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Chronic allograft dysfunction is a major cause of late graft failure after kidney transplantation. One of the histological changes is interstitial fibrosis, which is associated with epithelial-mesenchymal transition. Bortezomib has been reported to prevent the progression of fibrosis in organs. We used rat renal transplantation model and human kidney 2 cell line treated with tumor necrosis factor-α (TNF-α) to examine their response to bortezomib. To explore the mechanism behind it, we assessed the previously studied TNF-α/protein kinase B (Akt)/Smad ubiquitin regulatory factor 2 (Smurf2) signaling and performed RNA sequencing. Our results suggested that bortezomib could attenuate the TNF-α-induced epithelial-mesenchymal transition and renal allograft interstitial fibrosis in vitro and in vivo. In addition to blocking Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase/Smurf2 signaling, bortezomib's effect on the epithelial-mesenchymal transition was associated with inhibition of nuclear factor kappa B (NF-κB) pathway by stabilizing inhibitor of NF-κB. The study highlighted the therapeutic potential of bortezomib on renal allograft interstitial fibrosis. Such an effect may result from inhibition of NF-κB/TNF-α/Akt/mTOR/p70S6 kinase/Smurf2 signaling via stabilizing protein of inhibitor of NF-κB., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Objectives: Antibody-mediated injury in chronic active antibody-mediated rejection, possibly with other effector T cells, may play a role in graft injury. The role of inflammatory cells in the inflammation and fibrosis and tubular atrophy region has been recently advocated in the progression of injury. Cytotoxic T cells play a prominent role in T-cell-mediated rejection; however, the possible role of cytotoxic T cells in circulation and the intragraft compartment in chronic active antibody-mediated rejection, a common immunological cause of long-term graft failure, has not been well-studied., Materials and Methods: We measured the frequency of circulating cytotoxic T cells with flow cytometry, serum granzyme B level by enzyme-linked immunosorbent assay and intragraft granzyme B+ cell, and mRNA by immunohistochemistry and real-time polymerase chain reaction in biopsy tissue from living donor renal allograft recipients with stable graft function and chronic active antibody-mediated rejection., Results: The frequency of CD3+ and CD3+CD8+ T cells was similar in both stable graft function patients and chronic active antibody-mediated rejection patients. The frequency of CD3+CD8+granzyme B+ cytotoxic T cells was significantly lower in peripheral blood. Serum granzyme B level and intragraft number of granzyme B+ cells (counts/mm²) were also significantly higher in the chronic active antibody-mediated rejection group compared with that of patients with stable graft function. The intragraft granzyme B+ T cell count was positively correlated with serum creatinine and 24-hour urine proteinuria but negatively correlated with estimated glomerular filtration rate., Conclusions: Granzyme B mediates covert graft injury in patients with chronic active antibody-mediated rejection in addition to antibody-mediated injury.

The identification of risk factors for acute rejection (AR) may lead to strategies to improve success of kidney transplantation. Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides into nucleosides, modulating the purinergic signaling. Some members of the Ectonucleotidase family have been linked to transplant rejection processes. However, the association of Ectonucleotide Pyrophosphatase / Phosphodiesterase 1 (ENPP1) with AR has not yet been evaluated. The aim of this study was to evaluate the association between the K121Q polymorphism of ENPP1 gene and AR in kidney transplant patients. We analyzed 449 subjects without AR and 98 with AR from a retrospective cohort of kidney transplant patients from Southern Brazil. K121Q polymorphism was genotyped using allelic discrimination-real-time PCR. Cox regression analysis was used to evaluate freedom of AR in kidney transplant patients according to genotypes. Q allele frequency was 17.6% in recipients without AR and 21.9% in those with AR (P = 0.209). Genotype frequencies of the K121Q polymorphism were in Hardy-Weinberg equilibrium in non-AR patients (P = 0.70). The Q/Q genotype (recessive model) was associated with AR (HR = 2.83, 95% CI 1.08-7.45; P = 0.034) after adjusting for confounders factors. Our findings suggest a novel association between the ENPP1 121Q/Q genotype and AR in kidney transplant recipients., Competing Interests: The authors have declared that no competing interests exist.

In kidney transplantation acute allograft rejection is the most common cause of late allograft loss. Changes in indoleamine 2,3 dioxygenase (IDO) activity, which catabolizes the degradation of tryptophan to kynurenine, may predict rejection. However, exogenous IDO is immunosuppressive in rodent kidney transplantation. Thus, the increase in IDO activity observed in acute allograft rejection is insufficient to prevent rejection. To address this question, we assessed the regulation of IDO and its role in acute rejection in a porcine model of kidney transplant. In tissue samples from rejecting kidney allografts, we showed a 13-fold increase in IDO gene transcription and 20-fold increase in IDO enzyme activity when compared with autotransplanted kidneys. Allografts also demonstrated an over fourfold increase in tissue interferon (IFN)-γ, with marked increases in tumor necrosis factor (TNF)-α, TNF-β and interleukin 1β. Gene transcription and protein levels of kynurenine 3-monooxygenase (KMO) were decreased. KMO generates the immunosuppressive kynurenine, 3-hydroxykynurenine. The results of these studies demonstrate a clear association between rejection and increased allograft IDO expression, likely driven in part by IFN-γ and facilitated by other cytokines of the allogeneic response. Moreover, the loss of downstream enzymatic activity in the IDO metabolic pathway may suggest novel mechanisms for the perpetuation of rejection., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: We aim to identify the key biomarker of acute rejection (AR) after kidney transplantation via bioinformatics methods., Methods: The gene expression data GSE75693 of 30 samples with stable kidney transplantation recipients and 15 AR samples were downloaded and analyzed by the limma package to identify differentially expressed genes (DEGs). Then, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were done to explore the biological functions and potential important pathways of DEGs. Finally, protein-protein interactions (PPIs) and literature mining were applied to construct the cocitation network and to select the hub protein., Results: A total of 437 upregulated genes and 353 downregulated genes were selected according to P < 0.01 and |log 2 (fold change)| > 1.0. DEGs of AR are mainly located on membranes and impact the activation of receptors in immune responses. In the PPI network, Src kinase, lymphocyte kinase (LCK), CD3G, B2M, interferon- γ , CD3D, tumor necrosis factor, VAV1, and CD3E in the T cell receptor signaling pathway were selected as important factors, and LCK was identified as the hub protein., Conclusion: LCK, via acting on T-cell receptor, might be a potential therapeutic target for AR after kidney transplantation.

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Chronic antibody-mediated rejection is the major cause of fading allograft function and loss after renal transplantation. Currently, pharmacological agents for the suppression of chronic antibody-mediated rejection are lacking. Non-selective proteasome inhibitors suppress antibody-mediated allograft rejection. However, extensive adverse side effects of these inhibitors severely limit their application. In contrast, immunoproteasome inhibition is effective in preclinical models of autoimmune diseases and was applied over weeks without obvious adverse side effects. ONX 0914, an immunoproteasome subunit LMP7 (β5i)-selective inhibitor, impeded the chronic rejection of kidneys transplanted from Fischer to allogeneic Lewis rats. ONX 0914 inhibited immunoproteasome induction both in immune organs and renal allografts. Selective immunoproteasome inhibition reduced the numbers of B and plasma cells, and suppressed donor-specific alloantibody production. The infiltration of T cells, B cells and macrophages as well as interferon-γ, interleukin-17, IgG and complement deposition were reduced in renal allografts of ONX 0914-treated recipients. Chronic nephropathy was ameliorated and renal allograft function preserved, enabling long-term survival of recipients. Thus, our studies define a critical role of the immunoproteasome in chronic kidney allograft rejection and suggest immunoproteasome inhibition as a promising therapeutic approach to suppress chronic antibody-mediated rejection., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Glucocorticoid (GC)-refractory acute rejection (AR) is a risk factor for inferior renal allograft outcome. We investigated genetic predisposition to the response to steroid treatment of acute allograft rejection., Methods: Single nucleotide polymorphisms of genes involved in GC signaling (GR, GLCCI1) and drug metabolism and transport (CYP3A5, ABCB1, and PXR) were analyzed in kidney transplant recipients (1995-2005, Leiden cohort, n = 153) treated with methylprednisolone. Significant associations were verified in a second cohort (Berlin cohort, n = 66)., Results: Patients who received a CYP3A5*1 allele expressing allograft had a lower risk of resistance to methylprednisolone during AR (odds ratio, 0.29; 95% confidence interval, 0.11-0.79; P = 0.016 in combined cohorts analysis). No differences were observed for GC signaling or other drug metabolism/transport-related genes. Both before transplantation (n = 69) and at time of AR (n = 88), tissue CYP3A5 mRNA expression was significantly higher in CYP3A5*1 allele expressing donor kidneys than in CYP3A5*3/*3 allografts (P < 0.00001). Moreover, steroid-responsive patients (n = 64) expressed significantly higher intragraft CYP3A5 mRNA levels compared to steroid-refractory patients (n = 42) in AR (P = 0.006)., Conclusions: CYP3A5 protein expression was detected in tubular epithelial cells and inflammatory cells within the grafts. Our findings show that steroid resistance during AR is associated with donor genotype and intragraft expression levels of CYP3A5.

