Efficacy of poly(adenosine diphosphate-ribose) polymerase inhibition in extracorporeal shock wave-induced renal injury

Abstract

Objectives: Extracorporeal shock wave lithotripsy (ESW) induces renal damage by excessive production of free oxygen radicals. Free Oxygen radicals cause cellular injury by inducing nicks in DNA. The enzyme poly(adenosine diphosphate-ribose) polymerase (PARP) involved in the process of repair of DNA in damaged cells. However, its activation in damaged cells can lead to adenosine triphosphate depletion and death. Thus, we designed a study to evaluate the efficacy of 3-aminobenzamide (3-AB), a PARP inhibitor, against extracorporeal shock wave induced renal injury. Methods: Twenty-four Sprague-Dawley rats were divided into three groups: control, ESW, ESW + 3-AB groups. All groups except control group were subjected to ESW procedure. ESW + 3-AB group received 20 mg/kg/day 3-aminobenzamide intraperitoneally at 2 h before ESW and continued once a day for consecutive 3 days. The surviving animals were sacrificed at the 4th day and their kidneys were harvested for biochemical and histopathologic analysis. Blood samples from animals were also obtained. Results: Serum ALT and AST levels, serum neopterin and tissue oxidative stress parameters were increased in the ESW group and almost came to control values in the treatment group (p < 0.05, ESW vs. ESW + 3-AB). Histopathological injury score were significantly lower in treatment group than the ESW group (p < 0.05, ESW vs. ESW + 3-AB). Conclusion: Our data showed that PARP inhibition protected renal tissue against ESW induced renal injury. These findings suggest that it would be possible to improve the outcome of ESW induced renal injury by using PARP inhibitors as a preventive therapy.

• 3-aminobenzamide
• extracorporeal shock wave lithotripsy
• oxidative stress
• PARP inhibition
• renal injury

Introduction

Extracorporeal shock wave lithotripsy (ESW) is the preferred treatment modality for uncomplicated kidney stones, because it is noninvasive and has a high success rate for stone removal. ESW also induces oxidative stress and inflammation resulting in scar formation and the loss of functional renal tissue.1,2 After ESW, the most frequently observed sign of kidney is hematuria, while less often perirenal, subcapsular, or intrarenal hematomas or other symptomatic fluid collections are observed.3,4 In recent reports, one of the mechanism discussed for tissue damage in ESW is free oxygen radical production.2

Concerning the mechanism of the adverse effects of ESW, although initially they were attributed to renal damage resulting only from the direct action of cavitation bubbles or shear stress originating from shock-wave energy, more recently free oxygen and nitrogen radical production originating from transient ischemia due to ESW application was considered to be an integral element in shock wave-induced renal damage through an indirect mechanism.5,6 Transient ischemia and following reperfusion causes release of cytokines/inflammatory cellular mediators, production of oxygen-derived (reactive oxygen species ROS) free radicals, such as superoxide (), peroxide (H₂O₂) and hydroxyl radicals (OH⁻), production of nitrogen-derived free radicals (reactive nitrogen species RNS), such as nitric oxide (NO) peroxynitrite (ONOO⁻), and infiltration of tissue by inflammatory response cells and this process initiates the cellular damage and death. Which treatment with free radical scavengers has been found to be effective in terms of survival and renal function in experimental and clinical studies supports this hypothesis.7–9 There is evidence that free oxygen and/or nitrogen radicals induce cellular injury by inducing nicks in DNA. Deoxyribonucleic acid damage is repaired via the activity of several DNA repair enzymes, including poly (adenosine diphosphate-ribose) polymerase (PARP). Poly(ADP-ribose) polymerase-1 (PARP-1) also known as poly(ADP-ribose) synthetase or poly(ADP-ribose) transferase is a nuclear enzyme present in eukaryotic cells.10,11 Extensive DNA damage may lead to excessive PARP activation that consumes large quantities of cellular nicotinamide adenine dinucleotide (NAD⁺), rapid depletion of NAD⁺ due to PARP activation leads to cellular ATP depletion and functional alteration of the cell, and eventually necrotic-type cell death occurs.12,13

We hypothesized that inhibition of activity of PARP prevents depletion of cellular ATP levels resulting in surviving of cells in inflammatory area. This study was designed to investigate whether 3-amino benzamide (3-AB), a PARP inhibitor, has protective effects on renal damage in an experimental model of extracorporeal shock wave induced renal damage in rats.

