Background. Evidence on the optimal maintenance of immunosuppressive regimen in kidney transplantation recipients is limited. Methods. The Amsterdam, LEiden, GROningen trial is a randomized,... Show moreBackground. Evidence on the optimal maintenance of immunosuppressive regimen in kidney transplantation recipients is limited. Methods. The Amsterdam, LEiden, GROningen trial is a randomized, multicenter, investigator-driven, noninferiority, open-label trial in de novo kidney transplant recipients, in which 2 immunosuppression minimization strategies were compared with standard immunosuppression with basiliximab, corticosteroids, tacrolimus, and mycophenolic acid. In the minimization groups, either steroids were withdrawn from day 3, or tacrolimus exposure was reduced from 6 mo after transplantation. The primary endpoint was kidney transplant function at 24 mo. Results. A total of 295 participants were included in the intention-to-treat analysis. Noninferiority was shown for the primary endpoint; estimated glomerular filtration rate at 24 mo was 45.3 mL/min/1.73 m(2) in the early steroid withdrawal group, 49.0 mL/ min/1.73 m2 in the standard immunosuppression group, and 44.7 mL/min/1.73 m2 in the tacrolimus minimization group. Participants in the early steroid withdrawal group were significantly more often treated for rejection (P = 0.04). However, in this group, the number of participants with diabetes mellitus during follow-up and total cholesterol at 24 mo were significantly lower. Conclusions. Tacrolimus minimization can be considered in kidney transplant recipients who do not have an increased immunological risk. Before withdrawing steroids the risk of rejection should be weighed against the potential metabolic advantages. Show less
The ESOT TLJ 3.0. consensus conference brought together leading experts in transplantation to develop evidence-based guidance on the standardization and clinical utility of pre-implantation kidney... Show moreThe ESOT TLJ 3.0. consensus conference brought together leading experts in transplantation to develop evidence-based guidance on the standardization and clinical utility of pre-implantation kidney biopsy in the assessment of grafts from Expanded Criteria Donors (ECD). Seven themes were selected and underwent in-depth analysis after formulation of PICO (patient/population, intervention, comparison, outcomes) questions. After literature search, the statements for each key question were produced, rated according the GRADE approach [Quality of evidence: High (A), Moderate (B), Low (C); Strength of Recommendation: Strong (1), Weak (2)]. The statements were subsequently presented in-person at the Prague kick-off meeting, discussed and voted. After two rounds of discussion and voting, all 7 statements reached an overall agreement of 100% on the following issues: needle core/wedge/punch technique representatively [B,1], frozen/paraffin embedded section reliability [B,2], experienced/non-experienced on-call renal pathologist reproducibility/accuracy of the histological report [A,1], glomerulosclerosis/other parameters reproducibility [C,2], digital pathology/light microscopy in the measurement of histological variables [A,1], special stainings/Haematoxylin and Eosin alone comparison [A,1], glomerulosclerosis reliability versus other histological parameters to predict the graft survival, graft function, primary non-function [B,1]. This methodology has allowed to reach a full consensus among European experts on important technical topics regarding pre-implantation biopsy in the ECD graft assessment. Show less
Background The aetiology of idiopathic nephrotic syndrome (INS) remains partially unknown. Viral infections have been associated with INS onset. Since we observed fewer first onset INS cases during... Show moreBackground The aetiology of idiopathic nephrotic syndrome (INS) remains partially unknown. Viral infections have been associated with INS onset. Since we observed fewer first onset INS cases during the Covid-19 pandemic, we hypothesised that lower INS incidence was the result of lockdown measures. Therefore, the aim of this study was to evaluate the incidence of childhood INS before and during the COVID-19 pandemic in two independent European INS cohorts.Methods Children with new INS in the Netherlands (2018-2021) and Paris area (2018-2021) were included. We estimated incidences using census data for each region. Incidences were compared using two proportion Z-tests.Results A total of 128 and 324 cases of first onset INS were reported in the Netherlands and Paris area, respectively, corresponding to an annual