Background: Organ transplantation into sensitized patients with preexisting donor-specific antibodies (DSA) is very challenging. Spleen tyrosine kinase (Syk) promotes leukocyte recruitment and activation via signaling through various cell surface receptors. We investigated whether a selective Syk inhibitor (GS-492429) could suppress antibody-mediated rejection (AMR) in a rat model of AMR in sensitized recipients., Methods: Recipient Lewis rats (RT1) were immunized with donor (Dark Agouti, RT1) spleen cells (day -5). Recipients underwent bilateral nephrectomy and orthotopic renal transplantation (day 0). Cellular rejection was minimized by tacrolimus treatment from day -1. Groups received GS-492429 (30 mg/kg, twice a day) (n = 11) or vehicle (n = 12) from 1 hour before transplantation until being killed on day 3., Results: Vehicle-treated recipients developed graft dysfunction on day 1 which rapidly worsened by day 3. Histology showed severe damage (thrombosis, acute tubular injury, capillaritis) and infiltration of many Syk leukocytes. GS-492429 did not affect graft dysfunction on day 1, but treatment reduced allograft damage and prevented the rapid deterioration of graft function on day 3. GS-492429 reduced the prominent macrophage infiltrate and reduced the M1 proinflammatory response. Neutrophil and NK cell infiltration and capillary thrombosis were also significantly reduced by GS-492429 treatment. Serum DSA levels and the deposition of IgG and C4d in the allograft were equivalent in the 2 groups., Conclusions: Treatment with a Syk inhibitor significantly reduced renal allograft injury in a model of severe antibody-mediated damage in highly sensitized recipients. Further studies are warranted to determine whether Syk inhibition is a potential adjunctive treatment in clinical AMR.

Objectives: This review focuses on the current limited evidence of graft function and graft survival in various immunosuppressive regimens involving mammalian target of rapamycin inhibitors with or without calcineurin inhibitors., Materials and Methods: We evaluated the current literature for describing the role of mammalian target of rapamycin inhibitors as an alternative to calcineurin inhibitors by searching the PubMed, EMBASE, Cochrane, Crossref, and Scopus databases using medical subject heading terms., Results: Our detailed analyses of all relevant literature showed use of mammalian target of rapamycin inhibitor-based de novo regimens, early calcineurin inhibitor withdrawal with subsequent introduction of mammalian target of rapamycin inhibitor-based regimens, and late conversion from a calcineurin inhibitor-based regimen to mammalian target of rapamycin inhibitor-based regimens. Notably, early calcineurin inhibitor withdrawal with subsequent introduction of mammalian target of rapamycin inhibitor-based regimen seemed to be a more practical and realistic approach toward immunosuppressive treatment of renal transplant recipients. However, in view of the high rejection rate observed in these studies, it is advisable not to offer these regimens to patients with moderate to high immunologic risk., Conclusions: The present evidences suggest that treatment with mammalian target of rapamycin inhibitors allows early and substantial calcineurin inhibitor minimization. The mammalian target of rapamycin inhibitors everolimus and sirolimus are preferred due to their complementary mechanisms of action and favorable nephrotoxicity profile, which have opened the way for calcineurin inhibitor reduction/withdrawal in the early posttransplant period.

Background: Serum lactate dehydrogenase (LDH) levels may help to distinguish ischemic acute tubular necrosis (ATN) from acute rejection after kidney transplantation., Methods: All kidney biopsies performed in the years 2010 to 2012 were reviewed. Serum LDH, creatinine level, clinical variables, and presence of donor-specific antibodies were recorded before the biopsy., Results: Overall 150 biopsies were included. Ischemic ATN was diagnosed in 45 biopsies and acute cellular-mediated rejection and/or antibody-mediated rejection in 59 biopsies, 38 of which were accompanied by ATN. Serum LDH was elevated in 23 (51%) of 45 cases with ischemic ATN versus 15 (14%) of 105 cases with other diagnoses ( P < .0001). Median serum LDH was 478 U/L (range 277-2018) for ischemic ATN and 372 U/L (range 191-748) for all other diagnoses ( P < .001). When delayed graft function or primary nonfunctioning grafts were caused by ischemic ATN, serum LDH was elevated in 58% of cases, but when caused by acute rejection, LDH was normal in 88% of cases ( P = .02)., Conclusions: There is a strong association between elevated serum LDH 1 to 3 days before performing kidney biopsy and the diagnosis of ischemic ATN after kidney transplantation, especially at the immediate posttransplantation period. Normal serum LDH at this period should raise a suspicion of acute rejection.

The profound involvement of cytokines in allograft rejection makes the molecules that control their actions, members of the Jak-Stat pathway, ideal targets for pharmacological intervention. Numerous studies have demonstrated that Jak3 is widely involved in the activation cascade and function of most immune cells. Tofacitinib, an oral Janus kinase inhibitor that targets Jak1/Jak3 dependent Stat activation, has been assessed as a substitute for calcineurin inhibitor therapy after low-to-moderate risk kidney transplantation in 3 randomized trials. Results using fixed-dose regimens showed a low incidence of rejection and better renal function with less interstitial fibrosis/tubular atrophy versus calcineurin inhibitor therapy. However, the safety profile of tofacitinib was poor, including increased incidences of cytomegalovirus disease, herpes zoster, BK virus, and nephropathy, which led to the discontinuation of its development for transplantation. High tofacitinib concentrations were independently associated with serious infection. Dosing according to exposure levels, coupled with pharmacodynamic monitoring based on phosphorylation of Stat5, could improve safety compared to the early fixed-dose regimens. Future studies could assess individualized dosing based on pharmacokinetic and pharmacodynamic monitoring. Additionally, because the increase of viral infections under tofacitinib may have been influenced by overlapping toxicity with concomitant mycophenolic acid, exploration of alternative adjunctive therapies (eg, a mammalian target of rapamycin inhibitor or belatacept) may demonstrate a better efficacy/safety profile. We believe that Jak inhibitors are a good and useful addition to the immunosuppressive armentarium for kidney transplant patients, and that new studies with personalized drug dosing, improved immune monitoring, and better patient selection should be performed.

Objectives: Cytomegalovirus infection and disease remain an issue in solid-organ transplant. Universal prophylaxis is more cost-effective than a preemptive strategy and is associated with significantly less Cytomegalovirus resistance after kidney transplant, especially in Cytomegalovirus-seropositive donors and Cytomegalovirus-seronegative recipients., Materials and Methods: Registry data and meta-analyses have shown that mammalian target of rapamycin inhibitors (sirolimus- and everolimus-based immunosuppression) are associated with significantly less Cytomegalovirus events in de novo kidney transplant patients than in patients who are treated with calcineurin inhibitors plus mycophenolate-based immunosuppression., Results: Recent pooled analyses of 3 randomized controlled trials in de novo kidney transplant patients, where immunosuppression was based on cyclosporine with either mycophenolate or everolimus, showed that patients who received everolimus had significantly less Cytomegalovirus events (Cytomegalovirus viremia, Cytomegalovirus infection/disease) than those who received mycophenolate, with or without cytomegalovirus as prophylaxis. An even more recent prospective randomized controlled study on de novo kidney transplant patients with no anticytomegalovirus prophylaxis demonstrated that everolimus-based immunosuppression plus low-dose tacrolimus was associated with significantly less Cytomegalovirus infection than standard-dose tacrolimus plus mycophenolate., Conclusions: The potential benefits are not fully known of such a therapeutic strategy to limit the long-term indirect effects mediated by Cytomegalovirus infections.

