Original Article: Laboratory Investigation
iju_2924257..263Role of angiotensin and endothelin in testicular ischemia
reperfusion injury
Burak Turkili,
1Zehra Kurcer,
2Gunnur Ozbakis Dengiz,
2Nilufer Onak Kandemir,
3Gorkem Mungan,
4Veysel Haktan Ozacmak
5and Zekiye Nur Banoglu
21Department of Pharmacology, Institute of Health Sciences, Departments of2Pharmacology,3Pathology,4Medical Biochemistry and 5Physiology, Faculty of Medicine, Zonguldak Karaelmas University, Zonguldak, Turkey
Abbreviations & Acronyms
A-II= angiotensin II ACE= angiotensin converting enzyme ARB= angiotensin receptor type 1 blockers
AT1= angiotensin II type I
CAT= catalase ET1= endothelin-1 ETA= endothelin receptor
ETA/B= dual endothelin
receptor GSH= glutathion GSHPx= glutathion peroxidase IR= ischemia reperfusion MBP= mean blood pressure
ROS= reactive oxygen species
SOD= superoxide dismutase
Correspondence:Zehra Kurcer
M.D., Department of Pharmacology, Faculty of Medicine, Zonguldak Karaelmas University, Zonguldak 67600, Turkey. Email:
zykurcer@yahoo.com Received 3 August 2011; accepted 14 November 2011. Online publication 14 December 2011
Objectives: To determine whether angiotensin and endothelin have any role in tes-ticular ischemia reperfusion injury by investigating the effects of the angiotensin con-verting enzyme inhibitor enalapril, selective non-peptide angiotensin-II type I blocker losartan and dual endothelin receptor blocker bosentan.
Methods: Rats were anesthetized with thiopental sodium (50 mg/kg i.p.) before the operation. The left testicular artery and vein of rats were occluded for 1 h; before the bilateral orchiectomy, the organ was allowed to reperfuse for 3 h or 24 h. Enalapril (20 mg/kg i.p.), losartan (30 mg/kg i.p.), bosentan (10 mg/kg i.p.) or vehicle (saline) were given 30 min before reperfusion. Malondialdehyde level was measured in testicular tissue after 3 h of reperfusion. Histological examination was carried out after 24 h of reperfusion.
Results: Ischemia reperfusion caused a significant increase in malondialdehyde level of ipsilateral testis, and histopathological injury in both ipsilateral and contralateral testes. Enalapril, losartan and bosentan treatments prevented the ischemia reperfusion-induced augmentation in malondialdehyde levels. Only bosentan treatment ameloriated ischemia reperfusion-induced histopathological alterations.
Conclusions: Endothelin might play a more important role in pathogenesis of testicu-lar ischemia reperfusion injury when compared with angiotensin.
Key words: Bosentan, enalapril, ischemia-reperfusion injury, losartan, testis.
Introduction
Testicular torsion is a medical emergency that usually requires surgical intervention to counter-rotate the testis and spermatic cord to allow for reperfusion.1It seems that the main
pathophysiology of testicular torsion-detorsion is IR injury. Although the basic pathological mechanism underlying testicular injury has not been completely understood , it has been shown that ROS formed during IR play an important role in this process.2–4
Evidence that activation of the renin–angiotensin system can exacerbate IR-induced injury has been provided by many studies. It has been shown that A-II levels are elevated in ischemic heart disease, hepatic ischemia, renal injury and cerebral ischemia.5–9Additionally,
ACE inhibition, which decreases production of A-II, has been shown to improve post-ischemic injury in both animal and human studies.10,11Thus, inhibition of A-II synthesis
might have a protective effect on IR injury. Several studies have reported that selective ARB and ACE inhibitors might reduce various IR-induced tissue damage, such as myocardial, hepatic, renal, cerebral and intestinal.12–17The effects of these drugs were also investigated
by Gokce et al. in a testicular IR model. They reported that administration of lisinopril, an ACE inhibitor, and losartan, an ARB, decreased the histopathological injury and apoptosis in the contralateral testes (non-ischemic), but they did not present the results of ipsilateral testes (ischemic).18
ET1 is a powerful endothelium-derived vasoconstrictor agent.19Interactions between ET1
It has been suggested that some cardiovascular effects of A-II are mediated by ET1 production,20and that both the
angiotensin and endothelin systems are involved in the pathophysiology of myocardial21 and cerebral22 ischemia.
