Allogeneic Kidney Transplantation Research Articles

De Novo Mutations in Mitochondrial DNA of iPSCs Produce Immunogenic Neoepitopes in Humans

Autologous induced pluripotent stem cells (iPSC)-based therapies are considered to be promising in the transplantation field, however their immunological potential is unclear. These therapies require expansion, reprogramming and differentiation of the cells in vitro. Mitochondrial (mt) DNA mutations can arise from those procedures and they have been considered to be involved in the rejection of the cells after transplantation. We therefore hypothesized that single nucleotide polymorphisms (SNPs) in the mtDNA can contribute to autologous and allogeneic graft immunoreactivity and we assessed their immunogenicity in transplantation. We studied neoantigen formation in vitro by reprogramming PBMCs from three volunteers to iPSC and differentiating them to endothelial cells (iEC). We challenged the iECs carrying mtDNA mutations with 20-residue oligomer peptides specifically designed around the SNPs. We quantified the immunological reactions with ELISpot assays. We detected nonsynonymous SNPs in mtDNA in 15 donor-recipient pairs undergoing allogeneic liver or kidney transplantation. To test the allogenicity of the SNPs we challenged the recipients' peripheral blood mononuclear cells (PBMCs) with 20-residue oligomer peptides that we generated around the SNPs. We assessed the Th1 cell response via ELISpot assays. DNA sequencing of the iEC revealed mtDNA mutations and their associated peptides induced specific immune responses quantified by IFNγ release, which increased significantly compared to the reactions against other peptides. Our analyses using the PBMCs collected from the 15 recipients that underwent solid organ transplantation, revealed that nonsynonymous SNPs in mtDNA can cause an immune reaction observed by an increase in IFNγ production in ELISpot assays. Long-term culture and differentiation of iPSC lead to mtDNA mutations that are not naturally selected against by the immune system. A single nucleotide mutation in the mtDNA giving rise to neoantigens triggers a strong immune response and may contribute to the rejection of grafted cells and thus could be of significant relevance to transplantation approaches in regenerative medicine.

Murine cytomegalovirus dissemination but not reactivation in donor-positive/recipient-negative allogeneic kidney transplantation can be effectively prevented by transplant immune tolerance

Cytomegalovirus (CMV) reactivation from latently infected donor organs post-transplantation and its dissemination cause significant comorbidities in transplant recipients. Transplant-induced inflammation combined with chronic immunosuppression has been thought to provoke CMV reactivation and dissemination, although sequential events in this process have not been studied. Here, we investigated this process in a high-risk donor CMV-positive to recipient CMV-negative allogeneic murine kidney transplantation model. Recipients were either treated with indefinite immunosuppression or tolerized in a donor-specific manner. Untreated recipients served as controls. Kidney allografts from both immunosuppressed and tolerized recipients showed minimal alloimmunity-mediated graft inflammation and normal function for up to day 60 post-transplantation. However, despite the absence of such inflammation in the immunosuppressed and tolerized groups, CMV reactivation in the donor positive kidney allograft was readily observed. Interestingly, subsequent CMV replication and dissemination to distant organs only occurred in immunosuppressed recipients in which CMV-specific CD8 T cells were functionally impaired; whereas in tolerized recipients, host anti-viral immunity was well-preserved and CMV dissemination was effectively prevented. Thus, our studies uncoupled CMV reactivation from its dissemination, and underscore the potential role of robust transplantation tolerance in preventing CMV diseases following allogeneic kidney transplantation.

Natural Killer Cells Promote Kidney Graft Rejection Independently of Cyclosporine A Therapy.

