NAD(P)H OXIDASE ACTIVITY IN CULTURED HUMAN PODOCYTES - EFFECTS OF ADENOSINE-TRIPHOSPHATE

Reactive oxygen species contribute to glomerular damage and proteinuri a. In this study, we show that cultured human podocytes produce supero xide in response to extracellular adenosine triphosphate (ATP), and we identified the oxidases involved in this process. Adenosine triphosph ate (10(-4) M far 4 hr) raised superoxide production from 1.28 +/- 0.1 5 to 2.67 +/- 0.34 nmol/mg protein/min. Studies with podocyte homogena tes revealed activation of both nicotinamide adenine dinucleotide (NAD H; from 2.65 +/- 0.23 to 7.43 +/- 0.57) and nicotinamide adenine dinuc leotide phosphate (NADPH) dependent oxidases [from 1.74 +/- 0.13 to 4. 05 +/- 0.12 (nmol O-2(.)/mg protein/min)] by ATP. Activity of xanthine -oxidases was low and unchanged by ATP. Activation of the plasma-membr ane bound NAD(P)H oxidases by ATP was time and dose dependent. Reverse transcribed-polymerase chain reaction (RT-PCR) studies with primers d erived from monocyte sequences amplified mRNA for the NADPH oxidase su bunits p22phox, p47phox, gp91phox and p67phox, and the latter was tran siently increased by ATP. Experiments with actinomycin D and cyclohexi mide suggested that ATP modulates enzyme activity at the transcription al and translational levels. In conclusion, NAD(P)H dependent, membran e associated oxidases represent the major superoxide source in human p odocytes. Activation of NAD(P)H oxidase by ATP might be secondary to i ncreased mRNA expression of the NADPH oxidase subunit gp67phox.