Mitochondria-targeted antioxidant MitoQ ameliorates ischaemia-reperfusion injury in kidney transplantation models.

BACKGROUND: Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation. Oxidative damage to mitochondria is an early event in IR injury. Therefore, the uptake, safety, and efficacy of the mitochondria-targeted antioxidant MitoQ were investigated in models of transplant IR injury. METHODS: MitoQ uptake by warm and cooled pairs of pig and declined human kidneys was measured when preserved in cold static storage or by hypothermic machine perfusion. Pairs of pigs' kidneys were exposed to defined periods of warm and cold ischaemia, flushed and stored at 4°C with or without MitoQ (50 nmol/l to 250 µmol/l), followed by reperfusion with oxygenated autologous blood in an ex vivo normothermic perfusion (EVNP). Pairs of declined human kidneys were flushed and stored with or without MitoQ (5-100 µmol/l) at 4°C for 6 h and underwent EVNP with ABO group-matched blood. RESULTS: Stable and concentration-dependent uptake of MitoQ was demonstrated for up to 24 h in pig and human kidneys. Total blood flow and urine output were significantly greater in pig kidneys treated with 50 µmol/l MitoQ compared with controls (P = 0.006 and P = 0.007 respectively). In proof-of-concept experiments, blood flow after 1 h of EVNP was significantly greater in human kidneys treated with 50 µmol/l MitoQ than in controls (P ≤ 0.001). Total urine output was numerically higher in the 50-µmol/l MitoQ group compared with the control, but the difference did not reach statistical significance (P = 0.054). CONCLUSION: Mitochondria-targeted antioxidant MitoQ can be administered to ischaemic kidneys simply and effectively during cold storage, and may improve outcomes after transplantation.

Ex vivo normothermic perfusion of isolated segmental porcine bowel: a novel functional model of the small intestine.

BACKGROUND: There is an unmet need for suitable ex vivo large animal models in experimental gastroenterology and intestinal transplantation. This study details a reliable and effective technique for ex vivo normothermic perfusion (EVNP) of segmental porcine small intestine. METHODS: Segments of small intestine, 1.5-3.0 m in length, were retrieved from terminally anaesthetized pigs. After a period of cold ischaemia, EVNP was performed for 2 h at 37°C with a mean pressure of 80 mmHg using oxygenated autologous blood diluted with Ringer's solution. The duration of EVNP was extended to 4 h for a second set of experiments in which two segments of proximal to mid-ileum (1.5-3.0 m) were retrieved from each animal and reperfused with whole blood (control) or leucocyte-depleted blood to examine the impact of leucocyte depletion on reperfusion injury. RESULTS: After a mean cold ischaemia time of 5 h and 20 min, EVNP was performed in an initial group of four pigs. In the second set of experiments, five pigs were used in each group. In all experiments bowel segments were well perfused and exhibited peristalsis during EVNP. Venous glucose levels significantly increased following luminal glucose stimulation (mean(s.e.m.) basal level 1.8(0.6) mmol/l versus peak 15.5(5.8) mmol/l; P < 0.001) and glucagon-like peptide 1 (GLP-1) levels increased in all experiments, demonstrating intact absorptive and secretory intestinal functions. There were no significant differences between control and leucocyte-depleted animals regarding blood flow, venous glucose, GLP-1 levels or histopathology at the end of 4 h of EVNP. CONCLUSIONS: This novel model is suitable for the investigation of gastrointestinal physiology, pathology and ischaemia reperfusion injury, along with evaluation of potential therapeutic interventions.

Risk factors for transplant renal artery stenosis after live donor transplantation.

BACKGROUND: Renal transplant surgeons are making increasing use of live donor kidneys with multiple renal arteries. This study aimed to identify independent risk factors for the development of transplant renal artery stenosis (TRAS) in the modern era of complex arterial reconstruction for multiple vessels. METHODS: Multivariable logistic regression analysis with a stepwise variable deletion model was used to identify risk factors for the development of TRAS in a consecutive series of live donor kidney transplants. RESULTS: Of 506 kidney transplants, 19 (3·8 per cent) had evidence of significant TRAS on CT angiography. Functional TRAS, defined by improvement in BP control or renal function after correction of a stenosis by angioplasty, occurred in 13 of 506 patients (2·6 per cent). Independent risk factors for TRAS were: use of an explanted internal iliac artery graft from the recipient (odds ratio (OR) 4·95; P = 0·020) and total ischaemia time (OR 1·82; P = 0·010). TRAS was associated with a lower 5-year allograft survival rate (79 versus 88·7 per cent; P = 0·020) but only one graft loss was attributed directly to TRAS. The 5-year allograft survival rate after internal iliac artery grafting was 86 per cent. CONCLUSION: Although use of an internal iliac artery graft is an independent risk factor for TRAS after live donor kidney transplantation, this technique is still a useful option for complex arterial reconstruction.

