Kidney organoid models reveal cilium-autophagy metabolic axis as a therapeutic target for PKD both in vitro and in vivo.

Human pluripotent stem cell-derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both in vitro and in vivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single-cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA-approved drug, effectively attenuated cyst formation in vivo. This in vivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation.

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.

Myc Supports Self-Renewal of Basal Cells in the Esophageal Epithelium.

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence.

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.

Influence of Persistent Inflammation in Follow-Up Biopsies After Antibody-Mediated Rejection in Kidney Transplantation.

Background: Despite recent advances in immunosuppression treatment, antibody-mediated rejection (ABMR) remains the leading cause of kidney graft loss. Information about prognostic markers and the efficacy of treatment is scarce. Methods: Retrospective study with kidney recipients diagnosed an active ABMR from January 1, 2004 to December 31, 2019 to explore the influence of persistent inflammation in follow-up biopsies on graft survival after ABMR treatment. Results: About 116 patients were included. Active ABMR were treated with a combination of plasma exchange (PE), intravenous immunoglobulin (IVIg), rituximab, and steroids. At 6 months of treatment, 63 (54.3%) patients presented a stabilization or improvement in kidney-graft function. The effectiveness varied depending on the timepoint of the presentation between transplantation and rejection, which is lower for those with late ABMR (63 vs. 21% for early vs. late ABMR, respectively). Ninety patients (77%) underwent a control biopsy after ABMR treatment, from which 46 (51%) responded to the treatment. Microvascular inflammation (MVI) persisted in 64 (71%) biopsies, whereas tubulitis persisted in 17 (19%) biopsies. Death-censored graft survival at 1 year was significantly lower in patients with persistent MVI (86% vs. 95% without persistent MVI, P = 0.002), or with persistent tubulitis (44% vs. 66% without tubulitis, P = 0.02). In the Cox Regression analysis, the persistence of MVI [hazard ratio (HR), 4.50 (95%CI, 1.35-14.96), P = 0.01] and tubulitis [HR 2.88 95%CI (1.24-6.69), P = 0.01) in follow-up biopsies significantly increased the risk of graft failure. Conclusion: Persistent inflammation in follow-up biopsies after ABMR treatment was associated with an increased risk of graft loss, even without meeting Banff rejection criteria. Study Registration: Agencia Española de Medicamentos y Productos Sanitarios (AEMPS): 14566/RG 24161. Study code: UTRINM-2017-01.

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.

Outcomes From Brain Death Donors With Previous Cardiac Arrest Accepted for Pancreas Transplantation: A Single-center Retrospective Analysis.

OBJECTIVE: The aim of the study was to evaluate the effect of cardiac arrest time (CAT) in donors after brain death (DBD) donors on pancreas transplant outcome. SUMMARY OF BACKGROUND DATA: Results from donors after circulatory death report good outcomes despite warm ischemia times up to 57 minutes. Previous cardiac arrest in DBD has been addressed as a potential risk factor, but duration of the CAT has never been evaluated. METHODS: We conducted a retrospective analysis including 342 pancreas transplants performed at our center from 2000 to 2016, and evaluated the effect of previous cardiac arrest in DBD (caDBD) on pancreas transplant outcomes. RESULTS: A total of 49 (14.3%) caDBD were accepted for transplantation [median CAT of 5.0 min (IQR 2.5-15.0)]. Anoxic encephalopathy was most frequent and P-PASS higher (16.9 vs 15.6) in caDBD group when compared with other DBD. No differences were found in all other characteristics evaluated.Graft survival was similar between both groups, as was the incidence of early graft failure (EGF). CAT increased the risk for EGF [OR 1.09 (95% CI, 1.01-1.17)], and the duration of CPR discriminated for EGF [AUC of 0.86 (95% CI, 0.74-0.98)], with a sensitivity and specificity of 100% and 75% at a cutoff of 15 minutes. When evaluated separately, caDBD >15 min increased over 5 times the risk for EGF [HR 5.80 (95% CI, 1.82-18.56); P = 0.003], and these presented fewer days on the ICU (1.0 vs 3.0 d). CONCLUSION: CaDBD donors are suitable for routine pancreas transplantation without increasing EGF risk, and in those with longer CAT it may be prudent to postpone donation a few days to allow a thorough evaluation of organ damage following cardiac arrest.

