The Sodium-Glucose Cotransporter 2 Inhibitor Dapagliflozin Prevents Renal and Liver Disease in Western Diet Induced Obesity Mice

Abstract

Obesity and obesity related kidney and liver disease have become more prevalent over the past few decades, especially in the western world. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with promising effects on cardiovascular and renal function. Given SGLT2 inhibitors exert both anti-diabetic and anti-obesity effects by promoting urinary excretion of glucose and subsequent caloric loss, we investigated the effect of the highly selective renal SGLT2 inhibitor dapagliflozin in mice with Western diet (WD) induced obesity. Low fat (LF) diet or WD-fed male C57BL/6J mice were treated with dapagliflozin for 26 weeks. Dapagliflozin attenuated the WD-mediated increases in body weight, plasma glucose and plasma triglycerides. Treatment with dapagliflozin prevented podocyte injury, glomerular pathology and renal fibrosis determined by second harmonic generation (SHG), nephrin, synaptopodin, collagen IV, and fibronectin immunofluorescence microscopy. Oil Red O staining showed dapagliflozin also decreased renal lipid accumulation associated with decreased SREBP-1c mRNA abundance. Moreover, renal inflammation and oxidative stress were lower in the dapagliflozin-treated WD-fed mice than in the untreated WD-fed mice. In addition, dapagliflozin decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), hepatic lipid accumulation as determined by H&E and Oil Red O staining, and Coherent Anti-Stokes Raman Scattering (CARS) microscopy, and hepatic fibrosis as determined by picrosirius red (PSR) staining and TPE-SHG microscopy in WD-fed mice. Thus, our study demonstrated that the co-administration of the SGLT2 inhibitor dapagliflozin attenuates renal and liver disease during WD feeding of mice.

Keywords: SGLT2; dapagliflozin; fibrosis; inflammation; lipid; obesity.

Figure 1

Effect of western diet and dapagliflozin on body weight and food intake. (A) Body weight was higher in the WD-fed group than in low fat group during the study. Body weight in the WD-fed with dapagliflozin group was lower than in the WD-fed group from 10 to 36 weeks of age. (B) Food intake in WD + dapagliflozin group was slightly higher than in the WD group. Results are expressed as means ± SEM (n = 6 mice per experimental and treatment group). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05), & LF + dapagliflozin group vs. LF group (p < 0.05).

Figure 2

Dapagliflozin prevented glomerular pathology and increased urinary albumin excretion in WD-fed mice. (A) Representative periodic acid-Schiff (PAS) staining of kidney sections. Mesangial expansion index was defined as the percentage of mesangial area in glomerular tuft area. The mesangial area was determined by assessment of PAS-positive and nucleus-free areas in the mesangium. Scale bar = 30 µm; (B) Urine albumin vs. creatinine ratio. Results are expressed as means ± SEM (n = 6 mice per group). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05).

Figure 3

Dapagliflozin prevented glomerular pathology and renal fibrosis in WD-fed mice. Shown are representative images of Immunofluorescence staining of kidney sections for (A) collagen IV; (B) Fibronectin; (C) Representative merged two-photon excitation (green)-SHG (red) images of kidney sections; (D) synaptopodin; and (E) nephrin. Scale bar = 20 µm in A, B, D and E, 100 µm in C. Results are expressed as means ± SEM (n = 6 mice per group). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05).

Figure 4

Dapagliflozin decreased renal lipid accumulation in WD-fed mice. (A) Oil red O staining of kidney sections. Scale bar = 20 µm; (B) Kidney triglyceride content analysis; (C) Quantitative real-time PCR (qRT-PCR) analysis of fatty acid synthesis master gene (SREBP-1c). Results are expressed as means ± SEM (n = 6 mice per group). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05).

Figure 5

Dapagliflozin decreased renal inflammation and oxidative stress in WD-fed mice. (A–E) Quantitative real-time PCR (qRT-PCR) analysis of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), intercellular adhesion molecule-1 (ICAM-1), toll-like receptor 2 (TLR2), monocyte chemotactic protein-1 (MCP-1) and osteopontin (OPN); (F) Immunofluorescence staining of kidney sections for CD68 (green) and the quantification of the green florescence. Scale bar = 20 µm; (G) Quantitative real-time PCR (qRT-PCR) analysis of Nox-2; (H) Western blot of Nox-2 in kidney and quantification of the observed levels. Equal protein loading and transfer was verified by using an anti-β-actin antibody. The detection was performed by an ECL western blotting analysis system. Results are expressed as means ± SEM (n = 6 mice per group, n = 4 for western blotting). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05), & LF + dapagliflozin group vs. LF group (p < 0.05).

Figure 6

Dapagliflozin reduced hepatic injury and lipid accumulation in liver of WD-fed mice. (A,B) Serum ALT and AST activity; (C) H&E staining, CV, central vein and PT, portal triad; (D) Oil red O staining of liver sections; (E) lipid droplets were visualized by Coherent Anti-Stokes Raman Scattering (CARS) Microscopy of liver sections; (F,G) Quantitative real-time PCR (qRT-PCR) analysis of fatty acid synthesis master gene (SREBP-1c) and carbohydrate-responsive element-binding protein-β (ChREBP-β). Scale bars = 100 µm. Results are expressed as means ± SEM (n = 6 mice per group). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05).

Figure 7

Dapagliflozin decreased inflammation in the liver of WD-fed mice. (A–E) Quantitative real-time PCR (qRT-PCR) analysis of monocyte chemoattractant protein-1 (MCP1), tumor necrosis factor alpha (TNFα), Toll-like receptor 4 (TLR4), interleukin-1 beta (IL-1β) and Osteopontin (OPN) in liver. Results are expressed as means ± SEM (n = 6 mice per group). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05).

Figure 8

Dapagliflozin decreased liver fibrosis in WD-fed obesity mice. (A) Picro Sirius Red staining for fibrillar collagens in liver sections, when visualized using either brightfield or polarized light microscopy of the same field; (B) Representative merged two-photon excitation (green)-SHG (red) images of liver sections; (C–E) Quantitative real-time PCR (qRT-PCR) analysis of profibrotic factor TGF-β, collagen1a1 and collagen3a1 gene expression in the liver. Scale bars = 200 µm in (A), 100 µm in (B). Results are expressed as means ± SEM (n = 6 mice per group). Statistical analysis was performed with one-way ANOVA. * vs. low fat group (p < 0.05), # vs. Western diet group (p < 0.05).