of fatty acid oxidation enzymes in HG-induced HK-2 cells. (A) Lipid droplets (LD) accumulation was stained using BODIPY 493/503 and dvisualized under a confocal microscope. (B) Rate of LD-positive cells in cultured HK-2 cells. (C) The triglyceride levels of HK-2 cells. (D,E) The expression level of CPT1, ACOX1 and PPAR-α in HK-2 cells was analyzed by western blotting. (F) The mRNA level of CPT1, ACOX1 and PPAR-α in the renal cortex tissues was analyzed by RT-PCR. HK-2 cells were treated with 5.6 mM glucose (NG), NG+30 µM BBR (NG + BBR), 30 mM glucose (HG) or HG+30 µM BBR (HG + BBR) for 4 days; (n = 6). Data are expressed as means ± SD. **p < 0.01 versus the NG group; #p < 0.05, compared with the HG group by ANOVA.

FIGURE 6

BBR ameliorates the metabolism reprogramming induced by HG in HK-2 cells. (A–D) The metabolic status of HK-2 cells was determined by evaluating the oxygen consumption rates (OCR) and the extracellular acidification rate of the media (ECAR) using the Agilent Seahorse XF technology. Left panels show representative OCR (A) and ECAR (C) curves from HK-2 cells treated with NG, M, HG, HG + BBR for 4 days. Right panels show quantified data from replicate studies (B,D); n = 3 per group.(E,F) FA-driven oxygen consumption rate (PA-dependent OCR) curve (E) and OCR (F) quantified data from HK-2 cells. HK-2 cells were treated with 5.6 mM glucose (NG), NG+30 µM BBR (NG + BBR), 30 mM glucose (HG) or HG+30 µM BBR (HG + BBR) for 4 days; (n = 6). Data are expressed as means ± SD. **p < 0.01 versus the NG group; #p < 0.05, compared with the HG group by ANOVA.

FIGURE 7

BBR restored mitochondrial morphology, mitochondrial function and increased mitochondrial biogenesis in HK-2 cells. (A) Mitochondrial morphology were stained by Mitotracker red and observed using confocal microscopy. (B) Mitochondrial membrane potential (ΔΨ) was measured using a fluorescent probe JC-1. The ratio of red/green fluorescence represented ΔΨ m. (C) Subcellular extracts of CYCS was evaluated by western blotting analysis. (D) ATP levels were measured using an ATP bioluminescent assay kit. (E) Mitochondrial ROS was detected using MitoSOX by flow cytometry. (F) The expression levels of PGC-1α and p-AMPK were analyzed by western blotting, and the relative intensity of PGC-1α and p-AMPK was normalized to the β-actin and total AMPK, respectively. (G). A summary figure showing presumptive molecular targets of berberine in tubular epithelial cells of diabetes. HK-2 cells were treated with 5.6 mM glucose (NG), NG+30 µM BBR (NG + BBR), 30 mM glucose (HG) or HG+30 µM BBR (HG + BBR) for 4 days; (n = 6). Data are expressed as means ± SD. **p < 0.01 versus the NG group; #p < 0.05, compared with the HG group by ANOVA.