Establishing an objective quality assessment of an organ prior to transplantation can help prevent unnecessary discard of the organ and reduce the probability of functional failure. In this regard, normothermic machine perfusion (NMP) offers new possibilities for organ evaluation. However, to date, few studies have addressed the identification of markers and analytical tools to determine graft quality. In this study, function and injury markers were measured in blood and urine during NMP of 26 porcine kidneys and correlated with ex vivo inulin clearance behavior. Significant differentiation of kidneys according to their function could be achieved by oxygen consumption, oxygen delivery, renal blood flow, arterial pressure, intrarenal resistance, kidney temperature, relative urea concentration, and urine production. In addition, classifications were accomplished with supervised learning methods and histological analysis to predict renal function ex vivo. Classificators (support vector machines, k-nearest-neighbor, logistic regression and naive bayes) based on relevant markers in urine and blood achieved 75% and 83% accuracy in the validation and test set, respectively. A correlation between histological damage and function could not be detected. The measurement of blood and urine markers provides information of preoperative renal quality, which can used in future to establish an objective quality assessment.

The increased utilization of high-risk renal grafts for transplantation requires optimization of pretransplant organ assessment strategies. Current decision-making methods to accept an organ for transplantation lack overall predictive power and always contain an element of subjectivity. Normothermic machine perfusion (NMP) creates near-physiological conditions, which might facilitate a more objective assessment of organ quality before transplantation. NMP is rapidly gaining popularity, with various transplant centers developing their own NMP protocols and renal viability criteria. However, to date, no validated sets of on-pump viability markers exist nor are there unified NMP protocols. This review provides a critical overview of the fundamentals of current renal NMP protocols and proposes a framework to approach further development of ex vivo organ evaluation. We also comment on the potential logistical implications of routine clinical use of NMP, which is a more complex procedure compared with static cold storage or even hypothermic machine perfusion.

The increasing reliance on marginal donors has driven research to investigate ways to repair and improve the quality of kidneys for transplantation. Normothermic perfusion technologies provide an opportunity for improved preservation, organ assessment and resuscitation/repair of damaged kidneys. This review describes the evolution of normothermic perfusion in kidney transplantation in the United Kingdom.

One hour of normothermic perfusion can be used to restore function and improve early graft function of extended criteria donor kidneys. A large multicentre trial is investigating the impact of normothermic perfusion on delayed graft function in a series of donation after circulatory death kidneys. Normothermic perfusion is also a platform for the delivery of therapies to the kidney to upregulate and modulate repair mechanisms or prevent injurious processes, such as activation of caspase-3 with the delivery of caspase-3 targeted small interfering RNAs. Normothermic perfusion can also be used to assess the quality and anatomical structure of a kidney to judge suitability for transplantation.

Normothermic perfusion technology is a useful adjunct in kidney transplantation. It can improve early graft function by upregulating protective mechanisms. It also has the advantage of providing a functional assessment of the kidney and as a platform for the delivery of therapies or graft manipulation to target ischaemia reperfusion injury or the immune response. This technology can be used to expand the organ donor pool and prevent the unnecessary discard of kidneys.

A prolonged cold ischaemia time (CIT) is suspected to be associated with an increased ischaemia and reperfusion injury (IRI) resulting in an increased damage to the graft. In total, 91 patients were evaluated for a delayed graft function within 7 days after kidney transplantation (48 deceased, 43 living donors). Blood and urine samples were collected before, immediately after the operation, and 1, 3, 5, 7 and 10 days later. Plasma and/or urine levels of total keratin 18 (total K18), caspase-cleaved keratin 18 (cc K18), the soluble receptor for advanced glycation end products (sRAGE), tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein-7 (IGFBP7) were measured. As a result of prolonged CIT and increased IRI, deceased donor transplantations were shown to suffer from a more distinct cell cycle arrest and necrotic cell death. Plasmatic total K18 and urinary TIMP-2 and IGFBP7 were therefore demonstrated to be of value for the detection of a delayed graft function (DGF), as they improved the diagnostic performance of a routinely used clinical scoring system. Plasmatic total K18 and urinary TIMP-2 and IGFBP7 measurements are potentially suitable for early identification of patients at high risk for a DGF following kidney transplantation from deceased or living donors.

