Maurits H. Booster

OVERCOMING SEVERE RENAL ISCHEMIA: THE ROLE OF EX VIVO WARM PERFUSION

BACKGROUND The ability to effectively utilize kidneys damaged by severe (2 hr) warm ischemia (WI) could provide increased numbers of kidneys for transplantation. The present study was designed to examine the effect of restoring renal metabolism after severe WI insult during ex vivo warm perfusion using an acellular technology. After warm perfusion for 18 hr, kidneys were reimplanted and evaluated for graft function. METHODS Using a canine autotransplant model, kidneys were exposed to 120 min of WI. They were then either reimplanted immediately, hypothermically machine perfused (4 degrees C) for 18 hr with Belzers solution, or transitioned to 18 hr of warm perfusion (32 degrees C) with an acellular perfusate before implantation. RESULTS Warm perfused kidneys with 120 min of WI provided life-sustaining function after transplantation, whereas the control kidneys immediately reimplanted or with hypothermic machine perfusion did not. The mean peak serum creatinine in the warm perfused kidneys was 3.7 mg/dl, with the mean peak occurring on day 2 and normalizing on day 9 posttransplant. CONCLUSIONS These results indicate that 18 hr of ex vivo warm perfusion of kidneys is feasible. Furthermore, recovery of renal function during warm perfusion is demonstrated, resulting in immediate function after transplantation. The use of ex vivo warm perfusion to recover function in severe ischemically damaged kidneys could provide the basis for increasing the number of transplantable kidneys.

Hypothermia – a Limiting Factor in Using Warm Ischemically Damaged Kidneys

A study was performed to determine the limiting factors to expanding the donor pool with warm ischemically (WI) damaged kidneys. Canine kidneys were damaged by 30 min of WI, and then either cold stored (CS) in ViaSpan (4 °C) for 18 h, or warm perfused with exsanguineous metabolic support (EMS) technology (32 °C) for 18 h, or subjected to combinations of both techniques. The kidneys were autotransplanted with contralateral nephrectomy. In kidneys with WI and CS alone, the mean peak serum creatinine value was 6.3 mg/dL and took 14 days to normalize. In contrast, kidneys where renal metabolism was resuscitated ex vivo during 18 h of warm perfusion demonstrated mild elevations in the serum chemistries (2.6 mg/dL). The damage in kidneys CS for 18 h was ameliorated with 3 h of subsequent warm perfusion and eliminated by 18 h of warm perfusion. In contrast, reversing the order with CS following WI and 18 h of warm perfusion resulted in a time‐dependent increase in damage. These results identify hypothermia as a major limiting factor to expanding indications for kidney donation. While hypothermia represents the foundation of preservation in the heart‐beating donor, its use in WI damaged organs appears to represent a limiting factor.

University Of Wisconsin Solution Is Superior To Histidine Tryptophan Ketoglutarate For Preservation Of Ischemically Damaged Kidneys

The current shortage of transplantable organs has renewed interest in kidneys obtained from non-heart-beating donors. Kidneys from these donors have suffered warm ischemia (WI). The effectiveness of two preservation solutions, i.e., the University of Wisconsin (UW) and the histidine tryptophan ketoglutarate (HTK) solutions, for preservation of kidneys that have been subjected to WI was tested in dogs. The left kidney was autotransplanted after 30 min of WI, and subsequent 24-hr cold storage (CS) in either UW (n = 6) or HTK (n = 6), with immediate contralateral nephrectomy. Surgical biopsies from the cortex were taken before WI, after 30 min of WI, after 24 hr of CS, and after 1 hr of reperfusion for electron microscopy and for analysis of energy metabolites. At 2 weeks after transplantation in the UW group, 4 out of 6 and, in the HTK group, 1 out of 6 dogs survived. As from day 2, serum creatinine was lower in the UW group as compared with the HTK group (P < 0.05). After 24 hr of CS, in the HTK group the luminal membranes of proximal tubular cells were partly denuded of microvilli. Moreover, the tubular lumen was filled with blebs and debris. In the UW group, the brush borders remained intact, although microvilli were swollen. Energy metabolites were analyzed with HPLC. Thirty minutes of WI resulted in a +/- 45% reduction of total adenine nucleotide (TAN) content. During CS, TAN levels further decreased in both groups; however, after 24 hr of CS, the levels of adenosine, inosine, hypoxanthine, and xanthine were significantly higher in the UW group as compared with the HTK group (P < 0.05, P < 0.01, P < 0.01, P < 0.01). At 1 hr of reperfusion, TAN levels were higher in the UW group as compared with the HTK group (4.66 +/- 0.16 vs. 4.02 +/- 0.28, P < 0.05). Our results show that UW is a superior solution compared with HTK in the preservation of ischemically damaged kidneys, demonstrating better survival, better recovery of kidney function, better protection against ischemia-induced ultrastructural damage, and better preservation of energy metabolism indicated by (a faster) regeneration of TAN levels after reperfusion.