Natural Killer (NK) cells have recently been recognized as key players in antibody-mediated chronic allograft failure, thus requiring a comprehensive understanding whether NK cells can escape conventional immunosuppressive regimens. Influence of cyclosporine A (CyA) on NK cell function was studied in a mouse model of allogeneic kidney transplantation (KTX, BALB/c to C57BL/6). Recipients were treated daily with CyA (10 mg/kg) for seven or 14 days for long term survival (day 56). Administration of CyA in recipients resulted in significantly reduced frequencies of intragraft and splenic CD8+ T cells, whereas the latter illustrated reduced IFNγ production. In contrast, intragraft and splenic NK cell frequencies remained unaffected in CyA recipients and IFNγ production and degranulation of NK cells were not reduced as compared with controls. Depletion of NK cells in combination with CyA resulted in an improvement in kidney function until day 7 and prolonged graft survival until day 56 as compared to untreated controls. Surviving animals demonstrated higher intragraft frequencies of proliferating CD4+FoxP3+Ki67+ regulatory T (TREG) cells as well as higher frequencies of CD8+CD122+ TREG. We here demonstrate that NK cell depletion combined with CyA synergistically improves graft function and prolongs graft survival, suggesting that NK cell targeting constitutes a novel approach for improving KTX outcomes.

RIOCIGUAT IN PATIENTS WITH CHRONIC THROMBOEMBOLIC PULMONARY HYPERTENSION AND SEVERE RENAL IMPAIRMENT

Objective: To demonstrate the efficiency and safety of using riociguat in patients with chronic thromboembolic pulmonary hypertension (CTEPH) and severe renal impairment with creatinine clearance (CrCL) <15 ml/min. Design and method: A 51-year old female was admitted to pulmonary hypertension department with shortness of breath during minimal exertion and cough. At the age of 37 she underwent allogeneic kidney transplantation due to terminal renal impairment, which developed as a result of chronic glomerulonephritis. Nephropathy of transplanted kidney progressed within following 3 years to creatinine level 350–400 μmol/l. At the age of 49 the patient had pulmonary embolism. After 1 year of anticoagulation therapy there was diagnosed CTEPH, WHO-FC III. Due to severity of renal impairment thrombendarterectomy and balloon pulmonary angioplasty weren’t performed. In department echocardiography showed increased systolic pulmonary artery pressure (PAP) (80 mmHg), mean PAP (44 mmHg). 6-minute walking distance (6MWD) was 360 m. Right heart catheterization and angiopulmonography were not performed due to the high risk of worsening of renal function. The patient was administered riociguat 0.5 mg with gradual increase to 1 mg 3 times a day. Because the product label states, that patients with renal impairment have increased riociguat exposure, daily dose wasn’t enlarged more. Results: After 2 weeks with riociguat we revealed disappearance of cough, increased physical activity (6MWD 416 m), decreased systolic and mean PAP (72 and 41 mmHg). In control blood tests there were no signs of renal function worsening: creatinine before treatment 356 vs after 387 μmol/l, CrCL by Cockroft-Golt – 14 vs 13 ml/min, urea – 18.4 vs 20.3 mmol/l. At 6 months follow-up we observed further reduction of systolic and mean PAP (67 and 39 mmHg), increase of 6MWD (430 m). The sings of renal impairment were at the same level: creatinine 385 μmol/l, CrCL 13 ml/min, urea 21.9 mmol/l. Conclusions: The administration of riociguat for patients with CTEPH and severe renal impairment with CrCL <15 ml/min can be effective and safe, if provided strict control of renal function and carefully adjusted dose.

