Differential Modulation of Markers of Oxidative Stress and DNA Damage in Arterial Hypertension.

Patients with arterial hypertension have an increased risk of developing tumors, particularly renal cell carcinoma. Arterial hypertension is linked to DNA damage via the generation of oxidative stress, in which an upregulated renin-angiotensin-aldosterone system plays a crucial role. The current study investigated surrogates of oxidative stress and DNA damage in a group of hypertensive patients (HypAll, n = 64) and subgroups of well (HypWell, n = 36) and poorly (HypPoor, n = 28) controlled hypertensive patients compared to healthy controls (n = 8). In addition, a longitudinal analysis was performed with some of the hypertensive patients. Markers for oxidative stress in plasma (SHp, D-ROM, and 3-nitrotyrosine) and urine (8-oxodG, 15-F2t-isoprostane, and malondialdehyde) and markers for DNA damage in lymphocytes (γ-H2AX and micronuclei) were measured. In HypAll, all markers of oxidative stress except malondialdehyde were increased compared to the controls. After adjustment for age, this association was maintained for the protein stress markers SHp and 3-nitrotyrosine. With regard to the markers for DNA damage, there was no difference between HypAll and the controls. Further, no significant differences became apparent in the levels of both oxidative stress and DNA damage between HypWell and HypPoor. Finally, a positive correlation between the development of blood pressure and oxidative stress was observed in the longitudinal study based on the changes in D-ROM and systolic blood pressure. In conclusion, we found increased oxidative stress in extensively treated hypertensive patients correlating with the level of blood-pressure control but no association with DNA damage.

Nrf2 Activation Does Not Protect from Aldosterone-Induced Kidney Damage in Mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is downregulated in chronic kidney disease (CKD). Activation of Nrf2 might be a therapeutic option in CKD. Here we investigate the effect of Nrf2 activation on aldosterone (Aldo)-induced renal injury. Wild-type (WT) mice, transgenic Keap1 hypomorphic (Nrf2ꜛ, genotype results in upregulation of Nrf2 expression) mice and WT mice treated with the Nrf2 activator sulforaphane (Sulf) received Aldo for 4 weeks. In Aldo-treated mice, kidneys were significantly heavier and pathologically altered, reflected by increased urinary albumin levels and tissue damage. In Nrf2ꜛ-Aldo mice the tubule damage marker NGAL was significantly decreased. Increased oxidative damage markers (8-OHdG, 15-isoprostane F2t) were measured in all Aldo-treated groups. Aldo-increased Nrf2 amounts were mainly found in the late tubule system. The amount of phosphorylated and thus putatively active Nrf2 was significantly increased by Aldo only in WT mice. However, expression of Nrf2 target genes NQO1 and HO1 was decreased in all Aldo-infused mice. GSK3ß, which promotes Nrf2 degradation, was significantly increased in the kidneys of Aldo-treated WT mice. Neither genetic nor pharmacological Nrf2 activation was able to prevent oxidative injury induced by Aldo, probably due to induction of negative regulators of Nrf2.

Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins.

Proximal tubular epithelial cells (PTEC) are constantly exposed to potentially toxic metabolites and xenobiotics. The regenerative potential of the kidney enables the replacement of damaged cells either via the differentiation of stem cells or the re-acquisition of proliferative properties of the PTEC. Nevertheless, it is known that renal function declines, suggesting that the deteriorated cells are not replaced by fully functional cells. To understand the possible causes of this loss of kidney cell function, it is crucial to understand the role of toxins during the regeneration process. Therefore, we investigated the sensitivity and function of human induced pluripotent stem cells (hiPSC), hiPSC differentiating, and hiPSC differentiated into proximal tubular epithelial-like cells (PTELC) to known nephrotoxins. hiPSC were differentiated into PTELC, which exhibited similar morphology to PTEC, expressed prototypical PTEC markers, and were able to undergo albumin endocytosis. When treated with two nephrotoxins, hiPSC and differentiating hiPSC were more sensitive to cisplatin than differentiated PTELC, whereas all stages were equally sensitive to cyclosporin A. Both toxins also had an inhibitory effect on albumin uptake. Our results suggest a high sensitivity of differentiating cells towards toxins, which could have an unfavorable effect on regenerative processes. To study this, our model of hiPSC differentiating into PTELC appears suitable.

