Kidney Project

     Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It results in lower rates of mortality and higher quality of life when compared to dialysis. As of today, kidneys are the most frequently transplanted organ. However, the organ donor shortage represents a major problem worldwide. Currently, more than 100,000 patients are waiting for a kidney transplant in the United States. Therefore, research focuses on increasing the donor pool by making more grafts available for transplantation.


     Current storage techniques for kidneys prior to transplantation are based on cold temperatures, which are known to be cause injury to renal grafts during the storage period. Prolonged storage times, which are sometimes necessary, can affect the patients’ outcome negatively following kidney transplantation. Furthermore, cold techniques do not allow the assessment or improvement of renal grafts during storage, as the graft is metabolically inactive. Therefore, we have developed a novel storage technique – called normothermic ex vivo kidney perfusion- that provides a more physiologic surrounding for the renal graft from recovery until transplantation. In a recently published manuscript in the journal “Transplantation”, we could demonstrate that normothermic kidney graft preservation is feasible and safe in good quality organs. Porcine kidneys were recovered and either stored on ice (common technique) or stored with our novel perfusion technique at normal temperatures providing oxygen and nutrition. Following  transplantation, pigs that received kidneys storage using our novel technique demonstrated superior outcomes when compared to kidneys stored with the currently established technique of cold storage.

      Our findings demonstrate that normothermic ex vivo kidney perfusion can be safely used in kidney transplantation. Based on these results we will initiate a clinical trial at the Toronto General Hospital, allowing the progression towards a larger donor pool and ultimately reducing the gap between supply and demand for renal grafts.

 


The Kidney Team

Figure 1. (A) Serum creatinine of transplanted animals during 8 days follow-up. Values presented as mean standard deviation (SD) in mg/dL and lmol/L. Overall, significant differences were observed at hour 10 posttransplant and days 1–4 between groups. Peak serum creatinine was significantly lower in the NEVKP 16-h group when compared to all other groups (p < 0.001). (B) Creatinine clearance of transplanted animals during 8 days follow-up. Values presented as mean ` SD in mL/min. The creatinine clearance between groups was significantly different on postoperative day 3 (p < 0.001) and significantly higher in the NEVKP 16-h group versus all other groups. NEVKP, normothermic ex vivo kidney perfusion; KTx, kidney transplant; DCD, donation after circulatory death; SCS, static cold storage; pod, postoperative day.

Eight Hour Continuous Normothermic Ex Vivo Kidney Perfusion is a Safe Preservation Technique for Kidney Transplantation: A New Opportunity for the Storage, Assessment and Repair of Kidney Grafts

Figure 2: (A) pH during normothermic ex vivo kidney perfusion. Values are presented as mean ± D. Dashed line and gray area represent mean pH with SD based on measurements performed in situ in 20 anesthetized pigs. *Significant differences. (B) HCO3– during normothermic ex vivo kidney perfusion (NEVKP). Values are presented as mean ± SD in mmol/L. Dashed line and ray area represent mean HCO3 – with SD based on measurements performed in situ in 20 anesthetized pigs. *Significant differences. (C) Base excess (BE) during NEVKP. Values are presented as mean ± SD in mmol/L. Dashed line and gray area represent mean BE with SD based on measurements performed in situ in 20 anesthetized pigs. *Significant differences. DCD, donation after circulatory death; HBD, heart- beating donor

Normothermic Ex Vivo Kidney Perfusion Following Static Cold Storage – Brief, Intermediate, or Prolonged Perfusion for Optimal Renal Graft Reconditioning?

Figure 1.  Schematic of the NEVKP circuit. The circuit consists of neonatal cardiopulmonary bypass technology. The perfusion solution is collected in the venous reservoir. A centrifugal pump propels the solution into the oxygenator, where it is enriched with oxygen and warmed to 37°C. After passing the arterial filter, the perfusate is driven with a pressure of 65 mm Hg through the renal artery into the graft located in the customized double-walled kidney chamber. The venous outflow (0-3 mm Hg) leads the perfusate back into the venous reservoir. Syringe and infusion pumps secure the supply with additional compounds. The urine is collected throughout the perfusion. Control panel and DMS indicate and record perfusion parameters continuously. DMS, Data Management System.

