Ir J Med Sci DOI 10.1007/s11845-015-1328-z

ORIGINAL ARTICLE

Administration of hydrogen sulfide protects ischemia reperfusioninduced acute kidney injury by reducing the oxidative stress F. Azizi1 • B. Seifi2 • M. Kadkhodaee2 • P. Ahghari3

Received: 2 March 2015 / Accepted: 20 June 2015 Ó Royal Academy of Medicine in Ireland 2015

Abstract Background Renal ischemia–reperfusion injury (IRI) is a major cause of acute kidney injury. Hydrogen sulfide (H2S) has been known as a novel gaseous signaling molecule. Aims The aim of this study was to investigate whether the efficacy of H2S in protecting against renal IRI is through its antioxidative effect. Method In this study, rats were randomized into Sham, IR, or sodium hydrosulfide (NaHS, an H2S donor) groups. To establish a model of renal IRI, both renal arteries were occluded for 55 min and then declamped to allow reperfusion for 24 h. Rats in the NaHS group received intraperitoneal injections of 75 lmol/kg NaHS 10 min before the onset of ischemia and immediately after the onset of reperfusion. Sham group underwent laparotomy without crossclamping of renal pedicles. After reperfusion, plasma and renal tissue samples were collected for functional, histological, and oxidative stress evaluation. & B. Seifi [email protected] F. Azizi [email protected] M. Kadkhodaee [email protected] P. Ahghari [email protected] 1

Department of Neurosciences and Addiction, School of Advanced in Medicine, Tehran University of Medical Sciences, Tehran, Iran

2

Department of Physiology, Faculty of Medicine, School of Medicine, Tehran University of Medical Sciences, Poorsina Ave., Tehran, Iran

3

Department of Physiology, School of Medicine, Hamedan University of Medical Sciences, Hamadan, Iran

Results The IR group exhibited significant rise in plasma creatinine, blood urea nitrogen (BUN), renal malondialdehyde (MDA) concentration, and significant reduction of renal superoxide dismutase (SOD) activity. Treatment with NaHS reduced the levels of plasma creatinine, BUN, renal MDA concentration, and increased SOD activity in the kidneys. NaHS improved renal histological changes in comparison to IR group. Conclusion Our data demonstrated that H2S can protect against renal IRI and that its therapeutic effects may be mediated by reducing oxidative stress. Keywords sulfide

Renal
Ischemia
Reperfusion
Hydrogen

Introduction Acute kidney injury (AKI) is common in intensive care unit and is associated with significantly increased mortality, morbidity, and length of stay [1]. Renal ischemia reperfusion injury (IRI) is well known as a main cause of AKI [2] that may occur because of an abrupt decrease of renal blood flow, low flow state, diverse surgical procedures, or during the organ transplantation [3]. IRI can be an early outcome of a transplanted organ that may lead to acute rejection of graft and has adverse effects on long term graft survival [4, 5]. Renal IRI induces renal dysfunction characterized by increasing levels of creatinine (Cr) and blood urea nitrogen (BUN) [6]. The search for a treatment to recover IRI is ongoing, but unfortunately, innovative interventions beyond supportive therapy are not currently available for IRI [3, 4]. Oxidative stress plays an important role in the development of AKI. Oxidative stress is caused by increased production of ROS (reactive oxygen species) and defective

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antioxidant capacity [7, 8]. A key mediator of cellular damage during the acute phase of IR injury is overproduction of reactive oxygen species and increased levels of oxidative stress mediated by damaged mitochondria. Hosgood and Nicholson demonstrated that oxidative damage was reduced by hydrogen sulfide in an ex vivo pig study after kidney transplantation [5]. The kidney is particularly vulnerable to oxidative stress because of the higher content of polyunsaturated fatty acids that are susceptible to lipid peroxidation [9]. ROS mediate a spiraling vicious cycle that can lead to progressive renal injury, by attacking membranes through peroxidation of polyunsaturated fatty acids [10]. Under physiological condition, ROS are generated in cells by several enzymes in mitochondria, endoplasmic reticulum, peroxisomes, and other cell compartments [11]. Endogenous antioxidant enzymes are systems that defense against ROS in physiological state. However, in pathological condition such as ischemia, these defense systems are not enough protective against ROS [12]. Superoxide dismutase (SOD), as a scavenger, causes rapid removal of ROS. SOD inhibits free radical generation and protects renal function after ischemia [13]. MDA is also an important product of lipid peroxidation. Studies have shown that renal MDA levels were also significantly increased, indicating the presence of enhanced lipid peroxidation due to IRI [14]. Therefore, intervention dietaries and pharmacological antioxidants should be able to attenuate or prevent the oxidative stress. Development of new pharmacological drugs affecting multiple targets such as oxidative stress and mitochondria may afford better protection against IRI [9]. H2S has been known as a novel gaseous signaling molecule synthesized by cystathionine b-synthase (CBS), cystathionine c-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Production of H2S in mammalian tissues has been known for a long time. H2S exhibits many cytoprotective effects. These may include the cell signaling pathways, which may play a role in anti-inflammation and anti-apoptotic actions, modulation of ion channels and metabolism (i.e., mitochondrial ATP production) [15, 16]. H2S supplementation was associated with the suppressions of oxidative stress and inflammation [17]. H2S was proved to have a direct effect on scavenging ROS and inhibiting ROS formation via different pathways [18]. Thus, in this study, we attempted to investigate whether the efficacy of H2S in protecting against renal IRI is through its antioxidative effect.

