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Introduction Diabetic kidney disease (DKD) is a common cause of chronic kidney disease with around 25-40% of patients with diabetes being affected. The course of DKD is variable and estimated glomerular filtration rate (eGFR) and albuminuria, the currently used clinical markers, are not able to accurately predict the individual disease trajectory, in particular in early stages of the disease. The aim of this study was to assess the association of urine levels of selected protein biomarkers with the progression of DKD at an early stage of disease. Methods We measured 22 protein biomarkers using the Mesoscale Discovery platform in 461 urine samples of the PROVALID cohort, an observational study of patients with type 2 diabetes mellitus followed at the primary health care level for a minimum of four years. Odds ratios (OR) were estimated for the effect of marker values above median on fast progression using unadjusted and adjusted logistic regression models. RNA expression at the single cell level in kidney biopsy samples obtained from a cohort of young persons with type 2 diabetes mellitus was in addition determined for markers showing significant associations with disease progression. Results Increased urinary levels of epidermal growth factor (EGF) were linked to lower odds of fast progression (defined as annual eGFR decline greater than 2.58 ml/min per 1.73 m2) with an odds ratio (OR) of 0.60 (95% CI 0.46, 0.78). The association with outcome was even stronger when adjusting for a set of 14 baseline clinical parameters including age, biological sex, eGFR, body mass index, albuminuria, and HbA1c. Elevated urinary levels of fatty acid binding protein 3 (FABP3) and vascular cell adhesion molecule 1 (VCAM1) were each significantly associated with fast progression with an OR of 1.44 (95% CI 1.11, 1.87) and an OR of 1.41 (95% CI 1.08, 1.83), respectively. Enriched expression of EGF and FABP3 was observed in distal convoluted tubular cells and VCAM1 in parietal epithelial cells at single cell level from biopsies of patients with early DKD. Conclusion In summary we show that lower urinary levels of EGF and higher urinary levels of FABP3 and VCAM1 are significantly associated with DKD progression in early-stage disease.

Summary: Background: The response to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccination is severely impaired in patients on maintenance immunosuppression after kidney transplantation. Methods: We conducted a prospective cohort study of 194 kidney transplant recipients (KTR) who exhibited no response to SARS-CoV-2 vaccinations (i.e., SARS-CoV-2 spike protein antibodies ≤264 U/mL) and had no prior documented infection. Patients received 300 mg of cilgavimab/tixagevimab as SARS-CoV-2 pre-exposure prophylaxis (PrEP) between March 4, 2022, and May 3, 2022 and were contrasted to a matched cohort of 186 KTRs also without immunization again defined as SARS-CoV-2 spike protein antibodies ≤264 U/mL and no documented prior infection. The primary outcome was the serum kinetics of cilgavimab/tixagevimab, the secondary endpoints were time to SARS-CoV-2 breakthrough infection, severity of disease and variant specific live viral in vitro neutralization tests of patient sera. Findings: Longitudinal serum level monitoring showed a half-life of 91 days for both antibodies (95% CI 86–95 days for cilgavimab and 85–96 days for tixagevimab) in KTRs. In vitro neutralization tests showed effectiveness against the BA.2 omicron subvariant but not BA.5. The cumulative incidence of SARS-CoV-2 infections until May 15, 2022, (BA.2 dominance) was 15/194 vs 36/186 in the PrEP and control group respectively (OR = 0.35, 95% CI 0.18–0.66) but was not different thereafter (BA.4/5 dominance). The number of severe infections during the BA.2 period was lower in the prophylaxis than in the control group (OR = 0.37, 95% CI 0.17–0.79). Interpretation: This study showed that SARS-CoV-2 PrEP with cilgavimab/tixagevimab demonstrated clinical effectiveness against variants that are neutralised (BA.2) but not against BA.4/5. Funding: This study was funded by the Medical University of Vienna and an unrestricted grant from AstraZeneca (ESR-21-21585).

Summary: Background: Induction of donor-specific tolerance is a promising approach to achieve long-term graft patency in transplantation with little to no maintenance immunosuppression. Changes to the recipient’s T cell receptor (TCR) repertoire are understood to play a pivotal role in the establishment of a robust state of tolerance in chimerism-based transplantation protocols. Methods: We investigated changes to the TCR repertoires of patients participating in an ongoing prospective, controlled, phase I/IIa trial designed to evaluate the safety and efficacy of combination cell therapy in living donor kidney transplantation. Using high-throughput sequencing, we characterized the repertoires of six kidney recipients who also received bone marrow from the same donor (CKBMT), together with an infusion of polyclonal autologous Treg cells instead of myelosuppression. Findings: Patients undergoing combination cell therapy exhibited partial clonal deletion of donor-reactive CD4+ T cells at one, three, and six months post-transplant, compared to control patients receiving the same immunosuppression regimen but no cell therapy (p = 0.024). The clonality, R20 and turnover rates of the CD4+ and CD8+ TCR repertoires were comparable in both groups, showing our protocol caused no excessive repertoire shift or loss of diversity. Treg clonality was lower in the case group than in control (p = 0.033), suggesting combination cell therapy helps to preserve Treg diversity. Interpretation: Overall, our data indicate that combining Treg cell therapy with CKBMT dampens the alloimmune response to transplanted kidneys in humans in the absence of myelosuppression. Funding: This study was funded by the Vienna Science and Technology Fund (WWTF).

