Renal Graft Rejection Research Articles

Hepatitis C Eradication and Improvement of Cryoglobulinemia-Associated Rash and Membranoproliferative Glomerulonephritis with Interferon and Ribavirin after Kidney Transplantation

Postrenal transplant hepatitis C is increasing in frequency due to the high prevalence of hepatitis C among patients with renal failure. Despite this, there is still no standard hepatitis C treatment available for renal transplanted recipients. Combination antiviral hepatitis C therapy, the standard of care in the nontransplant population, is generally avoided because of documented renal graft rejection secondary to interferon treatment. A case of a male patient with postrenal transplant hepatitis C, which was associated with cryoglobulinemia and glomerulonephritis of the graft, is presented. He was treated with standard interferon with ribavirin. Sustained viral clearance was achieved despite ongoing evidence of cryoglobulinemia. Renal function, which had been deteriorating before treatment, improved as evidenced by the stabilization of serum creatinine and marked improvement of proteinuria. In conclusion, in selected patients, combination antiviral therapy may still be a viable option postrenal transplant.

Immune Response Gene Polymorphisms in Renal Transplant Recipients

T-cell activation and regulation are under genetic control and vary between individuals. However, the influence of functional immune response gene polymorphisms on transplant outcomes remains controversial. A case-control design compared 100 white renal transplant recipients with or without acute graft rejection during the first year posttransplant and 50 normal controls. The polymorphic frequencies of the T-cell signaling genes CD45, CD40L and CTLA-4, and the cytokine genes TNF-alpha, IFN-gamma, IL-10 and TGF-beta1 were studied. The primary analysis examined rejection risk, and subsidiary analyses graft failure and patient death. Multivariate analysis showed no significant association between acute rejection and single nucleotide polymorphisms in CTLA-4, TGF-beta1, IL-10 or TNF-alpha genes or dinucleotide repeat polymorphisms in IFN-gamma and CD40L genes. Allele CD40L-147 was associated with reduced graft failure (P=0.004), and TGFb-25pro with increased graft failure (P=0.0007), although the latter showed a bidirectional dose effect. There was no significant association between patient death and any polymorphisms in the genes examined. The variant (G) allele of the CD45 gene was not detected in the study population. Minor differences in carriage rates observed by univariate analysis did not predict graft or patient outcome in multivariate analysis. The primary analysis demonstrated no significant association between the immune response gene polymorphisms examined and acute renal graft rejection in Caucasian patients receiving triple immunosuppression. Subsidiary analyses suggesting an influence of CD40L and TGFbeta1 genes on graft survival require independent confirmation.

Prior renal transplantation does not facilitate subsequent canine islet allograft survival

Successful islet allografts can be established in rats bearing long surviving renal allografts, without additional immunosuppression, when kidney and islet donor animals are of the same strain. The applicability of such a scheme to clinical practice has been investigated in a large animal model of diabetes: the pancreatectomized dog. Eight dogs with previously established renal allografts and immunosuppressed with cyclosporin A received islet allografts from their respective original kidney donors. The median islet graft functional survival was only 10.5 days, significantly less than for six similarly immunosuppressed dogs receiving islet allografts alone (48.5 days, P less than 0.05). Three of the sequentially transplanted dogs had had no renal graft rejection episodes before islet transplantation, yet their islet grafts were all rejected within 19 days. In the pancreatectomized dog, prior donor specific renal transplantation has an adverse effect upon subsequent islet graft survival.

Effects of Photopheresis on Mononuclear Cells from Healthy Individuals.

