Molecular Mechanisms by Which Iron Induces Nitric Oxide Synthesis in Cultured Proximal Tubule Cells

Abstract

Nitric oxide (NO) levels are increased after exposure of cultured proximal tubule cells (PTC) to non-haem iron, potentially contributing to PTC injury in disease states associated with increased iron exposure, including proteinuric renal disease. The mechanisms underlying this observed increase were investigated. After 3 h exposure to 400 µM nitrilotriacetate (NTA)-Fe, inducible nitric oxide synthase (iNOS) mRNA expression was significantly increased, with a corresponding increase in iNOS protein after 12 h. The nuclear binding activity of NFĸB with 400 µM NTA-Fe was increased, and pyrrolidine dithiocarbamate (PDTC), an antioxidant inhibitor of NFĸB, prevented both activation of NFĸB and NO production in response to NTA-Fe. Inhibition of protein tyrosine kinase reduced iNOS mRNA, iNOS protein levels and NO production in response to NTA-Fe. The effect of tyrosine kinase inhibition on NFĸB activation was variable, with herbimycin but not genistein having an inhibitory effect. Activation of either protein kinase A or C increased iNOS mRNA and protein levels, and NO production in response to NTA-Fe, whereas only the protein kinase C activator phorbol dibutyrate (PDBu) had a stimulatory effect on NFĸB activation. The protein kinase A activator forskolin did not alter iron-induced activation of NFĸB. These data suggest that the observed increase in NO production by PTC in response to iron is due to increased transcription of iNOS. The transcriptional regulation of this response is complex and involves NFĸB, protein tyrosine kinase and the protein kinases A and C.