The impact of functional delayed graft function in the modern era of kidney transplantation - A retrospective study.

The dialysis-based definition of Delayed Graft Function (dDGF) is not necessarily objective as it depends on the individual physician's decision. The functional definition of DGF (fDGF, the failure of serum creatinine to decrease by at least 10% daily on 3 consecutive days during the first week post-transplant), may be more sensitive to reflect recovery after the ischemia-reperfusion injury. We retrospectively analyzed both definitions in 253 deceased donor kidney transplant recipients for predicting death-censored graft failure as primary outcome, using eGFR < 25 ml/min/1.73 m2 as a surrogate end-point for graft failure. Secondary outcome was a composite outcome that included graft failure as above and also patient's death. Median follow-up was 3.22 [2.38-4.21] years. Seventy-nine patients developed dDGF (31.2%) and 127 developed fDGF (50.2%). Sixty-three patients fulfilled criteria for both definitions (24.9%). At multivariable analysis, the two definitions were significantly associated with the primary [HR (95%CI) 2.07 (1.09-3.94), P = 0.026 for fDGF and HR (95%CI) 2.41 (1.33-4.37), P = 0.004 for dDGF] and the secondary composite outcome [HR (95%CI) 1.58 (1.01-2.51), P = 0.047 for fDGF and HR (95%CI) 1.67 (1.05-2.66), P = 0.028 for dDGF]. Patients who met criteria for both definitions had the worst prognosis, with a three-year estimates (95%CI) of survival from the primary and secondary outcomes of 2.31 (2.02-2.59) and 2.20 (1.91-2.49) years for fDGF+/dDGF+, in comparison with the other groups (P < 0.01 for trend). fDGF provides supplementary information about graft outcomes on top of the dDGF definition in a modern series of kidney transplantation.

Complement as the enabler of carfilzomib-induced thrombotic microangiopathy.

Carfilzomib has been associated with the development of thrombotic microangiopathy (TMA) in relapsed/refractory multiple myeloma patients, a severe disease with no currently available aetiological treatment. We evaluated the potential role of terminal complement pathway in four patients with carfilzomib-induced TMA. Membrane attack complex (C5b-9) deposition on endothelial cells in culture exposed to plasma from patients during the acute phase of the disease suggests complement overactivation as a mechanism of potential endothelial damage in three out of four patients. If confirmed in larger cohorts, C5b-9 evaluation will allow early identification of patients who could benefit from complement blockade and treatment monitoring.

A novel assay to measure calcification propensity: from laboratory to humans.

Cardiovascular calcification (CVC) contributes to morbidity and mortality in patients undergoing dialysis. We examined the pharmacodynamic effects of SNF472, a calcification inhibitor, on plasma calcium phosphate crystallization using spectrometric measurements, and its correlations with effects on CVC in rats or humans. Rats (N = 38) injected with vitamin D (days 1-3) to induce CVC were infused with saline or SNF472 (days 1-12). Inhibition of CVC was 50-65% with SNF472 3 mg/kg and ~ 80% with SNF472 10 or 30 mg/kg. SNF472 dose-dependently inhibited calcium phosphate crystallization, which correlated with inhibition of CVC (r = 0.628, P = 0.005). In patients with calciphylaxis (N = 14), infusion of SNF472 (~ 7 mg/kg) during hemodialysis for 12 weeks inhibited calcium phosphate crystallization by nearly 70%. In patients with CVC (N = 274), infusion of SNF472 during hemodialysis for 52 weeks inhibited calcium phosphate crystallization (placebo: 15%; 300 mg: 61%; 600 mg: 75%), which correlated with inhibition of CVC (r = 0.401, P = 0.003). These findings show a direct correlation between inhibition of calcium phosphate crystallization in plasma and inhibition of CVC both in a rat model and in humans, supporting the use of the pharmacodynamic assay in clinical trials as a potentially predictive tool to evaluate the activity of calcification inhibitors.