Previous studies have reported that ETAand ETA/Bblockers
might reduce IR-induced damage in some tissues, such as myocardium, liver, kidney and skeletal muscle.14,23–26
In light of the aforementioned literature, in testicular IR injury, the roles of ACE, AT1and ETA/Breceptors have not
been fully investigated , and thus it remains unclear whether ACE inhibitors, ARB and endothelin receptor blockers exert protective effects in testicular torsion/detorsion injury. The present study was designed to determine whether angio-tensin and endothelin play any role in testicular IR injury. To examine this, in the present study, we evaluated the effects of the ACE inhibitor, enalapril, the selective non-peptide AT1receptor blocker, losartan, and ETA/Bblocker, bosentan,
on MDA level, which is an indicator for lipid peroxidation as a result of increased ROS production, and histopathologi-cal alterations in testicular IR injury.
Methods
Animals
Male Wistar albino rats weighing 200–250 g were placed in a temperature- (21⫾ 2°C) and humidity- (60 ⫾ 5%) con-trolled room in which a 12 h/12 h light–dark cycle was maintained. All experiments in the present study were carried out in accordance with Principles of Laboratory
Animal Care (NIH publication no. 86-23, revised 1984) and
were approved by the Committee on Animal Research at Zonguldak Karaelmas University, Zonguldak.
Surgery and experimental protocol
Under anesthesia induced by i.p. injection 50 mg/kg of sodium thiopental (Pental, I˙E Ulugay I˙laç Sanayi AS¸, Istan-bul, Turkey), an abdominal incision was made. The testicular artery and vein of the left testis were occluded with a vas-cular clamp for 1 h, later the clamp was removed and the organ was allowed to reperfuse (3 h or 24 h).27The control
group underwent a sham operation of the left testis. The rats were treated with ACE inhibitor enalapril (20 mg/kg i.p.), AT1 receptor blocker losartan (30 mg/kg i.p.), dual ETA/B
receptor blocker bosentan (10 mg/kg i.p.) and vehicle at 30 min before reperfusion. Enalapril (Deva Holding AS¸, Istanbul, Turkey), losartan (Merck & Co, Whitehouse Station, NJ, USA) and bosentan (Sequoia Research Prod-ucts, Reading, UK) were dissolved in saline.
Measurement of tissue MDA level
The rats were killed at 3 h of reperfusion for evaluation of biochemical parameters. The left and right testis tissue
samples were stored at -70°C for measurement of MDA levels. The testis tissues were homogenized in 1.15% KCl and the consequent tissue volume was 10%. The homoge-nates were centrifuged at 1500 g for 10 min. The superna-tant was used for the determination malondialdehyde kit (Recipe Chemicals and Instruments Labortechnik, Munich, Germany) by HPLC (Zivak Technologies P100, TÜBI˙TAK MAM, Kocaeli, Turkey).28
Histological analysis
The rats were killed at 24 h of reperfusion for histopatho-logical evaluation. For light microscopic evaluation, testes were fixed in 10% neutral buffered formalin, processed routinely by automatic tissue processor and embedded in paraffin wax. Then, 4-mm sections were stained with hematoxylin–eosin before investigation under light micros-copy (DMLB, Leica, Germany). Histological findings in seminiferous tubules were evaluated according to the Johns-en’s scoring system.29,30 Tubules in 10 consecutive ¥400
field areas were scored and mean values were determined. The Johnsen’s score is based on the premise that with tes-ticular damage there is successive disappearance of the most mature cell type, with progressive degeneration of germinal epithelium, with the disappearance of sperm and sperma-tids, then spermatocytes and finally Sertoli cells, in that order. A score of 1 indicates no seminiferous epithelial cells and tubular sclerosis; a score of 2 indicates no germ cells, only Sertoli cells; a score of 3 indicates spermatogonia only; a score of 4 indicates no spematids, since spermatocytes and arrest of spermatogenesis at the primary spermatocyte stage; a score of 5 indicates no spermatids and many spermato-cytes; a score of 6 indicates no late spermatids with few early spermatids, arrest of spermatogenesis at the spermatid stage and disturbance of spermatid differentiation; a score of 7 indicates no late spermatids and many early spermatids; a score of 8 indicates few late spermatids; a score of 9 indictes late spermatids and disorganised tubuler epithelium; and a score of 10 indicates full spermatogenesis.
