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Am J Physiol Renal Physiol 292: F1427-F1434, 2007. First published January 16, 2007; doi:10.1152/ajprenal.00458.2006
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Angiotensin II-induced genomic damage in renal cells can be prevented by angiotensin II type 1 receptor blockage or radical scavenging

Nicole Schupp,1 Ursula Schmid,1 Przemyslaw Rutkowski,2 Ursula Lakner,1 Nilesh Kanase,1 August Heidland,3 and Helga Stopper1

1Institute of Pharmacology and Toxicology, and 3Department of Internal Medicine, University of Würzburg, Würzburg, Germany; and 2Department of Nephrology Transplantology and Internal Diseases, Medical University, Gdansk, Poland

Submitted 16 November 2006 ; accepted in final form 9 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypertensive patients exhibit elevated cancer incidence, especially of cancers of the kidney. Elevated levels of ANG II, the active peptide of the renin-angiotensin system, regulating blood pressure and cardiovascular homeostasis, are known to cause hypertension and kidney diseases. There is evidence that ANG II is an activator of NAD(P)H oxidase, leading to the formation of free radicals, which are known to participate in the induction of DNA damage. This study was undertaken to characterize ANG II-induced DNA damage. DNA damage was measured by comet assay and micronucleus frequency test. Incubation of pig kidney cells (LLC-PK1) in vitro with ANG II concentrations between 85 and 340 nM led to a 6- to 15-fold increase of DNA damage compared with the control as revealed by comet assay analysis. Micronuclei were induced about fourfold compared with the control in pig and rat kidney cells (LLC-PK1, NRK) and in human promyelocytic cells (HL-60). ANG II-induced DNA damage could be prevented by coincubation with the ANG II type 1 receptor blocker candesartan and the antioxidants N-acetylcysteine and {alpha}-tocopherol. The ANG II type 2 receptor antagonist PD123319 could not reduce ANG II-induced DNA damage. Measurement of reactive oxygen species (ROS) by flow cytometry showed an enhanced formation after exposure to ANG II and a reduction of ROS after candesartan, N-acetylcysteine, and {alpha}-tocopherol. The present findings support our hypothesis that ANG II causes DNA damage via ANG II type 1 receptor binding and subsequent formation of oxidative stress.

kidney cells; deoxyribonucleic acid damage; oxidative stress; candesartan; N-acetylcysteine

EPIDEMIOLOGICAL STUDIES EXPLORING the connection between hypertension and cancer incidence found a higher cancer mortality in hypertensive patients (9) and an increased risk to develop kidney cancer (6, 19). In most patients with hypertension, the activity of renin is either inappropriately normal (in relation to sodium balance) or even elevated (14). Elevated levels are a typical feature of patients with renoparenchymal, especially renal vascular, hypertension. In particular, the renoprotection by blockade of the renin-angiotensin-aldosterone system in hypertension supports the role of a stimulated renin-angiotensin system (RAS) (12). ANG II is one of the oldest known peptide hormones. It is generated from the precursor protein angiotensinogen, predominantly by the actions of renin and angiotensin-converting enzyme. ANG II, the main effector of the RAS, is a major regulator of blood pressure and cardiovascular homeostasis and can cause hypertension and kidney diseases. Beside the systemic RAS, which produces the plasma ANG II, a functional local RAS, and accordingly a local ANG II production, has been found in various organs, for example in the kidney, heart, and brain (2, 17).

The actions of ANG II are mediated by two receptor molecules, the ANG II type 1 receptor (AT1) and the ANG II type 2 receptor (AT2). AT1 receptors are expressed in many tissues, whereas AT2 receptors are highest in fetal tissues (25). All classic physiological effects of ANG II, such as vasoconstriction, aldosterone and vasopressin release, sodium and water retention, are mediated by the AT1 receptor. Via this receptor, ANG II is also involved in cell proliferation, nephrosclerosis, endothelial dysfunction, and processes leading to atherothrombosis. The AT2 receptor often functions as a counterregulatory receptor and is involved, for example, in cell differentiation and apoptosis (4, 13).

