Human Kidney Allografts Research Articles

Urinary cell mRNA profiles predictive of human kidney allograft status

Kidney allograft status is currently characterized using the invasive percutaneous needle core biopsy procedure. The procedure has become safer over the years, but challenges and complications still exist including sampling error, interobserver variability, bleeding, arteriovenous fistula, graft loss, and even death. Because the most common type of acute rejection is distinguished by inflammatory cells exiting the intravascular compartment and gaining access to the renal tubular space, we reasoned that a kidney allograft may function as an in vivo flow cytometer and sort cells involved in rejection into urine. To test this idea, we developed quantitative polymerase chain reaction (PCR) assays for absolute quantification of mRNA and pre-amplification protocols to overcome the low RNA yield from urine. Here, we review our single center urinary cell mRNA profiling studies that led to the multicenter Clinical Trials in Organ Transplantation (CTOT-04) study and the discovery and validation of a 3-gene signature of 18S rRNA-normalized measures of CD3ε mRNA and IP-10 mRNA and 18S rRNA that is diagnostic and predictive of acute cellular rejection in the kidney allograft. We also review our development of a 4-gene signature of mRNAs for vimentin, NKCC2, E-cadherin, and 18S rRNA diagnostic of interstitial fibrosis/tubular atrophy (IF/TA).

A Higher Risk of Acute Rejection of Human Kidney Allografts Can Be Predicted from the Level of CD45RC Expressed by the Recipients’ CD8 T Cells

Although transplantation is the common treatment for end-stage renal failure, allograft rejection and marked morbidity from the use of immunosuppressive drugs remain important limitations. A major challenge in the field is to identify easy, reliable and noninvasive biomarkers allowing the prediction of deleterious alloreactive immune responses and the tailoring of immunosuppressive therapy in individuals according to the rejection risk. In this study, we first established that the expression of the RC isoform of the CD45 molecule (CD45RC) on CD4 and CD8 T cells from healthy individuals identifies functionally distinct alloreactive T cell subsets that behave differently in terms of proliferation and cytokine secretion. We then investigated whether the frequency of the recipients CD45RC T cell subsets before transplantation would predict acute graft rejection in a cohort of 89 patients who had undergone their first kidney transplantation. We showed that patients exhibiting more than 54.7% of CD8 CD45RChigh T cells before transplantation had a 6 fold increased risk of acute kidney graft rejection. In contrast, the proportions of CD4 CD45RC T cells were not predictive. Thus, a higher risk of acute rejection of human kidney allografts can be predicted from the level of CD45RC expressed by the recipients’ CD8 T cells.

MicroRNA Sequence Profiles of Human Kidney Allografts With or Without Tubulointerstitial Fibrosis

MicroRNA (miRNA) alterations accompanying interstitial fibrosis and tubular atrophy (IFTA) in kidney allografts may point toward pathologic mechanisms. Small-RNA sequencing provides information on total miRNA abundance and specific miRNA expression and allows analysis of differential expression based on read counts. MiRNA expression profiles of 8 human kidney allograft biopsies (4 IFTA and 4 normal biopsies, discovery set) were characterized using barcoded deep-sequencing of a cDNA library prepared from multiplexed RNA. Statistical analysis of the sequence data guided selection of miRNAs for validation and the levels of selected miRNAs were quantified in 18 biopsies (10 IFTA and 8 normal) using real-time quantitative PCR assays (RT-QPCR). Total miRNA content was 50% lower in RNA from IFTA compared with normal biopsies. Global miRNA profiles clustered in partial agreement with diagnosis. Several miRNAs, including miR-21, 142-3p, and 5p and the cluster comprising miR-506 on chromosome X had twofold to sevenfold higher expression in IFTA compared with normal biopsies, whereas miRNA miR-30b and 30c were lower in IFTA biopsies (miR-30a, -30d, -30e, and all respective star sequences showed similar trends). IFTA and normal biopsy distinction was also noted in the pattern of miRNA nucleotide sequence variations. Differentially expressed miRNAs were confirmed on the larger set of allograft biopsies using RT-QPCR, and the levels of miRNAs were found to be associated with allograft function and survival. Differentially expressed miRNAs and their predicted targets identified by deep sequencing are candidates for further investigation to decipher the mechanism and management of kidney allograft fibrosis.