Background: Extracorporeal photopheresis (ECP) has been proposed as an alternative therapy for immune-mediated disease including graft-versus-host-disease (GVHD) and transplant rejection (Photopheresis for the treatment of refractory renal graft rejection, Transplantation 2005; 79(1):123–5, Kumlien G et al). The mechanisms of photopheresis are still poorly understood but induction of tolerogenic dendritic cells and regulatory T cells as well as a shift of cytokine profile from Th1 to Th2 have been observed in patients treated with ECP. We wanted to investigate some basic effects of ECP on mononuclear cells from healthy individuals unaffected by pathogenic mechanisms and immunosuppressive drugs.Method: Mononuclear cells (MNCs) were collected from six healthy volunteer apheresis donors using CobeSpectra apheresis machine (Gambro BCT, Lakewood, CO, USA) and peripheral blood was drawn immediately before apheresis. Apheresis products were thoroughly mixed and divided into three equal parts. One part was left untreated, one part was irradiated with UVA 365 nm, 2J/cm2 (BioGenic, Vilber Lourmat, Marne-la-Vallée, France) and one part was treated with psoralen (8-methoxypsoralen 200 ng/ml) plus UVA-irradiation (PUVA). Peripheral MNCs and aliquots of apheresis MNCs were analysed simultaneously regarding incorporation of 3H Thymidine after stimulation with mitogens (PHA, ConA, SpA) and production of cytokines correlated with GVHD pathogenesis (IL-2, IL-6, IL-12, TNFα, IFNγ) after stimulation with antigens (BG, PHA, LPS) using commercial ELISA-kits (Quantikine, R&D Systems, Minneapolis, MN, USA). Statitical analysis was performed using a nonparametric method (one-sided ANOVA).Results: Mitogenic capacity was reduced to baselevel after PUVA-treatment but not affected by the apheresis procedure or UVA-irradiation alone. Table 1. Statistically significant decrease of cytokine production was seen only after PUVA-treatment but production of IL-2 and IL-6 was reduced after both apheresis and UVA-irradiation alone. Small amounts of IL-2, IL-6, TNFα and IFNγ were produced even after PUVA-treatment.Conclusion: Significant reduction of mitogenic capacity only after PUVA-treatment was the expected finding. Apheresis can theoretically effect cells through mechanic damage (centrifugation) and/or interaction with apheresis harness and UVA is known to have immunomodulatory effects which can explain the reduction of cytokine production. Cytokines detected after PUVA-treatment may be explained by release from apoptotic cells or by production in cells that escape apoptosis.Lymphocyte proliferation assayConAPHASpAperipheral vs apheresis MNCsnsnsnsperipheral vs UVA-irradiated MNCsnsnsnsapheresis vs UVA-irradiated MNCsnsnsnsperipheral vs PUVA-treated MNCsp<0.05p<0.05p<0.05apheresis vs PUVA-treated MNCsp<0.05p<0.05p<0.05UVA -irradiated vs PUVA-treated MNCsp<0.05p<0.05p<0.05MNCs = mononuclear cells

148. The Transgene Cassette Is Not Fully Responsible for Alterations of Renal Cytochrome P450 Expression after Systemic Administration of Recombinant Adenovirus

Gene transfer to the kidney has shown promise in treating severe renal disease and prevention of acute renal graft rejection in several animal models. To date, recombinant adenovirus (Ad) is the most efficient vector for gene delivery to the kidney. Although Ad does not infect the kidney efficiently when administered systemically, transgene products secreted from the liver can reach the kidney at therapeutic levels. We have found that systemic administration of recombinant Ad significantly alters the expression of the cytochrome P450 (CYP) enzymes, largely responsible for the clearance of toxicants and the metabolism of xenobiotics and endogenous compounds, in the kidney. In order to determine if the transgene cassette could be partially responsible for this effect, Sprague-Dawley rats received one of four treatments via a surgically implanted catheter: 0.5 ml of phosphate-buffered saline (control) or 5.7|[times]|1012 viral particles/kg of a first generation Ad expressing either E.coli beta-galactosidase (AdlacZ), murine erythropoietin (Epo) or no transgene (Null). Animals were sacrificed 0.25, 1, 4 and 14 days after injection. Renal CYP protein expression and mRNA levels were analyzed by Western blot and RT-PCR. All treatments significantly induced protein levels of the CYP4A family. Following AdlacZ administration, CYP4A increased by 50%, 32% and 64% above control at 0.25, 1 and 4 days, respectively (p<0.01). The vector expressing Epo raised CYP4A protein levels by 42%, 24% and 64% and the Null vector produced elevations of 74%, 25% and 49% above the control at the same timepoints (p<0.01). CYP4A protein expression recovered by 14 days in each group. RT-PCR revealed that mRNA levels of CYP4A2, the primary CYP4A isoform responsible for renal fatty acid metabolism, increased by 16.8% and 19% above control 24 hours after administration of AdlacZ and Epo, respectively (p<0.01). CYP4F1, an isoform responsible for metabolism of fatty acids, prostaglandins and leukotrienes, was unaffected in all treatment groups. In contrast, renal CYP2E1, which metabolizes small lipophilic compounds and carcinogenic agents, was significantly reduced throughout the study. CYP2E1 mRNA levels in animals treated with AdlacZ were 74%, 78%, 69% and 66% of control at 0.25, 1, 4 and 14 days, respectively (p<0.01). Similar reductions were seen with Epo and Null vectors although levels form animals treated with Epo began to recover by 14 days. Real time quantitative PCR revealed that viral genomes (|[sim]|850/100 ng DNA) were initially present in the kidney and declined throughout the study for all treatments. These results suggest that systemic administration of Ad significantly altered the transcription and translation of several key CYP enzymes in the kidney regardless of the transgene expression cassette. This should be considered for accurate drug monitoring when employing recombinant Ad vectors in the clinic. Additional mechanistic studies assessing the effect of expression of Ad early genes and local and systemic eicosanoid biosynthesis on the expression of renal CYP will allow further explanation of these results.

