NDT Advance Access originally published online on July 29, 2007
Nephrology Dialysis Transplantation 2007 22(11):3147-3153; doi:10.1093/ndt/gfm365
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High glucose induces macrophage inflammatory protein-3
in renal proximal tubule cells via a transforming growth factor-β1 dependent mechanism
1Kolling Institute, Department of Medicine, Royal North Shore Hospital and University of Sydney, Sydney, 2Department of Medicine, St Vincent's Hospital, University of Melbourne, Melbourne, Australia and 3Department of Medicine, University of Toronto, St. Michael's Hospital, Canada
Correspondence and offprint requests to: Prof Carol A Pollock, Department of Medicine, Level 3, Wallace Freeborn Professorial Block, Royal North Shore Hospital, St Leonards, NSW, Australia 2065. Email: carpol{at}med.usyd.edu.au
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Abstract |
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Background. Hyperglycaemia is a causative factor in the pathogenesis of diabetic nephropathy, known to induce chemokines in the kidney. Macrophage inflammatory protein-3
(MIP-3) is a CC chemokine that has been reported to attract memory T lymphocytes. Our previous microarray study showed significant increased level of MIP-3 in high glucose-induced transcriptional profile in renal proximal tubule cells. Transforming growth factor-β1 (TGF-β1) is a key regulator in inflammation and fibrosis in diabetes mellitus setting.
Methods. This study aimed to determine the role of TGFβ1 in high glucose-induced MIP-3 expression. An in vitro model of human proximal tubular cells (HK-2 cells) and an in vivo model of the transgenic (mRen-2)27 diabetic rat, well characterized as a model of human diabetic nephropathy, were used. Small interfering RNA technology was used to silence TGF-β1 gene in HK-2 cells and subsequent experiments were performed to measure mRNA and protein levels of MIP-3 using real time reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to measure the protein level of MIP-3 and CD3 a marker of T lymphocytes in the in vivo model.
Results. MIP-3 mRNA and protein expression was increased in HK-2 cells by high glucose and TGF-β1. MIP-3 was up-regulated in the dilated tubules of diabetic rats compared with non-diabetic control animals and CD3 was found to be present around the dilated tubules expressing MIP-3. This up-regulation was attenuated in the presence of an angiotensin-converting enzyme (ACE) inhibitor. MIP-3 expression significantly decreased in cells in which the TGF-β1 gene was silenced using small interfering RNA. Furthermore, exposure to high glucose did not induce MIP-3 expression in TGF-β1 gene silenced cells compared with wild-type cells.
Conclusions. In summary, we have uniquely demonstrated that high glucose increases MIP-3 through a TGFβ1 dependent pathway, suggesting the centrality of TGF-β1 in both the inflammatory and previously demonstrated fibrotic responses in diabetic nephropathy.
Keywords: high glucose; macrophage inflammatory protein-3 (MIP-3); renal tubular cells; transforming growth factor-β1 (TGF-β1)
*The authors wish it to be known that, in their opinion, the first two authors contributed equally to this work.
Received for publication: 11.11.06
Accepted in revised form: 11. 5.07
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