Caspase inhibitor therapy enhances marginal mass islet graft survival and preserves long-term function in islet transplantation

Islet transplantation can provide insulin independence in patients with type 1 diabetes, but islets derived from two or more donors are often required. A significant fraction of the functional islet mass is lost to apoptosis in the immediate posttransplant period. The caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-FMK) has been used therapeutically to prevent apoptosis in experimental animal models of ischemic injury, autoimmunity, and degenerative disease. In the current study, zVAD-FMK therapy was examined in a syngeneic islet transplant model to determine whether caspase inhibition could improve survival of transplanted islets. zVAD-FMK therapy significantly improved marginal islet mass function in renal subcapsular transplantation, where 90% of zVAD-FMK-treated mice became euglycemic with 250 islets, versus 27% of the control animals (P < 0.001). The benefit of zVAD-FMK therapy was further demonstrated after intraportal transplantation, where 75% of zVAD-FMK-treated animals established euglycemia with only 500 islets, and all of the controls remained severely diabetic (P < 0.001). zVAD-FMK pretreatment of isolated islets in the absence of systemic therapy resulted in no significant benefit compared with controls. Long-term follow-up of transplanted animals beyond 1 year posttransplant using glucose tolerance tests confirmed that a short course of zVAD-FMK therapy could prevent metabolic dysfunction of islet grafts over time. In addition, short-term zVAD-FMK treatment significantly reduced posttransplant apoptosis in islet grafts and resulted in preservation of graft insulin reserve over time. Our data suggest that caspase inhibitor therapy will reduce the islet mass required in clinical islet transplantation, perhaps to a level that would routinely allow for insulin independence after single-donor infusion.     

Reduction of early graft loss after intraportal porcine islet transplantation in monkeys

BACKGROUND: Pig islets constitute a possible resolution to the shortage of human islets for transplantation. After intraportal infusion of porcine islets in primates, many islets are lost through what has been termed the instant blood-mediated inflammatory reaction (IBMIR). We report on our experience with IBMIR. METHODS: Ten monkeys underwent intraportal porcine islet transplantation. Immunosuppressive therapy was with conventional agents (n=3) or based on costimulation blockade (n=7). Treatment specific for IBMIR was administered in eight monkeys; two additional monkeys received no such therapy (group 1). Cobra venom factor completely inhibited complement activity in four (group 2) and dextran sulfate provided anticoagulation in four (group 3). Islet graft function was monitored by following blood glucose, insulin requirement, and porcine C-peptide values. RESULTS: In monkeys that received neither cobra venom factor nor dextran sulfate (group 1), there was rapid destruction of islets indicated by severe hypoglycemia and the need for dextrose infusion; C-peptide levels were initially low and further reduction occurred within the first five days. In both groups 2 and 3, there was significantly less destruction of islets and some reversal of diabetes. However, when 40,000 IEQ/kg were infused, normoglycemia was lost within five days, but when 80,000 IEQ/kg were infused in one case, normoglycemia was more persistent. We observed that even when C-peptide levels were in the normal range for healthy nondiabetic pigs, these were not sufficient to maintain normoglycemia in the monkeys. CONCLUSIONS: Although pretransplantation complement depletion or anticoagulation reduces porcine islet xenograft loss significantly, neither alone is sufficient to prevent IBMIR.     

Progressive deterioration of endocrine function after intraportal but not kidney subcapsular rat islet transplantation

In inbred streptozocin-induced diabetic rats, the long-term function of different endocrine pancreatic isografts was compared. Isolated islets transplanted into the portal vein showed a progressive deterioration of function over time. In contrast, islets under the kidney capsule sustained a constant long-term function controlling all clinical signs of diabetes. Recipients of kidney subcapsular islets displayed normal growth rate, peripheral serum glucose and insulin levels, and metabolic parameters. However, their functional reserve was markedly reduced as revealed by diminished glucose tolerance and reduced insulin-secreting capacity after an intravenous glucose challenge. Vascularized whole-organ pancreatic grafts with portal venous drainage led to complete normalization of all parameters determined in this study. This study showed that the long-term function of islets transplanted under the kidney capsule is superior compared with islets transplanted into the portal vein.     

