Induction of Monocyte Chemoattractant Protein-1 by Albumin Is Mediated by Nuclear Factor κB in Proximal Tubule Cells

Abstract

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