Current immunosuppressive medications used after transplantation have significant toxicities. Foxp3(+) T-regulatory cells can prevent allograft rejection without compromising protective host immunity. Interestingly, inhibiting the class III histone/protein deacetylase Sirtuin-1 can augment Foxp3(+) T-regulatory suppressive function through increasing Foxp3 acetylation. Here we determined whether Sirtuin-1 targeting can stabilize biological allograft function. BALB/c kidney allografts were transplanted into C57BL/6 recipients with a CD4-conditional deletion of Sirtuin-1 (Sirt1(fl/fl)CD4(cre)) or mice treated with a Sirtuin-1-specific inhibitor (EX-527), and the native kidneys removed. Blood chemistries and hematocrit were followed weekly. Sirt1(fl/fl)CD4(cre) recipients showed markedly longer survival and improved kidney function. Sirt1(fl/fl)CD4(cre) recipients exhibited donor-specific tolerance, accepted BALB/c, but rejected third-party C3H cardiac allografts. C57BL/6 recipients of BALB/c renal allografts that were treated with EX-527 showed improved survival and renal function at 1, but not 10 mg/kg/day. Pharmacologic inhibition of Sirtuin-1 also improved renal allograft survival and function with dosing effects having relevance to outcome. Thus, inhibiting Sirtuin-1 can be a useful asset in controlling T-cell-mediated rejection. However, effects on non-T cells that could adversely affect allograft survival and function merit consideration., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Expression of both platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) is increased during the development of chronic rejection which remains the major reason for late allograft loss in clinical kidney transplantation. Sunitinib is a tyrosine kinase inhibitor which inhibits both VEGF and PDGF receptors. Here we investigated its effect on the development of chronic rejection., Methods: Rat aortic denudation model was used to define sunitinib dose. In vitro studies were done to investigate the effect of sunitinib on smooth muscle cell proliferation and migration. Kidney transplantations were performed from dark agouti rat strain (DA) to Wistar furth rat strain rats and syngenic DA-DA grafts were used as controls. Allografts were immunosuppressed either with cyclosporine or with cyclosporine and sunitinib. Grafts were harvested at 5 and 90 days for histology and immunohistochemistry. Serum creatinine levels were measured weekly to monitor graft function., Results: Sunitinib decreased neointimal formation and smooth muscle cell proliferation and migration in a dose-dependent manner. Sunitinib was well tolerated and almost completely prevented chronic rejection changes and preserved significantly better renal graft function after transplantation. Sunitinib also inhibited chronic PDGF-A and -B and VEGF-A and -B expressions., Conclusions: These results demonstrate that combined inhibition of PGDF and VEGF with sunitinib prevents chronic rejection changes in experimental kidney transplantation which indicates that sunitinib could be a potential intervention also in clinical kidney transplantation.

Mammalian target of rapamycin inhibitors, such as rapamycin and more recently everolimus, have substituted calcineurin inhibitors in many minimization strategies. Despite their acclaimed renal safety profile, several lines of evidence are emerging on their potential nephrotoxic effect. Predisposing conditions for nephrotoxicity involve a complex interplay between several environmental and genetic factors in the donor-recipient pair. Renal injury may be enhanced by pharmacodynamic interactions when combined with other drugs such as calcineurin inhibitors or nutrients that are predominantly related to an increase in local tissue exposure. These toxic interactions may occur within adequate doses and therapeutic blood levels. This explains the occurrence of nephrotoxicity in some but not all cases. Here, we postulated that activity of a low permeability glycoprotein efflux pump related to low protein expression and/or inhibition enhanced immunosuppressive drug entry in different cells. A rise in intracellular drug concentration increases bioactivity, leading to greater immunosuppression and more immune-related, nonrenal adverse events in the recipient and increased nephrotoxicity in the kidney graft. Under specific isolated or combined environmental and/or genetic conditions in both the recipient and donor affecting the glycoprotein efflux pump and/or the mammalian target of rapamycin pathway, these renal injuries may be aggravated by heightened drug tissue concentrations despite adherence to therapeutic drug and blood levels. Mammalian target of rapamycin inhibitors may induce predominantly a dose-dependent renal epithelial cell injury affecting either the glomerular or the renal tubular epithelial cells, leading to cell death and apoptosis. Epithelial mesenchymal transition mediated interstitial fibrosis and tubular atrophy observed with these drugs may be the result of a cumulative toxic renal tubular injury induced by the direct insult of the drug itself and/or podocytopathy-associated proteinuria. The resulting glomerular tubular damage will ultimately lead to graft failure and loss, if exposure persists.

Background: The immunoregulatory properties of mesenchymal stem cells (MSCs) have been well characterized in vitro. However, the role of MSCs in organ transplantation remains unclear. The purpose of this study was to examine the role of indoleamine 2, 3-dioxgenase (IDO)-transfected MSCs in immunoregulation both in vitro and in vivo., Methods: Wild-type (WT) MSCs, empty lentivirus-transfected MSCs (Lenti-MSCs) or IDO-lentivirus-transfected MSCs (IDO-MSCs) were cocultured with peripheral blood mononuclear cells (PBMCs) or CD4CD25 regulatory T (Treg) cells to examine the impact of IDO on the immunoregulatory properties of MSCs in vitro. WT-MSCs, Lenti-MSCs or IDO-MSCs (2 × 10/kg) were intravenously injected into rabbit renal transplant recipients immediately after surgery to examine the role of IDO-MSCs in tolerance induction in vivo., Results: Lentivirus infection of MSCs resulted in stable expression of IDO. The IDO-MSCs inhibited the proliferation of CD4CD25 effector T cells to a greater extent than WT-MSCs. Coculture of PBMCs and IDO-MSCs induced a higher percentage of CD4CD25Foxp3 Treg cells in PBMCs. Additionally, the antigen-specific suppressive function of these CD4CD25 Treg cells was increased. The IDO-MSCs-treated Treg cells showed upregulated expression of cytotoxic T-lymphocyte-associated antigen 4 and increased secretion of IL-10 and TGF-β. Low doses of IDO-MSCs prolonged graft survival and induced tolerance by inducing antigen-specific CD4CD25 Treg cells, as evidenced by the finding that IDO-MSCs-treated kidney transplant recipients accepted donor-specific skin grafts but rejected third-party grafts., Conclusions: The IDO increased the direct immunoregulatory properties of MSCs. The IDO-MSCs enhanced the expression and function of CD4CD25 Foxp3 Treg cells and induced allograft tolerance.

Background: There is a need for new immunosuppression strategies to minimize calcineurin inhibitor (CNI) toxicity while effectively preventing antibody-mediated rejection (AMR)., Methods: We tested the efficacy of an investigational proteasome inhibitor, ixazomib, alone and in a CNI minimization strategy in a rat kidney transplant model of transfusion-elicited acute AMR. Nonsensitized (naïve) and sensitized allograft recipients were randomized into 4 treatment groups (8 groups total, n = 3 to 6 in each group) and treated for 1 week. Groups included: no treatment, full dose cyclosporine (CsA, 10 mg/kg per day), ixazomib (0.25 mg/kg on days -5, -2 and +2) alone, and half dose CsA (5 mg/kg per day) + ixazomib., Results: Compared to untreated animals, ixazomib alone or in combination with ½ dose CsA reduced donor-specific antibody, intragraft transcripts for chemokines CCL-21 and CXCL-13, and CD19 expression in both sensitized and naïve transplants. Compared to full dose CsA, the CNI minimization strategy with ixazomib inhibited AMR and allograft injury as evidenced by reduced C4d staining in peritubular capillaries, microcirculation inflammation, splenic plasma cells, circulating B cell activating factor, and intragraft transcripts for major histocompatibility complex class II, Toll-like receptors (TLR-1, TLR-10, and TLR-12) and CCL-21 and CXCL-13 in sensitized animals, indicating downregulation of B cell activation, antigen presentation and T-cell and B-cell signaling., Conclusions: These studies suggest that CNI minimization strategies including ixazomib are effective to prevent AMR including in sensitized kidney allograft recipients. Clinical studies are needed to determine the role of novel proteasome inhibitors for the prevention and treatment of AMR.