Desmopressin impairs microcirculation in donor pancreas and early graft function after experimental pancreas transplantation

BACKGROUND: Protective effects of desmopressin in brain dead organ donors oppose reports on a hypercoagulatory potential and an increased leukocyte-endothelial interaction (LEI) after application of the drug. The aim was to evaluate the effect of desmopressin on organ donor's pancreas and early graft function. METHODS: Donor microcirculation was evaluated via intra-vital microscopy (IVM) in 24 BR (di/di) rats with central diabetes insipidus, randomly assigned to groups I (control without desmopressin application), II (single i.v. application, no pretreatment) or group III (single i.v. desmopressin application, s.c. pretreatment for 3 days). Microcirculation in recipients was evaluated 1 hr and 6 hr after syngenic pancreas transplantation. Groups III and I served as organ donors. After IVM specimens were taken for histology and immunohistochemistry. RESULTS: Desmopressin in II vs. I led to temporarily (30') increased LEI (Sticker 274.3+/-87.7 vs. 76.5+/-31.1/mm2 endothelial surface; P<0.01) and impaired microcirculation (MCEV 0.43+/-0.07 vs. 0.99+/-0.06 mm/s; P<0.01). Repeated application reduced MCEV and increased LEI for up to 12 hr. Histology in I vs. III showed increased inflammation (n.s.), necrosis (P<0.05) and vacuolization (P<0.01). Immunohistochemistry revealed increased endothelial P-selectin 20' after application. 6 hr after reperfusion organs from III showed reduced MCEV and increased LEI (P<0.01). CONCLUSION: Repeated application of desmopressin impairs graft microcirculation. Perfusion of the pancreas is significantly reduced at the beginning of organ tissue conservation as well as after reperfusion. These disturbances might partly be due to observed endothelial P-selectin expression. Application of desmopressin up to 12 hr prior to organ explantation may impact graft quality.     

A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site

Intraportal site is the standard for grafting in clinical islet transplantation. In the mouse model, the whole liver has been used as the grafting site to mimic clinical islet transplantation. However, this model lacks the potency to directly assess the contribution of the islet graft to diabetes control. Only demonstrating the immediate recurrence of diabetes in a surviving recipient after the removal of the islet graft can validate this assessment. In this study, we develop a mouse model of intraportal islet transplantation equipped with the potency of this assessment by injecting islets selectively into the right hepatic lobe under temporal clamp of the left portal vein. The mouse of this model survives after the right hepatectomy by which the islet graft is removed. This model can be applied to investigate both the specific graft-recipient interaction in the liver and the islet graft contribution to the control of diabetes.     

Importance of renal mass on graft function outcome after 12 months of living donor kidney transplantation.

BACKGROUND: Few studies have directly measured the kidney weight and investigated donor parameters related to it. The aim of this study was to evaluate the kidney weight and its relationship to creatinine clearance (CrCl) after 12 months post-transplantation. METHODS: A total of 123 recipients of renal transplantation from living donors were evaluated. Demographic and anthropometric data from donors and recipients were collected in the pre-operative phase. Data about kidney weight were obtained through kidney measurement using an electronic weighing machine at the moment of transplantation. Glomerular filtration rate (GFR) was estimated through CrCl (modification of diet in renal disease formula) at the 1st, 6th, 12th and 18th month post-transplantation. RESULTS: The mean value of kidney weight was 170 +/- 31 g (166.4 +/- 29.2 g in women and 177.5 +/- 32.5 g in men). The kidney weight had a correlation with the donor's BMI (r = 0.43, P < 0.001) and with the CrCl on the 12th month (r = 0.31, P = 0.001). Using multiple linear regression, the kidney weight could be predicted through the BMI and donor's gender (R(2) = 0.21; P < 0.01). The CrCl after 12 months had a significant correlation with the graft weight/recipient weight ratio and with the donor age (R(2) = 0.22; P < 0.01). CONCLUSION: The kidney weight can be estimated using the donor's gender and BMI. The kidney weight significantly influences the CrCl 12 months after transplantation.     

