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Effects of hydrogen sulphide in an experimental model of renal ischaemia–reperfusion injury

Effects of hydrogen sulphide in an experimental model of renal ischaemia–reperfusion injury Abstract Background Renal ischaemia–reperfusion injury (IRI) is a major cause of acute renal failure and renal transplant dysfunction. The aim of this study was to investigate the efficacy of the endogenous gaseous signalling molecule hydrogen sulphide in protecting against renal IRI. Methods Large White female pigs underwent laparotomy and cross-clamping of the left renal pedicle for 60 min. Animals were allocated randomly to treatment with either intravenous hydrogen sulphide (n = 6) or saline control (n = 6) 10 min before clamp release, and then underwent a right nephrectomy. Staff were blinded to treatment allocation and animals were recovered for 7 days. Results Hydrogen sulphide therapy resulted in a marked reduction in kidney injury with reduced serum creatinine levels on days 1–5, in a reduced area under the creatinine–time curve, and a halving of the time to achieve a creatinine level of less than 250 µmol/l, compared with the control. Hydrogen sulphide also preserved glomerular function, as shown by the urinary protein/creatinine ratio, which, compared with baseline, increased on days 1 and 3 in the control group (mean(s.e.m.) 3·22(1·43), P = 0·016 and 2·59(1·27), P = 0·031), but not in the treatment group (0·99(0·23), P = 0·190 and 1·06(0·44), P = 0·110, respectively). Mean(s.e.m.) tumour necrosis factor α levels at 6 h postreperfusion increased in the control animals (56(6) versus 115(21) pg/ml; P = 0·026), but not in the hydrogen sulphide-treated animals (61(7) versus 74(11) pg/ml; P = 0·460). Renal neutrophil infiltration at 30 min (myeloperoxidase staining) was also significantly reduced by treatment with hydrogen sulphide (P = 0·016). Conclusion Hydrogen sulphide offers a promising new approach to ameliorating renal IRI with potential translation into a number of clinical settings, including renal transplantation. Surgical relevance Porcine renal models are established as an important transition between small-animal studies and human trials. Hydrogen sulphide has an increasing body of evidence as a therapy in reducing ischaemia–reperfusion injury (IRI) in many biological tissues, but without much evidence in renal models. This large-animal study examined the use of hydrogen sulphide in acute kidney injury resulting from direct renal ischaemia. The study demonstrated an improvement in renal function in hydrogen sulphide-treated animals without any deleterious effects. This study highlights the potential of hydrogen sulphide as a therapy that ameliorates renal IRI and provides a step towards its use in clinical trials in renal transplantation. Introduction Deciding on the suitability of kidneys for transplantation that have been retrieved from marginal donors and donation after circulatory death (DCD) donors presents a challenge for the clinician. DCD donor kidneys have to be harvested rapidly following the cessation of circulation and often undergo prolonged warm ischaemia1. This results in DCD kidneys having the highest rate of delayed graft function and primary non-function. In DCD donors, the exact duration of warm ischaemia is often unclear and the warm ischaemic tolerance of the human kidney is not completely understood. Warm ischaemic injury is due to ongoing cellular metabolism in the absence of oxygen, the accumulation of toxic metabolites, and cellular damage. Further injury occurs on reperfusion of the damaged organ, which leads to the cascade of events termed ischaemia–reperfusion injury (IRI). IRI is a complex symphony of events that is pivotal in the early outcome of the transplanted organ. Delayed graft function is an independent risk factor for acute rejection and adversely affects long-term graft survival2,3. The search for a treatment that ameliorates IRI is ongoing, and hydrogen sulphide has shown promise. Historically it has been known as a gas with a fetid odour that is highly toxic even in moderate doses4. However, at low doses it has been shown to reduce IRI in a variety of biological tissues, including brain, lung, liver, skeletal muscle, heart and kidney5–11. Hydrogen sulphide is an endogenously produced gaseous signalling molecule that can permeate the cell membrane and act directly as a second messenger12. It has been isolated in renal tissue and its production is upregulated in rodent models of IRI13. Furthermore it has a role in mitochondrial protection, platelet aggregation, inflammation and apoptosis14–17. Recent research suggests a role for hydrogen sulphide in reducing the effects of IRI in models of cardiac surgery and aortic occlusion18,19. The aim of this study was to assess the role of hydrogen sulphide in IRI in a porcine model of acute kidney injury. Methods Animals and materials Animal welfare and experimental procedures were in adherence with Home Office codes of practice and the Animals (Scientific Procedures) Act 1986. Study design was ratified and ethical approval was obtained through standard Home Office procedures. Large White female pigs (36–50 kg) were used following a minimum of 3 weeks of acclimatization at the experimental site. Study design Pigs were allocated randomly into one of three groups: sham, control or treatment. Sham animals (n = 2) underwent a surgical procedure identical to that of the control and treatment groups but without cross-clamping of the renal pedicle. Animals in control and treatment groups (n = 6) had the standard surgical procedure described below and received either 0·9 per cent saline (control) or hydrogen sulphide (treatment). Surgeons, veterinary staff and technical staff were blinded to the treatment. Anaesthesia was induced and maintained in all animals by one of two veterinarians. All procedures were carried out over a 1-week interval. Pigs were recovered for 7 days and then killed by lethal injection