Corrigendum: Ex vivo normothermic preservation of a kidney graft from uncontrolled donation after circulatory death over 73 hours.

[This corrects the article DOI: 10.3389/fbioe.2023.1330043.].

Inhibition of BRD4 Attenuates ER Stress-induced Renal Ischemic-Reperfusion Injury.

Renal ischemia-reperfusion injury (IRI) leads to endoplasmic reticulum (ER) stress, thereby initiating the unfolded protein response (UPR). When sustained, this response may trigger the inflammation and tubular cell death that acts to aggravate the damage. Here, we show that knockdown of the BET epigenetic reader BRD4 reduces the expression of ATF4 and XBP1 transcription factors under ER stress activation. BRD4 is recruited to the promoter of these highly acetylated genes, initiating gene transcription. Administration of the BET protein inhibitor, JQ1, one hour after renal damage induced by bilateral IRI, reveals reduced expression of ATF4 and XBP1 genes, low KIM-1 and NGAL levels and recovery of the serum creatinine and blood urea nitrogen levels. To determine the molecular pathways regulated by ATF4 and XBP1, we performed stable knockout of both transcription factors using CRISPR-Cas9 and RNA sequencing. The pathways triggered under ER stress were mainly XBP1-dependent, associated with an adaptive UPR, and partially regulated by JQ1. Meanwhile, treatment with JQ1 downmodulated most of the pathways regulated by ATF4 and related to the pathological processes during exacerbated UPR activation. Thus, BRD4 inhibition could be useful for curbing the maladaptive UPR activation mechanisms, thereby ameliorating the progression of renal disease.

Midnight Cortisol is Associated with Changes in Systolic Blood Pressure and Diabetic Neuropathy in Subjects with Type 1 Diabetes Undergoing Simultaneous Kidney-Pancreas Transplantation.

INTRODUCTION: An increased midnight cortisol (MC) has been described in end-stage kidney disease (ESKD) and type 1 diabetes (T1D). Lower circulating levels of the cytokine soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (sTWEAK) have been found in T1D and ESKD and associated with cardiovascular (CV) events in the latter. We aimed to study MC and sTWEAK in simultaneous pancreas-kidney transplant (SPKT) recipients, and the association of these markers with CV risk factors and transplant outcomes. METHODS: This was a retrospective cohort study including subjects with T1D who received a first SPKT between 2008 and 2020. MC and sTWEAK at baseline were correlated with CV risk factors and evolution 1 year after SPKT. RESULTS: We included 29 subjects (58.6% women, mean age 43.5 ± 7.5 years, diabetes duration 31.9 ± 9.4 years). Systolic blood pressure (SBP) increased directly with MC quartiles, despite similar hypertension prevalence (p < 0.05). At 1 year, antihypertensive treatment was deintensified in those in lower MC quartiles (p < 0.05). Diabetic neuropathy prevalence decreased progressively in higher cortisol quartiles (p for trend = 0.005). Low MC was associated with delayed kidney graft function (p for trend = 0.044), and high sTWEAK with kidney graft rejection (p for trend = 0.018). In multivariate analyses, MC (standardized-ß 0.505, p = 0.004) and age (standardized-ß - 0.460, p = 0.040) were independently correlated with SBP, and MC was independently associated with the presence of diabetic neuropathy (OR 0.633, 95% CI 0.425-0.944, p = 0.025), adjusted for confounders. CONCLUSIONS: In this exploratory study, lower MC was associated with a lower baseline SBP, an improvement of antihypertensive treatment 1 year after transplant, and a higher diabetic neuropathy prevalence in SPKT recipients.

Physiopathological role of extracellular vesicles in alloimmunity and kidney transplantation and their use as biomarkers.

Antibody-mediated rejection is the leading cause of kidney graft dysfunction. The process of diagnosing it requires the performance of an invasive biopsy and subsequent histological examination. Early and sensitive biomarkers of graft damage and alloimmunity are needed to identify graft injury and eventually limit the need for a kidney biopsy. Moreover, other scenarios such as delayed graft function or interstitial fibrosis and tubular atrophy face the same problem. In recent years, interest has grown around extracellular vesicles, specifically exosomes actively secreted by immune cells, which are intercellular communicators and have shown biological significance. This review presents their potential as biomarkers in kidney transplantation and alloimmunity.