Photopheresis for the Treatment of Refractory Renal Graft Rejection

Acute rejection episodes still occur after kidney transplantation in spite of modern immunosuppressive protocols including combined tacrolimus, mycophenolate mofetil, and prednisolone. The authors present seven cases of biopsy-proven acute rejection after kidney transplantation refractory to conventional rejection therapy with repeated pulses of high-dose steroids followed by polyclonal or monoclonal antibodies that responded well to photopheresis treatment. Photopheresis is an atoxic immunomodulatory apheresis-based treatment with no generalized immunosuppressive action; rather, it is directed at suppressing donor-specific T-cell clones. At the last follow-up, 9 to 43 months after transplantation, all patients had functioning grafts, with serum creatinine levels ranging from 105 to 312 microM. The authors conclude that photopheresis treatment contributed to the favorable outcome. Therefore, the authors are presently designing a prospective, randomized trial to evaluate the effect of photopheresis as an adjuvant prophylactic treatment after renal transplantation.

Estudo dos polimorfismos dos sistemas de aloantígenos plaquetários humanos (HPA) em doenças hemorrágicas e trombóticas

Abstract Human platelet alloantigens (HPA) are polymorphicsequences of the platelet membrane glycoproteins, whichare associated with immune platelet disorders, such asneonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), and also to platelet transfusionrefractoriness or occlusive vascular disease. Severaldistinct biallelic systems are known and a heterogeneousdistribution of HPA alleles has been described amongdistinct ethnic groups. In this study, the five HPA systemsmost frequently involved in alloimmunization werecharacterized using molecular biology methods, in threedistinct ethnic groups which compose the Brazilianpopulation: Caucasians, Blacks and native Indians. TheHPA-1 to -5 allele frequencies obtained with Caucasiansand Blacks were similar. The data obtained contrasts withthose reported for similar ethnic groups in other countries.Among the native Indians, no b alleles (low frequencyallele) of the HPA-1, -4 and -5 systems were identified. Toevaluate the possible clinical impact of these data, aprospective study was performed among neonates duringa period of three years. In this study, the occurrence ofneonatal thrombocytopenia, as well as its causes wasanalyzed. Maternal-fetal HPA incompatibility wasinvestigated among normal and thrombocytopenicneonates in a case-control study. The results obtainedshowed that the most frequently involved systems wereHPA-3 and -2, corresponding to almost 50% ofincompatibilities. No NAIT cases were identified in thisperiod, suggesting that other factors (like genetic orenvironmental factors) may be associated with thedevelopment of the disease.Another study was carried out in order to determinewhether the HPA-5 system could be involved with acuteimmune thrombocytopenic purpura (ITP). The etiology ofITP remains unknown. The HPA-5 system is associated toimmune-mediated diseases such as chronic ITP and renalgraft rejection, so it could be involved in the pathogenesisof acute-ITP. The result of this study was the detection ofa two-fold higher frequency of allele HPA-5b among acute-ITP patients compared to a control group. These datasuggest that the presence of HPA-5b allele could be relatedto an increased risk of acute ITP in our population.Finally, the association between HPA-1b allele andthe development of vascular diseases was evaluated. Thisallele in GpIIIa is known to result in a higher aggregationresponse of platelets to immobilized fibrinogen, andconsequently, to an enhanced clot retraction. Three groupswere evaluated: a control group, one carrying venousthrombosis and another with arterial disease. In the venousthrombosis group, no association was found between theHPA-1b allele and an increased risk of the disease inaccordance to previously published data. However, ourdata contrast with those reported for arterial diseasepatients as the frequencies obtained between patients andcontrols were similar. Although, when we compared theHPA-1b frequency between over 45-year-old myocardialinfarction survivors to those under 45-year-old, theyoungest group showed a higher frequency of the allele.These data reinforce the idea of a distinct pathogenesis ofthe early onset of coronary disease among HPA-1bcarriers.

