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Cancer Research 68, 9239, November 15, 2008. doi: 10.1158/0008-5472.CAN-08-1310
© 2008 American Association for Cancer Research
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Endocrinology

Angiotensin II Induces DNA Damage in the Kidney

Ursula Schmid1, Helga Stopper1, Frank Schweda2, Nina Queisser1 and Nicole Schupp1

1 Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany and 2 Institute of Physiology, University of Regensburg, Regensburg, Germany

Requests for reprints: Nicole Schupp, Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany. Phone: 49-931-20-148760; Fax: 49-931-20-148446; E-mail: nicole.schupp{at}toxi.uni-wuerzburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
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. [Cancer Res 2008;68(22):9239–46]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The renin angiotensin aldosterone system plays an integral role in the homeostasis of arterial pressure, tissue perfusion, and extracellular volume. Increased levels of angiotensin II can cause hypertension and contribute to the progression of kidney diseases by stimulating growth, inflammation, and fibrosis (1–3). Epidemiologic studies exploring the connection between hypertension and cancer incidence found a higher cancer mortality in hypertensive patients (4) and an increased risk to develop kidney cancer (5, 6). We observed the formation of DNA damage following treatment with angiotensin II in vitro (7). This damaging effect could be ascribed to the activation of the angiotensin II type 1 (AT1) receptor with subsequent release of reactive oxygen species (ROS).

The pivotal enzyme responsible for angiotensin II–mediated ROS generation in the kidney is the NADPH oxidase complex, which is composed of two membrane-bound components (p22phox and Nox1, Nox2, or Nox4), three cytoplasmic subunits (p40phox, p47phox, and p67phox), and the small GTPase Rac 1/2 (8–10). In contrast to the neutrophil NADPH oxidase, the enzyme complex in nonphagocytic cells continuously forms low levels of superoxide intracellularly and can be further activated by agonists such as angiotensin II (11). Angiotensin II activates NADPH oxidase either by up-regulating subunits, like Nox1, p22phox, p47phox, and p67phox (3, 12), or by facilitating the assembly of the subunits (13, 14). Nox4 is unique because it seems to operate constitutively, being regulated at the mRNA level (15, 16). The ROS produced on stimulation with angiotensin II is mainly superoxide, which is converted enzymatically to hydrogen peroxide and can also react to hypochlorous acid and the hydroxyl free radical (3).

ROS on their part are able to evoke DNA damage (17). More than 100 oxidative DNA adducts have been identified; in addition, ROS directly induce single- and double-strand breaks, abasic sites, and DNA cross-links (18). Estimations exist that a human cell is exposed to up to 105 oxidative hits a day from ROS, which can result in permanent modifications representing the beginning of carcinogenesis (17).

To transfer our results of the genotoxic effect of angiotensin II in cell culture to the more physiologic context of an intact kidney, we examined the effect of angiotensin II in the isolated perfused mouse kidney. This model is widely used for studies of renal physiology and pathophysiology in the absence of interindividual influences of systemic confounding factors (19, 20). Whereas the application of angiotensin II in vivo is accompanied by marked hemodynamic effects, which might indirectly induce genotoxicity, the isolated kidney allows the observation of effects under controlled conditions (e.g., under a constant perfusion pressure).

The alkaline comet assay was done for the first time with cells extracted from isolated perfused kidneys. The comet assay is a rapid and quantitative method to measure DNA damage in any tissue, provided that a single-cell suspension can be obtained (21, 22). Its greatest advantage is its applicability to terminally differentiated nondividing cells, such as cells out of the isolated perfused kidney.

