Techniques to Ameliorate the Impact of Second Warm Ischemic Time on Kidney Transplantation Outcomes

Anastomosing the renal artery and vein in transplant recipients without a cooling mechanism exposes the kidney to temperatures exceeding the metabolic threshold (15°C to 18°C), at which the protective effects of renal hypothermia are lost. This anastomotic time, or second warm ischemic time, can be deleterious to graft outcomes, especially if it is prolonged. Techniques to ameliorate organ warming prior to reperfusion have been designed, and range from simpler surface cooling techniques, to organ immersion in bags of ice slush, and the application of ‘jackets’ that incorporate their own internal cooling mechanism. The efficacy of these methods with respect to the minimization of kidney temperature prior to reperfusion and subsequent effects on graft outcomes are discussed using clinical and experimental data, in the setting of open, laparoscopic, and robotic kidney transplantation.     

Evaluating a New Device for Reducing Second Warm Ischemia During Organ Transplantation in a Porcine Model of Kidney,Heart, and Pancreas Transplantation

BackgroundSecond warm ischemia (SWI) injury between the completion of vascular anastomosis and graft reperfusion has been a longstanding issue in organ transplantation. This type of SWI injury is more severe in transplanted organs more sensitive to temperature changes. This study aimed to present the newly developed OrganPocket, an organ protector made from a proprietary elastomer material, and to demonstrate its efficacy in mitigating SWI injury in clinical kidney transplantation.MethodsWe used an ex vivo porcine organ model to evaluate OrganPocket. After removal, donor organs were immersed and cryopreserved in an organ preservation solution at 4°C before being placed in an OrganPocket. The organ graft and OrganPocket were held for 30 minutes in a 37°C environment mimicking intra-abdominal conditions while temperatures were recorded. Control organs were evaluated under the same conditions without an OrganPocket. In addition, we tested OrganPocket in an intra-abdominal porcine allograft transplant model.ResultsThe control organ group temperature reached ≥16°C after 30 minutes, while the mean core temperature in the OrganPocket organ group remained at no more than 10°C. Despite an SWI time of approximately 30 minutes, the surface organ temperature upon removal of OrganPocket was 20°C. Cardiac grafts also exhibited a normal heartbeat after reperfusion.ConclusionsOrganPocket is the world's first device designed to prevent SWI and should also prove useful for heart transplantation.     

Impact of Ex Vivo Administration of Mesenchymal Stem Cells on the Function of Kidney Grafts From Cardiac Death Donors in Rat

Background

Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia–reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue–derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD).

Methods

Using male Lewis rats (8–10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase–LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10⁶ cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 μL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, which were then preserved (1 hour; 22°C) before being transplanted into bilaterally nephrectomized recipient rats (n = 8). Serum was collected to assess the therapeutic effects of AT-MSC administration, and the recipients of rats surviving to Day 14 were separately evaluated histopathologically. Follow-up was by in vivo imaging and histological LacZ staining, and tumor formation was evaluated in MSC-injected rats at 3 months.

Results

Systemic injection of MSC did not improve recipient survival. In vivo imaging showed MSCs trapped in the lung that later became undetectable. Ex vivo injection of MSCs did show a benefit without adverse effects. At Day 14 after RTx, 75% of the rats in the AT-MSC–injected group (MSC[+]) had survived, whereas 50% of the rats in the AT-MSC–non-injected group (MSC[−]) had died. Renal function in the MSC(+) group was improved compared with that in the MSC(−) group at Day 4. LacZ staining revealed AT-MSCs attached to the renal tubules at 24 hours after RTx that later became undetectable. Histopathologic examination showed little difference in fibrosis between the groups at Day 14. No teratomas or other abnormalities were seen at 3 months.     