This study was designed to explore the clinical significance of dynamically monitoring the serum level of matrix metalloproteinase 9 (MMP-9) before and after renal transplantation. Before transplantation and 1, 3, 5, 7, 10, 15, and 20 days after transplantation, the peripheral blood was collected from 102 renal transplant recipients, including 8 with acute rejection (ARs) and 94 non-ARs. The serum MMP-9 level was detected by Luminex 200 analyzer (Luminex Corporation, Austin, TX, USA). By day 3 post-transplantation, the serum MMP-9 level in non-ARs had significantly reduced as compared to the pretransplantation level, and reached the lowest value on day 20 post-transplantation. In contrast, the serum MMP-9 level in ARs had significantly increased by day 3, reached the highest value on day 7, and remained significantly higher on day 20 as compared to the pretransplantation level. The receiver operating characteristic curve was plotted to evaluate the power of serum MMP-9 level on day 20 post-transplantation to differentiate the non-AR and AR groups. Our data revealed that with a threshold of 8473.26 pg/mL, the area under the curve was 0.758 (0.661, 0.856); the sensitivity and specificity of the diagnostic were 78.40% and 61.30%, respectively; the positive and the negative predictive values were 74.60% and 66.67%, respectively; and the accuracy rate was up to 71.57%. Taken together, the results indicated that dynamically monitoring serum MMP-9 levels in renal allograft recipients might be a convenient and safe method to diagnose ARs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Kidney allografts induce strong T-cell and antibody responses which mediate acute rejection. Spleen tyrosine kinase (Syk) is expressed by most leucocytes, except mature T cells, and is involved in intracellular signalling following activation of the Fcγ-receptor, B-cell receptor and some integrins. A role for Syk signalling has been established in antibody-dependent native kidney disease, but little is known of Syk in acute renal allograft rejection. Sprague-Dawley rats underwent bilateral nephrectomy and received an orthotopic Wistar renal allograft. Recipient rats were treated with a Syk inhibitor (CC0482417, 30 mg/kg/bid), or vehicle, from 1 h before surgery until being killed 5 days later. Vehicle-treated recipients developed severe allograft failure with marked histologic damage in association with dense leucocyte infiltration (T cells, macrophages, neutrophils and NK cells) and deposition of IgM, IgG and C3. Immunostaining identified Syk expression by many infiltrating leucocytes. CC0482417 treatment significantly improved allograft function and reduced histologic damage, although allograft injury was still clearly evident. CC0482417 failed to prevent T-cell infiltration and activation within the allograft. However, CC0482417 significantly attenuated acute tubular necrosis, infiltration of macrophages and neutrophils and thrombosis of peritubular capillaries. In conclusion, this study identifies a role for Syk in acute renal allograft rejection. Syk inhibition may be a useful addition to T-cell-based immunotherapy in renal transplantation., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Background: Production of reactive oxygen species (ROS) and thereby induction of oxidative stress seem to be one of the major mediators of inflammatory adverse outcomes after renal transplantation. p22(phox) is a polymorphic subunit of NAD(P)H-oxidase that is critical for activation and stabilization of the enzyme. This enzyme is involved in the production of superoxide that triggers inflammatory injuries to the kidney. So in this study, the association between donors and recipients' C242T polymorphism of p22(phox) and acute rejection (AR), delayed graft function (DGF), creatinine clearance (CrCl), and blood pressure in renal-allograft recipients was studied., Methods: One hundred ninety six donor-recipient pairs were studied. The C242T polymorphism of p22(phox) was determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). According to p22 genotype, the subjects were divided in wild-type (CC) and T allele carriers (CT+TT). Transplantation outcomes were determined using acute rejection and delayed graft function criteria. The mean arterial pressure was also measured monthly after transplantation., Results: There was a significant association between the recipients' p22(phox) polymorphism and DGF occurrence (OR=2.5, CI: 1.2-4.9, p=0.0009). No significant association was detected between donors' p22(phox) polymorphism and AR and DGF events. CrCl during the six months follow-up after transplantation was lower in the patients who received allograft from donors carrying 242T allele (B=-12.8, CI: -22.9-12.8 (-22.9 to -2.6)). Changes in the blood pressure were not different among the patients having different genotypes of p22(phox)., Conclusion: These results suggest that the recipients' p22(phox) C242T polymorphism may be a major risk factor for DGF in renal transplantation. Moreover, the donors' 242T allele seems to affect the rate of CrCl in the renal allograft recipients., (Copyright © 2014. Published by Elsevier B.V.)

Acute rejection is a major risk factor for chronic allograft injury (CAI). Blood leukocytes interacting with allograft endothelial cells during acute rejection were suggested to contribute to the still enigmatic pathogenesis of CAI. We hypothesize that tissue transglutaminase (Tgm2), a multifunctional protein and established marker of M2 macrophages, is involved in acute and chronic graft rejection. We focus on leukocytes accumulating in blood vessels of rat renal allografts (Fischer-344 to Lewis), an established model for reversible acute rejection and CAI. Monocytes in graft blood vessels overexpress Tgm2 when acute rejection peaks on day 9 after transplantation. Concomitantly, caspase-3 is activated, suggesting that Tgm2 expression is linked to apoptosis. After resolution of acute rejection on day 42, leukocytic Tgm2 levels are lower and activated caspase-3 does not differ among isografts and allografts. Cystamine was applied for 4 weeks after transplantation to inhibit extracellular transglutaminase activity, which did, however, not reduce CAI in the long run. In conclusion, this is the first report on Tgm2 expression by monocytes in vivo. Tgm2 may be involved in leukocytic apoptosis and thus in reversion of acute rejection. However, our data do not support a role of extracellular transglutaminase activity as a factor triggering CAI during self-limiting acute rejection.

Aim: The purpose of this study was to develop a population pharmacokinetic and pharmacodynamic (PK-PD) model for mycophenolic acid (MPA) in paediatric renal transplant recipients in the early post-transplant period., Methods: A total of 214 MPA plasma concentrations-time data points from 24 patients were available for PK model development. In 17 out of a total of 24 patients, inosine monophosphate dehydrogenase (IMPDH) enzyme activity measurements (n = 97) in peripheral blood mononuclear cells were available for PK-PD modelling. The PK-PD model was developed using non-linear mixed effects modelling sequentially by 1) developing a population PK model and 2) incorporating IMPDH activity into a PK-PD model using post hoc Bayesian PK parameter estimates. Covariate analysis included patient demographics, co-medication and clinical laboratory data. Non-parametric bootstrapping and prediction-corrected visual predictive checks were performed to evaluate the final models., Results: A two compartment model with a transit compartment absorption best described MPA PK. A non-linear relationship between dose and MPA exposure was observed and was described by a power function in the model. The final population PK parameter estimates (and their 95% confidence intervals) were CL/F, 22 (14.8, 25.2) l h(-1) 70 kg(-1) ; Vc /F, 45.4 (29.6, 55.6) l; Vp /F, 411 (152.6, 1472.6)l; Q/F, 22.4 (16.0, 32.5) l h(-1) ; Ka , 2.5 (1.45, 4.93) h(-1) . Covariate analysis in the PK study identified body weight to be significantly correlated with CL/F. A simplified inhibitory Emax model adequately described the relationship between MPA concentration and IMPDH activity. The final population PK-PD parameter estimates (and their 95% confidence intervals) were: E0 , 3.45 (2.61, 4.56) nmol h(-1)  mg(-1) protein and EC50 , 1.73 (1.16, 3.01) mg l(-1) . Emax was fixed to 0. There were two African-American patients in our study cohorts and both had low IMPDH baseline activities (E0 ) compared with Caucasian patients (mean value 2.13 mg l(-1) vs. 3.86 mg l(-1) )., Conclusion: An integrated population PK-PD model of MPA has been developed in paediatric renal transplant recipients. The current model provides information that will facilitate future studies and may be implemented in a Bayesian algorithm to allow a PK-PD guided therapeutic drug monitoring strategy., (© 2014 The British Pharmacological Society.)