Further experiments served to characterize the DNA damage caused by angiotensin II. As an alternative marker for DNA damage, the phosphorylation of histone H2AX ({gamma}-H2AX) was used, which marks chromatin at the damage site and serves to recruit DNA repair and signaling molecules (23). Cleavage at oxidatively altered bases by the enzyme formamidopyrimidine DNA glycosylase (FPG; ref. 24) is another sign for the involvement of ROS in the development of the DNA damage. To show the base modifications after angiotensin II treatment of cultured cells, the comet assay with FPG preincubation was carried out. In addition, the repairability of the angiotensin II–induced genomic lesions was investigated using the comet assay and the detection of {gamma}-H2AX.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Materials. If not mentioned otherwise, all chemicals were purchased from Sigma Aldrich. Candesartan was provided by AstraZeneca.

Isolated perfused mouse kidneys. Male C57BL/6 mice (20–26 g body weight, Charles River) with free access to commercial pellet chow and tap water were used as kidney donors. Perfusion of isolated mouse kidneys was at a constant pressure of 100 mm Hg conducted as described earlier (20). Stock solutions of the test substances were added to the perfusate for 1 h with a latency of 10 min. After perfusion, the isolated mouse kidney was minced to small pieces in 3 mL buffer [RPMI 1640, 15% DMSO, 1.8% (w/v) NaCl], sifted through a cell strainer with a mesh pore size of 100 µm (Becton Dickinson), precipitated at 1,000 rpm and 4°C for 5 min (Heraeus Labofuge 400e), and resuspended in 1 mL buffer.

Cell culture. LLC-PK1, an epithelial porcine kidney cell line with proximal tubule properties, was obtained from American Type Culture Collection and grown in DMEM (1.0 g/L glucose) supplemented with 10% FCS, 1% glutamine, 25 mmol/L HEPES buffer, and antibiotics. Cells were split routinely twice a week to ensure exponential growth and were cultured for no more than 10 passages after thawing them from stock.

Determination of cell vitality. The fluorescein-ethidium bromide assay was carried out according to Yang and colleagues (25), with the following modifications: The staining solution contained 30 µg/mL fluorescein diacetate and 12 µg/mL ethidium bromide. Cells (17.5 µL) were stained with 7.5 µL staining solution. Ten microliters of this mixture were applied to the slide and the fractions of green and red cells in a total of 2 x 200 cells were counted under a microscope at a 200-fold magnification. Vital cells stain green because their active esterases cleave the acetate groups from fluorescein diacetate, which then starts to fluoresce. Dead cells stain red because ethidium bromide can cross their defective membrane and intercalates in the DNA. Cells with residual esterase activity and defective membranes appear brownish green and were not included in the quantification.

Quantification of apoptotic cells. Apoptotic cells were quantified with the NucView 488 Caspase-3 Assay Kit for Live Cells (Biotium, Inc.). In brief, 50 µL of the kidney cell suspension were centrifuged at 1,000 rpm for 5 min in a microfuge. The cells were resuspended in 200 µL PBS, and 5 µL caspase-3 substrate was added. After 30-min incubation at room temperature, the cells were washed twice with PBS (4,000 rpm, 5 min, microfuge) and resuspended in 10 µL PBS. The number of green fluorescent apoptotic cells was counted under a microscope at a 200-fold magnification in a total of 200 cells observed under transmitted light. Apoptotic cells stained green because the substrate of caspase-3 included in this kit, on cleavage, migrates into the nucleus and starts to fluoresce after binding to the DNA.

Comet assay. LLC-PK1 cells (3.5 x 105), seeded the day before in small culture flasks, were treated for 4 h with test substances in 5 mL medium. Afterward, the cells were harvested. Cell suspensions of extracted primary kidney cells or LLC-PK1 cells were used in the comet assay as described earlier (26) using a fluorescence microscope at a 200-fold magnification and computer-aided image analysis (Komet 5, Kinetic Imaging Ltd.). After DNA staining with propidium iodide (20 µg/mL), 25 cells from each of two slides were measured, with percent tail DNA being the evaluation parameter.