Exsanguinous metabolic support perfusion--a new strategy to improve graft function after kidney transplantation.

Background. The compounding damage of warm ischemia (WI) followed by cold preservation is a major barrier in renal transplantation. Although the relative effect of WI is not yet well understood, therapeutic strategies have mostly focused on minimizing the pathology seen upon reperfusion from the cold.Our study was designed to examine the effect of restoration of renal metabolism by warm perfusion on graft survival and to investigate the compounding damage of WI. Methods. Using a known critical canine autotransplantation model (1), kidneys were exposed to 30 min WI followed by 24 hr cold storage in Viaspan. They were then either reimplanted directly or first transitioned to 3 hr of warm perfusion with an acellular perfusate before reimplantation. Contralateral kidneys were subjected to 0, 30, or 60 min WI; 24 hr cold storage, and 3 hr warm perfusion. Results. Transplanted kidneys that were warm perfused before reimplantation had both lower 24 hr posttransplant serum creatinine (median of 3.2 vs. 4.1 mg/dl) and lower peak serum creatinine (median of 4.95 vs. 7.1 mg/dl). Survival rate for warm perfused kidneys was 90% (9/10) vs. 73% (8/11). In the contralateral kidneys, metabolism was affected by the compounding damage of WI. Renal oxygen and glucose consumption diminished significantly, whereas vascular resistance and lactate dehydrogenase-release rose significantly with increasing WI. Conclusions. The results demonstrate a reduction of reperfusion damage by an acellular ex vivo restoration of renal metabolism. Furthermore, data from the contralateral kidneys substantiates the relative role of WI on metabolism in renal transplantation.

NOS: The Underlying Mechanism Preserving Vascular Integrity and During Ex Vivo Warm Kidney Perfusion

Research involving metabolically active and functioning organs, maintained ex vivo in culture‐like conditions, could provide numerous opportunities for medical innovations and research. We report successful perfusion of isolated canine and human kidneys ex vivo at near physiologic temperature for 48 h. During the perfusions parameters of metabolism and function remained stable. Nitric oxide synthase (NOS) was identified as the underlying mechanism preserving vascular integrity. Most importantly, when the canine kidneys were reimplanted there was immediate normal renal function. This report highlights the potential significance of whole organ culture using a warm temperature ex vivo perfusion and discusses medical applications that could be developed.

PRETRANSPLANTATION PROGNOSTIC TESTING ON DAMAGED KIDNEYS DURING EX VIVO WARM PERFUSION 1

Background. Further expansion of the donor pool with ischemically damaged kidneys will be predicated on the ability to develop prognostic testing. Using a well-established canine autotransplantation injury model, we assessed whether actual restoration of renal metabolism by ex vivo warm perfusion could be used to predict the status of an organ before transplantation. Methods. Kidneys were subjected to 30 min of warm ischemia followed by 24 hr of static storage in ViaSpan at 4°C. After warm ischemia and static storage the kidneys were transitioned to 3 hr of warm perfusion using Exsanguinous Metabolic Support technology. During this period, parameters indicative of renal metabolism and vascular function were used to predict outcomes prospectively. Parameters included measures of oxidative metabolism, perfusion characteristics, and vascular condition. A Viability Score (VS) was calculated as the sum of the three parameters mentioned above. Results were grouped by a VS>2 and a VS<2. Results. A clear association between the severity and duration of graft dysfunction and the VS was observed. Organs with a VS>2 had a significantly milder period of acute tubular necrosis, with both a less severe rise in serum creatinine (mean of 4.4 vs. 11 mg/dl) and a shorter recovery period (mean of 8 vs. 18 days) than those with a VS<2. Conclusions. Results indicate the possibility of utilizing warm perfusion to evaluate kidneys before transplantation. The VS developed demonstrated efficacy in classifying the severity of the acute tubular necrosis and the occurrence of primary nonfunction, offering a sensitive assay for prospective organ testing.

Retrograde oxygen persufflation in combination with UW solution enhances adenine nucleotide contents in ischemically damaged rat kidney during cold storage

Retrograde oxygen persufflation (ROP) has been reported to be beneficial to kidney preservation. The purpose of this study was to investigate whether use of ROP during cold storage (CS) with Universita of Wisconsin (UW) solution could ameliorate energy metabolism and functional recovery of ischemically injured rat kidneys and, moreover, to study the particular role of adenosine (ADO) in CS with ROP. Kidneys subjected to 30 min of warm ischemia (WI) were preserved for 24 h in 4°C UW solution with or without ROP and with or without ADO. Measurements of tissue highenergy phosphate levels showed that reduced total adenine nucleotides (TAN) after 30 min of WI further declined during the subsequent CS. In ROP kidneys, however, TAN were less reduced, suggesting that even during CS, TAN can still be regenerated in the injured kidneys when ROP is combined with UW solution. When UW did not contain ADO, regeneration of TAN by ROP was slightly less than in the case of UW with ADO. This indicates that the supply of molecular oxygen is a significant factor in TAN resynthesis during CS. There was no statistically significant difference in survival rate between the ROP and CS group, indicating that an improved energy status is not the sole determinant of functional recovery. We conclude that the gaseous oxygen supply provided by ROP during CS in UW solution ameliorates the energy state of ischemically injured rat kidneys and that exogenous ADO from the UW solution contributes to the improvement of energy metabolism to a limited extent.