Noninvasive Diagnosis of Acute Antibody Mediated Rejection of Human Kidney Transplant Recipients by Measurement of Serum MicroRNAs

Introduction: Micro(mi)RNAs are single stranded RNAs that have a profound impact on mRNA/protein expression and have been advanced as cancer biomarkers. Herein, we investigated the hypothesis that extracellular/circulating levels of miRNAs are diagnostic biomarkers of acute antibody mediated rejection (AMR) of human kidney allografts. Methods: We isolated total RNA from 36 individuals; 12 patients with AMR biopsies, 20 patients with normal biopsies and 4 healthy volunteers. Discovery: We did global expression profiling of serum miRNAs from 7 kidney transplant recipient (3 AMR and 4 Normal) and 4 healthy volunteers. Differentially expressed (P< 0.1) miRNAs were queried and a panel of miRNAs associated with endothelial and immune cell functions were identified. The miRNA with least variability within 4 healthy volunteers was used for normalizing other miRNAs to adjust for biological variability. Verification: The differential expression was verified by measuring miRNAs in the same serum samples by targeted miRNA specific realtime (RT) PCR assays. Validation: We did targeted miRNA specific RT-PCR assay of the miRNAs in the serum of 25 kidney transplant recipients; 9 AMR and 16 Normal. Sera were spiked with C. Elegans miR-54 synthetic RNA and were used for normalizing other miRNAs to adjust for technical variability. The fold differences between AMR and Normal were calculated. Results: Of the 377 mature miRNAs analyzed in the Discovery set, 38% were measureable (Ct < 35) in sera from AMR patients and 41% in sera of normal patients. Unsupervised hierarchical clustering of circulating miRNA levels correctly classified the two groups and circulating levels of 17 miRNAs were significantly different between AMR and Normal (Figure 1).[Figure 1]Six of the 17 distinguishing miRNAs were associated with endothelial and/or immune cells. We measured their levels in the sera from 25 kidney transplant recipients (AMR=9 and Normal=16). We validated that the circulating levels 5 of the 6 miRNAs are significantly higher in AMR than Normal (Figure 2).[Figure 2]Conclusion: Our data demonstrate that extracellular/circulating levels of miRNA expression are associated with AMR of human renal allografts. Our discovery and validation of circulating levels of miRNAs as biomarkers of AMR introduces a novel strategy to noninvasively diagnose AMR.

Unique and Shared Molecular Pathways Driving Acute T-Cell Mediated Rejection or Acute Antibody Mediated Rejection of Human Kidney Allografts Unmasked by Urinary Cell mRNA Profiling

Introduction: Neither clinical nor biochemical characteristics differentiate acute T-cell mediated rejection (ACR) from acute antibody mediated rejection (AMR) and the invasive kidney allograft biopsy remains the procedure of choice. The pathophysiology of these two major types of acute rejection may involve shared as well as unique molecular pathways and the trafficking of cells to the graft undergoing ACR may be different from those trafficking to the grafts undergoing AMR. We therefore investigated the hypothesis that urinary cell mRNA expression patterns could distinguish ACR from AMR in kidney transplant recipients. Methods: A customized and hypothesis based mRNA panel comprised of 26 unique mRNAs encoding immunoregulatory proteins was designed and absolute levels of the 26 mRNAs were measured in urinary cells from 40 kidney allograft recipients whose for-cause biopsies were classified as ACR (n=20) or AMR (n=20). Urine samples were collected at the time of biopsy prior to any treatment. Total RNA was isolated from urinary cells and reverse transcribed to cDNA. Absolute mRNA copy numbers were quantified by a standard curve method with a synthetic amplicon using a pre-amplification enhanced real-time PCR assay developed and validated in our laboratory. Results: Kidney allograft function, evaluated by eGFR, was comparable between ACR and AMR groups at the time of rejection (27.20 ± 12 .10 ml/min.1.73 m2 in ACR group and 31.12 ± 18.07 ml/min.1.73 m2 in AMR group, P=0.42). In support of the notion that unique pathways drive AMR and ACR, we found the levels of 5 of the 26 mRNAs measured (CXCL13, Foxp3, OX40, IL-6 and vimentin) were significantly different between the ACR group and AMR group (Figure 1)[Figure 1]We also found evidence for shared pathogenetic pathways for AMR and ACR; the levels of the 21 of the 26 mRNAs measured were similar between the two groups (Figure 2).[Figure 2]Conclusions: Our results demonstrate that urinary cell mRNA expression profiles distinguish AMR from ACR in patients with no clinically distinguishing characteristics. Furthermore, we found evidence for unique as well as shared pathogenetic pathways despite biopsies being dichotomized histologically as ACR only or AMR only.