Normothermic machine perfusion for the assessment and transplantation of declined human kidneys from donation after circulatory death donors.

BACKGROUND: A significant proportion of donation after circulatory death (DCD) kidneys are declined for transplantation because of concerns over their quality. Ex vivo normothermic machine perfusion (NMP) provides a unique opportunity to assess the quality of a kidney and determine its suitability for transplantation. METHODS: In phase 1 of this study, declined human DCD kidneys underwent NMP assessment for 60 min. Kidneys were graded 1-5 using a quality assessment score (QAS) based on macroscopic perfusion, renal blood flow and urine output during NMP. In phase 2 of the study, declined DCD kidneys were assessed by NMP with an intention to transplant them. RESULTS: In phase 1, 18 of 42 DCD kidneys were declined owing to poor in situ perfusion. After NMP, 28 kidneys had a QAS of 1-3, and were considered suitable for transplantation. In phase 2, ten of 55 declined DCD kidneys underwent assessment by NMP. Eight kidneys had been declined because of poor in situ flushing in the donor and five of these were transplanted successfully. Four of the five kidneys had initial graft function. CONCLUSION: NMP technology can be used to increase the number of DCD kidney transplants by assessing their quality before transplantation.

Ex vivo normothermic perfusion for quality assessment of marginal donor kidney transplants.

BACKGROUND: A significant proportion of kidneys procured for transplantation are discarded because of concerns about their suitability. In this study ex vivo normothermic perfusion (EVNP) was used as a quality assessment device before renal transplantation. METHODS: Seventy-four human kidneys deemed unsuitable for transplantation following retrieval underwent 60 min of EVNP with an oxygenated red cell-based solution at 36°C. Receiver operating characteristic (ROC) curves were used to identify thresholds of renal blood flow and urine output. These thresholds and a grading of macroscopic appearance were incorporated into an EVNP assessment score (highest quality, 1; lowest, 5). This was applied to a series of 36 kidneys transplanted after EVNP. RESULTS: In the discarded kidney series, 60 (81 per cent) scored 1-4 and 14 (19 per cent) scored 5. Although none of these kidneys was transplanted, those with a score from 1 to 4 were considered suitable for transplantation. In the 36 transplanted kidneys, the score ranged between 1 and 3 (score 1, 17; score 2, 11; score 3, 8). All of these kidneys were transplanted without any complications or primary non-function. The delayed graft function rate was 6 per cent (1 of 17) in kidneys scoring 1, 0 per cent (0 of 11) in those scoring 2 and 38 per cent (3 of 8) in those scoring 3 (P = 0·024). The mean(s.d.) estimated glomerular filtration rate at 12 months was 51(16), 63(15) and 38(21) ml in kidneys scoring 1, 2 and 3 respectively (P = 0·015). CONCLUSION: EVNP combined with a simple scoring system is an innovative technology for pretransplant assessment of kidney quality and acceptability for transplantation. This study suggests that a high percentage of retrieved kidneys are being discarded unnecessarily.

Ischaemic conditioning reduces kidney injury in an experimental large-animal model of warm renal ischaemia.

BACKGROUND: Ischaemic conditioning, using short repeated sequences of intermittent ischaemia, is a strategy that may ameliorate ischaemia-reperfusion injury. The aim of the study was to assess the effects of direct and remote ischaemic conditioning in a porcine model of renal warm ischaemia-reperfusion injury. METHODS: Pigs (50 kg) underwent laparotomy and 60-min occlusion of the left renal pedicle followed by right nephrectomy. Animals were divided into three groups: untreated controls (n = 8); direct postconditioning involving six 15-s cycles of clamping then releasing of the left renal artery (n = 7); or remote periconditioning involving four 5-min cycles of clamping then releasing of the left common iliac artery (n = 8). After 7 days kidney tissue was harvested, and blood and urine samples were collected on postoperative days 1, 3 and 7. RESULTS: The direct postconditioning group had a lower area under the serum creatinine curve (mean(s.d.) 1378(157) versus 2001(1022) µmol/l · day respectively; P = 0.036) and peak creatinine level (316(46) versus 501(253) µmol/l respectively; P = 0.033) compared with values in control animals. There was a significant increase in serum levels of tumour necrosis factor α on day 1 in control animals but not in the conditioning groups (P = 0.013). Urinary levels of neutrophil gelatinase-associated lipocalin increased over the study period in both the control and remote groups (P = 0.001 for both), but not in the direct group (P = 0.176). There was no mortality and no complications related to either conditioning technique. CONCLUSION: In this in vivo large-animal model, direct renal artery ischaemic postconditioning protected kidneys against warm ischaemia injury. This straightforward technique could readily be translated into clinical practice. Surgical relevance Ischaemic conditioning has been shown to improve outcomes in both experimental studies and clinical trials in cardiac surgery. Evidence from small-animal and human studies assessing ischaemic conditioning techniques in renal transplantation have not yet established the optimal technique and timing of conditioning. In this study, a large-animal model of renal warm ischaemia was used to compare different conditioning techniques. Postconditioning applied directly to the renal artery was shown to reduce renal injury. Furthermore, new evidence is provided that shorter cycles of ischaemic postconditioning than previously described can protect against renal injury. Evidence from a large-animal model is provided for different conditioning techniques. The beneficial postconditioning technique described is straightforward to perform and provides an alternative method of conditioning following renal transplantation, with potential for application in clinical practice.

