Published online before print August 3, 2007

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(American Journal of Pathology. 2007;171:744-754.)
© 2007 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2007.060813

High Glucose-Induced Thioredoxin-Interacting Protein in Renal Proximal Tubule Cells Is Independent of Transforming Growth Factor-ß1

Weier Qi^*, Xinming Chen^*, Richard E. Gilbert, Yuan Zhang, Mark Waltham, Maria Schache, Darren J. Kelly and Carol A. Pollock^*

From the Department of Medicine,^* Kolling Institute, Royal North Shore Hospital and University of Sydney, Sydney, New South Wales, Australia; the Department of Medicine, St. Vincent’s Hospital, Melbourne, Victoria, Australia; St. Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia; and the Department of Medicine, University of Toronto, St. Michael’s Hospital, Toronto, Ontario, Canada

Hyperglycemia is a causative factor in the pathogenesis of diabetic nephropathy. Here, we demonstrate the transcriptional profiles of the human proximal tubule cell line (HK-2 cells) exposed to high glucose using cDNA microarray analysis. Thioredoxin-interacting protein (Txnip) was the gene most significantly increased among 10 strongly up-regulated and 15 down-regulated genes. Txnip, heat shock proteins 70 and 90, chemokine (C-C motif) ligand 20, and matrix metalloproteinase-7 were chosen for verification of gene expression. Real-time reverse transcriptase-polymerase chain reaction confirmed the mRNA expression levels of these five genes, consistent with microarray analysis. The increased protein expression of Txnip, CCL20, and MMP7 were also verified by Western blotting and enzyme-linked immunosorbent assay. Increased expression of Txnip and of nitrotyrosine, as a marker of oxidative stress, were confirmed in vivo in diabetic Ren-2 rats. Subsequent studies focused on the dependence of Txnip expression on up-regulation of transforming growth factor (TGF)-ß1 under high-glucose conditions. Overexpression of Txnip and up-regulation of Txnip promoter activity were observed in cells in which the TGF-ß1 gene was silenced in HK-2 cells using short interfering RNA technology. High glucose further increased both Txnip expression and its promoter activity in TGF-ß1 silenced cells compared with wild-type cells exposed to high glucose, suggesting that high glucose induced Txnip through a TGF-ß1-indepen-dent pathway.

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