Rat Renal Transplantation Research Articles

Use of Sphingosine-1-Phosphate 1 Receptor Agonist, KRP-203, in Combination with a Subtherapeutic Dose of Cyclosporine A for Rat Renal Transplantation

We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts. We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-1-phosphate (S1P)1 and S1P3 receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model. Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P1 and S1P3 receptors, KRP-203-P selectively activated S1P1, but not the S1P3 receptor (EC50:>1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P<0.05, survival time: 9.8 days (CsA) vs. >27.4 days (CsA+KRP)). Although a higher dose of CsA (3 mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P<0.05, creatinine clearance: 0.41+/-0.25 ml/min [CsA] vs. 1.15+/-0.16 ml/min [CsA+KRP]). The selectivity of KRP-203 for S1P1 reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.

NADPH oxidase subunits (NOX-1, p22phox, Rac-1) and tacrolimus-induced nephrotoxicity in a rat renal transplant model

TGF-beta and oxidative stress are known mediators of renal injury. However, the precise mechanisms by which TGF-beta and oxidative stress may be involved in the development of nephrotoxicity are not known. We examined whether anti-TGF-beta antibody limits nephrotoxicity produced by tacrolimus (TAC) and whether this altered genes that regulate oxidative stress. Renal transplants were performed in Wistar-Furth and Lewis rat strains. Groups included: isograft controls; untreated allografts; allografts treated with 0.25 mg/kg TAC till 90 days with or without 1.0 mg/kg anti-TGF-beta antibody or control antibody. Serum creatinine and BUN levels and renal histology were determined. Real time PCR and western analysis were used to quantify mRNA and protein expression. BUN and creatinine were elevated in TAC-treated rats. TAC increased expression of TGF-beta (37-fold) and NADPH oxidase subunits, NOX-1 (18-fold), p22(phox) (31-fold) and Rac-1 mRNA (20-fold), respectively. Contrariwise, expression of antioxidant genes, superoxide dismutase (SOD) and thioredoxin (TRX) was decreased. Anti-TGF-beta antibody but not control antibody reversed the TAC-induced changes in gene expression, renal histology and function. Our findings suggest a potential for anti-TGF-beta antibody as a novel adjunct therapeutic tool to prevent TAC-induced nephrotoxicity in transplant recipients. The mechanism of protection involves suppression of TGF-beta and the expression of genes that regulate oxidative stress. Moreover, the specific up-regulation of NOX-1, a non-phagocytic NADPH oxidase subunit and its reversal by anti-TGF-beta antibody strongly implicates for the first time the up-regulation of renal parenchymal cell NADPH oxidase in the aetiology of immunosuppression-induced nephrotoxicity.

Prolongation of Renal Allograft Survival in Rats by Replication-Defective Recombinant Adenovirus-Mediated Coexpression of CD40L and CTLA4Ig

Background Blockade of costimulatory signals has been shown to prolong allograft survival. The aim of the present study was to investigate the effect of simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways by replication-defective adenovirus-mediated expression of secretable extracellular domain of human CD40L (shCD40L) and CTLA4Ig to prolong rats renal allograft survival. Methods We constructed Adv-shCD40L-IRES 2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig. Coexpression of shCD40L and CTLA4Ig was evaluated by confocal laser scanning microscopy. The function of these two molecules was examined in human mixed lymphocyte reactions (MLRs) in vitro and in experimental BN-to-LEWIS rat renal transplantation in vivo. Results Successful construction of Adv-shCD40L-IRES 2-CTLA4Ig was confirmed by polymerase chain reaction. Coexpression of shCD40L and CTLA4Ig on human kidney cell line HK-2 cells after transfection was detected by direct immunofluorescence staining. Human MLR was inhibited to 52.2% ± 0.6% and 42.1% ± 0.2% of the vehicle control by Adv-shCD40L and Adv-CTLA4Ig, respectively. Adv-shCD40L-IRES 2-CTLA4Ig resulted in further inhibition of MLR to 22.0% ± 0.2% of vehicle control. Transfection with Adv-shCD40L or Adv-CTLA4Ig alone prolonged renal graft survival to 24.8 ± 2.5 days and 27.3 ± 3.6 days, respectively, as compared to vehicle-treated controls (7.8 ± 0.3 days). Cotransfection of both genes extended graft survival to 41.8 ± 3.7 days. Conclusions Adv-shCD40L-IRES 2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig, achieved simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways, Adv-shCD40L-IRES 2-CTLA4 by Ig synergistically inhibited human T-cell proliferation in MLR, and prolonged rats renal allograft survival.