Nonmodifiable factors including pre-operative renal function and amount of healthy renal tissue preserved are the most important predictive factors that determine renal function after partial nephrectomy. Ischaemia time is an important modifiable risk factor and cold ischaemia time should be used if longer ischaemia time is anticipated. New techniques may have a role in maximising postoperative kidney function, but more robust studies are required to understand their potential benefits and risks.

The optimal hypothermic machine perfusion (HMP) solution has not yet been developed. An adenosine and lidocaine (AL) solution has been shown to be protective in cardiac preservation. The aim of the present study was to examine a modified AL solution with low Ca2+, 16 mM Mg2+, and 4% albumin on kidney preservation compared with University Wisconsin solution (UW).

Twenty donation of organs after cardiac death porcine kidneys underwent HMP for 10 h (AL, n = 10; UW, n = 10) and then 2 h of normothermic reperfusion. Perfusion dynamics, functional parameters, histology, and real-time microdialysis were used to assess kidney responses and viability.

During HMP, modified AL-perfused kidneys maintained higher flow rates (21.5 versus 17.9 mL/min/100 g, P = 0.01), with perfusion flow index during the first 3 h 25% greater than with UW (AL = 0.50 ± 0.2, UW = 0.40 ± 0.17 mL/min/100 g/mmHg; P = 0.03), followed by an increase in UW kidneys which was not significantly different to AL over the remaining 7 h (0.54 versus 0.55 mL/min/100 g/mmHg, respectively). During warm reperfusion, there were no significant differences between the two HMP groups in creatinine clearance, oxygen, and glucose consumption between groups. Modified AL kidneys had significantly lower perfusate lactates (3.1 versus 4.1 mmol/L, P = 0.04) during reperfusion and lower cortical lactate levels (AL = 0.66 ± 0.31, UW = 0.89 ± 0.53 mM, P = 0.33). Histology showed similar degrees of reperfusion injury.

We conclude that HMP with modified AL solution showed improved perfusion compared with UW and lower perfusate lactate levels during warm reperfusion. Further modification of the AL composition is warranted and may lead to more rapid kidney stabilization and improved graft viability assessment, potentially expanding donor pools.

Recent investigations have indicated that reactive metabolites and AGE-RAGE-mediated inflammation might play an important role in the pathogenesis of ischemia-reperfusion injury in liver transplantation. In this observational clinical study, 150 patients were enrolled following liver transplantation from deceased donors. The occurrence of short-term complications within 10 days of transplantation was documented. Blood samples were collected prior to transplantation, immediately after transplantation, and at consecutive time points, for a total of seven days after transplantation. Plasma levels of methylglyoxal were determined using HPLC, whereas plasma levels of L-arginine, asymmetric dimethylarginine, advanced glycation endproducts-carboxylmethyllysine, soluble receptor for advanced glycation endproducts, and total antioxidant capacity were measured by ELISA. Patients following liver transplantation were shown to suffer from increased RAGE-associated inflammation with an AGE load mainly dependent upon reactive carbonyl species-derived AGEs. In contrast, carboxylmethyllysine-derived AGEs were of a minor importance. As assessed by the ratio of L-arginine/asymmetric dimethylarginine, the bioavailability of nitric oxide was shown to be reduced in hepatic IRI, especially in those patients suffering from perfusion disorders following liver transplantation. For the early identification of patients at high risk of perfusion disorders, the implementation of asymmetric dimethylarginine measurements in routine diagnostics following liver transplantation from deceased donors should be taken into consideration.

Prolonged warm ischemia (WI) occurring in marginal kidney donors together with reperfusion injury determines allograft survival, in which apoptosis and inflammation play crucial roles. There is no single valid biomarker, so far, to assess the degree of kidney donor injury. To define new biomarkers for detecting initial donor ischemic injury, caspase-3, caspase-7, apoptosis, inflammation, HSP70 and renal histological changes were examined in porcine kidneys subjected to 7- 15- 25- or 40-min WI, two-hour cold storage and six-hour hemoreperfusion. Caspase-3 activity was gradually increased by prolonged reperfusion, with a decrease trend against WI time. This result was verified by raised 17 kDa active caspase-3 in postreperfusion kidneys, with elevated 12 kDa active caspase-3 and lowered precursor at seven-minute WI. Active caspase-7 was also doubled by reperfusion with decreased precursor at seven-minute WI, but declined against prolonged WI. Apoptotic cells in tubular and interstitial areas were greatly increased by reperfusion at seven-minute WI, but decreased against prolonged WI. In addition, myeloperoxidase (MPO)+ cells were dramatically increased by reperfusion and presented as a bell-shape against WI time, while HSP70 was significantly increased at 7-min WI, but decreased at 40-min WI after reperfusion. In postreperfusion kidneys, tubular dilation and cell shedding were observed at 7- and 15-min WI, while tubular vacuolation and cell debris were found in tubular lumens at longer WI times. At 40-min WI, early nuclear pyknosis, tubular epithelia detachment and peri-tubular capillary dilation were detected. Furthermore, caspase-3, caspase-7, apoptosis, but not MPO+ cells or HSP70, were correlated with renal function. In conclusion, caspase-3, caspase-7 and apoptosis appear to be better biomarkers than MPO+ cells or HSP70 for assessing warm ischemic injury in donor kidneys. Hemoreperfusion activates caspase-3 and caspase-7, promotes apoptosis of damaged cells in kidneys only with limited WI, which might be beneficial to renal structural re-modeling and functional recovery.