Use of De Novo mTOR Inhibitors in Hypersensitized Kidney Transplant Recipients: Experience From Clinical Practice.

BACKGROUND: It is commonly believed that mTOR inhibitors (mTORi) should not be used in high-immunological risk kidney transplant recipients due to a perceived increased risk of rejection. However, almost all trials that examined the association of optimal-dose mTORi with calcineurin inhibitor (CNI) have excluded hypersensitized recipients from enrollment. METHODS: To shed light on this issue, we examined 71 consecutive patients with a baseline calculated panel reactive antibody (cPRA) ≥50% that underwent kidney transplantation from June 2013 to December 2016 in our unit. Immunosuppression was based on CNI (tacrolimus), steroids and alternatively mycophenolic acid (MPA; n = 38), or mTORi (either everolimus or sirolimus, n = 33, target trough levels 3-8 ng/mL). RESULTS: Demographic and immunological risk profiles were similar, and almost 90% of patients in both groups received induction with lymphocyte-depleting agents. Cox-regression analysis of rejection-free survival revealed better results for mTORi versus MPA in terms of biopsy-proven acute rejection (hazard ratio [confidence interval], 0.32 [0.11-0.90], P = 0.031 at univariable analysis and 0.34 [0.11-0.95], P = 0.040 at multivariable analysis). There were no differences in 1-year renal function, Banff chronicity score at 3- and 12-month protocol biopsy and development of de novo donor-specific antibodies. Tacrolimus trough levels along the first year were not different between groups (12-mo levels were 8.72 ± 2.93 and 7.85 ± 3.07 ng/mL for MPA and mTORi group respectively, P = 0.277). CONCLUSIONS: This single-center retrospective cohort analysis suggests that in hypersensitized kidney transplant recipients receiving tacrolimus-based immunosuppressive therapy similar clinical outcomes may be obtained using mTOR inhibitors compared to mycophenolate.

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.

IgA Nephropathy Recurrence after Kidney Transplantation: Role of Recipient Age and Human Leukocyte Antigen-B Mismatch.

BACKGROUND: Recurrence of immunoglobulin (Ig)A nephropathy (rIgAN) is a growing cause of kidney allograft dysfunction. This study was aimed at investigating factors associated with rIgAN and the subsequent progression to end-stage renal disease (ESRD). METHODS: Retrospective study including consecutive patients with IgA nephropathy (IgAN) who received a kidney transplant in our center between 1992 and 2016 and had a renal biopsy by clinical indication. The date of detection of chronic kidney disease (CKD) 5 was used as renal outcome. RESULTS: Eighty-six kidney transplants were performed in patients with IgAN, 38 (44%) were from living donors (related n = 26). rIgAN was diagnosed in 23 allografts (27%). Renal function and proteinuria at the end of the follow-up period were worst in the rIgAN patients compared to those without rIgAN (2.2 vs. 1.4 mg/dL, p = 0.014, and 1.16 vs. 0.49 g/day, p = 0.005, respectively). Risk of rIgAN and progression to CKD 5 decreased with patient's age (hazard ratio [HR] 0.95, 95% CI 0.92-0.98, p = 0.002, and HR 0.97, 95% CI 0.83-0.97, p = 0.008 per year, respectively). Patients with rIgAN had a higher risk of progression to CKD 5 (HR 6.7, 95% CI 1.3-35.7, p = 0.025). Full donor-recipient mismatch in the human leukocyte antigen (HLA)-B loci decreased the risk of rIgAN (HR 0.22, 95% CI 0.06-0.76, p = 0.017). CONCLUSIONS: rIgAN was an independent risk factor for ESRD after renal allograft. Younger age increased the risk of rIgAN and CKD 5. Conversely, HLA-B mismatching was a potential protective factor for rIgAN of this glomerular disease.