Materials and methods

Animals and study groups

All animal procedures were approved by the Institutional Committee on the Care and Use of Animals of our institution (Issue; 11/34, 01 July 2011). Twenty-four male Sprague-Dawley rats (200–250 g) provided by the animal laboratory of our institute were randomly assigned into three groups containing eight rats each: control, ESW and ESW + 3-AB groups. Before the experiment, animals were fed standard rat chow and water ad libitum and housed in cages with controlled temperature and 12-hour light/dark cycle for at least 1 week.

Induction of ESW-induced renal injury

All animals were fasted overnight and anesthetized by an intramuscular injection of 50 mg/kg ketamine (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and 10 mg/kg xylazine (Rompun; Bayer AG, Leverkusen, Germany), and were operated at room temperature (24 °C). Through a midline laparotomy, left kidneys of all rats nephrectomized, and then radiopaque materials were placed the rear wall of the abdomen on the back of remaining right kidneys in ESW and ESW + 3-AB groups in order to focus renal tissue. The control group was underwent laparotomy with just nephrectomy. Finally, the abdominal incision was closed, and for the adaptation of animals to single kidney after nephrectomy, the animals were returned to their cages to recover for two weeks. Water and food were available ad libitum. The weight of rats was recorded throughout the experimental period.

Under anesthesia (intramuscular injection of 50 mg/kg ketamine (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and 10 mg/kg xylazine (Rompun; Bayer AG, Leverkusen, Germany), rats in the ESW and ESW + 3-AB groups were fixed in supine position on the platform of lithotriptor and applied with shock wave lithotripsy at the left kidney under the guidance of X-Rays. Each rat received 2000 shocks, 18 kV and total 15 joule energy delivered with a Siemens Lithoskop^TM (Germany). The focal size is 16 mm, the pressure range is not known according to technical brochure and the focal depth is 16 cm. The shock wave rate was 60 shock waves/min. The shock waves were applied with ramping similar to human application. Applied the number of shock waves and the level of energy were decided based on previous studies and our preliminary studies.14–17 After ESW procedure, rats were returned to their cages.

Treatment modalities

Two hours before the ESW procedure, the rats in the ESW + 3-AB group were administrated 3-AB at a dose of 20 mg/kg daily via intraperitoneal route and continued for three consecutive days.

Harvesting the samples

After 24 hour of last dose of agent, animals were sacrificed by cervical dislocation. At the time of death, blood was collected by heart puncture for biochemical analyses and left kidneys were harvested for histopathological evaluation and biochemical examination. Harvested renal tissue samples stored in 10% formalin solution for histopathological evaluation and in −80 °C to study antioxidant enzyme activity and tissue lipid peroxidation levels. Blood samples also obtained for biochemical analysis and centrifuged to study other biochemical parameters in serum.

Tissue preparation and biochemical analysis

The frozen tissues were homogenized in phosphate buffer (pH 7.4) by means of a homogenizer (Heidolph Diax 900; Heidolph Elektro GmbH, Kelhaim, Germany) on an ice cube. The supernatant was used for entire assay. Initially, the protein content of tissue homogenates was measured by the method of Lowry with bovine serum albumin as the standard18 was used for all assays.

Lipid peroxidation level was measured with the thiobarbituric acid (TBA) reaction by the method of Ohkawa.19 This method was used to obtain a spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with malondialdehyde (MDA) at 535 nm. The calculated MDA levels were expressed as mmol/g-protein.

Superoxide dismutase (SOD) activity was assayed using the nitroblue tetrazolium (NBT) method of Sun et al and modified by Durak et al.20 In this method, NBT was reduced to blue formazan by , which has a strong absorbance at 560 nm. One unit (U) of SOD is defined as the amount of protein that inhibits the rate of NBT reduction by 50%. The estimated SOD activity was expressed as Units per gram protein.

The glutathione peroxidase (GSH-Px) activity was measured using the method described by Paglia and Valentine21 in which GSH-Px activity was coupled with the oxidation of NADPH by glutathione reductase. The oxidation of NADPH was spectrophotometrically followed up at 340 nm at 37 °C. The absorbance at 340 nm was recorded for 5 min. The activity was the slope of the lines as mmol of NADPH oxidized per minute. GSH-Px activity was presented as U/g-protein.

Serum creatinine, uric acid, alkaline phosphatase (ALP) and electrolytes (sodium, potassium and chloride) concentrations were measured with a spectrophotometric technique by the Olympus AU-2700 autoanalyzer using commercial kits (Olympus, Hamburg, Germany) and presented as U/L.

Serum neopterin (NP) levels were determined by using a High Pressure Liquid Chromatography (HPLC) system with a fluorescence detector (AgilentTechnologies 1200 Series System, Santa Clara, CA) as prescribed previously22,23 and presented as nmol/L.