Ischemia-Reperfusion Injury of Rat Kidney Relates More to Tubular Than to Microcirculatory Disturbances

Several pathophysiological mechanisms have been purported to be involved in the development of acute ischemic renal failure, such as impairment of tubular function and/or of the renal microcirculation. However, it has not been elucidated as yet which of these mechanisms relates to the extent of kidney damage. Besides, little is known about the time course relationship between tubular and microcirculatory disturbances during the development of ischemia-reperfusion injury. We therefore performed intravital videomicroscopy of the proximal tubules as well as the peritubular microcirculation of the rat renal cortex during the first 24 hr of reperfusion after varying lengths of warm ischemia (30 min, 30 WI group; 60 min, 60 WI group; 90 min, 90 WI group). In a separate group of animals subjected to the same protocol, the survival rate (SR) was determined. The SR in these groups were 100%, 20% and 0%, respectively. Initially, the tubular and microcirculatory changes (i.e., increased tubular diameter and reduced capillary blood flow) relate well to the length of warm ischemia as well as the SR. At a later stage of reperfusion, however, we observed that peritubular capillary blood flow and tubular diameter recovered more quickly in the 90 WI group than in the 30 WI and 60 WI groups. As a result, these parameters as obtained at 24 hr of reperfusion did not relate anymore to the survival rate. Besides, at this stage a severe loss of integrity of the tubular wall was noted in the 60 WI and 90 WI groups. These findings suggest that kidney viability is not determined by the extent of recovery of microcirculatory blood flow and/or tubular diameter during early reperfusion, but by the integrity of the tubular wall.

I: Negative effect of cold ischemia on initial renal function.

Correlation between post-transplant function and exposure to cold ischemia (CI) during preservation has been reported. We attempted to identify the effect of CI on renal function using exsanguinous metabolic support (EMS) technology, to eliminate effects of reperfusion complications. Small bovine kidneys were used to evaluate 4 vs. 24 hours of CI, after warm ischemic (WI) exposure of <15, 30 or 60 minutes. After CI, kidneys were warm perfused (30 degrees C to 32 degrees C) ex vivo using EMS technology. Restored renal metabolism and function were quantified by oxygen consumption, urine production, glomerular filtration rate (GFR), and hemodynamic characteristics. The results demonstrate a CI-associated lag phase in the restoration of metabolism, in which the longer cold-preserved kidneys exhibit a lower initial rate of oxygen consumption. However, after 3 hours of EMS perfusion there was no significant difference in the O2 consumed, urine flow, GFR, perfusion flow, or pressure between the kidneys stored for 4 or 24 hours. An initial reduction in metabolism after longer CI may influence the severity of actual reperfusion injury during transplantation. Therefore, these results provide preliminary evidence suggesting that an acellular warm temperature reperfusion ex vivo may enhance restoration of cellular metabolism and minimize damage from the cold seen upon actual reperfusion.

Protection of canine renal grafts by reninangiotensin inhibition through nucleoside transport blockade

The aims of this study were (1) to investigate the effect of R 75231, a nucleoside transport inhibitor, on renin-angiotensin release after renal ischemia-reperfusion and (2) to establish a possible protective effect of this drug on renal function. We used a canine model for auto-transplantation of kidneys that had been subjected to 30 min of warm ischemia and subsequently to 24h of cold storage in HTK preservation solution, with immediate contralateral nephrectomy. R 75231 was injected intravenously into six dogs in two equal portions of 0.05 mg/kg both 30 min and 10 min before reanastomosis was established. Another six dogs were used as a control group. At 2 weeks post-transplantation, five out of six dogs in the R 75231 group and one out of six in the control group were still alive. Starting on day 4, serum creatinine was lower in the R 75231 group than in the control group (p<0.05). In contrast to the control group, an inversion of the median preischemia adenosine/inosine ratio was observed in the R 75231 group after reperfusion (0.4 preischemia vs 4.0 after 60 min of reperfusion). Reperfusion of the graft resulted in an immediate increase in renin, angiotensin I, and angiotensin II venous blood levels in the control group. In the R 75231 group, renin, angiotension I, and angiotensin II levels were significantly lower. We conclude that administration of R 75231 before reperfusion has a protective effect on post-transplant function of kidneys that have been subjected to prolonged warm ischemia. This effect may, at least in part, be ascribed to inhibition of the breakdown and disposal of endogenous adenosine which, in turn, inhibits the excessive stimulation of the renin-angiotensin system in the early phase of reperfusion.