Bart M. Stubenitsky

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.

Repair of Damaged Organs in Vitro

Treatments that can be performed ex vivo following ischemia to accelerate cellular recovery and ameliorate reperfusion injury could have major impact. An acellular, near‐normothermic perfusion was employed to deliver growth factors to ischemically damaged kidneys. During the treatment oxidative metabolism was sufficiently restored to support up‐regulation of cellular processes with the potential to modulate both injury and repair proteins in damaged kidneys. The results suggest that growth factor administration, without concomitant inflammation, triggers pathways for new synthesis leading to cellular recovery rather than cell death.

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.

Induction of heme oxygenase-1 in kidneys during ex vivo warm perfusion

Background. Reperfusion injury plays a pivotal role in the occurrence of delayed graft function and chronic rejection. Heme oxygenase-1 (HO-1), an inducible heat shock protein, is known to have cytoprotective effects against reperfusion injury. We report on the potential for ex vivo induction of HO-1 expression during acellular warm perfusion of canine kidneys, using cobalt protoporphyrin (CoPP) as an HO-1 inducer and zinc protoporphyrin as an HO-1 inhibitor. Methods. Canine kidneys were used to evaluate HO-1 increase after exposure to warm ischemia (WI), hypothermic perfusion (mechanical perfusion [MP], 4°C), warm perfusion (exsanguineous metabolic support [EMS], 32°C), or various combinations. Results. WI alone, MP, or EMS with or without WI, had no effect on HO-1 activity. However, the presence of CoPP during EMS perfusion resulted in a significant increase in kidney HO-1 activity, whereas zinc protoporphyrin reduced HO-1 activity. The presence of CoPP during MP did not induce elevated HO-1 expression. The results of our study demonstrate that sufficient metabolism supporting new protein synthesis resulting in the expression of the protective gene, HO-1, can be accomplished during an acellular near-normothermic perfusion using CoPP. Most importantly, the time required for ex vivo HO-1 induction with this method is compatible with the current time frame for which organs are preserved clinically. Conclusions. The ability to induce HO-1 expression ex vivo eliminates the need for donor therapy to induce HO-1 increase before retrieving organs and also prevents the potential of decreasing HO-1 enzyme activity that is known to occur with temperature-mediated inhibition of oxidative metabolism during hypothermic organ storage.

Pretransplant Kidney-Specific Treatment to Eliminate the Need for Systemic Immunosuppression

Background. Despite significant side effects, chronic systemic immunosuppression remains the backbone of clinical transplantation. We investigated the feasibility of preventing early allorecognition in canine renal allografts using a nonsystemic pretreatment. Methods. The renal vasculature was treated with a bioengineered interface consisting of a nano-barrier membrane during 3 hr of ex vivo warm perfusion. Results. Preliminary feasibility of the immunocloaking technology was established by the following criteria: it is possible to achieve approximately 90% coverage of the vasculature with nano-barrier membrane after 3 hr of ex vivo warm perfusion; covering the luminal surfaces prevents allorecognition as determined by mixed lymphocyte-vascular endothelial reaction; covering the luminal surfaces does not negatively affect renal function as determined by autotransplant outcomes; and graft rejection is significantly postponed in canine kidneys treated with the immunocloaking technology. In the absence of systemic immunosuppression, untreated control dogs experienced a mean onset of rejection on day 6, whereas in the treated dogs with modified renal vascular luminal surfaces, the mean onset of rejection was significantly delayed until day 30. Conclusions. The ability to postpone, or eventually eliminate, the allorecognition that occurs immediately on reperfusion could provide a new window of opportunity to introduce adjunct therapies to support tolerance induction. To our knowledge, this is the first time significantly prolonged canine renal allograft survival has been achieved in the absence of systemic immunosuppression or immunologic manipulation of the recipient.

Horseshoe kidney transplantation: an overview

The horseshoe kidney is the most common anatomical renal variation. It represents a fusion anomaly, mainly at the lower poles, occurring between the 4th and 6th week of gestation. Horseshoe kidneys display a great variation in origin, number and size of the vasculature. Transplantation of these deviant kidneys can be done en bloc or they can be split into two halves and transplanted into two recipients, depending on the number of vessels and the anatomy of the urinary collecting system. A literature review reveals 31 case histories, published between 1975 and 1998. Of these 21 were transplanted into 38 recipients after division and ten were implanted en bloc. Nineteen grafts (41%) showed immediate function and 21 grafts (46%) showed delayed function. Thrombosis and acute rejection, leading to non‐function was seen in six grafts (13%). The overall success rate was 87% with a mean follow‐up of 22 months. The results of horseshoe kidney transplantation are good, provided that attention is paid to certain technical details. Because of donor scarcity, horseshoe kidneys should be used for 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.

The modified Honig velopharyngoplasty – a new technique to treat hypernasality by palatal lenghtening

BACKGROUND Palatal lengthening by pushback with a pharyngeal flap is a commonly used operative technique for the treatment of velopharyngeal insufficiency. The conventional Honig velopharyngoplasty uses full thickness mucoperiosteal flaps for the oral lining of the defect. PURPOSE A modification is described using only mucosal flaps, thus preserving the periosteum and the palatine arteries. Vascularisation of the hard palate is preserved and bone is not exposed, avoiding potential detrimental scar formation overlying the hard palate, which may affect normal outgrowth of the maxilla. STUDY DESIGN Eight patients with persisting hypernasality were included. Velopharyngeal closure was evaluated by nasendoscopy, nasometry and a Dutch speech test for cleft patients. RESULTS Hypernasality was corrected in all cases. One patient developed a light hyponasality. In four patients the overall speech normalised and in the remaining four cases small errors persisted, but speech was well understandable. CONCLUSION The procedure yields satisfactory speech results in this preliminary study, comparable to the conventional Honig velopharyngeaplasty. Long term follow up regarding maxillary growth and comparative studies with other operative techniques are now warranted.