of intravenous pentobarbital. Randomization Administration staff at the animal institution performed randomization and were independent of all staff directly involved with the study. A computer-generated random number sequence was used to allocate each animal to a group. A sealed-envelope technique revealed the allocation to a single investigator, who prepared and administered the infusion. Group allocation was unblinded to the investigators at the end of the study interval, once the animals had been killed. Anaesthesia Animals were sedated and anaesthetized following standard protocols for the study centre. Anaesthesia was maintained with isofluorane in oxygen via positive-pressure ventilation. All animals received intravenous co-amoxiclav 25 mg/kg (GlaxoSmithKline, Brentford, UK) at induction. Remifentanil 2 µg/ml (GlaxoSmithKline) was given as a continuous intravenous infusion for analgesia, and maintenance fluids were delivered at 2 ml per kg per h (Hartmann's solution, Aqupharm; Animalcare, York, UK) throughout surgery. Oxygen saturation, heart rate, respiratory rate, expired carbon dioxide level, body temperature, electrocardiography and blood metabolic parameters were monitored throughout surgery. Surgical technique The pig was placed supine and the abdomen opened through a midline incision. The left renal pedicle was exposed and cross-clamped for 60 min. During the period of warm renal ischaemia, a double-lumen cuffed silicone vascular access catheter (Tyco Healthcare, Rugby, UK) was placed in the right external jugular vein via surgical cut-down. The lumens of the central line were locked with 1·5 ml heparin 1000 units/ml (Multiparin®; CP Pharmaceuticals, Wrexham, UK). After 60 min the left renal clamp was released and the kidney reperfused. A right nephrectomy was then performed, and the renal artery and vein were suture-ligated with polypropylene (Prolene™; Ethicon, Livingston, UK). The ureter was ligated and divided. The abdomen was mass closed using loop polydioxanone (PDS™; Ethicon) and the skin was closed using polyglactin 910 (Vicryl Rapide™; Ethicon). Hydrogen sulphide administration Sodium sulphide (Sigma-Aldrich, Poole, UK) was used as a hydrogen sulphide donor, prepared by dissolution in 0·9 per cent saline. The infusion was prepared immediately before administration and was given intravenously by an investigator independent of the surgical team. A bolus of 100 µg/kg was given 10 min before reperfusion, followed by an infusion of 1 mg/kg given continuously for 30 min after reperfusion, resulting in a total duration of infusion of 40 min. The control group received an equivalent volume of 0·9 per cent saline, both as a bolus and as an infusion, over the same duration. Postoperative care Animal recovery was standard. To ensure adequate hydration, Ringer's lactate solution (40 ml per kg per 24 h) (GlaxoSmithKline) was administered intravenously for 48 h after surgery, and animals had free access to water immediately; food was introduced on the first postoperative day. Buprenorphine 30 µg/kg (Schering-Plough, Welwyn Garden City, UK) was administered intravenously every 8–10 h for up to 4 days as postoperative analgesia. Additional analgesia was provided at the discretion of the veterinary surgeon responsible for the care of the animals. Sampling Blood samples were taken before the operation, at 1 and 6 h after operation, and daily thereafter. Urine was collected directly from the urinary bladder on entry to the peritoneal cavity and at 30 min after reperfusion. Urine samples on days 1, 3, 5 and 7 were collected using either clean-catch sampling or temporary placement of the animals in a metabolic cage. Renal biopsies were taken following entry into the peritoneal cavity, 30 min after reperfusion (30 min after clamp release) and at autopsy, immediately after exsanguination. Tumour necrosis factor α Tumour necrosis factor (TNF) α was used as an early marker of inflammation; plasma levels were determined by the quantitative sandwich enzyme immunoassay technique (R&D Systems, Minneapolis, Minnesota, USA). Samples and standards were added in duplicate to the precoated enzyme-linked immunosorbent assay (ELISA) plate and analysed according to the manufacturer's instructions. Myeloperoxidase As a measure of neutrophil infiltration, immunohistochemical staining with myeloperoxidase, a marker mainly for neutrophil granulocytes, was undertaken on paraffin sections using a ChemMate™ DAKO EnVision™ Detection Kit (DAKO, Glostrup, Denmark), as described previously20. Renal biopsies were taken before cross-clamping, 30 min after reperfusion and at autopsy on day 7. For each biopsy, 20 fields at × 400 magnification were scored for positive cells by two separate technicians who were blinded to the allocation of the animals. Neutrophil gelatinase-associated lipocalin Neutrophil gelatinase-associated lipocalin (NGAL) was used as a biomarker of kidney injury; urinary levels were determined using a pig NGAL sandwich ELISA kit (BioPorto Diagnostics, Gentofte, Denmark). The samples were diluted 1 in 5000, then added in duplicate to the precoated wells and analysed as per the manufacturer's instructions. Statistical analysis Data are presented as mean(s.e.m.). Normally distributed data were compared using the unpaired t test, and the Mann–Whitney U test was used for data that were not normally distributed. Statistical analysis was done using InStat and Prism 5 software (GraphPad Software, San Diego, California, USA). P < 0·050 was considered statistically significant. Results There was no significant difference between the two groups in relation to the mean weight of animals (43·1(2·1) versus 48·8(2·2) kg; P = 0·126) or duration of operation (128(7) versus 126(5) min; P = 0·936). All animals recovered from the operation and there was no difference in haemodynamic parameters during surgery. There was no metabolic disturbance in either group during surgery on blood pH measured before cross-clamping of the renal pedicle and 30 min after