Association of Fc gamma receptor IIA polymorphisms with acute renal-allograft rejection.

Acute rejection is a leading cause of early renal-allograft failure. The human Fc gamma receptor IIA (FcgammaRIIA) forms an essential link between the humoral branch and the effector cells of the immune system. In this study, we examined FcgammaRIIA genotypes in renal-allograft recipients (rejectors) with acute graft rejection and in a number of control groups to investigate a possible association between FcgammaRIIA polymorphism and acute renal-allograft rejection. The distribution of the genotypes in the study patient group differed from the control groups. The frequency of homozygosity for FcagammaRIIA-R/R131 in the rejectors was significantly higher than that in the recipients (nonrejectors) with well-functioning renal allografts and in blood donors (P< 0.05). In comparison with the control groups, the rejectors displayed a higher R131 allele frequency (P< 0.05) and a lower H131 allele frequency (P< 0.05). These results reveal a significant association between FcgammaRIIA-R/R131 and acute renal-graft rejection, and it is likely that FcgammaRIIA polymorphisms could be useful markers for potential risk of rejection.

Expression of TRAIL, DR4, and DR5 in kidney and serum from patients receiving renal transplantation

Renal transplantation is the best treatment of some end-stage renal diseases. Unfortunately, not every transplant is successful due to the rejection or dysfunction of the transplanted kidney. Many cytokines participate in rejection by inducing inflammation or apoptosis. In this study, the expressions of TRAIL, DR4, and DR5 in rejected renal tissue and of serum soluble TRAIL (sTRAIL) in patients with kidney rejection were investigated by immunohistochemical staining and sandwich enzyme-linked immunosorbent assay, respectively. The results showed that the expression of TRAIL, DR4 and DR5, and serum sTRAIL levels were markedly upregulated among renal transplant patients. Since both membrane and soluble forms of TRAIL can induce apoptosis of DR4/DR5-expressing cells via recruiting FADD and caspase 8, elevated TRAIL and its receptors may participate in renal graft rejection.

Gene therapy: how to target the kidney. Promises and pitfalls.

The success of gene therapy strongly depends on an efficient delivery system to allow local transfer and expression of the therapeutic gene in the target organ or tissue. Vector systems have been improved and many show promise. There are two different categories of delivery vehicles: non-viral and viral vectors, both with advantages and disadvantages that must be taken into consideration in view of the final aim. Compared to other solid organs, the kidney offers the main advantage of access by different routes that dictate different sites of transfection. Thus, the choice of the delivery vehicle and administration route has to take account which cells are to be specifically targeted by the gene transfer approach. This concept will be discussed in the first part of the review. Using a gene therapy approach, improvements of renal function and interstitial inflammation have been achieved in experimental models of glomerulonephritis and tubulo-interstitial damage. Gene therapy applied to renal transplantation has shown promising results in rodents, almost controlling acute rejection. Finally, the development of animal models resembling the clinical features of human genetic renal disorders offers a first step towards new treatments among which gene therapy could become reality in the near future. The main findings concerning the suitability of gene therapy for slowing the progression of kidney diseases, and preventing acute renal graft rejection, or treating genetic disorders, are discussed.