Early kinetics of donor-derived cell-free DNA after transplantation predicts renal graft recovery and long-term function.

BACKGROUND: Ischemia-reperfusion injury (IRI) upon transplantation is one of the most impactful events that the kidney graft suffers during its life. Its clinical manifestation in the recipient, delayed graft function (DGF), has serious prognostic consequences. However, the different definitions of DGF are subject to physicians' choices and centers' policies, and a more objective tool to quantify IRI is needed. Here, we propose the use of donor-derived cell-free DNA (ddcfDNA) for this scope. METHODS: ddcfDNA was assessed in 61 kidney transplant recipients of either living or deceased donors at 24 h, and 7, 14 and 30 days after transplantation using the AlloSeq cfDNA Kit (CareDx, San Francisco, CA, USA). Patients were followed-up for 6 months and 7-year graft survival was estimated through the complete and functional iBox tool. RESULTS: Twenty-four-hour ddcfDNA was associated with functional DGF [7.20% (2.35%-15.50%) in patients with functional DGF versus 2.70% (1.55%-4.05%) in patients without it, P = .023] and 6-month estimated glomerular filtration rate (r = -0.311, P = .023). At Day 7 after transplantation, ddcfDNA was associated with dialysis duration in DGF patients (r = 0.612, P = .005) and worse 7-year iBox-estimated graft survival probability (ß -0.42, P = .001) at multivariable analysis. Patients with early normalization of ddcfDNA (<0.5% at 1 week) had improved functional iBox-estimated probability of graft survival (79.5 ± 16.8%) in comparison with patients with 7-day ddcfDNA ≥0.5% (67.7 ± 24.1%) (P = .047). CONCLUSIONS: ddcfDNA early kinetics after transplantation reflect recovery from IRI and are associated with short-, medium- and long-term graft outcome. This may provide a more objective estimate of IRI severity in comparison with the clinical-based definitions of DGF.

High-Intensity Exercise Promotes Deleterious Cardiovascular Remodeling in a High-Cardiovascular-Risk Model: A Role for Oxidative Stress.

Although the benefits of moderate exercise in patients at high cardiovascular risk are well established, the effects of strenuous exercise remain unknown. We aimed to study the impact of strenuous exercise in a very high cardiovascular risk model. Nephrectomized aged Zucker obese rats were trained at a moderate (MOD) or high (INT) intensity or were kept sedentary (SED) for 10 weeks. Subsequently, echocardiography and ex vivo vascular reactivity assays were performed, and blood, aortas, perivascular adipose tissue (PVAT), and left ventricles (LVs) were harvested. An improved risk profile consisting of decreased body weight and improved response to a glucose tolerance test was noted in the trained groups. Vascular reactivity experiments in the descending thoracic aorta demonstrated increased endothelial NO release in the MOD group but not in the INT group, compared with SED; the free radical scavenger TEMPOL improved endothelial function in INT rats to a similar level as MOD. An imbalance in the expression of oxidative stress-related genes toward a pro-oxidant environment was observed in the PVAT of INT rats. In the heart, INT training promoted eccentric hypertrophy and a mild reduction in ejection fraction. Obesity was associated with LV fibrosis and a transition toward ß-myosin heavy chain and the N2Ba titin isoform. Exercise reverted the myosin imbalance, but only MOD reduced the predominance of the N2Ba titin isoform. In conclusion, moderate exercise yields the most intense cardiovascular benefits in a high-cardiovascular-risk animal model, while intense training partially reverts them.

Chimeric HLA antibody receptor T cells for targeted therapy of antibody-mediated rejection in transplantation.

The presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal long-term outcomes after solid organ transplantation. The development of chimeric autoantibody receptor T lymphocytes has been postulated for targeted therapy of autoimmune diseases. We aimed to develop a targeted therapy for DSA desensitization and ABMR, generating T cells with a chimeric HLA antibody receptor (CHAR) that specifically eliminates DSA-producing B cells. We have genetically engineered an HLA-A2-specific CHAR (A2-CHAR) and transduced it into human T cells. Then, we have performed in vitro experiments such as cytokine measurement, effector cell activation, and cytotoxicity against anti-HLA-A2 antibody-expressing target cells. In addition, we have performed A2-CHAR-Tc cytotoxic assays in an immunodeficient mouse model. A2-CHAR expressing T cells could selectively eliminate HLA-A2 antibody-producing B cells in vitro. The cytotoxic capacity of A2-CHAR expressing T cells mainly depended on Granzyme B release. In the NSG mouse model, A2-CHAR-T cells could identify and eradicate HLA-A2 antibody-producing B cells even when those cells are localized in the bone marrow. This ability is effector:target ratio dependent. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA class I antibody-producing B cells. Thus, we consider that CHAR technology may be used as a selective desensitization protocol or an ABMR therapy in transplantation.