clamp release. Renal function Serum creatinine There was no difference in mean preoperative creatinine levels between the control and treatment animals (107(3) versus 116(5) µmol/l; P = 0·253). All control and treatment animals demonstrated a significant rise in serum creatinine level and all animals showed an improvement in renal function by day 6. The hydrogen sulphide-treated group demonstrated a significant improvement in renal function with a lower serum creatinine level on postoperative days 1–5 (Fig. 1). The area under the curve (AUC) for creatinine was also significantly lower in sulphide-treated animals (2219(106) versus 3769(762) µmol/l·days in the control group; P = 0·026), as was time to achieve a creatinine level below 250 µmol/l (3(1) versus 6(1) days; P = 0·007). Mean peak creatinine levels were also significantly lower in the hydrogen sulphide-treated group (396(33) versus 647(170) µmol/l; P = 0·026). Fig. 1 Open in new tabDownload slide Mean(s.e.m.) serum creatinine levels over time in pigs subjected to 60 min left renal ischaemia followed by right nephrectomy and treatment with either hydrogen sulphide (n = 6) or saline as control (n = 6). Sham animals underwent laparotomy, exposure of the left renal pedicle and right nephrectomy (n = 2) Serum urea There was no difference in mean preoperative serum urea levels between control and treatment animals (3·7(0·2) versus 4·5(0·2) mmol/l; P = 0·060). The hydrogen sulphide-treated group had lower serum urea levels on postoperative days 4–6. The AUC for urea was significantly lower in the sulphide-treated group (133(40) versus 64(3) mmol/l·days; P = 0·015). Inflammation Tumour necrosis factor α Compared with baseline values, mean serum levels of TNF-α were significantly increased in the control group at 6 h (56(6) versus 115(21) pg/ml; P = 0·026); however, there was no such increase in the treatment group at 6 h (61(7) versus 74(11) pg/ml; P = 0·460). Levels of TNF-α were numerically lower in the treatment group in comparison with the control group, but the difference was not statistically significant (AUC: 416(40) versus 314(42) µmol/l·days for control versus treatment respectively; P = 0·100) (Fig. 2). Fig. 2 Open in new tabDownload slide Mean(s.e.m.) levels of tumour necrosis factor (TNF) α over time in pigs subjected to 60 min left renal ischaemia followed by right nephrectomy and treatment with either hydrogen sulphide (n = 6) or saline as control (n = 6) Neutrophil infiltration There was no difference between groups at baseline, but at 30 min more cells stained positive in the control group than in the treatment group (6(3) versus 2(2) cells per field; P = 0·016). Glomerular injury There was no difference in the mean protein/creatinine ratio between control and treatment groups in the baseline samples (0·19(0·10) versus 0·25(0·09); P = 0·680). There was an increase in the ratio on days 1 and 3 in the control group in comparison with the baseline value (3·22(1·43), P = 0·016 and 2·59(1·27), P = 0·031), but no increase in the treatment group at the same time points (0·99(0·23), P = 0·190 and 1·06(0·44), P = 0·110) (Fig. 3). Fig. 3 Open in new tabDownload slide Mean(s.e.m.) protein/creatinine ratio at different time points in pigs subjected to 60 min left renal warm ischaemia and right nephrectomy, and given either hydrogen sulphide or 0·9 per cent saline as control Biomarkers of kidney injury Neutrophil gelatinase-associated lipocalin/creatinine ratio Numerically, the treatment group demonstrated a lower NGAL/creatinine ratio, but the difference was not statistically significant (AUC: 407(165) versus 147(51); P = 0·590) (Fig. 4). Fig. 4 Open in new tabDownload slide Mean(s.e.m.) urinary neutrophil gelatinase-associated lipocalin (NGAL)/creatinine ratio over time in pigs subjected to 60 min left renal ischaemia followed by right nephrectomy and treatment with either hydrogen sulphide or saline as control Discussion This study demonstrated that hydrogen sulphide was protective against acute kidney injury in a randomized large-animal model of renal warm ischaemia. Exogenous hydrogen sulphide therapy improved renal function, glomerular function and reduced inflammation. Hydrogen sulphide, in the form of sodium sulphide, has been trialled, as a sulphide donor, in human subjects in the USA by Ikaria (Hampton, New Jersey, USA). Trials included intravenous hydrogen sulphide in both healthy subjects and patients with varying degrees of renal failure, although as yet the results are unpublished. Hydrogen sulphide has shown promise as an agent that attenuates the ischaemia–reperfusion response in a variety of tissues, although most of the work to date has been in rodent models21–26. The findings from rodent models translate poorly into human subjects but provide proof of principle. There is a paucity of evidence on the effect of hydrogen sulphide in IRI in large-animal models. Previous work in an ex vivo model, using porcine kidneys subjected to both cold and warm ischaemic injury, showed an improvement in renal function (AUC for creatinine: 1640(248) versus 2322(154) µmol/l·days; P = 0·001) in sulphide-treated kidneys. Tubular function was also preserved in sulphide-treated kidneys, shown by a reduction in fractional excretion of sodium (P < 0·050) and increased urine output (P < 0·050)7. The present study was the natural progression of the ex vivo work and is the first large-animal model demonstrating that sulphide therapy has a protective effect on renal function in direct renal ischaemia. Hydrogen sulphide treatment has previously been investigated in a porcine model of acute kidney injury following aortic occlusion. A direct comparison cannot be made with the kidney injury in the present study; however, sulphide-treated animals demonstrated a reduction in serum creatinine levels and improvement in creatinine clearance18. Simon and colleagues18 did not demonstrate any improvement in tubular function, although the sulphide-treated animals had a significantly increased urine