Renal Transplant Recipient with Chronic Hepatitis C Who Obtained Sustained Viral Response after Interferon-.BETA. Therapy

A 44-year-old Japanese woman with a history of living-related renal transplantation was treated with interferon-beta (IFN-beta) for chronic infection with sero-group 2 hepatitis C virus (HCV). Serum HCV-RNA titer was 160 kilo-international units/ml. Treatment with intravenous IFN-beta daily was given for 6 weeks. Serum HCV-RNA was undetectable at 3 weeks after initiating therapy. Renal graft rejection did not occur. Six months after completing therapy, she obtained sustained viral response. This case demonstrates that IFN-beta therapy safely induced clearance of HCV in a renal transplant recipient with stable renal function, low viral load and/or HCV sero-group 2.

Clinical graft evolution of lymphocytes, polymorphonuclear cells, and antigen expression in tubular renal cells in the urine sediment of 20 renal allograft recipients

Urinary samples from 20 kidney transplant recipients were studied to determine the cellular composition of the sediments using an immunocytological (IC) technique. The expression of HLA class I (A, B, C) and class II (DR, DQ, DP), CD2, CD3, CD4, CD8, and interleukin (IL)-2 receptor (IL-2R) on lymphocytes was assessed using a panel of monoclonal antibodies. The results were correlated with graft function and with the number of episodes of acute renal graft rejection (AR) during a period of 6 months posttransplantation. The cellular infiltration of lymphocytes (LC) and polymorphonuclear cells (PMNC) also was studied using a standard cytology (SC) technique. During this period, 17 of 30 episodes of graft dysfunction due to AR occurred in 12 patients: 8 to acute tubular necrosis (ATN) (n = 8); 4 to cyclosporine (CsA) toxicity (n = 4) and 1 to amphotericin toxicity (n = 1). The diagnosis of AR was made clinically by 3 independent observers, using biopsy in some cases. The immunocytology showed a significantly increased expression of HLA-DR, DO, and DP namely, greater than 20% positivity in 10% of samples on the tubular epithelial cells (TEC) of patients presenting with versus without AR ( P ≤ .001). In addition, a high correlation was observed between the expression of IL-2R and the presence of AR (p ≤ .002). The standard cytology results showed a significantly increased percentage of LC and decreased percentage of PMNCs in samples obtained 2 days prior to the clinical manifestations of patients who developed AR ( P = .001). A greater level of expression of antigen determinants was observed prior to AR. These results suggest that immunocytology of urinary sediments, which is a noninvasive technique, has enormous clinical potential for the differential diagnosis of AR, ATN, and CsA toxicity. In our study, the use of HLA class IL–specific monoclonal antibodies (Abs) gave a 100% specificity, 95% sensitivity, and 95% predictability. Although our results also indicate a potential value in the increased IL-2R expression, these findings must be confirmed by further studies. Furthermore, the combination of both immunologic and SC techniques in urinary sediments allows early detection of AR and is cost effective and simple features that could be used routinely for follow-up of renal transplant recipients.

Sirolimus: its discovery, biological properties, and mechanism of action

Sirolimus is the USAN-assigned generic name for the natural product rapamycin. Sirolimus is produced by a strain of Streptomyces hygroscopicus, isolated from a soil sample collected from Rapa Nui commonly known as Easter Island. Although sirolimus was isolated as an antifungal agent with potent anticandida activity, subsequent studies revealed impressive antitumor and immunosuppressive activities. Sirolimus demonstrates activity against several murine tumors, such as B16 43 melanocarcinoma, Colon 26 tumor, EM ependymoblastoma, and mammary and colon 38 solid tumors. Sirolimus is a potent inhibitor of antigen-induced proliferation of T cells, B cells, and antibody production. Demonstration of the potent immunosuppressive activity of sirolimus in animal models of organ transplantation led to clinical trials and subsequent approval by regulatory authorities for prophylaxis of renal graft rejection. Interest in sirolimus as an immunosuppressive therapy in organ transplantation derives from its unique mechanism of action, its unique side-effect profile, and its ability to synergize with other immunosuppressive agents. The molecular mechanism underlying the antifungal, antiproliferative, and immunosuppressive activities of sirolimus is the same. Sirolimus forms an immunosuppressive complex with intracellular protein, FKBP12. This complex blocks the activation of the cell-cycle–specific kinase, TOR. The downstream events that follow the inactivation of TOR result in the blockage of cell-cycle progression at the juncture of G1 and S phase.

New model for simultaneous heart and kidney transplantation in mice.

