The Banff Digital Pathology Working Group (DPWG) was established with the goal to establish a digital pathology repository; develop, validate, and share models for image analysis; and foster... Show moreThe Banff Digital Pathology Working Group (DPWG) was established with the goal to establish a digital pathology repository; develop, validate, and share models for image analysis; and foster collaborations using regular videoconferencing. During the calls, a variety of artificial intelligence (AI)-based support systems for transplantation pathology were presented. Potential collaborations in a competition/trial on AI applied to kidney transplant specimens, including the DIAGGRAFT challenge (staining of biopsies at multiple institutions, pathologists' visual assessment, and development and validation of new and pre-existing Banff scoring algorithms), were also discussed. To determine the next steps, a survey was conducted, primarily focusing on the feasibility of establishing a digital pathology repository and identifying potential hosts. Sixteen of the 35 respondents (46%) had access to a server hosting a digital pathology repository, with 2 respondents that could serve as a potential host at no cost to the DPWG. The 16 digital pathology repositories collected specimens from various organs, with the largest constituent being kidney (n = 12,870 specimens). A DPWG pilot digital pathology repository was established, and there are plans for a competition/trial with the DIAGGRAFT project. Utilizing existing resources and previously established models, the Banff DPWG is establishing new resources for the Banff community. Show less
Gaykema, L.H.; Nieuwland, R.Y. van; Lievers, E.; Moerkerk, W.B.J.; Klerk, J.A. de; Dumas, S.J.; ... ; Rabelink, T.J. 2023
Immune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as “stealth” organoids, hold promise for clinical transplantation. To address immune rejection, we investigated... Show moreImmune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as “stealth” organoids, hold promise for clinical transplantation. To address immune rejection, we investigated the impact of genetically modifying human leukocyte antigen (HLA) class I in kidney organoids prior to transplantation. By using CRISPR-Cas9, we successfully knocked out beta-2-microglobulin (B2M), resulting in iPSCs devoid of HLA class I surface expression. In vitro, the B2M knockout protected kidney organoids derived from these iPSCs against T-cell rejection. To assess in vivo protection, unmodified (control) and B2M–/– kidney organoids were transplanted into humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs). Successful engraftment of human PBMCs was confirmed, and after 4 weeks, we observed no discernible difference in the infiltration rate, proliferation, or cytotoxicity of CD4+ and CD8+ T cells between control and B2M–/– organoids. Both groups of organoids showed compromised tissue integrity, displaying tubulitis and loss of tubule integrity. Notably, while B2M–/– organoids failed to express HLA class I on their cell surface, there was preexisting expression of HLA class II in both control and B2M–/– organoids transplanted into mice with human PBMCs. HLA class II expression was not limited to antigen-presenting cells but also evident in epithelial cells of the kidney organoid, posing an additional immunological challenge to its transplantation. Consequently, we conclude that B2M knockout alone is insufficient to protect iPSC-derived kidney organoids from T-cell-mediated immune rejection. Additionally, our findings suggest that modulating HLA class II signaling will be necessary to prevent rejection following transplantation. Show less
Gritter, M.; Wei, K.Y.; Wouda, R.D.; Musterd-Bhaggoe, U.M.; Dijkstra, K.L.; Kers, J.; ... ; Rotmans, J. 2023
Background Dietary potassium (K+) has emerged as a modifiable factor for cardiovascular and kidney health in the general population, but its role in people with chronic kidney disease (CKD) is... Show moreBackground Dietary potassium (K+) has emerged as a modifiable factor for cardiovascular and kidney health in the general population, but its role in people with chronic kidney disease (CKD) is unclear. Here, we hypothesize that CKD increases the susceptibility to the negative effects of low and high K+ diets.Methods We compared the effects of low, normal and high KChloride (KCl) diets and a high KCitrate diet for 4 weeks in male rats with normal kidney function and in male rats with CKD using the 5/6th nephrectomy model (5/6Nx).Results Compared with rats with normal kidney function, 5/6Nx rats on the low KCl diet developed more severe extracellular and intracellular K+ depletion and more severe kidney injury, characterized by nephromegaly, infiltration of T cells and macrophages, decreased estimated glomerular filtration rate and increased albuminuria. The high KCl diet caused