Statistics
All data were expressed as the arithmetic mean⫾ SEM. A
P-value<0.05 was considered statistically significant.
Dis-tribution of the samples in the groups was analyzed with one sample by the Kolmogorov–Smirnov test. The MDA results were statistically analyzed using analysis of variance to assess the differences between multiple groups. If statisti-cally significant, the mean values obtained from each group were then compared by the least significant difference multiple comparisons test. The histological results were statistically analyzed by the Kruskal–Wallis H-test. The dif-ferences between the groups were evaluated by the Mann– Whitney U-test. Paired Student’s t-test and Wilcoxon
test were used to assess the differences between ipsilateral and contralateral testis.
Results
The left (ipsilateral) and right (contralateral) testis MDA levels in all groups are shown in Table 1. The MDA levels of the left testes in the IR group were higher than the ipsilteral testes of the sham group (P= 0.020). The MDA levels of the
left testes in the IR group were not significantly higher than the contralateral testes. There were no significant differences in the MDA levels of contralateral testes in all of the groups. Enalapril, losartan and bosentan treatments prevented the IR-induced augmentation in MDA levels of ipsilateral testes. The mean Johnsen’s scores of ipsilateral and contralateral testes are shown in Table 2. Normal testicular morphology and mature spermatogenesis in the ipsilateral (Fig. 1a) and contralateral testes of the sham group were observed. Uncer-tain of border of seminiferous tubules, deterioration of cohe-sion in germ cells, picnosis and coagulative necrosis were observed in ipsilateral IR testes. The IR group of ipsilateral testis showed a general decrease in maturation of sper-matogonia (Fig. 1b). The changes observed in the contralat-eral testes of the IR group were similar to them in the ipsilateral testes; however, the intensity of histological changes was slightly lower than the ipsilateral testes. The Johnsen’s scores between the ipsilateral and contralateral testes were significantly different from each other, except in the sham group (Table 2). Enalapril (Fig. 1c) and losartan (Fig. 1d) treatments did not improve these changes observed in both ipsilateral and contralateral testes of the IR group. Bosentan treatment significantly showed positive effects on sperm maturation in ipsilateral testes, but it did not reverse
(a) (b) (c)
(d) (e) (f)
Fig. 1 Light microscopic appearances in ipsilateral testes of the control and IR groups (hematoxylin–eosin; magnification: ¥400). (a)
Spermatogenic maturation is seen on a sample from the control group and numerous spermatozoa are observed within the tubule lumen (arrow). (b) Coagulative necrosis on germ cells desquamating into the lumen; ischemic alterations and maturation arrest (arrow) are observed within a testicular tissue sample of the IR group. (c) After enalapril treatment, it is observed that ischemic alterations are underway and that maturation in testicular tissue has stopped. (d) After losartan treatment, immature spermatogonia and a few early-stage spermatids in the lumen are observed within seminiferous tubules. (e) After bosentan treatment, it is observed that the effect of ischemic injury continues in some seminiferous tubules. (f) After bosentan teratment, restarting spermatogenic maturation and a few mature spermatids in the lumen are observed within seminiferous tubules.