Abstract Background Chronic kidney disease (CKD) is a common comorbidity in people with diabetes mellitus, and a key risk factor for further life-threatening conditions such as cardiovascular disease. The early prediction of progression of CKD therefore is an important clinical goal, but remains difficult due to the multifaceted nature of the condition. We validated a set of established protein biomarkers for the prediction of trajectories of estimated glomerular filtration rate (eGFR) in people with moderately advanced chronic kidney disease and diabetes mellitus. Our aim was to discern which biomarkers associate with baseline eGFR or are important for the prediction of the future eGFR trajectory. Methods We used Bayesian linear mixed models with weakly informative and shrinkage priors for clinical predictors (n = 12) and protein biomarkers (n = 19) to model eGFR trajectories in a retrospective cohort study of people with diabetes mellitus (n = 838) from the nationwide German Chronic Kidney Disease study. We used baseline eGFR to update the models’ predictions, thereby assessing the importance of the predictors and improving predictive accuracy computed using repeated cross-validation. Results The model combining clinical and protein predictors had higher predictive performance than a clinical only model, with an $$R^{2}$$ R 2 of 0.44 (95% credible interval 0.37–0.50) before, and 0.59 (95% credible interval 0.51–0.65) after updating by baseline eGFR, respectively. Only few predictors were sufficient to obtain comparable performance to the main model, with markers such as Tumor Necrosis Factor Receptor 1 and Receptor for Advanced Glycation Endproducts being associated with baseline eGFR, while Kidney Injury Molecule 1 and urine albumin-creatinine-ratio were predictive for future eGFR decline. Conclusions Protein biomarkers only modestly improve predictive accuracy compared to clinical predictors alone. The different protein markers serve different roles for the prediction of longitudinal eGFR trajectories potentially reflecting their role in the disease pathway.

Summary: Despite the similar clinical outcomes after renin-angiotensin system (RAS) inhibitor (RASi) continuation or withdrawal in COVID-19, the effects on angiotensin-converting enzyme 2 (ACE2) and RAS metabolites remain unclear. In a substudy of the randomized controlled Austrian Corona Virus Adaptive Clinical Trial (ACOVACT), patients with hypertension and COVID-19 were randomized 1:1 to either RASi continuation (n = 30) or switch to a non-RASi medication (n = 29). RAS metabolites were analyzed using a mixed linear regression model (n = 30). Time to a sustained clinical improvement was equal and ACE2 did not differ between the groups but increased over time in both. Overall ACE2 was higher with severe COVID-19. ACE-S and Ang II levels increased as expected with ACE inhibitor discontinuation. These data support the safety of RASi continuation in COVID-19, although RASi were frequently discontinued in our post hoc analysis. The study was not powered to draw definite conclusions on clinical outcomes using small sample sizes.

IntroductionAntibody mediated rejection (ABMR) is a major factor limiting outcome after organ transplantation. Anti-HLA donor-specific antibodies (DSA) of the IgG isotype are mainly responsible for ABMR. Recently DSA of the IgE isotype were demonstrated in murine models as well as in a small cohort of sensitized transplant recipients. In the present study, we aimed to determine the frequency of pre-existing and de novo anti-HLA IgE antibodies in a cohort of 105 solid organ transplant recipients.MethodsWe prospectively measured anti-HLA IgE antibodies in a cohort of kidney (n=60), liver, heart and lung (n=15 each) transplant recipients before and within one-year after transplantation, employing a single-antigen bead assay for HLA class I and class II antigens. Functional activity of anti-HLA IgE antibodies was assessed by an in vitro mediator release assay. Antibodies of the IgG1-4 subclasses and Th1 and Th2 cytokines were measured in anti-HLA IgE positive patients.ResultsPre-existing anti-HLA IgE antibodies were detected in 10% of renal recipients (including 3.3% IgE-DSA) and in 4.4% of non-renal solid organ transplant recipients (heart, liver and lung cohort). Anti-HLA IgE occurred only in patients that were positive for anti-HLA IgG, and most IgE positive patients had had a previous transplant. Only a small fraction of patients developed de novo anti-HLA IgE antibodies (1.7% of kidney recipients and 4.4% of non-renal recipients), whereas no de novo IgE-DSA was detected. IgG subclass antibodies showed a distinct pattern in patients who were positive for anti-HLA IgE. Moreover, patients with anti-HLA IgE showed elevated Th2 and also Th1 cytokine levels. Serum from IgE positive recipients led to degranulation of basophils in vitro, demonstrating functionality of anti-HLA IgE.DiscussionThese data demonstrate that anti-HLA IgE antibodies occur at low frequency in kidney, liver, heart and lung transplant recipients. Anti-HLA IgE development is associated with sensitization at the IgG level, in particular through previous transplants and distinct IgG subclasses. Taken together, HLA specific IgE sensitization is a new phenomenon in solid organ transplant recipients whose potential relevance for allograft injury requires further investigation.

Abstract SARS-CoV-2 gains cell entry via angiotensin-converting enzyme (ACE) 2, a membrane-bound enzyme of the “alternative” (alt) renin-angiotensin system (RAS). ACE2 counteracts angiotensin II by converting it to potentially protective angiotensin 1–7. Using mass spectrometry, we assessed key metabolites of the classical RAS (angiotensins I–II) and alt-RAS (angiotensins 1–7 and 1–5) pathways as well as ACE and ACE2 concentrations in 159 patients hospitalized with COVID-19, stratified by disease severity (severe, n = 76; non-severe: n = 83). Plasma renin activity (PRA-S) was calculated as the sum of RAS metabolites. We estimated ACE activity using the angiotensin II:I ratio (ACE-S) and estimated systemic alt-RAS activation using the ratio of alt-RAS axis metabolites to PRA-S (ALT-S). We applied mixed linear models to assess how PRA-S and ACE/ACE2 concentrations affected ALT-S, ACE-S, and angiotensins II and 1-7. Median angiotensin I and II levels were higher with severe versus non-severe COVID-19 (angiotensin I: 86 versus 30 pmol/L, p

BackgroundRenal operational tolerance is a rare and beneficial state of prolonged renal allograft function in the absence of immunosuppression. The underlying mechanisms are unknown. We hypothesized that tolerance might be driven by inherited protein coding genetic variants with large effect, at least in some patients.MethodsWe set up a European survey of over 218,000 renal transplant recipients and collected DNAs from 40 transplant recipients who maintained good allograft function without immunosuppression for at least 1 year. We performed an exome-wide association study comparing the distribution of moderate to high impact variants in 36 tolerant patients, selected for genetic homogeneity using principal component analysis, and 192 controls, using an optimal sequence-kernel association test adjusted for small samples.ResultsWe identified rare variants of HOMER2 (3/36, FDR 0.0387), IQCH (5/36, FDR 0.0362), and LCN2 (3/36, FDR 0.102) in 10 tolerant patients vs. 0 controls. One patient carried a variant in both HOMER2 and LCN2. Furthermore, the three genes showed an identical variant in two patients each. The three genes are expressed at the primary cilium, a key structure in immune responses.ConclusionRare protein coding variants are associated with operational tolerance in a sizable portion of patients. Our findings have important implications for a better understanding of immune tolerance in transplantation and other fields of medicine.ClinicalTrials.gov, identifier: NCT05124444.