The NADPH Oxidase Isoform 1 Contributes to Angiotensin II-Mediated DNA Damage in the Kidney.

In higher concentrations, the blood pressure regulating hormone angiotensin II leads to vasoconstriction, hypertension, and oxidative stress by activating NADPH oxidases which are a major enzymatic source of reactive oxygen species (ROS). With the help of knockout animals, the impact of the three predominant NADPH oxidases present in the kidney, i.e., Nox1, Nox2 and Nox4 on angiotensin II-induced oxidative damage was studied. Male wildtype (WT) C57BL/6 mice, Nox1-, Nox2- and Nox4-deficient mice were equipped with osmotic minipumps, delivering either vehicle (PBS) or angiotensin II, for 28 days. Angiotensin II increased blood pressure and urinary albumin levels significantly in all treated mouse strains. In Nox1 knockout mice these increases were significantly lower than in WT, or Nox2 knockout mice. In WT mice, angiotensin II also raised systemic oxidative stress, ROS formation and DNA lesions in the kidney. A local significantly increased ROS production was also found in Nox2 and Nox4 knockout mice but not in Nox1 knockout mice who further had significantly lower systemic oxidative stress and DNA damage than WT animals. Nox2 and Nox4 knockout mice had increased basal DNA damage, concealing possible angiotensin II-induced increases. In conclusion, in the kidney, Nox1 seemed to play a role in angiotensin II-induced DNA damage.

Aldosterone Induces DNA Damage and Activation of Nrf2 Mainly in Tubuli of Mouse Kidneys.

Hypertensive patients have an increased risk of developing chronic kidney disease (CKD). Many of these patients have increased levels of the blood pressure regulating mineralocorticoid aldosterone. As a protection against aldosterone-induced damage, kidney cells can upregulate key regulators of the antioxidant defense, such as nuclear factor-erythroid-2-related factor 2 (Nrf2). In the present study aldosterone-induced kidney damage and Nrf2 activation in kidney cells of mice treated with three different concentrations of aldosterone for 4 weeks was localized. Increased albumin and neutrophil gelatinase-associated lipocalin (NGAL) in urine revealed an impaired kidney function of the aldosterone-infused mice. Localization of aldosterone-induced oxidative damage (in the form of DNA lesions) in specific kidney cells showed an increase in proximal tubuli and to an even greater extend in distal tubuli. Phosphorylated Nrf2 was increased in distal tubule cells after aldosterone-infusion. Nrf2 activation in proximal tubuli or in glomeruli after aldosterone-treatment could not be observed. Nrf2 target genes and proteins analyzed, paradoxically, showed a downregulation in the whole kidney. Aldosterone-treated mice exhibited an increased kidney injury and DNA damage in distal and proximal tubuli. Nrf2 seemed only to be specifically activated in distal tubule cells, where we also detected the highest amount of oxidative damage.

Effect of infliximab, a tumor necrosis factor-alpha inhibitor, on doxorubicin-induced nephrotoxicity in rats.

Treatment with the chemotherapeutic agent, doxorubicin (DOX), is limited by nephrotoxicity. We investigated the possible protective effect of infliximab, a tumor necrosis factor alpha (TNF-α) inhibitor on DOX-induced nephrotoxicity. Rats were treated with a single intraperitoneal (ip) injection of DOX (17.5 mg/kg) in the absence or presence of infliximab (5 mg/kg, i.p.). Plasma and urinary markers of kidney function, oxidative stress, and inflammation were measured. Kidney and heart tissue was evaluated histopathologically. DOX-induced nephrotoxicity was confirmed by increased plasma urea, creatinine, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), and clusterin concentrations. In addition, DOX increased urinary albumin/creatinine ratio, N-acetyl-ß-D-glucosaminidase (NAG) activity, kidney injury molecule (KIM-1) concentrations, and reduced creatinine clearance. DOX significantly reduced renal antioxidants and increased plasma inflammatory markers and adiponectin concentrations. Concomitant treatment with infliximab did not significantly affect DOX-induced changes in plasma creatinine, cystatin C, or creatinine clearance. However, infliximab significantly reduced DOX-induced action on plasma urea, NGAL, clusterin, and adiponectin. Infliximab also significantly reduced urinary albumin/creatinine ratio, NAG activity, and KIM-1 concentrations, as well as the occurrence of fibrotic lesions in kidney tissue. Fibrosis detected in the heart was unchanged. In addition, infliximab reduced DOX-induced effects on plasma inflammatory markers, renal superoxide dismutase (SOD) and total antioxidant capacity. Our results show that infliximab is partially effective in mitigating DOX-induced nephrotoxicity in rats.