Effect of prolonged warm ischemia and pneumoperitoneum on renal function in a rat syngeneic kidney transplantation model

Laparoscopic donor nephrectomy is associated with several advantages for the donor. However, graft function may be impaired due to use of pneumoperitoneum and prolonged warm ischemia. This study investigated the impact of pneumoperitoneum and prolonged warm ischemia on long-term graft function in a syngeneic rat renal transplant model. A total of 27 Brown Norway rats were randomized for transplantation of kidneys after three different procedures: no insufflation and no warm ischemia (group 1), no insufflation with 20 min of warm ischemia (group 2), and CO2 insufflation and 20 min of warm ischemia (group 3). Glomerular filtration rate (GRF), serum creatinine, urine volume, urine creatinine, and proteinuria were determined monthly for 1 year. One year after transplantation, the grafts were removed for histomorphologic analysis. No significant differences in GRF, serum creatinine, urine volume, and proteinuria were found among the three groups. Histologic analysis also showed no differences between the groups. Warm ischemia in combination with CO2 pneumoperitoneum, as used in laparoscopic donor nephrectomy, does not result in a negative effect on long-term graft function.

Kidney transplantation procedures in rats: Assessments, complications, and management

Kidney transplantation in rats is an experimental model often used for the development of general microsurgical or transplantation techniques, for immunologic studies, and for analyzing transplant-associated long-term arterial blood-pressure changes. The aim of the present study was to analyze different surgical techniques of kidney transplantation in rats, with emphasis on minimizing surgical complications and establishing guidelines for their prevention and management. Complications were categorized into general (e.g., core body temperature drop, ischemic time) and surgically related vascular and urinary tract complications. In conclusion, a significant reduction of the complication rate in renal transplantation in rats can be achieved by placing the animal on a heating pad at an appropriate temperature. To reduce the risk of vascular thrombosis, ice-cold saline with heparin and careful flushing of the donor kidneys are recommended. Vascular complications can be avoided by performing "end-to-end" anastomosis techniques. The use of stents and cannulas in the urinary tract is associated with a high risk of urinary tract obstruction, and therefore is not recommended.

Acute Rejection After Rat Renal Transplantation Leads to Downregulation of Na+ and Water Channels in the Collecting Duct

Renal transplantation is associated with alterations of tubular functions and of the renin-angiotensin-aldosterone system. The underlying cellular and molecular mechanisms are unclear. We used an allogeneic rat renal transplantation model of acute rejection with and without immunosuppression by cyclosporine A (CsA) and a syngeneic model as control. Uninephrectomized Lewis or Lewis-Brown-Norway (LBN) rats received a kidney from LBN-rats. Renal transporters and receptors were analyzed by immunohistochemistry, semiquantitative RT-PCR and Western-blot analysis. Intracellular Na(+) was analyzed microfluorimetrically in isolated cortical collecting ducts. mRNA expression and function of the epithelial Na(+)-channel (ENaC) and mRNA and protein expression of the water-channel AQP2 were downregulated in transplanted kidneys undergoing rejection. Expression of the serum- and glucocorticoid-kinase (Sgk1) was decreased and that of the ubiquitin-protein ligase Nedd4-2 was increased. These changes were absent under CsA-therapy and in syngeneic model. Expression and function of the Na(+)-K(+)-ATPase, expression of the secretory K(+)-channel and of the mineralocorticoid receptor remained unchanged. Reduced ENaC function is likely due to decreased Sgk1- and increased Nedd4-2 mRNA expression leading to reduced ENaC expression in the membrane. These acute downregulations of ENaC and AQP2 may be triggered to reduce energy consumption in the distal nephron to protect the kidney immediately after transplantation.

