Bart M. Stubenitsky – Warm Perfusion In Renal Transplantation

Conclusions

The results of the studies support the concept that a warm temperature ex vivo organ preservation technology is feasible and could play an important role in transplantation. Hypothermic preservation technology is at present the foundation of clinical kidney preservation. The ability to procure kidneys distally and distribute them over wide geographical areas is dependant upon hypothermic preservation techniques. However, the ability to expand the donor organ pool, to asses the functional status of a kidney prospectively, and eventually the future ability to immuno-modify or even repair grafts will be dependent upon facilitating organ metabolism. Since hypothermic preservation has by definition the goal of inhibiting metabolism by more than 95% at 40C, the only way further developments can be made in the field of organ preservation is by raising the temperature at which kidneys are stored. In contrast to hypothermic techniques, the warm preservation technology described in these studies are predicated upon continued organ metabolism during ex vivo preservation. These two approaches of organ preservation are not mutually exclusive. As can be seen from the studies, a period of warm perfusion following hypothermic preservation may in fact combine both of the technologies. Warm preservation may be implemented in the future following harvest from a non-heartbeating donor to assess the degree of damage or likelihood of eventual recovery of function. Once assessed, the kidney could be stored or transported using traditional hypothermic preservation techniques. Alternatively, warm preservation technology could be employed following hypothermic preservation to evaluate or treat organs to avoid subsequent reperfusion injury when implanted. Therefore, the future of organ preservation technology may represent a synergy between the abilities to support ongoing metabolic functions and the ability to inhibit them. This projection is based on the following conclusions:

Impact of cold ischemia on renal function

Although hypothermic preservation is one of the foundations of clinical transplantation, it is a general consensus that with increasing cold ischemia (CI) times, damage to the kidney seen after reperfusion increases. Nevertheless, uncertainty exists concerning the mechanism by which CI influences the severity of the reperfusion injury. We studied the negative effect of hypothermia on renal metabolism and function using an ex vivo warm perfusion technology. The results indicated that renal function was compromised with CI greater than 24 hours and preceded the loss of cell viability following 48 hours of CI.

A CI associated lag-phase in the restoration of metabolism was also seen, where the longer cold stored kidney showed a diminished metabolic rate. We concluded that these observations might provide preliminary evidence suggesting that ex vivo restoration of metabolism could minimize damage from actual reperfusion from the cold.

Reduction of reperfusion injury

Reperfusion from a metabolically inactive state results in endothelial cell injury, fluid accumulation and inflammatory cell infiltration. The vasculature seems to have one of the most prominent positions in this inflammatory cascade, with reactive metabolites of oxygen as important modulators. Our experiments focused on an inhibition of the formation of radical oxygen and their activation of inflammatory cells. We achieved this by restoring metabolism with a perfusate containing scavengers and lacking inflammatory cells (EMS perfusate). The results indicated that ex vivo restoration of sufficient metabolism appeared to be a key factor in reduction of the reperfusion injury. We furthermore postulated that the initial warm ischemic injury, as seen in non-heartbeating donors, plays an important rate-limiting factor in the actual restoration of a sufficient level of metabolism.

Impact of warm ischemia on renal function

It is well appreciated that warm ischemic damage reduces the duration of effective cold preservation. This means that tolerable warm ischemia (WI) times are reduced when preservation and transplantation follow. To enhance the use of non-heartbeating donors as a source of kidneys for transplantation, the compounding damage of WI on the graft must be understood. In our experiments, kidneys were transitioned to a warm temperature perfusion following WI and CI. Increasing the WI time, reduced the renal metabolism substantially, and was interpreted as the loss of cellular viability. One of the known effects of ischemia on a kidney is an increase in vascular resistance and LDH release. This was confirmed in our experiments.

The data showed that WI plays a rate-limiting role in the restoration of metabolism after cold preservation. We furthermore believe that these studies indicate the potential of ex vivo warm perfusion as a useful tool in prospective organ evaluation.

Viability testing

Viability testing in clinical transplantation has gained renewed interest with the resurgence of the non-heartbeating donor. Despite efforts there is currently no objective method to evaluate the potential function of a kidney. This can, in part, be attributed to the severe inhibition of renal metabolism that occurs during hypothermic preservation. Warm perfusion seems to create an opportunity for viability testing through the restoration of metabolism prior to actual transplantation.

The degree of metabolism and the integrity of the vasculature both play a major role in the viability of an organ. In view of this, three parameters for the viability testing were developed, forming a score that could predict posttransplant outcome. Our results show that it is possible to use the ongoing metabolism during the warm temperature perfusion to effectively assess the kidney before it is transplanted.

In conclusion, these findings indicate that a period of warm perfusion reduces reperfusion injury upon implantation and provides a means for viability testing, that will allow for use of kidneys with longer warm ischemic times. We postulate that if, following severe WI, actual resuscitation of metabolism to a sufficient level were possible, we might one day be able to transplant an allograft with a warm ischemic exposure of a magnitude that is currently never considered.

Summary

The present thesis was designed to examine the effect of restoration of renal metabolism by ex vivo warm perfusion with an acellular perfusate on (1) the reduction of the damage to the kidney seen upon reperfusion from the cold (2) the development of prognostic testing to evaluate the functional status of kidneys prior to implantation.

Chapter 1 is a brief overview of the development of organ preservation and immunosuppression in the field of organ transplantation. It discusses the organ shortage, the major obstacle facing today’s clinical transplantation, and formulates the general introduction to this thesis.

In chapter 2 a historical overview of previous attempts at warm preservation, in the context of the current status of kidney preservation, is presented. The principle of preservation at warmer temperatures is not new, but with the present developments and approaches, successful clinical application appears to be within reach. The potential for prospective testing of the function and enhanced resistance to ischemic damage are discussed.