Histopathologic evaluation

One-half of each kidneys were taken for histopathologic evaluation. In all groups, samples of kidney were placed in 10% tamponed formalin and send to pathology for routine automatic tissue processing. After paraffin embedding, blocks were subsequently sectioned at 5 μm thickness and stained with hematoxylin and eosin (H&E). The sections were scored with a semiquantitative scale designed to evaluate the degree of renal damage. The kidneys were evaluated in terms of glomerular changes (congestion, hemorrhage, necrosis, intracapillary and extracapillary proliferation crescent formation), tubular changes (epithelial vacuolar degeneration, necrosis and regeneration) interstitial changes (edema, peritubular capillary congestion, hemorrhage and inflammation) and vascular changes (fibrinoid necrosis, and fibrointimal thickening). All section areas for each kidney slide were examined and assigned for severity of changes. The scoring system used was 0, normal; 1, focal and mild; 2, focal and severe; 3, diffuse and mild; 4, diffuse and severe. Total histopathologic injury score per kidney was calculated by addition of all scores. Blind analysis of the histological samples was performed by two independent experts.

Statistical analysis

Results were expressed as median ± standard deviation (SD). In the other analyses, differences among the groups were analyzed by the Kruskal–Wallis test. Dual comparisons among groups with significant values were evaluated with the Mann–Whitney U-test. p < 0.05 were considered significant. All analyses were performed with the Statistical Package for the Social Sciences (SPSS) statistical program (Software version 11.0, SPSS Inc., Chicago, IL).

Results

Serum biochemical values

All subjects survived throughout the study period and there was no statistical difference between animal and study groups in terms of body weight. All serum biochemical values are summarized in the Table 1. Serum creatinine and serum electrolyte levels (Na⁺, K⁺, Cl⁻) were significantly lower in the ESW and ESW + 3-AB groups than the control group (p < 0.05). Serum AST and ALP levels were significantly higher in the ESW group than the other groups, suggesting increased renal injury (p < 0.05). On the other hand, serum AST and ALp were came to control values in the ESW + 3-AB group.

Table 1. Biochemical values in serum.

Groups	Control (n = 8)	ESW (n = 8)	ESW + 3-AB (n = 8)
Creatinine (mg/dL)	0.66 ± 0.02	0.44 ± 0.05^a	0.46 ± 0.07^a
Urea (mg/dL)	1.25 ± 0.34	1.28 ± 0.43	0.97 ± 0.35
AST (IU/L)	84.2 ± 14.4	241.7 ± 32.5^a	127.4 ± 26.8^a,b
ALP (IU/L)	97.7 ± 34.5	327.4 ± 69.5^a	209.7 ± 39.8^a,b
Na (mEq/L)	139.9 ± 5.1	109.2 ± 13.6^a	112.6 ± 12.7^a
K (mEq/L)	5.9 ± 0.4	4.2 ± 0.6^a	5.1 ± 0.6^a
Cl (mEq/L)	98.4 ± 3.4	80.6 ± 7.9^a	83.6 ± 9.5^a
Neopterin (nmol/L)	6.1 ± 0.7	8.9 ± 1.6^a	6.8 ± 0.8^a

^ap < 0.05 Statistically different from control group.

^bp < 0.05 Statistically different from ESW group.

Data are expressed as median ± (SD).

Serum neopterin levels

Serum NP level was significantly increased in the ESW group than the control group (p < 0.05). It was significantly decreased in the ESW + 3-AB group when compared to the ESW group (p < 0.05; Table 1).

Tissue lipid peroxidation levels

The MDA levels in the ESW group were significantly higher than the other groups indicating increased renal cellular damage (p < 0.05, ESW vs. the other groups). The MDA levels were decreased in the treatment group, but still higher than the control group (p < 0.05, ESW + 3-AB group vs. the other groups; Table 2).

Table 2. Values of lipid peroxidation and antioxidant enzymes.

Groups	Control (n = 8)	ESW (n = 8)	ESW + 3-AB (n = 8)
MDA (nmol/g-pro)	0.25 ± 0.24	1.13 ± 0.32^a	0.47 ± 0.26^b
SOD (U/g-pro)	854 ± 145	1875 ± 327^a	1220 ± 445^a,b
GSH-Px (U/g-pro)	41.8 ± 10.2	98.5 ± 15.7^a	57.5 ± 14.3^a

^ap < 0.05 Statistically different from control group.

^bp < 0.05 Statistically different from ESW group.

Data are expressed as median ± (SD).

MDA: Malondialdehyde.

SOD: Superoxide dismutase.

GSH-Px: The glutathione peroxidase.