Table 1 Levels of MDA in all groups at 3 h of reperfusion
Groups MDA (ng/g tissue)
Ipsilateral testis Contralateral testis
Sham (n= 9) 2.27⫾ 0.206 2.84⫾ 0.304
IR (n= 8) 3.05⫾ 0.292* 2.83⫾ 0.288
E+IR (n = 7) 2.70⫾ 0.317 3.04⫾ 0.340
L+IR (n = 9) 2.48⫾ 0.155 2.64⫾ 0.198
B+IR (n = 7) 2.37⫾ 0.154 3.01⫾ 0.146
*P< 0.05 versus sham. Data shown as mean values ⫾ SEM. B, bosentan; E, enalapril; IR, ischemia/reperfusion; L, losartan.
the scores back to normal levels (Table 2, Fig. 1e,f). In the group treated with bosentan, the spermatogenesis restarted in some tubules and the presence of a few spermatids within the lumen was observed (Fig. 1e). In the contralateral testes of this group, bosentan treatment reversed sperm maturation to the sham (Table 2).
Discussion
In the present study, it was shown that 1 h ischemia/3 h reperfusion significantly increased the MDA level in ipsilat-eral testes and that 1 h ischemia/24 h reperfusion caused histopathological injury in both ipsilateral and contralateral testes. Enalapril, losartan and bosentan treatments prevented the IR-induced augmentation in MDA levels. However, only bosentan treatment ameliorated IR-induced histopathologi-cal alterations.
In mammalian testes, ACE, A-II and ET1 are produced. Also, AT1, AT2, A(1–7), ETAand ETBreceptors are located in
testes tissues.31–38A-II is produced in seminiferous tubules of
rats, and it causes the transport of spermatozoa and sperm release during spermiation, through the contraction of semi-niferous tubules through AT1 receptors within peritubular
myoid cells.31Similarly, it has been reported that endothelin,
produced in Sertoli and seminiferous tubule cells, causes the contraction of seminiferous tubules by binding to ETAand
ETBreceptors in the peritubular myoid cells.35It has been
suggested that ACE is involved in the endothelium of tes-ticular vessels and is inhibited by perindopril, an ACE inhibi-tor.34ET
AmRNA was expressed in seminiferous tubules and
all testicular tissues, except for testicular vessels. ETB
mRNA was expressed in all testicular tissues, including endothelium and smooth muscle cells of testicular vessels.37
The aforementioned studies have shown that the physiologi-cal effects of A-II and ET1 on transport of spermatozoa and sperm release in testes are similar to each other.
A-II is a potent vasoconstrictor peptide. AT1 receptors
mediate the known effects of A-II, including smooth muscle cell proliferation and growth, enhancement of inflammation through macrophage activation and cell migration, and ROS production.39–42 ET1, endothelial-derived vasoconstricting
peptide, is more potent than A-II. It stimulates several acti-vation mechanisms on neutrophils, and induces ROS and the pro-inflammatory tumor necrosis factor-a production.43–46
A-II- and ET1-induced processes contribute to acute testicu-lar ischemic events.2–4,27,47 It has been postulated that the
protective effects of ACE inhibitors, ARB and ETA/ETB
receptor blockers on IR injury might be related to opposing the vasoconstrictor, pro-inflammatory and oxidant effects of A-II and ET1.17,48,49 In the present study, effects of ACE
inhibitor enalapril, ARB losartan and ETA/ETB receptor
blocker bosentan were investigated on ROS production, which is one of these processes in testicular IR injury. ROS levels increase during testicular IR and have an important role.2–4Elevation of lipid peroxidation in testicular IR injury
has been related to augmentation in ROS production in testes tissues.4,50MDA is used widely as an indicator of lipid
peroxidation in IR-induced tissue injury. In the present study, although MDA levels significantly increased at 3 h of reperfusion, they were decreased by enalapril, losartan and bosentan treatments.
