Angiotensin II induces p27(Kip1) expression in renal tubules in vivo: role of reactive oxygen species.

Previous studies have demonstrated that angiotensin II (ANG II) mediates cell cycle arrest of cultured renal tubular cells by induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. However, it is not known whether ANG II exerts similar effects in vivo. Infusion of ANG II into naive rats for 7 days increased formation of reactive oxygen species in tubular cells of the kidney. Furthermore, ANG II infusion stimulated protein expression of p27(Kip1) as detected by western blotting of tubular lysates and immunohistochemistry. Infusion of ANG II reduced tubular proliferation as detected by proliferating-cell nuclear antigen (PCNA) immunohistochemistry. The increase in p27(Kip1) expression was not due to an increase in mRNA. Immunoprecipitation experiments revealed that the increased p27(Kip1) protein associates with cyclin-dependent kinase 2. Coadministration of the radical scavenger dimethylthiourea abolished this ANG II mediated p27(Kip1) expression without reducing systemic blood pressure. Furthermore, dimethylthiourea infusion attenuates the ANG II mediated G(1)-phase arrest of tubular cells. However, infusion of norepinephrine did not induce reactive oxygen species or p27(Kip1) expression, despite a significant increase in blood pressure. Thus ANG II induces p27(Kip1) expression in renal tubular cells in vivo. This effect is mediated by reactive oxygen species. Since tubular hypertrophy depends on G(1)-phase arrest and may promote subsequent development of interstitial fibrosis, administering oxygen radical scavenger may be a therapeutic tool to counteract ANG II dependent remodeling of renal tubular cells.

Antioxidant treatment induces transcription and expression of transforming growth factor beta in cultured renal proximal tubular cells.

Transforming growth factor beta (TGF-beta) plays an important role in the development of tubulointerstitial fibrosis in chronic renal disease. We were interested whether interference with oxygen radicals may modulate TGF-beta expression. Unexpectedly, we discovered that diphenylene iodine (DIP), an inhibitor of NADP(H) oxidase, induces a robust increase in TGF-beta transcript expression in cultured mouse proximal tubular cells (MCT cells). A similar increase was seen with EUK-8, a synthetic salen-manganese complex with high oxyradical scavenger activities. This induction of TGF-beta1 mRNA was paralleled by increasing protein expression. Transient transfection of MCT cells with a reporter construct in which murine TGF-beta1 enhancer/promoter elements were cloned in front of the luciferase gene, revealed that DIP, EUK-8, and Tiron all stimulated transcription of the TGF-beta1 gene whereas exogenous H2O2 suppressed transcription. Antisense oligonucleotides against p22phox, but not sense oligonucleotides, also increased transcriptional activity of TGF-beta1. Mutagenesis of Sp1 binding sites in the mouse TGF-beta1 enhancer/promoter abolished the stimulatory effect of the antioxidants. Gel shift experiments revealed that DIP as well as EUK-8 activated binding of nuclear proteins to Sp1 consensus sequence. Our data provide evidence that TGF-beta1 transcription is negatively regulated in MCT cells under basal conditions by NADP(H) oxidase-mediated oxygen radicals. Thus, antioxidant therapy may increase local synthesis of TGF-beta1 in the tubulointerstitium.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Atrial natriuretic peptide attenuates ANG II-induced hypertrophy of renal tubular cells.

ANG II arrests LLC-PK1 cells in the G1 phase of the cell cycle and induces hypertrophy, an effect mediated by induction of p27Kip1. We studied whether atrial natriuretic peptide (ANP) may modulate ANG II-induced hypertrophy and p27Kip1 expression in tubular LLC-PK1 cells. ANP, through its fragments 3---28 and 4---27, prevented ANG II-induced cell cycle arrest. ANP inhibited >80% of ANG II-induced p27Kip1 protein expression (Western blots). ANP stimulated expression of MKP-1, a phosphatase involved in dephosphorylation of p44/42 mitogen-activated protein (MAP) kinase, up to 12 h. ANP prevented the ANG II-mediated phosphorylation peak of MAP kinase after 12 h of stimulation. 8-Bromo-cGMP mimicked all the effects of ANP. Transfection with MKP-1 antisense, but not sense, oligonucleotides abolished the modifying role of ANP on ANG II-mediated cell cycle arrest. The effect of ANP on ANG II-mediated hypertrophy of LLC-PK1 cells is regulated on the level of MAP kinase phosphorylation, a key step in the induction of p27Kip1. Although ANP and ANG II both stimulate generation of reactive oxygen species, ANP additionally induces expression of MKP-1, leading to interference with ANG II-mediated MAP kinase phosphorylation.

Involvement of metabotropic and ionotropic glutamate receptors in inositol polyphosphate formation in carp retinal slices.

The contribution of ionotropic and metabotropic glutamate receptors to inositol polyphosphate accumulation in carp retinal slices was investigated using myo-[2-3H]inositol prelabelling. In the presence of the glutamate agonists quisqualate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and trans-(+/-)-1-amino-1,3-cyclopentane-dicarboxylic acid (t-ACPD), formation of [3H]inositol phosphate was significantly increased in a dose-dependent manner, with EC50 values of 350 nM, 1.5 microM and 10 microM respectively. The complete AMPA-induced response and a large component of the quisqualate-induced response were inhibited in a competitive manner when the ionotropic antagonist 6-cyano-7-nitroquinoxalin- 2,3-dione (CNQX) was present. Furthermore, the remaining level of quisqualate-induced [3H]inositol phosphate formation closely matched that produced by ACPD alone, and coincubation of AMPA and ACPD showed additive effects, suggesting that the quisqualate-induced response resulted from coactivation of metabotropic and ionotropic glutamate receptors. The ionotropic component was partially reduced in the presence of cobalt, suggesting indirect effects resulting from synaptic interactions. We could exclude indirect effects through depolarization-induced release of other neurotransmitters. Only serotonin (EC50 1 microM) and carbachol (at a concentration of 1 mM) stimulated [3H]inositol phosphate formation, but their antagonists did not affect the quisqualate response and coactivation with quisqualate and serotonin or carbachol resulted in additive effects. The ionotropic component was completely suppressed when Ca2+ was omitted from the medium and cobalt was present. This makes it likely that the ionotropic component resulted from Ca2+ entry through AMPA-gated channels and subsequent Ca(2+)-dependent activation of phospholipase C.