Mammalian target of rapamycin inhibitors (mTOR-I), everolimus and sirolimus, are immunosuppressive drugs extensively used in renal transplantation. Their main mechanism of action is the inhibition of cell signaling through the PI3 K/Akt/mTOR pathway. This interesting mechanism of action confers to these medications both great immunosuppressive potential and important anti-neoplastic properties. Although the clinical utility of this drug category, as with other antineoplastic/immunosuppressants, is clear, the use of mTOR-I commonly results in the development of several complications. In particular, these agents may determine severe renal toxicity that, as recent studies report, seems clearly correlated to dose and duration of drug use. The mTOR-I-induced renal allograft spectrum of toxicity includes the enhanced incidence of delayed graft function, nephrotoxicity in particular when co-administered with calcineurin inhibitors (CNI) and onset of proteinuria. The latter effect appears highly frequent in patients undergoing mTOR-I treatment and significantly associated with a rapid graft lost. The damage leading to this complication interests both the glomerular and tubular area. mTOR-I cause an inhibition of proliferation in podocytes and the epithelial-to-mesenchymal transition in tubular cells. Interestingly, all these side effects are mostly reversible and dose related. Therefore, it is unquestionable that these particular drugs should be administered at the lowest dose able to maintain relatively low trough levels, in order to maximize their important and specific therapeutic effects while minimizing or avoiding drug toxicities. Utilization of low dosages of mTOR-I should be encouraged not only in CNI-combined schemas, but also when administered alone in a CNI-free immunosuppressive protocol.

Background: Cells of the monocyte/macrophage lineage have been implicated as effectors in acute allograft rejection based on short-term depletion studies. However, the therapeutic potential of targeting monocyte/macrophages in acute rejection is unknown. We investigated the potential of a c-fms kinase inhibitor (fms-I) in acute renal allograft rejection., Methods: Lewis rats underwent bilateral nephrectomy and received an orthotopic Dark Agouti renal allograft. Recipients received fms-I or vehicle from the time of transplantation until being killed on day 5., Results: Vehicle-treated rats developed severe allograft rejection with massive macrophage and T-cell infiltration. In contrast, fms-I substantially inhibited renal allograft dysfunction and structural damage with abrogation of macrophage and dendritic cell infiltration but had only a minor effect on the T-cell infiltrate. However, fms-I suppressed T-cell activation within the allograft, whereas systemic T- and B-cell activation was not affected. In a longer-term study to assess therapeutic potential, fms-I-treated rats developed severe antibody-mediated rejection on day 8 after transplantation. These transplants exhibited features of antibody-mediated rejection including capillaritis with thrombosis, acute tubular injury, IgG and C4d deposition, and neutrophil infiltration and activation. Interestingly, T-cell activation within these rejecting allografts remained suppressed, indicating separation of T-cell and antibody-mediated rejection., Conclusion: This study demonstrates the ability of c-fms kinase blockade to selectively deplete monocyte/macrophages in acute allograft rejection, although this did not result in significant prolongation of allograft survival. Furthermore, we identify contrasting roles for macrophages in cellular and humoral mechanisms of acute renal allograft rejection.

Background: Both acute rejection (AR) and major infection events (MIE) can reduce long-term allograft survival. We assessed the simultaneous efficacy of serum and urine biomarker indoleamine 2,3-dioxygenase (IDO) enzyme activity and peripheral blood CD4-ATP levels for AR and MIE association, respectively., Methods: We prospectively tested 217 blood and 167 urine serial samples, collected monthly for 12 months after transplantation from 29 consecutive children receiving a kidney transplant. The indoleamine 2,3-dioxygenase activity was assessed by mass spectrometry assays using the ratio of product L-kynurenine (kyn) to substrate tryptophan (trp). Kyn/trp ratios and blood CD4 T-cell ATP levels were correlated with AR, MIE, or stable group (no events) in the next 30 days., Results: Using absolute cutoffs and allocating to samples to AR, MIE, or stable group, mean serum kyn/trp ratios were significantly elevated in the group that experienced AR (P=0.0007). Similarly, peripheral blood CD4-ATP levels were significantly lower in the group experiencing MIE (P=0.0351). Urine kyn/trp ratios and blood tacrolimus levels were not different between AR and stable groups. Within-subject analyses, accounting for repeated measures in subjects, also showed that, over time, serum kyn/trp ratios were higher before AR (P=0.031) and blood CD4-ATP levels were lower before MIE (P=0.008)., Conclusions: These results from our pilot discovery group suggest that a panel of biomarkers together can predict overimmunosuppression or underimmunosuppression. Further independent validation in a multicenter cohort is suggested.

Objective: The infiltration of mononuclear cells and replication and migration of smooth muscle cells (SMCs) from media into the intima in the vascular wall are the cardinal pathological changes in the early stage of chronic allograft nephropathy (CAN). But the mechanism is unclear. Therefore we investigated the role of matrix metalloproteinase 9 (MMP-9) and its interaction with TGF-beta1, tubulointerstitial mononuclear cells infiltration and migration of SMCs in the early stage of CAN., Methods: Kidneys of Fisher (F334) rats were orthotopically transplanted into bilaterally nephrectomized Lewis (LEW) recipients. To suppress an initial episode of acute rejection, rats were briefly treated with cyclosporine A (1.5 mg/kg/day) for the first 10 days. Animals were harvested at 12 weeks after transplantation for histological, immunohistochemistry and molecular biological analysis., Results: The expression of MMP-9 was up-regulated in interstitium and vascular wall in the early stage of CAN, where there were interstitial mononuclear cells infiltration and SMCs migration and proliferation. Moreover the expression of MMP-9 were positively correlated with the degree of interstitial mononuclear cells infiltration, the quantity of SMCs in arteriolar wall, and also the increased TFG-beta1 expression in the tubulointerstitium and arteriolar wall., Conclusions: MMP-9 may play an important role in the mechanism of pathological changes during the earlier period of CAN., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1582313332832700.

Objective: To investigate the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metallopropteinase-1 (TIMP-1) in the renal allografts of patients with chronic active antibody-mediated rejection (AMR), and to explore their role in the pathogenesis of AMR., Methods: Immunohistochemistry assay and computer-assisted image analysis were used to detect the expression of MMP-2 and TIMP-1 in the renal allografts with interstitial fibrosis and tubular atrophy (IF/TA) in 46 transplant recipients and 15 normal renal tissue specimens as the controls. The association of the expression level of either MMP-2 or TIMP-1 with the pathological grade of IF/TA in AMR was analyzed., Results: The expression of either MMP-2 or TIMP-1 was significantly increased in the renal allografts of the recipients as compared with the normal renal tissue (P < 0.05). MMP-2 expression tended to decrease, while TIMP-1 and serum creatinine increased along with the increase of pathological grade of IF/TA (P < 0.05). In IF/TA groups, the expression of TIMP-1 was positively correlated to serum creatinine level (r = 0.718, P < 0.05)., Conclusions: It is suggested by the results that abnormal expressions of MMP-2 and TIMP-1 might play roles in the development of renal fibrosis in chronic AMR., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1128474926172838.

Background: Interindividual variation in inosine monophosphate dehydrogenase (IMPDH) enzyme activity and adverse effects caused by mycophenolate mofetil (MMF) inhibition may be genetically determined, and if so, transplant recipients should receive personalized dosing regimens of MMF, which would maximize efficacy and minimize toxicity. Some studies have demonstrated a relationship between the single nucleotide polymorphism and the risk of acute rejection with IMPDH I variants rs2278293 and rs2278294 and IMPDH II variant rs11706052, whereas others have failed to exhibit an effect. The aim of this work was to investigate the influence of these polymorphisms on acute rejection rates, graft survival and function, and MMF doses in a large cohort of patients., Methods: A random sample of 1040 recipients from the Collaborative Transplant Study DNA bank was genotyped for the variants IMPDH I rs2278293 and rs2278294 and IMPDH II rs11706052., Results: The presence of the T (rs2278293) and G alleles (rs2278294) in the IMPDH I variants and carriage of the G allele (rs11706052) in the IMPDH II variant did not increase the risk of rejection or affect graft function by 1 year after transplantation. There was no association with MMF dose tolerated at 1 year. Furthermore, these polymorphisms did not impact graft or patient survival at 5 years., Conclusion: This study represents the largest cohort of patients with the longest follow-up to date and does not support previous evidence for an association between these IMPDH variants and renal allograft rejection and graft survival.