Remote preconditioning (rPeC) is a phenomenon by which short-time intermittent ischaemia-reperfusion (I/R) of a remote organ during ischaemia protects other organs from I/R injury (IRI). The aim of the present study was to investigate the protective effect of rPeC on renal IRI in rats. Rats were subjected to right nephrectomy and randomized as into a sham group (no additional intervention), an I/R group (subjected to 45 min left renal pedicle occlusion) and an rPeC group (subjected to four cycles of 5 min I/R of the left femoral artery administered at the beginning of renal ischaemia). After 24 h, blood, urine and tissue samples were collected. Compared with the sham group, I/R resulted in renal dysfunction, as evidenced by significantly lower creatinine clearance (CCr; 0.52 ± 0.06 vs 0.11 ± 0.02 mL/min, respectively) and higher fractional excretion of sodium (FE(Na) ; 0.80 ± 0.07% vs 2.46 ± 0.20%, respectively). This was accompanied by decreased superoxide dismutase (SOD; 6.9 ± 1.7 vs 26.7 ± 2.7 U/g tissue) and catalase (CAT; 20.2 ± 8.8 vs 32.2 ± 8.7 K/g tissue) activity in the I/R group, as well as decreased levels of reduced glutathione (GSH; 21.7 ± 8.1 vs 81.2 ± 20.2 μmol/g tissue) and increased malondialdehyde levels (MDA; 1.2 to 0.1 vs 0.5 ± 0.2 μmol/100 mg), cyclo-oxygenase (COX)-2 expression and histological damage. In the rPeC group, renal histology and function were significantly improved (CCr 0.32 ± 0.02 mL/min; FE(Na) 1.33 ± 0.12%) compared with the I/R group. Furthermore, compared with the I/R group, the rPeC group exhibited increases in SOD and CAT activity (22.8 ± 3.8 U/g tissue and 21.7 ± 8.6 K/g tissue, respectively), increased GSH levels (74.0 ± 4.9) and decreased MDA levels (1.1 ± 0.3 μmol/100 mg) and COX-2 expression. In conclusion, rPeC appears to exert protective effects against renal IRI. This protection may be a consequence of reductions in lipid peroxidation, intensification of anti-oxidant systems and downregulation of COX-2 expression. A simple approach, rPeC may be a promising strategy for protection against IRI in clinical practice.

Oxidatively damaged DNA is implicated in various diseases, including neurodegenerative disorders, cancer, diabetes, cardiovascular and inflammatory diseases as well as aging. Several methods have been developed to detect oxidatively damaged DNA. They include chromatographic techniques, the Comet assay, (32)P-postlabelling and immunochemical methods that use antibodies to detect oxidized lesions. In this review, we discuss the detection of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), the most abundant oxidized nucleoside. This lesion is frequently used as a marker of exposure to oxidants, including environmental pollutants, as well as a potential marker of disease progression. We concentrate on studies published between the years 2000 and 2011 that used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry to detect 8-oxodG in humans, laboratory animals and in cell lines. Oxidative damage observed in these organisms resulted from disease, exposure to environmental pollutants or from in vitro treatment with various chemical and physical factors.

Kidneys from marginal or donation after cardiac death (DCD) donors are particularly susceptible to injury during hypothermic preservation and may benefit from alternative methods of preservation. Normothermic preservation can be adapted to improve the quality of kidneys for transplantation by a variety of techniques.