Angiotensin II-induced hypertension increases the mutant frequency in rat kidney.

Epidemiological studies revealed an increased risk for kidney cancer in hypertensive patients. In many of these patients, the blood pressure regulating renin-angiotensin-aldosterone system (RAAS) is activated. A stimulated RAAS leads to oxidative stress and increases markers of DNA damage, both in vitro and in animal models of hypertension. However, the mutagenic potential of RAAS activation has not been investigated yet. To quantify hypertension-induced mutations, BigBlue®+/- rats, which carry a transgenic lacI gene for mutation analysis, were treated for 20 weeks with a mean dose of 400 µg angiotensin II/kg × day. Angiotensin II-treated animals showed significantly increased blood pressure and impaired kidney function. Urinary excretion of oxidized nucleobases was raised. Additionally, in the renal cortex, oxidative stress, oxidatively generated DNA lesions and DNA strandbreaks were significantly increased. Further, a significant elevation of the mutant frequency in kidney DNA was detected. Sequencing revealed the presence of GC → T:A transversions in the mutated lacI genes of the angiotensin II-treated animals as a result of unrepaired oxidatively modified DNA bases, while no such transversions were found in the mutated lacI genes from control animals. The results demonstrate that the oxidative stress and DNA damage previously observed in kidney cells in vitro and in vivo after angiotensin II treatment indeed is associated with the accumulation of mutations in rat kidneys, providing further evidence for a cancer-initiating potential of elevated angiotensin II concentrations.

Effects of the SGLT-2 Inhibitor Canagliflozin on Adenine-Induced Chronic Kidney Disease in Rats.

BACKGROUND/AIMS: SGLT-2 inhibitors have been shown to be nephroprotective in diabetes. Here, we examined if one of these drugs (canagliflozin) could also ameliorate non-diabetic chronic kidney disease (CKD). METHODS: CKD was induced in rats by feeding them adenine (0.25%w/w for 35 days) and canagliflozin (10 or 25 mg/kg, by gavage) was given with or without adenine. Several conventional and novel plasma and urine biomarkers and tissues morphology were used to investigate the canagliflozin effect on kidney structure and function. RESULTS: Rats fed adenine showed the typical features of CKD that included elevation of blood pressure, decreased food intake and growth, increased water intake and urine output, decrease in creatinine clearance, and increase in urinary albumin/creatinine ratio, liver-type fatty acid binding protein, N-acetyl-beta-D-glucosaminidase, and plasma urea, creatinine, uric acid, calcium, indoxyl sulfate and phosphorus concentrations. Adenine also increased concentrations of several biomarkers of inflammation such as neutrophil gelatinase-associated lipocalin, interleukin-6, tumor necrosis factor alpha, clusterin, cystatin C and interleukin-1ß, and decreased some oxidative biomarkers in kidney homogenate, such as superoxide dismutase, catalase, glutathione reductase, total antioxidant activity, and also urinary 8-isoprostane and urinary 8-hydroxy-2-deoxy guanosine. Adenine significantly increased the renal protein content of Nrf2, caused renal tubular necrosis and fibrosis. Given alone, canagliflozin at the two doses used did not significantly alter any of the parameters mentioned above. When canagliflozin was given concomitantly with adenine, it significantly and dose - dependently ameliorated all the measured adenine - induced actions. CONCLUSION: Canagliflozin ameliorated adenine - induced CKD in rats, through reduction of several inflammatory and oxidative stress parameters, and other indices such as uremic toxins, and by antagonizing the increase in the renal content of the transcription factor Nrf2. The drug caused no overt or significant untoward effects, and its trial in patients with CKD may be warranted.