Gene transfer to the rat kidney in vivo and ex vivo using an adenovirus vector: factors influencing transgene expression

The characteristics of adenovirus-mediated gene transfer into the kidney are not well examined. We studied the effects of contact time and temperature on adenovirus-mediated transgene expression in rat kidneys, using catheter-based in vivo gene transfer and a rat renal transplant model ex vivo. An adenovirus vector containing the luciferase (Ad-Luc) or beta-galactosidase (Ad-LacZ) gene was introduced in vivo into the kidney via a renal artery catheter. Various contact times and temperatures were evaluated. Ex vivo, the renal graft was injected with Ad-Luc through the renal artery, chilled for 60 min and then transplanted. Luciferase expression was evaluated periodically by a non-invasive bioimaging system or histology. Cells expressing the LacZ gene were identified by immunoelectron microscopy. In in vivo gene transfer, successful transgene expression was achieved; however, its efficiency was independent of contact time or temperature. In ex vivo gene transfer, transgene expression in the renal graft peaked early and gradually decreased. Strong gene expression was observed in the recipients' livers. LacZ expression was detected in fibroblasts, parietal epithelial cells of Bowman's capsule, mesangial cells, podocytes and tubular cells. This study generated new information about in vivo and ex vivo gene transfer into the kidney, which would be useful for renal gene therapy.

A New Alloantigen-Independent Control for Chronic Allograft Nephropathy Rat Models

Isografts are used as controls in many transplant experiments. Our laboratory and others have noticed histological changes in control isograft groups of rats similar to allograft groups, suggesting alloantigen-independent factors contributing to chronic allograft nephropathy. However, the isograft model as a nonalloantigen control is flawed because of the potential of unrecognized minor antigen differences between rats. We designed a study using autografts to isolate alloantigen-independent factors in the rat renal transplant allograft model. Male Lewis rats weighing 150-250 g underwent a procedure designed to mimic the injury of renal transplant, in which the left kidney was perfused with cold University of Wisconsin solution and subjected to similar cold and warm ischemic times as Lewis isograft rats undergoing renal transplanation. Six autograft rats were compared to five isograft and three single nephrectomy rats. Autograft rats showed normal kidney function according to serum BUN, Cr, and urinary protein. At 360 days, four of six autografts displayed normal renal parenchymal histology, whereas the remaining two autografts displayed histological changes scored as Banff acute rejection 1a and 1b. At sacrifice time, four of five isografts showed histological changes scored as Banff chronic rejection 1 and the single nephrectomy group showed normal histology in the remaining native kidney. Our data demonstrate that the chronic nephropathy observed in the isograft cannot be completely freed from suspicion of immunological origin. We propose that the autograft model for rat renal transplant research is a better nonimmunologic control than the isograft model for chronic allograft nephropathy.

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1)-mediated pathway and vascular oxidative injury in older-age rat renal transplants

Older-age renal allografts are associated with inferior survival; however, the mechanisms are unclear. Reactive oxygen species participate in aging and in chronic vascular disease. We investigated how mediators of oxidative stress may increase allograft susceptibility to vascular injury. We employed the low-responder allogeneic F344-to-Lew rat renal transplantation model. We used nonimmunosuppressed young (donors and recipients aged 12 weeks), old (donors and recipients aged 52 weeks), and old-to-young animal (donors aged 52 weeks and recipients aged 12 weeks) combinations. Grafts were transplanted after 2 hours cold preservation in University of Wisconsin solution and harvested 1, 2, 7 and 10 days later. Additionally, old animals receiving continuous 1.5 mg/kg cyclosporine (CyA) immunosuppression were included. Renal allograft pathology was scored according to Banff criteria. We studied intragraft vascular adhesion molecule-1 (VCAM-1), lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), and hypochlorite-modified LDL expression as well as ED-1+ monocytes/macrophages and CD8+ lymphocyte infiltration. Intragraft in situ superoxide anion radical production was determined with dihydroethidium assay on cryosections. During the first 2 posttransplant days, old transplants demonstrated higher functional impairment and increased oxidative stress, while young transplant had higher ED-1+ monocytes/macrophage infiltration and VCAM-1 expression. The degree of VCAM-1 expression and ED-1+ monocytes/macrophage and CD8+ lymphocyte infiltration correlated at later time points directly with the transplant age. VCAM-1 and LOX-1 staining were localized predominantly on the endothelium of arterial vessels, shifting the distribution to vascular smooth muscle layer strongly dependent on donor age and the grade of vascular injury. LOX-1 staining colocalized with hypochlorite-modified epitopes in the media of injured arteries. We measured increased in situ superoxide anion radical production in corresponding areas. Immunosuppression with CyA had no protective effect on vascular injury and LOX-1 expression. Induction of LOX-1-related oxidation pathways and increased susceptibility to oxidative stress could play an important role in promoting vascular injury in old renal transplants independent of the recipient age.