Objectives: Chronic allograft nephropathy remains one of the main causes of late graft loss after kidney transplant owing to multifactorial development of kidney scarring. Chronic allograft nephropathy is characterised by an excess accumulation of extracellular matrix. A key system regulating extracellular matrix homeostasis are matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. This study sought to determine if a change in the matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases system contributes to chronic allograft nephropathy-associated progressive kidney scarring., Materials and Methods: Examination of sequential renal biopsies was done at implantation, acute rejection, and subsequent chronic allograft nephropathy. In situ localisation of matrix metalloproteinase activity was assessed with a high-resolution in situ zymography technique using gelatin and collagen 1 substrates. Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases were localised using immunohistochemistry., Results: In situ zymography showed over a 50% reduction in matrix metalloproteinase activity against both collagen 1 and gelatin substrates in chronic allograft nephropathy biopsies. A similar loss of matrix metalloproteinase activity was seen in the glomerular and tubulointerstitial compartments. Immunoreactive matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases were observed intracellularly in mesangial and tubular epithelial cells. Matrix metalloproteinases-1, -2, -3, and -9 were significantly reduced in acute rejection and later in chronic allograft nephropathy. However, glomerular matrix metalloproteinases 1 and 9 and tubulointerstitial matrix metalloproteinase-2 were reduced at implantation. Tissue inhibitors of matrix metalloproteinase-2 and -3 were elevated from implantation onwards. We were unable to stain reproducibly for tissue inhibitors of matrix metalloproteinase-1., Conclusions: Kidney scarring underlying chronic allograft nephropathy is associated with a reduction in matrix metalloproteinases activity that may be due to reduced expression of matrix metalloproteinases -1, -2, -3, and -9, and up-regulation of tissue inhibitors of matrix metalloproteinases -2 and -3. Some of these changes originate from implantation.

The balance between oxidative stress and anti-oxidant defense systems after renal transplantation may explain the development and progression of allograft dysfunction. Glutathione S-transferase (GST) decreases the damage from oxidative stress. In contrast, recipient antibodies against GSTT1 expressed on the graft are believed to cause its dysfunction. The aim of our research was to study the probable relationship to rejection between GST gene polymorphisms and anti-GSTT1 antibodies. We included 122 patients transplantations from living donors and 51 healthy individuals as controls group in our study. The patient groups were comprised of 57 patients who did and 65 who did not experienced rejection episode. Polymerase chain reactions were used to detect GSTM1 and GSTT1 polymorphisms, whereas PCR-RFLP (restriction fragment length polymorphism), for GSTP1 polymorphism. An enzyme-linked immunosorbent assay method was used for anti-GSTT1 antibody scans. There was no significant difference between the groups for allele and genotype frequencies of GSTT1, GSTM1, GSTP1 polymorphisms of the recipients, donors, and controls. Within the rejection group the frequency of patients with the GSTM1 null genotype was higher among subjects prescribed cyclosporine A versus tacrolimus (P = .029). Among the entire patient group, 46 subjects with GSTT1 null genotype were scanned for anti-GSTT1 antibody which was detected in 5 of 8 patients with an acute rejection episode (P = .04). Anti-GSTT1 antibody was observed more frequently albeit not significantly, among the cyclosporine versus tacrolimus patient group (P = .16). This study suggested that GSTM1 genotype may be important for cyclosporine detoxification and for allograft outcomes due to drug nephrotoxicity. After transplantation, antigens distinct from the HLA system such as GSTT1 protein may also be targets for alloimmune responses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The development of donor-specific anti-human leukocyte antigen antibodies (DSAs) following renal transplantation significantly reduces long-term renal graft function and survival. The traditional therapies for antibody-mediated rejection (AMR) have provided inconsistent results and transient effects that may be due to a failure to deplete mature antibody-producing plasma cells. Proteasome inhibition (PI) is a novel AMR therapy that deletes plasma cells. Initial reports of PI-based AMR treatment in refractory rejection demonstrated the ability of bortezomib to deplete plasma cells producing DSA, reduce DSA levels, provide histological improvement or resolution, and improve renal allograft function. These results have subsequently been confirmed in a multicenter collaborative study. PI has also been shown to provide effective primary AMR therapy in case reports. Recent studies have demonstrated that PI therapy results in differential responses in early and late post-transplant AMR. Additional randomized studies are evaluating the role of PI in transplant induction, acute AMR, and chronic rejection in renal transplantation. An important theoretical advantage of PI-based regimens is derived from several potential strategies for achievement of synergy.

Antibody-mediated rejection (AMR) following renal transplantation is less responsive to conventional anti-rejection therapies. Plasmapheresis (PP), intravenous immunoglobulin (IVIg), rabbit antithymocyte globulin (rATG) and rituximab deplete immature B-cells but not mature plasma cells. The proteasome inhibitor bortezomib has activity against mature plasma cell, the source of damaging donor-specific antibody (DSA).We present the successful use of bortezomib in 2 patients who developed AMR following kidney transplantation. The first patient was a 54-year-old white female who received living-unrelated kidney transplantation from her husband. She developed severe AMR early after transplantation with rising DSA titers consistent with an anamnestic immune response by memory cells to the donor antigens. Renal function deteriorated despite treatment with pulse methylprednisolone (MP), PP and IVIg. After initiation of therapy with bortezomib, DSA titers became negative and serum creatinine returned to baseline with histological resolution of AMR. The second patient was a 19-year-old white male who received deceased donor kidney transplantation and developed AMR within 2 weeks, refractory to therapy with pulse MP, PP and IVIg with rising DSA. Bortezomib use resulted in disappearance of DSA and renal function improvement. Both patients tolerated the treatment well with stable renal function at last follow-up. The novel mechanisms of action and preliminary results with bortezomib are encouraging, but require larger studies and longer follow-up.

The use of mammalian target of rapamycin inhibitor (mTOR-I) after kidney transplantation has been associated with a higher incidence of proteinuria compared with calcineurin inhibitors (CNIs). This review will focus on mTOR-I-associated proteinuria in different settings after kidney transplantation: de novo mTOR-I treatment in combination with CNI, de novo mTOR-I-containing and CNI-free treatment, early conversion from a CNI-based regimen to an mTOR-I-based regimen, and late conversion. Some possible mechanisms of mTOR-I-induced proteinuria will also be reviewed., (Copyright © 2012. Published by Elsevier Inc.)

Purpose of Review: This review will discuss the mechanism of action and important clinical trial data in renal transplantation for the small molecule Janus kinase (JAK) 3 inhibitor tofacitinib, formerly known as CP-690,550 and tasocitinib., Recent Findings: JAKs are cytoplasmic tyrosine kinases that participate in the signaling of a broad range of cell surface receptors, particularly members of the cytokine receptor common gamma (cγ) chain family. JAK3 inhibition has immunosuppressive effects and treatment with tofacitinib in clinical trials has demonstrated efficacy in autoimmune disorders such as psoriasis and rheumatoid arthritis. Nonhuman primate models of renal transplantation demonstrated prolonged graft survival with tofacitinib compared with vehicle control. Renal transplant clinical trials in humans have demonstrated tofacitinib to be noninferior to cyclosporine in terms of rejection rates and graft survival. There was also a lower rate of new-onset diabetes after transplant. However, there was a trend toward more infections, including cytomegalovirus and BK virus nephritis., Summary: Tofacitinib may be a promising alternative to calcineurin inhibitors. The optimal therapeutic window is still being determined.

Background: UDP-glucuronosyltransferases (UGTs) are a group of enzymes involved in the detoxification and excretion of xeno- and endobiotics. Polymorphic variants of the UGT1A9 gene were shown to influence exposition to mycophenolate mophetil (MMF), a common immunosuppressive drug used in kidney allograft recipients. Therefore, the aim of this study was to evaluate an association between key clinical features of kidney post-transplant course in patients receiving MMF therapy and UGT1A9-2152C>T and -275 T>A SNPs, known to induce UGT1A9 gene expression and UGT1A9 98T>C, resulting in reduced enzyme activity., Material/methods: DNA was isolated from peripheral blood of kidney allograft recipients (n=103) and a control group representing the background population of Poland (n=450). Presence of the analyzed SNP was detected using the PCR restriction fragment length polymorphism (RFLP) method. Accuracy of the applied method was confirmed by DNA sequencing., Results: In patients carrying the UGT1A9-2152T and -275A minor alleles we observed a trend of increased risk of acute allograft rejection within 3 months after transplantation, but this difference was at the border of significance. However, the UGT1A9 98C allele was found to be associated with diminished estimated glomerular filtration rate (eGFR) during the first year after engraftment and transient proteinuria in the first and second month post-transplantation. This association was not observed for UGT1A9-2152C>T and -275 T>A. Our data show that transplanted kidney function may be affected in patients carrying UGT1A9 98C allele and receiving MMF., Conclusions: Genotyping of the functional UGT1A9 SNP may be of practical use in kidney transplant recipients.

