Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals

Abstract

Angiotensin II (Ang II) induces hypertrophy of cultured proximal tubular cells. We have previously demonstrated that this Ang II-mediated hypertrophy occurs in the G1-phase of the cell cycle and depends on the induction of p27Kip1, an inhibitor of G1-phase cyclin/cyclin-dependent kinase complexes. The present study was undertaken to investigate whether Ang II may stimulate superoxide anions (O2.) formation in cultured LLC-PK1 and cultured mouse proximal tubule (MCT) cells, and to gain further insight into a potential relationship between O2. and cell cycle regulation. Reactive oxygen species were measured with the lucigenin method in intact cells. The effects of various inhibitors were tested on Ang II-induced O2. production. Cells were transiently transfected with phosphorothioate-modified rat p22phox antisense oligonucleotides to investigate the potential role of NAD(P)H oxidase. Expression of p22phox mRNA after Ang II-treatment was detected with Northern blots. Incorporation of [3H]leucine into de novo synthesized proteins was used as a parameter of cell hypertrophy. Expression of p27Kip1 was evaluated in cell lysates by Western blotting. Ang II stimulated the accumulation of O2. in tubular cells; however, an addition of two different antioxidants completely abolished measurable O2. This effect was transduced by angiotensin receptor type-1 (AT1) and was inhibited by a flavoprotein inhibitor (DIP) or p22phox antisense oligonucleotides, indicating the involvement of membrane NAD(P)H oxidase. Ang II-stimulated de novo protein synthesis was attenuated by DIP, antioxidants, and p22phox antisense oligonucleotides. The Ang II-induced expression of p27Kip1 protein and cellular hypertrophy were reduced by similar treatments. Generation of O2. by xanthine supplementation also stimulated p27Kip1 expression and induced hypertrophy in LLC-PK1 cells. This study provides the first evidence, to our knowledge, that Ang II induces O2. in cultured tubular cells. Ang II-mediated activation of membrane bound NAD(P)H oxidase, probably by an increase in p22phox transcripts, is likely responsible for this induction. Generation of O2. subsequently induces p27Kip1 expression and stimulates hypertrophy, suggesting a novel mechanism of how Ang II can modulate cell cycle regulation.