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Preconditioning donor with a combination of tacrolimus and rapamacyn to decrease ischaemia–reperfusion injury in a rat syngenic kidney transplantation model

Reperfusion injury remains one of the major problems in transplantation. Repair from ischaemic acute renal failure (ARF) involves stimulation of tubular epithelial cell proliferation. The aim of this exploratory study was to evaluate the effects of preconditioning donor animals with rapamycin and tacrolimus to prevent ischaemia–reperfusion (I/R) injury. Twelve hours before nephrectomy, the donor animals received immunosuppressive drugs. The animals were divided into four groups, as follows: group 1 control: no treatment; group 2: rapamycin (2 mg/kg); group 3 FK506 (0, 3 mg/kg); and group 4: FK506 (0, 3 mg/kg) plus rapamycin (2 mg/kg). The left kidney was removed and after 3 h of cold ischaemia, the graft was transplanted. Twenty‐four hours after transplant, the kidney was recovered for histological analysis and cytokine expression. Preconditioning treatment with rapamycin or tacrolimus significantly reduced blood urea nitrogen and creatinine compared with control [blood urea nitrogen (BUN): P < 0·001 versus control and creatinine: P < 0·001 versus control]. A further decrease was observed when rapamycin was combined with tacrolimus. Acute tubular necrosis was decreased significantly in donors treated with immunosuppressants compared with the control group (P < 0·001 versus control). Moreover, the number of apoptotic nuclei in the control group was higher compared with the treated groups (P < 0·001 versus control). Surprisingly, only rapamycin preconditioning treatment increased anti‐apoptotic Bcl2 levels (P < 0·001). Finally, inflammatory cytokines, such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐6, showed lower levels in the graft of those animals that had been pretreated with rapamycin or tacrolimus. This exploratory study demonstrates that preconditioning donor animals with rapamycin or tacrolimus improves clinical outcomes and reduce necrosis and apoptosis in kidney I/R injury.

Donor preconditioning with rabbit anti-rat thymocyte immunoglobulin ameliorates ischemia reperfusion injury in rat kidney transplantation.

A major concern in transplantation is the preservation of organ function. Ischemia time and microcirculatory disturbance of the organ cannot be avoided and may result in ischemia reperfusion injury (IRI), increasing the risk of delayed graft function (DGF) and acute and chronic rejection. Anti-thymocyte immunoglobulin (rATG) is a polyclonal antibody preparation with multiple effects when administered to recipients. Our objective has been to evaluate whether the administration of rATG to kidney donors instead of recipients, in an experimental model of syngeneic rat transplantation, ameliorates IRI and facilitates immediate graft function recovery. Urea and creatinine levels and necrosis severity scores were significantly lower in kidneys from donors that had received rATG (urea: control: 211±8mg/dl vs. treatment: 110±15mg/dl, p<0.001; creatinine: control: 4.6±0.24mg/dl vs. treatment: 2.6±0.22mg/dl, p<0.001; necrosis severity scores: control: 2.3 vs. treatment: 1.6, p<0.05). TUNEL staining showed 80±13 positive cells in control group and 9±3 (p<0.001) in treatment group. In situ expression of proinflammatory cytokines TNF-α, IL-6, IL-21 and TGF-β1 was reduced in rATG group (p<0.01); the same was observed for KIM-1 and caspase 8 (p<0.001). Cytoprotective genes Bcl2 and HO-1 were upregulated in situ in treatment group (p<0.001). In situ expression of IL-17, caspase 9, IL-23a, CxCl3 and ICAM1 showed no difference between groups (p>0.05). Findings suggest ATG administered to donors may ameliorate the IRI process in kidney transplantation, expressed by lower necrosis and apoptosis scores and the improvement of renal function, which may be explained through the diminished in situ expression of inflammatory mediators.

Defining the Nonreturn Time for Intestinal Ischemia Reperfusion Injury in Mice

Among the abdominal organs, the intestine is probably the most sensitive to ischemia reperfusion injury (IRI), a phenomenon that occurs in many intestinal disorders. Few studies have reported in detail the impact of intestinal ischemia time in mice. We evaluated the effect of various warm intestinal ischemia times in an intestinal IRI model in mice. Adult male Balb/c mice were divided into 4 groups that differed in intestinal ischemia time: G1, 30; minutes; G2, 35 minutes; G3, 40 minutes; and G4, 45 minutes. Histological evaluation showed average Park scores as follows: G1 0.6 ± 0.55; G2 1.8 ± 0.45; G3 4.8 ± 2.25; and G4 5 ± 1.79. All animals from G1 survived 30 hours. G2 animals showed intermediate behavior with all succumbing between 18 and 30 hours postprocedure. G3 and G4 displayed similar survival results with animals succumbing before 6 hours after intestinal reperfusion. These data showed that Park index scores of 3 or higher were related to early death. We concluded that the 5 minutes between 35 and 40 minutes is the critical limit, after which all mice die after reperfusion. This result may represent a valuable tool for future research in mice.