Materials and methods Male Wistar rats, weighing 250–300 g, were used in this study. All rats were housed in a climate-controlled and light-regulated facility with 12:12-h day–night cycles.

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Standard laboratory chow and drinking water were provided. All experiments were approved by the animal ethics committee of Tehran University of Medical Sciences. Surgical technique Eighteen rats were randomly divided into 3 groups (n = 6, each): sham group, IR group, and sodium hydrosulfide (NaHS) group. All animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Rats in the IR group received IR injury to both renal arteries for 55 min followed with 24 h reperfusion without any treatment. In NaHS group, rats were placed supine while NaHS (75 lmol/kg, ip) was administered to them. After 10 min, the abdomen was opened through a midline incision. Renal pedicles were exposed and crossclamped for 55 min. Then renal clamps were released and the kidneys were reperfused. Next dose of NaHS was administered immediately at the beginning of reperfusion. Animals in sham group underwent a surgical procedure identical to that of the ischemia and treatment groups but without cross-clamping of the renal pedicle. However, they received an equal volume of saline. Body temperature and blood pressure were monitored throughout the surgery. Rats were recovered for 1 day and then anesthetized to collect blood samples via inferior vena cava. Right kidneys were immediately removed, frozen in liquid nitrogen, and stored at -70 °C until processed. Left kidneys were removed and fixed in 10 % formalin for histological examination. Tissue sections were fixed by formalin (10 % phosphate buffered) and then dehydrated. Paraffin-embedded renal sections (4 mm) were stained by hematoxylin and eosin. Evaluation of the renal histology was based on the presence and extent of necrosis, cellular degeneration and vacuolization, tubular obstruction, and formation of luminal debris and casts. The following grading scale was used: 0 = minimal or no lesion; 1 = less than 25 % of tubules are involved; 2 = 25–50 % of tubules are involved; and 3 = more than 50 % of tubules are involved. During the performance of these experiments, all animals were survived. At the end of the sampling, animals were sacrificed by bleeding during anesthesia. Malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were determined in the supernatant of renal tissue. MDA level was determined according to the methods of Esterbauer and Cheeseman. Briefly, the renal tissue was mixed with 2 volumes of 10 % trichloroacetic acid (TCA) for protein precipitating. After centrifugation of the precipitate, supernatant is separated and reacted with thiobarbituric acid in boiling water for 10 min followed by cooling. The concentration of MDA was measured at 532 nm [19]. SOD activity was determined according to the method of Paoletti and Mocali [20]. In this assay,

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superoxide anion is generated from O2 in the presence of EDTA, mercaptoethanol, and manganese (II) chloride. Oxidation of NADPH is linked to the availability of superoxide anions in the medium. Statistical analysis For multiple comparisons, statistical analysis was performed by one-way ANOVA followed by post hoc Turkey’s test. p \ 0.05 was considered statistically significant. All data are expressed as mean ± SEM (standard error of the mean).

Results

reduced the levels of renal MDA compared with the levels in the IR group (ANOVA, 1.7 ± 0.03 vs 2 ± 0.13; p = 0.027) (Fig. 2b). In the kidneys of the sham group, there were no detectable changes observed by light microscopy. In the kidney sections of IR group, there were severe changes in the tubules comparing to sham (Fig. 3). The changes were included the destruction of the tubular cells. In some cases, there were severe flattening of the tubules, and in some others, necrosis especially in the straight proximal tubules was seen. Significant tubular obstructions were present, especially in the more distal tubules. Administration of H2S was able to attenuate the severity of the structural changes. No detectable cellular necrosis was seen and lower amount of tubular cast formation was present.

Compared to the sham group, IR animals demonstrated a significant rise in BUN level (ANOVA, 107.5 ± 17.2 vs 18 ± 0.7; p = 0.001). Treatment with H2S reduced the levels of BUN compared with the IR group (ANOVA, 56 ± 6.6 vs 107.5 ± 17.2; p = 0.007) (Fig. 1a). Compared to the sham group, IR animals demonstrated a significant rise in plasma creatinine level (ANOVA, 1.6 ± 0.2 vs 0.6 ± 0.03; p = 0.001). Treatment with H2S reduced the levels of plasma creatinine compared with the IR group (ANOVA, 0.87 ± 0.04 vs 1.6 ± 0.2; p = 0.01) (Fig. 1b). IR group showed significant reduction in the renal SOD activity compared to the sham group (ANOVA, 22.1 ± 4.3 vs 31.8 ± 2.3; p = 0.016). Treatment with H2S significantly increased the levels of kidney SOD activity compared with the levels in the IR group (ANOVA, 34.3 ± 0.39 vs 22.1 ± 4.3; p = 0.013) (Fig. 2a). IR group showed marked increases in the renal MDA concentration compared to the sham group (ANOVA, 3 ± 0.13 vs 1.7 ± 0.06; p = 0.034). Treatment with H2S