Chapter 3 discusses the original concept underlying the development of warm perfusion using Exsanguinous Metabolic Support (EMS) technology. EMS functions by supporting two major ongoing and critical processes: the support of continued cellular metabolism and the maintenance of the integrity of the vasculature in order to provide continued barrier functions. If these two requirements can be met, it seems feasible to preserve an organ at a near physiologic temperature in conditions that mirrored cells in tissue culture. The warm perfusion solution and perfusion system are reviewed in detail.

In chapter 4, using a bovine calf slaughter house model, the effect of varying cold ischemia times on the metabolism and function of the kidney is described. It is shown that renal function is compromised prior to the loss of cell viability with increasing cold ischemia times. This finding suggests that an acellular warm temperature reperfusion ex vivo may enhance restoration of metabolism and thereby reduce the damage seen upon actual reperfusion from the cold.

The influences of warm ischemia on renal metabolism and function are investigated in chapter 5. The studies in the bovine calf slaughterhouse model form a basis for the development of a sensitive viability test prior to transplantation.

Using a critical autotransplantation model in the dog, the effect of restoring renal metabolism by warm perfusion on reperfusion injury was investigated in chapter 6. As compared to reperfusion from the cold, the results showed a clear reduction in the severity of delayed graft function following transplantation.

Chapter 7 discusses the renewed interest in viability testing with the resurgence of the NHB donor. With the development of a more physiologic preservation technology that supports adequate metabolism at a warm temperature, it appears feasible to develop viability testing that can distinguish between viable and non-viable organs.

In chapter 8 the general conclusions are discussed and interpreted, indicating the possible synergy between the cold and the warm in the field of organ transplantation.

Samenvatting

In dit proefschrift zijn de effecten beschreven van een ex vivo herstel van niermetabolisme op (1) de reductie van nierschade zoals waargenomen bij reperfusie vanuit de koude preservatie, (2) de ontwikkeling van prognostische testen om de functionele status van de nieren nog voor transplantatie te bepalen. Het metabolisme werd hersteld middels warme perfusie met een acellulair perfusaat

Hoofdstuk 1 vormt de algemene introductie voor dit proefschrift. Het geeft een kort overzicht van de ontwikkeling van nierpreservatie en immunosuppressie op het gebied van de orgaantransplantatie. Tevens wordt het tekort aan nieren, een van de voornaamste obstakels in de hedendaagse klinische transplantatiegeneeskunde, besproken.

In hoofdstuk 2 wordt een historisch overzicht gegeven van eerdere pogingen tot warme perfusie van nieren. Het principe van warme perfusie is niet nieuw, maar met de huidige technieken en ontwikkelingen, lijkt een succesvolle klinische toepasbaarheid binnen handbereik. De potentie van warme perfusie voor het prospectief testen van de nierfunctie en de toegenomen weerbaarheid tegen ischemische schade wordt besproken.

Hoofdstuk 3 bespreekt het oorspronkelijke concept hetgeen ten grondslag ligt aan de ontwikkeling van warme perfusie met ‘Exsanguinous Metabolic Support’ (EMS) technologie. EMS werkt via het ondersteunen van twee, tijdens preservatie, kritieke processen: te weten het cellulair metabolisme en de vasculaire integriteit teneinde respectievelijk de functionaliteit van de niercellen en de barrière functies van het vaatbed te behouden.

Indien aan deze twee voorwaarden is voldaan, lijkt het haalbaar een orgaan te bewaren bij een bijna fysiologische temperatuur onder omstandigheden die gelijk zijn aan die van cellen in een celkweek. De perfusie vloeistof en het perfusie systeem komen in detail aan de orde.

In hoofdstuk 4 wordt met behulp van een kalver-slachthuismodel het effect beschreven van verschillende periodes van koude preservatie op het metabolisme en de functie van de nier. Er wordt aangetoond dat met toenemende periodes van koude ischemie de nierfunctie wordt gecompromitteerd vóórdat er verlies optreedt van de levensvatbaarheid van de niercellen. De bevindingen van deze studies wijzen op een mogelijk positief effect van ex vivo herstel van het metabolisme. Dit herstel van metabolisme zou kunnen leiden tot een vermindering van de nierschade, welke gezien wordt bij reperfusie na koude preservatie.

De invloeden van warme ischemie op het niermetabolisme en –functie worden onderzocht in hoofdstuk 5 (kalver-slachthuismodel). Deze onderzoeken vormen de basis voor de ontwikkeling van prognostische testen om de functionele status van de nieren vóór transplantatie te bepalen.

Middels een kritisch auto-transplantatie model bij honden is in hoofdstuk 6 het effect van het ex vivo herstel van niermetabolisme op reperfusieschade onderzocht. Vergeleken met reperfusie na uitsluitend koude preservatie, tonen de resultaten dat de functie van een tijdelijk warm geperfuseerde nier na transplantatie minder gecompromitteerd wordt.

Hoofdstuk 7 (auto-transplantatie model bij honden) bespreekt het belang van kwaliteitstesten naar de levensvatbaarheid van nieren. Met de toepassing van een meer fysiologische preservatie technologie, welke een adequaat metabolisme ex vivo ondersteunt, lijkt het haalbaar om kwaliteitstesten voor nieren te ontwikkelen die kunnen differentiëren tussen al dan niet levensvatbare nieren vóór transplantatie.

In hoofdstuk 8 worden de algemene conclusies besproken en geïnterpreteerd. Deze wijzen op een mogelijk synergisme tussen koude en warme preservatie op het gebied van orgaantransplantatie in de toekomst.