Combination of calcineurin and mTOR inhibitors in kidney transplantation: a propensity score analysis based on current clinical practice.

INTRODUCTION: The TRANSFORM study demonstrated that an immunosuppression based on a combination of calcineurin inhibitors and de-novo mTOR inhibitors (mTORi) is safe and effective in kidney transplant recipients. However, data that validate this approach in clinical practice are currently missing. MATERIALS AND METHODS: Analysis of 401 kidney transplant recipients transplanted from June 2013 to December 2016. All patients received tacrolimus with prednisone in combination with either mycophenolate (n = 186) or mTORi (either everolimus or sirolimus, n = 215). A propensity score to receive mTORi was calculated based on the inverse probability of treatment weighting (IPTW) from the following parameters: age and sex of donor and recipient, BMI, previous transplants, diabetes, cPRA, dialysis before transplantation, dialysis vintage, type of donor, ABO-incompatibility, HLA-mismatches, induction and ischemia time. Median follow-up was 2.6 [1.9; 3.7] years. RESULTS: Cox-regression analysis suggests good results for mTORi versus MPA in terms of 1-year biopsy-proven acute rejection (BPAR, P = 0.063), 1-year graft loss (P = 0.025) and patient survival (P < 0.001). Results observed for BPAR and graft failure were largely attributed to those patients that would have been excluded by the TRANSFORM because of some exclusion criteria (52.9% of the population, P = 0.003 for 1-year BPAR and P = 0.040 for graft loss). In patients who met selection criteria for TRANSFORM, no effect of treatment for BPAR or graft failure was observed, while the beneficial effect on overall survival persisted. CONCLUSIONS: In a real-life setting, a protocol based on de-novo mTORi with tacrolimus and prednisone could be employed as a standard immunosuppressive regimen and was associated with good outcomes.

Complement Activation and Thrombotic Microangiopathies.

BACKGROUND AND OBJECTIVES: Atypical hemolytic uremic syndrome is a form of thrombotic microangiopathy caused by dysregulation of the alternative complement pathway. There is evidence showing complement activation in other thrombotic microangiopathies. The aim of this study was to evaluate complement activation in different thrombotic microangiopathies and to monitor treatment response. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Complement activation was assessed by exposing endothelial cells to sera or activated-patient plasma-citrated plasma mixed with a control sera pool (1:1)-to analyze C5b-9 deposits by immunofluorescence. Patients with atypical hemolytic uremic syndrome (n=34) at different stages of the disease, HELLP syndrome (a pregnancy complication characterized by hemolysis, elevated liver enzymes, and low platelet count) or severe preeclampsia (n=10), and malignant hypertension (n=5) were included. RESULTS: Acute phase atypical hemolytic uremic syndrome-activated plasma induced an increased C5b-9 deposition on endothelial cells. Standard and lower doses of eculizumab inhibited C5b-9 deposition in all patients with atypical hemolytic uremic syndrome, except in two who showed partial remission and clinical relapse. Significant fibrin formation was observed together with C5b-9 deposition. Results obtained using activated-plasma samples were more marked and reproducible than those obtained with sera. C5b-9 deposition was also increased with samples from patients with HELLP (all cases) and preeclampsia (90%) at disease onset. This increase was sustained in those with HELLP after 40 days, and levels normalized in patients with both HELLP and preeclampsia after 6-9 months. Complement activation in those with malignant hypertension was at control levels. CONCLUSIONS: The proposed methodology identifies complement overactivation in patients with atypical hemolytic uremic syndrome at acute phase and in other diseases such as HELLP syndrome and preeclampsia. Moreover, it is sensitive enough to individually assess the efficiency of the C5 inhibition treatment.

Impact of Discards for Living Donor Kidney Transplantation in a Transplant Program.