Real-time PCR. Total RNA was isolated from the frozen kidneys as described earlier (27). For amplification of the cDNAs, the following primers were used: glutathione peroxidase, 5'-GTCCACCGTGTATGCCTTCT-3' (sense), 5'-CCTGAGAGAGACGCGACATT-3' (antisense); heme oxygenase, 5'-CAGGTGATGCTGACAGAGGA-3' (sense), 5'-TCTCTGCAGGGGCAGTATCT-3' (antisense); NADPH oxidase subunit p47phox (p47), 5'-GTCCCTGCATCCTATCTGGA-3' (sense), 5'-GGGACATCTCGTCCTCTTCA-3' (antisense); NADPH oxidase subunit Nox4, 5'-CTGGAAGAACCCAAGTTCCA-3' (sense), 5'-TGACAGGTTTGTTGCTCCTG-3' (antisense); β-actin, 5'-TACAGCTTCACCACCACAGC-3' (sense), 5'-CCTCAGAGAGACGCGACATT-3' (antisense). All amplificates were sequenced and found to be 99% to 100% identical to the respective mouse gene.

Detection of phosphorylated {gamma}-H2AX sites. For immunofluorescence staining of phosphorylated {gamma}-H2AX foci, LLC-PK1 cells treated with test substances for 4 h and cells from perfused mouse kidneys were transferred onto glass slides by cytospin centrifugation and fixed in ice-cold methanol for at least 2 h. After washing, rabbit polyclonal antibody to {gamma}-H2AX (Abcam) was added 1:100 in 5% PBS/FCS for 1 h at 37°C to LLC-PK1 cells, and phospho-histone H2AX (pS139) rabbit monoclonal antibody (Epitomics) was added 1:100 in 5% PBS/FCS for overnight at 37°C to the mouse cells. After washing with PBS + 0.2% Tween, the FITC-conjugated secondary antibody was added for 30 min at room temperature. The slides were mounted with Antifade Gold (Invitrogen). Images of LLC-PK1 cells were visualized and captured using an Axioskop 2 (Zeiss) with a 100-fold oil immersion lens. {gamma}-H2AX foci of cells from perfused mouse kidneys were visualized and captured using an Eclipse 55i (Nikon) with a 40-fold lens and quantified by measuring gray values of 200 cells per perfused kidney with ImageJ 1.40g (28).3

For flow cytometric quantification of {gamma}-H2AX, 106 LLC-PK1 cells, seeded the day before in medium culture flasks, were treated with test substances for 4 h, washed twice with PBS, and supplied with fresh medium. After 0, 15, 30, 60, 120 min, and 24 h, cells were harvested, washed with cold PBS, and fixed for 1 h in ice-cold ethanol (70%). Cells were washed twice with cold PBS and permeabilized with 0.5% Triton X in PBS for 10 min at room temperature. Unspecific antibody binding sites were blocked for 30 min with 5% FCS in PBS. Thereafter, cells were centrifuged and incubated with 1.5 µL of 1:100 anti–phospho-histone H2AX (clone JBW301, Millipore) in 5% PBS/FCS overnight at 4°C. Cells were washed twice with FCS/PBS and incubated with a FITC-conjugated secondary antibody for 45 min in the dark. After washing twice with PBS/FCS, 740 µL PBS, 50 µL saponin (1% in PBS), and 10 µg propidium iodide were added and phosphorylated {gamma}-H2AX foci were detected by flow cytometry (FACS LSR I, Becton-Dickinson). Geometric means were assessed using the "histogram statistics" function of CellQuest Pro 4.0.

