Protein overload-induced NF-κB activation in proximal tubular cells requires H2O2 through a PKC-dependent pathway

Marina Morigi, Daniela Macconi, Carla Zoja, Roberta Donadelli, Simona Buelli, Cristina Zanchi, Marina Ghilardi, Giuseppe Remuzzi

Research output: Contribution to journalArticle

Abstract

Abnormal traffic of proteins through the glomerular capillary has an intrinsic toxicity that results in tubular dysfunction and interstitial inflammation. It has been previously shown that in porcine proximal tubular cells high concentrations of albumin activated NF-κB, which is responsible for the enhanced synthesis of the inflammatory chemokine RANTES. This study investigates whether reactive oxygen species (ROS) served as second messengers in protein overload-induced NF-κB activation. Human proximal tubular cells (HK-2) were incubated (5 to 60 min) with human albumin and IgG (1 to 30 mg/ml). Both proteins induced a rapid or significant increase in hydrogen peroxide (H2O2) production at 5 min and persisting at 60 min. This effect was dose-dependent. The contribution of H2O2 in regulating NF-κB activation was evaluated by using the antioxidants dimethyl-thiourea and pyrrolidine dithiocarbamate in protein-overloaded HK-2 cells. Both agents, by preventing H2O2 generation, induced human albumin or IgG inhibited NF-κB activation. Stimulation of HK-2 with exogenous H2O2 resulted in the activation of a NF-κB subunit pattern similar to that obtained after protein challenge. Specific inhibitors of protein kinase C (PKC) activity significantly prevented H2O2 production and consequent NF-κB activation, suggesting that ROS generation in HK-2 cells occurs downstream of PKC activation. Either antioxidants or PKC inhibitor almost completely abolished the upregulation of the monocyte chemoattractant protein-1 gene induced by excess albumin, as evaluated by real-time PCR, thus supporting a role for PKC and ROS as critical signals for the expression of NF-κB-dependent inflammatory genes. To identify the enzymatic sources responsible for the increased H2O2 production, the effect of dyphenyleneiodonium, an inhibitor of the membrane NADP(H) oxidase, was studied, as was the effect of rotenone, which blocks complex I of the mitochondrial respiratory chain. It was found that both agents significantly reduced the exaggerated H2O2 induced by protein overload. These data indicate that exposure to excess proteins in proximal tubular cells induces the formation of ROS, which are responsible for NF-κB activation and consequent induction of NF-κB-dependent inflammatory signals.

Original languageEnglish
Pages (from-to)1179-1189
Number of pages11
JournalJournal of the American Society of Nephrology
Volume13
Issue number5
Publication statusPublished - 2002

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ASJC Scopus subject areas

  • Nephrology

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