incidence of 1.21 and 2.58 per 100,000 children/year. Boys and young children (< 7 years) were more frequently affected. Incidence before and during the pandemic did not differ. When schools were closed, incidence was lower in both regions: 0.53 vs. 1.31 (p = 0.017) in the Netherlands and 0.94 vs. 2.63 (p = 0.049) in the Paris area. During peaks of hospital admissions for Covid-19, no cases were reported in the Netherlands or Paris area.Conclusions Incidence of INS before and during the Covid-19 pandemic was not different, but when schools were closed during lockdown, incidence was significantly lower. Interestingly, incidences of other respiratory viral infections were also reduced as was air pollution. Together, these results argue for a link between INS onset and viral infections and/or environmental factors. Show less
Background: Proteasome-associated autoinflammatory syndromes (PRAASs) form a family of recently described rare autosomal recessive disorders of disturbed proteasome assembly and proteolytic... Show moreBackground: Proteasome-associated autoinflammatory syndromes (PRAASs) form a family of recently described rare autosomal recessive disorders of disturbed proteasome assembly and proteolytic activity caused by mutations in genes coding for proteasome subunits. The treatment options for these proteasome disorders consist of lifelong immunosuppressive drugs or Janus kinase inhibitors, which may have partial efficacy and noticeable side effects. Because proteasomes are ubiquitously expressed, it is unknown whether hematopoietic stem cell transplantation (HSCT) may be a sufficient treatment option. Objective: Our aim was to report the case of a young boy with a treatment-resistant cutaneous vasculitis that was initially suspected to be associated with a gene variant in SH2D1A. Methods: Whole-exome sequencing was performed to identify the genetic defect. Molecular and functional analyses were performed to assess the impact of variants on proteasomal function. The immune characterization led to the decision to perform HSCT on our patient and conduct follow-up over the 7-year period after the transplant. Because loss of myeloid chimerism after the first HSCT was associated with relapse of autoinflammation, a second HSCT was performed. Results: After the successful second HSCT, the patient developed mild symptoms of lipodystrophy, which raised the suspicion of a PRAAS. Genetic analysis revealed 2 novel heterozygous variants in PSMB4 (encoding proteasomal subunit beta 7). Retrospective analysis of patient cells stored before the first HSCT and patient cells obtained after the second HSCT demonstrated that HSCT successfully rescued proteasome function, restored protein homeostasis, and resolved the interferon-stimulated gene signature. Furthermore, successful HSCT alleviated the autoinflammatory manifestations in our patient. Conclusion: Patients with treatment-resistant PRAAS can be cured by HSCT. Show less
Background Histopathological assessment of transplant biopsies is currently the standard method to diagnose allograft rejection and can help guide patient management, but it is one of the most... Show moreBackground Histopathological assessment of transplant biopsies is currently the standard method to diagnose allograft rejection and can help guide patient management, but it is one of the most challenging areas of pathology, requiring considerable expertise, time, and effort. We aimed to analyse the utility of deep learning to preclassify histology of kidney allograft biopsies into three main broad categories (ie, normal, rejection, and other diseases) as a potential biopsy triage system focusing on transplant rejection.Methods We performed a retrospective, multicentre, proof-of-concept study using 5844 digital whole slide images of kidney allograft biopsies from 1948 patients. Kidney allograft biopsy samples were identified by a database search in the Departments of Pathology of the Amsterdam UMC, Amsterdam, Netherlands (1130 patients) and the University Medical Center Utrecht, Utrecht, Netherlands (717 patients). 