Preoperative administration of FTY720 prolonged renal allograft survival

Background FTY729 is an immunomodulator obtained by chemical modification of Myriocin(ISI-1) which exists in the culture filtrate of an ascomycete, Isaria sinclairii. It has been reported that postoperative administration of FTY720 prolonged survival of various kinds of transplanted organs. In the present study, we evaluated the effect of 2-day preoperative administration of FTY 720 on graft survival. Materials and methods We used a rat renal transplantation model in which Wistar King Aptekman Hokkaido (WKAH, RT1 K) served as the organ donor and Lewis (LEW, RT l) as the recipient. FTY720 was given to the recipients consecutively 2 days (day-2, day-1) before transplantation at the doses of 1, 3 or 5 mg/kg/day. Renal allograft survivals, hematological parameters of recipient blood and phenotypic analysis of recipient splenic cells and graft infiltrate were evaluated. Results Consecutive 2-day preoperative oral administration of FTY 720 at the doses of 1, 3 or 5 mg/kg/day significantly prolonged WKAH allograft survivals compared with those of the untreated recipients. The number of peripheral blood lymphocytes was markedly decreased in the recipients treated with FTY720 at the doses of 3 mg/kg/day or 5 mg/kg/day on the 5th postoperative day. Preoperative FTY 720 administration significantly decreased the number of CD4 positive cells and the percentage of interleukin 2 receptor (IL-2 R) positive cells infiltrating both spleen and allograft at the dose of 3 mg/kg/day or 5 mg/kg/day. Conclusion FTY 720 could act as a safe and potent immunomodulator by decreasing the number of peripheral lymphocytes, especially CD4 positive cells and IL-2R positive cells when it is given to the recipient preoperatively.

Peritransplant treatment with cobalt protoporphyrin attenuates chronic renal allograft rejection

Allogen-independent injury contributes to chronic rejection in renal allografts and heme oxygenase-1 (HO-1) has been shown to be protective in a number of settings. This study evaluated the effect of renal allograft recipient HO-1 up-regulation on chronic rejection in a rat model. Rat (F344 to Lewis) renal transplantation recipients were grouped: (i) cyclosporine (CsA) alone (0.75 mg/kg s.c. x 10 day; n = 5); (ii) CsA + low dose cobalt protoporphyrin (CoPP) an HO-1 inducer (0.5 mg/kg i.p. on days -5,0,5; n = 13) and (iii) CsA + high dose CoPP (5.0 mg/kg i.p. on days -5,0,5; n = 8). Renal function was assessed by serum creatinine levels on day 140. Histopathologic changes in allografts were graded. Morphometric analyses performed to objectively quantify the vascular changes and glomerulosclerosis. HO-1 expression quantified by Western blot and both HO-1 and endothelin (ET-1) localized using immunohistochemistry. Recipients treated with CsA + high dose CoPP had significantly decreased cortical scarring, vascular hyalinization and intimal thickness. They also had a significant, dose-dependent, reduction in luminal obliteration and glomerulosclerosis by morphometric analyses. This freedom from chronic rejection in recipients treated with CoPP translated into quiescent grafts at postoperative day 140 with immunostaining and Western blot demonstrating decreased level of HO-1 versus controls (P = 0.012). In summary, the peritransplant up-regulation of HO-1 in renal allograft recipients significantly attenuates chronic rejection in rat renal allografts by inhibiting transplant vasculopathy.

