Nina Queisser

Effect of Gum Arabic on Oxidative Stress and Inflammation in Adenine–Induced Chronic Renal Failure in Rats

Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in chronic renal failure (CRF) in rats. The aim of this work was to study the role of inflammation and oxidative stress in adenine-induced CRF and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in feed (0.75%, w/w), GA in drinking water (15%, w/v) and adenine+GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration). In addition, the concentrations of the pro-inflammatory cytokine TNF-α and the oxidative stress markers glutathione and superoxide dismutase, renal apoptosis, superoxide formation and DNA double strand break frequency, detected by immunohistochemistry for γ-H2AX, were measured. Adenine significantly increased the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance and induced significant increases in the concentration of the measured inflammatory mediators. Further, it caused oxidative stress and DNA damage. Treatment with GA significantly ameliorated these actions. The mechanism of the reported salutary effect of GA in adenine-induced CRF is associated with mitigation of the adenine-induced inflammation and generation of free radicals.

Mineralocorticoid receptor-mediated DNA damage in kidneys of DOCA-salt hypertensive rats

Epidemiological studies exploring the connection between hypertension and cancer demonstrate a higher cancer incidence, especially of kidney cancer, and a higher cancer mortality in hypertensive patients. Hormones elevated in hypertension, i.e., aldosterone and angiotensin II, which exert genotoxic effects in vitro, could contribute to carcinogenesis in hypertension. The present study was conducted to investigate the possible DNA‐damaging effect of aldosterone receptor activation in vivo. Crl:CD (Sprague‐Dawley) rats were treated for 6 wk with desoxycorticosterone acetate (DOCA) and salt to induce a mineralocorticoid‐dependent hypertension. DOCA‐salt treatment caused increased blood pressure (+26 mmHg) compared to untreated rats, elevated markers of kidney failure (up to 62‐fold for Kim‐1), and the induction of several proinflammatory genes and proteins (up to 2.6‐fold for tissue MCP‐1). The mineralocorticoid receptor (MR) antagonist spironolactone (MR IC50 24 nM) and the novel nonsteroidal antagonist BR‐4628 (MR IC50 28 nM) decreased these damage markers. DOCA‐salt treatment also caused 8.8‐fold increased structural DNA damage, determined with the comet assay, double‐strand breaks (3.5‐fold), detected immunohistochemically, and oxidative stress. Furthermore, the oxidatively modified mutagenic DNA base 7,8‐dihydro‐8‐oxo‐guanine (8‐oxodG), quantified by LC‐MS/MS, was almost 2‐fold higher in DOCA‐salt‐treated kidneys. Our results suggest a mutagenic potential of high mineralocorticoid levels, frequent in hypertensive individuals.—Schupp, N., Kolkhof, P., Queisser, N., Gartner, S., Schmid, U., Kretschmer, A., Hartmann, E., Oli, R G., Schäfer, S., Stopper, H. Mineralocorticoid receptor‐mediated DNA damage in kidneys of DOCA‐salt hypertensive rats. FASEB J. 25, 968–978 (2011). www.fasebj.org

Angiotensin II Induces DNA Damage in the Kidney

Increased activity of the renin angiotensin system with enhanced levels of angiotensin II leads to oxidative stress with endothelial dysfunction, hypertension, and atherosclerosis. Epidemiologic studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. Because elevated angiotensin II levels might contribute to carcinogenesis, we tested whether angiotensin II induces DNA damage in the kidney. In isolated perfused mouse kidneys, as little as 1 nmol/L angiotensin II caused a significant increase in DNA strand breaks, measured with the comet assay. This damage was independent of the hemodynamic effect of angiotensin II and mediated by the angiotensin II type 1 receptor. Angiotensin II also caused double-strand breaks in the cells of the isolated perfused kidney, detected with an antibody against the double-strand break marker gamma-H2AX. Studies in cell culture allowed further characterization of the DNA damage induced by angiotensin II. Single- and double-strand breaks, abasic sites, and 7,8-dihydro-8-oxo-guanine, all types of oxidative DNA lesions, were detected in angiotensin II-treated renal cells. The majority of detected strand breaks was repaired within 1 hour, but double-strand breaks increased and persisted for at least 24 hours.

