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J Am Soc Nephrol 10:1204-1213, 1999
© 1999 American Society of Nephrology

REGULAR ARTICLES

Induction of Monocyte Chemoattractant Protein-1 by Albumin Is Mediated by Nuclear Factor {kappa}B in Proximal Tubule Cells

YIPING WANG, GOPALA K. RANGAN, YUET-CHING TAY, YAO WANG and DAVID C. H. HARRIS

Department of Renal Medicine, The University of Sydney at Westmead Hospital, Westmead, Australia.

Correspondence to A/Prof. David C. H. Harris, Department of Renal Medicine, Westmead Hospital, Westmead NSW 2145, Australia. Phone: 61-2-9845-7388; Fax: 61-2-9633-9351; E-mail: dch{at}renal.wh.usyd.edu.au

Abstract. 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 {kappa}B (NF{kappa}B)/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. NF{kappa}B, the NF{kappa}B inhibitory protein (I{kappa}B), and MCP-1 transcription were assessed using electrophoretic mobility shift assays, Western immunoblotting, semiquantitative reverse transcription-PCR, and ribonuclease protection assays. Activation of NF{kappa}B 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 I{kappa}B{alpha} levels were decreased 30 min after stimulation and returned to unstimulated levels by 4 to 8 h. I{kappa}Bß levels were decreased at 2 h and there was no recovery until 8 h. Inhibition of NF{kappa}B with pharmacologic agents (N-tosyl-phenylalanine chloromethyl ketone and dexamethasone) and an antisense oligonucleotide to the rat p65 subunit of NF{kappa}B 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 {kappa}B binding sites were identified within the enhancer region. Therefore, albumin increased NF{kappa}B and reduced I{kappa}B levels in PTC, and MCP-1 expression was dependent on NF{kappa}B activation. It is concluded that the activation of NF{kappa}B/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.




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