Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice

Abstract

Recent studies have demonstrated that chronic inflammation-induced lymphangiogenesis plays a crucial role in the progression of various renal diseases, including diabetic nephropathy. SAR131675 is a selective vascular endothelial cell growth factor receptor-3 (VEGFR-3)-tyrosine kinase inhibitor that acts as a ligand for VEGF-C and VEGF-D to inhibit lymphangiogenesis. In this study, we evaluated the effect of SAR131675 on renal lymphangiogenesis in a mouse model of type 2 diabetes. Male C57BLKS/J db/m and db/db mice were fed either a regular chow diet or a diet containing SAR131675 for 12 weeks from 8 weeks of age. In addition, we studied palmitate-induced lymphangiogenesis in human kidney-2 (HK2) cells and RAW264.7 monocytes/macrophages, which play a major role in lymphangiogenesis in the kidneys. SAR131475 ameliorated dyslipidemia, albuminuria, and lipid accumulation in the kidneys of db/db mice, with no significant changes in glucose and creatinine levels and body weight. Diabetes-induced systemic inflammation as evidenced by increased systemic monocyte chemoattractant protein-1 and tumor necrosis factor-α level was decreased by SAR131475. SAR131475 ameliorated the accumulation of triglycerides and free fatty acids and reduced inflammation in relation to decreased chemokine expression and pro-inflammatory M1 macrophage infiltration in the kidneys. Downregulation of VEGF-C and VEGFR-3 by SAR131475 inhibited lymphatic growth as demonstrated by decreased expression of LYVE-1 and podoplanin that was further accompanied by reduced tubulointerstitial fibrosis, and inflammation in relation to improvement in oxidative stress and apoptosis. Treatment with SAR131475 improved palmitate-induced increase in the expression of VEGF-C, VEGFR-3, and LYVE-1, along with improvement in cytosolic and mitochondrial oxidative stress in RAW264.7 and HK2 cells. Moreover, the enhanced expression of M1 phenotypes in RAW264.7 cells under palmitate stress was reduced by SAR131475 treatment. The results suggest that modulation of lymphatic proliferation in the kidneys is a new treatment approach for type 2 diabetic nephropathy and that SAR131675 is a promising therapy to ameliorate renal damage by reducing lipotoxicity-induced lymphangiogenesis.

Fig. 1. SAR131675 treatment decreases circulating monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) level in db/db mice.

Circulating MCP-1 and TNF-α level were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. *P < 0.05 and ^#P < 0.001 vs. other groups

Fig. 2. SAR131675 attenuates intrarenal lipid accumulation, inflammation, and macrophage M1 polarization in db/db mice.

Lipid accumulation in the kidneys, inflammatory cell infiltration, and macrophage polarization were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative images of oil red O staining. b Intrarenal total cholesterol, free fatty acids, and triglycerides. c Monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) levels. d Representative images of immunohistochemical staining for F4/80-positive cells. e Representative western blots for CD68, arginase I, arginase II, inducible nitric oxide synthase (iNOS), and β-actin and quantitative data. *P < 0.05, **P < 0.01, and ^#P < 0.001 vs. other groups

Fig. 2. SAR131675 attenuates intrarenal lipid accumulation, inflammation, and macrophage M1 polarization in db/db mice.

Lipid accumulation in the kidneys, inflammatory cell infiltration, and macrophage polarization were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative images of oil red O staining. b Intrarenal total cholesterol, free fatty acids, and triglycerides. c Monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) levels. d Representative images of immunohistochemical staining for F4/80-positive cells. e Representative western blots for CD68, arginase I, arginase II, inducible nitric oxide synthase (iNOS), and β-actin and quantitative data. *P < 0.05, **P < 0.01, and ^#P < 0.001 vs. other groups

Fig. 3. SAR131675 attenuates transforming growth factor (TGF-β) expression and lymphangiogenesis in the kidneys in db/db mice.

Effects of SAR131675 on the expression of TGF-β, vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, LYVE-1, and podoplanin in the cortex and medulla were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative immunohistochemistry for TGF-β and VEGF-C. b Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin in the cortex and medullar and quantitative data. c Representative images of immunohistochemical staining for LYVE-1 and podoplanin, and d western blots for LYVE-1, podoplanin, and β-actin in the cortex and medullar and quantitative data. *P < 0.05, **P < 0.01, and ^#P < 0.001 vs. other groups

Fig. 3. SAR131675 attenuates transforming growth factor (TGF-β) expression and lymphangiogenesis in the kidneys in db/db mice.