Expression of nitric oxide and inducible nitric oxide synthase in acute renal allograft rejection in the rat.

Recent studies have shown that nitric oxide (NO) synthases, particularly inducible nitric oxide synthase (i-NOS), are induced in acute rejection episodes following heart, liver, pancreas and kidney allotransplantation. Furthermore, tissue and cellular injury has been demonstrated to be mediated by peroxynitrite (ONOO-), a metabolite of NO as well as a potent oxidant. However, a detailed relationship between NO, i-NOS and graft injury in transplantation remains elusive. The present study used the following models of renal transplantation in rats: allografts (n = 5, Brown-Norway to Lewis [LEW] rats), isografts (n = 5, LEW to LEW) and allografts treated with aminoguanidine (AG), an i-NOS inhibitor (n = 5). Blood urea nitrogen (BUN), serum creatinine (SCr) and urinary and serum nitrosocompounds (NOx) were measured on days 2, 4 and 7 post-transplant. Western blot analysis of i-NOS protein expression and measurement of i-NOS activity were carried out in grafts harvested on Day 7, along with immunohistochemical and histopathological examinations. In the allograft group, both BUN and SCr levels increased markedly on Day 7, in parallel with a sharp increase in NOx. A band stained by anti-i-NOS antibody was detected at approximately 130 kDa, along with high levels of i-NOS activity and diffusely distributed i-NOS-positive cells (macrophages). Histologically, an acute rejection episode was confirmed (Grade 3 according to Banff classifications). In the AG group, reduced renal function and graft injury were significantly less severe than in the allograft group. In rat renal allograft acute rejection, markedly increased levels of serum NOx were observed, along with enhanced tissue i-NOS activity, together resulting in graft injury. AG administration suppressed the increase of serum NOx levels, with concomitant mitigation of tissue injury and renal function impairment.

NITRIC OXIDE KINETICS AND EARLY INTERVENTION IN ACUTE RENAL ALLOGRAFT REJECTION IN RAT

P1114 Aims: Recent studies have shown that nitric oxide (NO) synthases,particularly inducible nitric oxide synthase (i-NOS),are massively induced in acute rejection episodes following heart,liver,pancreas and kidney allotransplantation. Furthermore, tissue and cellular injury in grafts has been demonstrated to be mediated by peroxynitrite anion (ONOO−), a metabollite of NO as well as a potent biological oxident. On the other hand, in chronic rejection of human renal allografts, ONOO− has been implicated in nitration and inactivation of manganese superoxide dismutase (MnSOD), a radical scavenger. However, a detailed relationship between NO, i-NOS, ONOO− and MnSOD in acute rejection episodes remains elusive. Methods: As models of renal transplantation in rats, allografts (Brown-Norway to Lewis), isografts (Lewis to Lewis), and allografts treated with aminoguanidine (AG), an i-NOS inhibitor, at a daily dose of 400 mg/kg of bodyweight,i.p., were used. Blood urea itrogen (BUN), serum creatinine (SCr) and urinary and serum nitrosocompounds (NOx) were measured on days 2,4 and 7 post transplant. Western blot analyses of i-NOS, MnSOD and nitrotyrosine-immunoprecipitated MnSOD protein expression, and measurement of i-NOS and MnSOD activities were performed in grafts harvested on day 7, along with immunohistochemical and histopathological examinations. Results: 1) In the allograft group, both BUN and SCr levels increased markedly on day 7, in parallel with a sharp increase in NOx. A band stained by anti-rat i-NOS antibody was detected at around 130 kDa, along with high levels of i-NOS activity and diffusely distributed i-NOS-positive cells (macrophages). Histologically, acute rejection episode was confirmed (Grade 3 according to Banff classifications). In the AG group, reduced renal function and graft injury were significantly less severe than in the allograft group. 2) In the allograft and isograft groups, a band stained by anti-rat MnSOD antibody was detected with equal intensity at around 23 kDa. In the allograft group, another strongly stained band was detected at around 25 kDa (inactivated tyrosine-nitrated MnSOD protein), along with significantly lower MnSOD activity than in the isograft group (p=0.0012). Increased nitrotyrosine reactivity detected by anti-rat nitrotyrosine antibody, was localized to inside tubular epithelial cells with no glomerular involvement. Conclusions: 1) In the allograft group, markedly increased levels of serum NOx were observed, along with enhanced tissue i-NOS activity, together resulting in graft injury. 2) In the allograft group, MnSOD activity was significantly decreased compared to the isograft group, in parallel with increased inactivated tyrosine-nitrated MnSOD, thus leading to enhanced accumulation of ONOO− products (nitrotyrosine) in tubules accounting for promotion of severe graft injuries. 3) AG administration suppressed the increase in serum NOx levels, with concomitant mitigation of tissue injury and renal function impairement.