Continuous Normothermic Ex Vivo Kidney Perfusion Improves Graft Function in Donation after Circulatory Death Kidney Transplantation: A Novel Technique to Expand the Kidney Donor Pool

      Severe graft shortage represents a major limitation in solid organ transplantation. Kidneys from extended criteria donors (ECD) and donation after circulatory death (DCD) are increasingly used worldwide in order to extend the donor pool. However, delayed graft function (DGF) and primary non function (PNF) represent a major challenge when such organs are transplanted. At this moment assessment of graft quality is done mainly based on the donor characteristics, which results in a high percentage of organ disposal, especially in DCD donors, sometimes also of grafts that might be suitable for transplantation. Normothermic ex vivo kidney perfusion (NEVKP) represents a novel approach for preservation and functional improvement in kidney transplantation. The aim of this study was to investigate whether NEVKP can also offer the possibility to assess different parameters during perfusion, which reflect graft quality and depending on these parameters decide, whether an organ is suitable for transplantation on not. Grafts from three different donor models – (A) noninjured heart-beating donor (HBD) kidneys, (B) moderately injured DCD kidneys, 30 min of warm ischemia (WI) and (C) severely injured DCD kidneys, 60 min of WI – were preserved with 8 hours of continuous NEVKP. After contralateral kidney resection and heterotopic graft autotransplantation, the pigs were followed for 3 days. For analysis, we correlated peak serum creatinine levels, a marker for posttransplantation renal graft function, with perfusion characteristics and clinically available biomarkers. Posttransplantation serum creatinine and serum BUN were significantly different among groups. Also, routinely available assessment parameters such as intrarenal resistance, lactate clearance, acid-based homeostasis (pH, HCO3¯, base excess) and AST were significantly different among groups and correlated with the peak serum creatinine. However, urine production and LDH did not significantly differ among groups and did not correlate with posttransplantation renal function. These data demonstrate that perfusion characteristics during NEVKP correlates with posttransplantation graft function, therefore could be used for graft quality assessment before transplantation. However, a generalisation of the assessment parameters we identified might prove difficult in a different perfusion model. Also, the setup we used only enables a short follow-up, which does not allow an assessment of delayed graft function. Further clinical trials need to asses perfusion parameter thresholds to identify DGF and PNF. 

Figure 2. Serum creatinine of transplanted animals during 8-days’ follow-up. Values presented as mean standard deviation in mL/min. DCD indicates donation after circulatory death; KTx, kidney transplant; NEVKD, normothermic ex vivo kidney perfusion; pod, postoperative day; SCS, static cold storage.

Figure 1: Intrarenal resistance during normothermic ex vivo kidney perfusion (NEVKP). Values are presented as mean ±SD in mm Hg/mL per minute. Dashed line and gray area represent mean intrarenal resistance with SD based on measurements performed in situ in 30 anesthetized pigs. *Significant differences among groups (P < .05). DCD, donation after circulatory death; HBD, heart- beating donor. 

Figure 1. Following 30 min of warm ischemia (WI), renal grafts were flushed with 4°C cold HTK (Histidine-tryptophan-ketoglutarate) solution and stored on ice (static cold storage, SCS) for 8 h. Afterwards, kidneys were transplanted immediately, preserved with 1 h normothermic ex vivo kidney perfusion (NEVKP) (brief), 8 h NEVKP (intermediate), or 16 h NEVKP (prolonged). Following NEVKP, kidneys were flushed cold for heterotopic autotransplantation, reperfusion, and 8-day follow-up.