In clinical settings, combined heart and kidney transplantation results in a lower incidence of cardiac graft and renal graft rejection as compared to isolated heart and kidney transplantation. To study the phenomenon in an experimental setting, we developed a model of combined heart and kidney transplantation in mice. According to our technique, we kept the patch of the infrarenal aorta and inferior vena cava (IVC) as long as possible during the donor's kidney explantation, and then, after finishing the kidney implantation with conventional techniques, we anastomosed the innominate artery and pulmonary artery of the heart graft to the patch of the infrarenal aorta and IVC of the renal graft, respectively, instead of the recipient's aorta and IVC. With the use of our method, combined heart and kidney transplantation in mice can be performed to get enough suture room for the second graft, to avoid prolonging the occlusion time of the recipient's circulation, and to profoundly decrease postoperative complications such as paraplegia and mortality. Of the 14 recipients with combined heart-kidney isografting, 10 have been successful (71.4%), surviving over 100 days with normal function of both grafts, and with lack of change in histological appearance. This suggests that the technique is feasible and reliable.

Nonfunctioning renal allograft embolization as an alternative to graft nephrectomy: report on seven years' experience.

Graft nephrectomy is the treatment of choice in patients with graft intolerance syndrome, but it is associated with high morbidity and mortality rates. Renal vascular embolization has been suggested as a possible alternative. The aim of this study was to evaluate the efficacy and safety of arterial embolization of these nonfunctioning transplanted kidneys. Twenty-six transplanted kidneys in 25 patients with irreversible renal graft rejection and graft intolerance who underwent arterial embolization at our center from August 1994 to April 2001 were analyzed for procedural success and long-term outcome. Embolization was performed with absolute alcohol or with polyvinyl alcohol (Ivalon) and coils. Twenty-four of the 26 (92%) procedures were technically successful, but in one patient only partial occlusion of one of two renal arteries was achieved, and in another the renal artery was already completely occluded. There were two major complications: emphysematous pyelonephritis necessitating nephrectomy and groin abscess that was drained. Follow-up ranged from 8 to 84 months. Clinical success was achieved in 24 of the 26 procedures (92%), and only in one patient did embolization fail to relieve the symptoms, and nephrectomy was performed 3 months later. Renal vascular embolization is a simple, safe and effective technique for the treatment of nonfunctioning renal allografts associated with graft intolerance syndrome. We suggest that it be considered the treatment of choice.

In vivo detection of acute rat renal allograft rejection by MRI with USPIO particles

Magnetic resonance imaging (MRI) for non-invasively detecting renal rejection was developed by monitoring the accumulation of macrophages labeled with dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles at the rat renal allografts during acute rejection. Five groups of male rats with DA-->BN renal allografts and one group with BN-->BN renal isografts were investigated by MRI before, immediately after, and 24 hr after intravenous infusion with different doses of USPIO particles. All infusions were done on post-operative day 4. MRI experiments were carried out in a 4.7-Tesla instrument using a gradient echo sequence. MR signal intensity (MRSI) of the cortex was found to decrease with higher dosages of USPIO particles. In the absence of USPIO infusion, a decrease in MRSI was seen in the medulla region, presumably due to hemorrhage associated with renal graft rejection, while no significant change was observed in the cortex. The optimal dose of USPIO particles for visualizing rejection-associated changes in our rat kidney model appears to be 6 mg Fe/kg body weight. Iron staining results correlated with the MRSI data, indicating that the signal reduction in the MR images was due to the presence of iron. Immunohistochemical results indicated that USPIO particles were mostly taken up by infiltrating macrophages in the rejecting grafts. Our results suggest that MRI with intravenous administration of dextran-coated USPIO particles appears to be a valuable and promising tool that can be used as a non-invasive and sensitive method to detect graft rejection in renal transplantation.

Combination therapy of N-acetylcysteine, sodium nitroprusside and phosphoramidon attenuates ischemia-reperfusion injury in rat kidney.