Protective effect of immunosuppressive treatment before orthotopic kidney autotransplantation

BACKGROUND Ischemia reperfusion injury (IRI) is one of the risk factors for delayed graft function, acute rejection and long term allograft survival after kidney transplantation. IRI is an independent antigen inflammatory process that produces tissue damage. Our objective was to study the impact of immunosuppressive treatment (IS) on IRI applying only one dose of IS before orthotopic kidney autotransplantation. METHODS Twenty-four rats allocated in four groups were studied. One group served as control (G1: autotransplanted rats without IS) and the rest received IS 12 h before kidney autotransplantation (G2: Rapamycin, G3: Mycophenolate mofetil and G4: Tacrolimus). RESULTS Improved renal function and systemic inflammatory response were found among IS groups compared to the control group (Delta Urea p<0.0001; Delta Creatinine p<0.0001; Delta C3 p<0.001). The number of apoptotic nuclei in renal medulla in G1 was higher than in IS groups (p<0.0001). Tubular damage was less severe in IS groups respecting G1 (p<0.001). C3, TNF-α and IL-6 expression in kidney samples was reduced when IS was used compared to the control group. No differences were observed among the different immunosuppressive drugs tested. However, Heme oxygenase-1(HO-1) was increased only in Rapamycin treatment. CONCLUSIONS These data suggest that the use of IS administered before transplant attenuates the IRI process after kidney transplantation in an animal model.

Amelioration of renal damage by administration of anti-thymocyte globulin to potential donors in a brain death rat model

Brain death (BD), a non‐immunological factor of renal injury, triggers an inflammatory process causing pathological signs of cell death in the kidney, such as necrosis and apoptosis. Kidneys from brain dead donors show lower success rates than kidneys from living donors and one strategy to improve transplantation outcome is to precondition the donors. For the first time, anti‐rat thymoglobulin (rATG) was administered in an experimental brain death animal model to evaluate if it could ameliorate histopathological damage and improve organ function. Animals were divided into three groups: V (n = 5) ventilated for 2 h; BD (n = 5) brain death and ventilated for 2 h; and BD+rATG (n = 5) brain death, ventilated for 2 h, rATG was administered during brain death (10 mg/kg). We observed lower creatinine levels in treatment groups (means): V, 0·88 ± 0·22 mg/dl; BD, 1·37 ± 0·07 mg/dl; and BD+rATG, 0·64 ± 0·02 mg/dl (BD versus BD+rATG, P < 0·001). In the BD group there appeared to be a marked increase of ATN, whereas ATN was decreased significantly in the rATG group (V, 2·25 ± 0·5 versus BD, 4·75 ± 0·5, P < 0·01; BD+rATG, 2·75 ± 0·5 versus BD 4·75 ± 0·5 P < 0·01). Gene expression was evaluated with reverse transcription–polymerase chain reaction; tumour necrosis factor (TNF)‐α, interleukin (IL)‐6, C3, CD86 showed no significant difference between groups. Increased IL‐10 and decreased CCL2 in BD+rATG compared to BD (both cases P < 0·01). Myeloperoxidase was increased significantly after the brain death setting (V: 32 ± 7·5 versus BD: 129 ± 18). Findings suggest that rATG administered to potential donors may ameliorate renal damage caused by BD. These findings could contribute in the search for specific cytoprotective interventions to improve the quality and viability of transplanted organs.

Evaluation of histological damage of solid organs after donor preconditioning with thymoglobulin in an experimental rat model.

Rabbit anti-rat thymoglobulin (rATG) administered to donors with brain death (BD) may improve organs quality. We explored the effects of rATG administered to BD donors in the histology of heart, lungs and small bowel in a rat experimental model. Animals were randomly assigned to 3 groups: V (n=5) no BD, 2h ventilation; BD (n=5) BD and 2h ventilation; BD and rATG: BD, 2h ventilation, rATG (10mg/kg) after BD diagnosis. Histopathological damage scores were based on neutrophil infiltration, airway epithelial cell damage, interstitial edema, hyaline membrane formation, and pulmonary hemorrhage (lungs); neutrophil infiltration and interstitial edema (heart); Park score (bowel). Lung damage was significantly lower in BD+rATG group: V 5 ± 1.6; BD 11.25 ± 0.5, BD+rATG 6.5 ± 1.9 (p<0.01). Heart: V 2.0 ± 0.81; BD 4.75 ± 1.25 and BD+rATG 3.5 ± 1.7 (p>0.05). Small bowel: BD 2.25 ± 0.96 vs. BD+rATG 1.00 ± 1.15 (n.s.). Histological damage amelioration in lung and attenuation tendency in heart and small bowel encourages research of cytoprotective strategies to improve organ viability.

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Background Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function. Methods ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. Results The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. Conclusions Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.