The renoprotective effect of the dipeptidyl peptidase-4 inhibitor sitagliptin on adenine-induced kidney disease in rats.

We assessed the effect of treatment with the dipeptidyl peptidase-4 inhibitor, sitagliptin, on adenine-induced chronic kidney disease (CKD). Six equal groups of rats were given either normal food or food mixed with adenine (0.25% w/w for five weeks) to induce CKD. Some of these groups were also simultaneously treated with sitagliptin (2.5 and 10 mg/kg/day, by gavage). Rats given adenine showed elevation of blood pressure, decreased body weight and increased relative kidney weight. Adenine also significantly increased plasma urea, creatinine, cystatin C, liver-type fatty acid-binding protein concentrations and neutrophil gelatinase-associated lipocalin activity by 404%, 354%, 667%, 91% and 281% respectively and reduced plasma α-Klotho by 50%. In addition, adenine significantly increased albumin/creatinine ratio and N-acetyl-ß-d-glucosaminidase activity by 3553% and 400% respectively and reduced creatinine clearance by 91%. Adenine feeding also significantly elevated the plasma concentration of inflammatory cytokines (plasma tumor necrosis factor-alpha, interleukin-1beta and transforming growth factor beta-1) and significantly reduced antioxidant indices (catalase, glutathione reductase and superoxide dismutase). Histopathologically, adenine caused renal fibrosis, inflammation and atrophy. When given concomitantly with adenine, sitagliptin ameliorated all the measured adenine-induced physiological and biochemical changes but not the histopathological changes. Sitagliptin (10 mg/kg/day) reduced plasma urea and creatinine by 32% and 25% respectively and increased creatinine clearance by 248%. These findings suggest a renoprotective action of sitagliptin on adenine-induced CKD.

Hepatic Rac1 GTPase contributes to liver-mediated basal immune homeostasis and LPS-induced endotoxemia.

BACKGROUND: The Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis. METHODS: We employed a constitutive Alb-Cre-driven rac1 knock-out and a poly I:C-inducible Mx1-Cre-based knock-out model and analyzed cytokine expression profiles in liver and other organs under basal situation and following LPS-induced endotoxemia by flow cytometry, qRT-PCR and immunocytochemistry. RESULTS: Constitutive Alb-Cre-driven rac1 knockout in hepatocytes altered the basal distribution and activation of immune cells in the liver and likewise in kidney and lung. Early systemic alterations in cytokine serum levels following LPS treatment remained unaffected by Rac1. Furthermore, lack of Rac1 in hepatocytes of untreated animals shifted the liver to a chronic inflammatory state, as depicted by an enhanced mRNA expression of marker genes related to activated macrophages. Upon acute LPS-induced endotoxemia, increased IL-10 mRNA expression in the liver of Alb-Cre Rac1-deficient mice provided an anti-inflammatory response. Employing a poly I:C-inducible Mx1-Cre-based rac1 knock-out, which allows a more widespread rac1 deletion in both hepatocytes and non-hepatocytes, we observed substantial differences regarding both basal and LPS-stimulated cytokine expression profiles as compared to the Alb-Cre system. CONCLUSIONS: Rac1-dependent mechanisms in hepatocytes and non-hepatocytes contribute to the maintenance of liver immune homeostasis under basal situation and following LPS-induced endotoxemia. Disturbed Rac1-regulated hepatocyte functions may promote liver damage under pathophysiological situation involving inflammatory stress.

Curcumin Ameliorates Kidney Function and Oxidative Stress in Experimental Chronic Kidney Disease.