Tissue antioxidant enzyme activities

The tissue SOD and GSH-Px activity were significantly increased in both groups subjected to ESW procedure (p < 0.05, ESW and ESW + 3-AB groups vs. control group). Antioxidant enzyme activities were significantly decreased in the treatment group when compared the ESW group (p < 0.05, ESW + 3-AB vs. ESW). However, antioxidant enzyme activities were still high in the ESW + 3-AB group when compared to the control group (p < 0.05, ESW + 3-AB groups vs. control group; Table 2).

Histological findings

Microscopic examination of the kidneys revealed obvious mild to moderate glomerular congestion, and severe tubular injury, which consisted of epithelial vacuolization and necrosis with necrotic luminal debris, diminishing or loss of brush borders, and moderate to severe interstitial edema, inflammation, and peritubular capillary congestion in the ESW group (Figure 1A) compared with the ESW + 3-AB and control group (Figure 1B and C). The highest histopathological scores were in the ESW group. These scores were significantly different than the other groups (p < 0.01, Table 3). Tubular injury was severe (Figure 1A). In the ESW + 3-AB group, glomerular, tubular and interstitial changes were better than the ESW and control group (Figure 1B and C).

Figure 1. Light microscopic examination of the kidneys. (A) Shows the kidney of control group. (B) There was mild to moderate glomerular congestion, and severe tubular dilatation and injury (arrows) in ESW group. (C) Tubular structures were preserved in the kidneys of ESW + 3-AB groups compared with the ESW group (H & E, Scale bars; 150 µm).

Table 3. Pathologic scores.

Groups	Sham-operated (n = 8)	ESW (n = 8)	ESW + 3-AB (n = 8)
Glomerular changes
Congestion	1 (1–2)	4 (3–4)^a	3 (2–3)^a
Hemorrhage	0 (0–0)	0 (0–1)	0 (0–0)
Necrosis	0 (0–0)	0 (0–0)	0 (0–0)
Cresent formation	0 (0–0)	0 (0–0)	0 (0–0)
Tubular changes
Vacuolar degeneration	1 (0–1)	4 (3–4)^a	2 (1–3)^b
Necrosis	0 (0–0)	2 (1–3)^a	1 (0–2)^b
Regeneration	0 (0–0)	2 (1–3)^a	1 (0–2)^b
Interstitial changes
Edema	0 (0–1)	1(0–2)	1 (0–2)
Congestion	2 (1–2)	4 (2–4)^a	3 (1–3)^a
Hemorrahage	0 (0–0)	0 (0–0)	0 (0–0)
Inflammation	0 (0–0)	1 (0–2)	0 (0–0)
Vascular changes
Fibrinoid changes	0 (0–0)	0 (0–0)	0 (0–0)
Fibrointimal thickening	0 (0–0)	0 (0–0)	0 (0–0)
Total score	5 (3–8)	18 (10–23)^a	11 (4–13)^a,b

^ap < 0.05 Statistically different from control group.

^bp < 0.05 Statistically different from ESW group.

Data are expressed as median (Min-Max).

Discussion

To the best of our knowledge, this is the first report investigating the effect of PARP inhibition on ESW induced renal injury in an experimental rat model. Our objective was to test the hypothesis that increased oxidative stress in the renal cells due to ESW leads to marked PARP activation, and that treatment with 3-AB, a well-known PARP inhibitor, attenuates renal injury in a rat model of ESW-induced renal injury. Our data revealed that ESW increased oxidative stress markers and histopathologic injury scores in the kidney. On the other hand, PARP inhibition with 3-AB significantly decreased renal injury and oxidative stress markers in the rats subjected to ESW.

PARP detects DNA strand breaks induced by binding to DNA of a variety of genotoxic insults, including ionizing radiation, alkylating agents, oxidants and free radicals.24,25 Upon binding to DNA, strand breaks occur, and PARP transfers ADP-ribose units from the respiratory coenzyme NAD⁺ to various nuclear proteins. From a physiological viewpoint, PARP activity and poly (ADP-ribosyl)ation reactions are implicated in DNA repair processes, the maintenance of genomic stability, the regulation of gene transcription, and DNA replication. An important function of PARP is to allow DNA repair and cell recovery under conditions associated with a low level of DNA damage. In case of severe cellular oxidative stress and DNA injury, over activation of PARP depletes the cellular stores of NAD⁺, an essential cofactor in principal energy production mechanisms, including the glycolytic pathway, the tricarboxylic acid cycle, and the mitochondrial electron transport chain. This may lead to cell death by two possible mechanisms. First, significant PARP activation may lead to a marked reduction in the cellular pools of NAD⁺/ATP, resulting in cellular dysfunction and death via the necrotic pathway. This is known as the “suicide hypothesis” of PARP activation and seems to be a regulatory mechanism to eliminate cells after irreversible DNA injury.24,25 Second, in the presence of adequate cell energy stores, increased PARP activation can lead to apoptosis via apoptotic inducing factor from the mitochondria or by caspase dependant mechanisms.26