黄芪甲苷孵育的脂肪源性干细胞对顺铂诱导的急性肾损伤小鼠的保护作用

目的：建立顺铂诱导的急性肾损伤（AKI）小鼠模型,尾静脉输注人脂肪源性干细胞（hADSC）及黄芪甲苷孵育的人脂肪源性干细胞（Ast-hADSC）,探讨其对AKI小鼠肾损伤的修复作用.方法：应用PKH26标记hADSC及Ast-hADSC;雄性BALB/C裸鼠随机分为4组（normal组、model组、hADSC及Ast-hADSC干预组）,干预组及模型组腹腔注射顺铂建立AKI模型,于建模后24 h尾静脉输注100 μl hADSC及Ast-hADSC悬液,模型组给予等量生理盐水.分别留取建模后24 h及静脉输注后1 d、3 d、7 d、14 d小鼠血清及肾组织标本,检测血Scr、BUN水平,HE染色观察肾组织病理变化,TUNEL法检测肾小管上皮细胞凋亡,激光共聚焦观察hADSC及Ast-hADSC 在小鼠肾组织的分布.结果：模型组小鼠Scr、BUN、肾组织病理改变及细胞凋亡区域均显著高于正常组及细胞干预组,其中Scr、BUN分别在第3天、第7天达到最高值,肾小管损伤严重;相同时间点Ast-hADSC组肾组织病理改变及细胞凋亡区域较hADSC组明显降低,至14 d可基本恢复至正常水平;激光共聚焦显示PKH26标记的hADSC及Ast-hADSC于14 d在小鼠肾组织有少量表达,其中少量红色荧光与肾小管上皮细胞呈融合状态.结论：hADSC移植可减轻AKI小鼠肾脏结构和功能的损伤;经Ast孵育后,可在一定程度上提高hADSC对受损肾组织的修复作用.     

Cytokine Response to Ischemia/Reperfusion Injury in an Ex Vivo Perfused Porcine Liver Model

Background

We evaluated the degree of inflammatory response after ischemia/reperfusion injury by an extracorporeal normothermic autologous hemoperfusion of porcine livers.

Materials and Methods

Livers explanted from 7 pigs were perfused extracorporeally at 39°C with autologous blood. Serum samples were obtained hourly until 6 hours from the beginning of reperfusion and assayed for 9 different cytokines.

Results

Significant elevations in interleukin 6 (IL-6) and IL-8 were noted following reperfusion (P < .001), with both demonstrating an increase which followed a sigmoid curve; other cytokines that were assessed showed no significant change.

Conclusions

The ex vivo model excludes the liver from the influence of external systemic factors such as hormones, the autonomic nervous system, and other regulatory molecules produced elsewhere in the body, allowing the response to the ischemia/reperfusion injury to be studied in isolation and in considerable detail. Although this study examined a relatively short period, the increases in only IL-6 and IL-8 suggested that these are important molecules in the early phase after reperfusion.     

Reduction of Warm Ischemia Using a Thermal Barrier Bag in Kidney Transplantation: Study in a Pig Model

BackgroundWith recent advances in surgical technologies, minimally invasive endoscopic and robot-assisted surgical procedures have been introduced. However, prolonged warm ischemic time of the kidneys remains a concern after the organ is removed from a donor and during transplantation into a recipient. We developed a Thermal Barrier Bag (TBB) to prevent warm ischemia during transplantation. To confirm the effectiveness of the TBB, adenosine triphosphate (ATP) activity in the kidney was measured during an ex vivo warming test. An ischemia model porcine kidney was also used as the donor kidney and placed into the TBB; thereafter, the change in temperature at the time of transplantation was examined.Main FindingsThe purse-like design of the TBB efficiently suppressed heat conduction. A simulation was conducted that allowed the calculation of organ heat transfer condition. In the ex vivo experiment, temperature increases were suppressed in the group whose kidneys were placed in the TBB (30 minutes after transplantation: with TBB = 30°C, without TBB = 35°C). ATP measurements showed that the residual rate was substantially higher in the TBB group (P = .056). Moreover, a temperature suppression effect was demonstrated during the renal transplantation experiment (30 minutes after transplantation: with TBB = 27°C, without TBB = 31°C).ConclusionThe ex vivo warming experiment demonstrated that use of TBB slows down the rate of ATP decay in fresh kidneys. In addition, when an ischemic model porcine kidney was placed into the TBB and the temperature change at the time of transplantation was measured, an in vivo temperature-suppressing effect was observed.     

The Development of a Multiorgan Ex Vivo Perfused Model: Results With the Porcine Liver‐Kidney Circuit Over 24 Hours

We already developed an ex vivo liver‐kidney model perfused for 6 h in which the kidney acted as a homeostatic organ to improve the circuit milieu compared to liver alone. In the current study, we extended the multiorgan perfusions to 24 h to evaluate the results and eventual pitfalls manifesting with longer durations. Five livers and kidneys were harvested from female pigs and perfused over 24 h. The extracorporeal circuit included a centrifugal pump, heat exchanger, and oxygenator. The primary end point of the study was the evaluation of the organ functions as gathered from biochemical and acid‐base parameters. In the combined liver‐kidney circuit, the organs survived and maintained an acceptable homeostasis for different lengths of time, longer for the liver (up to 19–23 h of perfusions) than the kidney (9–13 h of perfusions). Furthermore, glucose and creatinine values decreased significantly over time (from the 5th and 9th hour of perfusion onward). The addition of a kidney to the perfusion circuit improved the biochemical environment by removing excess products from ongoing metabolic processes. The consequence is a more physiological milieu that could improve results from future experimental studies. However, it is likely that long perfusions require some nutritional support over the hours to maintain the organ's vitality and functionality throughout the experiments.     