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme for de novo synthesis of guanine nucleotides, is required for lymphocyte proliferation. Inhibition of IMPDH by mycophenolic acid (MPA) constitutes part of an immunosuppressive therapy in kidney allograft recipients. The 3757T>C polymorphic variant (rs11706052) of the IMPDH2 gene, which encodes 1 of 2 IMPDH isoenzymes, has been associated with increased IMPDH activity and reduced ability of MPA to exert antiproliferative effects on lymphocytes. The association of IMPDH2 3757T>C SNP with posttransplant courses of kidney allograft recipients remains unclear. Therefore, the aim of the present study was to evaluate associations between this single nucleotide polymorphism and common posttransplant complications among Polish kidney allotransplant recipients. We observed that the frequency of IMPDH2 3757C allele in this group (n=177) did not differ significantly from a control cohort representing the background population of Poland (n=550). There were no significant differences between patients carrying the IMPDH2 3757CT and TT genotypes with respect to acute rejection risk, neutropenia, or incidences of serious infections or gastrointestinal side effects. However, we noted that the 3757C allele was associated with higher lymphocyte counts and a reduced incidence of lymphopenia among kidney allograft recipients. Our findings may be of practical significance to tailor immunosuppressive regimens in kidney transplant recipients., (Copyright © 2011. Published by Elsevier Inc.)

Background: PTPN22 gene, located on the long arm of chromosome 1, encodes PTPN22 protein, known as lymphoid tyrosine phosphatase (Lyp). The function of Lyp is suggested to be negative regulation of T-cell reaction through dephosphorylation of Src family kinases--Lck and Fyn, negative regulatory kinase Csk and other signaling molecules. Several studies suggested that individuals lacking the C allele of the C1858T PTPN22 gene polymorphism may have reduced capacity to downregulate T-cell response. Therefore, they may present changes in immunological reaction and be more susceptible to autoimmunity. The aim of this study was to examine the association of the rs2476601 (C1858T) PTPN22 gene polymorphism with transplanted kidney function., Material/methods: The study enrolled 269 Caucasian renal transplant recipients (166 males, 103 females, mean age 47.63±12.96 years). Genotyping of the rs2476601 (C1858T) PTPN22 gene polymorphism was performed using the PCR-RFLP method., Results: Comparison of the distribution of genotypes and alleles of the rs2476601 PTPN22 gene polymorphism among patients with delayed graft function (DGF) and without DGF revealed no statistically significant differences (OR=0.69, 95%CI=0.37-1.28, p=0.29) for TT+CT vs. CC genotypes. Similarly, there were no statistically significant differences in regard to the studied polymorphism and acute rejection (OR=0.79, 95%CI=0.41-1.52, p=0.52) or chronic allograft nephropathy (CAN) occurrence (OR=1.22, 95%CI=0.64-2.32, p=0.61)., Conclusions: We found no association between rs2476601 (C1858T) PTPN22 gene polymorphism and transplanted kidney function.

Purpose: Epithelial-mesenchymal transition (EMT) plays an important role in progress of renal allograft fibrosis. The adenovirus-mediated anti-sense extracellular signal-regulated kinase 2 (Adanti-ERK2) gene therapy was used to block ERK signaling pathway, and its effect on EMT and renal allograft fibrosis both in vivo and in vitro was explored., Methods: We first generated an in vitro EMT model by connective tissue growth factor (CTGF) stimulation in a HK-2 cell culture system, and then applied Adanti-ERK2 gene therapy on it. The transition of epithelial marker (E-cadherin) to mesenchymal markers (α-SMA, Vimentin) and the cell mobility function alteration were monitored for the observation of EMT progress. In vivo, a rat renal transplant model with Fisher-Lewis combination was employed and the Adanti-ERK2 gene therapy was given. The tubular EMT changes and pathology of allograft fibrosis were examined., Results: In vitro, Adanti-ERK2 gene therapy inhibited CTGF-induced tubular EMT and attenuated the cell motility function induced by CTGF. In vivo, Adanti-ERK2 gene therapy attenuated tubular EMT, modulated the infiltration of macrophages and CD8(+), CD4(+)T lymphocytes, and ameliorated fibrosis effectively in the renal allografts 24weeks after transplantation., Conclusions: Adanti-ERK2 gene therapy inhibits tubular EMT and attenuates renal allograft fibrosis. It is possible to develop promising molecular drug(s) in the future based on ERK signaling pathway., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Matrix metalloproteinase-9 (MMP-9) is the most important member of the MMP family responsible for the development and progression of various renal diseases. Our study aims to investigate the localization of MMP-9 in human renal allografts and to assess whether MMP-9 immunostaining is contributory to detect pathological change in renal biopsy., Methods: We examined 150 renal allograft biopsies (48 baseline and 102 follow-up) from 49 transplants and analyzed the associations of clinical and histopathological data with the MMP-9 staining intensity using a semi-quantitative scoring., Results:   MMP-9 immunostaining in proximal tubule epithelium was negative before transplantation, but positive in biopsies with episodes, particularly with acute cellular rejection (ACR) and acute calcineurin inhibitor (CNI) toxicity. Tubulitis was the most significant association factor (p < 0.0001) with increased MMP-9 staining intensity. The expression in proximal tubules remained augmented in allografts recovered from ACR episodes, while it was disappeared or diminished in those recovered from acute CNI toxicity or ischemia/reperfusion effects., Conclusion: These findings suggest the necessary participation of MMP-9 in the pathogenesis of tubulitis and the subsequent stage of pathogenesis in ACR. Up-regulation of MMP-9 expression in the proximal tubule could be a new indicator of tubular injury and a predictive factor for the prognosis of renal allograft., (© 2011 John Wiley & Sons A/S.)

Background: Catalase is an intracellular antioxidant enzyme that is mainly located in cellular peroxisomes and in the cytosol. This enzyme plays a significant role in the development of tolerance to oxidative stress in the adaptive response of cells and tissues. The aim of the present study was to examine the association between the -262C/T polymorphism in the catalase gene and delayed graft function (DGF), acute rejection and chronic allograft nephropathy of kidney allografts., Methods: One hundred eighty-seven recipients of first renal transplants were included in the study. The histories of the patients were analysed regarding DGF, acute rejection and chronic allograft nephropathy. The polymorphism -262C/T in the catalase gene was analysed using the polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) method., Results: The risk of DGF was significantly lower in T allele carriers compared with CC homozygotes: odds ratio = 0.34, 95% confidence interval = 0.17-0.67, P = 0.001. There were no statistically significant associations between the studied polymorphism and acute rejection or chronic allograft nephropathy., Conclusion: The results of this study suggest that -262C/T polymorphism in the catalase gene is associated with DGF in kidney allograft recipients.

The risk of acute rejection in patients with higher exposure to mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), might be due to inosine 5'-monophosphate dehydrogenase (IMPDH) polymorphisms. The correlations with subclinical acute rejection, IMPDH1 polymorphisms and MPA exposure on day 28 post-transplantation were investigated in 82 Japanese recipients. Renal transplant recipients were given combination immunosuppressive therapy consisting of tacrolimus and 1.0, 1.5 or 2.0 g/day of MMF in equally divided doses every 12 hr at designated times. There were no significant differences in the incidence of subclinical acute rejection between IMPDH1 rs2278293 or rs2278294 polymorphisms (p = 0.243 and 0.735, respectively). However, in the high MPA night-time exposure range (AUC > 60 microg x h/ml and C(0 )> or = 1.9 microg/ml), there was a significant difference in the incidence of subclinical acute rejection between IMPDH1 rs2278293 A/A, A/G and G/G genotypes (each p = 0.019), but not the IMPDH1 rs2278294 genotype. In the higher daytime MPA exposure range, patients with the IMPDH1 rs2278293 G/G genotype also tended to develop subclinical acute rejection. In patients with the IMPDH rs2278293 A/A genotype, the risk of subclinical acute rejection episode tends to be low and the administration of MMF was effective. The risk of subclinical acute rejection for recipients who cannot adapt in therapeutic drug monitoring (TDM) of MPA seems to be influenced by IMPDH1 rs2278293 polymorphism. The prospective analysis of IMPDH1 rs2278293 polymorphism as well as monitoring of MPA plasma concentration after transplantation might help to improve MMF therapy.