Extracorporeal Photopheresis Improves Graft Survival in a Full-Mismatch Rat Model of Kidney Transplantation.

Extracorporeal photopheresis (ECP) is an immunomodulatory therapy based on the infusion of autologous cellular products exposed to ultraviolet light (UV) in the presence of a photosensitizer. The study evaluates the ECP efficacy as induction therapy in a full-mismatch kidney transplant rat model. Dark Agouti to Lewis (DA-L) kidney transplant model has been established. ECP product was obtained from Lewis rat recipients after DA kidney graft transplantation (LewDA). Leukocytes of those LewDA rats were exposed to 8-methoxy psoralen, and illuminated with UV-A. The ECP doses assessed were 10 × 106 and 100 × 106 cells/time point. Lewis recipients received seven ECP infusions. DA-L model was characterized by the appearance of donor-specific antibodies (DSA) and kidney function deterioration from day three after kidney transplant. The dysfunction progressed rapidly until graft loss (6.1 ± 0.5 days). Tacrolimus at 0.25 mg/kg prolonged rat survival until 11.4 ± 0.7 days (p = 0.0004). In this context, the application of leukocytes from LewDA sensitized rats accelerated the rejection (8.7 ± 0.45, p = 0.0012), whereas ECP product at high dose extended kidney graft survival until 26.3 ± 7.3 days, reducing class I and II DSA in surviving rats. ECP treatment increases kidney graft survival in full-mismatch rat model of acute rejection and is a suitable immunomodulatory therapy to be explored in kidney transplantation.

Ex vivo normothermic preservation of a kidney graft from uncontrolled donation after circulatory death over 73 hours.

The transplant community is focused on prolonging the ex vivo preservation time of kidney grafts to allow for long-distance kidney graft transportation, assess the viability of marginal grafts, and optimize a platform for the translation of innovative therapeutics to clinical practice, especially those focused on cell and vector delivery to organ conditioning and reprogramming. We describe the first case of feasible preservation of a kidney from a donor after uncontrolled circulatory death over a 73-h period using normothermic perfusion and analyze hemodynamic, biochemical, histological, and transcriptomic parameters for inflammation and kidney injury. The mean pressure and flow values were 71.24 ± 9.62 mmHg and 99.65 ± 18.54 mL/min, respectively. The temperature range was 36.7°C-37.2°C. The renal resistance index was 0.75 ± 0.15 mmHg/mL/min. The mean pH was 7.29 ± 0.15. The lactate concentration peak increased until 213 mg/dL at 6 h, reaching normal values after 34 h of perfusion (8.92 mg/dL). The total urine output at the end of perfusion was 1.185 mL. Histological analysis revealed no significant increase in acute tubular necrosis (ATN) severity as perfusion progressed. The expression of KIM-1, VEGF, and TGFß decreased after 6-18 h of perfusion until 60 h in which the expression of these genes increased again together with the expression of ß-catenin, Ki67, and TIMP1. We show that normothermic perfusion can maintain a kidney graft viable ex vivo for 3 days, thus allowing a rapid translation of pre-clinical therapeutics to clinical practice.

Immune Profiling of Peripheral Blood Mononuclear Cells at Pancreas Acute Rejection Episodes in Kidney-Pancreas Transplant Recipients.

Profiling of circulating immune cells provides valuable insight to the pathophysiology of acute rejection in organ transplantation. Herein we characterized the peripheral blood mononuclear cells in simultaneous kidney-pancreas transplant recipients. We conducted a retrospective analysis in a biopsy-matched cohort (n = 67) and compared patients with biopsy proven acute rejection (BPAR; 41%) to those without rejection (No-AR). We observed that CD3+ T cells, both CD8+ and CD4+, as well as CD19+ B cells were increased in patients with BPAR, particularly in biopsies performed in the early post-transplant period (<3 months). During this period immune subsets presented a good discriminative ability (CD4+ AUC 0.79; CD8+ AUC 0.80; B cells AUC 0.86; p < 0.05) and outperformed lipase (AUC 0.62; p = 0.12) for the diagnosis of acute rejection. We further evaluated whether this could be explained by differences in frequencies prior to transplantation. Patients presenting with early post-transplant rejection (<3 months) had a significant increase in T-cell frequencies pre-transplant, both CD4+ T cells and CD8+ T cells (p < 0.01), which were associated with a significant inferior rejection-free graft survival. T cell frequencies in peripheral blood correlated with pancreas acute rejection episodes, and variations prior to transplantation were associated with pancreas early acute rejection.