FcγRIIIb Inhibits Renal Transplant Rejection by Inducing Neutrophils Apoptosis

Fcγ receptors (FcI³R) have been proposed to be involved in the pathogenesis of acute rejection (AR) after organ transplantation. However, the mechanisms by which FcγRIIIB mediate this process are still unclear. Previous studies suggested that the FCGR3B gene copy number variation (CNV) is associated with the risk of autoimmune diseases. In this study, we delineated the association of FCGR3B-CNV with end-stage renal disease (ESRD) and risk of rejection post kidney transplantation in 414 kidney transplant recipients and 219 healthy subjects. Our data showed that the percentage of kidney transplant recipients with high copy number of FCGR3B gene was significantly lower than that of healthy controls, suggesting that individuals with low copy of FCGR3B gene are more prone to develop ESRD. Besides, patients with low copy of the FCGR3B gene are associated with susceptibility to AR following kidney transplantation. In mice, transgenic expression of human FcγRIIIB effectively inhibited the AR and improved the function of the transplanted kidney in a model of allogeneic kidney transplantation. This protective effect was associated with an inhibition of T lymphocytes infiltration and Th1 cell immune response. Finally, we showed that FcγRIIIB cross-linking combined with TNF-α treatment in vitro promoted neutrophil apoptosis and was associated with increased activation of cleaved caspase-8 and caspase-3. The combined treatment also increased the arginase-1 expression in neutrophils, which may explain FcγRIIIB-mediated immunosuppression. Funding: This work was supported by the National Fund Committee of China (2012CB517603, 2012AA02A512). Declaration of Interest: No conflicts of interest exist for any authors. Ethical Approval: The study was performed in adherence with the Declaration of Helsinki, and was approved by the Ethics Committee of the First Affiliated Hospital of College of Medicine of Zhejiang University. All patients and healthy controls provided written informed consent.

Chemokine CXCL13 as a New Systemic Biomarker for B-Cell Involvement in Acute T Cell-Mediated Kidney Allograft Rejection.

The presence of B-cell clusters in allogenic T cell-mediated rejection (TCMR) of kidney allografts is linked to more severe disease entities. In this study we characterized B-cell infiltrates in patients with TCMR and examined the role of serum CXCL-13 in these patients and experimentally. CXCL-13 serum levels were analyzed in 73 kidney allograft recipients at the time of allograft biopsy. In addition, four patients were evaluated for CXCL13 levels during the first week after transplantation. ELISA was done to measure CXCL-13 serum levels. For further mechanistic understanding, a translational allogenic kidney transplant (ktx) mouse model for TCMR was studied in BalbC recipients of fully mismatched transplants with C57BL/6 donor kidneys. CXCL-13 serum levels were measured longitudinally, CD20 and CD3 composition and CXCL13 mRNA in tissue were examined by flow cytometry and kidneys were examined by histology and immunohistochemistry. We found significantly higher serum levels of the B-cell chemoattractant CXCL13 in patients with TCMR compared to controls and patients with borderline TCMR. Moreover, in patients with acute rejection within the first week after ktx, a >5-fold CXCL13 increase was measured and correlated with B-cell infiltrates in the biopsies. In line with the clinical findings, TCMR in mice correlated with increased systemic serum-CXCL13 levels. Moreover, renal allografts had significantly higher CXCL13 mRNA expression than isogenic controls and showed interstitial CD20+ B-cell clusters and CD3+ cell infiltrates accumulating in the vicinity of renal vessels. CXCL13 blood levels correlate with B-cell involvement in TCMR and might help to identify patients at risk of a more severe clinical course of rejection.

Hypothermic Oxygenated Perfusion: A Simple and Effective Method to Modulate the Immune Response in Kidney Transplantation.

Hypothermic oxygenated perfusion (HOPE) has been shown to protect liver recipients from acute rejection in an allogeneic model of liver transplantation in rats. Here we investigate the impact of HOPE on the T cell-mediated immune response following kidney transplantation. Kidneys from Lewis rats were transplanted into Brown Norway recipients to trigger acute rejection (allogeneic untreated group). Next, Brown Norway recipients were treated either with tacrolimus,= or donor kidneys underwent 1h-HOPE-treatment before implantation without additional immunosuppression in recipients. Syngeneic kidney transplants (Brown Norway to Brown Norway) served as controls. In a second set of experiments, the immune response was assessed in a donation after circulatory death model of kidney transplantation comparing standard cold storage with subsequent HOPE treatment and hypothermic nitrogenated perfusion, where oxygen was replaced during cold perfusion. Allogeneic kidney transplantation led to death in all untreated recipients within 10 days due to severe acute rejection. In contrast, immune activation was prevented by tacrolimus with significantly improved recipient survival. Similarly, HOPE treatment, without any immunosuppression, protected recipients from acute immune response, as measured by less cytokine release, T-cell, and macrophage activation. Additionally, HOPE-treated kidneys showed better function and less early fibrosis leading to a significantly improved recipient survival, compared with untreated allogeneic controls. Similarly, HOPE treatment protected recipients of extended donation after circulatory death kidneys from immune activation. This effect was lost when deoxygenated perfusate was used. In summary, this is the first study demonstrating the beneficial effects of HOPE on the immune response following kidney transplantation in an allogeneic rodent model.