Combined Kidney and Vascularized Total Bladder Transplantation: Experience in an Animal Model

Background

Few reports have described a partial bladder graft with an en bloc kidney transplantation, mainly to facilitate reconstruction of the urinary tract, but also to augment the native bladder. The present study assessed the feasibility to graft vascularized total bladder in association with a renal transplantation.

Methods

The right kidney, in continuity with the ureter and the entire bladder, was retrieved from three female pigs weighing 20 g. The visceral bloc was transplanted to three recipient pigs of the same weight. The entire bladder was transplanted with its vascular connection to ensure a better blood supply. After 3 days of observation, one recipient was humanely killed to examine the bladder graft. Oxygen saturation in the bladder graft monitored for 8 hours was compared with the native bladder in the other two recipients. All three bladder grafts were examined by a pathologist.

Results

All bladder grafts seemed to be macroscopically well-perfused upon removal of the vascular clamps. In case 1, the recipient was clinically well with good urinary output over the first 2 days of observation; is contrast, on day 3 the animal displayed an acute reduced urinary output. Laparotomy on day 3 of observation showed recent thrombosis of the bladder and renal graft vessels. In cases 2 and 3, oxygen saturations of the bladder graft were normal during the 8-hour observation period, without any difference between the graft and the native bladder.

Conclusions

According to our results, vascularized total bladder transplantation is feasible. In combination with renal transplantation, it could be applied as an alternative to bladder augmentation or total bladder replacement.     

Isolation and Ex Vivo Expansion of Bone Marrow-Derived Porcine Mesenchymal Stromal Cells: Potential for Application in an Experimental Model of Solid Organ Transplantation in Large Animals

Pharmacological aspecific immunosuppression, despite being widely used in solid organ transplantation recipients, is unable to completely prevent allograft rejection. It promotes the occurrence of sometimes life-threatening infections. Due to their immunosuppressive and anti- inflammatory properties, there is great interest in the therapeutic use of bone marrow (BM)-derived mesenchymal stromal cells (MSC). Large animal models play a crucial role to investigate the biological and functional properties of MSCs as novel cellular therapy. In the current study we sought to isolate expand ex vivo, and phenotypically characterize MSC derived from BM of 4 Large White 6-month-old piglets. Porcine MSC (pMSC) were characterized for their in vitro differentiation capacity. pMSC were successfully isolated from all BM samples. They showed spindle-shaped morphology and a stable doubling time on culture. They were positive for CD90, CD29, CD105, and negative for CD45 and CD11b. Furthermore, they differentiated, upon specific in vitro conditions toward adipogenic and osteogenic lineages. The optimization of methods for the isolation and characterization of pMSC may be useful to elucidate their biological and functional properties. The anatomy and physiology of the pig, which is similar to humans, make this animal model more attractive than small animals to test the safety and efficacy of MSC in the context of solid organ transplantation.     

微透析技术实时检测肾热缺血后葡萄糖变化的动物实验研究

目的探讨应用微透析技术研究兔肾热缺血再灌注损伤后肾皮质葡萄糖浓度变化规律的可行性。方法新西兰大耳白兔20只,其中实验组10只,麻醉后游离出右肾及其动静脉,将微透析探针置入肾背侧皮质内,复方氯化钠溶液持续灌注平衡60min后,连接电化学分析系统,阻断肾动静脉60min,然后开放动静脉,观察60min。利用电化学方法实时在线检测肾透析液中葡萄糖含量的变化。对照组10只,仅游离出肾动静脉而不结扎,同样留置微透析针取样,连续检测葡萄糖浓度,比较2组在正常灌注期、缺血期和再灌注期葡萄糖浓度的变化。结果葡萄糖电极的电流响应与其浓度呈良好的线性关系,微透析针的回收率在（63.6±2.1）%。实验组兔肾皮质正常状态下测得的透析液葡萄糖浓度为（1.89±0.37）mmol/L,缺血期葡萄糖浓度为（0.69±0.12）mmol/L（LSD检验,P=0.000）,缺血期较自身正常灌注期降低（36.7±2.4）%;再灌注期葡萄糖浓度为（0.62±0.14）mmol/L（LSD检验,P=0.000）。与对照组比较,实验组缺血期（t=-11.975,P=0.000）和再灌注期（t=-11.993,P=0.000）葡萄糖浓度显著降低。结论应用微透析与活体在线电化学技术相结合的方法可以方便、灵敏检测肾缺血再灌注损伤后葡萄糖水平,可以较好地实时反映肾皮质缺血状态。     