OBJECTIVE: Living donor kidney transplantation (LDKT) is the best treatment for end-stage renal disease. In this setting, a significant percentage of transplants are not undertaken because of medical and nonmedical reasons of both donors and recipients. However, the impact of these discards in a transplant program has not been identified thoroughly so far. Our objective was to clarify key reasons for exclusion of LDKTs and the consequences for the discarded transplant candidates in the following 5 years. METHODS: Analysis of donors' and recipients' characteristics of 781 couples evaluated in our hospital from January 2005 to December 2013. The consequences of discards in transplant candidates were analyzed in the cohort 2012 to 2013 (n = 106) and followed up until October 2018. RESULTS: In our study group, 402 (51.5%) LDKT couples were successfully donated, and 379 (48.5%) were excluded. Donor and transplant recipient candidates discarded were older at the evaluation (55.07 ± 12.14 years vs 51.73 ± 10.93 years, P < .001; 48.81 ± 14.05 years vs 44.62 ± 13.91 years, P < .001, respectively). The most frequent reason for kidney discard was medical contraindication found in the potential donor (47.5%; low eGFR, diabetes mellitus, impaired glucose tolerance, high blood pressure, cardiovascular pathology casually found during evaluation, and proteinuria). Of the discarded candidates from 2012 to 2013, 36.8% received a deceased donor kidney transplant, 17% a LDKT with another donor, 7.5% stayed on the waiting list, 18.9% died, 3.8% were excluded from the waiting list, and 14.2% were lost to follow-up. CONCLUSIONS: In most cases, transplantation was not undertaken because of donor pathology. Fifty-three percent of the discarded patients were eventually transplanted, with a 31.4% probability to receive an organ from another living donor.

Effect of immunosuppression in miRNAs from extracellular vesicles of colorectal cancer and their influence on the pre-metastatic niche.

Colorectal cancer (CRC) occurs with more aggressiveness in kidney transplant recipients compared to the general population. Immunosuppressive therapy plays a crucial role in the development of post-transplant malignancy. Concretely, cyclosporine A (CsA) has intrinsic pro-oncologic properties, while several studies report a regression of cancer after the introduction of rapamycin (RAPA). However, their effect on the extracellular vesicle (EV) content from CRC cell lines and their relevance in the pre-metastatic niche have not yet been studied. Here, we investigated the effect of RAPA and CsA in EV-miRNAs from metastatic and non-metastatic CRC cell lines and the role of relevant miRNAs transferred into a pre-metastatic niche model. EV-miRNA profiles showed a significant upregulation of miR-6127, miR-6746-5p, and miR-6787-5p under RAPA treatment compared to CsA and untreated conditions in metastatic cell lines that were not observed in non-metastatic cells. From gene expression analysis of transfected lung fibroblasts, we identified 22 shared downregulated genes mostly represented by the histone family involved in chromatin organization, DNA packaging, and cell cycle. These results suggest that EV-miR-6127, miR-6746-5p and miR-6787-5p could be a potential epigenetic mechanism induced by RAPA therapy in the regulation of the pre-metastatic niche of post-transplant colorectal cancer.

Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction.

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a De Novo Vascular Network.

Human pluripotent stem cell-derived kidney organoids recapitulate developmental processes and tissue architecture, but intrinsic limitations, such as lack of vasculature and functionality, have greatly hampered their application. Here we establish a versatile protocol for generating vascularized three-dimensional (3D) kidney organoids. We employ dynamic modulation of WNT signaling to control the relative proportion of proximal versus distal nephron segments, producing a correlative level of vascular endothelial growth factor A (VEGFA) to define a resident vascular network. Single-cell RNA sequencing identifies a subset of nephron progenitor cells as a potential source of renal vasculature. These kidney organoids undergo further structural and functional maturation upon implantation. Using this kidney organoid platform, we establish an in vitro model of autosomal recessive polycystic kidney disease (ARPKD), the cystic phenotype of which can be effectively prevented by gene correction or drug treatment. Our studies provide new avenues for studying human kidney development, modeling disease pathogenesis, and performing patient-specific drug validation.