In proximal tubule cells, activation of the AT1 receptor by ANG II led to the induction of NAD(P)H-oxidase, a multi-enzyme complex which enhances intracellular synthesis of reactive oxygen species (ROS) (10). Increased formation of ROS, of various etiology, is associated with the induction of genomic damage (11).

The present study was undertaken to characterize the genotoxic effects of ANG II in vitro in the epithelial porcine kidney cell line LLC-PK1. We hypothesize these effects to be mediated by the AT1 receptor, which upon activation causes oxidative stress. To prove this, we modulated the genotoxicity of ANG II with an AT1 receptor antagonist and with antioxidants. Two standard genotoxicity assays were employed: the micronucleus frequency test, which detects a subset of chromosomal aberrations, inherited to the first generation of daughter cells after mitosis and the comet assay, which detects structural DNA damage, which may partially or completely be transient, then leading to repair before mitosis or to cell death.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. If not mentioned otherwise, all chemicals were purchased from Sigma (Taufkirchen, Germany). Candesartan was provided by AstraZeneca (Wedel, Germany).

Cell culture. LLC-PK1 cells, an epithelial porcine kidney cell line with proximal tubule properties, were obtained from ATCC and grown as described in Ref. 22. For experiments, 106 cells were treated with test compounds in 5 ml culture medium. The cells were harvested after 24-h incubation with tested compounds for the comet assay and after 48 h for the micronucleus frequency test.

HL-60 cells (human promyelocytic cells) were kindly donated by Prof. R. Schinzel (Vasopharm, Würzburg, Germany). They were grown in RPMI medium, supplemented with 10% fetal calf serum, 1% glutamine, and antibiotics. NRK cells, an epithelial rat kidney cell line with proximal tubule properties, were obtained from ECACC and grown in DMEM medium (4,500 mg glucose/l), supplemented with 10% fetal calf serum, 1% glutamine, 1% nonessential amino acids and antibiotics.

All cells were routinely split twice a week to keep the exponential growth conditions and except for HL-60 (40 passages) were cultured for no more than 20 passages after thawing them from stock.

Comet assay. The comet assay was carried out according to Singh et al. (23), with slight modifications, as described earlier (22). A fluorescence microscope at 200-fold magnification and a computer-aided image analysis system (Komet 5, Kinetic Imaging LTD, Liverpool, UK) were used for analysis. Fifty cells in total (25 per slide) were analyzed, and results were expressed as percentage of DNA in the tail region.

Micronucleus frequency test. Cells were incubated for 48 h with the tested compounds, and after 24 h 5 µg/ml cytochalasin B was added to obtain binucleated cells (BN). For the analysis of the effect of {alpha}-tocopherol on ANG II-induced micronuclei, the cells were incubated for 24 h simultaneously with test compounds and cytochalasin B. After this incubation, the cells were brought onto glass slides by cytospin centrifugation and fixed with methanol (–20°C, 1 h). For staining, the slides were incubated with acridine orange (62.5 µg/ml in Sørensen buffer, pH 6.8) for 5 min, washed twice with Sørensen buffer for 5 min, and mounted for microscopy. The frequency of micronuclei was obtained after scoring 1,000 BN cells on each of two slides. The averages of three independent experiments are shown.

Quantification of apoptotic cells. During the analysis of the slides prepared for the micronucleus frequency test, cells with nuclei that showed characteristics of apoptotic nuclei (highly condensed chromatin) were also counted and their number was referred to 1,000 BN cells.

Proliferation index. Furthermore, the slides prepared for the micronucleus frequency test were used to calculate the proliferation index of the cells treated with ANG II, using the following formula

Formula
The result is the proliferation index (PI).

RT-PCR experiments. The expression of mRNA was detected using RT-PCR. Total RNA was isolated from LLC-PK1 cells with the RNeasy mini kit (Qiagen, Hilden, Germany), and 2.5 µg of RNA was used for cDNA synthesis using RevertAid First-Strand cDNA Synthesis Kit (Fermentas GmbH, St. Leon-Rot, Germany).

Table 1 shows the primers and conditions used during amplification. All primers were designed with the programme Primer3 (21).