Prevention of early islet graft failure by selective inducible nitric oxide synthase inhibitors after pig to nude rat intraportal islet transplantation

BACKGROUND: Clinical and experimental data indicate that early failure of intraportally grafted islets is caused by inflammation including secretion of cytokines and nitric oxide. Direct inducible nitric oxide synthase suppression may avoid detrimental effects associated with steroid administration. We compared the efficiency of selective and unselective inducible nitric oxide synthase inhibitors with dexamethasone to suppress nitric oxide generation after intraportal islet xenotransplantation into nude rats. METHODS: Nonfasting serum glucose levels were daily evaluated after intraportal transplantation of 4000 freshly isolated pig islets into diabetic nude rats (85 mg/kg streptozotocin) either sham-treated with saline (n=21) or continuously infused for 7 days with L-NG-monomethyl-arginine (n=7), S-methyl-isothiourea (n=15), or S-(2-aminoethyl)-isothiourea (n=19) in a dosage of 240, 100, or 50 mg/kg/day, respectively. Dexamethasone was injected i.p. twice as a daily bolus of 20 mg/kg (n=10) starting 1 day pretransplant. The nitrate/nitrite serum level was quantified colorimetrically 0, 24, and 48 hr posttransplant. RESULTS: Saline treatment partially resulted in graft function (4/21) throughout the observation period (21 days). L-NG-monomethyl-arginine-treated rats showed sustained hyperglycemia (0/7) not different from diabetic controls. Normoglycemia was observed after treatment with dexamethasone (6/10, P<0.05 versus saline and L-NG-monomethyl-arginine), S-methyl-isothiourea (10/15, P<0.01), or S-(2-aminoethyl)-isothiourea (15/19, P<0.001). Graft function was associated with complete suppression of nitric oxide generation after S-methyl-isothiourea and S-(2-aminoethyl)-isothiourea treatment (P<0.001 versus saline) and partial suppression after dexamethasone treatment (P<0.05). CONCLUSIONS: Our observation of long-term function of xenogeneic islets in an inflammatory environment without interference of reactive T cells revealed the potency of highly selective isothioureas to completely suppress inducible nitric oxide synthase making reduction of islet-toxic immunosuppression feasible.     

Identification of in vitro parameters predictive of graft function: a study in an animal model of islet transplantation

The quality of human islets is one of the factors decisive for the success of human islet transplantation. Several parameters have been proposed to characterize islet quality, but none of them has been able to predict the fate of a transplant. The aim of our study was to correlate a panel of in vitro parameters for islet viability with their in vivo function after transplantation in nude mice. Islets were obtained after enzymatic digestion of a human pancreas; they were purified from exocrine tissue using a continuous-density gradient. Two aliquots of islets (1000 and 2000 islets) were transplanted under the kidney capsule of diabetic nude mice. The animals were followed for 1 month with repeated measurements of blood glucose and body weight. One month after transplantation, mice were killed and their graft harvested for histologic analysis. In parallel we studied in vitro islet viability with propidium iodide and fura-2, their insulin content, their purity, and their insulin response to glucose upon static incubation. Ten islet preparations were transplanted: 3 out of 10 preparations did not restore normoglycemia; 4 out of 10 normalized glycemia only in mice receiving 2000 islets, and 3 out of 10 fully restore normoglycemia in all mice. The purity of preparations (R(2) = 0.63 and 0.85, respectively, with 1000 and 2000 islets) and the insulin content (R(2) = 0.75 with 2000 IE) correlated with transplant success. These data show that purity of islet preparations and their insulin content should be useful parameters for the selection of islet preparations for transplant purposes.     

