Helen L. Waller

Biomarkers of oxidative damage to predict ischaemia-reperfusion injury in an isolated organ perfusion model of the transplanted kidney.

Ischaemia-reperfusion (IR) injury is known to be a risk factor influencing both short and long-term graft function following transplantation. The pathophysiology of IR injury is suggested to involve elevated reactive oxygen species production resulting in oxidative damaged cellular macromolecules. The objective of this study was to evaluate oxidative damage following IR using an isolated organ perfusion model of the transplanted kidney, in order to determine a simple, preferably non-invasive biomarker for IR injury. Porcine kidneys were retrieved with 10 or 40 min warm ischaemic (WI) time and haemoperfused for 6 h on an isolated organ perfusion machine. ELISA was used to detect carbonyls, 8-isporostane and 8-hydroxy-2′-deoxyguanosine, representing protein, lipid and DNA damage respectively in pre and post reperfusion samples of plasma, urine and biopsy material. Plasma carbonyl and 8-isporostane and were significantly increased in the 40 min group compared to pre-perfusion (0.96 ± 0.10 vs. 0.62 ± 0.06, P < 0.001 and 1.57(1.28–4.9) vs. 0.36(0.09–0.59), P < 0.05). The levels also correlated with creatinine clearance used to determine renal function (r = − 0.6150, P < 0.01 and r = − 0.7727, P < 0.01). The results of this study suggest both plasma carbonyl and 8-isporostane to be reliable biomarkers to predict the level IR injury.

The Effect of Warm Ischemic Time on Renal Function and Injury in the Isolated Hemoperfused Kidney

Background. The precise effect of warm ischemia on renal allograft function remains unclear and leads to variable warm ischemic time (WIT) limits advocated by transplant programs. This study aims to investigate the relationship between WIT, renal ischemia reperfusion injury, and graft function using a hemoperfused kidney model. Methods. Porcine kidneys were perfused with normothermic blood on an isolated organ perfusion system. Kidneys were divided into four groups (n=6) and subjected to 7, 15, 25, and 40 min WIT. Physiological parameters were measured throughout the 6 hr perfusion period. Serum, tissue, and urine samples were analyzed for histological and biochemical markers of ischemia reperfusion injury. Results. Creatinine clearance, urine output, renal hemodynamics, and oxygen consumption deteriorated proportionally with increasing WIT. A significant increase in plasma carbonyl levels during perfusion was seen after 25 and 40 min WIT only. Plasma 8-isoprostane levels were higher after 40 min WIT (2.5±1.6) vs. 7, 15, and 25 min WIT (0.65±0.43, 0.25±0.12, and 0.62±0.21, respectively; P<0.05). A negative correlation was shown between urine output and plasma carbonyls (r=−0.415, P<0.05) and between 8-isoprostane levels and creatinine clearance (r=−0.649, P<0.005). Caspase-3 activity was significantly higher after 7 min WIT compared with the other groups, correlating positively with creatinine clearance, urine output, and renal blood flow. Conclusion. The isolated organ perfusion system successfully delineated a clear WIT-dependent variation in renal function which correlated accurately with oxidative injury markers. This model may represent a clinically applicable tool for assessing graft viability.

Differential expression of cytoprotective and apoptotic genes in an ischaemia‐reperfusion isolated organ perfusion model of the transplanted kidney

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.

Static normothermic preservation of renal allografts using a novel nonphosphate buffered preservation solution

The aim of this study was to assess the viability and function of renal allografts under normothermic conditions using a novel nonphosphate buffered preservation solution AQIX®RS‐I. Porcine kidneys were flushed at 30 °C with AQIX®RS‐I at 100 mmHg pressure after 5–10 min warm ischaemic time and stored statically at either 4 °C or 30 °C for 2 h (n = 6 per group). Assessment of renal function by physiological and biochemical parameters was performed by perfusing the organs with autologous blood at 37 °C, with an initial circulating serum creatinine concentration of 1000 μmol/l on an isolated organ perfusion system for 6 h. Although the hypothermic group demonstrated overall superior renal function, the normothermic stored kidneys displayed a statistically comparable acid‐base balance (7.37 ± 0.15 vs. 7.3 ± 0.09, P = 0.24). Furthermore, renal function was still evident after 6 h perfusion with increasing oxygen consumption, renal blood flow and reduced renal vascular resistance. The effectiveness and versatility of AQIX®RS‐I as a preservation solution under both normothermic and hypothermic conditions has been demonstrated. Renal viability was maintained after 2 h static normothermic storage. This study provides a foundation for further analysis utilizing normothermic preservation.

Validation of a novel ELISA for measurement of MDA-LDL in human plasma.

The involvement of oxidatively modified low density lipoprotein (LDL) in the development of CHD is widely described. We have produced two antibodies, recognizing the lipid oxidation product malondialdehyde (MDA) on whole LDL or ApoB-100. The antibodies were utilized in the development of an ELISA for quantitation of MDA-LDL in human plasma. Intra- and inter-assay coefficients of variation (% CV) were measured as 4.8 and 7.7%, respectively, and sensitivity of the assay as 0.04 micro g/ml MDA-LDL. Recovery of standard MDA-LDL from native LDL was 102%, indicating the ELISA to be specific with no interference from other biomolecules. Further validation of the ELISA was carried out against two established methods for measurement of lipid peroxidation products, MDA by HPLC and F(2)-isoprostanes by GC-MS. Results indicated that MDA-LDL is formed at a later stage of oxidation than either MDA or F(2)-isoprostanes. In vivo analysis demonstrated that the ELISA was able to determine steady-state concentrations of plasma MDA-LDL (an end marker of lipid peroxidation). A reference range of 34.3 +/- 8.8 micro g/ml MDA-LDL was established for healthy individuals. Further, the ELISA was used to show significantly increased plasma MDA-LDL levels in subjects with confirmed ischemic heart disease, and could therefore possibly be of benefit as a diagnostic tool for assessing CHD risk.

Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo.

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.