Thrombospondin 1 mediates renal dysfunction in a mouse model of high-fat diet-induced obesity

Abstract

Obesity is prevalent worldwide and is a major risk factor for many diseases including renal complications. Thrombospondin 1 (TSP1), a multifunctional extracellular matrix protein, plays an important role in diabetic kidney diseases. However, whether TSP1 plays a role in obesity-related kidney disease is unknown. In the present studies, the role of TSP1 in obesity-induced renal dysfunction was determined by using a diet-induced obese mouse model. The results demonstrated that TSP1 was significantly upregulated in the kidney from obese mice. The increased TSP1 was localized in the glomerular mesangium as well as in the tubular system from obese wild-type mice. Obese wild-type mice developed renal hypertrophy and albuminuria, which was associated with increased kidney macrophage infiltration, augmented kidney inflammation, and activated transforming growth factor (TGF)-β signaling and renal fibrosis. In contrast, obese TSP1-deficient mice did not develop these kidney damages. Furthermore, in vitro studies demonstrated that leptin treatment stimulated the expression of TSP1, TGF-β1, fibronectin, and collagen type IV in mesangial cells isolated from wild-type mice. These leptin-stimulated effects were abolished in TSP1-deficient mesangial cells. Taken together, these data suggest that TSP1 is an important mediator for obesity- or hyperleptinemia-induced kidney dysfunction.

Keywords: kidney; leptin; obesity; thrombospondin 1.

Fig. 1.

Renal thrombospondin 1 (TSP1) levels were increased in high-fat diet-induced obesity mouse model. Wild-type mice (C57BL/6J) were fed with a low-fat (LF; 10% fat) or a high-fat (HF; 60% fat) diet for 16 wk. Lean mice were referred as LF feeding group. Obese mice were referred as HF feeding group. At the end of the study, mice were killed and kidneys were harvested. Kidney TSP1 mRNA (A) and protein (B) levels were determined by real-time PCR and immunoblotting, respectively. Data are presented as means ± SE (n = 10 mice/group). *P < 0.05 vs. lean mice. C: immunohistochemical staining showing the increased TSP1 in glomeruli as well as in the tubular system. The representative light photomicrograph was shown. The positive TSP1 staining was shown as brown color. The scale bar represents 10 μm.

Fig. 2.

TSP1 deficiency protected mice from obesity-induced kidney functional changes in vivo. Male TSP1−/− mice at 8 wk of age and age-matched littermate controls were fed with LF or HF diet for 16 wk. At the end of the study, glomerular filtration rate (GFR; A) and urinary albumin-to-creatinine ratio (ACR; B) were examined. Data are presented as means ± SE (n = 10 mice/group). *P < 0.05 vs. wild-type (WT) lean mice. #P < 0.05 vs. WT obese mice. &P < 0.05 vs. lean TSP1 knockout (KO) mice.

Fig. 3.

TSP1 deficiency protected mice from obesity-induced kidney structural changes in vivo. Male TSP1−/− mice at 8 wk of age and age-matched littermate controls were fed with LF or HF diet for 16 wk. At the end of the study, mice were killed and kidneys were harvested. A: body weight. B: left kidney weight. C: left kidney-to-body weight ratio. D: representative light micrographs of periodic acid-Schiff (PAS)-stained kidney sections from 4 groups of mice. E: glomerular area was analyzed by computer image analysis software. Data are means ± SE (n = 10 mice/group). *P < 0.05 vs. WT lean mice. #P < 0.05 vs. WT obese mice. The scale bar represents 10 μm.

Fig. 4.

Lipid accumulation in the kidney from both wild-type and TSP1-deficient mice. A: representative photograph of Oil Red O staining of frozen kidney sections from 4 groups of mice. Lipid droplets were shown as red spots indicated by arrows. B: triglyceride contents were measured in lipid extracts from kidney samples. Data are presented as means ± SE (n = 4 mice/group). *P < 0.05.

Fig. 5.

TSP1 deficiency protected mice from obesity-induced kidney fibrotic changes in vivo. Kidney phosphor-Smad2 (A) and phosphor-Smad3 (B) levels were detected using immunohistochemical staining, followed by semiquantitative analysis. Fibronectin (C) and collagen type IV (D) protein levels in the kidney cortex were examined by immunoblotting. Data are means ± SE (n = 6–10 mice/group). *P < 0.05 vs. WT lean mice. #P < 0.05 vs. WT obese mice. The scale bar represents 10 μm.

Fig. 6.

TSP1 deficiency protected mice from obesity-induced hypertension. At the end of the study, blood pressure was measured in 4 groups of mice using tail-cuff method. Data are means ± SE (n = 6 mice/group). *P < 0.05 vs. WT lean mice. #P < 0.05 vs. WT obese mice.

Fig. 7.

TSP1 deficiency protected mice from obesity-induced renal inflammation. After 16 wk of LF or HF feeding, mice were killed and kidneys were harvested. A: representative image of kidney sections from 4 groups of mice stained with anti-F4/80 antibody to identify macrophage accumulation in kidney tissue. The positive staining showed brown color and was indicated by arrows. The scale bar represents 10 μm. B–E: mRNA levels of F4/80, TNF-α, IL-6, and monocyte chemotactic protein-1 (MCP-1) levels in the kidney were determined by real-time PCR and normalized to 18sRNA. Data are means ± SE (n = 6 mice/group). *P < 0.05 vs. WT lean mice. #P <0.05 vs. WT obese mice.

Fig. 8.

Plasma leptin levels from wild-type and TSP1−/− mice. Male TSP1−/− mice at 8 wk of age and age-matched littermate controls were fed with a LF or HF diet for 16 wk. At the end of the study, mice were killed and blood samples were collected. Plasma leptin levels were measured. Data are means ± SE (n = 6 mice/group). *P < 0.05.

Fig. 9.

Leptin stimulated wild-type mesangial cell fibrotic phenotype change in vitro. Primary mesangial cells from wild-type mice were exposed either to leptin in a final concentration of 100 ng/ml or to PBS as control for 3–24 h. TSP1 (A, B), TGF-β1 (C, D), fibronectin (E, F), and collagen type IV (G, H) mRNA and protein levels were detected by real-time PCR and immunoblotting, respectively. Experiments were repeated at least 3 times and the data are presented as means ± SE. *P < 0.05 vs. control group.

Fig. 10.

TSP1-deficient mesangial cell lost leptin-induced fibrotic phenotype. Primary mesangial cells from TSP1-deficient mice were exposed either to leptin in a final concentration of 100 ng/ml or to PBS as control for 3–24 h. TGF-β1 (A, B), fibronectin (C, D), and collagen type IV (E, F) mRNA and protein levels were detected by real-time PCR and immunoblotting, respectively. Experiments were repeated 3 times and the data are means ± SE.