hyperkalemia, hyperaldosteronism, hyperchloremic metabolic acidosis and severe hypertension in 5/6Nx but not in sham rats. The high KCitrate diet caused hypochloremic metabolic alkalosis but attenuated hypertension despite higher abundance of the phosphorylated sodium chloride cotransporter (pNCC) and similar levels of plasma aldosterone and epithelial sodium channel abundance. All 5/6Nx groups had more collagen deposition than the sham groups and this effect was most pronounced in the high KCitrate group. Plasma aldosterone correlated strongly with kidney collagen deposition.Conclusions CKD increases the susceptibility to negative effects of low and high K+ diets in male rats, although the injury patterns are different. The low K+ diet caused inflammation, nephromegaly and kidney function decline, whereas the high K+ diet caused hypertension, hyperaldosteronism and kidney fibrosis. High KCitrate attenuated the hypertensive but not the pro-fibrotic effect of high KCl, which may be attributable to K+-induced aldosterone secretion. Our data suggest that especially in people with CKD it is important to identify the optimal threshold of dietary K+ intake.Graphical Abstract Show less
Background Amiodarone is associated with a range of unwanted effects on pulmonary, thyroid, and liver function. However, the nephrotoxic side effect caused by renal phospholipidosis has hardly... Show moreBackground Amiodarone is associated with a range of unwanted effects on pulmonary, thyroid, and liver function. However, the nephrotoxic side effect caused by renal phospholipidosis has hardly received any attention up to now.Case summary This is a case of an 86-year-old Caucasian male with an acute on chronic kidney disease 4 months after the initiation of amiodarone. A renal biopsy demonstrated the intracellular accumulation of phospholipids that have previously been demonstrated in association with organ dysfunction because of amiodarone use. Serum creatinine levels subsequently improved from 388 to 314 & mu;mol/L after stopping amiodarone over the course of 2 months.Discussion In this case, a diagnosis of partially reversible acute on chronic kidney disease caused by lysosomal phospholipidosis due to amiodarone use was deemed highly likely. Lysosomal dysfunction leads to the accumulation of intra-lysosomal phospholipids (phospholipidosis). This accumulation is accompanied by progressive organ damage and dysfunction, including renal dysfunction, in rare instances. Guidelines advise regular surveillance for liver, lung, and thyroid toxicity during amiodarone treatment but do not mention the potential for renal toxicity. This case suggests that it might be prudent to include screening for renal toxicity in this surveillance. Show less
Veltkamp, D.M.J.; Nijhoff, M.F.; Broek, D.A.J. van den; Buntinx, M.; Kers, J.; Engelse, M.A.; ... ; Vries, A.P.J. de 2023
The purpose of pancreas or islet transplantation is to restore glycemic control in order to mitigate diabetes-related complications and prevent severe hypoglycemia. Complications from chronic... Show moreThe purpose of pancreas or islet transplantation is to restore glycemic control in order to mitigate diabetes-related complications and prevent severe hypoglycemia. Complications from chronic pancreas allograft rejection may lead to transplantectomy, even when the endocrine function remains preserved. We present first evidence of a successful HLA incompatible islet re-transplantation with islets isolated from a rejecting pancreas allograft after simultaneous kidney pancreas transplantation. The pancreas allograft was removed because of progressively painful pancreatic panniculitis from clinically uncontrolled chronic rejection. The endocrine function was preserved. Induction treatment for this “islet alloautotransplantation” consisted of plasmapheresis, IVIg and alemtuzumab. At 1 year, the patient retained islet graft function with good glycemic control and absence of severe hypoglycemia, despite persistent low-grade HLA donor-specific antibodies. His panniculitis had resolved completely. In our point of view, islet alloautotransplantation derived from a chronically rejecting pancreas allograft is a potential option to salvage (partial) islet function, despite preformed donor-specific antibodies, in order to maintain stable glycemic control. Thereby it protects against severe hypoglycemia, and it potentially mitigates kidney graft dysfunction and other diabetes-related complications in patients with continued need for immunosuppression and who are otherwise difficult to retransplant. 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
Meziyerh, S.; Gelder, T. van; Kers, J.; Helm, D. van der; Boog, P.J.M. van der; Fijter, J.W. de; ... ; Vries, A.P.J. de 2023
