High glucose induces macrophage inflammatory protein-3 alpha in renal proximal tubule cells via a transforming growth factor-beta 1 dependent mechanism.
BACKGROUND
Hyperglycaemia is a causative factor in the pathogenesis of diabetic nephropathy, known to induce chemokines in the kidney. Macrophage inflammatory protein-3 alpha (MIP-3 alpha) is a CC chemokine that has been reported to attract memory T lymphocytes. Our previous microarray study showed significant increased level of MIP-3 alpha in high glucose-induced transcriptional profile in renal proximal tubule cells. Transforming growth factor-beta1 (TGF-beta1) is a key regulator in inflammation and fibrosis in diabetes mellitus setting.


METHODS
This study aimed to determine the role of TGF beta 1 in high glucose-induced MIP-3 alpha 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-beta1 gene in HK-2 cells and subsequent experiments were performed to measure mRNA and protein levels of MIP-3 alpha 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 alpha and CD3 a marker of T lymphocytes in the in vivo model.


RESULTS
MIP-3 alpha mRNA and protein expression was increased in HK-2 cells by high glucose and TGF-beta1. MIP-3 alpha 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 alpha. This up-regulation was attenuated in the presence of an angiotensin-converting enzyme (ACE) inhibitor. MIP-3 alpha expression significantly decreased in cells in which the TGF-beta1 gene was silenced using small interfering RNA. Furthermore, exposure to high glucose did not induce MIP-3 alpha expression in TGF-beta1 gene silenced cells compared with wild-type cells.


CONCLUSIONS
In summary, we have uniquely demonstrated that high glucose increases MIP-3 alpha through a TGF beta 1 dependent pathway, suggesting the centrality of TGF-beta1 in both the inflammatory and previously demonstrated fibrotic responses in diabetic nephropathy.
Qi
Weier
W
Kolling Institute, Department of Medicine, Royal North Shore Hospital, St. Leonards, NSW, Australia.
Chen
Xinming
X
Zhang
Yuan
Y
Holian
John
J
Mreich
Ellein
E
Gilbert
Richard E
RE
Kelly
Darren J
DJ
Pollock
Carol A
CA
D000818:Animals; D002460:Cell Line; D054418:Chemokine CCL20 / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers; D003921:Diabetes Mellitus, Experimental / Q000503:physiopathology; D003928:Diabetic Nephropathies / Q000503:physiopathology; D005260:Female; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000502:physiology*
D054418:Chemokine CCL20; D017931:DNA Primers; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D005947:Glucose