Humoral and Cellular Immune Responses After a 3-dose Course of mRNA-1273 COVID-19 Vaccine in Kidney Transplant Recipients: A Prospective Cohort Study.

In kidney transplant recipients, there is discordance between the development of cellular and humoral response after vaccination against SARS-CoV-2. We sought to determine the interplay between the 2 arms of adaptive immunity in a 3-dose course of mRNA-1273 100 µg vaccine. Methods: Humoral (IgG/IgM) and cellular (N- and S-ELISpot) responses were studied in 117 kidney and 12 kidney-pancreas transplant recipients at the following time points: before the first dose, 14 d after the second dose' and before and after the third dose, with a median of 203 and 232 d after the start of the vaccination cycle, respectively. Results: After the second dose, 26.7% of naive cases experienced seroconversion. Before the third dose and in the absence of COVID-19, this percentage increased to 61.9%. After the third dose, seroconversion occurred in 80.0% of patients. Naive patients who had at any time point a detectable positivity for S-ELISpot were 75.2% of the population, whereas patients who maintained S-ELISpot positivity throughout the study were 34.3%. S-ELISpot positivity at 42 d was associated with final seroconversion (odds ratio' 3.14; 95% confidence interval' 1.10-8.96; P = 0.032). Final IgG titer was significantly higher in patients with constant S-ELISpot positivity (P < 0.001). Conclusions: A substantial proportion of kidney transplant recipients developed late seroconversion after 2 doses. Cellular immunity was associated with the development of a stronger humoral response.

Preemptive simultaneous pancreas kidney transplantation has survival benefit to patients.

Several organ allocation protocols give priority to wait-listed simultaneous kidney-pancreas (SPK) transplant recipients to mitigate the higher cardiovascular risk of patients with diabetes mellitus on dialysis. The available information regarding the impact of preemptive simultaneous kidney-pancreas transplantation on recipient and graft outcomes is nonetheless controversial. To help resolve this, we explored the influence of preemptive simultaneous kidney-pancreas transplants on patient and graft survival through a retrospective analysis of the OPTN/UNOS database, encompassing 9690 simultaneous transplant recipients between 2000 and 2017. Statistical analysis was performed applying a propensity score analysis to minimize bias. Of these patients, 1796 (19%) were transplanted preemptively. At ten years, recipient survival was significantly superior in the preemptive group when compared to the non-preemptive group (78.9% vs 71.8%). Dialysis at simultaneous kidney-pancreas transplantation was an independent significant risk for patient survival (hazard ratio 1.66 [95% confidence interval 1.32-2.09]), especially if the dialysis duration was 12 months or longer. Preemptive transplantation was also associated with significant superior kidney graft survival compared to those on dialysis (death-censored: 84.3% vs 75.4%, respectively; estimated half-life of 38.57 [38.33 -38.81] vs 22.35 [22.17 - 22.53] years, respectively). No differences were observed between both groups neither for pancreas graft survival nor for post-transplant surgical complications. Thus, our results sustain the relevance of early referral for pancreas transplantation and the importance of pancreas allocation priority in reducing patient mortality after simultaneous kidney-pancreas transplantation.

Donor-derived Cell-free DNA Shows High Sensitivity for the Diagnosis of Pancreas Graft Rejection in Simultaneous Pancreas-kidney Transplantation.