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

An increase of the mineralocorticoid aldosterone is induced by a stimulated renin–angiotensin system in a subgroup of hypertensive patients. Epidemiological studies find higher cancer mortality in hypertensive patients and an increased risk to develop kidney cancer. This work investigated the involvement of oxidants in the genotoxicity of aldosterone and on a potential activation of transcription factor nuclear factor‐κB (NF‐κB) in kidney tubule cells. Aldosterone, at concentrations as low as 1 nM caused a significant increase of DNA damage, as assessed by comet assay and micronucleus frequency test. Aldosterone also led to a dose‐dependent activation of NF‐κB. Time courses of DNA damage and NF‐κB‐activation showed that these effects already occurred after 5 and 30 min of aldosterone exposure, respectively, suggesting non‐genomic events of the hormone. Antioxidants prevented aldosterone‐induced DNA damage and NF‐κB‐activation, indicating the involvement of oxidants. In fact, aldosterone caused an increase in intracellular oxidant levels, and in particular of superoxide anions. As a consequence, increased levels of the oxidized DNA modification 7,8‐dihydro‐8‐oxo‐guanine were observed in aldosterone‐treated kidney cells. Aldosterone‐induced DNA damage and NF‐κB‐activation was dependent on the involvement of the mineralocorticoid receptor. The induction of oxidant‐mediated genotoxic effects, and of a long‐term activation of the potentially oncogenic cell signal NF‐κB by aldosterone could contribute to the increased kidney cancer incidence in hypertensive patients. ©2010 Wiley‐Liss, Inc.

Superoxide anion and hydrogen peroxide-induced signaling and damage in angiotensin II and aldosterone action

Abstract The formation of reactive oxygen species (ROS) can be induced by xenobiotic substances, such as redox cycling molecules, but also by endogenous substances such as hormones and cytokines. Recent research shows the importance of ROS in cellular signaling. Here, the signaling pathways of the two blood pressure-regulating hormones angiotensin II and aldosterone are presented, focusing on both their physiological effects and the change of signaling owing to the action of increased concentrations or prolonged exposure. When present in high concentrations, both angiotensin II and aldosterone, as various other endogenous substances, activate NADPH oxidase, which produces superoxide. In this review the generation of superoxide anions and hydrogen peroxide in cells stimulated with angiotensin II or aldosterone, as well as the subsequently induced signaling processes and DNA damage is discussed.

Aldosterone, oxidative stress, and NF-κB activation in hypertension-related cardiovascular and renal diseases.

The mineralocorticoid aldosterone regulates electrolyte and fluid balance and is involved in blood pressure homoeostasis. Classically, it binds to its intracellular mineralocorticoid receptor to induce expression of proteins influencing the reabsorption of sodium and water in the distal nephron. Aldosterone gained special attention when large clinical studies showed that blocking its receptor in patients with cardiovascular diseases reduced their mortality. These patients present increased plasma aldosterone levels. The exact mechanisms of the potential toxic effects of aldosterone leading to cardiovascular damage are not known yet. The observation of reduced nitric oxide bioavailability in hyperaldosteronism implied the generation of oxidative stress by aldosterone. Subsequent studies confirmed the increase of oxidative stress markers in patients with chronic heart failure and in animal models of hyperaldosteronism. The effects of reactive oxygen species have been related to the activation of transcription factors, such as NF-κB. This review summarizes the present-day knowledge of aldosterone-induced oxidative stress and NF-κB activation in humans and different experimental models.

Blood pressure has only minor influence on aldosterone-induced oxidative stress and DNA damage in vivo.