output. The period of 60 min ischaemia to a single functioning kidney leads to acute kidney injury, resulting in a degree of damage that was significant but from which all animals recovered. Previous work in a porcine autotransplant model assessing a variety of different warm ischaemia durations illustrated a similar degree of kidney injury when 60 min was used18. This degree of damage is a greater insult than in many human DCD kidneys and in a proportion of kidneys from marginal donation after brain death donors. Such kidneys are increasingly being used for transplantation to cover the shortfall in donor organs. The present model demonstrated a marked difference in renal function between the two groups. Serum creatinine was used as a surrogate measure of renal function as it is the simplest and most widely used marker in clinical practice. Tubular and glomerular damage is common following warm ischaemic injury, and the present study demonstrated a reduction in the protein/creatinine ratio, a surrogate marker of glomerular function, in the treated animals on days 1 and 3. The exact mechanism of action by which hydrogen sulphide prevents renal injury is unclear. Previous work has suggested that the properties of sulphide in vitro are: anti-inflammatory, antiplatelet, mitochondrial protective, antiapoptotic and vasodilatory7,14–17,26. Exogenous hydrogen sulphide has been shown to improve renal blood flow and to preserve urine output when the organ is subjected to IRI7. It has been suggested that the release of endogenous hydrogen sulphide is oxygen-dependent, and in ischaemic conditions the intracellular concentration of hydrogen sulphide increases, leading to activation of downstream pathways27. In particular, the opening of adenosine 5′-triphosphate-sensitive potassium channels, which lead to vasodilatation, increased renal blood flow and improved function28–30. Oxidative damage, caused by the production of free radicals, has long been implicated as a pivotal process in renal IRI. Free radicals cause damage to a variety of cellular components, including proteins, DNA and lipids. In addition there is an increasing body of work highlighting the anti-inflammatory effects of hydrogen sulphide therapy31. The present study lends some weight to the anti-inflammatory effects of hydrogen sulphide, although the effect was short-lived. TNF-α is an acute inflammatory cytokine that is well validated as an early marker of injury in ischaemia–reperfusion models. The present study demonstrated a significant increase in the control group after 6 h, but not the treatment group, although this difference did not persist to 24 or 48 h. Simon et al.18 also demonstrated a significant difference between the two groups in TNF-α levels, with increased values in the control arm. In addition, work performed in porcine myocardial ischaemia–reperfusion studies demonstrated a reduction also in myocardial inflammatory TNF-α levels32. Neutrophil infiltration, as a marker of acute inflammation, was determined using myeloperoxidase and, much like previous work, demonstrated a decrease in neutrophil numbers in the tissue in hydrogen sulphide-treated groups, albeit only in the 30-min biopsy samples32. Follow-up in the present study was of only short duration and more extensive studies are required to elucidate the renal outcome and potential longer-term side-effects of sulphide therapy. The study was designed to provide further evidence on the effect of sulphide therapy on renal function, and potentially confounding factors involved in a transplant model, such as cold ischaemia, perfusion and immunosuppression, were not included. Furthermore, the authors acknowledge that serum creatinine levels are affected by variables such as muscle mass and hydration status, and these factors were accounted for in the study design. Young animals were selected for their lean bodyweight and there was no statistical difference in animal weights between the two groups. All animals received a standard intraoperative and postoperative fluid regimen including administration of intravenous crystalloid 40 ml per kg per 24 h for the first 48 h. There was no difference in duration of surgery, intraoperative haemodynamic parameters or acid–base balance. Moreover, urea is a useful indicator of hydration and its level is disproportionately increased in relation to that of creatinine when an animal is dehydrated. The trend in levels of urea in the two groups was comparable and mirrored that of creatinine. This supports the conclusions drawn above and largely eliminates the likelihood that the results were due to a difference in the hydration status of the two groups. This study demonstrates the progression of hydrogen sulphide from an ex vivo into an in vivo model, and provides the foundation for translation into a renal transplant model. It strengthens the possibility of future clinical trials of hydrogen sulphide in transplantation. Acknowledgements The project was funded using departmental monies. Disclosure: The authors declare no conflict of interest. References 1 Summers DM , Johnson RJ, Allen J, Fuggle SV, Collett D, Watson CJ et al. 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Hydrogen sulfide therapy attenuates the inflammatory response in a porcine model of myocardial ischemia/reperfusion injury . J Thorac Cardiovasc Surg 2009 ; 138 : 977 – 984 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Winner of the Moynihan Medal at the 2012 International Surgical Congress of the Association of Surgeons of Great Britain and Ireland, Liverpool, UK, May 2012 Copyright © 2012 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Copyright © 2012 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Surgery Oxford University Press

Effects of hydrogen sulphide in an experimental model of renal ischaemia–reperfusion injury

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Oxford University Press
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0007-1323
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DOI