Renal ischemia is of clinical interest because of its role in renal failure and also renal graft rejection. To evaluate the effect of the combination of N-acetylcysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor, and phosphoramidon (P), an endothelin converting enzyme inhibitor, on tissue protection against ischemia-reperfusion injury, we studied the biochemical and morphological changes due to 90 min of renal ischemia-reperfusion in the rat model. Ninety min of ischemia caused very severe injury and the animals could not survive after 4 days without any treatment. Whereas, animals in the treated groups survived i.e. the NAC group (25%), NAC + SNP group (43%) and in the NAC + SNP + P group (100%), 2 weeks after 90 min of ischemia. A significant increase in the serum levels of creatinine and urea nitrogen was shown in the untreated group and to a much lesser extent in the treated group, especially in the NAC + SNP + P group. The protective effect was also supported by light microscopic studies on renal tissue sections. We also measured the activities of antioxidant enzymes in tissue homogenates. With the exception of Mn-superoxide dismutase, the activities of antioxidant enzymes (catalase, glutathione peroxidase, CuZn-superoxide dismutase) were decreased in the untreated kidney. The administration of NAC alone and NAC + SNP protected against the loss of activities. Treatment with a combination of NAC, SNP and P showed a synergistic effect as evidenced by the best protection. These results suggest that pre-administration of a combination of antioxidant (NAC) with endothelin derived vasodilators (sodium nitroprusside and Phosphoramidon) attenuates renal ischemia-reperfusion injury, e.g. in donor kidney for transplantation, by protecting cells against free radical damage.

Prevalence of transfusion transmitted virus infection and its effect on renal graft survival in renal transplant recipients.

Little is known about the prevalence of transfusion transmitted virus (TTV) infection in renal transplant recipients (RTxs) and its effects on allograft survival. We investigated the prevalence of TTV and its effects on liver injury and graft survival in RTxs. The study was performed in 33 consecutive RTxs (8 females, 25 males) and 100 blood donors (35 females, 65 males). A nested polymerase chain reaction was used to detect TTV DNA in serum. Serum creatinine and alanine aminotransferase (ALT) levels and 24-h protein excretion were determined in both TTV-positive and-negative patients. The total number of blood transfusions, the duration of hemodialysis and the total duration after transplantation were recorded in RTxs. In addition, hepatitis B surface antigen (HbsAg), anti-hepatitis C virus (HCV) and hepatitis G virus DNA antibodies were determined in all patients. TTV DNA was detected in 51.5% of RTxs and in 7% of the control group and this difference was statistically significant (p < 0.01). In the RTx group, 64.7% of TTV-positive and 56.2% of TTV-negative patients had undergone a previous blood transfusion. However, the blood transfusion replacement rate, total duration of dialysis therapy and posttransplant period did not differ between these two groups. Five (15.1%) patients in the RTx group had abnormal liver function tests (ALT >40 IU/l). Of these patients, 2 were anti-HCV-positive, 1 was HBsAg-positive and anti-HCV- plus TTV DNA-positive and the serologic tests of the remaining 2 patients were all negative. Among the TTV-positive patients, 2 (11.7%) were anti-HCV-positive, 1 (5.8%) was HBsAg-positive and 3 (17.6%) were HGV DNA-positive. The baseline serum creatinine levels did not differ significantly between the TTV-positive and-negative patients, being 1.5 +/- 0.6 and 1.4 +/- 0.6 mg/dl, respectively ( p > 0.05). Two of the TTV-positive patients and 1 of the TTV-negative patients had proteinuria. A 1-year follow-up of TTV-positive and-negative patients demonstrated neither acute nor chronic graft rejection. In RTxs, TTV infection was more prevalent than in the normal population. In our patients the virus did not have an important effect on renal graft rejection and did not cause liver injury. However, the question of whether TTV infection may affect graft survival requires further long-term investigation in larger groups.

