In-vitro and in-vivo induction of interleukin-8 expression by albumin in proximal tubular epithelial cells: an extended study

Abstract

Introduction: We have previously demonstrated that albumin superinduces the expression of IL-8, a potent chemokine, in cultured human renal proximal tubular epithelial cells (PTEC). In this study, we explored the intracellular signaling mechanisms leading to heightened IL-8 production in the nephrotic mileu. Method: Human PTEC was obtained by primary culture. Endocytosis of albumin was assayed by flow cytometry. IL-8 gene and protein expression was measured by RT-PCR and ELISA, respectively. The role of NFkappaB was determined by electrophoretic mobility shift assay, and confirmed with the NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), and a decoy peptide. Intracellular generation of reactive oxygen species (ROS) was detected by a specific fluorescence probe. IL-8 expression in kidney biopsies of nephrotic subjects was studied by immunohistochemistry and in-situ hybridization. Results: Receptor-mediated endocytosis of albumin by PTEC significantly induced IL-8 gene expression, which was localized by in situ hybridization to the proximal tubules in nephrotic kidney tissues. Apical albumin stimulated IL-8 secretion in both directions, with basolateral secretion predominating. Albumin activated nuclear translocation of both the p65 and p50 subunits of NF-kappaB. There was corresponding upregulation of intracellular ROS. Albumin-induced tubular IL-8 secretion was abrogated by PDTC, or the cell-permeable peptide, SN50, but not the mutant sequence, SN50M. Histologic scores, graded in an observer-blinded fashion, for IL-8 immunostaining were higher in nephrotic than non-nephrotic subjects (P = 0.039), and correlated with the magnitude of proteinuria (r2 = 0.332, b = 0.116 [95% CI, 0.029-0.203], P = 0.012). Conclusion: Albumin is a strong stimulus for tubular IL-8 expression both in vivo and in vitro, which occurs via NF-κB-dependent pathways. Our findings provide evidence that tubule-secreted IL-8 is important in the pathogenesis of tubulointerstitial inflammation in the proteinuric state. *Supported by the UDM Research Grant 00/01.