Peri-Operative Complement Activation During Early Phase of I/R Injury Does not Seem to Create a Pro-Regenerative Biochemical Signaling for Regeneration of Human Kidney Allografts. A Preliminary Report

Peri-Operative Complement Activation During Early Phase of I/R Injury Does not Seem to Create a Pro-Regenerative Biochemical Signaling for Regeneration of Human Kidney Allografts. A Preliminary Report

Clinical Analysis of Perioperative Complement Activity during Ischemia/Reperfusion Injury following Renal Transplantation

The complement cascade seems to be an important mediator modulating renal ischemia/reperfusion injury. This study analyzed whether significant changes occur in the levels of a terminal panel of complement molecules (C3a, C5a, and C5b-9/membrane attack complex) during the early phase of human kidney allograft reperfusion and evaluated the potential association of these changes with clinical post-transplant graft function in kidney transplant recipients. Seventy-five renal transplant recipients undergoing transplantation between 2004 and 2006 were enrolled in the study and divided into early, slow, and delayed graft function groups. Blood samples were collected perioperatively during consecutive minutes of allograft reperfusion from the renal vein. Levels of complement molecules were measured using ELISA. Analysis revealed no significant changes in C3a and C5a levels throughout reperfusion. The main complement molecule that was significantly associated with post-transplant graft function was C5b-9/membrane attack complex; throughout the reperfusion period, perioperative levels of C5b-9/membrane attack complex were around two to three times higher in delayed graft function patients than early and slow graft function individuals (P<0.005). In addition, C5b-9/membrane attack complex levels had a relatively high clinical sensitivity and specificity (70%-87.5%) for the prediction of early and long-term (1 year) post-transplant allograft function. This clinical study supports a role for the complement cascade in delayed graft function development. However, additional studies are needed to elucidate the exact mechanisms responsible for this phenomenon. In addition, perioperative measurements of C5b-9/membrane attack complex are highlighted as promising potential clinical markers of post-transplant renal allograft function.

Interferon-γ +874 Polymorphism in the First Intron of the Human Interferon-γ Gene and Kidney Allograft Outcome

Background Despite advances in immunosuppressive therapy in the past decade, allograft rejection remains an important cause of kidney graft failure. Cytokines play a major role in the inflammatory and immune responses that mediate allograft outcomes. Several studies have shown that the production of cytokines varies among individuals. These variations are determined by genetic polymorphisms, most commonly within the regulatory region of cytokine genes. The aim of the present study was to assess the effect of allelic variation on acute rejection episodes (ARE) or chronic allograft nephropathy (CAN) after kidney transplantation. Methods To determine a possible correlation between the interferon (INF)-γ +874 polymorphism and kidney allograft outcome, we isolated genomic DNA from 74 patients who underwent isolated kidney allografts and were classified into 2 groups—a rejection and a nonrejection group—for comparison with a control group of 163 healthy subjects. Results We genotyped INF-γ +874 polymorphisms in all groups. The transplant group showed a significantly increased homozygous genotype T/T ( P = .0118) compared with healthy controls. Similarly, considering only patients with CAN, the homozygous genotype T/T ( P = .0067) was significantly increased compared with the healthy controls. The rejection group indicated a significant increased homozygous genotype T/T compared with the control group ( P = .0061). Conclusion Homozygous genotype T/T was associated with increased levels of INF-γ and greater numbers among the rejection and CAN cohorts.