Renal Allograft Rejection: Noninvasive Ultrasound- and MRI-Based Diagnostics.

To date, allogeneic kidney transplantation remains the best available therapeutic option for patients with end-stage renal disease regarding overall survival and quality of life. Despite the advancements in immunosuppressive drugs and protocols, episodes of acute allograft rejection, a sterile inflammatory process, continue to endanger allograft survival. Since effective treatment for acute rejection episodes is available, instant diagnosis of this potentially reversible graft injury is imperative. Although histological examination by invasive core needle biopsy of the graft remains the gold standard for the diagnosis of ongoing rejection, it is always associated with the risk of causing substantial graft injury as a result of the biopsy procedure itself. At the same time, biopsies are not immediately feasible for a considerable number of patients taking anticoagulants due to the high risk of complications such as bleeding and uneven distribution of pathological changes within the graft. This can result in the wrong diagnosis due to the small size of the tissue sample taken. Therefore, there is a need for a tool that overcomes these problems by being noninvasive and capable of assessing the whole organ at the same time for specific and fast detection of acute allograft rejection. In this article, we review current state-of-the-art approaches for noninvasive diagnostics of acute renal transplant inflammation, i.e., rejection. We especially focus on nonradiation-based methods using magnetic resonance imaging (MRI) and ultrasound.

Thalidomide ameliorate graft chronic rejection in an allogenic kidney transplant model

Chronic T cell mediated rejection (TCMR), which is characterized by infiltration of the interstitium by T cells and macrophages, still remains a major barrier to the long-term survival of kidney transplantation. Our recent report indicated that thalidomide can attenuate graft arteriosclerosis in an aortic transplant model. In this study, we investigated the effect of thalidomide on chronic TCMR in a rat model of kidney transplantation.Fischer or Lewis kidney allografts were transplanted into Lewis recipient rats. After kidney transplantation, recipient rats were divided into 3 groups: the isograft (Iso) group, allograft (Allo) group, and thalidomide (Tha) group. Rats were sacrificed at 8 weeks after kidney transplantation, and blood and kidney samples were collected. Serum concentrations of creatinine (SCr),interleukin (IL)-2, IL-6, IL-17, and TNF-α in recipients were determined, and flow cytometry was used to detect the percentages of CD4+CD25+, CD4+ Foxp3+and CD4+Th17+ cell subsets in the peripheral blood. Grafts were procured for histopathological examination, and the expressions of α-SMA, transforming growth-β1 (TGF-β1), and VEGF in kidney grafts were investigated using Western blot. Thalidomide treatment significantly ameliorated chronic rejection, reduced renal allograft tissue damage, and decreased serum creatinine levels. Attenuation of chronic TCMR was due to the prohibited production of inflammatory cytokines, altered distribution of the CD4+ CD25+ FoxP3+ regulatory T (Treg) and CD4+ Th17+ cells in the peripheral blood, and decreased expression of TGF-β1, α-SMA, and VEGF in the kidney graft. These results demonstrated that thalidomide could effectively ameliorate chronic TCMR in a rat kidney transplant model.

Correlation Between Stem and Progenitor Cells Number and Immune Response in Patients After Allogeneic Kidney Transplant.