Extracorporeal membrane support to maintain circulation before cooling and organ retrieval has been used to improve the condition of DCD donor kidneys, with lower rates of delayed graft function (DGF) compared with standard retrieval conditions. Experimentally, normothermic perfusion has been used in conjunction with hypothermic techniques as a resuscitation technique to improve graft outcome. An ex-vivo porcine kidney model showed that energy levels could be replenished to improve tissue perfusion during reperfusion. This technique was translated into a porcine transplant model demonstrating that it was a feasible and safe method of preservation.

Normothermic preservation techniques have the potential to be adapted into an improved method of retaining tissue viability compared with hypothermic techniques. Furthermore, they may be used as a device to enhance and assess the condition of the kidney which would be particularly beneficial for kidneys from DCD donors.

The Two layer method (TLM) has been extremely successful in the preservation of the pancreas. However, this has not been thoroughly investigated in other organs or in clinically relevant large animal models. The aim of this study was to assess the effects of TLM in a large animal model of kidney preservation.

Porcine kidneys were retrieved after 10 minutes of warm ischaemic injury and flushed with 300 ml UW solution at 4°C. Kidneys were then either placed in University of Wisconsin solution (UW) or TLM using pre-oxygenated perfluorodecalin and UW. Kidneys were stored for 18 hours at 4°C then reperfused with oxygenated autologous blood to assess renal function.

Renal blood flow (RBF) was significantly lower and intra-renal resistance (IRR) higher in TLM compared to UW group [Area under the curve (AUC) RBF, UW; 427±168 vs TLM; 247±55 ml/min/100g.h; P=0.041, AUC IRR, UW; 7.7±2.2 vs TLM; 10.5±1.9 ml/min/mmHg; P=0.041]. Levels of creatinine clearance (CrCl) were significantly lower in TLM group [AUC CrCl, UW; 1.8±1.0 vs TLM; 0.6±0.4 ml/min/100 g.h; P=0.034]. Levels of lipid peroxidation were significantly lower in TLM group [8-isoprostane/Cr ratio 3h; UW 3338±896 vs TLM 2072±886 pg/ml/mmol/L; P=0.04]. Levels of total nitric oxide were significantly higher in TLM group (P=0.009).

TLM did not improve the preservation condition of porcine kidneys. Furthermore, there appeared to be increased inflammation, endothelial injury and reduced renal function compared to preservation with UW. Further experimental work is needed to determine the role of PFC in kidney preservation.

Cold ischaemic (CI) injury is associated with reduced renal graft function and survival. However, there is little evidence on the benefits of reducing CI injury within a 24 h period in donation after cardiac death (DCD) kidney transplantation.

Porcine kidneys subjected to 10-min warm ischaemia were retrieved and flushed with hyperosmolar citrate (HOC) preservation solution at 4 °C. They were stored on ice for periods of 2, 6, 18 or 24 h (n = 6). Renal function and injury were assessed during 3 h of ex-vivo reperfusion with oxygenated autologous blood.

Area under the curve (AUC) serum creatinine (Cr) levels were significantly higher in the 18- and 24-h groups and creatinine clearance (CrCl) lower compared to the 2-h group (P = 0·001, 0·003). Urinary biomarkers of ischaemic damage (Endothelin-1, Total nitric oxide) and renal and tubular cell function significantly correlated with the duration of CI time (r = 0·726, 0·642; P ≤ 0·001).

This study demonstrated the progressive effects of CI injury in DCD porcine kidneys over a 24 h hypothermic storage period. This highlights the need to minimise the cold storage period to improve graft function in DCD kidney transplantation.

There is increasing support for the use of hypothermic machine perfusion (HMP) in an attempt to reduce preservation injury. However, experimental evidence is needed to further examine the effects of HMP on renal ischemia reperfusion injury.

Porcine kidneys were subjected to 10 min of warm ischemia followed by 18 hr of static cold storage with hyperosomolar citrate (HOC), histidine-tryptophan-ketoglutarate (HTK), or University of Wisconsin (UW) solutions or 18 hr HMP with Kidney Perfusion Solution using the Lifeport perfusion system. Renal function, oxidative damage, and morphology were assessed during 3 hr of reperfusion with autologous blood using an isolated organ perfusion system.