Chronic Humoral Rejection of Human Kidney Allografts Is Associated with MMP-2 Accumulation in Podocytes and its Release in the Urine

Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP-2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP-2 staining was detected by immunohistochemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP-2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non-CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP-2 correlated with higher levels of proteinuria in both CHR and non-CHR patients. Longitudinal analysis indicated that increase in urine MMP-2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP-2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP-2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR.

Mycophenolic Acid May Delay Allograft Fibrosis by Inhibiting Transforming Growth Factor-β1-Induced Activation of Nox-2 Through the Nuclear Factor-κB Pathway

We evaluated the role of renal tubular Nox-2 in the pathogenesis of epithelial-to-mesenchymal transition (EMT) in kidney allografts. We examined this question in the human kidney allografts with interstitial fibrosis and tubular atrophy not otherwise specified (IFTANOS), in the Fisher to Lewis rat transplant model, and in the in vitro model of transforming growth factor-beta1-induced EMT in normal rat kidney epithelial cells (NRK52E). We first demonstrated that Nox-2 and alpha-smooth muscle actin (SMA) were increased in renal tubules from kidney transplant recipients on calcineurin inhibitors, mycophenolic acid (MPA), and prednisone with IFTANOS, suggestive of EMT (n=6). Next, we examined Nox-2 expression and fibrogenesis in syngeneic transplants, allogeneic transplants treated with MPA 40 mg/kg per 24 hr, and untreated allogeneic transplants for 6 months (n=14 in each group). Immunofluorescent and immunohistochemical studies for Nox-2, alpha-SMA, and E-cadherin showed that similar to patients with IFTANOS, rat allografts had greater tubulointerstitial staining for Nox-2 and alpha-SMA. MPA therapy prevented these changes. Immunoblot analyses examining Nox-2 signaling (phospho-nuclear factor [NF]-kappaB), redox signaling (phospho-smad2), and fibrosis (alpha-SMA and fibronectin) demonstrated that MPA treatment prevented the up-regulation of Nox-2, inhibited p-NF-kappaB and p-smad2, and down-regulated alpha-SMA and fibronectin levels. Finally, we examined Nox-2 signaling in vitro and confirmed that MPA inhibited phospho-NF-kappaB, Nox-2, phospho-smad2, and alpha-SMA during transforming growth factor-beta1-induced EMT of NRK52E cells while reducing Nox-2, vimentin, and fibronectin mRNA levels. MPA may down-regulate Nox-2 activation and EMT through the NF-kappaB pathway in the tubular epithelial cells, suggesting a novel role for this drug independent of its immunosuppressive properties.

JNK signalling in human and experimental renal ischaemia/reperfusion injury

Ischaemia/reperfusion (I/R) is an important factor in delayed graft function in renal transplantation and is a determinant of long-term graft outcome. This study examined the role of c-Jun N-terminal kinase (JNK) signalling in human and experimental renal I/R injury. Biopsies obtained 15-20 min after reperfusion of human renal allografts were examined for JNK signalling by immunostaining for phospho-c-Jun. To examine the pathologic role of JNK signalling, a selective JNK inhibitor (CC-401) was administered to rats before or after the induction of a 30-min period of bilateral renal ischaemia followed by reperfusion. Renal function and tubular damage were analysed. Substantial JNK activation was evident in tubular epithelial cells in kidneys from deceased donors (n = 30) which was less prominent in kidneys from live donors (n = 7) (44.6 +/- 24.8% vs 29.1 +/- 20% p-c-Jun+, respectively; P < 0.05), whereas biopsies of thin basement membrane disease exhibited little, or no, p-c-Jun staining. The degree of p-c-Jun staining correlated with ischaemic time in deceased donor allografts, but not with graft function. Administration of CC-401 to rats prior to bilateral renal I/R prevented acute renal failure and largely prevented tubular damage, leucocyte infiltration and upregulation of pro-inflammatory molecules. However, delaying CC-401 treatment until 1 h after reperfusion (after the peak of JNK activation) had no protective effect. We have identified acute activation of the JNK signalling pathway following I/R in human kidney allografts. Experimental studies indicate that blockade of JNK signalling, commenced prior to this activation, can prevent acute tubular necrosis and renal dysfunction secondary to I/R injury.