BACKGROUND: Pancreas graft status in simultaneous pancreas-kidney transplant (SPKTx) is currently assessed by nonspecific biochemical markers, typically amylase or lipase. Identifying a noninvasive biomarker with good sensitivity in detecting early pancreas graft rejection could improve SPKTx management. METHODS: Here, we developed a pilot study to explore donor-derived cell-free DNA (dd-cfDNA) performance in predicting biopsy-proven acute rejection (P-BPAR) of the pancreas graft in a cohort of 36 SPKTx recipients with biopsy-matched plasma samples. dd-cfDNA was measured using the Prospera test (Natera, Inc.) and reported both as a fraction of the total cfDNA (fraction; %) and as concentration in the recipient's plasma (quantity; copies/mL). RESULTS: In the absence of P-BPAR, dd-cfDNA was significantly higher in samples collected within the first 45 d after SPKTx compared with those measured afterward (median, 1.00% versus 0.30%; median, 128.2 versus 35.3 cp/mL, respectively with both; P = 0.001). In samples obtained beyond day 45, P-BPAR samples presented a significantly higher dd-cfDNA fraction (0.83 versus 0.30%; P = 0.006) and quantity (81.3 versus 35.3 cp/mL; P = 0.001) than stable samples. Incorporating dd-cfDNA quantity along with dd-cfDNA fraction outperformed dd-cfDNA fraction alone to detect active rejection. Notably, when using a quantity cutoff of 70 cp/mL, dd-cfDNA detected P-BPAR with a sensitivity of 85.7% and a specificity of 93.7%, which was more accurate than current biomarkers (area under curve of 0.89 for dd-cfDNA (cp/ml) compared with 0.74 of lipase and 0.46 for amylase). CONCLUSIONS: dd-cfDNA measurement through a simple noninvasive blood test could be incorporated into clinical practice to help inform graft management in SPKTx patients.

Impact of insulin therapy before donation on graft outcomes in pancreas transplantation: An analysis of the OPTN/UNOS database.

AIMS: Information on the impact of insulin therapy before pancreas donation on pancreas outcomes is scarce. We aim to explore the influence of insulin therapy before donation on recipient and pancreas graft survival. METHODS: Registry study including 12,841 pancreas recipients from the OPTN/UNOS registry performed between 2000 and 2017. Inverse probability of treatment weighting (IPTW) was used to account for covariate imbalance between recipients from a donor with and without insulin requirements. RESULTS: A total of 7765 (60%) patients received a pancreas from a donor with insulin before donation (IBD). Pancreas graft survival (death-censored) was similar between recipients from IBD and non-IBD donors at 1, 5 and 10 years (89% vs 89%, 78% vs 79 and 69% vs 70%, respectively, P = 0.35). Recipients from IBD donors presented a similar 90-days pancreas graft survival. After IPTW weighting, IBD donors were neither associated with any post-transplant surgical complication (HR 1.11 [95% CI 0.98-1.24], P = 0.06), nor with risk for recipient death (HR 0.94 [95% CI 0.85-1.04], P = 0.26), nor pancreas graft failure (HR 1.06 [95% CI 0.98-1.16], P = 0.15). CONCLUSIONS: Insulin therapy before donation in accepted pancreas donors was not associated, per se, with an impaired pancreas graft and patient survival.

Cellular and humoral response after MRNA-1273 SARS-CoV-2 vaccine in kidney transplant recipients.

According to preliminary data, seroconversion after mRNA SARS-CoV-2 vaccination might be unsatisfactory in Kidney Transplant Recipients (KTRs). However, it is unknown if seronegative patients develop at least a cellular response that could offer a certain grade of protection against SARS-CoV-2. To answer this question, we prospectively studied 148 recipients of either kidney (133) or kidney-pancreas (15) grafts with assessment of IgM/IgG spike (S) antibodies and ELISpot against the nucleocapside (N) and the S protein at baseline and 2 weeks after receiving the second dose of the mRNA-1273 (Moderna) vaccine. At baseline, 31 patients (20.9%) had either IgM/IgG or ELISpot positivity and were considered to be SARS-CoV-2-pre-immunized, while 117 (79.1%) patients had no signs of either cellular or humoral response and were considered SARS-CoV-2-naïve. After vaccination, naïve patients who developed either humoral or cellular response were finally 65.0%, of which 29.9% developed either IgG or IgM and 35.0% S-ELISpot positivity. Factors associated with vaccine unresponsiveness were diabetes and treatment with antithymocytes globulins during the last year. Side effects were consistent with that of the pivotal trial and no DSAs developed after vaccination. In conclusion, mRNA-1273 SARS-CoV-2 vaccine elicits either cellular or humoral response in almost two thirds of KTRs.