Introduction: Globally, there are more women with chronic kidney disease (CKD), yet they comprise only 40% of patients receiving kidney replacement therapy by dialysis. We aimed to describe the perspectives of nephrologists on gender disparities in access to care and outcomes in CKD and dialysis. Methods: We conducted semistructured interviews with 51 nephrologists (28, 55% women) from 22 countries from October 2019 to April 2020. Transcripts were analyzed thematically. Results: We identified 6 themes. Related to women were primary commitment to caregiving (with subthemes of coordinating care, taking charge of health management, deprioritizing own health, centrality of family in decision-making); vigilance and self-reliance (diligence and conscientiousness, stoicism and tolerating symptoms, avoiding burden on family, isolation and coping alone); and stereotyping, stigma, and judgment (body image, dismissed as anxiety, shame and embarrassment, weakness and frailty). Related to men was protecting masculinity (safeguarding the provider role, clinging to control, self-regard, and entitled). Decisional power and ownership included men’s dominance in decision-making and women’s analytical approach in treatment decisions. Inequities compounded by social disadvantage (financial and transport barriers, without social security, limited literacy, entrenched discrimination, vulnerability) were barriers to care for women, particularly in socioeconomically disadvantaged communities. Conclusion: Nephrologists perceived that women with CKD faced many challenges in accessing care related to social norms and roles of caregiving responsibilities, disempowerment, lack of support, stereotyping by clinicians, and entrenched social and economic disadvantage. Addressing power differences, challenging systemic patriarchy, and managing unconscious bias may help to improve equitable care and outcomes for all people with CKD.

Abstract Background Next-generation sequencing (NGS) is nowadays the most used high-throughput technology for DNA sequencing. Among others NGS enables the in-depth analysis of immune repertoires. Research in the field of T cell receptor (TCR) and immunoglobulin (IG) repertoires aids in understanding immunological diseases. A main objective is the analysis of the V(D)J recombination defining the structure and specificity of the immune repertoire. Accurate processing, evaluation and visualization of immune repertoire NGS data is important for better understanding immune responses and immunological behavior. Results ImmunoDataAnalyzer (IMDA) is a pipeline we have developed for automatizing the analysis of immunological NGS data. IMDA unites the functionality from carefully selected immune repertoire analysis software tools and covers the whole spectrum from initial quality control up to the comparison of multiple immune repertoires. It provides methods for automated pre-processing of barcoded and UMI tagged immune repertoire NGS data, facilitates the assembly of clonotypes and calculates key figures for describing the immune repertoire. These include commonly used clonality and diversity measures, as well as indicators for V(D)J gene segment usage and between sample similarity. IMDA reports all relevant information in a compact summary containing visualizations, calculations, and sample details, all of which serve for a more detailed overview. IMDA further generates an output file including key figures for all samples, designed to serve as input for machine learning frameworks to find models for differentiating between specific traits of samples. Conclusions IMDA constructs TCR and IG repertoire data from raw NGS reads and facilitates descriptive data analysis and comparison of immune repertoires. The IMDA workflow focus on quality control and ease of use for non-computer scientists. The provided output directly facilitates the interpretation of input data and includes information about clonality, diversity, clonotype overlap as well as similarity, and V(D)J gene segment usage. IMDA further supports the detection of sample swaps and cross-sample contamination that potentially occurred during sample preparation. In summary, IMDA reduces the effort usually required for immune repertoire data analysis by providing an automated workflow for processing raw NGS data into immune repertoires and subsequent analysis. The implementation is open-source and available on https://bioinformatics.fh-hagenberg.at/immunoanalyzer/ .

Abstract Background Chronic kidney disease (CKD) is a well-established complication in people with diabetes mellitus. Roughly one quarter of prevalent patients with diabetes exhibit a CKD stage of 3 or higher and the individual course of progression is highly variable. Therefore, there is a clear need to identify patients at high risk for fast progression and the implementation of preventative strategies. Existing prediction models of renal function decline, however, aim to assess the risk by artificially grouped patients prior to model building into risk strata defined by the categorization of the least-squares slope through the longitudinally fluctuating eGFR values, resulting in a loss of predictive precision and accuracy. Methods This study protocol describes the development and validation of a prediction model for the longitudinal progression of renal function decline in Caucasian patients with type 2 diabetes mellitus (DM2). For development and internal-external validation, two prospective multicenter observational studies will be used (PROVALID and GCKD). The estimated glomerular filtration rate (eGFR) obtained at baseline and at all planned follow-up visits will be the longitudinal outcome. Demographics, clinical information and laboratory measurements available at a baseline visit will be used as predictors in addition to random country-specific intercepts to account for the clustered data. A multivariable mixed-effects model including the main effects of the clinical variables and their interactions with time will be fitted. In application, this model can be used to obtain personalized predictions of an eGFR trajectory conditional on baseline eGFR values. The final model will then undergo external validation using a third prospective cohort (DIACORE). The final prediction model will be made publicly available through the implementation of an R shiny web application. Discussion Our proposed state-of-the-art methodology will be developed using multiple multicentre study cohorts of people with DM2 in various CKD stages at baseline, who have received modern therapeutic treatment strategies of diabetic kidney disease in contrast to previous models. Hence, we anticipate that the multivariable prediction model will aid as an additional informative tool to determine the patient-specific progression of renal function and provide a useful guide to early on identify individuals with DM2 at high risk for rapid progression.

