NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate

Kidney Int. 1998 Mar;53(3):654-63. doi: 10.1046/j.1523-1755.1998.00796.x.

Abstract

Reactive oxygen species contribute to glomerular damage and proteinuria. In this study, we show that cultured human podocytes produce superoxide in response to extracellular adenosine triphosphate (ATP), and we identified the oxidases involved in this process. Adenosine triphosphate (10-4 M for 4 hr) raised superoxide production from 1.28 +/- 0.15 to 2.67 &/- 0.34 nmol/mg protein/min. Studies with podocyte homogenates revealed activation of both nicotinamide adenine dinucleotide (NADH; from 2.65 +/- 0.23 to 7.43 +/- 0.57) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidases [from 1.74 +/- 0.13 to 4.05 +/- 0.12 (nmol O2/mg protein/min)] by ATP. Activity of xanthine-oxidases was low and unchanged by ATP. Activation of the plasma-membrane bound NAD(P)H oxidases by ATP was time and dose dependent. Reverse transcribed-polymerase chain reaction (RT-PCR) studies with primers derived from monocyte sequences amplified mRNA for the NADPH oxidase subunits p22phox, p47phox, gp91phox, and p67phox, and the latter was transiently increased by ATP. Experiments with actinomycin D and cycloheximide suggested that ATP modulates enzyme activity at the transcriptional and translational levels. In conclusion, NAD(P)H dependent, membrane associated oxidases represent the major superoxide source in human podocytes. Activation of NAD(P)H oxidase by ATP might be secondary to increased mRNA expression of the NADPH oxidase subunit gp67phox.

MeSH terms

  • Adenosine Triphosphate / pharmacology*
  • Cell Membrane / enzymology
  • Cells, Cultured
  • Gene Expression Regulation, Enzymologic / drug effects
  • Humans
  • Kidney Glomerulus / cytology
  • Kidney Glomerulus / drug effects*
  • Kidney Glomerulus / metabolism*
  • Membrane Glycoproteins / genetics
  • Membrane Transport Proteins*
  • Monocytes / metabolism
  • NADH, NADPH Oxidoreductases / genetics
  • NADH, NADPH Oxidoreductases / metabolism*
  • NADPH Dehydrogenase / genetics
  • NADPH Oxidase 2
  • NADPH Oxidases*
  • Phosphoproteins / genetics
  • Polymerase Chain Reaction
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Reactive Oxygen Species / metabolism
  • Superoxides / metabolism

Substances

  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • Phosphoproteins
  • RNA, Messenger
  • Reactive Oxygen Species
  • neutrophil cytosol factor 67K
  • Superoxides
  • Adenosine Triphosphate
  • NADH, NADPH Oxidoreductases
  • CYBB protein, human
  • NADPH Oxidase 2
  • NADPH Oxidases
  • CYBA protein, human
  • neutrophil cytosolic factor 1
  • NADPH Dehydrogenase