Determination of FPG-sensitive sites. FPG-sensitive sites were determined by comet assay including an incubation step with FPG. After treatment with test substances, embedding the cells in agarose, and lysis of the cell membranes, the slides were washed thrice for 5 min in cold buffer (40 mmol/L HEPES-KOH, 100 mmol/L KCl, 0.5 mmol/L Na2EDTA, and 0.2 mg/mL bovine serum albumin) to remove the lysis solution. The FPG-sensitive sites were detected by incubation of the nuclei embedded in agarose with 0.005 µg/mL FPG protein (kindly donated by Professor Bernd Epe, Institute for Pharmacy, University of Mainz, Mainz, Germany) for 60 min at 37°C. Afterward, the comet assay was carried out as described above. In this case, ethidium bromide (20 µg/mL) was used for DNA staining. The net level of FPG-sensitive sites was obtained as the difference in score between samples incubated with FPG protein and samples incubated with buffer.

Detection of abasic sites. Cells were treated with test substances for 4 h, harvested, and subsequently fixed in ice-cold ethanol (100%) for 10 min at 4°C. The cells were washed once with cold PBS and incubated for 2 h at 25°C with 1 mL staining solution [5 µmol/L Alexa Fluor 488 C5-aminooxyacetamide, bis(triethylammonium) salt (Alexa Fluor 488 hydroxylamine, Invitrogen), 3% acetic acid, 2% dimethylformamide, 0.25 mol/L MgCl2 in PBS] per sample. Abasic sites were detected by flow cytometry. The medians of the resulting histograms were evaluated using the freeware WinMDI 2.8.4

For microscopic detection of abasic sites, cells were treated with test substances for 4 h, harvested, and brought to slides by cytospin centrifugation. After fixation in ice-cold methanol for at least 2 h, cells were stained with 10 µL staining solution for 2 h at 25°C. Hereupon, the slides were washed extensively with PBS and mounted with Antifade Gold. Images were visualized and captured using an Axioskop 2 (Zeiss) with a 40-fold magnification. For better comparison, these pictures were magnified 2.5 times in Fig. 2B.


Figure 2
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  		Figure 2. A, incidence of {gamma}-H2AX foci in LLC-PK1 cells after 4-h treatment with 200 nmol/L angiotensin II, 5 µmol/L candesartan, 200 nmol/L angiotensin II together with 5 µmol/L candesartan, or 100 µmol/L genistein (positive control). Representative images of the localization of {gamma}-H2AX foci (green fluorescence), the Hoechst 33258–stained nuclei (blue fluorescence), and the overlay of both stains, captured at a 100-fold magnification. B, incidence of abasic sites in LLC-PK1 cells after 4-h treatment with 200 nmol/L angiotensin II, 5 µmol/L candesartan, 200 nmol/L angiotensin II together with 5 µmol/L candesartan, or 750 µmol/L hydrogen peroxide (positive control). Representative images of the localization of the green fluorescing abasic sites, captured at a 40-fold magnification and enlarged 2.5 times afterward for better comparison with A.

 
Statistics. If not mentioned otherwise, data from at least three independent experiments ± SD or SE (flow rate of the kidneys) are depicted. Statistical significance among multiple groups was tested with Kruskal-Wallis over all groups, and Mann-Whitney test was used to determine significance between two groups. For calculations, SPSS 16.0 was used. Results were considered significant if P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Isolated perfused mouse kidney. Angiotensin II–induced DNA damage in isolated perfused mouse kidneys was measured with the comet assay (Fig. 1A ). Compared with perfused control kidneys, a dose-dependent, significant increase in DNA damage after 1 hour of perfusion with 1, 10, and 50 nmol/L angiotensin II was observed. The dose dependency indicates the effect to be dependent on the substance. Damage distribution analysis did not reveal cell populations with different sensitivities (distribution not shown). Because the kidneys were perfused at a constant pressure of 100 mm Hg, the vasoconstrictor angiotensin II dose-dependently reduced the perfusate flow (milliliters per gram of kidney). The perfusion procedure itself caused only a small amount of DNA strand breaks in comparison with nonperfused kidneys processed the same way. The following experiments were conducted with the angiotensin II concentration of 10 nmol/L to ensure a sufficiently high damage, which has the potential to be decreased significantly.