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  		Table 1. Sequences, annealing temperatures, and cycle times of used primer for the amplification of genes of the angiotensin II receptors type 1 and type 2

 
PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide. For quantification of the mRNA, the density of the bands was measured using the Gel Doc 2000 (Bio-Rad, Hercules, CA).

PCR products destined for sequencing were cut out of the gel, eluated with the MinElute Gel Extraction Kit (Qiagen), and ligated into the pGEM-T-easy vector (Promega, Madison, WI) and transformed into DH5{alpha} cells (Invitrogen, Carlsbad, CA). After overnight culture, the plasmids were isolated using the HighSpeed Plasmid Midi Kit (Qiagen) and were sent to MWG-Biotech (Ebersberg, Germany) for sequencing.

Flow cytometric analysis of oxidative stress. 2',7'-Dichlorodihydrofluorescein diacetate (H2DCF-DA) was used to detect ROS production in cells. LLC-PK1 cells were preincubated with 10 µM H2DCF-DA for 5 min at 37°C and then ANG II alone, or together with N-acetylcysteine (NAC) or candesartan, was added for an additional 4 h. As a positive control 0.5 M hydrogen peroxide was used after an incubation time of 30 min. For the analysis of the potential antioxidative capacity of candesartan, after the preincubation with H2DCF-DA, 1.25 µM hydrogen peroxide, which yields a similar amount of ROS as 170 nM ANG II, alone, or together with NAC, {alpha}-tocopherol, or candesartan, was added for an additional 30 min. Cells were harvested, washed three times with PBS/1% BSA, and analyzed (3 x 105 cells/sample) by flow cytometry using a FACS LSR I (Becton-Dickinson, Mountain View, CA) after incubation for 10 min on ice with 1 µg/ml propidium iodide.

Statistical analysis. If not mentioned otherwise, data from three independent experiments are shown ± SE. Statistical significance among multiple groups was tested with the nonparametric Kruskal-Wallis test. Individual groups were then tested using the Mann-Whitney U-test. A P value of ≤0.05 was considered significant. For calculations SPSS 13.0 was used. Analysis of flow cytometry histograms was done with the free software WinMDI ver. 2.8 (Scripps Research Institute Cytometry Software page at http://facs.scripps.edu/software.html).


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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The presence of the expression of the AT1 and the AT2 receptor in LLC-PK1 cells was verified by RT-PCR. The obtained sequences were compared with the database GenBank, yielding 99% identity of the LLC-PK1 AT1 receptor to the database sequence of Sus sp. mRNA for AT1 receptor (accession number D11340) and 100% identity of the LLC-PK1 AT2 receptor to the database sequence of Sus sp. mRNA for AT2 receptor (accession number AF195509).

ANG II, in our experiments with LLC-PK1, did not change cell proliferation (Table 2). Also, ANG II did not induce apoptosis, since the number of apoptotic cells observed under the microscope did not rise when increasing doses of ANG II were applied (Table 2). Of the two other cell lines HL-60 (human promyelocytic cells) and NRK (rat proximal tubule cells) also tested for apoptosis and proliferation after incubation with ANG II, only the NRK cells showed an induction of both apoptosis and proliferation (Table 2).


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  		Table 2. Effect of 170 nM ANG II on the apoptosis and proliferation rate in human promyelocytic cells (HL-60), porcine kidney cells (LLC-PK1), and rat kidney cells (NRK)

 
ANG II-induced DNA damage was first measured in LLC-PK1 cells with the comet assay (Fig. 1). Upon treatment with ANG II, a dose-dependent increase in genomic damage was demonstrated (Fig. 1B). A statistically significant genomic damage appeared at 85 nM ANG II and increased with higher doses.


Figure 1
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  		Fig. 1. A: representative images of a healthy (1) and a highly damaged (2) cell in the comet assay, stained with propidium iodide. A computer-aided analysis system determines the extent of migrated DNA. B: DNA damage in LLC-PK1 cells as measured by the comet assay, after a 24-h treatment with various concentrations of ANG II. Control, no treatment; n.s., not statistically significant. The positive control methyl methanesulfonate yielded a DNA damage of 50.69 ± 5.30% DNA in tail. *P ≤ 0.05 vs. control. **P < 0.01 vs. control.