10.1002/bjs.8956
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Abstract

Abstract Background Renal ischaemia–reperfusion injury (IRI) is a major cause of acute renal failure and renal transplant dysfunction. The aim of this study was to investigate the efficacy of the endogenous gaseous signalling molecule hydrogen sulphide in protecting against renal IRI. Methods Large White female pigs underwent laparotomy and cross-clamping of the left renal pedicle for 60 min. Animals were allocated randomly to treatment with either intravenous hydrogen sulphide (n = 6) or saline control (n = 6) 10 min before clamp release, and then underwent a right nephrectomy. Staff were blinded to treatment allocation and animals were recovered for 7 days. Results Hydrogen sulphide therapy resulted in a marked reduction in kidney injury with reduced serum creatinine levels on days 1–5, in a reduced area under the creatinine–time curve, and a halving of the time to achieve a creatinine level of less than 250 µmol/l, compared with the control. Hydrogen sulphide also preserved glomerular function, as shown by the urinary protein/creatinine ratio, which, compared with baseline, increased on days 1 and 3 in the control group (mean(s.e.m.) 3·22(1·43), P = 0·016 and 2·59(1·27), P = 0·031), but not in the treatment group (0·99(0·23), P = 0·190 and 1·06(0·44), P = 0·110, respectively). Mean(s.e.m.) tumour necrosis factor α levels at 6 h postreperfusion increased in the control animals (56(6) versus 115(21) pg/ml; P = 0·026), but not in the hydrogen sulphide-treated animals (61(7) versus 74(11) pg/ml; P = 0·460). Renal neutrophil infiltration at 30 min (myeloperoxidase staining) was also significantly reduced by treatment with hydrogen sulphide (P = 0·016). Conclusion Hydrogen sulphide offers a promising new approach to ameliorating renal IRI with potential translation into a number of clinical settings, including renal transplantation. Surgical relevance Porcine renal models are established as an important transition between small-animal studies and human trials. Hydrogen sulphide has an increasing body of evidence as a therapy in reducing ischaemia–reperfusion injury (IRI) in many biological tissues, but without much evidence in renal models. This large-animal study examined the use of hydrogen sulphide in acute kidney injury resulting from direct renal ischaemia. The study demonstrated an improvement in renal function in hydrogen sulphide-treated animals without any deleterious effects. This study highlights the potential of hydrogen sulphide as a therapy that ameliorates renal IRI and provides a step towards its use in clinical trials in renal transplantation. Introduction Deciding on the suitability of kidneys for transplantation that have been retrieved from marginal donors and donation after circulatory death (DCD) donors presents a challenge for the clinician. DCD donor kidneys have to be harvested rapidly following the cessation of circulation and often undergo prolonged warm ischaemia1. This results in DCD kidneys having the highest rate of delayed graft function and primary non-function. In DCD donors, the exact duration of warm ischaemia is often unclear and the warm ischaemic tolerance of the human kidney is not completely understood. Warm ischaemic injury is due to ongoing cellular metabolism in the absence of oxygen, the accumulation of toxic metabolites, and cellular damage. Further injury occurs on reperfusion of the damaged organ, which leads to the cascade of events termed ischaemia–reperfusion injury (IRI). IRI is a complex symphony of events that is pivotal in the early outcome of the transplanted organ. Delayed graft function is an independent risk factor for acute rejection and adversely affects long-term graft survival2,3. The search for a treatment that ameliorates IRI is ongoing, and hydrogen sulphide has shown promise. Historically it has been known as a gas with a fetid odour that is highly toxic even in moderate doses4. However, at low doses it has been shown to reduce IRI in a variety of biological tissues, including brain, lung, liver, skeletal muscle, heart and kidney5–11. Hydrogen sulphide is an endogenously produced gaseous signalling molecule that can permeate the cell membrane and act directly as a second messenger12. It has been isolated in renal tissue and its production is upregulated in rodent models of IRI13. Furthermore it has a role in mitochondrial protection, platelet aggregation, inflammation and apoptosis14–17. Recent research suggests a role for hydrogen sulphide in reducing the effects of IRI in models of cardiac surgery and aortic occlusion18,19. The aim of this study was to assess the role of hydrogen sulphide in IRI in a porcine model of acute kidney injury. Methods Animals and materials Animal welfare and experimental procedures were in adherence with Home Office codes of practice and the Animals (Scientific Procedures) Act 1986. Study design was ratified and ethical approval was obtained through standard Home Office procedures. Large White female pigs (36–50 kg) were used following a minimum of 3 weeks of acclimatization at the experimental site. Study design Pigs were allocated randomly into one of three groups: sham, control or treatment. Sham animals (n = 2) underwent a surgical procedure identical to that of the control and treatment groups but without cross-clamping of the renal pedicle. Animals in control and treatment groups (n = 6) had the standard surgical procedure described below and received either 0·9 per cent saline (control) or hydrogen sulphide (treatment). Surgeons, veterinary staff and technical staff were blinded to the treatment. Anaesthesia was induced and maintained in all animals by one of two veterinarians. All procedures were carried out over a 1-week interval. Pigs were recovered for 7 days and then killed by lethal injection of intravenous pentobarbital. Randomization Administration staff at the animal institution performed randomization and were