Modified Spectrometry (O2C Device) of Intraoperative Microperfusion Predicts Organ Function After Kidney Transplantation: A Pilot Study

Background Delayed graft function (DGF) is defined as posttransplantation dialysis within 1 week, which might be associated with impaired long-term graft survival. The aim of our pilot study was to establish the ability of intraoperative spectrometry of allograft microperfusion to predict DGF. Methods Twenty human kidney allografts transplanted from deceased donors were evaluated intraoperatively after reperfusion using modified organ spectrometry (O2C device). We examined hemoglobin oxygen saturation, intravascular amount of hemoglobin, and microperfusion flow/velocity. Results Retrospectively, 10/20 (50%) allografts with measurable impairment of cortical hemoglobin oxygen saturation and microperfusion flow/velocity developed DGF. Retrospectively, we found that if the intravascular amount of hemoglobin was increased upon intraoperative measurement, the kidney was prone to develop DGF. Conclusions Spectrometry data predicted DGF. Our results supported the thesis that impaired microperfusion is the key to DGF and might be related to postcapillary endothelial damage or intravascular sludge.

Isoforms of human leukocyte antigen-G and their inhibitory receptors in human kidney allograft acceptance

Novel therapeutic strategies such as the modulation of dendritic cell and T-cell function have exhibited great potential in clinical transplantation. Human leukocyte antigen (HLA)-G is a molecule that plays a significant role in establishing complex mechanisms to protect semiallogeneic fetuses from rejection by the maternal immune system. The unique characteristics of both cell-surface and soluble isoforms of HLA-G, the formation of disulfide-bonded dimers with the potential to augment inhibitory receptor signaling, and the function of HLA-G as a preferential ligand for the immunoglobulin-like transcript receptors make HLA-G very important in fundamental approaches for the modulation of immune responses to improve allogeneic graft survival in clinical transplantation. Experimental data from several groups as well as our data from experiments involving HLA-G-mediated human tolerogenic dendritic cells in vitro and receptor transgenic mice in vivo indicate that different isoforms of HLA-G have various immunomodulatory effects through the inhibitory receptors. This knowledge is crucial in understanding mechanisms of prolongation of allograft survival. The analyses of HLA-G isoforms and inhibitory receptors in patients with kidney allograft and the relationship among different isoforms of HLA-G, inhibitory receptors, their mediated immunoregulation, and graft acceptance or failure will be discussed here.

Nox-2 Is a Modulator of Fibrogenesis in Kidney Allografts

We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox-2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox-2 also colocalized with alpha-SMA in areas of injury, suggestive of epithelial-to-mesenchymal transition (EMT). Interstitial macrophages (CD68(+)) and myofibroblasts (alpha-SMA(+)) expressed Nox-2 while graft infiltrating T cells (CD3(+)) and mature fibroblasts (S100A4(+)) were Nox-2(-). These results were confirmed in the Fisher-to-Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox-2 and alpha-SMA, suggestive of EMT. Immunoblot analyses showed that Nox-2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (alpha-SMA and phospho-Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for 1 week showed reduced fibronectin and phospho-Smad2 and increased E-cadherin levels. Cyclosporine A, TGF-beta1 and angiotensin II increased Nox-2 mRNA levels 2- to 7-fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E-cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox-2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts.