EFFICIENCY OF ADENOVIRUS-MEDIATED GENE TRANSDUCTION ON VARIOUS GRAFT ORGANS IN RATS

P1161 Aims: Gene therapy has been considered as a potential strategy for various diseases and tested in both animals and humans. Adenovirus vector is widely employed by its high efficiency of transgene expression and the ability of transducing both dividing and non-dividing cells. Systemic inflammation is the major negative effect, and local gene transfer is applied to overcome this problem. In organ transplantation, gene transfer into the isolated organs seems to be easier than in vivo gene transfer, and ex vivo gene transfer is a matter of concern for investigators as a method of graft modulation. Actually, there are some reports demonstrating the efficiency of ex vivo gene transfer in liver, heart, and renal transplantation. However marker genes such as LacZ gene inevitably require tissue samplings for gene expression evaluation and the time course of gene expression in individual animals has not been studied. In this study, we demonstrated the characteristics of transgene expression in ex vivo gene transfer using adenovirus vector in heart, small bowel, and renal transplantation by assessing the gene expression periodically in the same animals using non-invasive bioimaging system. Methods: Syngenic heart, small bowel and renal transplants were performed in Lewis rats. Five times 108 plaque-forming units (pfu) of adenovirus vector containing firefly luciferase gene (Ad-Luc) was infused into the harvested graft, and it was preserved at 4 °C until transplant. Transgene expression was visualized and quantified by non-invasive bioimaging system periodically without sacrifice. Results: In rat heart transplant model, transgene expression in the graft was observed at 1 week after transplantation after prolonged contact time. However, cardiac arrest was confirmed in some grafts about 1 week after transplantation. In rat small bowel transplantation model, gene expression in the graft was observed from day 1 to day 14 after the operation. In rat renal transplantation model, transgene expression reached the peak at day 2 or 3 and lasted about 1 week, although it was weaker than that of in vivo gene transfer by transarterial infusion of the same amount of Ad-Luc. Conclusions: We demonstrated the basic information of gene expression patterns in adenovirus-mediated ex vivo gene transduction for various graft organs.

Induction of epidermal fatty acid binding protein in intravascular monocytes of renal allografts.

During acute rejection of rat renal allografts, numerous activated monocytes accumulate in the vasculature of the graft. These monocytes seem to be involved in allograft destruction. Proinflammatory and effector functions of monocytes and macrophages can be down-regulated by peroxisome proliferators, which are probably transported in the cytoplasm by fatty acid binding proteins (FABPs). We performed renal transplantation in rats in the Dark Agouti-to-Lewis strain combination. Intravascular graft leukocytes were harvested 4 days posttransplantation. Epidermal (E)-FABP mRNA and protein expression were investigated by reverse-transcriptase polymerase chain reaction and immunoblotting, respectively. E-FABP-expressing cells were identified by immunofluorescence. After allogeneic transplantation, intravascular graft leukocytes expressed E-FABP mRNA and protein. In isografts, significantly lower expression levels were observed. E-FABP protein was detected in monocytes expressing ED1 and in alphabeta-T-cell receptor positive T lymphocytes. E-FABP might regulate monocyte activation and may represent a promising target for a therapeutic intervention in allograft rejection.