Effects of SAR131675 on the expression of TGF-β, vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, LYVE-1, and podoplanin in the cortex and medulla were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative immunohistochemistry for TGF-β and VEGF-C. b Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin in the cortex and medullar and quantitative data. c Representative images of immunohistochemical staining for LYVE-1 and podoplanin, and d western blots for LYVE-1, podoplanin, and β-actin in the cortex and medullar and quantitative data. *P < 0.05, **P < 0.01, and ^#P < 0.001 vs. other groups

Fig. 4. SAR131675 decreases glomerulosclerosis and tubulointerstitial fibrosis in db/db mice.

Effects of SAR131675 on glomerulosclerosis and tubulointerstitial fibrosis were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative images of periodic acid Schiff (PAS) and trichrome staining and immunohistochemistry for type IV collagen (Col IV) and quantitative data. b Representative images of trichrome staining and immunohistochemistry for transforming growth factor (TGF-β) and Col IV and quantitative data. c Representative images of immunohistochemistry for α-smooth muscle actin (α-SMA) and western blots for α-SMA and β-actin in the cortex and medullar and quantitative data. d Representative western blots for fibronectin and β-actin in the cortex and medullar and quantitative data. *P < 0.05, **P < 0.01, and ^#P < 0.001 vs. other groups

Fig. 4. SAR131675 decreases glomerulosclerosis and tubulointerstitial fibrosis in db/db mice.

Effects of SAR131675 on glomerulosclerosis and tubulointerstitial fibrosis were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative images of periodic acid Schiff (PAS) and trichrome staining and immunohistochemistry for type IV collagen (Col IV) and quantitative data. b Representative images of trichrome staining and immunohistochemistry for transforming growth factor (TGF-β) and Col IV and quantitative data. c Representative images of immunohistochemistry for α-smooth muscle actin (α-SMA) and western blots for α-SMA and β-actin in the cortex and medullar and quantitative data. d Representative western blots for fibronectin and β-actin in the cortex and medullar and quantitative data. *P < 0.05, **P < 0.01, and ^#P < 0.001 vs. other groups

Fig. 5. SAR131675 attenuates apoptosis and oxidative stress in the kidneys in db/db mice.

Glomerulosclerosis and tubulointerstitial fibrosis were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative images of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining in the glomeruli and tubules and quantitative data. b Representative western blots for B cell leukemia/lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and β-actin in the kidneys and quantitative data. c Twenty-four hour urinary 8-hydroxy-deoxyguanosine (8-OH-dG) and isoprostane levels. *P < 0.05, **P < 0.01, and ^#P < 0.001 vs. other groups

Fig. 6. SAR131675 decreases high-glucose and palmitate-induced vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-3 (VEGFR-3), and LYVE-1 expression in human kidney-2 (HK2) cells.

To determine whether the addition of SAR131675 might modulate lymphangiogenesis in human proximal tubular epithelial HK2 cells, the cells were stimulated with palmitate (500 μM) and exposed to SAR131675 at 1, 10, or 100 nM in low-glucose (LG; 5 mmol/L d-glucose) or high-glucose (HG; 30 mmol/L d-glucose) medium. Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin and quantitative data. *P < 0.05, **P < 0.01 vs. LG control

Fig. 7. SAR131675 decreases high-glucose and palmitate-induced vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-3 (VEGFR-3), and LYVE-1 expression and M polarization in RAW264.7 cells.

To determine whether SAR131675 might modulate lymphangiogenesis and macrophage polarization in RAW264.7 murine macrophage cells, the cells were stimulated with palmitate (500 μM) in high-glucose medium (HG; 30 mmol/l d-glucose) and exposed to SAR131675 at 1, 10, and 100 nM. a Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin and quantitative data. b Representative western blots for CD68, arginase I, and arginase II, inducible nitric oxide synthase (iNOS), and β-actin and quantitative data. c Representative confocal microscopy images showing dihydroethidium (DHE) and MitoSOX fluorescence in the human kidney-2 (HK2) and RAW264.7 cells and quantitative data. *P < 0.05, **P < 0.01, ^#P < 0.001 vs. control

Fig. 7. SAR131675 decreases high-glucose and palmitate-induced vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-3 (VEGFR-3), and LYVE-1 expression and M polarization in RAW264.7 cells.

To determine whether SAR131675 might modulate lymphangiogenesis and macrophage polarization in RAW264.7 murine macrophage cells, the cells were stimulated with palmitate (500 μM) in high-glucose medium (HG; 30 mmol/l d-glucose) and exposed to SAR131675 at 1, 10, and 100 nM. a Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin and quantitative data. b Representative western blots for CD68, arginase I, and arginase II, inducible nitric oxide synthase (iNOS), and β-actin and quantitative data. c Representative confocal microscopy images showing dihydroethidium (DHE) and MitoSOX fluorescence in the human kidney-2 (HK2) and RAW264.7 cells and quantitative data. *P < 0.05, **P < 0.01, ^#P < 0.001 vs. control