Clinical experience with mixed chimerism to induce transplantation tolerance

Lymphohematopoietic chimerism was first shown to be associated with donor-specific allograft tolerance more than 60 years ago. However, early clinical experience with bone marrow transplantation soon revealed that conventional, myeloablative approaches were far too toxic and the risk of graft-versus-host disease too great to justify using this technology for the purpose of organ allograft tolerance induction in the absence of malignant disease. In this review, we discuss a step-wise approach that has been applied by several centers to establish less toxic approaches to using hematopoietic cell transplantation (HCT) for tolerance induction. These steps include (i) feasibility and efficacy data for tolerance induction in large animal models; (ii) safety data in clinical trials for patients with hematologic malignancies; and (iii) pilot trials of combined HCT and kidney transplantation for tolerance induction. Thus far, only one published trial conducted at the Massachusetts General Hospital in Boston has achieved long-term acceptance of human leukocyte antigen-mismatched kidney allografts without chronic immunosuppressive therapy. Alternative protocols have been successful in large animals, but long-term organ allograft tolerance has not been reported in patients. Thus, proof-of-principle that nonmyeloablative induction of mixed chimerism can be used intentionally to induce organ allograft tolerance has now been achieved. Directions for further research to make this approach applicable for a broader patient population are discussed.

Monitoring of human liver and kidney allograft tolerance: a tissue/histopathology perspective

Several factors acting together have recently enabled clinicians to seriously consider whether chronic immunosuppression is needed in all solid organ allograft recipients. This has prompted a dozen or so centers throughout the world to prospectively wean immunosuppression from conventionally treated liver allograft recipients. The goal is to lessen the impact of chronic immunosuppression and empirically identify occasional recipients who show operational tolerance, defined as gross phenotype of tolerance in the presence of an immune response and/or immune deficit that has little or no significant clinical impact. Rare operationally tolerant kidney allograft recipients have also been identified, usually by single case reports, but only a couple of prospective weaning trials in conventionally treated kidney allograft recipients have been attempted and reported. Pre- and postweaning allograft biopsy monitoring of recipients adds a critical dimension to these trials, not only for patient safety but also for determining whether events in the allografts can contribute to a mechanistic understanding of allograft acceptance. The following is based on a literature review and personal experience regarding the practical and scientific aspects of biopsy monitoring of potential or actual operationally tolerant human liver and kidney allograft recipients where the goal, intended or attained, was complete withdrawal of immunosuppression.

Tim-3 expression in human kidney allografts

Background Tim-3 was recently described as a Th1-specific molecule, participating in the regulation of immune responses and in the induction of allograft tolerance. Here, we studied Tim-3 mRNA expression together with molecular markers of T-cell activation and cytotoxicity, in rejected human kidney grafts. Methods Twenty human kidney grafts that had undergone nephrectomy due to an irreversible acute rejection episode were studied. We quantified intragraft expression of Tim-3, granzyme B, perforin, IFN-γ and Fas–ligand mRNA by real-time RT-PCR, with probes and primers TaqMan. Protocol biopsies were studied as controls. Statistical analyses were performed to compare groups, and to investigate the potential association with gene transcripts measures and rejection. Results All molecules studied were up-regulated in the rejection group compared with controls ( p < 0.001). Acute rejection type III (Banff 97) profiles were associated with higher values, where granzyme B and perforin presented the highest (5672.51 ± 9002.16 and 1866.59 ± 2426.38, respectively) and Tim-3 had the lowest ones (166.62 ± 174.94). Tim-3 had also a lower expression in those patients that did not respond to anti-rejection therapy. There was a positive correlation between Tim-3 and IFN-γ mRNA expression levels ( r 2 = 0.73; p < 0.001). Conclusions Our results corroborate the concept that acute rejection is an active process, where inflammatory as well as regulatory factors have their roles. Severe episodes of acute rejection were associated with higher expression of cytotoxic molecules and lower expression of potential regulatory molecule.