During reperfusion, intrarenal resistance was significantly lower in the HMP group compared with HOC and UW (area under the curve; HMP 3.8+/-1.7, HOC 9.1+/-4.3, UW 7.7+/-2.2, HTK 5.6+/-1.9 mm Hg/min; P=0.006), and creatinine clearance was significantly higher compared with the UW group (area under the curve creatinine clearance; HMP 9.8+/-7.3, HOC 2.2+/-1.7, UW 1.8+/-1.0, HTK 2.1+/-1.8 mL/min/100 g; P=0.004). Tubular function was significantly improved in the HMP group (P<0.05); however, levels of lipid peroxidation were significantly higher (P=0.005).

HMP demonstrated a reduced level of preservation injury compared with the static techniques resulting in improved renal and tubular function and less tubular cell inflammation during reperfusion.

: 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.

: 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).

: 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).

: Hydrogen sulphide ameliorated the renal dysfunction associated with ischaemic damage, and has potential as a therapy against IR injury in NHB donor kidney transplantation.

Especially for preservation of marginal donor organs, machine perfusion (MP) and retrograde oxygen persufflation (ROP) are alternatives to cold storage (CS). Using a porcine kidney autotransplantation model we compared metabolic and morphologic effects of CS, ROP, and MP on kidneys exposed to warm ischemia.

Kidneys of 21 pigs were exposed in situ to warm ischemia for 60min. The kidneys were randomly allocated to three experimental groups, each receiving a 4-h treatment of either cold storage, machine perfusion, or retrograde oxygen persufflation. Tissue samples were examined for malondialdehyde and histological changes. Daily blood samples were examined for creatinine levels.

Seven days after transplantation, the plasma creatinine levels in the CS and MP groups were still significantly elevated above the baseline values. In the ROP group, all animals exhibited nearly normal creatinine levels. Malondialdehyde, an indicator of lipidperoxidation, was dramatically increased in the machine perfused kidneys on day 7, whereas the malondialdehyde levels in the other two groups were near normal values. The MP kidneys exhibited the most striking histological changes.

Though MP has been well introduced in organ transplantation, in our opinion, it must still be optimized and standardized. It is necessary to clarify questions such as whether there is a need for oxygenation during perfusion, the length of perfusion, the impact of pressure, and the effects of additional scavengers. The results of the present study suggest the reconsideration of the ROP-technique for the preservation of predamaged donor grafts especially of NHBD and further studies, comparing MP and ROP upon long term preservation are strongly encouraged.

The optimal kidney preservation system and methods to ameliorate reperfusion injury are major factors in accomplishing successful graft function following transplantation. Ischaemia and reperfusion lead to cellular stress and the adaptive response may include the activation of genes involved in cellular protection and/or cell death by apoptosis. We investigated the expression of cytoprotective heme oxygenase-1 (HO-1), anti-apoptotic Bcl-2 and pro-apoptotic Bax after 6 h isolated organ perfusion in porcine kidneys that had been given 10 and 40 min warm ischaemic time. The level of HO-1 was shown to be significantly higher in the 10-min warm ischaemic group compared with 40-min group (0.90 +/- 0.03 vs. 0.83 +/- 0.03; P = 0.002). The levels of HO-1 showed a significant positive correlated with parameters of renal function, creatinine clearance, and renal blood flow and urine output (AUC; r = 0.8042, P = 0.03; r = 0.6028, P = 0.04; r = 0.6055, P = 0.04), demonstrating a possible protective role of this gene in this model of renal transplantation.

A methodology has been developed and validated for quantifying 8-hydroxydeoxyguanosine (8-OHdG) in both commercial DNA and DNA isolated from livers of male Sprague-Dawley rats by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry. The analytical method conditions, including conditions for stabilizing 8-OHdG during complex nuclease P1 enzymatic digestion, were also evaluated. The limit of detection for 8-OHdG was 1.0 ng/mL (17.6 fmol on-column), and the linearity of the calibration curve was greater than 0.998 from 1.0 to 500 ng/mL. The intraday assay precision relative standard deviation (RSD) value for quality control (QC) samples was < or =5.59% with accuracies ranging from 91.84 to 117.61%. The interday assay precision (RSD) value was < or =1.76% with accuracies ranging from 91.84 to 116.67%. This method, combined with the LC/UV analysis of deoxyguanosine (dG), was used for determination of the levels of 8-OHdG/10(6) dG in DNA nuclease P1 enzymatic hydrolysates from both commercial DNA and rat liver DNA.