The first clinical case of intermediate ex vivo normothermic perfusion in renal transplantation.

A short period of ex vivo normothermic perfusion (EVNP) immediately before transplantation can revive the kidney and reduce the effects of cold ischemic (CI) injury. Herein, we report a clinical case of EVNP carried out at an intermediate period of the preservation interval. The kidney was retrieved from a 63-year-old extended criteria donor. After 10 h 29 min of CI the kidney underwent EVNP with 1 unit of compatible packed red blood cells mixed with a priming solution at 35.0°C while the recipient was being prepared for surgery. The mean renal blood flow was 93.6 mL/min/100 g and the kidney produced 60 mL of urine. Shortly after the start of surgery the first intended recipient became unfit for transplantation. After 60 min EVNP the kidney was flushed with cold preservation solution and re-packed in ice. The second period of CI was 5 h and 21 min. The kidney was transplanted without any complications into a 54-year-old predialysis patient. The recipient had immediate graft function with serum creatinine levels falling from 315 to 105 µmol/L by day 7. This is the first report of an intermediate period of EVNP in clinical renal transplantation. This case demonstrates the feasibility and safety of the technique.

Pain management in laparoscopic donor nephrectomy: a review.

The management of postoperative pain is a key to patient early recovery, in particular, where the surgery was performed to benefit another human being. In recent years it has been recognized that multimodal analgesic methods are superior for postoperative pain relief. It is also imperative to remember that inadequately managed acute postoperative pain opens the doorway to possible suffering from chronic postoperative pain later. Although the laparoscopic donor nephrectomy has reduced the disincentives associated with open surgery, still significant percentage of donors suffers from postoperative pain. In the UK, patient-controlled analgesic system (PCAS) using morphine for postoperative pain relief is being used in majority of the transplant centres. Though opioids provide good analgesia, they are far from being an ideal analgesic due to their adverse effects. This paper pragmatically looks in depth on different modalities of pain management in patients undergoing laparoscopic live donor nephrectomy.

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

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.

Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2).

AIMS/HYPOTHESIS: Rapamycin (sirolimus) is one of the primary immunosuppressants for islet transplantation. Yet there is evidence that the long-term treatment of islet-transplant patients with rapamycin may be responsible for subsequent loss of islet graft function and viability. Therefore, the primary objective of this study was to elucidate the molecular mechanism of rapamycin toxicity in beta cells. METHODS: Experiments were performed on isolated rat and human islets of Langerhans and MIN6 cells. The effects of rapamycin and the roles of mammalian target of rapamycin complex 2 (mTORC2)/protein kinase B (PKB) on beta cell signalling, function and viability were investigated using cell viability assays, insulin ELISA assays, kinase assays, western blotting, pharmacological inhibitors, small interfering (si)RNA and through the overproduction of a constitutively active mutant of PKB. RESULTS: Rapamycin treatment of MIN6 cells and islets of Langerhans resulted in a loss of cell function and viability. Although rapamycin acutely inhibited mTOR complex 1 (mTORC1), the toxic effects of rapamycin were more closely correlated to the dissociation and inactivation of mTORC2 and the inhibition of PKB. Indeed, the overproduction of constitutively active PKB protected islets from rapamycin toxicity whereas the inhibition of PKB led to a loss of cell viability. Moreover, the selective inactivation of mTORC2 using siRNA directed towards rapamycin-insensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment. CONCLUSIONS/INTERPRETATION: This report provides evidence that rapamycin toxicity is mediated by the inactivation of mTORC2 and the inhibition of PKB and thus reveals the molecular basis of rapamycin toxicity and the essential role of mTORC2 in maintaining beta cell function and survival.