Normothermic Ex Vivo Kidney Perfusion for Graft Quality Assessment Prior to Transplantation

Figure 2. Tubular injury as identified by brush border loss, luminal ectasia, and sloughing is slightly worse in 16-h SCS pigs (A) com- pared to the other groups (B, C, and D) but not significantly different between the groups at 8 days after transplantation (periodic acid–Schiff, 1009); TUNEL staining shows many more apoptotic cells in 16-h SCS pigs (E) compared to the NEVKP groups (F, G, and H), with the fewest numbers in the 16-h NEVKP group (H) at the same time point (TUNEL stain, 1009). SCS, static cold storage; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling; NEVKP, normothermic ex vivo kidney perfusion. 

Continuous Normothermic Ex Vivo Kidney Perfusion Improves Graft Function in Donation After Circulatory Death Pig Kidney Transplantation

Figure 2. A, Serum creatinine of the transplanted animals during 10-day postoperative follow-up for autologous kidney transplantation after SCS and NEVKP. Values presented as mean ± SD in mg/dL and μmol/L. NEVKP demonstrated significantly lower serum creatinine on postoperative days 1 to 7 (P < 0.05) and lower peak values when compared to grafts preserved at 4°C (NEVKP 5.5 ± 1.7 mg/dL vs SCS 11.1 ± 2.1 mg/dL, P = 0.002). B, Serum BUN/urea during 10-day postoperative follow-up for autologous kidney transplantation after SCS and NEVKP. Values presented as mean ± SD in mg/dL and μmol/L. Serum BUN was significantly lower in the normothermic perfused kidneys on day 2 till 4 after transplant (P < 0.05, respectively) with a significantly lower peak value of 47 ± 6.2 mg/dL versus 68 ± 24.2 mg/dL (P = 0.007).

       Kidney grafts of lower quality recovered from extended criteria donors (ECD) and donation after circulatory death (DCD) are increasingly used for transplantation to increase the donor pool. However, several studies demonstrate that transplantation of ECD and DCD kidney grafts can lead to increased rates of primary non-function (PNF), delayed graft function (DGF), and reduced long-term outcomes. These outcomes after ECD and DCD kidney transplantation are linked to hypothermic preservation and their poor tolerance to cold ischemia. 

 following SCS or prolonged, continuous NEVKP is superior in DCD kidney transplantation. Following 30min of renal warm ischemia, grafts were exposed to one of four different preservation protocols: (A) 16 h of SCS, (B) 15h of SCS + 1 h of NEVKP, (C) 8 h of SCS + 8 h of NEVKP, and (D) 16 h of NEVKP. After contralateral kidney resection, preserved grafts were auto-transplanted and followed for 8 days. 
       The aim of this study was to investigate whether an additional, short period of Normothermic Ex vivo Kidney Perfusion[NEVKP] following SCS or prolonged, continuous NEVKP is superior in DCD kidney transplantation. Following 30min of renal warm ischemia, grafts were exposed to one of four different preservation protocols: (A) 16 h of SCS, (B) 15h of SCS + 1 h of NEVKP, (C) 8 h of SCS + 8 h of NEVKP, and (D) 16 h of NEVKP. After contralateral kidney resection, preserved grafts were auto-transplanted and followed for 8 days. 
          In groups C and D, hourly assessed perfusate injury markers, AST and LDH, remained low and lactate decreased significantly from the baseline until the end of perfusion. Grafts in group D demonstrated a significantly lower serum creatinine peak and the 24-h creatinine clearance at post operative day 3 was significantly higher when compared to all other groups. A histological assessment on day 8 demonstrated that tubular injury and interstitial inflammation were lowest in group D with fewer apoptotic cells. 

         In conclusion, prolonged, continuous NEVKP provides superior graft function and reduced renal injury following DCD kidney transplantation versus SCS or short additional NEVKP following SCS. Future clinical trials should aim to replace SCS with NEVKP and minimize cold storage periods. Furthermore, 16 h NEVKP versus 8 h NEVKP demonstrated lower serum creatinine following transplantation, suggesting that prolonged NEVKP might provide potential benefits itself, not only by exclusion of SCS. This study also postulates the availability of a portable normothermic kidney perfusion device that facilitates placing the graft on normothermic preservation immediately following retrieval. 