 
To analyze the role of the AT1 receptor in ANG II-induced comet formation, the AT1 receptor antagonist candesartan was added simultaneously with ANG II to the cells (Fig. 2). Candesartan prevented the ANG II-induced genomic damage completely.


Figure 2
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  		Fig. 2. DNA damage in LLC-PK1 cells as measured by the comet assay, after 24-h treatment with 2 concentrations of ANG II with and without coincubation with either 6 mM N-acetylcysteine (NAC) or 5 µM candesartan (Cand). Control, no treatment. Six millimolar NAC or 5 µM candesartan alone had no effect on the DNA (1.04 ± 0.07 and 1.11 ± 0.35% DNA in tail, respectively). The positive control methyl methanesulfonate yielded a DNA damage of 62.30 ± 2.00% DNA in tail. *P ≤ 0.05 vs. control. °P ≤ 0.05 vs. ANG II treatment.

 
ANG II is known to induce oxidative stress via its AT1 receptor, which can lead to genomic damage. To demonstrate the participation of oxidative stress in ANG II-induced comet formation, the cells were coincubated with ANG II and the antioxidant NAC. NAC, like candesartan, prevented the ANG II-induced genomic damage (Fig. 2). Incubation with NAC or candesartan alone did not lead to the formation of comets (data included in the figure legends).

As a second method to measure DNA damage, we chose the micronucleus frequency test. ANG II caused the induction of micronuclei in LLC-PK1 cells (Fig. 3), which could also be reduced by NAC and candesartan. The number of micronuclei in cells treated with 170 nM ANG II combined with NAC and candesartan and in cells treated with 340 nM ANG II combined with candesartan did not differ from the control cells. NAC was not able to prevent the induction of micronuclei caused by 340 nM ANG II completely; however, it did reduce them significantly.


Figure 3
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  		Fig. 3. A: representative micronuclei-containing binucleated cells, stained with acridine orange. B: micronuclei frequencies in binucleate LLC-PK1 cells after treatment for 48 h with 2 ANG II concentrations with and without coincubation with either 6 mM NAC or 5 µM Cand. After 24 h, 5 µg/ml cytochalasin B was added to all samples to yield binucleated cells by inhibiting cytokinesis. Control, treatment only with cytochalasin B. The positive control methyl methanesulfonate yielded 34.13 ± 4.49 micronuclei per 1,000 binucleated cells (not shown). *P ≤ 0.05 vs. control. °P ≤ 0.05 vs. ANG II treatment.

 
Since it was reported that NAC inhibits the binding of ANG II to AT1 receptor due to its ability to reduce disulfide bonds (28), the effect of a second antioxidant, which contains no free sulfhydryl groups, {alpha}-tocopherol, on ANG II-induced DNA damage was examined. As can be seen in Fig. 4A, comet formation due to ANG II incubation was prevented completely. Also, the formation of micronuclei by 170 nM ANG II was inhibited (Fig. 4B).


Figure 4
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  		Fig. 4. A: DNA damage in LLC-PK1 cells as measured by the comet assay, after 4-h treatment with 170 nM ANG II with and without coincubation with 750 µM {alpha}-tocopherol ({alpha}-TOC). Control, no treatment. Seven hundred fifty micromolar {alpha}-TOC alone had no effect on the DNA (2.20 ± 0.07% DNA in tail). B: micronuclei frequencies in binucleate LLC-PK1 cells after treatment for 24 h with 170 nM ANG II with and without 750 µM {alpha}-TOC. Control. no treatment. *P ≤ 0.05 vs. control. °P < 0.05 vs. ANG II treatment.

 
The application of the AT2 receptor antagonist PD123319 to ANG II-treated LLC-PK1 cells showed that this substance had no effect on the ANG II-induced comet formation (Fig. 5A). PD123319 lessened the ANG II-caused micronuclei number slightly but was not able to reduce them significantly (Fig. 5B).