Epidemiological studies found an increased kidney cancer risk in hypertensive patients. These patients frequently present an increase in the mineralocorticoid aldosterone (Ald) due to a stimulated renin angiotensin aldosterone system (RAAS). Recently, we showed pro-oxidative and genotoxic effects of Ald in vitro. Here, we investigated the influence of blood pressure on aldosterone-induced oxidative damage. To distinguish whether effects in Sprague-Dawley rats treated with Ald were caused by Ald or by increased blood pressure, the mineralocorticoid receptor (MR) antagonist spironolactone was administered in a subtherapeutical dose, not lowering the blood pressure, and hydralazine, a RAAS-independent vasodilator, was given to normalize the pressure. With the antioxidant tempol, oxidative stress-dependent effects were demonstrated. Ald treatment caused kidney damage and oxidative and nitrative stress. Structural DNA damage and the mutagenic oxidative base modification 7,8-dihydro-8-oxoguanine were increased, as well as DNA repair activity and nuclear NF-κB translocation. Spironolactone and tempol decreased all markers significantly, whereas hydralazine had just slight effects. These data comprise the first report of essentially blood pressure-independent tissue- and DNA-damaging effects of Ald. A fully activated MR and the production of reactive oxygen and nitrogen species were crucial for these effects.

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

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.

Aldosterone Activates Transcription Factor Nrf2 in Kidney Cells Both In Vitro and In Vivo

AIMS An increased kidney cancer risk was found in hypertensive patients, who frequently exhibit hyperaldosteronism, known to contribute to kidney injury, with oxidative stress playing an important role. The capacity of kidney cells to up-regulate transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2), a key regulator of the cellular antioxidative defense, as a prevention of aldosterone-induced oxidative damage was investigated both in vitro and in vivo. RESULTS Aldosterone activated Nrf2 and increased the expression of enzymes involved in glutathione (GSH) synthesis and detoxification. This activation depended on the mineralocorticoid receptor (MR) and oxidative stress. In vitro, Nrf2 activation, GSH amounts, and target gene levels decreased after 24 h, while oxidant levels remained high. Nrf2 activation could not protect cells against oxidative DNA damage, as aldosterone-induced double-strand breaks and 7,8-dihydro-8-oxo-guanine (8-oxodG) lesions steadily rose. The Nrf2 activator sulforaphane enhanced the Nrf2 response both in vitro and in vivo, thereby preventing aldosterone-induced DNA damage. In vivo, Nrf2 activation further had beneficial effects on the aldosterone-caused blood pressure increase and loss of kidney function. INNOVATION This is the first study showing the activation of Nrf2 by aldosterone. Moreover, the results identify sulforaphane as a substance that is capable of preventing aldosterone-induced damage both in vivo and in vitro. CONCLUSION Aldosterone-induced Nrf2 adaptive response cannot neutralize oxidative actions of chronically increased aldosterone, which, therefore could be causally involved in the increased cancer incidence of hypertensive individuals. Enhancing the cellular antioxidative defense with sulforaphane might exhibit beneficial effects.

Aldosterone induces fibrosis, oxidative stress and DNA damage in livers of male rats independent of blood pressure changes

Mineralocorticoid receptor blockers show antifibrotic potential in hepatic fibrosis. The mechanism of this protective effect is not known yet, although reactive oxygen species seem to play an important role. Here, we investigated the effects of elevated levels of aldosterone (Ald), the primary ligand of the mineralocorticoid receptor, on livers of rats in a hyperaldosteronism model: aldosterone-induced hypertension. Male Sprague-Dawley rats were treated for 4 weeks with aldosterone. To distinguish if damage caused in the liver depended on increased blood pressure or on increased Ald levels, the mineralocorticoid receptor antagonist spironolactone was given in a subtherapeutic dose, not normalizing blood pressure. To investigate the impact of oxidative stress, the antioxidant tempol was administered. Aldosterone induced fibrosis, detected histopathologically, and by expression analysis of the fibrosis marker, α-smooth muscle actin. Further, the mRNA amount of the profibrotic cytokine TGF-β was increased significantly. Fibrosis could be reduced by scavenging reactive oxygen species, and also by blocking the mineralocorticoid receptor. Furthermore, aldosterone treatment caused oxidative stress and DNA double strand breaks in livers, as well as the elevation of DNA repair activity. An increase of the transcription factor Nrf2, the main regulator of the antioxidative response could be observed, and of its target genes heme oxygenase-1 and γ-glutamylcysteine synthetase. All these effects of aldosterone were prevented by spironolactone and tempol. Already after 4 weeks of treatment, aldosteroneinfusion induced fibrosis in the liver. This effect was independent of elevated blood pressure. DNA damage caused by aldosterone might contribute to fibrosis progression when aldosterone is chronically increased.