Post-biopsy renal allograft compartment syndrome: Addressing the problem, illustrated with a case report.

INTRODUCTION: Renal allograft compartment syndrome (RACS) has recently been coined to describe early allograft dysfunction secondary to raised pressure in the retroperitoneal space. This may be caused by direct compression of the renal vessels or by a diffuse renal parenchymal compression. Herein, we report a renal allograft compartment syndrome secondary to a needle core transplant biopsy and discuss the management strategies in line with an updated literature review. PRESENTATION OF CASE: A retrospective case-note review was carried out where a 45-year-old male had a transplant renal biopsy at 4-weeks after transplant for raising creatinine. Following biopsy patient developed abdominal discomfort and had haematuria. DISCUSSION: Doppler ultrasound scanning of graft demonstrated good perfusion but a small haematoma (2 × 2 × 2 cm) in the upper pole of the kidney at the site of the biopsy. Patient was thereafter assessed conservatively with serial ultrasound monitoring. After 24 h, significant deterioration of graft function was observed. The third scan, demonstrated reversed flow in diastole in the upper pole of the kidney with a resistive index of 1.0 in the main renal vessel. With the above findings the kidney transplant was explored immediately and the transplant released from a 300 ml of liquefied haematoma, which was under considerable pressure. In the next 24-h, the patient showed an immediate return of graft function. CONCLUSION: We recommend sequential ultrasound Doppler scanning as an invaluable tool to help identify early RACS. The surgical exploration and adequate heamostasis with surgical glue should be sought out in all RACS.

Hypothermic machine perfusion after static cold storage does not improve the preservation condition in an experimental porcine kidney model.

BACKGROUND: Combining hypothermic techniques, as a more practical approach to preservation, may enhance the condition of kidneys donated after cardiac death. METHODS: Porcine kidneys were retrieved after 10 min in situ warm ischaemia, then preserved by either 18 h static cold storage (CS), hypothermic machine perfusion for 18 h (HMP) or 14 h static CS followed by 4 h HMP (4HMP). Kidneys were reperfused for 3 h with oxygenated autologous blood on an isolated organ perfusion system to assess renal function and injury. RESULTS: Intrarenal resistance was significantly higher in the 4HMP group than in the CS and HMP groups: mean(s.d.) area under the curve (AUC) 8·48(2·97), 3·41(1·80) and 3·78(1·68) mmHg/min.h respectively (P = 0·011). Creatinine clearance was lower after 4HMP and CS: AUC 2·3(0·6) and 2·2(1·7) ml per min per 100g.h respectively versus 9·8(7·3) ml per min per 100g.h in the HMP group (P = 0·022). Levels of endothelin 1 were higher in the 4HMP and CS groups: mean(s.d.) 21·6(4·0) and 24·2(2·3) pg/ml respectively versus 11·4(4·6) pg/ml in the HMP group (P = 0·002). Morphological damage was increased in the 4HMP group. CONCLUSION: This porcine kidney study demonstrated no advantage to the addition of 4 h of HMP after CS.

Complete resolution of oral Kaposi's sarcoma achieved by changing immunosuppression: a case report.

A 38-year-old Afro-Caribbean woman, who was pre-dialysis with polycystic kidney disease, received a live-donor kidney transplant from her 55-year-old mother. This study documents her imunosuppression therapy including resolution of an oral Kaposi's sarcoma and explores the many underlying problems with converting to an mTOR inhibitor.

Randomized clinical trial of laparoscopic versus open donor nephrectomy.

BACKGROUND: This randomized controlled trial was designed to determine the safety and efficacy of laparoscopic donor nephrectomy (LDN) in comparison with short-incision open donor nephrectomy (ODN). METHODS: Eighty-four live kidney donors were randomized in a 2 : 1 ratio to LDN (56 patients) or short-incision ODN without rib resection (28). Primary endpoints were pain relief and duration of inpatient stay. RESULTS: There was no donor death or allograft thrombosis in either group. The first warm ischaemic time median (range) 4 (2-7) versus 2 (1-5) min; P = 0.001) and the duration of operation (160 (110-250) versus 150 (90-200); P = 0.004) were longer for LDN. LDN led to a reduction in parenteral morphine requirement 59 (6-136) versus 90 (35-312) mg; P = 0.001) and hospital stay (4 (2-6) versus 6 (2-9) days; P = 0.001), and earlier return to employment (42 (14-84) versus 66.5 (14-112) days; P = 0.004). Postoperative respiratory function was improved after LDN. There were more postoperative complications per donor in the ODN group (0.6(0.7) versus 0.3(0.5); P = 0.033). At a median follow-up of 74 months, there were no differences in renal function or allograft survival between the groups. CONCLUSION: LDN removes some of the disincentives to live donation without compromising the outcome of the recipient transplant.