independent of all staff directly involved with the study. A computer-generated random number sequence was used to allocate each animal to a group. A sealed-envelope technique revealed the allocation to a single investigator, who prepared and administered the infusion. Group allocation was unblinded to the investigators at the end of the study interval, once the animals had been killed. Anaesthesia Animals were sedated and anaesthetized following standard protocols for the study centre. Anaesthesia was maintained with isofluorane in oxygen via positive-pressure ventilation. All animals received intravenous co-amoxiclav 25 mg/kg (GlaxoSmithKline, Brentford, UK) at induction. Remifentanil 2 µg/ml (GlaxoSmithKline) was given as a continuous intravenous infusion for analgesia, and maintenance fluids were delivered at 2 ml per kg per h (Hartmann's solution, Aqupharm; Animalcare, York, UK) throughout surgery. Oxygen saturation, heart rate, respiratory rate, expired carbon dioxide level, body temperature, electrocardiography and blood metabolic parameters were monitored throughout surgery. Surgical technique The pig was placed supine and the abdomen opened through a midline incision. The left renal pedicle was exposed and cross-clamped for 60 min. During the period of warm renal ischaemia, a double-lumen cuffed silicone vascular access catheter (Tyco Healthcare, Rugby, UK) was placed in the right external jugular vein via surgical cut-down. The lumens of the central line were locked with 1·5 ml heparin 1000 units/ml (Multiparin®; CP Pharmaceuticals, Wrexham, UK). After 60 min the left renal clamp was released and the kidney reperfused. A right nephrectomy was then performed, and the renal artery and vein were suture-ligated with polypropylene (Prolene™; Ethicon, Livingston, UK). The ureter was ligated and divided. The abdomen was mass closed using loop polydioxanone (PDS™; Ethicon) and the skin was closed using polyglactin 910 (Vicryl Rapide™; Ethicon). Hydrogen sulphide administration Sodium sulphide (Sigma-Aldrich, Poole, UK) was used as a hydrogen sulphide donor, prepared by dissolution in 0·9 per cent saline. The infusion was prepared immediately before administration and was given intravenously by an investigator independent of the surgical team. A bolus of 100 µg/kg was given 10 min before reperfusion, followed by an infusion of 1 mg/kg given continuously for 30 min after reperfusion, resulting in a total duration of infusion of 40 min. The control group received an equivalent volume of 0·9 per cent saline, both as a bolus and as an infusion, over the same duration. Postoperative care Animal recovery was standard. To ensure adequate hydration, Ringer's lactate solution (40 ml per kg per 24 h) (GlaxoSmithKline) was administered intravenously for 48 h after surgery, and animals had free access to water immediately; food was introduced on the first postoperative day. Buprenorphine 30 µg/kg (Schering-Plough, Welwyn Garden City, UK) was administered intravenously every 8–10 h for up to 4 days as postoperative analgesia. Additional analgesia was provided at the discretion of the veterinary surgeon responsible for the care of the animals. Sampling Blood samples were taken before the operation, at 1 and 6 h after operation, and daily thereafter. Urine was collected directly from the urinary bladder on entry to the peritoneal cavity and at 30 min after reperfusion. Urine samples on days 1, 3, 5 and 7 were collected using either clean-catch sampling or temporary placement of the animals in a metabolic cage. Renal biopsies were taken following entry into the peritoneal cavity, 30 min after reperfusion (30 min after clamp release) and at autopsy, immediately after exsanguination. Tumour necrosis factor α Tumour necrosis factor (TNF) α was used as an early marker of inflammation; plasma levels were determined by the quantitative sandwich enzyme immunoassay technique (R&D Systems, Minneapolis, Minnesota, USA). Samples and standards were added in duplicate to the precoated enzyme-linked immunosorbent assay (ELISA) plate and analysed according to the manufacturer's instructions. Myeloperoxidase As a measure of neutrophil infiltration, immunohistochemical staining with myeloperoxidase, a marker mainly for neutrophil granulocytes, was undertaken on paraffin sections using a ChemMate™ DAKO EnVision™ Detection Kit (DAKO, Glostrup, Denmark), as described previously20. Renal biopsies were taken before cross-clamping, 30 min after reperfusion and at autopsy on day 7. For each biopsy, 20 fields at × 400 magnification were scored for positive cells by two separate technicians who were blinded to the allocation of the animals. Neutrophil gelatinase-associated lipocalin Neutrophil gelatinase-associated lipocalin (NGAL) was used as a biomarker of kidney injury; urinary levels were determined using a pig NGAL sandwich ELISA kit (BioPorto Diagnostics, Gentofte, Denmark). The samples were diluted 1 in 5000, then added in duplicate to the precoated wells and analysed as per the manufacturer's instructions. Statistical analysis Data are presented as mean(s.e.m.). Normally distributed data were compared using the unpaired t test, and the Mann–Whitney U test was used for data that were not normally distributed. Statistical analysis was done using InStat and Prism 5 software (GraphPad Software, San Diego, California, USA). P < 0·050 was considered statistically significant. Results There was no significant difference between the two groups in relation to the mean weight of animals (43·1(2·1) versus 48·8(2·2) kg; P = 0·126) or duration of operation (128(7) versus 126(5) min; P = 0·936). All animals recovered from the operation and there was no difference in haemodynamic parameters during surgery. There was no metabolic disturbance in either group during surgery on blood pH measured before cross-clamping of the renal pedicle and 30 min after clamp release. Renal function Serum creatinine There was no difference in mean preoperative creatinine levels between the control and