101 consecutive kidney transplant biopsies were identified in the archive of the Institute of Pathology, RWTH Aachen University Hospital, Aachen, Germany. Convolutional neural networks (CNNs) were trained to classify allograft biopsies as normal, rejection, or other diseases. Three times cross-validation (1847 patients) and deployment on an external real-world cohort (101 patients) were used for validation. Area under the receiver operating characteristic curve (AUROC) was used as the main performance metric (the primary endpoint to assess CNN performance).Findings Serial CNNs, first classifying kidney allograft biopsies as normal (AUROC 0.87 [ten times bootstrapped CI 0.85-0.88]) and disease (0.87 [0.86-0.88]), followed by a second CNN classifying biopsies classified as disease into rejection (0.75 [0.73-0.76]) and other diseases (0.75 [0.72-0.77]), showed similar AUROC in cross-validation and deployment on independent real-world data (first CNN normal AUROC 0.83 [0.80-0.85], disease 0.83 [0.73-0.91]; second CNN rejection 0.61 [0.51-0.70], other diseases 0.61 [0.50-4.74]). A single CNN classifying biopsies as normal, rejection, or other diseases showed similar performance in cross-validation (normal AUROC 0.80 [0.73-0.84], rejection 0.76 [0.66-0.80], other diseases 0.50 [0.36-0.57]) and generalised well for normal and rejection classes in the real-world data. Visualisation techniques highlighted rejection-relevant areas of biopsies in the tubulointerstitium.Interpretation This study showed that deep learning-based classification of transplant biopsies could support pathological diagnostics of kidney allograft rejection. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd. Show less
Uil, M.; Hau, C.M.; Ahdi, M.; Mills, J.D.; Kers, J.; Saleem, M.A.; ... ; Roelofs, J.J.T.H. 2021
Background. Diabetic nephropathy (DN) is a major complication of diabetes and the main cause of end-stage renal disease. Extracellular vesicles (EVs) are small cell-derived vesicles that can alter... Show moreBackground. Diabetic nephropathy (DN) is a major complication of diabetes and the main cause of end-stage renal disease. Extracellular vesicles (EVs) are small cell-derived vesicles that can alter disease progression by microRNA (miRNA) transfer.Methods. In this study, we aimed to characterize the cellular origin and miRNA content of EVs in plasma samples of type 2 diabetes patients at various stages of DN. Type 2 diabetes patients were classified in three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. The concentration and cellular origin of plasma EVs were measured by flow cytometry. A total of 752 EV miRNAs were profiled in 18 subjects and differentially expressed miRNAs were validated.Results. Diabetic patients with microalbuminuria and/or macroalbuminuria showed elevated concentrations of total EVs and EVs from endothelial cells, platelets, leucocytes and erythrocytes compared with diabetic controls. miR-99a-5p was upregulated in macroalbuminuric patients compared with normoalbuminuric and microalbuminuric patients. Transfection of miR-99a-5p in cultured human podocytes downregulated mammalian target of rapamycin (mTOR) protein expression and downregulated the podocyte injury marker vimentin.Conclusions. Type 2 diabetes patients with microalbuminuria and macroalbuminuria display differential EV profiles. miR-99a-5p expression is elevated in EVs from macroalbuminuria and mTOR is its validated mRNA target. Show less
Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a... Show moreBackground Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a direct virus-induced effect, but also the immune response. The aims of this study were to assess the duration of viral presence, identify the extent of inflammatory response, and investigate the underlying cause of coagulopathy.Methods This prospective autopsy cohort study was done at Amsterdam University Medical Centers (UMC), the Netherlands. With informed consent from relatives, full body autopsy was done on 21 patients with COVID-19 for whom autopsy was requested between March 9 and May 18, 2020. In addition to histopathological evaluation of organ damage, the presence of SARS-CoV-2 nucleocapsid protein and the composition of the immune infiltrate and thrombi were assessed, and all were linked to disease course.Findings Our cohort (n=21) included 16 (76%) men, and median age was 68 years (range 41-78). Median disease course (time from onset of symptoms to death) was 22 days (range 5-44 days). In 11 patients tested for SARS-CoV-2 tropism, SARS-CoV-2 infected cells were present in multiple organs, most abundantly in the lungs, but presence in the lungs became sporadic with increased disease course. Other SARS-CoV-2-positive organs included the upper respiratory tract, heart, kidneys, and gastrointestinal tract. In histological analyses of organs (sampled from nine to 21 patients per organ), an extensive inflammatory response was present in the lungs, heart, liver, kidneys, and brain. In the brain, extensive inflammation was seen in the olfactory bulbs and medulla oblongata. Thrombi and neutrophilic plugs were present in the