Activation of the free-radical oxidation processes (FROP) in the environment of activity inhibition of antioxidant defense system (ADS) plays an important role in pathogenesis of an ischemic-reperfusion damage (IRD) of renal allotransplant (RAT), causes its dysfunction and shortens long-term survival. Along with standard measures there was proposed intraoperative intravenous administration of antioxidant "cocktail", containing preparations lipin and quercetin, to recipient before switching on the kidney into the blood circulation and further during 3-5 days postoperatively. There were studied up the impact of lipin and quercetin on indices of peroxidal oxidation of lipids, and ADS activity of the RAT recipients in early postoperative period. There was established, that these preparations inhibit activity of the FROP, raise the ADS potential in the recipient organism, promoting IRD severity reduction of RAT and acceleration of its function restoration,

Azathioprine (AZA) is a thiopurine prodrug commonly used in patients with kidney transplantation. The aim of this study is to explore in patients with kidney transplantation whether AZA-related side effects can be explained by the inosine triphophate pyrophosphatase (ITPA) or thiopurine S-methyltransferase (TPMT) polymorphisms using both pheno-and genotyping. Erythrocyte ITPA and TPMT activity of 155 patients with kidney transplantation and AZA therapy was determined by HPLC. The frequencies of ITPA and TPMT polymorphisms were detected. Among 155 patients, three cases with zero activity were homozygote for 94C>A. The allele frequency of the 94C>A polymorphism was 0.12. Allele for the IVS2+21A>C mutation in the patients of this study was not found. Thirty-five cases had stopped azathioprine medication or were on reduced dose due to AZA-related side effects, including hematotoxicity (n = 12), hepatotoxicity (n = 18), gastrointestinal toxicity (n = 5, one patient developed hepatotoxicity simultaneously) and flu-like symptoms (n = 1). No statistical significant associations between ITPA 94C>A phenotype or genotype and AZA-related hematotoxicity or hepatotoxicity could be detected. However, five patients who developed gastrointestinal disturbance, two patients were homozygote for 94C>A and other three patients had 94C>A heterozygous allele. The patient who experienced flu-like symptoms were the remaining homozygote for 94C>A. This study demonstrates that ITPA activity reduced in patients with 94C>A mutation (P < 0.01). Patients with ITPA 94C>A homozygous allele are at high risk to develop AZA-related gastrointestinal toxicity and flu-like symptoms (P < 0.01). TPMT wild-type/ITPA variant (homozygote) is closely related to the AZA-induced side effects (P < 0.01).

The peroxidal oxidation of lipids (POL) and antioxidant defense (AOD) system state in the patients blood before renal transplantation performing and on the fourth day after it were studied, depending on kind of familial or potential agonal with beating (DBH) or nonbeating (DNBH) heart donor applied and the presence of complications--delayed renal autotransplant (RAT) function and acute reaction of rejection (ARR). In chronic renal insufficiency, RAT transplantation from cadaver, DNBH and ARR the POL processes intensification and AOD system activity lowering were noted.

Background: Tumor necrosis factor receptors (TNFR) are differentially regulated in human rejecting renal transplants. The TNF-α converting enzyme (TACE) regulates the membrane shedding of both receptors and TNF itself. We analyzed the expression and regulation of TACE in human rejecting renal transplants., Methods: Samples from normal renal tissue or renal transplant undergoing severe acute rejection were immunostained for TACE using antibodies from different species. Human kidney epithelial cells were cultured and TACE plasmid was transfected to upregulate TACE expression. Cells were fractionated and immunoblotted for TACE, and ELISA was performed to test soluble TNFR2., Results: We showed that TACE was upregulated mainly in tubular epithelial cells in acute rejecting kidney, where it colocalized with TNFR2. Epithelial cells with increased levels of TACE shed more soluble TNFR2 into culture media and even more after TACE activation by phorbol-12-myristate-13-acetate stimulation. The shedding could be completely blocked by the TACE inhibitor TNF-α protease inhibitor., Conclusion: Upregulation of TACE in epithelial cells in acute rejecting kidney could lead to more TNFR2 shedding and, hence, antagonize the proinflammatory effect of local TNF., (Copyright © 2010 S. Karger AG, Basel.)

We describe the longitudinal follow-up of calcineurin activity and its clinical relevance in 4 de novo living-donor kidney transplant recipients treated with cyclosporine (n=1) or tacrolimus (n=3). The calcineurin activity in peripheral blood mononuclear cells was measured in combination with therapeutic drug monitoring during hospitalization. Serial blood samplings were performed after the oral administration of each drug to evaluate the temporal pharmacokinetic and pharmacodynamic profiles. Significant changes in enzyme activity were evaluated in relation to clinical outcomes. A nadir of calcineurin activity occurred at the maximum blood drug concentration within 4 h post-dose in most cases. Unlike cyclosporine, tacrolimus partially suppressed calcineurin activity throughout the dosing interval compared to the pre-dose level (cyclosporine, 62-67% inhibition; tacrolimus, 13-35% inhibition). Notably, calcineurin activity rapidly increased a few days before the onset of acute rejection in 2 patients, 1 receiving cyclosporine and 1 receiving tacrolimus, despite the achievement of therapeutic trough blood concentrations. These preliminary findings indicate that therapeutic monitoring of calcineurin activity in addition to the measurement of blood drug concentrations may be helpful to evaluate the pharmacodynamic effects of cyclosporine and tacrolimus early after renal transplantation.

Purpose of Review: The purpose of this review is to describe the biochemistry and physiology of proteasome inhibition and to discuss recent studies with proteasome inhibitor therapy in organ transplantation., Recent Findings: Traditional antihumoral therapies do not deplete plasma cells, the source of antibody production. Proteasome inhibition depletes both transformed and nontransformed plasma cells in animal models and human transplant recipients. Bortezomib is a first in a class proteasome inhibitor that has been shown to effectively treat antibody-mediated rejection in kidney transplant recipients. In this experience, bortezomib provided reversal of histologic changes and also induced a reduction in donor-specific anti-HLA antibody levels. Recent experiences have also shown that bortezomib reduces donor-specific anti-human leukocyte antigen antibody in the absence of rejection. Finally, evidence has been presented that bortezomib therapy depletes human leukocyte antigen-specific antibody producing plasma cells., Summary: Proteasome inhibition induces a complex series of biochemical events that results in pleiotropic effects on multiple cell populations, and plasma cells in particular. Initial clinical results have provided evidence that bortezomib effectively treats antibody-mediated rejection and acute cellular rejection and reduces or eliminates donor-specific anti-human leukocyte antigen antibody. Carefully designed clinical trials are needed to accurately define the role of proteasome inhibition in transplant recipients.

Antibodies against non-HLA targets are increasingly recognized in the context of transplant rejection. However, their specific role remains largely elusive, as evidence exists supporting both their occurrence as an epiphenomenon and their actual pathogenicity in the rejection process. Sutherland et al. describe protein kinase Czeta as a novel, non-HLA antigenic target in the setting of graft rejection.

Antibodies to human leukocyte antigens (HLAs) are a risk factor for acute renal allograft rejection and loss. The role of non-HLAs and their significance to allograft rejection have gained recent attention. Here, we applied protein microarray technology, with the capacity to simultaneously identify 5056 potential antigen targets, to assess non-HLA antibody formation in 15 pediatric renal transplant recipients during allograft rejection. Comparison of the pre- and post-transplant serum identified de novo antibodies to 229 non-HLA targets, 36 of which were present in multiple patients at allograft rejection. On the basis of its reactivity, protein kinase Czeta (PKCzeta) was selected for confirmatory testing and clinical study. Immunohistochemical analysis found PKCzeta both within the renal tissue and infiltrating lymphocytes at rejection. Patients who had an elevated anti-PKCzeta titer developed rejection, which was significantly more likely to result in graft loss. The absence of C4d deposition in patients with high anti-PKCzeta titers suggests that it is a marker of severe allograft injury rather than itself being pathogenic. Presumably, critical renal injury and inflammation associated with this rejection subtype lead to the immunological exposure of PKCzeta with resultant antibody formation. Prospective assessment of serum anti-PKCzeta levels at allograft rejection will be needed to confirm these results.