treatment animals (107(3) versus 116(5) µmol/l; P = 0·253). All control and treatment animals demonstrated a significant rise in serum creatinine level and all animals showed an improvement in renal function by day 6. The hydrogen sulphide-treated group demonstrated a significant improvement in renal function with a lower serum creatinine level on postoperative days 1–5 (Fig. 1). The area under the curve (AUC) for creatinine was also significantly lower in sulphide-treated animals (2219(106) versus 3769(762) µmol/l·days in the control group; P = 0·026), as was time to achieve a creatinine level below 250 µmol/l (3(1) versus 6(1) days; P = 0·007). Mean peak creatinine levels were also significantly lower in the hydrogen sulphide-treated group (396(33) versus 647(170) µmol/l; P = 0·026). Fig. 1 Open in new tabDownload slide Mean(s.e.m.) serum creatinine levels over time in pigs subjected to 60 min left renal ischaemia followed by right nephrectomy and treatment with either hydrogen sulphide (n = 6) or saline as control (n = 6). Sham animals underwent laparotomy, exposure of the left renal pedicle and right nephrectomy (n = 2) Serum urea There was no difference in mean preoperative serum urea levels between control and treatment animals (3·7(0·2) versus 4·5(0·2) mmol/l; P = 0·060). The hydrogen sulphide-treated group had lower serum urea levels on postoperative days 4–6. The AUC for urea was significantly lower in the sulphide-treated group (133(40) versus 64(3) mmol/l·days; P = 0·015). Inflammation Tumour necrosis factor α Compared with baseline values, mean serum levels of TNF-α were significantly increased in the control group at 6 h (56(6) versus 115(21) pg/ml; P = 0·026); however, there was no such increase in the treatment group at 6 h (61(7) versus 74(11) pg/ml; P = 0·460). Levels of TNF-α were numerically lower in the treatment group in comparison with the control group, but the difference was not statistically significant (AUC: 416(40) versus 314(42) µmol/l·days for control versus treatment respectively; P = 0·100) (Fig. 2). Fig. 2 Open in new tabDownload slide Mean(s.e.m.) levels of tumour necrosis factor (TNF) α over time in pigs subjected to 60 min left renal ischaemia followed by right nephrectomy and treatment with either hydrogen sulphide (n = 6) or saline as control (n = 6) Neutrophil infiltration There was no difference between groups at baseline, but at 30 min more cells stained positive in the control group than in the treatment group (6(3) versus 2(2) cells per field; P = 0·016). Glomerular injury There was no difference in the mean protein/creatinine ratio between control and treatment groups in the baseline samples (0·19(0·10) versus 0·25(0·09); P = 0·680). There was an increase in the ratio on days 1 and 3 in the control group in comparison with the baseline value (3·22(1·43), P = 0·016 and 2·59(1·27), P = 0·031), but no increase in the treatment group at the same time points (0·99(0·23), P = 0·190 and 1·06(0·44), P = 0·110) (Fig. 3). Fig. 3 Open in new tabDownload slide Mean(s.e.m.) protein/creatinine ratio at different time points in pigs subjected to 60 min left renal warm ischaemia and right nephrectomy, and given either hydrogen sulphide or 0·9 per cent saline as control Biomarkers of kidney injury Neutrophil gelatinase-associated lipocalin/creatinine ratio Numerically, the treatment group demonstrated a lower NGAL/creatinine ratio, but the difference was not statistically significant (AUC: 407(165) versus 147(51); P = 0·590) (Fig. 4). Fig. 4 Open in new tabDownload slide Mean(s.e.m.) urinary neutrophil gelatinase-associated lipocalin (NGAL)/creatinine ratio over time in pigs subjected to 60 min left renal ischaemia followed by right nephrectomy and treatment with either hydrogen sulphide or saline as control Discussion This study demonstrated that hydrogen sulphide was protective against acute kidney injury in a randomized large-animal model of renal warm ischaemia. Exogenous hydrogen sulphide therapy improved renal function, glomerular function and reduced inflammation. Hydrogen sulphide, in the form of sodium sulphide, has been trialled, as a sulphide donor, in human subjects in the USA by Ikaria (Hampton, New Jersey, USA). Trials included intravenous hydrogen sulphide in both healthy subjects and patients with varying degrees of renal failure, although as yet the results are unpublished. Hydrogen sulphide has shown promise as an agent that attenuates the ischaemia–reperfusion response in a variety of tissues, although most of the work to date has been in rodent models21–26. The findings from rodent models translate poorly into human subjects but provide proof of principle. There is a paucity of evidence on the effect of hydrogen sulphide in IRI in large-animal models. Previous work in an ex vivo model, using porcine kidneys subjected to both cold and warm ischaemic injury, showed an improvement in renal function (AUC for creatinine: 1640(248) versus 2322(154) µmol/l·days; P = 0·001) in sulphide-treated kidneys. Tubular function was also preserved in sulphide-treated kidneys, shown by a reduction in fractional excretion of sodium (P < 0·050) and increased urine output (P < 0·050)7. The present study was the natural progression of the ex vivo work and is the first large-animal model demonstrating that sulphide therapy has a protective effect on renal function in direct renal ischaemia. Hydrogen sulphide treatment has previously been investigated in a porcine model of acute kidney injury following aortic occlusion. A direct comparison cannot be made with the kidney injury in the present study; however, sulphide-treated animals demonstrated a reduction in serum creatinine levels and improvement in creatinine clearance18. Simon and colleagues18 did not demonstrate any improvement in tubular function, although the sulphide-treated animals had a significantly increased urine output. The period of 60 min ischaemia to a single functioning kidney leads to acute kidney injury, resulting in a degree of damage