lungs, heart, kidneys, liver, spleen, and brain and were most frequently observed late in the disease course (15 patients with thrombi, median disease course 22 days [5-44]; ten patients with neutrophilic plugs, 21 days [5-44]). Neutrophilic plugs were observed in two forms: solely composed of neutrophils with neutrophil extracellular traps (NETs), or as aggregates of NETs and platelets..Interpretation In patients with lethal COVID-19, an extensive systemic inflammatory response was present, with a continued presence of neutrophils and NETs. However, SARS-CoV-2-infected cells were only sporadically present at late stages of COVID-19. This suggests a maladaptive immune response and substantiates the evidence for immunomodulation as a target in the treatment of severe COVID-19. Show less
Yepes-Calderon, M.; Sotomayor, C.G.; Rasmussen, D.G.K.; Hijmans, R.S.; Velde-Keyzer, C.A.T.; Londen, M. van; ... ; Born, J. van den 2020
The PRO-C6 assay, a reflection of collagen type VI synthesis, has been proposed as a non-invasive early biomarker of kidney fibrosis. We aimed to investigate cross-sectional and longitudinal... Show moreThe PRO-C6 assay, a reflection of collagen type VI synthesis, has been proposed as a non-invasive early biomarker of kidney fibrosis. We aimed to investigate cross-sectional and longitudinal associations between plasma and urine PRO-C6 and proven histological changes after kidney transplantation. The current study is a post-hoc analysis of 94 participants of the MECANO trial, a 24-month prospective, multicenter, open-label, randomized, controlled trial aimed at comparing everolimus-based vs. cyclosporine-based immunosuppression. PRO-C6 was measured in plasma and urine samples collected 6 and 24 months post-transplantation. Fibrosis was evaluated in biopsies collected at the same time points by Banff interstitial fibrosis/tubular atrophy (IF/TA) scoring and collagen staining (Picro Sirius Red; PSR); inflammation was evaluated by the tubulo-interstitial inflammation score (ti-score). Linear regression analyses were performed. Six-month plasma PRO-C6 was cross-sectionally associated with IF/TA score (Std. beta = 0.34), and prospectively with 24-month IF/TA score and ti-score (Std. beta = 0.24 and 0.23, respectively) (p < 0.05 for all). No significant associations were found between urine PRO-C6 and any of the biopsy findings. Fibrotic changes and urine PRO-C6 behaved differentially over time according to immunosuppressive therapy. These results are a first step towards non-invasive fibrosis detection after kidney transplantation by means of collagen VI synthesis measurement, and further research is required. Show less
Tammaro, A.; Kers, J.; Scantlebery, A.M.L.; Florquin, S. 2020
Renal ischemia reperfusion injury (IRI), a common event after renal transplantation, causes acute kidney injury (AKI), increases the risk of delayed graft function (DGF), primes the donor kidney... Show moreRenal ischemia reperfusion injury (IRI), a common event after renal transplantation, causes acute kidney injury (AKI), increases the risk of delayed graft function (DGF), primes the donor kidney for rejection, and contributes to the long-term risk of graft loss. In the last decade, epidemiological studies have linked even mild episodes of AKI to chronic kidney disease (CKD) progression, and innate immunity seems to play a crucial role. The ischemic insult triggers an acute inflammatory reaction that is elicited by Pattern Recognition Receptors (PRRs), expressed on both infiltrating immune cells as well as tubular epithelial cells (TECs). Among the PRRs, Toll-like receptors (TLRs), their synergistic receptors, Nod-like receptors (NLRs), and the inflammasomes, play a pivotal role in shaping inflammation and TEC repair, in response to renal IRI. These receptors represent promising targets to modulate the extent of inflammation, but also function as gatekeepers of tissue repair, protecting against AKI-to-CKD progression. Despite the important considerations on timely use of therapeutics, in the context of IRI, treatment options are limited by a lack of understanding of the intra- and intercellular mechanisms associated with the activation of innate immune receptors and their impact on adaptive tubular repair. Accumulating evidence suggests that TEC-associated innate immunity shapes the tubular response to stress through the regulation of immunometabolism. Engagement of innate immune receptors provides TECs with the metabolic flexibility necessary for their plasticity during injury and repair. This could significantly affect pathogenic processes within TECs, such as cell death, mitochondrial damage, senescence, and pro-fibrotic cytokine secretion, well-known to exacerbate inflammation and fibrosis. This article provides an overview of the past 5 years of research on the role of innate immunity in experimental and human IRI, with a focus on the cascade of events activated by hypoxic damage in TECs: from programmed cell death (PCD) and mitochondrial dysfunction-mediated metabolic rewiring of TECs to maladaptive repair and progression to fibrosis. Finally, we will discuss the important crosstalk between metabolism and innate immunity observed in TECs and their therapeutic potential in both experimental and clinical research. Show less