Natural endogenous peptides have contributed to ROS production. ET1, through a ETAreceptor-NAD(P)H oxidase
pathway, has been implicated as a causative factor in the increase in vascular superoxide in chronic ET1-infused rats.44Interestingly, it was also reported that ROS can
stimu-late the synthesis of ET1 in endothelial and vascular smooth muscle cells by increasing preproendothelin-1 mRNA content, and that this effect is mediated predominantly by superoxide anions.45Similarly, A-II, through a AT
1receptor,
causes oxidative stress in the endothelium, smooth muscle and fibroblasts.41,42 In IR injury, the protective effects of
ACE inhibitors, AT1 and endothelin receptor blockers was
reported to be related to their anti-oxidant properties. It has been suggested that anti-oxidant properties of sulfhydryl ACE inhibitors, such as zofenopril and captopril, are more prominent than non-sulfhydryl ACE inhibitors.51–54
Sulfhy-dryl compounds have anti-oxidant effects and are able to neutralize oxygen radicals by either a hydrogen-donating or electron-transferring mechanism.53In addition, it has
previ-ously been reported that captopril is very effective in scav-enging free radicals in a manner similar to glutathione, N-2-mercaptoproppionylglycine and N-acetylcysteine, but this effect was not mimicked by enalapril.54 However, in
some studies, it was shown that enalapril, a non-sulfhydryl ACE inhibitor, reduced lipid peroxidation by increasing the anti-oxidant enzymes.15,39 Cavanagh et al. showed that
enalapril and captopril treatments for 11 weeks increased activities of SOD and GSHPx in liver cells of mice.55
Seujang et al. reported that enalapril and losartan, AT1 Table 2 Semiquantitative estimates of testicular injury at
24 h reperfusion
Groups Ipsilateral testis
score Contralateral testis score Sham (n= 10) 8.60⫾ 0.085 8.61⫾ 0.081 IR (n= 11) 7.64⫾ 0.073* 8.19⫾ 0.138*,*** E+IR (n = 8) 7.65⫾ 0.098* 8.25⫾ 0.154*,*** L+IR (n = 12) 7.48⫾ 0.087* 7.98⫾ 0.054*,*** B+IR (n = 8) 8.00⫾ 0.110*,** 8.51⫾ 0.052**,***
*P< 0.05 versus sham (ipsilateral or contralateral testis), **P< 0.05 versus IR (ipsilateral or contralateral testis), ***P< 0.05 between ipsilateral and contralateral testis. Data shown as mean values⫾ SEM. B, bosentan; E, enalapril; IR, ischemia/reperfusion; L, losartan.
receptor blockers, ameliorated IR-induced renal injury by reducing the increase of MDA level.15Khaper and Signal
observed that losartan treatment for 4 weeks after coronary ligation increased CAT and GSHPx activity, and reduced lipid peroxidation.56It was reported that losartan treatment
decreased superoxide production in vascular segments from A-II-infused rats.57Some studies relating to the anti-oxidant
properties of bosentan, an ETA/Breceptor blocker, were
pre-sented in the literature. Bosentan has been shown to reduce IR-induced myocardial lipid peroxidation58,59and to
amelio-rate IR-induced injury by increasing GSH, SOD and CAT levels48 in myocardium. BQ123, an ET
A receptor blocker,
has been reported to decrease the increase in lipid peroxi-dation, and to increase the decrease in nitric oxide level, intracellular GSH content, SOD and CAT enzymes activities in rats with myocardial IR-induced injury.60 The present
study and previous studies have reported that enalapril as non-sulfhydryl ACE inhibitors, losartan as an AT1receptor
blocker and bosentan as an ETA/B receptor blocker might
reduce the oxidative stress by modulating anti-oxidant mechanisms in testicular IR.
