Thrombospondin 1 Deficiency Ameliorates the Development of Adriamycin-Induced Proteinuric Kidney Disease

Accumulating evidence suggests that thrombospondin 1 (TSP1) is an important player in diabetic nephropathy. However, the role of TSP1 in podocyte injury and the development of non-diabetic proteinuric kidney disease is largely unknown. In the current study, by using a well-established podocyte injury model (adriamycin-induced nephropathy mouse model), we examined the contribution of TSP1 to the development of proteinuric kidney disease. We found that TSP1 was up-regulated in the glomeruli, notably in podocytes, in adriamycin injected mice before the onset of proteinuria. ADR treatment also stimulated TSP1 expression in cultured human podocytes in vitro. Moreover, increased TSP1 mediated ADR-induced podocyte apoptosis and actin cytoskeleton disorganization. This TSP1’s effect was through a CD36-dependent mechanism and involved in the stimulation of p38MAPK pathway. Importantly, in vivo data demonstrated that TSP1 deficiency protected mice from ADR induced podocyte loss and foot process effacement. ADR induced proteinuria, glomerulosclerosis, renal macrophage infiltration and inflammation was also attenuated in TSP1 deficient mice. Taken together, these studies provide new evidence that TSP1 contributes to the development of non-diabetic proteinuric kidney disease by stimulating podocyte injury and the progression of renal inflammation

CD47 Deficiency Protects Mice from Diet-Induced Obesity and Improves Whole Body Glucose Tolerance and Insulin Sensitivity

CD47 is a transmembrane protein with several functions including self-recognition, immune cell communication, and cell signaling. Although it has been extensively studied in cancer and ischemia, CD47 function in obesity has never been explored. In this study, we utilized CD47 deficient mice in a high-fat diet induced obesity model to study for the first time whether CD47 plays a role in the development of obesity and metabolic complications. Male CD47 deficient and wild type (WT) control mice were fed with either low fat (LF) or high fat (HF) diets for 16 weeks. Interestingly, we found that CD47 deficient mice were protected from HF diet-induced obesity displaying decreased weight gain and reduced adiposity. This led to decreased MCP1/CCR2 dependent macrophage infiltration into adipose tissue and reduced inflammation, resulting in improved glucose tolerance and insulin sensitivity. In addition, CD47 deficiency stimulated the expression of UCP1 and carnitine palmitoyltransferase 1b (CPT1b) levels in brown adipose tissue, leading to increased lipid utilization and heat production. This contributes to the increased energy utilization and reduced adiposity observed in these mice. Taken together, these data revealed a novel role for CD47 in the development of obesity and its related metabolic complications

Primer sequences for q-PCR.

<p>Primer sequences for q-PCR.</p

ADR or TSP1 treatment induced human podocyte apoptosis and actin cytoskeleton disorganization.

<p>Human podopcytes were treated with ADR (20μg/ml) or TSP1 (5μg/ml) for 24 hours. Then, TUNEL and F-actin staining was performed (A). TUNEL positive cells were calculated (B). Caspase 3 activity in cell lysates was measured (C). Data are presented as mean ± SE (n = 3 individual experiments). ***, <i>P</i> <0.001 vs. control.</p

ADR-induced renal macrophage infiltration and inflammation was attenuated in TSP1 deficient mice.

<p>(A). Kidney samples from ADR or saline injected WT or TSP1-/- mice were stained with anti-F4/80 antibody. Representative images were shown. The positive brown staining was indicated by red arrows. (B). mRNA levels of pro-inflammatory cytokines including F4/80, MCP-1, CD11c, TNF-α, IL-1β and CD31 in the glomeruli were determined by real-time PCR. Data are presented as mean ± SE (n = 3–5 mice /group). * <i>P</i> <0.05; ** P<0.01; ***P<0.001.</p

TSP1 expression was up-regulated in podocytes from adriamycin (ADR) treatment <i>in vivo</i> as well as <i>in vitro</i>.

<p>(A). Glomeruli TSP1 expression from ADR injected BALB/c mice was determined by immunoblotting. Data are represented as mean ± SE (n = 3 mice /group). * p<0.05, ** P<0.01 vs. control. (B). Colocolization of TSP1 with nephrin in glomeruli was determined by immunofluorescence staining of kidney samples from ADR injected mice. ADR (20μg/ml) treated human podocyte was harvested for analyzing TSP1 expression in mRNA (C) and protein (D) levels by real-time PCR and immunoblotting, respectively. Data are presented as mean ± SE (n = 3 individual experiments).</p

TSP1 treatment induced human podocyte apoptosis through activation of CD36-dependent p38 MAPK pathway.

<p>(A). Human podocytes were treated with different concentration of TSP1 (0.1, 1, and 5μg/ml) in the presence or absence of anti-CD36 antibody (15 μg/ml) for 15 minutes. Then phospho-p38MAPK level in cell lysates was determined by immunoblotting and normalized to total p38 levels. (B) Human podocytes were pre-incubated with SB202190 (p38 inhibitor, at 10⁻⁵ mol/L) and anti-CD36 antibody (15 μg/ml) for 2 h and then treated with TSP1 (5μg/ml) for 24 hours. Cells were then stained with TUNEL to calculate apoptotic cells. Data are presented as mean ± SE (n = 3 individual experiments). ***, <i>P</i> <0.001 vs. Ctrl; ##, P<0.01 vs.TSP1; ###, P<0.001 vs. TSP1 (5).</p

ADR induced podocyte apoptosis and actin cytoskeleton disorganization was attenuated in TSP1 deficient podocytes.

<p>(A). Podocytes were isolated from wild type or TSP1deficient mice and characterized by staining cells with WT1. (B). Cells were treated with ADR (20 μg/ml) for 24 h. After treatment, TUNEL staining and F-actin staining were performed. TUNEL positive cells were counted. Data are presented as mean ± SE (n = 3 individual experiments). *P<0.05.</p

ADR-induced proteinuria and glomerulosclerosis was attenuated in TSP1 deficient mice.

<p>ADR was injected into male TSP1-/- mice and wild type control mice. After 14 days of injection, urinary albumin and creatinine ratio (A) and serum creatinine (B) levels were determined. (C) Glomerular collagen type IV mRNA levels were determined by real-time PCR. Data are presented as mean ± SE (n = 3–5 mice /group). *, <i>P</i> <0.05 and **, P<0.01. (D) Representative images of immunohistochemical staining of kidney samples with anti-collagen IV antibody were shown. The positive staining was shown as brown.</p

ADR-induced podocyte injury was attenuated in TSP1 deficient mice.

<p>ADR was injected into male TSP1-/- mice and wild type control mice to induced nephropathy. (A) Representative overlay images of immunofluorescence staining of frozen kidney sections with WT1 (green) and DAPI (purple) were shown. WT1 positive cells in glomeruli were counted. Data are presented as mean ± SE (n = 3–5 mice /group). *, <i>P</i> <0.05. (B) Representative images of electron microscopy showed that ADR injected wild type mice had foot process effacement (red arrow).</p