Repeated vaccination against SARS-CoV-2 increases serological response in kidney transplant recipients (KTR) with high interindividual variability. No decision support tool exists to predict SARS-CoV-2 vaccination response to third or fourth vaccination in KTR. We developed, internally and externally validated five different multivariable prediction models of serological response after the third and fourth vaccine dose against SARS-CoV-2 in previously seronegative, COVID-19-naïve KTR. Using 20 candidate predictor variables, we applied statistical and machine learning approaches including logistic regression (LR), least absolute shrinkage and selection operator (LASSO)-regularized LR, random forest, and gradient boosted regression trees. For development and internal validation, data from 590 vaccinations were used. External validation was performed in four independent, international validation cohorts comprising 191, 184, 254, and 323 vaccinations, respectively. LASSO-regularized LR performed on the whole development dataset yielded a 20- and 10-variable model, respectively. External validation showed AUC-ROC of 0.840, 0.741, 0.816, and 0.783 for the sparser 10-variable model, yielding an overall performance 0.812. A 10-variable LASSO-regularized LR model predicts vaccination response in KTR with good overall accuracy. Implemented as an online tool, it can guide decisions whether to modulate immunosuppressive therapy before additional active vaccination, or to perform passive immunization to improve protection against COVID-19 in previously seronegative, COVID-19-naïve KTR.

Background Sotrovimab, a monoclonal antibody against SARS-CoV-2, is used as a pre-exposition prophylaxis (PrEP) against COVID-19, but monitoring strategies using routine test systems have not been defined. Methods Twenty kidney transplant recipients without antibodies after vaccination received 500 mg Sotrovimab. Antibody levels were quantified over eight weeks using live-virus neutralization (BA1 and BA2), antibody binding assays (TrimericS, Elecsys, QuantiVAC) and surrogate virus neutralization tests (sVNTs; TECOmedical, cPass and NeutraLISA). Results Sotrovimab neutralized both Omicron subvariants (BA1 NT titer 90 (+−50) > BA2 NT titer 33 (+−15) one hour post infusion). Sotrovimab was measurable on all used immunoassays, although a prior 1:100 dilution was necessary for Elecsys due to a presumed prozone effect. The best correlation with live-virus neutralization titers was found for QuantiVAC and TrimericS, with a respective R2 of 0.65/0.59 and 0.76/0.57 against BA1/BA2. Elecsys showed an R2 of 0.56/0.54 for BA1/BA2, respectively. sVNT values increased after infusion but had only a poor correlation with live-virus neutralization titers (TECOmedical and cPass) or did not reach positivity thresholds (NeutraLISA). Conclusion Antibody measurements by the used immunoassays showed differences in antibody levels and only a limited correlation with neutralization capacity. We do not recommend sVNTs for monitoring SARS-CoV-2 neutralization by Sotrovimab.

Response to SARS-CoV-2-vaccines in kidney-transplant recipients (KTR) is severely reduced. Heterologous3rd vaccination combining mRNA and vector vaccines did not increase seroconversion at 4 weeks after vaccination, but evolution of antibody levels beyond the first month remains unknown. We have recently completed a randomized-controlled trial on heterologous (Ad26COVS1) vs. homologous (BNT162b2 or mRNA-1273) 3rd vaccination in 201 KTR not developing SARS-CoV-2-spike-protein antibodies following two doses of mRNA vaccine (EurdraCT: 2021-002927-39). Here, we report seroconversion at the second follow-up at 3 months after the 3rd vaccination (prespecified secondary endpoint). In addition, higher cut-off levels associated with neutralizing capacity and protective immunity were applied (i.e., > 15, > 100, > 141, and > 264 BAU/ml). A total of 169 patients were available for the 3-month follow-up. Overall, seroconversion at 3 months was similar between both groups (45 vs. 50% for mRNA and the vector group, respectively; p = 0.539). However, when applying higher cut-off levels, a significantly larger number of individuals in the vector group reached antibody levels > 141 and > 264 BAU/ml at the 3-month follow-up (141 BAU/ml: 4 vs. 15%, p = 0.009 and 264 BAU/ml: 1 vs. 10%, p = 0.018 for mRNA vs. the vector vaccine group, respectively). In line, antibody levels in seroconverted patients further increased from month 1 to month 3 in the vector group while remaining unchanged in the mRNA group (median increase: mRNA = 1.35 U/ml and vector = 27.6 U/ml, p = 0.004). Despite a similar overall seroconversion rate at 3 months following 3rd vaccination in KTR, a heterologous 3rd booster vaccination with Ad26COVS1 resulted in significantly higher antibody levels in responders.

Secondary hyperparathyroidism in chronic kidney disease poses a major risk factor for vascular calcification and high bone turnover, leading to mineralization defects. The aim was to analyze the effect of active vitamin D and calcimimetic treatment on fibroblast growth factor 23 (FGF23), serum calcification propensity (T50), a surrogate marker of calcification stress and bone specific alkaline phosphatase (BAP) in hemodialysis. This is a subanalysis of a randomized trial comparing etelcalcetide vs. alfacalcidol in 62 hemodialysis patients for 1 year. We compared the change of BAP and serum calcification propensity between the two medications and assessed the influence of FGF23 change over time. We found no significant differences in the change of BAP or serum calcification propensity (T50) levels from baseline to study end between treatment arms (difference in change of marker between treatment with etelcalcetide vs. alfacalcidol: BAP : 2.0 ng/ml [95% CI-1.5,5.4], p = 0.3; T50: –15 min [95% CI –49,19], p = 0.4). Using FGF23 change over time, we could show that BAP levels at study end were associated with FGF23 change (–0.14 [95% CI –0.21, –0.08], p < 0.001). We did not observe the same association between FGF23 change and T50 (effect of FGF23 change on T50: 3.7 [95% CI –5.1, 12], p = 0.4; R2 = 0.07 vs. R2 = 0.06). No significant difference was found in serum calcification propensity (T50) values between treatment arms. FGF23 was not associated with serum calcification propensity (T50), but was negatively correlated with BAP underlying its role in the bone metabolism.Clinical Trial Registration[www.ClinicalTrials.gov], identifier [NCT03182699].

