Expression and Cellular Localization of Classic NADPH Oxidase Subunits in the Spontaneously Hypertensive Rat Kidney

Abstract

Phagocytes generate superoxide anion (O 2 − ) by a classic, 5-component NADPH oxidase. O 2 − contributes to hypertension in spontaneously hypertensive rats (SHR). Therefore, we tested the hypothesis that NADPH oxidase expression is enhanced in the SHR kidney. We also analyzed the localization of NADPH oxidase components in SHR kidney. Renal NADPH oxidase was quantified by reverse transcription-polymerase chain reaction and Western blotting and was localized in SHR and Wistar Kyoto rat (WKY) kidney by immunohistochemistry. The mRNA for 5 subunits of phagocyte NADPH oxidase, and also for MOX1 and RENOX (NOX4), was detected in adult rat kidney. Kidneys of adult (10 weeks old) SHR had a significantly ( P <0.01) greater mRNA for p47phox (SHR 0.81±0.05 versus WKY 0.37±0.01, arbitrary unit), which was confirmed by Western blotting (SHR 0.58±0.04 versus WKY 0.42±0.04, arbitrary unit; P <0.05) and by immunohistochemistry. This higher p47phox protein expression was also detected in young prehypertensive SHR (SHR 0.61±0.05 versus WKY 0.39±0.04, arbitrary unit; P <0.01). The 10-week-old SHR contained more modest but significantly ( P <0.05) greater protein for p67phox (SHR 0.54±0.02 versus WKY 0.46±0.02). Immunostaining localized p47phox, p67phox, and p22phox in vasculature, macula densa, distal convoluted tubule, cortical collecting duct, and outer and inner medullary collecting ducts. The kidney of SHR expresses genes for all the main components of phagocyte NADPH oxidase, RENOX, and MOX1. There is a prominent increase in the SHR kidney of the mRNA, and protein expression of p47phox in the vasculature, macula densa, and distal nephron, which precedes development of hypertension.