BackgroundStem and progenitor cells are of great interest in all medical procedures involving tissue regeneration. There is a consensus that the use of stem cells after solid organ transplantation may play a role in tissue repair and in immunosuppression. The aim of this study was to determine possible relations between stem cell count and the immune response in a group of patients after kidney transplantation.Material/MethodsThe study was conducted on a group of 100 patients who underwent kidney transplantation. The following phenotypic markers of the studied cell subpopulations were adopted: Treg cells (CD3+CD4+CD25high), circulating hematopoietic cells (CD34+CD133+CD45+CD38−), and non-hematopoietic cells (Lin−CXCR4+CD133−CD45−). Cell subpopulations were assessed using LSRII flow cytometer (BD Biosciences, San Jose, CA, USA).ResultsPositive correlation was observed between non-hematopoietic stem cells percentage and recipient’s platelets count (P=0.04). Moreover, a higher percentage of non-hematopoietic cells was accompanied by lower numbers of B lymphocytes (P=0.03) and Treg cells (P=0.02).ConclusionsOur study revealed significant associations between the intensity of ongoing immune response processes and tissue damage, and the release of stem and progenitor cells into circulation. These findings suggest their role in the stimulation of protective processes in terms of graft regeneration.

Facilitating cells: role in inducing transplantation tolerance.

This review discusses the role and mechanisms by which facilitating cells promote stem cell engraftment and induce tolerance in HLA-disparate kidney transplant recipients. Facilitating cells in both mice and human are heterogeneous, consisting of several subpopulations. They have been shown to enhance stem cell engraftment in allogeneic recipients. They also increase hematopoietic stem cells (HSC) clonogenicity, enhance migration and homing of stem cells via secretion of cytokines/chemokines/growth factors, prevent apoptosis of stem cells and induce regulatory cells. This review summarizes the findings that led to the development of chimerism-based induction of tolerance using FCRx (a mobilized blood product enriched in stem cells and facilitating cells) in allogenic kidney transplant patients. A phase-2 clinical trial based on FCRx therapy has been successful in inducing tolerance to living donor kidney allografts, leading to withdrawal of immunosuppression in over 70% of patients transplanted. The ultimate goal of establishing tolerance in the absence of immunosuppresive drugs can be achieved using FCRx therapy.

Diagnostic possibilities of using micro-RNA for kidney transplantation

Allogeneic kidney transplantation is the optimal approach for the treatment of patients with terminal stage of chronic kidney disease. Moreover, post-transplant monitoring and optimization of immunosuppressive therapy with early non-invasive molecular-biological markers can significantly improve the long-term outcome of transplantation. As markers of damage to the kidney transplant, it is proposed to use micro-RNAs that play a fundamental role in the regulation of the activity of various genes. The level of expression of micro-RNA in different tissues can correlate with certain pathological conditions. In this review, the literature data on the study of the perspectives for the use of micro-RNA as biomarkers of the post-transplantation period in kidney transplant recipients are considered.

Ex vivo expanded natural regulatory T cells from patients with end-stage renal disease or kidney transplantation are useful for autologous cell therapy

Novel concepts employing autologous, exvivo expanded natural regulatory T cells (nTreg) for adoptive transfer has potential to prevent organ rejection after kidney transplantation. However, the impact of dialysis and maintenance immunosuppression on the nTreg phenotype and peripheral survival is not well understood, but essential when assessing patient eligibility. The current study investigates regulatory T-cells in dialysis and kidney transplanted patients and the feasibility of generating a clinically useful nTreg product from these patients. Heparinized blood from 200 individuals including healthy controls, dialysis patients with end stage renal disease and patients 1, 5, 10, 15, 20 years after kidney transplantation were analyzed. Differentiation and maturation of nTregs were studied by flow cytometry in order to compare dialysis patients and kidney transplanted patients under maintenance immunosuppression to healthy controls. CD127 expressing CD4+CD25highFoxP3+ nTregs were detectable at increased frequencies in dialysis patients with no negative impact on the nTreg end product quality and therapeutic usefulness of the exvivo expanded nTregs. Further, despite that immunosuppression mildly altered nTreg maturation, neither dialysis nor pharmacological immunosuppression or previous acute rejection episodes impeded nTreg survival invivo. Accordingly, the generation of autologous, highly pure nTreg products is feasible and qualifies patients awaiting or having received allogenic kidney transplantation for adoptive nTreg therapy. Thus, our novel treatment approach may enable us to reduce the incidence of organ rejection and reduce the need of long-term immunosuppression.