IntroductionKidney transplant recipients (KTR) are at high risk of developing severe COVID-19. However, vaccine response in this population is severely impaired with humoral response rates of 36–54 and 55–69% after two or three doses of SARS-COV-2 vaccines, respectively. Triple immunosuppression and specifically the use of anti-proliferative agents such as mycophenolic acid (MPA) or azathioprine (AZA) have been identified as risk factors for vaccine hypo-responsiveness.MethodsWe hypothesized that in vaccine non-responders to at least three previous vaccine doses, pausing of MPA or AZA for 1 week before and 1 week after an additional vaccination would improve humoral response rates. We conducted an open-label, non-randomized controlled pilot study including 40 KTR with no detectable humoral response after three or four previous vaccine doses. Primary endpoint was seroconversion following SARS-CoV-2 vaccination. MPA and AZA was paused in 18 patients 1 week before until 1 week after an additional vaccine dose while immunosuppression was continued in 22 patients.ResultsThere was no difference in the humoral response rate between the MPA/AZA pause group and the control group (29 vs. 32%, p > 0.99). Absolute antibody levels were also not statistically significantly different between the two groups (p = 0.716).Renal function in the MPA/AZA pause group remained stable and there was no detection of new onset donor-specific antibodies or an increase of donor-derived cell-free DNA serving as a marker of allograft damage throughout the study period.ConclusionPausing of MPA/AZA for 2 weeks peri-vaccination did not increase the rate of seroconversion in kidney transplant. However, one in three KTR without humoral immune response to at least three previous vaccinations developed antibodies after an additional vaccine dose supporting continued vaccination in non-responders.

Fibroblast growth factor 23 (FGF23) is elevated in patients with chronic kidney disease and contributes to left ventricular hypertrophy (LVH). The aim of the analysis was to determine whether this effect is mediated by the renin-angiotensin-aldosterone system (RAAS) in hemodialysis. Serum samples from 62 randomized hemodialysis patients with LVH were analyzed for plasma renin activity (PRA-S), angiotensin II (AngII), and metabolites, angiotensin-converting enzyme-2 (ACE2) and aldosterone using a high throughput mass spectrometry assay. Compared to healthy individuals, levels of the RAAS parameters PRA-S, AngII and aldosterone were generally lower [median (IQR) PRA-S 130 (46–269) vs. 196 (98, 238) pmol/L; AngII 70 (28–157) vs. 137 (76, 201) pmol/L; Aldosterone 130 (54, 278) vs. 196 (98, 238) pmol/L]. We did not find an indication that the effect of FGF23 on LVH was mediated by RAAS parameters, with all estimated indirect effects virtually zero. Furthermore, FGF23 was not associated with RAAS parameter levels throughout the study. While there was a clear association between FGF23 levels and left ventricular mass index (LVMI) at the end of the study and in the FGF23 fold change and LVMI change analysis, no association between RAAS and LVMI was observed. Serum concentrations of PRA-S, AngII, and aldosterone were below the ranges measured in healthy controls suggesting that RAAS is not systemically activated in hemodialysis patients. The effect of FGF23 on LVMI was not mediated by systemic RAAS activity. These findings challenge the current paradigm of LVH progression and treatment with RAAS blockers in dialysis.Clinical Trial Registration[https://clinicaltrials.gov/ct2/show/NCT03182699], identifier [NCT03182699].

BackgroundPre-sensitized kidney transplant recipients have a higher risk for rejection following kidney transplantation and therefore receive lymphodepletional induction therapy with anti-human T-lymphocyte globulin (ATLG) whereas non-sensitized patients are induced in many centers with basiliximab. The time course of lymphocyte reconstitution with regard to the overall and donor-reactive T-cell receptor (TCR) specificity remains elusive.Methods/DesignFive kidney transplant recipients receiving a 1.5-mg/kg ATLG induction therapy over 7 days and five patients with 2 × 20 mg basiliximab induction therapy were longitudinally monitored. Peripheral mononuclear cells were sampled pre-transplant and within 1, 3, and 12 months after transplantation, and their overall and donor-reactive TCRs were determined by next-generation sequencing of the TCR beta CDR3 region. Overall TCR repertoire diversity, turnover, and donor specificity were assessed at all timepoints.ResultsWe observed an increase in the donor-reactive TCR repertoire after transplantation in patients, independent of lymphocyte counts or induction therapy. Donor-reactive CD4 T-cell frequency in the ATLG group increased from 1.14% + -0.63 to 2.03% + -1.09 and from 0.93% + -0.63 to 1.82% + -1.17 in the basiliximab group in the first month. Diversity measurements of the entire T-cell repertoire and repertoire turnover showed no statistical difference between the two induction therapies. The difference in mean clonality between groups was 0.03 and 0.07 pre-transplant in the CD4 and CD8 fractions, respectively, and was not different over time (CD4: F(1.45, 11.6) = 0.64 p = 0.496; CD8: F(3, 24) = 0.60 p = 0.620). The mean difference in R20, a metric for immune dominance, between groups was -0.006 in CD4 and 0.001 in CD8 T-cells and not statistically different between the groups and subsequent timepoints (CD4: F(3, 24) = 0.85 p = 0.479; CD8: F(1.19, 9.52) = 0.79 p = 0.418).ConclusionReduced-dose ATLG induction therapy led to an initial lymphodepletion followed by an increase in the percentage of donor-reactive T-cells after transplantation similar to basiliximab induction therapy. Furthermore, reduced-dose ATLG did not change the overall TCR repertoire in terms of a narrowed or skewed TCR repertoire after immune reconstitution, comparable to non-depletional induction therapy.