B Cell-Derived Extracellular Vesicles Reveal Residual B Cell Activity in Kidney Graft Recipients Undergoing Pre-Transplant Desensitization.

Background: Living-donor kidney transplant (LDKT) recipients undergoing desensitization for Human Leukocyte Antigen (HLA)-incompatibility have a high risk of developing antibody-mediated rejection (ABMR). The purpose of the study is to evaluate if residual B cell activity after desensitization could be estimated by the presence of circulating B cell-derived extracellular vesicles (BEVs). Methods: BEVs were isolated by Sepharose-based size exclusion chromatography and defined as CD19+ and HLA-II+ extracellular vesicles. We analyzed stored serum samples from positive crossmatch LDKT recipients before and after desensitization at first post-transplant biopsy and at 12-month protocol biopsy (n = 11). Control groups were formed by hypersensitized patients who were not submitted to desensitization (n = 10) and by low-risk recipients (n = 9). A prospective validation cohort of 11 patients also included the analysis of B cells subpopulations in recipients' blood and lymph nodes recovered upon graft implantation, along with BEVs analysis before and after desensitization. Results: We found out that CD19+ and HLA-II+BEVs dropped significantly after desensitization and relapse in patients who later developed ABMR was evident. We validated these findings in a proof-of-concept prospective cohort of 6 patients who received the same desensitization protocol and also in a control group of 5 LDKT recipients. In these patients, B cell subpopulations were also studied in recipients' blood and lymph nodes that were recovered before the graft implantation. We confirmed the significant drop in BEVs after desensitization and that this paralleled the reduction in CD19+cells in lymph nodes, while in peripheral blood B cells, this change was almost undetectable. Conclusions: BEVs reflected B cell residual activity after desensitization and this could be a valid surrogate of humoral alloreactivity in this setting.

Nephroprotective Potential of Mesenchymal Stromal Cells and Their Extracellular Vesicles in a Murine Model of Chronic Cyclosporine Nephrotoxicity.

BACKGROUND: Cell therapies and derived products have a high potential in aiding tissue and organ repairing and have therefore been considered as potential therapies for treating renal diseases. However, few studies have evaluated the impact of these therapies according to the stage of chronic kidney disease. The aim of this study was to evaluate the renoprotective effect of murine bone marrow mesenchymal stromal cells (BM-MSCs), their extracellular vesicles (EVs) and EVs-depleted conditioned medium (dCM) in an aggressive mouse model of chronic cyclosporine (CsA) nephrotoxicity in a preventive and curative manner. METHODS: After 4 weeks of CsA-treatment (75 mg/kg daily) mice developed severe nephrotoxicity associated with a poor survival rate of 25%, and characterized by tubular vacuolization, casts, and cysts in renal histology. BM-MSC, EVs and dCM groups were administered as prophylaxis or as treatment of CsA nephrotoxicity. The effect of the cell therapies was analyzed by assessing renal function, histological damage, apoptotic cell death, and gene expression of fibrotic mediators. RESULTS: Combined administration of CsA and BM-MSCs ameliorated the mice survival rates (6-15%), but significantly renal function, and histological parameters, translating into a reduction of apoptosis and fibrotic markers. On the other hand, EVs and dCM administration were only associated with a partial recovery of renal function or histological damage. Better results were obtained when used as treatment rather than as prophylactic regimen i.e., cell therapy was more effective once the damage was established. CONCLUSION: In this study, we showed that BM-MSCs induce an improvement in renal outcomes in an animal model of CsA nephrotoxicity, particularly if the inflammatory microenvironment is already established. EVs and dCM treatment induce a partial recovery, indicating that further experiments are required to adjust timing and dose for better long-term outcomes.

Outcomes of pancreas transplantation in older diabetic patients.