Hermsen, M.; Bel, T. de; Boer, M. den; Steenbergen, E.J.; Kers, J.; Florquin, S.; ... ; Laak, J.A.W.M. van der 2019
Background The development of deep neural networks is facilitating more advanced digital analysis of histopathologic images. We trained a convolutional neural network for multiclass segmentation of... Show moreBackground The development of deep neural networks is facilitating more advanced digital analysis of histopathologic images. We trained a convolutional neural network for multiclass segmentation of digitized kidney tissue sections stained with periodic acid-Schiff (PAS).Methods We trained the network using multiclass annotations from 40 whole-slide images of stained kidney transplant biopsies and applied it to four independent data sets. We assessed multiclass segmentation performance by calculating Dice coefficients for ten tissue classes on ten transplant biopsies from the Radboud University Medical Center in Nijmegen, The Netherlands, and on ten transplant biopsies from an external center for validation. We also fully segmented 15 nephrectomy samples and calculated the network's glomerular detection rates and compared network-based measures with visually scored histologic components (Banff classification) in 82 kidney transplant biopsies.Results The weighted mean Dice coefficients of all classes were 0.80 and 0.84 in ten kidney transplant biopsies from the Radboud center and the external center, respectively. The best segmented class was "glomeruli" in both data sets (Dice coefficients, 0.95 and 0.94, respectively), followed by "tubuli combined" and "interstitium." The network detected 92.7% of all glomeruli in nephrectomy samples, with 10.4% false positives. In whole transplant biopsies, the mean intraclass correlation coefficient for glomerular counting performed by pathologists versus the network was 0.94. We found significant correlations between visually scored histologic components and network-based measures.Conclusions This study presents the first convolutional neural network for multiclass segmentation of PAS-stained nephrectomy samples and transplant biopsies. Our network may have utility for quantitative studies involving kidney histopathology across centers and provide opportunities for deep learning applications in routine diagnostics. Show less
Barbour, S.J.; Coppo, R.; Zhang, H.; Liu, Z.H.; Suzuki, Y.; Matsuzaki, K.; ... ; Int IgA Nephropathy Network 2019
ImportanceAlthough IgA nephropathy (IgAN) is the most common glomerulonephritis in the world, there is no validated tool to predict disease progression. This limits patient-specific risk... Show moreImportanceAlthough IgA nephropathy (IgAN) is the most common glomerulonephritis in the world, there is no validated tool to predict disease progression. This limits patient-specific risk stratification and treatment decisions, clinical trial recruitment, and biomarker validation. ObjectiveTo derive and externally validate a prediction model for disease progression in IgAN that can be applied at the time of kidney biopsy in multiple ethnic groups worldwide. Design, Setting, and ParticipantsWe derived and externally validated a prediction model using clinical and histologic risk factors that are readily available in clinical practice. Large, multi-ethnic cohorts of adults with biopsy-proven IgAN were included from Europe, North America, China, and Japan. Main Outcomes and MeasuresCox proportional hazards models were used to analyze the risk of a 50% decline in estimated glomerular filtration rate (eGFR) or end-stage kidney disease, and were evaluated using the R-D(2) measure, Akaike information criterion (AIC), C statistic, continuous net reclassification improvement (NRI), integrated discrimination improvement (IDI), and calibration plots. ResultsThe study included 3927 patients; mean age, 35.4 (interquartile range, 28.0-45.4) years; and 2173 (55.3%) were men. The following prediction models were