Evidence to define target ranges for tacrolimus (Tac) and mycophenolic acid (MPA) exposure after the first year of kidney transplantation is limited. We investigated the association of measurements... Show moreEvidence to define target ranges for tacrolimus (Tac) and mycophenolic acid (MPA) exposure after the first year of kidney transplantation is limited. We investigated the association of measurements at 1 year and repeated measurements of real-world Tac-trough levels (C-0) and abbreviated area under the curve from zero to 12 hours (AUC(0-12h)) of Tac and MPA with biopsy-proven acute rejection (BPAR) between years 1 and 3 post-transplant in 968 kidney transplant recipients (KTRs). Thirty-five (3.6%) out of 968 KTRs experienced BPAR. Both Tac-AUC(0-12h) (hazard ratio (HR): 0.39, 95% confidence interval (CI): 0.30-0.50, P < 0.001), Tac-C-0 (HR: 0.46, 95% CI: 0.38-0.57, P < 0.001) and MPA-AUC(0-12h) at 1 year (HR: 0.80, 95% CI: 0.68-0.94, P = 0.006), as well as repeated measurements of Tac-C-0 (HR: 0.70, 95% credibility interval (CrI): 0.61-0.82, P < 0.001), and of MPA-AUC(0-12h) (HR: 0.75, 95% CrI: 0.62-0.93, P < 0.001) were associated with BPAR. In our population, the recommended target range for Tac-AUC(0-12h) at 1 year would be 75-95 ng*hour/mL and a Tac-C-0 5-7 ng/mL. The Tac-AUC(0-12h) predicted BPAR better than Tac-C-0 and identified KTRs with over- or underexposure despite supposedly adequate Tac-C-0. We did not find evidence to recommend another target than the consensus range of 30-60 mg*hour/L for MPA-AUC(0-12h) after the first year of transplantation. To our knowledge, this is a first study on the simultaneous exposure of Tac and MPA at year 1 and subsequent BPAR up to year 3, which may help define the therapeutic target window for the longer term. Show less
Russcher, A.; Molenaar-de Backer, M.; Brouwer, C. de; Dijkstra, K.; Kers, J.; Vries, A. de; ... ; Kroes, A. 2023
Parvovirus B19 (B19V) DNAemia appears to be a relatively common finding after kidney transplantation. However, not all DNAemia signifies active infection with replicating virus. This study screened... Show moreParvovirus B19 (B19V) DNAemia appears to be a relatively common finding after kidney transplantation. However, not all DNAemia signifies active infection with replicating virus. This study screened 134 patients posttransplantation for B19V DNAemia and identified 2 cases in which viral DNA was present after transplantation, with the donor kidney as probable source of the DNA. In both cases intact viral particles could not be detected using an endonuclease method, indicating the presence of noninfectious DNA remnants. Show less
Russcher, A.; Backer, M.M.D.; Brouwer, C. de; Dijkstra, K.; Kers, J.; Vries, A. de; ... ; Kroes, A. 2023
Parvovirus B19 (B19V) DNAemia appears to be a relatively common finding after kidney transplantation. However, not all DNAemia signifies active infection with replicating virus. This study screened... Show moreParvovirus B19 (B19V) DNAemia appears to be a relatively common finding after kidney transplantation. However, not all DNAemia signifies active infection with replicating virus. This study screened 134 patients posttransplantation for B19V DNAemia and identified 2 cases in which viral DNA was present after transplantation, with the donor kidney as probable source of the DNA. In both cases intact viral particles could not be detected using an endonuclease method, indicating the presence of noninfectious DNA remnants. Show less
Recently, the randomized phase-II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy facilitated early tacrolimus withdrawal in living donor kidney transplant recipients. The... Show moreRecently, the randomized phase-II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy facilitated early tacrolimus withdrawal in living donor kidney transplant recipients. The current sub-study analyzed formation of de novo donor-specific HLA antibodies (dnDSA) in the context of the degree of HLA eplet mismatches. At the time of protocol biopsy at 6 months, 7/29 patients (24%) in the MSC group and 1/27 patient (3.7%) in the control group had developed dnDSA. In the MSC group, all dnDSA were antiHLA-DQ; two patients had anti-DQ alone and five patients combined with anti-class I, HLA-DR or-DP. Despite excess dnDSA formation in the MSC-arm of the study, the evolution of eGFR (CKD-EPI) and proteinuria were comparable 2 years posttransplant. All dnDSA were complement-binding and three patients had antibody-mediated rejection in the protocol biopsy, but overall rejection episodes were not increased. Everolimus had to be discontinued in nine patients because of toxicity, and tacrolimus was reintroduced in six patients because of dnDSA formation. The HLA-DQ eplet mismatch load independently associated with dnDSA (adjusted hazard ratio = 1.07 per eplet mismatch, p = 0.008). A threshold of >= 11 HLA-DQ eplet mismatches predicted subsequent dnDSA in all 11 patients in the MSC group, but specificity was low (44%). Further research is warranted to explore HLA molecular mismatch load as a biomarker to guide personalized maintenance immunosuppression in kidney transplantation. Show less