Chronic kidney disease (CKD) is known to involve inflammation, oxidative stress and apoptosis. Here, we investigated the impact of curcumin (diferuloyl methane, a phenolic turmeric pigment), which has strong antioxidant, anti-inflammatory and anti-apoptotic activities on kidney structure and function in rats with adenine-induced CKD. Rats were treated for 5 weeks with adenine to induce CKD-like renal damage and combined with three doses of curcumin. Markers of kidney function and oxidative stress were quantified in plasma, urine, renal homogenates and on kidney tissue. Curcumin was found to significantly abate adenine-induced toxic effects such as reduced creatinine clearance, elevated neutrophil gelatinase-associated lipocalin levels and raised urinary N-acetyl-ß-D-glucosaminidase activities. Curcumin markedly reduced renal morphological damage and histopathological markers of inflammation, fibrosis and apoptosis. Curcumin further reduced adenine-induced hypertension, urinary albumin, the inflammatory cytokines IL-1ß, IL-6 and TNF-α, cystatin C and adiponectin. It restored plasma sclerostin concentrations and lowered oxidative stress in renal homogenates. In animals treated with the two higher curcumin concentrations, alone or in combination with adenine, an increased expression of the antioxidative transcription factor Nrf2 was found as well as up-regulation of the activity of its direct target glutathione reductase, and of an indirect target, the glutathione level. In conclusion, curcumin exhibits salutary effects against adenine-induced CKD in rats by reducing inflammation and oxidative stress via up-regulation of the transcription factor Nrf2.

Angiotensin II type 1a receptor-deficient mice develop angiotensin II-induced oxidative stress and DNA damage without blood pressure increase.

Hypertensive patients have an increased risk of developing kidney cancer. We have shown in vivo that besides elevating blood pressure, angiotensin II causes DNA damage dose dependently. Here, the role of blood pressure in the formation of DNA damage is studied. Mice lacking one of the two murine angiotensin II type 1 receptor (AT1R) subtypes, AT1aR, were equipped with osmotic minipumps, delivering angiotensin II during 28 days. Parameters of oxidative stress and DNA damage of kidneys and hearts of AT1aR-knockout mice were compared with wild-type (C57BL/6) mice receiving angiotensin II, and additionally, with wild-type mice treated with candesartan, an antagonist of both AT1R subtypes. In wild-type mice, angiotensin II induced hypertension, reduced kidney function, and led to a significant formation of reactive oxygen species (ROS). Furthermore, genomic damage was markedly increased in this group. All these responses to angiotensin II could be attenuated by concurrent administration of candesartan. In AT1aR-deficient mice treated with angiotensin II, systolic pressure was not increased, and renal function was not affected. However, angiotensin II still led to an increase of ROS in kidneys and hearts of these animals. Additionally, genomic damage in the form of double-strand breaks was significantly induced in kidneys of AT1aR-deficient mice. Our results show that angiotensin II induced ROS production and DNA damage even without the presence of AT1aR and independently of blood pressure changes.

Aldosterone activates the oncogenic signals ERK1/2 and STAT3 via redox-regulated mechanisms.

Epidemiological studies found an increased risk for kidney cancer in hypertensive patients, of which a subgroup has high aldosterone (Ald) levels. We recently showed that Ald is genotoxic both in kidney tubular cells and in rats with mineralocorticoid-mediated hypertension. The present work investigated in vitro and in vivo, if the oxidative stress-mediated activation of the ERK1/2 pathway, and its downstream target STAT3, could be one mechanism involved in the potential oncogenic capability of excess Ald exposure. The effects of excess Ald were investigated in LLC-PK1 cells and in Ald-induced hypertensive rats. Ald caused cRaf, MEK1/2, and ERK1/2 phosphorylation both in LLC-PK1 cells and in rat kidneys. ERK1/2 activation led to an increased phosphorylation of MSK1, p90RSK, and STAT3. The involvement of ERK1/2 in the activation of STAT3 was evidenced by the capacity of the MEK inhibitor U0126 to prevent Ald-mediated ERK1/2 and STAT3 phosphorylation. Both in vitro and in vivo, the activation of ERK1/2 and STAT3 by Ald was dependent on the mineralocorticoid receptor and was triggered by an increase in cellular oxidants. Ald-mediated oxidant increase was in part due to the activation of the enzymes NADPH oxidase and NO synthase. Proliferation was significantly enhanced and apoptosis decreased in Ald-treated rat kidneys and/or LLC-PK1 cells. Results support the concept that the oxidant-mediated long-term activation of ERK1/2/STAT3 by persistently high Ald levels could trigger proliferative and prosurvival events. Ald-mediated promotion of cell survival and DNA damage could result in kidney cell transformation and initiation of cancer in hypertensive patients with hyperaldosteronism.