Kidney and Pancreas Transplantation in the United States, 1995–2004

This article examines OPTN/SRTR data on kidney and pancreas transplantation for 2004 and the previous decade, and discusses recent changes in kidney-pancreas (KP) allocation policy and emerging issues in kidney donation after cardiac death (DCD). Although the number of kidney donors continues to increase, new waiting list registrations again outpaced the number of kidney transplants performed, rising by 11% between 2003 and 2004 and contributing to a 1-year increase of 8% in the number of patients active on the waiting list. DCD has increased steadily since 2000; 39% more DCD transplants were performed in 2004 than 2003. Both deceased donor and living donor kidney graft survival rates remain excellent and are improving. The number of people living with a functioning kidney transplant doubled between 1995 and 2004, to 101 440 with a functioning kidney-alone and 7213 with a functioning KP. Health care providers in all settings are more likely to be exposed to these transplant recipients. Patient survival following simultaneous pancreas-kidney (SPK) transplantation is excellent and has improved incrementally since 1995; death rates in the first year fell from 60 per 1000 patient-years at risk in 2001 to 45 in 2003. The number of solitary pancreas transplants increased dramatically in 2004.     

Kidney and Pancreas Transplantation in the United States, 1996–2005

Kidney and pancreas transplantation in 2005 improved in quantity and outcome quality, despite the increasing average age of kidney graft recipients, with 56% aged 50 or older. Geography and ABO blood type contribute to the discrepancy in waiting time among the deceased donor (DD) candidates. Allocation policy changes are decreasing the median times to transplant for pediatric recipients. Overall, 6% more DD kidney transplants were performed in 2005 with slight increases in standard criteria donors (SCD) and expanded criteria donors (ECD). The largest increase (39%) was in donation after cardiac death (DCD) from non-ECD donors. These DCD, non-ECD kidneys had equivalent outcomes to SCD kidneys. 1-, 3- and 5-year unadjusted graft survival was 91%, 80% and 70% for non-ECD-DD transplants, 82%, 68% and 53% for ECD-DD grafts, and 95%, 88% and 80% for living donor kidney transplants. In 2005, 27% of patients were discharged without steroids compared to 3% in 1999. Acute rejection decreased to 11% in 2004. There was a slight increase in the number of simultaneous pancreas-kidney transplants (895), with fewer pancreas after kidney transplants (343 from 419 in 2004), and a stable number of pancreas alone transplants (129). Pancreas underutilization appears to be an ongoing issue.     

Glycemic Stability Through Islet‐After‐Kidney Transplantation Using an Alemtuzumab‐Based Induction Regimen and Long‐Term Triple‐Maintenance Immunosuppression

Pancreatic islet transplantation is performed in a select group of patients with type 1 diabetes mellitus. Immunosuppressive regimens play an important role in long‐term islet function. We aimed to investigate the efficacy of islet transplantation in patients with type 1 diabetes and a previous kidney transplantation using an alemtuzumab‐based induction regimen and triple maintenance immunosuppression. Patients with type 1 diabetes, who had received a kidney transplant previously, were treated with alemtuzumab as induction therapy for their first islet transplantation and basiliximab induction therapy for subsequent islet transplantations. Maintenance immunosuppression consisted of triple immunosuppression (tacrolimus, mycophenolate mofetil, and prednisolone). Thirteen patients (age 50.9 ± 9.2 years, duration of diabetes 35 ± 9 years) received a total of 22 islet transplantations. One‐ and 2‐year insulin independence was 62% and 42%, respectively; graft function was 100% and 92%, respectively. HbA1c dropped from 57.2 ± 13.1 (7.4 ± 1.2%) to 44.5 ± 11.8 mmol/molHb (6.2 ± 0.9%) (p = 0.003) after 2 years. Six of 13 patients suffered from severe hypoglycemia before islet transplantation. After transplantation, severe hypoglycemia was restricted to the only patient who lost graft function. Creatinine clearance was unchanged. Islet‐after‐kidney transplantation in patients with type 1 diabetes using an alemtuzumab‐based induction regimen leads to considerable islet allograft function and improvement in glycemic control.