Evaluation of the rate of progression of chronic renal transplant rejection using the proliferating cell nuclear antigen

Since the introduction of kidney transplantation, the short-term patient and graft survival have been progressively improving, but the long-term graft survival and half-life of transplants have not. Beyond doubt, chronic rejection (CR) remains the major cause of chronic graft failure and is responsible for the loss over 10 years of 50% of grafts. (1-4). CR is characterized clinically by proteinuria, hypertension and declining renal function, and pathologically by arteriosclerosis, glomerulosclerosis and interstitial fibrosis. The progressive morphological changes of glomerular, vascular and tubulointerstitial (TIN) compartments in renal allografts experiencing chronic rejection correlate directly with declining renal function and the eventual graft loss [5]. In consideration of this fact and using proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation, a retrospective analysis of renal allograft biopsies was carried out with the aim to determine whether quantification of the cell proliferation activity in three tissue compartments may be used as a prognostic index of CR progression. Clinical course and biopsy specimens of 27 patients with clinically and morphologically confirmed (Banff schema) diagnosis of CR were retrospectively evaluated. Patients were transplanted between 1988 and 1994 at our Institute and regularly followed-up for at least 2 years after transplantation (14 patients), or till haemodialysis has been resumed (11 patients) or till death (2 patients). The progression rate of renal graft function deterioration was estimated by slopes of regression lines obtained by plotting 1/sCr vs time. According to the progression rate after biopsy patients were divided into two groups: fast progression group (10 patients) and slow progression group (17 patients). Immunosuppressive regimens included induction with anti-lymphocyte globulin followed with cyclosporine, steroids and azathioprine. Immunohistochemistry. Sections of formalin-fixed, paraffinembedded tissue were stained for PCNA with monoclonal mouse anti-proliferating cell nuclear antigen (clone PC 10, Dako) as follows in ref. [6]. PCNA immunoreactivity of smooth muscle cells and lymphocytes in vascular intima, lymphocytes in TIN and glomerular mesangium proliferation was assessed by semiquantitative scoring (scale 0-3). Normal tonsil was used as a positive control. Differences between groups were evaluated using Student's t-test. Pearson's correlation test was used to study the relationship between variables. P values < 0.05 were considered as significant. Data on patients are presented in Table 1. The only statistical difference was in progression of chronic graft failure presented by regression lines obtained by plotting 1/sCr vs time. PCNA protein expressions in vascular, glomerular and TIN compartments for both groups are presented in Figs. 1-3.. PC 10 immunoreactivity was confined to the nucleus. The pattern of staining was granular or diffuse. Individual values of the scores of PCNA immunoreactivity in different tissue compartments for the groups examined are presented in Tables 2 and 3. Comparison of the means of PCNA immunoreactivity scores in different tissue compartments of fast and slow progression group showed that cells proliferation in glomerular, TIN and vascular compartments are significantly higher in the fast than in the slow progression group (Table 4). Besides, significant positive correlation was found between the slopes of the regression lines plotting 1/sCr vs. time and cells proliferation scores for all compartments (Table 5). Chronic graft failure may occur due to CR, cyclosporine nephrotoxicity, repeated acute rejection, recurrent glomerular diseases, surgical and urological complications, but CR was recognized as the most common cause of chronic graft failure and renal graft loss [7]. Progression of renal disease reflects the interactive effects of changes in the structure and function. This notion has been examined many times in the native kidneys by studies correlating glomerular filtration rate with architectural deterioration in the glomerular or interstitial compartment [8-10]. Meanwhile, the similar studies in human renal allograft are scarce. Increased PCNA immunodetection in glomerular cells as well as in interstitial infiltrates was described in acute and chronic renal graft rejection and diagnostic value of this finding was analyzed [12, 15]. Similarly, PCNA was often examined immunohistochemically in lymphoma and solid tumours as a marker of cell proliferation, but its reliability as a prognostic factor was also evaluated [18-20]. In the present study the prognostic value of PCNA expression in different tissue compartments of renal grafts experiencing CR was examined for the first time. The present study revealed that two groups examined with the different chronic graft failure progression rate as the main difference had significantly different proliferation of cells in glomerular, TIN and vascular compartments. PCNA immunoreactivity scores in these three compartments were significantly higher in the fast than in slow progression group. The significant positive correlation between the slope of the regression line plotting 1/sCr vs. time and cell proliferation in glomerular, TIN and vascular compartments was also found. Therefore, it was suggested that the measurement of PCNA expression in renal graft tissue might be used as a prognostic index.

Simultaneous pancreas-kidney and pancreas transplantation.

The discovery of insulin in 1922 changed the treatment of type 1 diabetes mellitus (DM) forever. Insulin was the first effective therapy for type 1 DM; however, its success engendered a terrible paradox for patients with this disease. The use of insulin transformed type 1 DM from a rapidly fatal