Figure 1
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  		Figure 1. DNA damage in primary mouse kidney cells extracted out of isolated perfused kidneys, as measured by the comet assay (columns) and perfusate flow measured gravimetrically (dots). Kidneys were perfused for 1 h at a constant perfusion pressure of 100 mm Hg. Shown are average values with SD for the DNA damage and average values with SE for the perfusate flow. The treatments were carried out in parallel, so the values for the control and for 10 nmol/L angiotensin II are the same in A and B. A, dose dependence of angiotensin II–induced DNA damage. Shown are values for the unperfused control (n = 24); for the treatment with perfusate buffer (control, n = 10); and for perfusion with 1 nmol/L angiotensin II (Ang II; n = 4), 10 nmol/L angiotensin II (n = 10), and 50 nmol/L angiotensin II (n = 3). B, role of vasoconstriction and AT1 receptor in angiotensin II–induced DNA damage. Shown are values for perfusion with 10 nmol/L of the thromboxane mimeticum U46619 (n = 3), with 10 nmol/L of U46619 together with angiotensin II (n = 3), with 10 nmol/L angiotensin II (n = 10), and with 10 nmol/L angiotensin II and 5 µmol/L candesartan (Cand; n = 3), as well as the effect of 5 µmol/L candesartan alone (n = 3). *, P ≤ 0.05, versus perfused control. {circ}, P ≤ 0.05, versus angiotensin II treatment (DNA damage); +, P ≤ 0.05, versus control; {bigtriangleup}, P ≤ 0.05, versus angiotensin II treatment (perfusate flow).

 
To exclude artifacts in the comet assay due to cytotoxicity, a vitality assay was done with cells extracted from nonperfused, control-perfused, and angiotensin II–perfused kidneys. Samples from all these treatments showed no difference in the percentage of viable cells (Table 1 ). Induction of apoptosis was monitored by staining the cells for caspase-3 activity. Again, no significant difference was observed between cells from nonperfused, control-perfused, and angiotensin II–perfused kidneys (Table 1).


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  		Table 1. Quantification of viable and apoptotic cells, DNA double strand breaks, and expression levels of selected genes in cell extracts from unperfused control kidneys, perfused control kidneys and kidneys perfused with 10 nmol/L angiotensin II

 
To exclude the possibility that the DNA damage was caused by mechanical stress or hypoxia due to the angiotensin II–induced vasoconstriction, the thromboxane mimeticum U46619 was infused in the same concentration as angiotensin II. Although application of U46619 resulted in approximately the same vasoconstriction as application of angiotensin II, it did not induce DNA damage (Fig. 1B). A combination of U46619 and angiotensin II was not able to reduce the flow rate any further and resulted in a comparable DNA damage as induced by angiotensin II alone. These two vasoconstrictors therefore showed neither synergism nor antagonism in their action.

To examine whether the AT1 receptor is involved in the formation of angiotensin II–induced DNA strand breaks in ex vivo perfused mouse kidneys, the AT1 receptor antagonist candesartan was applied. In fact, whereas 5 µmol/L candesartan alone had no effect on DNA damage and perfusate flow, it completely prevented angiotensin II–induced DNA damage and restored the flow (Fig. 1B).

Quantification of double-strand breaks with an antibody against {gamma}-H2AX, an early marker of these lesions (23), showed a 1.4-fold increase in angiotensin II–perfused kidneys (Table 1).

Measurement of transcript expression of the oxidative stress marker genes glutathione peroxidase and heme oxygenase, as well as of the NADPH oxidase subunits Nox4 and p47, showed no change after 1-hour perfusion of the kidneys with angiotensin II compared with the control kidneys (Table 1).

Cell culture experiments. To characterize in more detail the angiotensin II–induced DNA damage, further experiments were conducted in LLC-PK1 cells, a porcine kidney cell line with proximal tubule properties.