Impact of renal transplantation on small vessel reactivity1

The function of large arteries is altered after renal transplantation. Whether transplantation also induces agonist-dependent functional changes in small arterial renal and extrarenal vessels has not yet been studied. Chronic rejection was induced by grafting Lewis rats with kidneys from Fischer rats (FL). Rats that underwent transplantation were bilaterally nephrectomized. Rats that underwent syngeneic transplantation, uninephrectomized rats, uninephrectomized rats with denervated kidneys or with kidneys made ischemic, and native rats served as controls. All animals were treated with cyclosporine for 10 days. Eighteen weeks after surgery, the reactivity of small arteries (220-270 microm) was tested by myography. Weight gain, glomerular filtration rate, and arterial pressure were similar in all groups, whereas proteinuria was elevated in FL. Only kidneys from FL showed glomerular lesions, tubular atrophy, and vasculopathy. Responsiveness of coronary, mesenteric, and femoral resistance vessels to both constrictor and dilator agonists was similar in transplanted and nontransplanted animals. Resistance vessels obtained from both allogeneically and syngeneically transplanted kidneys were more sensitive to norepinephrine, phenylephrine, angiotensin II, and vasopressin than renal vessels from weight-matched controls. Vasodilation in response to acetylcholine and sodium nitroprusside was mitigated in transplanted versus nontransplanted kidneys. In rat renal transplantation, renal resistance vessel responsiveness to constrictor or dilator stimuli is altered. Extrarenal small vessel function is not affected. The changes in function of renal resistance vessels are not explained by reduction of nephron mass, denervation, ischemia, or chronic rejection.

Kidney transplantation in rats: an appraisal of surgical techniques and outcome.

Renal transplantation in rats is an essential experimental tool in transplantation research. The surgical procedure per se could affect the outcome of an experiment, independent of the hypothesis addressed, therefore requiring a standardized method which should be comparable across studies. To date, however, there is little information on the optimal surgical technique. We performed a Medline search on original articles published between 1965-2001 in order to evaluate whether specific technical issues affecting the outcome of the procedure could be defined. Articles that reported on a novel microsurgical procedure, or whose main purpose was the outcome of a surgical technique itself, were included in the analysis. From 2,060 retrieved publications, 34 corresponded to the selection criteria (rats and microsurgery and technique and kidney or renal transplantation). Among the essential determining factors for a good outcome, body weight >200 g and warm ischemic time <30 min were identified. Other important factors were the techniques used for vascular (end-to-end and end-to-side procedure or sleeve technique) and ureteral (bladder patch or end-to-end procedure) anastomosis. Gender, animal strain, type of anesthesia, prophylactic administration of antibiotics, and type of flushing solution did not affect the success of renal allografts. In order to avoid a bias related to the surgical procedure in rat renal transplantation, a warm ischemia time <30 min in animals with a body weight >200 g seems to be essential. Also, end-to-end or end-to-side vascular anastomoses are preferable to the sleeve technique. Other factors do not influence the immediate function of the graft.

Genetic modification of cold-preserved renal grafts using HSP70 or bcl-2 HVJ-liposome method

Background: We tested the hypothesis that the best time for genetic modification is while the cell viability of the graft is reduced for long-term preservation. The hemagglutinating virus of Japan (HVJ)-liposome method, a nonviral gene transfer technique, was used with a luciferase gene to test the efficacy of protein induction under the critical preservation time. Furthermore, we tested this genetic modification with heat shock protein (HSP) 70 or bcl-2 genes to prevent primary nonfunction (PNF) after long-term preservation. Methods: Orthotopic rat renal transplantation (RT) was performed using the cuff technique in the syngeneic combination of Lew (major histocompatible complex, haplotype: RT1 l). Rat kidney grafts were preserved for 24 or 48 h in University of Wisconsin (UW) or Ringer's lactate solution using HVJ method with the luciferase gene. Rats with gene-transfected kidneys were re-laparotomized 48 h after transplantation to estimate the lack of arterial flow in the graft and killed for histological evaluation of the degree of PNF luciferase intensity assay. Then, two functional genes (HSP70 or bcl-2) were tested for the occurrence of PNF and histological and immunohistochemical analysis of the grafted kidneys preserved for 48 h in the UW solution. Results: In the kidneys preserved for 24 h, 50% of the Ringer's lactate group had PNF; but all of the UW group had sufficient blood flow. The graft viability was well corrected by the degree of luciferase intensity. The PNF rate was significantly suppressed in the bcl-2 gene-transfer group, and tended to be reduced in the HSP70 group. Conclusions: The HVJ-liposome method effectively induced the foreign gene for kidney grafts even in the cold-preservation solution. Induction of bcl-2 or the HSP70 gene reduced the occurrence of PNF in the rat renal graft. The results suggest that gene transfer not only maintains graft viability, but also graft activation.

