Expression of inter-α-trypsin inhibitor and tumor necrosis factor-stimulated gene 6 in renal proximal tubular epithelial cells

Abstract

Our previous studies have demonstrated that renal proximal tubular epithelial cells (PTCs) may contribute to renal interstitial fibrosis by the generation of transforming growth factor-beta1 (TGF-beta1). In these in vitro experiments, TGF-beta1 was, however, secreted in its latent form. Plasmin has been implicated as a potential physiological activator of TGF-beta1. The inter-alpha-trypsin inhibitor (IalphaI) family of serum protease inhibitors together with tumor necrosis factor-stimulated gene 6 (TSG-6) recently have been implicated in the regulation of this protease pathway. The aim of the current study was to examine PTC synthesis of these proteins and to relate it to alterations of plasmin-protease activity. PTCs were grown to confluence and stimulated under serum-free conditions with either interleukin-1beta (IL-1beta) or 25 mmol/L D-glucose. Alterations in IalphaI and TSG-6 generation were detected by Western analysis of both membrane extracts and supernatant samples. Alterations in gene expression were examined by reverse transcription-polymerase chain reaction. The effect of alteration in synthesis of TSG-6 on plasmin activity was determined by quantitating plasmin inhibitory activity of supernatant samples by in vitro calorimetric assay prior to and following TSG-6 immunoprecipitation. The data demonstrate that human PTCs constitutively express mRNA for bikunin and heavy chain 3 (H3) of IalphaI. Neither IL-1beta (1 ng/mL) nor 25 mmol/L D-glucose influenced their mRNA expression nor protein synthesis. In contrast, the addition of either IL-1beta or 25 mmol/L D-glucose increased TSG-6 mRNA expression. This was accompanied by an early up-regulation of TSG-6 protein expression following IL-1beta stimulation (24 h) and a late up-regulation after the addition of 25 mmol/L D-glucose (96 h) in the cell culture supernatant and associated with the cell membranes. Early induction of TSG-6 mRNA by IL-1beta was unaffected by the addition of the protein synthesis inhibitor cycloheximide. In contrast, the later glucose-stimulated induction of TSG-6 mRNA was abrogated by the addition of cycloheximide. Stimulation of TSG-6 by either IL-1beta or 25 mmol/L D-glucose was associated with an inhibition of total percentage plasmin activity. Immunoprecipitation of TSG-6 in these samples returned plasmin activity to control levels. : The data demonstrate that human PTCs constitutively express the bikunin and H3 components of the IalphaI family of serum protease inhibitors. Moreover, the addition of IL-1beta or 25 mmol/L D-glucose up-regulates the expression of TSG-6 in these cells, resulting in an inhibition of plasmin activity.