Original Contribution
Aldosterone increases kidney tubule cell oxidants through calcium-mediated activation of NADPH oxidase and nitric oxide synthase

https://doi.org/10.1016/j.freeradbiomed.2011.08.028Get rights and content

Abstract

Chronic hyperaldosteronism has been associated with an increased cancer risk. We recently showed that aldosterone causes an increase in cell oxidants, DNA damage, and NF-κB activation. This study investigated the mechanisms underlying aldosterone-induced increase in cell oxidants in kidney tubule cells. Aldosterone caused an increase in both reactive oxygen and reactive nitrogen (RNS) species. The involvement of the activation of NADPH oxidase in the increase in cellular oxidants was demonstrated by the inhibitory action of the NADPH oxidase inhibitors DPI, apocynin, and VAS2870 and by the migration of the p47 subunit to the membrane. NADPH oxidase activation occurred as a consequence of an increase in cellular calcium levels and was mediated by protein kinase C. The prevention of RNS increase by BAPTA-AM, W-7, and L-NAME indicates a calcium–calmodulin activation of NOS. A similar pattern of effects of the NADPH oxidase and NOS inhibitors was observed for aldosterone-induced DNA damage and NF-κB activation, both central to the pathogenesis of chronic aldosteronism. In summary, this paper demonstrates that aldosterone, via the mineralocorticoid receptor, causes an increase in kidney cell oxidants, DNA damage, and NF-κB activation through a calcium-mediated activation of NADPH oxidase and NOS. Therapies targeting calcium, NOS, and NADPH oxidase could prevent the adverse effects of hyperaldosteronism on kidney function as well as its potential oncogenic action.

Section snippets

Materials

MDCK and LLC-PK1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cell culture media and reagents were obtained from PAA Laboratories GmbH (Pasching, Austria) and Invitrogen Life Technologies (Carlsbad, CA, USA), respectively. The primary antibody against p47phox (sc-14015) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and α-tubulin (T6199) was purchased from Sigma (St. Louis, MO, USA). The oligonucleotide containing the consensus sequence

Aldosterone induces a calcium-mediated increase in ROS and RNS production via the mineralocorticoid receptor

Two kidney cell lines (LLC-PK1, MDCK) with different functional characteristics were chosen to study the mechanisms involved in the increased oxidant production associated with exposure to high aldosterone levels. LLC-PK1 cells are from pig kidney and resemble proximal tubule cells [29], and MDCK cells are from dog kidney and resemble distal tubule cells [30].

Mineralocorticoid and glucocorticoid receptors are expressed in LLC-PK1 [31] and MDCK cells (data not shown). To investigate if the

Discussion

We previously demonstrated that aldosterone is genotoxic in kidney tubule cells and causes the activation of NF-κB, with evidence that an increase in cellular oxidants in part triggers those effects [16], [31]. Both DNA damage and chronic NF-κB activation can lead to oncogenesis. Understanding the mechanism underlying aldosterone-induced oxidant production is essential in the development of therapies that could prevent the adverse effects of chronic aldosteronism, particularly the potential

Acknowledgments

This work was supported by grants from the University of California at Davis (USA) and by Deutsche Forschungsgemeinschaft Grant Schu 2367/1-1 (to N.S. and H.S.) (Germany). N.Q. was supported by a fellowship from the DAAD (Germany).

References (58)

  • N.L. Simmons

    Cultured monolayers of MDCK cells: a novel model system for the study of epithelial development and function

    Gen. Pharmacol.

    (1982)
  • J.P. Crow

    Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species

    Nitric Oxide

    (1997)
  • M.S. Forbes et al.

    Lack of endothelial nitric-oxide synthase leads to progressive focal renal injury

    Am. J. Pathol.

    (2007)
  • R.M. Losel et al.

    Nongenomic effects of aldosterone: cellular aspects and clinical implications

    Steroids

    (2002)
  • B.J. Harvey et al.

    Nongenomic effects of aldosterone on Ca2+ in M-1 cortical collecting duct cells

    Kidney Int.

    (2000)
  • E. Dopp et al.

    Induction of genotoxic effects and modulation of the intracellular calcium level in Syrian hamster embryo (SHE) fibroblasts caused by ochratoxin A

    Food Chem. Toxicol.

    (1999)
  • N. Xu et al.

    Ca2+ signal blockers can inhibit M/A transition in mammalian cells by interfering with the spindle checkpoint

    Biochem. Biophys. Res. Commun.

    (2003)
  • M. Jaiswal et al.

    Nitric oxide-mediated inhibition of DNA repair potentiates oxidative DNA damage in cholangiocytes

    Gastroenterology

    (2001)
  • S. Friis et al.

    Angiotensin-converting enzyme inhibitors and the risk of cancer: a population-based cohort study in Denmark

    Cancer

    (2001)
  • L.E. Moore et al.

    Lifestyle factors, exposures, genetic susceptibility, and renal cell cancer risk: a review

    Cancer Invest.

    (2005)
  • G. Corrao et al.

    Hypertension, antihypertensive therapy and renal-cell cancer: a meta-analysis

    Curr. Drug Safe

    (2007)
  • R.D. Gordon et al.

    Evidence that primary aldosteronism may not be uncommon: 12% incidence among antihypertensive drug trial volunteers

    Clin. Exp. Pharmacol. Physiol.

    (1993)
  • D.A. Calhoun et al.

    Resistant hypertension: diagnosis, evaluation, and treatment. A scientific statement from the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research

    Circulation

    (2008)
  • A.M. Marney et al.

    Aldosterone and end-organ damage

    Clin. Sci. (London)

    (2007)
  • J.M. Connell et al.

    The new biology of aldosterone

    J. Endocrinol.

    (2005)
  • D.W. Good

    Nongenomic actions of aldosterone on the renal tubule

    Hypertension

    (2007)
  • H. Hayashi et al.

    Aldosterone nongenomically produces NADPH oxidase-dependent reactive oxygen species and induces myocyte apoptosis

    Hypertens. Res.

    (2008)
  • N. Queisser et al.

    Aldosterone induces oxidative stress, oxidative DNA damage and NF-kappaB-activation in kidney tubule cells

    Mol. Carcinog.

    (2011)
  • W. Han et al.

    Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells

    Hypertension

    (2008)
  • Cited by (0)

    View full text