DNA Damage in Chronic Kidney Disease: Evaluation of Clinical Biomarkers.

Patients with chronic kidney disease (CKD) exhibit an increased cancer risk compared to a healthy control population. To be able to estimate the cancer risk of the patients and to assess the impact of interventional therapies thereon, it is of particular interest to measure the patients' burden of genomic damage. Chromosomal abnormalities, reduced DNA repair, and DNA lesions were found indeed in cells of patients with CKD. Biomarkers for DNA damage measurable in easily accessible cells like peripheral blood lymphocytes are chromosomal aberrations, structural DNA lesions, and oxidatively modified DNA bases. In this review the most common methods quantifying the three parameters mentioned above, the cytokinesis-block micronucleus assay, the comet assay, and the quantification of 8-oxo-7,8-dihydro-2'-deoxyguanosine, are evaluated concerning the feasibility of the analysis and regarding the marker's potential to predict clinical outcomes.

Therapeutic Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats.

BACKGROUND/AIMS: To study the therapeutic effect of chrysin, a flavonoid with strong antioxidant and anti-inflammatory activities, on adenine-induced chronic kidney diseases (CKD) in rats. METHODS: Chrysin, in three graded oral doses (10, 50 and 250 mg/kg), was given for 10 consecutive days to rats after the induction of CKD by feeding them adenine (0.25%(w/w) for 35 days). Several plasma and urine biomarkers and tissues morphology were used the investigate chrysin effect on kidney structure and function. RESULTS: Adenine lowered creatinine clearance and elevated the concentrations of urea, creatinine, plasma neutrophil gelatinase-associated lipocalin and urinary N-Acetyl-beta-D-glucosaminidase activity, and increased the concentrations of the uremic toxin indoxyl sulfate, in addition to some inflammatory cytokines. Renal histopathological markers of inflammation and fibrosis were significantly increased. Renal catalase and superoxide dismutase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine - induced actions were moderately mitigated by chrysin, especially at the highest dose. Compared to control, chrysin did not cause any overt adverse effects on the treated rats. CONCLUSION: Different doses of chrysin produce variable therapeutic salutary effects in rats with CKD, and that, pending further studies, its usability as a possible therapeutic agent in human CKD should be considered.

Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells.

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury.

Ameliorative effect of chrysin on adenine-induced chronic kidney disease in rats.

Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine - induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered.

Development of a new model for the induction of chronic kidney disease via intraperitoneal adenine administration, and the effect of treatment with gum acacia thereon.

Oral adenine (0.75% w/w in feed), is an established model for human chronic kidney disease (CKD). Gum acacia (GA) has been shown to be a nephroprotective agent in this model. Here we aimed at developing a new adenine-induced CKD model in rats via a systemic route (intraperitoneal, i.p.) and to test it with GA to obviate the possibility of a physical interaction between GA and adenine in the gut. Adenine was injected i.p. (50 or 100 mg/Kg for four weeks), and GA was given concomitantly in drinking water at a concentration of 15%, w/v. Several plasma and urinary biomarkers of oxidative stress were measured and the renal damage was assessed histopathologically. Adenine, at the two given i.p. doses, significantly reduced body weight, and increased relative kidney weight, water intake and urine output. It dose-dependently increased plasma and urinary inflammatory and oxidative stress biomarkers, and caused morphological and histological damage resembling that which has been reported with oral adenine. Concomitant treatment with GA significantly mitigated almost all the above measured indices. Administration of adenine i.p. induced CKD signs very similar to those induced by oral adenine. Therefore, this new model is quicker, more practical and accurate than the original (oral) model. GA ameliorates the CKD effects caused by adenine given i.p. suggesting that the antioxidant and anti-inflammatory properties possessed by oral GA are the main mechanism for its salutary action in adenine-induced CKD, an action that is independent of its possible interaction with adenine in the gut.

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-ß 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.

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.