Limits...
Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes

Tarabra E, Giunti S, Barutta F, Salvidio G, Burt D, Deferrari G, Gambino R, Vergola D, Pinach S, Perin PC, Camussi G, Gruden G - Diabetes (2009)

Bottom Line: In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism.The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy.In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.

Affiliation: Diabetic Nephropathy Laboratory, Department of Internal Medicine, University of Turin, Italy.

ABSTRACT

Objective: Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes.

Research design and methods: Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting.

Results: In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.

Conclusions: These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.

CCR2 staining of human glomeruli from control subjects and patients with diabetic nephropathy. CCR2 protein expression was evaluated in human glomeruli from control subjects (A and D) and diabetic patients with overt nephropathy (B and E) by immunohistochemistry as described in research design and methods. C: Nonspecific staining was determined by preabsorbing the anti-CCR2 antibody with a 10-fold excess of control peptide. F: Double immunofluorescence for CCR2 (F) and (G) the podocyte marker synaptopodin performed on the diabetic glomeruli showed colocalisation of the positive staining, as demonstrated by merging (H). Magnification ×400 (×80 D and E). Arrows and arrowhead indicate podocytes and mesangial cells, respectively. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy

Figure 4: CCR2 staining of human glomeruli from control subjects and patients with diabetic nephropathy. CCR2 protein expression was evaluated in human glomeruli from control subjects (A and D) and diabetic patients with overt nephropathy (B and E) by immunohistochemistry as described in research design and methods. C: Nonspecific staining was determined by preabsorbing the anti-CCR2 antibody with a 10-fold excess of control peptide. F: Double immunofluorescence for CCR2 (F) and (G) the podocyte marker synaptopodin performed on the diabetic glomeruli showed colocalisation of the positive staining, as demonstrated by merging (H). Magnification ×400 (×80 D and E). Arrows and arrowhead indicate podocytes and mesangial cells, respectively. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: In normal renal cortex only few glomerular cells per kidney biopsy, predominantly podocytes and mesangial cells, stained positively for CCR2, as assessed by immunohistochemistry (Fig. 4A and D). Specificity of the antibody binding was confirmed by disappearance of the signal when the antibody was preabsorbed with a 10-fold excess of control peptide (Fig. 4C).

View Similar Images In: Results  - Collection
View Article: Medline Plus - Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2731530&rFormat=json&query=the&fields=all&favor=none&it=none&sp=none&sub=none&req=5
Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes

Tarabra E, Giunti S, Barutta F, Salvidio G, Burt D, Deferrari G, Gambino R, Vergola D, Pinach S, Perin PC, Camussi G, Gruden G - Diabetes (2009)

CCR2 staining of human glomeruli from control subjects and patients with diabetic nephropathy. CCR2 protein expression was evaluated in human glomeruli from control subjects (A and D) and diabetic patients with overt nephropathy (B and E) by immunohistochemistry as described in research design and methods. C: Nonspecific staining was determined by preabsorbing the anti-CCR2 antibody with a 10-fold excess of control peptide. F: Double immunofluorescence for CCR2 (F) and (G) the podocyte marker synaptopodin performed on the diabetic glomeruli showed colocalisation of the positive staining, as demonstrated by merging (H). Magnification ×400 (×80 D and E). Arrows and arrowhead indicate podocytes and mesangial cells, respectively. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy
Figure 4: CCR2 staining of human glomeruli from control subjects and patients with diabetic nephropathy. CCR2 protein expression was evaluated in human glomeruli from control subjects (A and D) and diabetic patients with overt nephropathy (B and E) by immunohistochemistry as described in research design and methods. C: Nonspecific staining was determined by preabsorbing the anti-CCR2 antibody with a 10-fold excess of control peptide. F: Double immunofluorescence for CCR2 (F) and (G) the podocyte marker synaptopodin performed on the diabetic glomeruli showed colocalisation of the positive staining, as demonstrated by merging (H). Magnification ×400 (×80 D and E). Arrows and arrowhead indicate podocytes and mesangial cells, respectively. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: In normal renal cortex only few glomerular cells per kidney biopsy, predominantly podocytes and mesangial cells, stained positively for CCR2, as assessed by immunohistochemistry (Fig. 4A and D). Specificity of the antibody binding was confirmed by disappearance of the signal when the antibody was preabsorbed with a 10-fold excess of control peptide (Fig. 4C).

Bottom Line: In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism.The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy.In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.

Affiliation: Diabetic Nephropathy Laboratory, Department of Internal Medicine, University of Turin, Italy.

ABSTRACT

Background:

Objective: Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes.

Research design and methods: Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting.

Results: In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.

Conclusions: These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.

View Similar Images In: Results  - Collection
View Article: Medline Plus - Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2731530&rFormat=json&query=the&fields=all&favor=none&it=none&sp=none&sub=none&req=5