Summary: Background: Kidney allograft failure is a common cause of end-stage renal disease. We aimed to develop a dynamic artificial intelligence approach to enhance risk stratification for kidney transplant recipients by generating continuously refined predictions of survival using updates of clinical data. Methods: In this observational study, we used data from adult recipients of kidney transplants from 18 academic transplant centres in Europe, the USA, and South America, and a cohort of patients from six randomised controlled trials. The development cohort comprised patients from four centres in France, with all other patients included in external validation cohorts. To build deeply phenotyped cohorts of transplant recipients, the following data were collected in the development cohort: clinical, histological, immunological variables, and repeated measurements of estimated glomerular filtration rate (eGFR) and proteinuria (measured using the proteinuria to creatininuria ratio). To develop a dynamic prediction system based on these clinical assessments and repeated measurements, we used a Bayesian joint models—an artificial intelligence approach. The prediction performances of the model were assessed via discrimination, through calculation of the area under the receiver operator curve (AUC), and calibration. This study is registered with ClinicalTrials.gov, NCT04258891. Findings: 13 608 patients were included (3774 in the development cohort and 9834 in the external validation cohorts) and contributed 89 328 patient-years of data, and 416 510 eGFR and proteinuria measurements. Bayesian joint models showed that recipient immunological profile, allograft interstitial fibrosis and tubular atrophy, allograft inflammation, and repeated measurements of eGFR and proteinuria were independent risk factors for allograft survival. The final model showed accurate calibration and very high discrimination in the development cohort (overall dynamic AUC 0·857 [95% CI 0·847–0·866]) with a persistent improvement in AUCs for each new repeated measurement (from 0·780 [0·768–0·794] to 0·926 [0·917–0·932]; p

BackgroundAntigen recognition of allo-peptides and HLA molecules leads to the activation of donor-reactive T-cells following transplantation, potentially causing T-cell-mediated rejection (TCMR). Sequencing of the T-cell receptor (TCR) repertoire can be used to track the donor-reactive repertoire in blood and tissue of patients after kidney transplantation.Methods/DesignIn this prospective cohort study, 117 non-sensitized kidney transplant recipients with anti-CD25 induction were included. Peripheral mononuclear cells (PBMCs) were sampled pre-transplant and at the time of protocol or indication biopsies together with graft tissue. Next-generation sequencing (NGS) of the CDR3 region of the TCRbeta chain was performed after donor stimulation in mixed lymphocyte reactions to define the donor-reactive TCR repertoire. Blood and tissue of six patients experiencing a TCMR and six patients without rejection on protocol biopsies were interrogated for these TCRs. To elucidate common features of T-cell clonotypes, a network analysis of the TCR repertoires was performed.ResultsAfter transplantation, the frequency of circulating donor-reactive CD4 T-cells increased significantly from 0.86 ± 0.40% to 2.06 ± 0.40% of all CD4 cells (p < 0.001, mean dif.: -1.197, CI: -1.802, -0.593). The number of circulating donor-reactive CD4 clonotypes increased from 0.72 ± 0.33% to 1.89 ± 0.33% (p < 0.001, mean dif.: -1.168, CI: -1.724, -0.612). No difference in the percentage of donor-reactive T-cells in the circulation at transplant biopsy was found between subjects experiencing a TCMR and the control group [p = 0.64 (CD4+), p = 0.52 (CD8+)]. Graft-infiltrating T-cells showed an up to six-fold increase of donor-reactive T-cell clonotypes compared to the blood at the same time (3.7 vs. 0.6% and 2.4 vs. 1.5%), but the infiltrating TCR repertoire was not reflected by the composition of the circulating TCR repertoire despite some overlap. Network analysis showed a distinct segregation of the donor-reactive repertoire with higher modularity than the overall TCR repertoire in the blood. These findings indicate an unchoreographed process of diverse T-cell clones directed against numerous non-self antigens found in the allograft.ConclusionDonor-reactive T-cells are enriched in the kidney allograft during a TCMR episode, and dominant tissue clones are also found in the blood.Trial RegistrationClinicaltrials.gov: NCT: 03422224 (https://clinicaltrials.gov/ct2/show/NCT03422224).

Donor organ shortage still remains a serious obstacle for the access of wait-list patients to kidney transplantation, the best treatment for End-Stage Kidney Disease (ESKD). To expand the number of transplants, the use of lower quality organs from older ECD or DCD donors has become an established routine but at the price of increased incidence of Primary Non-Function, Delay Graft Function and lower-long term graft survival. In the last years, several improvements have been made in the field of renal transplantation from surgical procedure to preservation strategies. To improve renal outcomes, research has focused on development of innovative and dynamic preservation techniques, in order to assess graft function and promote regeneration by pharmacological intervention before transplantation. This review provides an overview of the current knowledge of these new preservation strategies by machine perfusions and pharmacological interventions at different timing possibilities: in the organ donor, ex-vivo during perfusion machine reconditioning or after implementation in the recipient. We will report therapies as anti-oxidant and anti-inflammatory agents, senolytics agents, complement inhibitors, HDL, siRNA and H2S supplementation. Renal delivery of pharmacologic agents during preservation state provides a window of opportunity to treat the organ in an isolated manner and a crucial route of administration. Even if few studies have been reported of transplantation after ex-vivo drugs administration, targeting the biological pathway associated to kidney failure (i.e. oxidative stress, complement system, fibrosis) might be a promising therapeutic strategy to improve the quality of various donor organs and expand organ availability.