Frequency of Human Papilloma Virus Occurrence Among Pathological Changes of the Oral Cavity in Kidney Allotransplant Recipients Undergoing Long-Term Pharmacological Immunosuppressive Therapy

Recipients of allotransplants are more susceptible to viral infections, among which the human papilloma virus infection is an independent factor inducing precancerous lesions and cancers of both the anogenital and the cervicocephalic region. Materials and methodsThe study included a group of 69 allogenic kidney transplantation recipients aged 20 to 70, who were treated with cyclosporine, azathioprine, and prednisone. The patients in whom the macroscopic examination of the oral mucosa revealed lesions were qualified for a biopsy. The infection with human papilloma virus (HPV) was confirmed by a histopathological examination and genotyping with the use of polymerase chain reaction (PCR) and Hybrid Capture II test. ResultsPapillomatous lesions in the oral cavity occurred in 36.1% of the research group participants. The HPV16 virus was the most common genotype in this group of patients (25%). The pathologic changes in the oral cavity were predominantly situated on the gingivae. In the group of transplant recipients, clinical changes resulting from HPV infection occurred within a period of 2 years following the transplantation.Cyclosporine used in the immunosuppression scheme has correlated in as many as 53.7% of cases of allogenic kidney transplant recipients with the appearance of clinical signs and symptoms of HPV infection. In 50% of cases there was a correlation with acute kidney transplant rejection. When induction therapy (anti-thymocyte globulin [ATG] and muromonab-CD3 [OKT3]) was applied, at least 1 oral cavity lesion in each case of allogenic transplant recipients was reported. ConclusionsTyping of HPV with the use of molecular methods should be a standard diagnostic technique.

CD47 blockade reduces ischemia/reperfusion injury in donation after cardiac death rat kidney transplantation.

Modulation of nitric oxide activity through blockade of CD47 signaling has been shown to reduce ischemia-reperfusion injury (IRI) in various models of tissue ischemia. Here, we evaluate the potential effect of an antibody-mediated CD47 blockade in a syngeneic and an allogeneic DCD rat kidney transplant model. The donor organ was subjected to 1hour of warm ischemia time after circulatory cessation, then flushed with a CD47 monoclonal antibody (CD47mAb) in the treatment group, or an isotype-matched immunoglobulin in the control group. We found that CD47mAb treatment improved survival rates in both models. Serum markers of renal injury were significantly decreased in the CD47mAb-treated group compared with the control group. Histologically the CD47mAb-treated group had significantly reduced scores of acute tubular injury and acute tubular necrosis. The expression of biomarkers related to mitochondrial stress and apoptosis also were significantly lower in the CD47mAb-treated groups. Overall, the protective effects of CD47 blockade were greater in the syngeneic model. Our data show that CD47mAb blockade decreased the IRI of DCD kidneys in rat transplant models. This therapy has the potential to improve DCD kidney transplant outcomes in the human setting.

BM-MSCs-derived microvesicles promote allogeneic kidney graft survival through enhancing micro-146a expression of dendritic cells

ObjectiveMicrovesicles (MVs) are plasmalemmal vesicles that are released from various cells and regarded as a mediator of intermolecular communication. In present study, we aimed to evaluate the therapeutic efficacy of the bone marrow mesenchymal stem cells (BM-MSCs)-derived MVs in the mice kidney transplant model and explored the underlying mechanism. MethodsBM-MSCs were isolated from C57BL/6 mice and identified using flow cytometry. In vivo allogenic kidney transplantation model of mice was performed between C57BL/6 mice (recipient) and BALB/c mice (donor). Recipient-type BM-MSC (0.1ml) or equal volume of medium as a control was injected i.v. 24h after kidney transplantation. Serum was collected for creatinine concentration detection at 14 d after transplantation. Dendritic cells (DCs) phenotype and miR-146a expression level in plant was identified. Immature DCs (iDCs) and mature DCs (mDCs) were derived from monocytes. MVs were separated from BM-MSCs. ResultsBM-MSCs positive for CD29 (95.8%) and CD44 (94.7%) were cultured and confirmed to prolong the allogenic kidney graft survival in mice. Importantly, the expression of miR-146a increased significantly in DCs of BM-MSCs-treated allogenic kidney. Moreover, both BM-MSCs and MVs derived from BM-MSCs enhanced miR-146a expression in iDCs and mDCs in vitro. Furthermore, MVs substantially reduced IL-12 mRNA expression and IL-12 production of mDCs whereas this action was reversed by miR-146a silencing. MiR-146a silencing also abrogated the MVs-induced decrease in serum creatinine, reduction of immature DCs phenotype in transplant and increase in miR-146a expression level. ConclusionIn summary, our data suggested that the BM-MSCs-derived MVs improved allogenic kidney transplantation survival through inhibiting DCs maturity by miR-146a.

