Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor kappaB in proximal tubule cells.
The transcription and translation of monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, are increased in proximal tubule epithelial cells (PTC) stimulated with pathophysiologically relevant concentrations of albumin. The purpose of this study was to investigate whether nuclear factor kappaB (NFkappaB)/Rel proteins play a role in albumin-induced MCP-1 transcription. Confluent monolayers of rat PTC in primary culture were stimulated with delipidated bovine serum albumin. NFkappaB, the NFkappaB inhibitory protein (IkappaB), and MCP-1 transcription were assessed using electrophoretic mobility shift assays, Western immunoblotting, semiquantitative reverse transcription-PCR, and ribonuclease protection assays. Activation of NFkappaB by delipidated bovine serum albumin (15 mg/ml) was detectable within 2 h, maximal after 8 h, and maintained for at least 16 h of continuous exposure. Supershift analysis showed that the activated proteins were composed of p50/p50, p50/p65, and p50/c-Rel dimers. dimers. Cytoplasmic IkappaBalpha levels were decreased 30 min after stimulation and returned to unstimulated levels by 4 to 8 h. IkappaBbeta levels were decreased at 2 h and there was no recovery until 8 h. Inhibition of NFkappaB with pharmacologic agents (N-tosyl-phenylalanine chloromethyl ketone and dexamethasone) and an antisense oligonucleotide to the rat p65 subunit of NFkappaB significantly reduced MCP-1 transcription. The 3.6-kb 5' flanking region of the rat MCP-1 gene was cloned and sequenced, and two putative kappaB binding sites were identified within the enhancer region. Therefore, albumin increased NFkappaB and reduced IkappaB levels in PTC, and MCP-1 expression was dependent on NFkappaB activation. It is concluded that the activation of NFkappaB/Rel proteins modulates chemokine production in PTC in response to albumin and is likely to have an important role in the mediation of tubulointerstitial injury in proteinuric renal disease.
Wang
Y
Y
Department of Renal Medicine, The University of Sydney at Westmead Hospital, NSW, Australia.
Rangan
G K
GK
Tay
Y C
YC
Wang
Y
Y
Harris
D C
DC
D000418:Albumins / Q000378:metabolism* / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured / Q000378:metabolism; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D012016:Reference Values; D012680:Sensitivity and Specificity
D000418:Albumins; D018932:Chemokine CCL2; D016328:NF-kappa B; D009841:Oligonucleotides