Abstract Background Fibroblast growth factor 23 (FGF23) is associated with left ventricular hypertrophy (LVH) in patients with chronic kidney disease, and calcimimetic therapy reduces plasma concentrations of FGF23. It remains unknown whether treatment with the calcimimetic etelcalcetide (ETL) reduces LVH in patients on hemodialysis. Methods/design This single-blinded randomized trial of 12 months duration will test the effects of ETL compared with alfacalcidol on LVH and cardiac fibrosis in maintenance hemodialysis patients with secondary hyperparathyroidism. Both treatment regimens will be titrated to equally suppress secondary hyperparathyroidism while alfacalcidol treatment causes an increase and ETL a decrease in FGF23, respectively. Patients treated thrice weekly with hemodialysis for ≥ 3 months and ≤ 3 years with parathyroid hormone levels ≥ 300 pg/ml and LVH will be enrolled in the study. The primary study endpoint is change from baseline to 12 months in left ventricular mass index (LVMI; g/m2) measured by cardiac magnetic resonance imaging. Sample size calculations showed that 62 randomized patients will be necessary to detect a difference in LVMI of at least 20 g/m2 between the two groups at 12 months. Due to the strong association of volume overload and LVH, randomization will be stratified by residual kidney function, and regular body composition monitoring will be performed to control the volume status of patients. Study medication will be administered intravenously by the dialysis nurses after every hemodialysis session, thus omitting adherence issues. Secondary study endpoints are cardiac parameters measured by echocardiography, biomarker concentrations of bone metabolism (FGF23, vitamin D, parathyroid hormone, calcium, phosphate, s-Klotho), cardiac markers (pro-brain natriuretic peptide, pre- and postdialysis troponin T) and metabolites of the renin–angiotensin–aldosterone cascade (angiotensin I (Ang I), Ang II, Ang-(1–7), Ang-(1–5), Ang-(1–9), and aldosterone). Discussion The causal inference and pathophysiology of LVH regression by FGF23 reduction using calcimimetic treatment has not yet been shown. This intervention study has the potential to discover a new strategy for the treatment of cardiac hypertrophy and fibrosis in patients on maintenance hemodialysis. It might be speculated that successful treatment of cardiac morphology will also reduce the risk of cardiac death in this population. Trial registration European Clinical Trials Database, EudraCT number 2017-000222-35; ClinicalTrials.gov, NCT03182699. Registered on

Abstract Background Kidney transplantation is the optimal treatment in end stage renal disease but the allograft survival is still hampered by immune reactions against the allograft. This process is driven by the recognition of allogenic antigens presented to T-cells and their unique T-cell receptor (TCR) via the major histocompatibility complex (MHC), which triggers a complex immune response potentially leading to graft injury. Although the immune system and kidney transplantation have been studied extensively, the subtlety of alloreactive immune responses has impeded sensitive detection at an early stage. Next generation sequencing of the TCR enables us to monitor alloreactive T-cell populations and might thus allow the detection of early rejection events. Methods/design This is a prospective cohort study designed to sequentially evaluate the alloreactive T cell repertoire after kidney transplantation. The TCR repertoire of patients who developed biopsy confirmed acute T cell mediated rejection (TCMR) will be compared to patients without rejection. To track the alloreactive subsets we will perform a mixed lymphocyte reaction between kidney donor and recipient before transplantation and define the alloreactive TCR repertoire by next generation sequencing of the complementary determining region 3 (CDR3) of the T cell receptor beta chain. After initial clonotype assembly from sequencing reads, TCR repertoire diversity and clonal expansion of T cells of kidney transplant recipients in periphery and kidney biopsy will be analyzed for changes after transplantation, during, prior or after a rejection. The goal of this study is to describe changes of overall T cell repertoire diversity, clonality in kidney transplant recipients, define and track alloreactive T cells in the posttransplant course and decipher patterns of expanded alloreactive T cells in acute cellular rejection to find an alternative monitoring to invasive and delayed diagnostic procedures. Discussion Changes of the T cell repertoire and tracking of alloreactive T cell clones after combined bone marrow and kidney transplant has proven to be of potential use to monitor the donor directed alloresponse. The dynamics of the donor specific T cells in regular kidney transplant recipients in rejection still rests elusive and can give further insights in human alloresponse. Trial registration Clinicaltrials.gov: NCT03422224, registered February 5th 2018.

BackgroundChronic kidney disease (CKD) is accompanied by increases in circulating fibroblast growth factor 23 (FGF23) and aldosterone levels. Here, we tested the hypothesis that aldosterone may be one of the driving forces behind increased FGF23 secretion in CKD.MethodsUsing data from a prospective study in humans, a retrospective study in dogs and cats, and an experimental study in 5/6-nephrectomized mice, we analyzed the relationship between circulating FGF23 and serum aldosterone levels in CKD across four species. To assess the effects of acute inhibition of aldosterone signaling on circulating FGF23, we acutely treated mice with established CKD with the mineralocorticoid receptor blocker canrenone (50 mg/kg iv/sc), and measured intact FGF23 before and 24 h as well as 72 h after start of administration of the drug.ResultsWe found a tight positive association between circulating intact FGF23 and serum aldosterone in human, canine, and feline CKD patients, as well as in experimental murine CKD (humans: rS = 0.57, p = 0.0368; dogs: rS = 0.66, p = 0.0019; cats: rS = 0.75, p = 0.0003; mice: rS = 0.49, p = 0.0004). Injection of canrenone in mice with established CKD did not lead to changes in FGF23 levels within 24 h, but reduced FGF23 in all mice at 72 h.ConclusionAldosterone may drive enhanced FGF23 secretion in CKD, possibly explaining the tight positive association between circulating intact FGF23 and aldosterone in human, canine, and feline CKD patients as well as in experimental CKD models.