The Timing of Immunomodulation Induced by Mesenchymal Stromal Cells Determines the Outcome of the Graft in Experimental Renal Allotransplantation

The immunomodulatory characteristics of mesenchymal stromal cells (MSCs) may lead to multifaceted strategies in rejection of organ transplantation. This study was designed to investigate, first, the effect of the donor-type MSCs from Wistar rats on the immune system of immunocompetent Lewis rats and, second, the rejection responses in a renal transplantation model of Wistar to Lewis. In the first experimental model, MSCs from the bone marrow induced a systemic immune response in the immunocompetent Lewis rats, characterized by two different phases. In the initial phase (days 1–3 after MSCs infusion), the main findings were a decrease in the percentage of the main peripheral blood (PB) lymphocyte subpopulations [T cells, B cells, and natural killer (NK) cells], an increase in the FOXP3 MFI in Tregs, and an elevated concentration of circulating proinflammatory cytokines (IL-1β and TNF-α). In the late phase (days 4–6), the percentage of T cells, B cells, and NK cells returned to baseline levels; the concentration of circulating IL-1β and TNF-α decreased; and the level of anti-inflammatory cytokines (IL-10 and IL-4) increased with respect to the initial phase. In the allogeneic kidney transplantation model, rats were randomized into four groups: nontreated, cyclosporine oral administration, and two groups of rats treated with two different schedules of MSC infusion: 4 days (MSCs-4) and 7 days (MSCs-7) before kidney transplantation and in both a further infusion at the day of transplantation. Both MSC treatments decreased the percentage of T, B, and NK cells in PB. Creatinine levels, survival, and histological parameters were better in MSCs-7 than in MSCs-4. We can conclude that MSCs, by themselves, produce changes in the immune system; they do not need a pathological condition to produce immunomodulatory responses. In the renal allograft model, the optimal time schedule for MSC infusion before grafting was 7 days to prevent acute rejection.

Antagonism of profibrotic microRNA-21 improves outcome of murine chronic renal allograft dysfunction

Chronic renal allograft dysfunction (CAD) is a major limiting factor of long-term graft survival. It is characterized by interstitial fibrosis and tubular atrophy. The underlying pathomechanisms are incompletely understood. MicroRNAs are powerful regulators of gene expression and may have an impact on various diseases by direct mRNA decay or translational inhibition. A murine model of allogenic kidney transplantation was used resulting in CADat 6 weeks after kidney transplantation. We identifiedfibrosis-associated miR-21a-5p by whole miRNAome expression analysis to be among the most highly upregulated miRNAs. Invitro in renal fibroblasts, miR-21a-5p was transcriptionally activated by interleukin 6-induced signal transducer and activator of transcription 3. Co-culture of LPS-activated macrophages with renal fibroblasts increased expression levels of miR-21a-5p and markers of fibrosis and inflammation. In addition, mature miR-21a-5p was secreted by macrophages in small vesicles, which were internalized by renal fibroblasts, thereby promoting profibrotic and proinflammatory effects. Notch2 receptor was identified as a potential target of miR-21a-5p and validated by luciferase gene reporter assays. Therapeutic silencing of miR-21a-5p in mice after allogenic kidney transplantation resulted in an amelioration of CAD, as indicated by a reduction in fibrosis development, inflammatory cell influx, tissue injury and BANFF lesion scoring. In a life-supporting model, miR-21a-5p antagonism had beneficial effects on kidney function. miR-21a-5p silencing may therefore be a viable therapeutic option in the treatment of patients following kidney transplantation to halt the development of CAD.