created in a derivation cohort of 2781 patients: a clinical model that included eGFR, blood pressure, and proteinuria at biopsy; and 2 full models that also contained the MEST histologic score, age, medication use, and either racial/ethnic characteristics (white, Japanese, or Chinese) or no racial/ethnic characteristics, to allow application in other ethnic groups. Compared with the clinical model, the full models with and without race/ethnicity had better R-D(2) (26.3% and 25.3%, respectively, vs 20.3%) and AIC (6338 and 6379, respectively, vs 6485), significant increases in C statistic from 0.78 to 0.82 and 0.81, respectively (Delta C, 0.04; 95% CI, 0.03-0.04 and Delta C, 0.03; 95% CI, 0.02-0.03, respectively), and significant improvement in reclassification as assessed by the NRI (0.18; 95% CI, 0.07-0.29 and 0.51; 95% CI, 0.39-0.62, respectively) and IDI (0.07; 95% CI, 0.06-0.08 and 0.06; 95% CI, 0.05-0.06, respectively). External validation was performed in a cohort of 1146 patients. For both full models, the C statistics (0.82; 95% CI, 0.81-0.83 with race/ethnicity; 0.81; 95% CI, 0.80-0.82 without race/ethnicity) and R-D(2) (both 35.3%) were similar or better than in the validation cohort, with excellent calibration. Conclusions and RelevanceIn this study, the 2 full prediction models were shown to be accurate and validated methods for predicting disease progression and patient risk stratification in IgAN in multi-ethnic cohorts, with additional applications to clinical trial design and biomarker research. Show less
Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by... Show moreUpon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury. To further test this, wild-type and S100A9 knockout mice (deficient for S100A8/A9 complex) were subjected to renal I/R. The expression of S100A8/A9 was significantly increased 1 day after I/R and was co-localized with Ly6G (mouse neutrophil marker)-positive cells. These knockout mice displayed similar renal dysfunction and damage and neutrophil influx compared with wild-type mice at this early time point. Interestingly, S100A9 knockout mice displayed altered tissue repair 5 and 10 days post I/R, as reflected by increased renal damage, sustained inflammation, induction of fibrosis, and increased expression of collagens. This coincided with enhanced expression of alternatively activated macrophage (M2) markers, while the expression of classically activated macrophage (M1) markers was comparable. Similarly, S100A9 deficiency affected M2, but not M1 macrophage polarization in vitro. During the repair phase following acute kidney injury, S100A9 deficiency affects M2 macrophages in mice leading to renal fibrosis and damage. Thus, S100A8/A9 plays a crucial part in controlling macrophage-mediated renal repair following I/R. Show less
Berge, I. ten; Heutinck, K.; Yapici, U.; Stokman, G.; Florquin, S.; Kooten, C. van; ... ; Claas, F. 2014
To study the predictive value of biopsy lesions in IgA nephropathy in a range of patient ages we retrospectively analyzed the cohort that was used to derive a new classification system for IgA... Show moreTo study the predictive value of biopsy lesions in IgA nephropathy in a range of patient ages we retrospectively analyzed the cohort that was used to derive a new classification system for IgA nephropathy. A total of 206 adults and 59 children with proteinuria over 0.5 g/24h/1.73 m(2) and an eGFR of stage-3 or better were followed for a median of 69 months. At the time of biopsy, compared with adults children had a more frequent history of macroscopic hematuria, lower adjusted blood pressure, and higher eGFR but similar proteinuria. Although their outcome was similar to that of adults, children had received more immunosuppressants and achieved a lower follow-up proteinuria. Renal biopsies were scored for variables identified by an iterative process as reproducible and independent of other lesions. Compared with adults, children had significantly more mesangial and endocapillary hypercellularity, and less segmental glomerulosclerosis and tubulointerstitial damage, the four variables previously identified to predict outcome independent of clinical assessment. Despite these differences, our study found that the cross-sectional correlation between pathology and proteinuria was similar in adults and children. The predictive value of each specific lesion on the rate of decline of renal function or renal survival in IgA nephropathy was not different between children and adults. Kidney International (2010) 77, 921-927; doi: 10.1038/ki.2010.43; published online 3 March 2010 Show less