Influence of donor and recipient gender on early graft function after living donor kidney transplantation

Long-term effects of donor and recipient gender on the outcome of living donor kidney transplantation have been examined but the impact on early graft function is less certain. In this study, we analyzed age, gender, body weight, height, body surface area (BSA), and lean body weight (LBW) of both donors and recipients. Preoperatively we collected 24-hour urine samples to measure creatinine excretion from donor and postoperatively we determined when the recipient serum creatinine (Scr) reached baseline levels. Variables included were ischemic times, kidney graft weight, duration of dialysis, cause of end-stage renal disease (ESRD), degree of HLA match, and mismatch, types of immunosuppression (cyclosporine or FK506, dual or triple), and episodes of acute rejection. The variables were analyzed by independent sample t tests and chi-square statistics using SPSS. Values of P < .05 were considered significant. Male patients of both donors and recipients were significantly taller and heavier (higher BSA and LBW) than female. Urinary 24-hour creatinine excretion was greater in male patients whether donors or recipients. There were no statistical differences in graft weight or creatinine clearance based on the gender of the donor or recipient. The creatinine of male donors or recipients was higher than that of females. The other variables were not significantly different. In conclusion, the effect of donor or recipient gender on early graft function depends on the metabolic demands, which are higher in male recipients.     

Metabolic demand and renal mass supply affecting the early graft function after living donor kidney transplantation

BACKGROUND: Graft mass has been demonstrated to be a determinant of outcome after kidney transplantation. An insufficient nephron might fail to meet the metabolic demands of the recipient and lead to hyperfiltration. METHODS: The study population was restricted to live donor transplants demonstrating immediate function that had neither ischemic injury, episodes of rejection, nor any complications that resulted in a functional decrease of the graft. The donated kidney was weighed just after cold flush, and the recipient's serum creatinine (Scr) was measured on a daily basis postoperatively. When the recipient's Scr reached the baseline, the recipient's 24-hour urine was collected for the amount of proteinuria (Upr), creatinine excretion (Ucr), and creatinine clearance (Ccr) calculation. As the parameters of the metabolic demands of donor and recipient, body weight, height, body surface area, lean body weight, and body mass index were noted. Pearson correlation and linear regression were carried out. RESULTS: The graft function, as measured by Scr, Ucr, and Upr, was not directly correlated with the graft weight but rather correlated with the ratios of graft weight to the parameters of recipient's metabolic demands. As recipient size increased, the metabolic demand has increased. The parameters of recipient's metabolic demands were directly correlated with Scr and Ucr, rather than with Upr. CONCLUSION: During living donor and recipient matching, both the potential sizes of the donated kidney and the recipient should be considered in terms of the early graft function after transplantation.     

Effect of intraportal infusion to improve small for size graft injury in living donor adult liver transplantation

The most important problem in the living donor adult liver transplantation (LDALT) is a small for size graft. Although a right lobe graft is used in many cases in order to avoid small for size graft, for a donor, the risk has few in left lobe graft. We evaluate the effect of an intraportal infusion treatment to the small for size graft. One hundred and twelve patients who underwent LDALT were studied. The graft weight recipient standard liver volume ratio (GV/SLV) of these patients were 50% or less. We divided the patients into following two groups; infusion group (n = 53) and control group (n = 59). For the infusion group, 16 G double lumen catheter was inserted into portal vein and nafamostat mesilate (protease inhibitor which stabilize coagulofibrinolytic state; 200 mg/day), prostaglandin E(1) (vasodilator and hepatoprotective effect; 500 microg/day) and thromboxane A(2) synthetase inhibitor (vasodilator and anticoagulant effect; 160 mg/day) were administrated continuously for 7 days. Small-for-size graft syndrome was defined as bilirubin >10 mg/dl and ascites >1000 cc on postoperative day (POD) 14. Comparison examination of a background factors and postoperative bilirubin and amount of ascites was carried out. The mean GV/SLV did not have the difference at 39.1% of infusion group, and 38.3% of control group (P = 0.58). By the control group, 15 patients (25.4%) were small-for-size graft syndrome, however, there was only two (3.8%) small-for-size graft syndrome in infusion group (P = 0.04). The bilirubin levels of infusion and control group on 7 and 14 POD were 9.9 and 7.8 vs. 9.5 and 10.5 mg/dl, respectively. The amount of ascites of infusion group on 7 and 14 POD were 870 and 430 cc, respectively. On the contrary, in control group, the amount of ascites on 7 and 14 POD were 1290 and 1070 cc, respectively. Bilirubin levels and the amount of ascites on 7 and 14 POD were lower in the patients with infusion group then those with control group. There were no differences between infusion group and control group in age, sex and Child's classification. The intraportal infusion had an effect in prevention of hyperbilirubinemia and loss in quality of excessive ascites in the patients with small for size graft. This was suggested to be what is depended on the improvement of the microcirculation insufficiency considered one of the causes of small-for-size graft syndrome.     