OBJECTIVE: Improvement in insulin alternatives is leading to a delayed presentation of microvascular and macrovascular complications of diabetes. The objective of this study was to evaluate the long-term outcomes of older (≥50 years) diabetic patients who receive a pancreas transplantation (PT). RESEARCH DESIGN AND METHODS: We retrospectively evaluated all 338 PTs performed at our center between 2000 and 2016 (mean follow-up 9.4±4.9 years). Recipient and graft survivals were estimated for up to 10 years after PT. Major adverse cardiovascular events (MACEs) before and after PT were included in the analysis. RESULTS: Thirty-nine patients (12%) were ≥50 years old (52.7±2.3 years) at the day of PT, of which 29 received a simultaneous pancreas-kidney transplantation (SPK) and 10 a pancreas after kidney transplantation (PAK). SPK recipients were first transplants, whereas in the PAK up to 50% were pancreas re-transplantations. Recipient and pancreas graft survivals at 10 years were similar between the group <50 years old and the older group for both SPK and PAK (log-rank p>0.05). The prevalence of MACE prior to PT was similar between both groups (31% vs 29%). Following PT, older recipients presented inferior post-transplant MACE-free survival. In a multivariate regression model, diabetes vintage (HR 1.054, p=0.03) and pre-transplantation MACE (HR 1.98, p=0.011), but not recipient age (HR 1.45, p=0.339), were associated with post-transplant MACE. CONCLUSIONS: Long-term survival of older pancreas transplant recipients are similar to younger counterparts. Diabetes vintage, but not age, increased the risk of post-transplantation MACE. These results suggest pancreas transplantation is a valuable treatment alternative to older diabetic patients.

Impact of Mesenchymal Stromal Cells and Their Extracellular Vesicles in a Rat Model of Kidney Rejection.

BACKGROUND: Mesenchymal stromal cells (MSCs) from different sources possess great therapeutic potential due to their immunomodulatory properties associated with allograft tolerance. However, a crucial role in this activity resides in extracellular vesicles (EVs) and signaling molecules secreted by cells. This study aimed to evaluate the immunomodulatory properties of donor and recipient MSCs isolated from adipose tissue (AD) or bone marrow (BM) and their EVs on kidney outcome in a rat kidney transplant model. METHODS: The heterotopic-kidney-transplant Fisher-to-Lewis rat model (F-L) was performed to study mixed cellular and humoral rejection. After kidney transplantation, Lewis recipients were assigned to 10 groups; two control groups; four groups received autologous MSCs (either AD- or BM- MSC) or EVs (derived from both cell types); and four groups received donor-derived MSCs or EVs. AD and BM-EVs were purified by ultracentrifugation. Autologous cell therapies were administered three times intravenously; immediately after kidney transplantation, 4 and 8 weeks, whereas donor-derived cell therapies were administered once intravenously immediately after transplantation. Survival and renal function were monitored. Twelve weeks after kidney transplantation grafts were harvested, infiltrating lymphocytes were analyzed by flow cytometry and histological lesions were characterized. RESULTS: Autologous AD- and BM-MSCs, but not their EVs, prolonged graft and recipient survival in a rat model of kidney rejection. Autologous AD- and BM-MSCs significantly improved renal function during the first 4 weeks after transplantation. The amelioration of graft function could be associated with an improvement in tubular damage, as well as in T, and NK cell infiltration. On the other side, the application of donor-derived AD-MSC was harmful, and all rats died before the end of the protocol. AD-EVs did not accelerate the rejection. Contrary to autologous MSCs results, the single dose of donor-derived BM-MSCs is not enough to ameliorate kidney graft damage. CONCLUSION: EVs treatments did not exert any benefit in our experimental settings. In the autologous setting, BM-MSCs prompted as a potentially promising therapy to improve kidney graft outcomes in rats with chronic mixed rejection. In the donor-derived setting, AD-MSC accelerated progression to end-stage kidney disease. Further experiments are required to adjust timing and dose for better long-term outcomes.

Isolation of Extracellular Vesicles Derived from Mesenchymal Stromal Cells by Ultracentrifugation.

Extracellular vesicles (EVs) are a heterogeneous group of membranous vesicles that differ on their biogenesis and release pathways, such as exosomes, microvesicles and apoptotic bodies. They are involved in cell-to-cell communication delivering signal molecules (proteins, nucleic acids, lipids, etc.) that can regulate different physiological processes, as well as the development and progression of several diseases. There are different methods and commercial kits to isolate EVs and depending on the methodology one could obtain EVs with different degrees of efficiency, purity and it can be more or less time-consuming. Then, the choice has to be according to the different advantages and disadvantages, and their use for downstream applications. Here, we describe the EVs isolation method from mesenchymal stromal cells by ultracentrifugation. This EVs isolation can be performed using common media and buffers, and only with the requirement of an analytical ultracentrifuge. Moreover, this method can be used to obtain large quantity of EVs with a good reproducibility for developing in vitro and in vivo experiments and studying their biological actions.