First, the occurrence of double-strand breaks induced by angiotensin II was investigated. Immunofluorescence staining showed that treatment with 200 nmol/L angiotensin II, a concentration that was chosen after previous experiments (7), resulted in an increased frequency of {gamma}-H2AX (Fig. 2A ). Flow cytometric quantification of {gamma}-H2AX revealed a significant increase in fluorescence after treatment with angiotensin II (Fig. 3A ). Addition of candesartan restored control levels.


Figure 3
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  		Figure 3. A, flow cytometric quantification of {gamma}-H2AX in LLC-PK1 cells after 4-h treatment with 200 nmol/L angiotensin II with and without coincubation with 5 µmol/L candesartan. Positive control: 100 µmol/L genistein. Depicted is the geometric mean of the resulting histograms normalized to control. *, P ≤ 0.05, versus control; {circ}, P ≤ 0.05, versus angiotensin II treatment. B, induction of abasic sites in LLC-PK1 cells measured by flow cytometry after treatment with 200 nmol/L angiotensin II with and without coincubation with 5 µmol/L candesartan. Positive control: 750 µmol/L hydrogen peroxide (H2O2). *, P ≤ 0.05, versus control; {circ}, P ≤ 0.05, versus angiotensin II treatment. C, DNA damage as measured by the FPG-modified comet assay in LLC-PK1 cells after 4-h treatment with 200 nmol/L angiotensin II with and without coincubation with 5 µmol/L candesartan. Positive control: 0.05 µg/mL methylene blue for 10 min, illuminated with a 60-W bulb in a distance of 10 cm on ice. Depicted is the difference ({Delta}) in the percentage of DNA in tail between nuclei treated with FPG enzyme and nuclei not treated with FPG enzyme. *, P ≤ 0.05, versus control; {circ}, P ≤ 0.05, versus angiotensin II treatment.

 
Abasic sites were detected by immunofluorescence staining and by flow cytometry. For this, the abasic sites were tagged with a fluorescent aldehyde-reactive probe. As can be seen in Fig. 2B, the nuclei of angiotensin II–treated cells are much brighter than those of the control or the candesartan-treated cells. Flow cytometric quantification revealed these differences in fluorescence-labeled abasic sites to be significant, whereas the cotreatment with candesartan yielded levels comparable to the untreated control (Fig. 3B).

Besides strand breaks and abasic sites, oxidative stress leads to the formation of base modifications such as 7,8-dihydro-8-oxo-guanine (8-oxodG). 8-OxodG is the main DNA lesion recognized by FPG in the FPG-modified comet assay. Whereas the normal alkaline comet assay detects strand breaks and abasic sites, the addition of FPG to it allows the quantification of the base modifications. The enzyme incises the DNA at the site of the lesion, thereby generating additional strand breaks (24). Treatment with angiotensin II significantly increased the occurrence of FPG-sensitive sites shown in Fig. 3C. Coincubation with candesartan prevented the formation of these oxidative base modifications.

To test if angiotensin II–induced DNA damage was only transient, DNA repair after treatment with angiotensin II was measured in a time course experiment by comet assay over a time period of 24 hours. Already after a recovery period of 15 minutes, the cells were able to repair approximately half of the induced DNA strand breaks, and after 24 hours, no more strand breaks than in the control were detected (Fig. 4 ). Quantification of double-strand breaks at the same time points by flow cytometry in contrast showed an increase of {gamma}-H2AX in the course of the recovery period, suggesting the persistence of double-strand breaks. FPG-sensitive sites were repaired even faster than the strand breaks detected in the normal comet assay; already after 30 minutes, control levels were reached (Fig. 4).


