High glucose-induced thioredoxin-interacting protein in renal proximal tubule cells is independent of transforming growth factor-beta1.
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)-beta1 under high-glucose conditions. Overexpression of Txnip and up-regulation of Txnip promoter activity were observed in cells in which the TGF-beta1 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-beta1 silenced cells compared with wild-type cells exposed to high glucose, suggesting that high glucose induced Txnip through a TGF-beta1-indepen-dent pathway.
Dept. of Medicine, University of Sydney, Sydney, New South Wales, Australia.
Weier
W
Qi
Xinming
X
Chen
Richard E
RE
Gilbert
Yuan
Y
Zhang
Mark
M
Waltham
Maria
M
Schache
Darren J
DJ
Kelly
Carol A
CA
Pollock
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D054418:Chemokine CCL20 / Q000235:genetics / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental; D005260:Female; D020869:Gene Expression Profiling; D005947:Glucose / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism; D019402:Macrophage Inflammatory Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D051381:Rats; D011919:Rats, Inbred Strains; D017382:Reactive Oxygen Species / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
C108355:CCL20 protein, human; D002352:Carrier Proteins; D054418:Chemokine CCL20; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D019402:Macrophage Inflammatory Proteins; D017382:Reactive Oxygen Species; C486715:TXNIP protein, human; D053773:Transforming Growth Factor beta1; D013879:Thioredoxins; D005947:Glucose