Longitudinal evaluation of perfusion changes in acute and chronic renal allograft rejection using arterial spin labeling in translational mouse models.

To examine the longitudinal changes of renal perfusion due to acute and chronic renal allograft rejection by using arterial spin labeling (ASL) MRI in translational mouse models of isogenic and allogenic kidney transplantation (ktx). Acute rejection was induced by allogenic ktx of C57BL/6 (B6)-kidney grafts to BALB/c-recipients with prolonged cold ischemia (CIT) of 60 minutes (n = 13). To induce chronic rejection BALB/c-kidneys were transplanted into B6-recipients with short CIT of 30 minutes (n = 22). Isogenic grafts without rejection (n = 14 with prolonged, n = 9 with short CIT) and normal kidneys (n = 22) were used for comparison. Perfusion was measured on a 7T small-animal magnetic resonance imaging (MRI) scanner using flow-sensitive alternating inversion recovery (FAIR) ASL-sequences at day 1 and 6 (acute) or at week 3 and 6 (chronic) after surgery. Histological analyses of grafts included inflammation, vascular changes, and fibrosis. In the acute ktx model, ASL showed perfusion impairment in isogenic and allogenic kidney grafts. Perfusion of allografts further decreased until day 6 and remained stable in isografts without rejection (allogenic ktx 62 ± 21 vs. isogenic ktx 181 ± 39 ml/min/100g, P < 0.01). In the chronic ktx model, perfusion in isografts was similar to normal kidneys over the entire observation period. Perfusion was severely reduced in allografts compared to isografts (week 3: 28 ± 7 vs. 310 ± 46 ml/min/100g, P < 0.001, week 6: 32 ± 5 vs. 367 ± 72 ml/min/100g, P < 0.001). Histological analysis revealed severe inflammation, vascular occlusion, and rejection in allografts. Chronic rejection grafts showed endothelialitis, peritubular capillaritis, interstitial fibrosis, and tubular atrophy. ASL allows longitudinal assessment of renal perfusion impairment due to acute and chronic renal allograft rejection in translational mouse models. 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017;46:1664-1672.

Prograf produces more benefits for CYP3A5 low expression patients in early stage after kidney transplantation.

This study is to analyze concentration changes of the prolonged-release and shorter-acting formulation of tacrolimus in patients with different CYP3A5 genotypes after kidney transplantation. A single-factor retrospective analysis was performed in patients underwent allogeneic kidney transplantation with postoperative administration of Advagraf or Prograf in our hospital from May 2013 to June 2014. The CYP3A5 genotypes were determined, and tacrolimus trough concentrations in whole blood were measured within 28days after transplantation. The rates of acute rejection rate, chronic rejection and infection were recorded and compared after one year follow-up after surgery. The study included 106 patients administered Advagraf (45 cases) or Prograf (61 cases). The low expression genotype of CYP3A5 was detected in 40 (37.7%) patients. A higher dose of Advagraf was required to increase the tacrolimus trough concentrations within 21days after transplantation. Moreover, a higher dose for Advagraf than Prograf was required to increase the tacrolimus trough concentrations in low expression patients. In the low expression patients, Prograf more frequently achieved the target tacrolimus trough concentrations within seven days after transplantation (five days: 7.14% vs. 84%, P=0.001; seven days: 33.33% vs. 77.78%, P=0.001). The patient and kidney graft survival rates one year after transplantation both were 100%. The estimated glomerular filtration rate showed no significant difference between different CYP3A5 phenotypes or formulations of tacrolimus (P>0.05). However, the incidence of infections was higher in the Advagraf group in low expression patients (P<0.05). Tacrolimus of different formulations had different impact on patients with different CYP3A5 genotypes after kidney transplantation.