Pancreas islet transplantation in the genitourinary tract associated with renal transplantation: an experimental study

Conceptually, pancreas islet transplantation (PIT) associated with renal transplantation (RT) should resolve not only chronic renal failure but also diabetes. Although the most frequently used site for PIT is the portal vein, genitourinary locations could be technically feasible during RT. Seventeen pigs (age 3 to 4 months; mean weight 34.5 kg) underwent the following experimental steps: On day 1 a left nephrectomy was performed and the kidney was perfused with cold Wisconsin solution. This was followed by a caudal pancreatectomy and islet isolation by means of digestion with intraductal collagenase. Islets were stained with Dithizone and cultured overnight al 37 degrees C and 5% CO(2). On day 2 a right nephrectomy and orthotopic RT of the preserved left kidney were performed. The islets were transplanted into four different sites: subcapsular in the kidney graft, in the bladder submucosa, in the testis by puncture, and in the testis by infusion through the vas deferens. On day 7 the animals were sacrificed. Islet viability was determined by histological examination with insulin immunostaining and determination of insulin in the blood of the veins draining the implantation sites. The mean weight of the pancreatic specimens was 27.8 g (13 to 46). The mean number of islets was 536,000 (16,600 to 1,5000,000). Islets were shown in the bladder submucosa and the testes after vas deferens infusion. The number of viable islets in the other implantation sites was very scarce. The insulin levels of the venous effluents were: 15.1 microU/mL for bladder submucosa, 10.2 microU/mL for intradeferential injection in the testis, 7.3 microU/mL for intratesticular injection by puncture, and 2.6 microU/mL for subcapsular implantation in the graft. In conclusion, the bladder submucosa and testis via the vas deferens might represent alternative sites for PIT. The latter route may benefit from the immunoprivileged and special trophic conditions of the testis. For the first time, the feasibility of the bladder submucosa as an implantation site for pancreas islets was demonstrated.     

Influence of laparoscopic live donor nephrectomy in ischemia-reperfusion syndrome and renal function after kidney transplantation: an experimental study

The increase of intra-abdominal pressure during laparoscopic techniques provokes oliguria and reduction of the renal blood flow (RBF). The aim of this study is to evaluate this effect during living donor nephrectomy and its influence in the ischemia-reperfusion syndrome and renal function after kidney transplantation. Autotransplantation was performed using 22 pigs (15 after conventional open nephrectomy and 7 after laparoscopic nephrectomy). During donor nephrectomy a significant reduction in RBF was observed in the laparoscopic group (70 mL/min) vs the open group (260 mL/min) (P<.05). After a cold ischemia period of 24 hours an autotransplantation was performed. During the first hour after revascularization RBF was lower for the laparoscopic than for the open group: 60 vs 180 mL/s at 1 minute and 160 vs 400 mL/s at 60 minutes (P<.05). The decrease of creatinine was slower for the laparoscopic than for the open group during the first posttransplant week (2 vs 1.3 mg/dL on the first day and 1.4 vs 0.8 mg/dL on the seventh day posttransplant, respectively) (P<.05).