The expression of NADPH oxidase components in human glomerular mesangial cells: detection of protein and mRNA for p47phox, p67phox, and p22phox.

Abstract

Previous work has shown that human mesangial cells (HMC) are capable of low rates of generation of reactive oxygen species for considerable periods of time. In this communication, the presence of components of an NADPH oxidase-like system, more commonly associated with phagocytic leukocytes, is shown. The ability of HMC to generate low levels of superoxide may have important implications in cellular signaling in general and may contribute to glomerular injury. Spectroscopic analysis of HMC membranes revealed a low-potential cytochrome b component, redox midpoint potential centered around -250 mV, which is present at 60 pmol/mg of membrane protein. Immunodetection studies suggested the presence of the p22phox, p47phox, and p67phox components of the NADPH oxidase, whereas the gp91phox was not detected. Further studies with oligonucleotide polymerase chain reaction primers showed that, in HMC the mRNA expression of the p67phox and p47phox was absent from growth-arrested cells but was present in HMC treated with interleukin-1 beta (1,000 pg/mL), whereas gp91phox could not be detected. Only mRNA corresponding to p22phox was present in growth-arrested cells; p47phox mRNA was induced by 2-h treatment with interleukin-1 beta but declined after 6-h treatment. These data illustrate for the first time that HMC are capable of expressing mRNA for several NADPH oxidase components. The apparent absence, or variation, of the gp91phox indicates the likelihood of an NADPH oxidase isoenzyme.