Potential of repairing ischemically damaged kidneys ex vivo.

Therapies that would accelerate recovery from ischemic injury could positively impact the number of kidneys procured from non-heart-beating donors. An acellular warm (32 degrees C) perfusion was used to deliver growth factors to canine kidneys damaged by 2 hours of warm ischemia. Fibroblast growth factors 1 and 2 were selected for activation of the tyrosine kinases because of their known receptor-specific binding in the kidney, metabolic regulation, and mitogenic effect. During 24 hours of ex vivo perfusion at near-normothermia, oxidative metabolism was sufficiently restored to the ischemically damaged tissue to support upregulation of cellular processes dependent on new synthesis. The junctional integrity protein, ZO-1 was used to determine recovery of cytoskeletal integrity. The upregulation of proliferating cell nuclear antigen was used as a marker for recovery of synthetic functions. This modulation of both injury and repair proteins in the damaged kidneys was dependent on new synthesis. The observed modulation resulting in normalization of the cytoskeletal integrity correlated with outcomes in that when the "repaired" kidneys were reimplanted, they provided life-sustaining function. In contrast, when warm ischemically damaged control kidneys without treatment, with subsequent hypothermic perfusion or warm perfused in the absence of growth factors, were reimplanted the result was nonviability. The results of this study suggest that the administration of growth factors during 24 hours of near-normothermic, acellular perfusion, in the absence of concordant inflammation, triggers pathways for new synthesis leading to cellular recovery rather than resulting in cell death.

The cadaveric kidney and the organ shortage--a perspective review.

Despite the technical and logistical hurdles that must be overcome with the reintroduction of non-heartbeating donor kidneys, the potential of these organs represents the only near-term solution for effectively alleviating the growing disparity between demand and supply. This review provides an argumentative overview of the history of cadaveric kidney transplantation. During the early years of transplantation retrieval of kidneys from non-heartbeating donors necessitated a prolonged period of warm ischemic exposure, with a corresponding minimal ex vivo period since organ preservation was in its infancy. Today we have the inverse situation where warm ischemic times are quite limited and hypothermic preservation times average 24 h because organs are shipped to remote centers due to mandated organ sharing algorithms. The recent experience with the reintroduction of non-heartbeating donors has necessitated combining the worst aspects from both eras: substantial warm ischemia with prolonged hypothermic preservation. Nevertheless, recent results from several transplant groups poignantly highlight the potential of this approach in expanding the organ donor pool.

Deletrious effect of prolonged cold ischemia on renal function.

The detrimental effect of prolonged cold ischemia (CI) on posttransplant renal function has long been recognized. However, the cellular consequences of CI have not been clearly defined. This study describes a model for the identification of CI-induced injury by evaluating ex-vivo renal metabolism and function prior to reperfusion. Small bovine kidneys were cold stored in Viaspan for 24-, 48-, 72-, and 96 h. Kidneys were then warm perfused (32 degrees C) using Exsangiunous Metabolic Support (EMS) technology and evaluated for oxidative metabolism, vascular dynamics and function. Oxygen consumption, vascular resistance, and diuresis were stable in kidneys with CI up to 48 h. After 72- and 96 h of CI, vascular resistance was increased while oxygen consumption and diuresis were reduced (P < 0.05). Glomerular filtration rate was diminished at CI greater than 24 h (P < 0.05). Results show that function was compromised with CI greater than 24 h and preceded the loss of cell viability following 48 h of CI.

Pretransplantation prognostic testing on damaged kidneys during ex vivo warm perfusion.

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 degrees 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.

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 degrees C) for 18 h, or warm perfused with exsanguineous metabolic support (EMS) technology (32 degrees C) for 18h, 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 6.3mg/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 (2.6mg/dL). The damage in kidneys CS for 18h 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 18h 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.

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.

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.

II: Ex vivo viability testing of kidneys after postmortem warm ischemia.

Future approaches to expand the organ donor pool with marginal and nonheartbeating donors, will be dependent upon prospective organ evaluation. Restoration of metabolism by preservation at warmer temperatures could potentially provide the window for such evaluation. Using a small bovine model, kidneys were subjected to either < 15, < 30 or < 60 minutes of warm ischemia (WI) followed by cold ischemia (CI) in ViaSpan. After WI and CI, kidneys were transitioned to a warm temperature perfusion (30 degrees C to 32 degrees C) using exsanguinous metabolic support (EMS) technology. Restored renal metabolism and function was assessed by oxygen consumption, glucose consumption, urine production, glomerular filtration rate, and hemodynamic characteristics. The results of this study suggest that it is feasible to distinguish viable from nonviable organs ex vivo by assessing renal metabolism and function during warm preservation using EMS technology.

Ex vivo evaluation of organ function after cold ischemia.

An ex vivo perfusion of kidneys was performed at 34 degrees C after cold ischemia of 24, 48, 72, and 96 hours to evaluate organ function prospectively. The prospective evaluation of organ function followed static hypothermic storage of the kidneys in a solution representative of clinical organ preservation. The warm perfusion was performed with an acellular solution that supports oxidative metabolism of sufficient magnitude to restore urine flow ex vivo. The parameters of organ function evaluated included oxygen consumption, vascular resistance, urine flow, and glomerular filtration rates, which were correlated with the histologic findings. The results of this study suggest that kidneys exposed to 24 and 48 hours of cold ischemia demonstrated oxygen consumption rates and vascular dynamics similar to control kidneys without exposure to cold ischemia, indicating cell viability. When the cold ischemic period was increased beyond 48 hours of preservation, substantially reduced rates of oxygen consumption and increased vascular resistances were observed, representing a loss of viability confirmed histologically. However, organ function was found to be impaired after exposure to cold ischemia at every time point. These results suggest that cold ischemic exposure had a negative impact on immediate renal function once oxidative metabolism was restored, which was exacerbated as the cold ischemic period was extended. Furthermore, these findings suggest that although the renal cells were viable after cold ischemic exposure, the viability status did not result in immediate function. Therefore, assessment of an organ based solely on cell viability may falsely indicate a functional organ. It will be necessary to identify parameters of organ function that can distinguish reversibility from non-reversibility of cellular impairment to distinguish permanent functional disturbances. The ability to predict organ function prospectively will be an important aspect of any effective future expansion of the organ donor pool.

Ex vivo resuscitation of kidneys after postmortem warm ischemia.

An ex vivo resuscitation of kidney function following substantial post mortem warm ischemia was attempted with the ultimate goal of overcoming the ischemic barriers in organ retrieval for transplantation. The resuscitation technology involved reperfusion at 32 degrees C with an acellular solution to reinstitute oxidative metabolism of sufficient magnitude to restore function after a substantial postmortem warm ischemic insult. The ability to resuscitate renal function at various time periods postmortem was evaluated. Resuscitation parameters included perfusion pressures, vascular flow rates, vascular resistances, restoration of diuresis with concordant urinary creatinine concentrations, and blinded histologic evaluations. The results of this study suggest that it may one day be feasible to resuscitate organs following as much as 2 hr of postmortem warm ischemia for clinical transplantation. An expanded donor pool consisting of allografts resuscitated post mortem from what is now considered to be the "non retrieval donor" could help alleviate the chronic organ shortage. Furthermore, since organs resuscitated ex vivo at 32 degrees C exhibited ongoing metabolism, which was artificially supported rather than inhibited by traditional hypothermia, diuresis was restored. The ability to collect and analyze urine during organ resuscitation and preservation may present the opportunity to assess organ function prospectively.