Promising effects of ischemic preconditioning in renal transplantation

Ischemic preconditioning, a phenomenon induced by brief ischemia and reperfusion periods, renders an organ tolerant to subsequent prolonged ischemia. This study evaluated different schedules of preconditioning the kidney to assess the role of nitric oxide (NO) and determine the effects of preconditioning on kidney transplantation. In study design A, to determine the optimum procedure of preconditioning, one-cycle schedules were assayed by occluding/releasing renal pedicles according to various warm ischemic (5, 10, 15, 20 min) and reperfusion (10, 20, 40 min) windows in Sprague-Dawley rats. Thereafter, warm renal ischemia was induced by clamping both pedicles for 40 minutes. Design B used the most suitable schedule found from the first study to obtain several groups, using either a direct nitric oxide donor (spermine NONOate) or two nitric oxide synthase (NOS) blockers (L-NAME and aminoguanidine), to determine whether NO mediates in renal preconditioning. To establish whether preconditioning reduces cold preservation damage, in Design C the optimum preconditioning schedule was used in syngeneic Lewis rats where preconditioned and non-preconditioned kidneys were transplanted after five hours of cold storage in Euro-Collins solution. The best preconditioning schedule consisted of 15 minutes of warm ischemia and 10 minutes of reperfusion (Prec 15/10), since it was the only schedule that offered both functional and histological protection. The NO donor reproduced the ischemic preconditioning. Non-selective NOS blockade abolished the preconditioning and exacerbated ischemic damage, which was overcome by the addition of the NO donor. Selective blocking of inducible NOS also abolished the effects of preconditioning. Renal NO increased at the end of preconditioning in the Prec 15/10 group. Prolongation of the reperfusion window (20 or 40 min) abolished the preconditioning protection, although it was associated with a further increase in renal NO. As renal DNA oxidative injury paralleled NO, increasing with prolongation of reperfusion, it may account for the disappearance of preconditioning. Finally, the one-cycle preconditioning schedule offered an effective functional and histological protection against cold preservation damage in rat renal transplantation. Fifteen minutes of warm ischemia and 10 minutes of reperfusion in the kidney is the most suitable one-cycle schedule for preconditioning since it protects from both warm and cold ischemia. The beneficial effect of preconditioning is related to the local production of NO, and we believe it has promising therapeutic value in clinical renal transplantation.

Hepatocyte growth factor (HGF) as a rapid diagnostic marker and its potential in the prevention of acute renal rejection

Several investigators have reported that hepatocyte growth factor (HGF) may be related to the protection or reconstruction of the kidney during acute renal rejection. To address this question, we examined the relationship between HGF and acute rejection in the following two studies with rat renal transplantation models. In study 1, the relationship between serum HGF levels and acute renal rejection in iso-, allo- and allograft with cyclosporine (CsA)-treated models was examined. In study 2, the focus was whether or not the injection of recombinant HGF can prevent acute renal rejection. Our results demonstrated that HGF levels were rapidly increased during acute rejection and that recombinant HGF effectively protected the kidney from acute rejection. These results suggest that HGF may be induced as a counter-response to the renal injury and that it can be used as a reliable indicator for the diagnosis of acute rejection. We suggest that recombinant HGF suppresses the onset of the pathological changes of acute rejection.