Figure 4
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  		Figure 4. DNA damage in LLC-PK1 cells as measured by the comet assay (white and black columns), by the FPG-modified comet assay (light and dark gray columns), and by flow cytrometric quantification of induction of {gamma}-H2AX fluorescence (diamonds) after 4-h treatment with 200 nmol/L angiotensin II and subsequent recovery periods of 0 min to 24 h. To show the repair of the FPG-sensitive sites, the difference ({Delta}) in the percentage of DNA in tail between nuclei treated with FPG enzyme and nontreated nuclei is depicted. {gamma}-H2AX fluorescence was normalized to control (4-h treatment with PBS). *, P ≤ 0.05, versus control; {circ}, P ≤ 0.05, versus 0 min repair time (DNA damage); +, P ≤ 0.05, versus control; {bigtriangleup}, P ≤ 0.05, versus 0 min repair time ({gamma}-H2AX fluorescence).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In the isolated perfused kidney, angiotensin II caused dose-dependent vasoconstriction and DNA strand breaks. Control kidneys perfused for 1 hour without any pharmacologic intervention showed a small increase in DNA damage compared with unperfused kidneys subjected to the comet assay immediately after sacrifice of the mice; thus, the perfusion per se had a slight effect on the integrity of the DNA. DNA strand breaks can also be caused by cytotoxic or apoptotic processes, which were observed in angiotensin II–treated cells by others (29, 30). Therefore, we tested the cells isolated out of the perfused kidney for their vitality and for an early apoptotic marker, the stimulation of caspase-3 activity. Although there was a certain amount of dead cells in the preparations, which was most likely due to the extraction procedure, there was no difference between the angiotensin II–treated kidneys and the control kidneys. We measured cytotoxicity and apoptosis only after 1 hour, whereas others report these effects of angiotensin II as late as after 16 to 72 hours (29, 31). Therefore, it cannot be excluded that angiotensin II would later induce cytotoxicity and apoptosis in the kidney at the concentration of 10 nmol/L. The short perfusion time could also be responsible for the lack of changes in the expression of the two analyzed oxidative stress marker genes, heme oxygenase and glutathione peroxidase, and the two analyzed NADPH oxidase subunit genes, p47 and Nox4. The reaction of glutathione peroxidase on increased angiotensin II levels was until now only studied in whole animals (32). Increase in heme oxygenase-1 expression in kidney cells was only seen after 16 to 48 hours (29, 33). Expression of p47 and Nox4 was higher after 1-hour incubation with 100 nmol/L angiotensin II in smooth muscle cells (34). In one report on kidney cells (podocytes), Nox4 was up-regulated, but details of incubation time and angiotensin II concentration are missing (35).

Angiotensin II is a potent vasoconstrictor that significantly reduced kidney perfusion in our study. Because ischemia is known to cause tubular epithelial necrosis and apoptosis (36) and both processes would generate DNA damage detectable in the comet assay, it is critical for our conclusion that the observed effects of angiotensin II are not related to its vasoconstrictor effects. The thromboxane mimetic U46619 generated a marked vasoconstriction, hereby drastically reducing kidney perfusion, but it did not induce DNA damage. This observation reinforces our hypothesis that the AT1 receptor–mediated DNA damage in the isolated perfused kidneys indeed is independent of the hemodynamic effects of angiotensin II.

Besides performing the comet assay, we quantified double-strand breaks in the same perfused kidney samples. The histone H2AX has been shown to be rapidly phosphorylated after induction of double-strand breaks and is thought to promote repair protein recruitment (23). {gamma}-H2AX can be visualized with the help of phospho-specific antibodies (37). Double-strand breaks are the most toxic form of DNA damage, resulting in cell death or genetic alterations like large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss (38). The chromosomal rearrangements that may occur after incorrect repair are considered a major initiating factor in carcinogenesis (39).

However, because double-strand breaks and DNA damage observed in the comet assay could nevertheless theoretically be completely repaired, further experiments were conducted in the porcine kidney cell line LLC-PK1 to be able to evaluate the relevance of the DNA damage induced by angiotensin II ex vivo.

