Homocysteine-induced macrophage inflammatory protein-2 production by glomerular mesangial cells is mediated by PI3 Kinase and p38 MAPKReport as inadecuate




Homocysteine-induced macrophage inflammatory protein-2 production by glomerular mesangial cells is mediated by PI3 Kinase and p38 MAPK - Download this document for free, or read online. Document in PDF available to download.

Journal of Inflammation

, 6:27

First Online: 26 September 2009Received: 12 May 2009Accepted: 26 September 2009

Abstract

BackgroundHomocysteine Hcy and inflammatory cytokines have been linked to adverse outcomes in persons with cardiovascular and kidney diseases and recent reports suggest that cytokine-mediated inflammatory infiltrates may be an important contributor to the pathogenesis the aforementioned diseases. Although some reports suggest that Hcy directly influences inflammatory cytokine production, this proposition has not been supported by data from other studies. The objective of the current study was to a utilize an in vitro cellular model to identify cytokines that may be affected by Hcy and b examine the role of mitogen activated protein kinase MAPK and phosphatidyl inositol 3- PI3 Kinase in Hcy modulated cytokine production.

MethodsPrimary rat glomerular mesangial cells MC, passage 8 to 15, isolated by standard sieving methodology, were exposed to Hcy 15, 50 or 100 μM with L-cysteine L-Cys; 100 μM serving as a control. An antibody array was used to identify cytokines that were modulated when MCs were exposed to Hcy. Gene expression was assessed by quantitative RT-PCR, while western blotting analysis was used to assess cellular protein levels in the presence and absence of inhibitors of MAPK and PI3 Kinase. Finally, leukocyte adhesion assay was used to examine the effect of Hcy on leukocyte adhesion to glomerular MCs that were maintained in media without, and with, kinase inhibitors.

ResultsWe identified macrophage inflammatory protein 2 MIP-2 as a key cytokine that manifested increases in both protein and mRNA following exposure of glomerular MC to pathophysiologic Hcy levels 50 μM. Further analyses revealed that Hcy-induced MIP-2 was dependent on activation of p38 MAPK and PI3 kinase. MIP-2 enhanced leukocyte adhesion to MC and this MIP-2-enhanced leukocyte adhesion was also dependent on activation of p38 MAPK and PI3K. Finally, we demonstrate that leukocyte adhesion to MC is specifically inhibited by anit-MIP2 antibody.

ConclusionThe data suggest that Hcy participates in inflammatory cytokines production by glomerular MC and that Hcy-induced MIP-2 mediates leukocyte adhesion to MC.

List of AbbreviationsCKDchronic kidney disease

Cyscysteine

ESRDendstage kidney disease

DMEMDulbecco-s Modified Eagle-s Medium

ESRDEndstage Renal Disease

FBSfetal bovine serum

GFRglomerular filtration rate

Hcyhomocysteine

Hhcyhyperhomocysteinemia

MCP-1marcophage chemoattractant protein 1

MCmesangial cells

MAPKmitogen activated protein kinase

NF-κBnuclear factor kappa B

PI3 Kinasephosphatidyl inositol 3-Kinase

PBSphosphate buffered saline

SDS - PAGEsodium dodecyl sulphate -polyacrylamide gel electrophoresis

TBSTris buffered saline

TTBSTween-Tris buffered saline

TIMP-1Tissue inhibitor of metalloproteinase 1.

Electronic supplementary materialThe online version of this article doi:10.1186-1476-9255-6-27 contains supplementary material, which is available to authorized users.

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Author: Suresh Shastry - Leighton R James

Source: https://link.springer.com/



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