Peter Urbanellis & Mátyás Hamar ​ 

        Normothermic ex vivo kidney perfusion (NEVKP) has demonstrated superior results compared to hypothermic storage in donation after circulatory death (DCD) kidney transplantation. However, continuous NEVKP is currently not feasible as portable kidney perfusion devices have not been developed as well as might include risk of graft loss due to machine failure during transport, higher health-care costs, and complications in surgical techniques. The aim of this study was to compare the outcome of brief, intermediate, and prolonged NEVKP following 8 h of SCS for graft transportation in a model of porcine heterotopic renal auto-transplantation. The optimal perfusion time following hypothermic storage to allow for the recovery of renal grafts from cold ischemic injury was also investigated. Following renal warm ischemia of 30 min, grafts were exposed to one of four different preservation protocols: Group A, 8h SCS only (control); group B, 8h SCS + 1h NEVKP (brief NEVKP); group C, 8h SCS + 8h NEVKP (intermediate NEVKP); and group D, 8h SCS + 16h NEVKP (prolonged NEVKP).
        Postoperative graft assessment during an 8 day follow-up demonstrated lowest levels of peak serum creatinine for intermediate and prolonged NEVKP. Histological assessment on post-operative day 8 demonstrated a significant difference in tubular injury, with highest values for group B, brief NEVKP. These results suggest that longer periods of NEVKP following SCS are feasible and safe for postponing surgical transplant procedure and superior to brief NEVKP, reducing the damage caused during cold ischemic storage of renal grafts. NEVKP improves outcomes by two different mechanisms: by avoiding SCS and facilitating graft reconditioning following SCS. Our data indicates that perfusion times longer than 1 h following SCS are necessary for optimal reconditioning processes.

       The findings of this study demonstrate that intermediate or prolonged NEVKP following hypothermic transportation is superior to brief NEVKP preimplantation and allows for postponing of renal transplant procedure to daytime safely. Complete exclusion of SCS using continuous NEVKP provides superior results, however, further research is needed to explore optimal perfusion times in normothermic preservation to design appropriate clinical studies. In addition, effort should focus on developing portable normothermic machine perfusion devices to provide optimal graft preservation.

Kidney transplantation offers several advantages for patients suffering from end-stage renal disease when compared with dialysis. However, due to a worldwide shortage of organs available for transplantation, current research explores various strategies to increase the pool of deceased donor kidney grafts. Current clinical practice to increase the donor pool and decrease a recipient’s time on the waiting list is to use kidneys donated after circulatory death (DCD). The most widely used preservation techniques are static cold storage (SCS) and hypothermic machine perfusion (HMP), however, deleterious effects on renal graft function have been shown after hypothermic preservation. Therefore, modification of current kidney preservation techniques represents a novel approach to improve renal graft function after DCD kidney transplantation.

This study compared a more physiological, continuous pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) with SCS in a porcine model of DCD autotransplantation. After 30 minutes of warm ischemia, the kidneys were preserved with either 8-hour NEVKP or SCS, followed by a kidney autotransplantation. The renal grafts preserved with NEVKP revealed results of improved renal function and reduced injury compared to SCS following autotransplantation. On post operative days 1 to 7, NEVKP demonstrated lower serum creatinine and urea levels, with a higher creatinine clearance on day 4. NGAL, a specific kidney injury marker, was significantly lower on day 3 after NEVKP versus SCS. 

The findings in this study demonstrate that continuous pressure-controlled NEVKP improves renal function in DCD kidney transplantation compared to SCS and may aid in decreasing post-transplant delayed graft function rates. In turn, extending the donor pool by allowing the use of more marginal grafts and reduce the recipient’s waiting list.
 

Toronto Organ Preservation Laboratory