High glucose induces macrophage inflammatory protein-3α in renal proximal tubule cells via a transforming growth factor-β1 dependent mechanism

Background. Hyperglycaemia is a causative factor in the pathogenesis of diabetic nephropathy, known to induce chemokines in the kidney. Macrophage inflammatory protein-3a (MIP-3a) is a CC chemokine that has been reported to attract memory T lymphocytes. Our previous microarray study showed significant increased level of MIP-3a in high glucose-induced transcriptional profile in renal proximal tubule cells. Transforming growth factor-b1 (TGF-b1) is a key regulator in inflammation and fibrosis in diabetes mellitus setting. Methods. This study aimed to determine the role of TGFb1 in high glucose-induced MIP-3a 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-b1 gene in HK-2 cells and subsequent experiments were performed to measure mRNA and protein levels of MIP-3a using real time reverse transcription– polymerase chain reaction (RT–PCR) and enzymelinked immunosorbent assay (ELISA). Immunohistochemistry was used to measure the protein level of MIP-3a and CD3 a marker of T lymphocytes in the in vivo model. Results. MIP-3a mRNA and protein expression was increased in HK-2 cells by high glucose and TGF-b1. MIP-3a 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-3a. This up-regulation was attenuated in the presence of an angiotensinconverting enzyme (ACE) inhibitor. MIP-3a expression significantly decreased in cells in which the TGF-b1 gene was silenced using small interfering RNA. Furthermore, exposure to high glucose did not induce MIP-3a expression in TGF-b1 gene silenced cells compared with wild-type cells. Conclusions. In summary, we have uniquely demonstrated that high glucose increases MIP-3a through a TGFb1 dependent pathway, suggesting the centrality of TGF-b1 in both the inflammatory and previously demonstrated fibrotic responses in diabetic nephropathy.