Characterization of Acute Cellular Rejection in the Different Layers of Rat Transplanted Intestines

Introduction Intestinal transplantation (IT) faces many challenges, among them, the necessity to understand and detect rejection processes. Rodent models of IT are used to provide evidence for intervention strategies as well as improve knowledge of IT biology. Our aim was to determine the kinetics of small bowel rejection with emphasis in the characterization of acute cellular rejection (ACR) in the different layers of the graft since most of the information coming from the clinics is on mucosal layer, the only one that is accessible by endoscopic biopsies. Methods Allogeneic (ALLO) heterotopic IT in rats was performed following standard procedure. ACR was diagnosed by H-E staining analysis. Also, real-time PCR from microdissected samples (epithelial, muscular and serosa layer) and whole graft to determine gene expression was performed at 5 and 10-12 postoperative days (POD). An Isogenic IT group was performed as a control. Results ACR was observed since 5 POD in ALLO group with mild rejection as the most characteristic grade. Severe ACR was diagnosed in all ALLO samples at 10-12 POD. Descriptive analysis showed a well-preserved architecture at 5 POD; confluent and loose apoptotic cells in the intestinal epithelium and perivascular infiltrate in all layers were evident. At 10-12 POD, significant cellular infiltrate, epithelial damage, ulcers and an increase of apoptotic cells were observed. Muscular and serosa layers showed inflammatory cell infiltrate and intercellular edema. At 5 POD, some markers were consistently increased in ALLO groups such as CXCL10 that showed a 120 ± 80-fold increase compared with nontransplanted tissue. Furthermore, IFNg and IDO showed a trend to be increased at these time points. Remarkably, other inflammation-related genes, such as CXCL1 and IL6 showed consistent increase in ALLO groups at 10-12 POD, when severe ACR was established. When a principal component analysis of the overall gene expression markers was performed, ALLO group at 10-12 POD clearly separated from the other conditions. The analysis of gene expression at different layers of the graft was coincident with whole tissue biopsies: higher levels of IL6 and CXCL1 were observed in ALLO groups at 10-12 POD with important activation of this response in serosa and muscular layer. Interestingly, IL22 expression was only measurable in epithelial layer in ALLO groups at 10-12 POD, indicating ACR-induced expression in this compartment. Serosa layer showed some of the highest relative increases in proinflammatory gene expression also in ALLO groups at 10-12 POD. Conclusion Although in the clinic mucosal rejection has been extensively characterized, in our animal model we could document that all graft layers are affected by ACR since the initial stages. Serosa and muscular layer show high expression of proinflammatory markers, with differential expression of IL22 in the epithelial compartment. This information could be useful in the search for early biomarkers of ACR.

Role of Adipose Tissue-Derived Mesenchymal Stromal Cells in Preventing Rejection in Rat Intestinal Transplantation

Aim and Background Standard immunosuppressants to prevent rejection in solid organ transplantation are not exempt from complications. Mesenchymal stromal cells (MSC) have been proposed as a promising alternative, either alone or combined, both for its safety and for its regenerative and immunomodulatory properties. In experimental models of intestinal transplantation, there is only one published article describing their advantages. Interestingly, we found different results in our study using adipose tissue-derived MSCs (Ad-MSC), more easily obtainable and richer in MSCs. Additionally, we compared the efficacy of allogeneic versus autologous Ad-MSCs. Also, we tested the feasibility of the intraarterial route to administer the cells, thus ensuring their arrival to the graft after reperfusion. Methods An acute rejection rat model after allogeneic intestinal transplantation was developed, using highly histoincompatible strains (Brown Norway as donors and Lewis as recipients). Four experimental groups were established: 1.-Control (n=6), 2.-Tacrolimus 5 days (n=4); 3.-Control + MSCs (n=11); 4.-Tacrolimus 5 days + MSCs (n=11), all of them euthanized in the 14th POD day. One additional group with daily Tacrolimus until euthanasia was used as negative control for rejection (n=6). MSCs were isolated from adipose tissue of Sprague rats (Third-party allogeneic cells) in half of them (n=6 and n=5), and from Lewis rats (Autologous MSCs) in the other half (n=5 and n=6, respectively). 1.5-2 x 106 MSCs diluted in 1 ml of Ringer Lactate were injected through the superior mesenteric artery, just before reperfusion. Non parametric tests were used to analyze differences between groups, regarding overall survival, development and severity of rejection, clinical features, immune cell subpopulations in the graft, and plasmatic cytokine expression. Results No surgical complications appeared after the intraarterial administration of the cells. We found some significant differences in the cell count of different immune cell subpopulations (macrophages CD68+, CD4+, CD8+, CD20+ and Foxp3+) supporting an immunomodulatory role of the Ad-MSCs (p<0.05). We also found differences in the plasmatic cytokine and chemokine secretion, as for those with proinflammatory as with antiinflammatory profile. However, we did not observe differences between the third party and the autologous groups. Neither did we find significant differences in survival or in the degree of rejection in those animals treated with additional MSCs. Conclusion Despite the findings supporting an immunomodulatory role, it is possible that the MSCs administered just during the transplant did not prevent clinical rejection due to the proinflammatory environment at that moment, redirecting the tolerogenic role of these cells towards one more proinflammatory, able to face the immunological insult of the transplant. This study opens the door to future studies which will probably give us the key to administer the MSCs in the optimal moment, and with the correct dose, administration route, and environmental conditions, to get the expected effect.

Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.