Flow cytometric measurements and immunofluorescence staining of {gamma}-H2AX in LLC-PK1 cells indicated that angiotensin II induced double-strand breaks in a significant number, which even increased over 24 hours. Comparison of these data with the additional repair comet assay and FPG-repair comet assay revealed that overall, the number of lesions decreased whereas that of double-strand breaks increased. This increase can be due to repair procedures during which DNA strands are incised at oxidative lesions.

In an earlier study, we have shown that, besides DNA damage, which is detectable by comet assay, a 24 hour-incubation with angiotensin II also caused micronuclei (7). Micronuclei, a subset of chromosomal aberrations, are chromatin-containing structures in the cytoplasm, surrounded by a separate membrane. They can be formed by chromatin with double-strand breaks, resulting in chromosome fragments lagging behind at anaphase during nuclear division (40). Thus, the double-strand breaks we detected after angiotensin II perfusion might lead to chromosomal aberrations and possibly also to initiation of carcinogenesis, further supported by enhanced cell proliferation and growth in the kidney, which is known to be caused by angiotensin II (41).

The alkaline comet assay detects a variety of lesions: single- and double-strand breaks, incomplete excision repair sites, and apurinic or apyrimidinic sites, which are alkali labile and therefore appear as breaks under the alkaline conditions of the assay (42). To detect base oxidation, the very sensitive FPG-modified comet assay was chosen. FPG is a bacterial DNA glycosylase/abasic site lyase enzyme that recognizes diverse but structurally related oxidative DNA base modifications like 7-hydro-8-oxoguanine (8-oxodG), formamidopyrimidines, N7-methyl formamidopyrimidines, and also abasic sites (43, 44). The significant increase in DNA damage observed with this assay supports the thesis that the angiotensin II–induced damage is of oxidative nature. Although the 8-oxodG lesions were repaired rather fast, 8-oxodG nevertheless has mutagenic potential, and repair errors or unrepaired 8-oxodGs lead to A:T->C:G and G:C->T:A transversions (43, 45). With an aldehyde-reactive probe, which reacts with aldehyde groups of abasic sites as well as with residual sugar moieties of DNA strand breaks (46), we could show that abasic sites, also a major class of oxidative DNA damage, increased after angiotensin II treatment. Additional experiments in the isolated perfused kidney could help to clarify the mechanism that causes the DNA damage (e.g., by identifying the involved radicals).

Summarizing, our study shows that angiotensin II induces DNA damage in the form of single- and double-strand breaks not only in cell culture but also in the intact whole kidney. In vitro, we could provide evidence for the possible destructive effect of the DNA strand breaks by showing that double-strand breaks persisted long after other comet assay–detectable lesions had been repaired. The AT1 receptor blocker candesartan was able to prevent all kinds of injury caused by angiotensin II, proving the damage to be mediated by this receptor.

Elevated concentrations of angiotensin II in humans may be either due to an activated renin angiotensin system in hypertension or formed locally in the kidney. Here, angiotensin II concentrations can be up to 1,000 times higher than in the plasma (47, 48) and therefore possibly have the potential to lead to DNA damage, followed by mutations and the onset of carcinogenesis. Further animal studies will have to show if angiotensin II indeed causes malignant transformations. An ongoing study examines the effect of an AT1 receptor inhibitor on the DNA damage of peripheral blood lymphocytes of patients whose medication was changed from another antihypertensive therapy. Finally, in humans, correlations between angiotensin II levels, blood pressure, and kidney damage should be investigated.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Deutsche Forschungsgemeinschaft Grant Schu 2367/1-1 (N. Schupp).

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

We thank Michaela Wolf, Thomas Büdel, Jessica Böhm, and Marlies Hamann for excellent technical assistance.


   Footnotes
 
3 http://rsb.info.nih.gov/ij/ Back

4 http://facs.scripps.edu/software.html Back

Received 4/ 8/08. Revised 9/10/08. Accepted 9/11/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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