Hydrogen sulphide ameliorates ischaemia-reperfusion injury in an experimental model of non-heart-beating donor kidney transplantation.

BACKGROUND: : Therapies to alleviate ischaemia-reperfusion (IR) injury have an important role in kidney transplantation. This study used a porcine model of non-heart-beating (NHB) donor kidneys to investigate the effects of hydrogen sulphide on IR injury. METHODS: : Porcine kidneys were subjected to 25 min of warm ischaemia and 18 h of cold storage. They were reperfused ex vivo with autologous oxygenated blood to assess renal function. A group treated with hydrogen sulphide (0.5 mmol/l) infused 10 min before and after reperfusion (n = 6) was compared with an untreated control group (n = 7). RESULTS: : Hydrogen sulphide significantly improved renal blood flow compared with control values (mean(s.d.) area under the curve (AUC) 614.9(165.5) versus 270.3(86.7) ml per min per 100 g.h; P = 0.001) and renal function (AUC creatinine: 1640(248) versus 2328(154) micromol/l.h; P = 0.001; AUC creatinine clearance: 6.94(5.03) versus 0.96(0.32) ml per min per 100 g.h; P = 0.004). Oxidative damage was also reduced by hydrogen sulphide (urinary 8-isoprostane at 1 h of reperfusion: 478.9(237.1) versus 1605.6(632.7) pg/ml per mmol/l creatinine; P = 0.032). CONCLUSION: : Hydrogen sulphide ameliorated the renal dysfunction associated with ischaemic damage, and has potential as a therapy against IR injury in NHB donor kidney transplantation.

Comparison of preservation solutions in an experimental model of organ cooling in kidney transplantation.

BACKGROUND: Viscous preservation solutions such as University of Wisconsin solution (UW) may be less effective at rapid removal of blood from an organ so that cooling takes longer. This study assessed the temperature changes of kidneys flushed with UW and hyperosmolar citrate (HOC). METHODS: Porcine kidneys were retrieved and flushed with 500 ml UW or HOC at 4 degrees C while monitoring kidney temperature at depths of 5 and 20 mm. Renal function was measured on an isolated organ preservation system. RESULTS: The mean(s.d.) rate of temperature fall was slower with UW (at 20 mm: 0.64(0.11) versus 1.01(0.56) degrees C per min per 100 g; P = 0.016). The perfusion flow rate required to reduce the temperature to less than 10 degrees C at a depth of 20 mm was lower in the UW group (P = 0.002). Kidneys flushed with HOC gained more weight than those flushed with UW (mean(s.d.) 50(8) versus 7(13) per cent; P = 0.002). Flushing with UW was associated with less histological injury but there were no significant differences in renal function parameters between the groups. CONCLUSION: UW cooled kidneys more slowly than HOC, but with no adverse effect on renal function. UW resulted in less oedema and histological injury than HOC.

Case-matched comparison of long-term results of non-heart beating and heart-beating donor renal transplants.

BACKGROUND: Function and survival of non-heart-beating donor (NHBD) renal transplants have been shown to be comparable to those from heart-beating donors (HBDs) up to 10 years after transplantation. However, there are few data on outcome after 10 years, particularly from uncontrolled NHBD donors. METHODS: All NHBD renal transplants (predominantly uncontrolled) performed between April 1992 and January 2002 were retrospectively matched with HBD renal transplants performed over the same period. RESULTS: Some 112 NHBD renal transplants were compared with 164 HBD renal transplants. Delayed graft function was significantly higher in the NHBD group (83.9 versus 22.0 per cent respectively; P < 0.001). Primary non-function rates were similar (5.4 versus 1.8 per cent respectively; P = 0.164). Overall serum creatinine was significantly higher in NHBDs (P < 0.001). Median graft and patient survival was 126 months for NHBD and 159 months for HBD kidneys. Death-censored graft survival at 1, 5, 10 and 15 years was respectively 91.8, 77.5, 61.0 and 44.2 per cent for NHBD, and 91.1, 86.3, 71.7 and 58.5 per cent for HBD kidneys (P = 0.108). CONCLUSION: Despite increased delayed graft function rates and serum creatinine levels, the long-term survival of NHBD renal transplants was similar to those from HBDs. However, there was a trend to poorer function and survival from 10 years after transplant.