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Protein Overload-Induced NF-B 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^* and Giuseppe Remuzzi^*

*Mario Negri Institute for Pharmacological Research, Bergamo, Italy; and Unit of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy.

Correspondence to: Dr. Marina Morigi, Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy. Phone: +39-035-319-888; Fax: +39-035-319-331; E-mail: morigi{at}marionegri.it

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-B, 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-B 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-B 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-B activation. Stimulation of HK-2 with exogenous H₂O₂ resulted in the activation of a NF-B subunit pattern similar to that obtained after protein challenge. Specific inhibitors of protein kinase C (PKC) activity significantly prevented H₂O₂ production and consequent NF-B 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-B-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-B activation and consequent induction of NF-B-dependent inflammatory signals.

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