In the present study, all three treatments reduced the lipid peroxidation, but enalapril and losartan did not ameloriate histopathological injury. Bosentan treatment reduced the injury in ipsilateral testes and also reversed histopathologi-cal alterations to the control group (sham) in controlateral testes. Gokce et al. reported that giving lisinopril (50 mg/kg i.p.) or losartan (30 mg/kg i.p.) 1 h before detorsion decreased the histopathological injury and apoptosis in the contralateral testes in the testicular IR model (4 h torsion/ 24 h detorsion), but they did not present the results of ipsi-lateral testes. These researchers has suggested that beneficial effects of lisinopril and losartan might arise from inhibition of the ischemic process resulting from increased sympa-thetic activity and elimination of insults subsequent to dys-regulation of the renin–angiotensin–aldosterone system.18
Similar to the present results, Jerkic et al. reported that bosentan was more effective than losartan in an experimen-tal acute renal failure model in rats. In the model reported by Jerkic et al., bosentan (10 mg/kg) or losartan (5 or 10 mg/ kg) had been given by i.v. infusion to rats 20 min before, during and 20 min after ischemia. Bosentan was found to have a more potent effect on the glomerular filtration rate and renal blood flow, and a more beneficial effect on renal tubular damage than losartan.14
In the present study, enalapril, losartan and bosentan were given as a single dose intraperitoneally at 30 min before reperfusion. Enalapril and losartan are prodrugs, but bosen-tan is not. As a result of all drugs having a positive effective on lipid peroxidation, we thought that enalapril and losartan transformed to their active metabolites. In the present study, 20 mg/kg enalapril, 30 mg/kg losartan and 10 mg/kg bosen-tan were used. The lipid peroxidation was decreased by enalapril and losartan, but the histopathological injury was
not. These results might be related to their hypotensive effects in using doses of these drugs. However, we did not measure the blood pressure of the animals. Pourdjabbar
et al. carried out myocardial ischemia after losartan (3 and
30 mg/kg) was given to rats for 1 week. The researchers found that MBP of the group treated with a 30 mg/kg dose of losartan at 24 h of reperfusion was significantly lower than the control and the group treated with a 3 mg/kg dose of losartan.61Similarly, Jerkic et al. reported that the MBP
value of the group given 10 mg/kg losartan at 24 h of rep-erfusion after renal ischemia was significantly lower than the control, and that MBP data of the groups given 5 mg/kg losartan or 10 mg/kg bosentan were similar to the control group. However, these investigators showed that bosentan had more beneficial effects on IR-induced renal injury than losartan used in an ineffective dose on MBP.14In the light of
previous studies and the present results, we believe that bosentan had more beneficial effect in comparison with losartan and enalapril, independent of their hypotensive effects. For understanding the action mechanism of these drugs in testicular IR, further studies are needed by using different doses of ACE inhibitors and ARB that have differ-ent chemical structures and pharmacokinetic properties.
In conclusion, the results of the present study show that the ETA/B receptor blocker, bosentan, has more beneficial
effects than the AT1 receptor blocker, losartan, and ACE
inhibitor, enalapril, on testicular IR injury. According to the results, in testicular IR pathophysiology, it seems that endot-helin and angiotensin might play a role, and that endotendot-helin might have a more important role than angiotensin.
Acknowledgments
This work was supported by a research fund from the Zon-guldak Karaelmas University (2009-2042-01-02). The authors thankfully acknowledge Askin Hekimoglu (Assis-tant Professor) for language support, to Derya Karaoglu (MD), Inci Turan (MD) and Zeynep Mehpare Eskici (MD) for measurements of the biochemical parameters, and to Hasan Tahsin and Bayram Meral for caring for the animals.
Conflict of interest
None declared.
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