that was significant but from which all animals recovered. Previous work in a porcine autotransplant model assessing a variety of different warm ischaemia durations illustrated a similar degree of kidney injury when 60 min was used18. This degree of damage is a greater insult than in many human DCD kidneys and in a proportion of kidneys from marginal donation after brain death donors. Such kidneys are increasingly being used for transplantation to cover the shortfall in donor organs. The present model demonstrated a marked difference in renal function between the two groups. Serum creatinine was used as a surrogate measure of renal function as it is the simplest and most widely used marker in clinical practice. Tubular and glomerular damage is common following warm ischaemic injury, and the present study demonstrated a reduction in the protein/creatinine ratio, a surrogate marker of glomerular function, in the treated animals on days 1 and 3. The exact mechanism of action by which hydrogen sulphide prevents renal injury is unclear. Previous work has suggested that the properties of sulphide in vitro are: anti-inflammatory, antiplatelet, mitochondrial protective, antiapoptotic and vasodilatory7,14–17,26. Exogenous hydrogen sulphide has been shown to improve renal blood flow and to preserve urine output when the organ is subjected to IRI7. It has been suggested that the release of endogenous hydrogen sulphide is oxygen-dependent, and in ischaemic conditions the intracellular concentration of hydrogen sulphide increases, leading to activation of downstream pathways27. In particular, the opening of adenosine 5′-triphosphate-sensitive potassium channels, which lead to vasodilatation, increased renal blood flow and improved function28–30. Oxidative damage, caused by the production of free radicals, has long been implicated as a pivotal process in renal IRI. Free radicals cause damage to a variety of cellular components, including proteins, DNA and lipids. In addition there is an increasing body of work highlighting the anti-inflammatory effects of hydrogen sulphide therapy31. The present study lends some weight to the anti-inflammatory effects of hydrogen sulphide, although the effect was short-lived. TNF-α is an acute inflammatory cytokine that is well validated as an early marker of injury in ischaemia–reperfusion models. The present study demonstrated a significant increase in the control group after 6 h, but not the treatment group, although this difference did not persist to 24 or 48 h. Simon et al.18 also demonstrated a significant difference between the two groups in TNF-α levels, with increased values in the control arm. In addition, work performed in porcine myocardial ischaemia–reperfusion studies demonstrated a reduction also in myocardial inflammatory TNF-α levels32. Neutrophil infiltration, as a marker of acute inflammation, was determined using myeloperoxidase and, much like previous work, demonstrated a decrease in neutrophil numbers in the tissue in hydrogen sulphide-treated groups, albeit only in the 30-min biopsy samples32. Follow-up in the present study was of only short duration and more extensive studies are required to elucidate the renal outcome and potential longer-term side-effects of sulphide therapy. The study was designed to provide further evidence on the effect of sulphide therapy on renal function, and potentially confounding factors involved in a transplant model, such as cold ischaemia, perfusion and immunosuppression, were not included. Furthermore, the authors acknowledge that serum creatinine levels are affected by variables such as muscle mass and hydration status, and these factors were accounted for in the study design. Young animals were selected for their lean bodyweight and there was no statistical difference in animal weights between the two groups. All animals received a standard intraoperative and postoperative fluid regimen including administration of intravenous crystalloid 40 ml per kg per 24 h for the first 48 h. There was no difference in duration of surgery, intraoperative haemodynamic parameters or acid–base balance. Moreover, urea is a useful indicator of hydration and its level is disproportionately increased in relation to that of creatinine when an animal is dehydrated. The trend in levels of urea in the two groups was comparable and mirrored that of creatinine. This supports the conclusions drawn above and largely eliminates the likelihood that the results were due to a difference in the hydration status of the two groups. This study demonstrates the progression of hydrogen sulphide from an ex vivo into an in vivo model, and provides the foundation for translation into a renal transplant model. It strengthens the possibility of future clinical trials of hydrogen sulphide in transplantation. Acknowledgements The project was funded using departmental monies. Disclosure: The authors declare no conflict of interest. References 1 Summers DM , Johnson RJ, Allen J, Fuggle SV, Collett D, Watson CJ et al. 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Hydrogen sulfide therapy attenuates the inflammatory response in a porcine model of myocardial ischemia/reperfusion injury . J Thorac Cardiovasc Surg 2009 ; 138 : 977 – 984 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Winner of the Moynihan Medal at the 2012 International Surgical Congress of the Association of Surgeons of Great Britain and Ireland, Liverpool, UK, May 2012 Copyright © 2012 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Copyright © 2012 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Journal

British Journal of Surgery – Oxford University Press

Published: Nov 6, 2012

Keywords: creatinine; tumor necrosis factors; hydrogen sulfide; kidney; reperfusion injury; suidae; sulfides; renal trauma; renal failure, acute; neutrophil infiltration; peroxidase; saline solutions; hydrogen; nephrectomy; serum creatinine level; renal pedicle; renal transplantation

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