Several clinical studies, measuring renal resistive index or followed renal blood velocity in the kidney subjected to SWT, showed that an increase in resistive index or a reduction in blood velocity indicative of vasoconstriction occurred.4,27,28 It has also been reported that there is a decrease in renal perfusion in kidney following ESW procedure by using dynamic gadolinium-DTPA enhanced magnetic resonance imaging.27 Studies focused on two possible explanations for the impairment of renal flow; either renal sympathetic nerves may be activated by shock waves or, alternatively, the vasoconstriction may be secondary to vasoconstrictors released from kidneys in response to the shock waves. As a result of this transient ischemic insult, it is thought that oxygen free radical generation due to ischemia/reperfusion injury may take place, which further contribute to parenchymal damage by lipid peroxidation and disruption of cellular membranes.7 We evaluated the tissue oxidative stress marker (MDA) and the tissue antioxidant system (SOD and GSH-Px) in the kidney. Our data revealed that oxidative stress is a pivotal role in the pathogenesis of ESW-induced renal injury as indicated previous studies.1,9,28,29 Moreover, our data displayed that PARP inhibition decreased oxidative stress status and attenued histopathological injury in the kidneys subjected to ESW. There are a few possible explanation for this observation. PARP inhibition is likely to down-regulate the activities of both extramitochondrial NADPH oxidase and mitochondrial NADH oxidase, which are the important superoxide generating enzymes, which may contribute to renal injury via depletion in production of SOD in the renal cells.30 In addition, PARP inhibition alters transcriptional regulation and deactivates nuclear factor κ-B and activator protein-1, a transcription factor that regulates proinflammatory gene expression.31 Such deactivation leads to decreased formation of endothelin-1 and inflammatory cytokines (e.g. tumor necrosis factor-α, interleukin-6, and interleukin-1β; all known to contribute to superoxide generation).32 Therefore, we assume that administration of 3-AB regulate oxidative stress resulting in attenuated histopathologic injury in kidney of rats subjected to ESW.

Neopterin, a compound belonging to the group of pteridine, is a marker associated with cell-mediated immunity and produced in monocytes/macrophages via the stimulation of interferon-γ. Neopterin measurements not only provide knowledge about the state of cell-mediated immunity but also allow monitoring of disease progression.22,33 In our study, there is a clear correlation between renal injury parameter levels and NP levels concluding that NP level might be a promising parameter to evaluation of renal injury in ESW procedure.

We found that serum creatinine level decreased in rats subjected to ESW procedure. It is known that following the glomerular filtration, water, electrolytes and other molecules (aminoacits, glucose, etc.) are handled at different rates depending on tubular regions.34 In addition, creatinine is secreted from proximal tubulus and not reabsorbed in distal tubules. Renal injury due to ESW is especially and intensively occurred in proximal tubulus as shown our histopathologic evaluation and our iNOS immunohistochemical staining in previous study.16 Therefore, we assume that creatinine is secreted more than normally from proximal tubules in rats ESW procedure resulting in lower serum creatinine level. Another explanation for this situation may be related to different protein channels for each reabsorbed molecules and this needs further studies.

To date, it has been shown that PARP is a contributor to both disease progression and severity in disease including myocardial infarction and inflammatory bowel disease and PARP is investigated as a therapeutic target in these states.35,36 In addition, inhibition of PARP attenuates acute inflammation and tissue damage in animal models of various pathological conditions including pleuritis, lung injury, inflammatory bowel diseases and arthritis.37–39 In our team’s previous works, beneficial effects of PARP inhibition were shown in different experimental inflammatory models.40–42 Therefore, it is possible that treatment with a PARP inhibitor may also attenuate renal injury in kidneys subjected to ESW.

Although our results indicate that PARP inhibitors are effective to improve ESW-induced renal injury in rats, it needs well-designed and documented clinical studies to examine the long term results of 3-AB on renal and the other system function.

In conclusion, inhibition of PARP with 3-AB reduced severity of pathological changes and oxidative stress in the experimental ESW induce renal injury of rat model. These findings suggest that it may be possible to improve the outcome of ESW induce renal injury by using novel and more effective PARP inhibitors as a promising therapeutic strategy.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.