Figure 5
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  		Fig. 5. A: DNA damage in LLC-PK1 cells as measured by the comet assay, after 24-h treatment of ANG II with and without coincubation with either 100 nM or 1 µM PD123319 (PD). Control, no treatment. One micromolar PD123319 alone had no effect on the DNA (6.24 ± 1.37% DNA in tail). The positive control methyl methanesulfonate yielded a 51.21 ± 2.79-fold induction of DNA damage. B: micronuclei frequencies in binucleate LLC-PK1 cells after treatment for 48 h with ANG II with and without coincubation with either 100 nM or 1 µM PD. Control, no treatment. The positive control methyl methanesulfonate yielded 54,33 ± 4,21 micronuclei. *P ≤ 0.05 vs. control.

 
The induction of micronuclei by ANG II was confirmed in two other cell lines, HL-60 and NRK, which also showed an increase in micronuclei-containing cells (Table 2). All cell lines (HL-60, LLC-PK1, and NRK) had a similar factor of micronuclei induction around 4 (Table 2).

Since we assume that the ANG II-induced genomic damage is caused by the formation of ROS upon AT1-mediated activation of the NAD(P)H oxidase, the generation of ROS was measured flow cytometrically. As shown in Fig. 6, ANG II led to a significant formation of ROS, which could be prevented either by candesartan, NAC, or {alpha}-tocopherol.


Figure 6
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  		Fig. 6. Flow cytometric analysis of ANG II-induced reactive oxygen species (ROS) production in LLC-PK1 cells. A: representative frequency histogram of the green fluorescence of H2DCF-positive cells of control cells (broken line), and cells after 4-h incubation with 170 nM ANG II without (fat solid line) and with coincubation with 6 mM NAC (light gray solid line). B: representative frequency histogram of the green fluorescence of H2DCF-positive cells of control cells (broken line), and cells after 4-h incubation with 170 nM ANG II without (fat solid line) and with coincubation with 5 µM Cand (dark gray solid line). C: quantification of the flow cytometry measurements by WinMDI 2.8. Relative fluorescence units are shown. Control, treatment with only H2DCF-DA. D: quantification of the flow cytometry measurements of cells incubated 4 h with 170 nM ANG II with and without 750 µM {alpha}-TOC. Control, treatment with only H2DCF-DA. *P ≤ 0.05 vs. control. °P < 0.05 vs. ANG II treatment.

 
To exclude the possibility of candesartan being a radical scavenger itself, a coincubation of LLC-PK1 cells with hydrogen peroxide (H2O2) and candesartan was performed (Fig. 7, A–D). An H2O2 concentration was chosen, which led to a shift to the right of the curve, comparable to the shift caused by 170 nM ANG II (see Fig. 6), and which also caused DNA damage in the comet assay (Fig. 7E). The flow cytometric analysis of this experiment showed that candesartan could not reduce the H2O2-induced oxidative stress in the cells, in contrast to NAC, which could.


Figure 7
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  		Fig. 7. Flow cytometric analysis of a potential antioxidative effect of Cand. A: representative frequency histogram of the green fluorescence of H2DCF-positive cells of control cells (broken line), and cells after 30-min incubation with 1.25 µM hydrogen peroxide (H2O2) without (fat solid line) and with coincubation with 6 mM NAC (light gray solid line). B: quantification of the flow cytometry measurements by WinMDI 2.8. Relative fluorescence units are shown. *Control, treatment with only H2DCF-DA. C: representative frequency histogram of the green fluorescence of H2DCF-positive cells of control cells (broken line), and cells after 30-min incubation with 1.25 µM H2O2 without (fat solid line) and with 5 µM Cand (dark gray solid line). D: quantification of the flow cytometry measurements by WinMDI 2.8. Relative fluorescence units are shown. Control, treatment with only H2DCF-DA. *P ≤ 0.05 vs. control. °P ≤ 0.05 vs. hydrogen peroxide treatment. E: DNA damage in LLC-PK1 cells as measured by the comet assay, after 30-min treatment with 1.25 µM H2O2. Control, no treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In LLC-PK1 pig kidney cells, we detected a direct DNA damaging potential of ANG II by the comet assay. Thus our data confirm and extend similar observations in cells of the ascending limb of the loop of Henle (20) and in microvessel endothelial cells (18). For the first time, chromosomal mutations caused by ANG II as revealed by micronucleus formation were observed. Micronuclei were induced in three different cell lines, in LLC-PK1, in rat kidney cells (NRK), and in human promyelocytic cells (HL-60).

