Monobody adapter for functional antibody display on nanoparticles for adaptable targeted delivery applications.

Vascular endothelial cells (ECs) play a central role in the pathophysiology of many diseases. The use of targeted nanoparticles (NPs) to deliver therapeutics to ECs could dramatically improve efficacy by providing elevated and sustained intracellular drug levels. However, achieving sufficient levels of NP targeting in human settings remains elusive. Here, we overcome this barrier by engineering a monobody adapter that presents antibodies on the NP surface in a manner that fully preserves their antigen-binding function. This system improves targeting efficacy in cultured ECs under flow by >1000-fold over conventional antibody immobilization using amine coupling and enables robust delivery of NPs to the ECs of human kidneys undergoing ex vivo perfusion, a clinical setting used for organ transplant. Our monobody adapter also enables a simple plug-and-play capacity that facilitates the evaluation of a diverse array of targeted NPs. This technology has the potential to simplify and possibly accelerate both the development and clinical translation of EC-targeted nanomedicines.

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.

Successful Transplantation of Human Kidneys Deemed Untransplantable but Resuscitated by Ex Vivo Normothermic Machine Perfusion.

We report the successful transplantation of a pair of human kidneys that were declined for transplantation due to inadequate in situ perfusion but subsequently transplanted after perfusion and assessment using ex vivo normothermic perfusion (EVNP). The kidneys were from a 35-year-old man, a donation after circulatory death donor. Both kidneys were declined by all UK transplant centers. On arrival, the kidneys had significant areas of incomplete clearance of blood from the microcirculation that did not clear after a further attempt to flush them. Kidneys underwent 60 min of EVNP with an oxygenated packed red blood cell-based solution warmed to 35.2°C. During EVNP, the patchy areas cleared in both kidneys. The mean renal blood flow and total urine output were 68.0 mL/min/100 g and 560 mL in the left kidney and 59.9 mL/min/100 g, 430 mL in the right, respectively. Based on the EVNP perfusion parameters, both kidneys were deemed suitable for transplantation. They were transplanted without any complications, and both recipients had initial graft function. The serum creatinine levels at 3 months were 1.2 mg/dl in the recipient of the left kidney and 1.62 mg/dl in the recipient of the right kidney. EVNP technology can be used to assess and rescue kidneys previously deemed unsuitable for transplantation.

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.

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.

Use of Ex Vivo Normothermic Perfusion for Quality Assessment of Discarded Human Donor Pancreases.

A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.

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.

Renal transplantation after ex vivo normothermic perfusion: the first clinical study.

Ex vivo normothermic perfusion (EVNP) is a novel method of preservation that restores circulation and allows an organ to regain function prior to transplantation. The aim of this study was to assess the effects of EVNP in kidneys from marginal donors. Eighteen kidneys from extended criteria donors (ECD) underwent a period of EVNP immediately before transplantation. Kidneys were perfused with a plasma free red-cell based solution at a mean temperature of 34.6°C. The outcome of these kidneys was compared to a control group of 47 ECD kidneys that underwent static cold storage (CS). The mean donor age was 61 ± 1 years in the EVNP and 62 ± 6 years in the CS group (p = 0.520). EVNP kidneys were perfused for an average of 63 ± 16 min and all were transplanted successfully. The delayed graft function rate (DGF), defined as the requirement for dialysis within the first 7 days was 1/18 patients (5.6%) in the EVNP group versus 17/47 (36.2%) in the CS group (p = 0.014). There was no difference in graft or patient survival at 12 months (p = 0.510, 1.000). This first series of EVNP in renal transplantation demonstrates that this technique is both feasible and safe. Our preliminary data suggests that EVNP offers promise as a new technique of kidney preservation.

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.

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.

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-control comparison of laparoscopic and open washout for peritoneal dialysis-associated peritonitis.

BACKGROUND: Peritonitis is the major complication of peritoneal dialysis (PD). Some 25-40 per cent of patients require surgical intervention, traditionally laparotomy, washout and removal of the PD catheter. The aim was to compare this open procedure with laparoscopic washout and catheter removal. METHODS: In a case-control comparison, 20 patients who had laparoscopic washout for PD-associated peritonitis were matched by age and causative organism with 20 patients who had open washout. RESULTS: The groups were well matched for age, sex, causative organism, preoperative C-reactive protein level, white cell count and presence of bowel wall sclerosis. Laparoscopic surgery was quicker than open operation (mean(s.d.) 49(13) versus 73(30) min; P = 0.006) and postoperative morphine requirements were significantly lower (median 0 versus 27 mg; P < 0.001). Bowel function recovered more quickly in the laparoscopic group, as measured by time to first passage of flatus (mean(s.d.) 2(1) versus 5(4) days; P = 0.004) and resumption of free fluids (median 2 versus 4 days; P = 0.044). Reoperation rates and 30-day mortality were identical in the two groups. CONCLUSION: This study suggests that laparoscopic washout for PD peritonitis is as effective as open washout, but is quicker and less painful with earlier return of bowel function.

Application of nitric oxide and carbon monoxide in a model of renal preservation.

BACKGROUND: Nitric oxide and carbon monoxide exert vasodilatory effects that minimize ischaemia-reperfusion injury. An isolated porcine kidney model was used to assess the effects of administering the nitric oxide donor sodium nitroprusside (SNP) and carbon monoxide-releasing molecule (CORM) 3 during a period of warm preservation followed by reperfusion. METHODS: Kidneys were perfused under warm preservation conditions after 10 min of warm ischaemia and 16 h of cold storage in four groups: SNP, control, CORM-3 and inactive CORM-3 (inactive control). Renal function and viability were assessed. RESULTS: SNP and CORM-3 increased renal blood flow (RBF) during warm preservation (P = 0.014). After reperfusion, RBF was significantly improved in the CORM-3 group compared with the control group (P = 0.019). The reduction in creatinine clearance was significantly less in the CORM-3 group than in the inactive CORM-3 group (P = 0.021), and serum creatinine levels were significantly lower (P = 0.029). There was a negative correlation between RBF during warm preservation and functional parameters during reperfusion (creatinine concentration: r(s) = - 0.722, P < 0.001; sodium excretion: r(s) = - 0.912, P < 0.001). CONCLUSION: The beneficial vasodilatory effects of CORM-3 during warm preservation improved renal function during reperfusion; SNP exerted similar, although less pronounced, effects.