The Universal NF-kappaB Inhibitor A20 Protects From Transplant Vasculopathy by Differentially Affecting Apoptosis in Endothelial and Smooth Muscle Cells

Transplant vasculopathy (TV) is an accelerated form of atherosclerosis resulting in chronic rejection of vascularized allografts. The causes of TV are multifactorial and integrate at the level of the vascular wall, leading to a phenotypic switch of endothelial cells (ECs) and smooth muscle cells (SMCs). A20 is a NF-κB-dependent stress response gene in ECs and SMCs with potent anti-inflammatory effect in both cell types through blockade of NF-κB. A20 expression in ECs and SMCs correlates with the absence of TV in rat kidney allografts and long-term functioning human kidney allografts. We demonstrate that A20 protects ECs from tumor necrosis factor, Fas, and natural killer cell-mediated apoptosis by inhibiting proteolytic cleavage of caspase 8. A20 also safeguards ECs from complement-mediated necrosis. Hence, effectively shutting down cell death pathways initiated by inflammatory and immune offenders associated with TV. In contrast, A20 sensitizes SMCs to cytokine and Fas-mediated apoptosis through a novel nitric oxide (NO)-dependent mechanism. The unexpected proapoptotic effect of A20 in SMCs translates in vivo by the regression of established neointimal carotid lesions following balloon angioplasty in rats. Antedating apoptosis of SMCs, expression of the inducible NO synthase increases in A20-expressing neointimal SMCs, corroborating the involvement of NO in causing the proapoptotic effect of A20 in SMCs. Combined anti-inflammatory and anti- or proapoptotic functions of A20 in ECs and SMCs respectively qualify the positive effect of A20 upon vascular remodeling and healing. We propose that A20-based therapies may be effective in prevention and treatment of TV.

Early phase of reperfusion of human kidney allograft does not affect an erythrocyte anti‐oxidative system

Generation of reactive oxygen specimens is the basic mechanism leading to ischaemia/reperfusion injury of the kidney graft. Oxygen burst is a trigger for sophisticated biochemical changes leading to generation of oxygenated lipids and changes in microcirculation, which recruit recipient's neutrophils and contribute to delayed graft function. It has been shown that the free radicals generation correlates with the activity of anti-oxidative system. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) are involved in protection against free radicals. To examine the activity of erythrocyte anti-oxidative system during reperfusion of the transplanted kidney allograft. The study included 40 renal transplant recipients. Blood was taken from the iliac vein before transplantation and from the graft's renal vein immediately, as well as 2 and 4 min after total reperfusion. The authors assessed the process of reperfusion using ThermaCAM SC500 termovision camera. Spectrophotometric methods were used to measure superoxide dismutase, glutathione peroxidase and catalase activity as well as glutathione concentrations in erythrocytes. There were no statistically significant differences in the activities of superoxide dismutase, catalase and glutathione peroxidase as well as glutathione concentrations during the first 4 min after total graft reperfusion. Nevertheless, there was a positive correlation between the activity of superoxide dismutase and glutathione peroxidase. The results suggest that the erythrocyte anti-oxidative system is stable during the early phase after reperfusion. An association between some anti-oxidative enzymes was noted.

Clinical Trial of Tripterygium Wilfordii Hook F. in Human Kidney Transplantation in China

Objective In this study, the effects of Triptergium Wilfordii Hook F.(T II) were assessed on human kidney allograft rejection and long-term survival. Methods This study compared treatment with T II(T II group, n = 121) to that without T II(control group, n = 102) among adult first cadaveric renal transplant recipients. The T II cohort of 121 recipients were divided into a regular dosage group ( n = 82) and a double dosage group ( n = 39). No antibody induction was administered to any patient. Results Biopsy-proven early acute allograft rejection occurred in 4.1% of patients in the T II group versus 24.5% of patients in the control group. No rejection or repeated rejections occurred in the double dosage group at 3 months after transplantation. Acute rejection episodes were milder in the T II than the control group. The incidence of CD25+ cells >10/ mm 3 in the allografts at 3 months after transplantation was lower in the T II group than the control group, 15% and 50%, respectively. All patients tolerated T II well over the 5 years of this study. The 5-year graft survival censored for death with function was 96.7% in the T II group and 80.4% in the control group. Conclusion T II was effective to prevent renal allograft rejection and increase long-term renal allograft survival among adult cadaveric renal transplant recipients.