Depending on the cell type, the stimulation of the ANG II receptor AT1 leads to cellular contraction, hypertrophy, proliferation, and/or apoptosis (31). In LLC-PK1, ANG II had no impact on the proliferation or the apoptosis rate in contrast to observations by Hannken et al. (10), who detected a cell cycle arrest in LLC-PK1. This difference might be due to the presence of serum in our experiments, while Hannken et al. incubated serum free. Of the other two cell lines we analyzed for micronuclei induction with ANG II, only the rat kidney cells showed enhanced proliferation and apoptosis.

Coincubation with the AT1 receptor antagonist candesartan prevented the appearance of genomic damage detected with the comet assay and the micronucleus frequency test, while the application of the AT2 receptor antagonist PD123319 had no effect.

It is well-known that ANG II binding to the AT1 receptor induces NAD(P)H oxidases, resulting in the generation of superoxide anions (7). This was also already demonstrated for LLC-PK1 cells (10). Here, it is shown for the first time that the genotoxic action of ANG II in the comet assay and its micronuclei-inducing effect could be prevented by the antioxidants NAC and {alpha}-tocopherol, linking the formation of ROS to the genotoxic effects.

DNA damage caused by ROS involves single- or double-strand DNA breaks, purine, pyrimidine, or deoxyribose modifications, and DNA cross-links (29). DNA repair, which starts after the oxidative attack on the chromosomes, often transforms the above-mentioned modifications to additional strand breaks. The comet assay detects such structural DNA damage (1), which, as we have shown, was also induced by incubation with ANG II. Micronuclei are formed for example after double-strand breaks which lead to chromosome fragments lagging behind at the anaphase during nuclear division (5). Hydrogen peroxide induces DNA double-strand breaks in a time- and dose-dependent manner, as revealed by an antibody specifically detecting double-strand breaks (15). Superoxide anions released after ANG II stimulation by NAD(P)H oxidase are rapidly converted to hydrogen peroxide (8), which can lead to the ANG II-induced micronuclei.

Our evidence for ANG II-induced genomic damage in renal tubular cells could be of significance with regard to the increased occurrence of kidney carcinomas under an activated renin-angiotensin-system (6, 19). In animal models, it was shown that the concentration of ANG II in the kidney is 25 to 1,000 times higher than in plasma (24, 30). Under pathological conditions, the ANG II concentration in the renal interstitial fluid of dogs can reach 800 nM (24) implying a physiological relevance of a genotoxic effect of 85 nM ANG II.

In the presence of hypertension, the incidence of renal cancer is enhanced (3). The potential involvement of a stimulated RAS in the kidney is underlined by the observation that long-term treatment with diuretics is associated with an increased risk of renal cancer (9). On the contrary, ANG I-converting enzyme inhibitors have been discussed as anticancer drugs (16), and ANG II type 1 receptor blockers have been tested in a pilot study with patients who suffer from advanced hormone-refractory prostate cancer (26, 27).

In conclusion, our in vitro results show that ANG II induces genomic damage in mammalian cells, most likely via oxidative mechanisms. This injury can be prevented by ANG II type 1 receptor blockade and by antioxidants.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Verein zur Bekämpfung der Nieren- und Hochdruckkrankheiten e.V., Würzburg, Germany. Dr. P. Rutkowski was supported by a Nephrocore fellowship from Fresenius Medical Care.


   ACKNOWLEDGMENTS
 
We thank M. Scheurich and T. Fischer for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Schupp, Institute of Pharmacology and Toxicology, Univ. of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany (e-mail: nicole.schupp{at}toxi.uni-wuerzburg.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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Angiotensin II-Dependent Superoxide: Effects on Hypertension and Vascular Dysfunction
Hypertension, July 1, 2008; 52(1): 51 - 56.
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