In the current study, administration of NaHS significantly attenuated IR injury in the kidney by improvement of functional indices and histopathological changes. Treatment with NaHS was able to preserve the levels of renal SOD and reduced the MDA level in the kidney. H2S possesses a number of signaling actions that are likely to attenuate the pathological aspects of IR injury [21]. We demonstrated that induction of 55 min ischemia to the functioning kidneys could lead to acute kidney injury. Treatment with NaHS was demonstrated to improve renal function and histological changes. Plasma creatinine and BUN are used as markers of renal function in clinical practice as they are the simplest and most widely used indices [22]. In the present study, we injected two doses of H2S before induction of ischemia and immediately after the onset of reperfusion. H2S has been demonstrated to have

Fig. 1 Effect of NaHS on plasma BUN (a) and creatinine (b) in different groups (n = 6). IR ischemia reperfusion group, NaHS administered NaHS (75 lmol/kg, ip) group. All data are expressed as

mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by post hoc Tukey test. *p \ 0.05 vs sham group, # p \ 0.05 vs IR group

Discussion

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Fig. 2 Effect of NaHS on renal SOD (a) and renal MDA (b) in different groups (n = 6). IR ischemia reperfusion group, NaHS administered NaHS (75 lmol/kg, ip). All data are expressed as

C

B

D 4 Renal histopathological score

A

mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by post hoc Tukey test. *p \ 0.05 vs sham group, # p \ 0.05 vs IR group

* 3

* #

2 1 0 Sham

IR

NaHS

Fig. 3 Effect of NaHS on renal histology in different groups. a Sham, b IR, c NaHS groups 9400. d Renal histopathological score in all three groups. In the sham group, there were no detectable changes. In the IR group, there was severe flattening of the tubules,

and in some cases, necrosis especially in the straight proximal tubules was seen. In NaHS group, there were no detectable cellular necrosis and lower amount of tubular cast formation. *p \ 0.05 vs sham group. #p \ 0.05 vs IR group

hypometabolism effects [16]. Therefore, the first injection was administered 10 min before onset of ischemia to decrease oxygen demand of tissue. On the other hand, H2S have a direct effect on scavenging ROS. Production of ROS is an essential destructive process during the ischemia and reperfusion [23]. Therefore, the second injection was administered immediately after the onset of reperfusion to inhibit their formation.

In the present study, we observed a marked difference in renal function between the IR and treatment groups. IR animals demonstrated a significant rise in BUN and Cr, while renal function was preserved in the NaHS group, shown by a reduction in BUN and Cr. These results were in consistent with the study of Hunter et al. in which they showed intravenous hydrogen sulphide ameliorate renal IRI in female pigs underwent laparotomy and cross-

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clamping of the left renal pedicle for 60 min [4]. Recently, it was shown that cystathionine c-lyase protects against renal ischemia reperfusion by modulating oxidative stress [24]. The present study is in agreement with previous studies demonstrating the highly protective effects of exogenously applied H2S treatment in similar models of ischemia [25, 26]. Renal IR injury is a complex pathophysiological process, involving many factors, such as oxidative-stress related free radical species [27] and pro-inflammatory cytokines. During oxidative damage to the tissues, lipid peroxidation occurs in polyunsaturated fatty acids [28]. MDA is an important product of lipid peroxidation. In the present study, the IR group showed marked increases in the MDA concentration, denoting the presence of oxidative stress. The H2S-treated group demonstrated a significant improvement in IRI with a lower MDA level as compared with the levels in the IR group. Liu et al. showed that NaHS significantly reduced intestinal IR injury and the levels of intestinal MDA and it dramatically increased the levels of serum and intestinal SOD activity [29]. Recently, it has shown that H2S alleviates diabetic nephropathy in streptozotocin-induced diabetic rats by attenuating oxidative stress [30]. Oxidative stress and higher levels of ROS production stimulate pathophysiological responses, which over-exhaust the antioxidant enzymes such as SOD that may lead to reduction of the levels of SOD in the body [31]. Our data showed that the levels of renal SOD in the IR group were significantly lower than those in the sham group. Treatment with H2S significantly increased the levels of renal SOD as compared with the levels in the IR group of rats. The present study shows that H2S protects cells from oxidative stress. The protective mechanism of H2S is multifaceted, but inhibition of apoptosis may be one of the major mechanisms that H2S ameliorate IRI [32]. Although reperfusion is essential for survival, it causes the release of ROS. Excess generation of ROS play major roles in the initiation of programmed cell death by activation of caspase-3. Both extrinsic and intrinsic apoptosis pathways culminate in the activation of the executor caspase-3 [33]. H2S may prevent apoptosis by inhibition of activation of the executor caspase-3 [34]. This study demonstrates that H2S protects against renal IR injury in rats and that its therapeutic effects may be mediated by increasing the activity of antioxidant SOD and reducing oxidative stress markers. This may help design new strategies to treat acute kidney injury in clinical implications. We are going to conduct future research about the molecular mechanisms of protective effect of H2S. Conflict of interest

None.

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