Background: The induction of donor-specific immunological tolerance could improve outcome after kidney transplantation. However, no tolerance protocol is available for routine clinical use. Chimerism-based regimens hold promise, but their widespread application is impeded in part by unresolved safety issues. This study tests the hypothesis that therapy with polyclonal recipient regulatory T cells (Tregs) and anti-IL6R (tocilizumab) leads to transient chimerism and achieves pro-tolerogenic immunomodulation in kidney transplant recipients also receiving donor bone marrow (BM) without myelosuppressive conditioning of the recipient.Methods/design: A prospective, open-label, controlled, single-center, phase I/IIa academic study is performed in HLA-mismatched living donor kidney transplant recipients.Study group: Recipients of the study group receive in vitro expanded recipient Tregs and a donor bone marrow cell infusion within 3 days after transplantation and tocilizumab for the first 3 weeks post-transplant. In addition they are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients.Control group: Recipients of the control group are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Co-primary endpoints of safety (impaired graft function [eGFR

Pure red cell aplasia (PRCA) after ABO-incompatible allogeneic hematopoietic stem cell transplantation (HSCT) is caused by persisting host-derived isohemagglutinins directed against donor red blood cell (RBC) antigens. ABO antigen-specific immunoadsorption (ABO-IA) with Glycosorb®, commonly used for desensitization therapy in ABO-incompatible living donor renal transplantation, specifically eliminates circulating isohemagglutinins and might represent a novel treatment option for post-HSCT PRCA. In this prospective observational (n = 3) and retrospective (n = 3) analysis of six adult HSCT-recipients with PRCA, ABO-IA was initiated at 159 (range: 104–186) days following HSCT. The median treatment frequency was 4.5 (range: 3.9–5.5) sessions/week. ABO-IA-treatment led to a continuous decrease in isohemagglutinin titers. Reticulocytes increased to ≥30 G/L after 17.5 (range: 4–37) immunoadsorption sessions over 28.5 (range: 6–49) days and continued to rise after that. By the end of the 3-month follow-up period after discontinuation of ABO-IA, all patients showed a sustained remission of PRCA and were independent of erythropoietin-stimulating agents and transfusions. No case of infection or graft-versus-host disease was observed. After a median follow-up of 22.03 (range: 6.08–149.00) months after ABO-IA-treatment, all patients were alive and showed a stable RBC engraftment of the donor blood group. Our data provide the first evidence for ABO-IA as an effective treatment for post-HSCT PRCA.

Pathological erythropoiesis with consequent anemia is a leading cause of symptomatic morbidity in internal medicine. The etiologies of anemia are complex and include reactive as well as neoplastic conditions. Clonal expansion of erythroid cells in the bone marrow may result in peripheral erythrocytosis and polycythemia but can also result in anemia when clonal cells are dysplastic and have a maturation arrest that leads to apoptosis and hinders migration, a constellation typically seen in the myelodysplastic syndromes. Rarely, clonal expansion of immature erythroid blasts results in a clinical picture resembling erythroid leukemia. Although several mechanisms underlying normal and abnormal erythropoiesis and the pathogenesis of related disorders have been deciphered in recent years, little is known about specific markers and targets through which prognosis and therapy could be improved in anemic or polycythemic patients. In order to discuss new markers, targets and novel therapeutic approaches in erythroid disorders and the related pathologies, a workshop was organized in Vienna in April 2017. The outcomes of this workshop are summarized in this review, which includes a discussion of new diagnostic and prognostic markers, the updated WHO classification, and an overview of new drugs used to stimulate or to interfere with erythropoiesis in various neoplastic and reactive conditions. The use and usefulness of established and novel erythropoiesis-stimulating agents for various indications, including myelodysplastic syndromes and other neoplasms, are also discussed.

Elderly patients represent a growing population among people suffering from ESRD. So far only limited data on actual survival benefits of elderly adults initiating dialysis have been published. Besides the high burden of preexisting comorbidities, dialysis treatment itself may be associated with a further deterioration in functional status in this population. We retrospectively analyzed the Austrian Dialysis and Transplant Registry and identified 8,622 patients who started maintenance hemodialysis after the age of 65 years between 2002 and 2009. We compared this data set to a cohort of 174 patients aged over 65 years with CKD stage 5 who progressed to an eGFR < 10ml/min/ and were managed conservatively in the same era. All patients who died of malignant disease were excluded from this analysis. The risk of mortality was analyzed using multivariable Cox proportional hazards models. Furthermore, a parametric model of time to event analysis was used for visualization of changing risk over time and precise calculation of time to equal risk assuming a Weibull distribution. Hemodialysis treatment was associated with a decreased risk for death with a HR of 0.23 (95% CI 0.18 to 0.29; p

To evaluate tacrolimus as therapeutic option for diabetic nephropathy (DN) based on molecular profile and network-based molecular model comparisons.We generated molecular models representing pathophysiological mechanisms of DN and tacrolimus mechanism of action (MoA) based on literature derived data and transcriptomics datasets. Shared enriched molecular pathways were identified based on both model datasets. A newly generated transcriptomics dataset studying the effect of tacrolimus on mesangial cells in vitro was added to identify mechanisms in DN pathophysiology. We searched for features in interference between the DN molecular model and the tacrolimus MoA molecular model already holding annotation evidence as diagnostic or prognostic biomarker in the context of DN.Thirty nine molecular features were shared between the DN molecular model, holding 252 molecular features and the tacrolimus MoA molecular model, holding 209 molecular features, with six additional molecular features affected by tacrolimus in mesangial cells. Significantly affected molecular pathways by both molecular model sets included cytokine-cytokine receptor interactions, adherens junctions, TGF-beta signaling, MAPK signaling, and calcium signaling. Molecular features involved in inflammation and immune response contributing to DN progression were significantly downregulated by tacrolimus (e.g. the tumor necrosis factor alpha (TNF), interleukin 4, or interleukin 10). On the other hand, pro-fibrotic stimuli being detrimental to renal function were induced by tacrolimus like the transforming growth factor beta 1 (TGFB1), endothelin 1 (EDN1), or type IV collagen alpha 1 (COL4A1).Patients with DN and elevated TNF levels might benefit from tacrolimus treatment regarding maintaining GFR and reducing inflammation. TGFB1 and EDN1 are proposed as monitoring markers to assess degree of renal damage. Next to this stratification approach, the use of drug combinations consisting of tacrolimus in addition to ACE inhibitors, angiotensin receptor blockers, TGFB1- or EDN1-receptor antagonists might warrant further studies.