Russcher, A.; Molenaar-de Backer, M.; Brouwer, C. de; Dijkstra, K.; Kers, J.; Vries, A. de; ... ; Kroes, A. 2023
Parvovirus B19 (B19V) DNAemia appears to be a relatively common finding after kidney transplantation. However, not all DNAemia signifies active infection with replicating virus. This study... Show moreParvovirus B19 (B19V) DNAemia appears to be a relatively common finding after kidney transplantation. However, not all DNAemia signifies active infection with replicating virus. This study screened 134 patients posttransplantation for B19V DNAemia and identified 2 cases in which viral DNA was present after transplantation, with the donor kidney as probable source of the DNA. In both cases intact viral particles could not be detected using an endonuclease method, indicating the presence of noninfectious DNA remnants. Show less
Wang, G.Q.; Heijs, B.; Kostidis, S.; Rietjens, R.G.J.; Koning, M.; Yuan, L.S.; ... ; Rabelink, T.J. 2022
Accumulating evidence demonstrates important roles for metabolism in cell fate determination. However, it is a challenge to assess metabolism at a spatial resolution that acknowledges both... Show moreAccumulating evidence demonstrates important roles for metabolism in cell fate determination. However, it is a challenge to assess metabolism at a spatial resolution that acknowledges both heterogeneity and cellular dynamics in its tissue microenvironment. Using a multi-omics platform to study cell-type-specific dynamics in metabolism in complex tissues, we describe the metabolic trajectories during nephrogenesis in the devel-oping human kidney. Exploiting in situ analysis of isotopic labeling, a shift from glycolysis toward fatty acid b-oxidation was observed during the differentiation from the renal vesicle toward the S-shaped body and the proximal tubules. In addition, we show that hiPSC-derived kidney organoids are characterized by a metabolic immature phenotype that fails to use mitochondrial long-chain fatty acids for energy metabolism. Further-more, supplementation of butyrate enhances tubular epithelial differentiation and maturation in cultured kid-ney organoids. Our findings highlight the relevance of understanding metabolic trajectories to efficiently guide stem cell differentiation. Show less
Infective endocarditis (IE) may be misdiagnosed as ANCA-associated vasculitis (AAV), especially when antineutrophil cytoplasmic antibodies (ANCA) are detected. Distinguishing IE from AAV is crucial... Show moreInfective endocarditis (IE) may be misdiagnosed as ANCA-associated vasculitis (AAV), especially when antineutrophil cytoplasmic antibodies (ANCA) are detected. Distinguishing IE from AAV is crucial to guide therapy. However, little is known about ANCA positivity in IE patients. We present a case report and systematic review of the literature on patients with ANCA-positive IE, aiming to provide a comprehensive overview of this entity and to aid clinicians in their decisions when encountering a similar case. A systematic review of papers on original cases of ANCA-positive IE without a previous diagnosis of AAV was conducted on PubMed in accordance with PRISMA-IPD guidelines. A predefined set of clinical, laboratory, and kidney biopsy findings was extracted for each patient and presented as a narrative and quantitative synthesis. A total of 74 reports describing 181 patients with ANCA-positive IE were included (a total of 182 cases including our own case). ANCA positivity was found in 18-43% of patients with IE. Patients usually presented with subacute IE (73%) and had positive cytoplasmic ANCA-staining or anti-proteinase-3 antibodies (79%). Kidney function was impaired in 72%; kidney biopsy findings were suggestive of immune complexes in 59%, while showing pauci-immune glomerulonephritis in 37%. All were treated with antibiotics; 39% of patients also received immunosuppressants. During follow-up, 69% of patients became ANCA-negative and no diagnosis of systemic vasculitis was reported. This study reviewed the largest series of patients with ANCA-positive IE thus far and shows the overlap in clinical manifestations between IE and AAV. We therefore emphasize that clinicians should be alert to the possibility of an underlying infection when treating a patient with suspected AAV, even when reassured by ANCA positivity. Show less
The application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical... Show moreThe application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical fields. One objective is to complement current diagnostic methods with new specific molecular information. An important goal is to achieve adequate and consistent protein recovery across and within large-scale studies. Here, we describe development of several protocols incorporating mass spectrometry compatible detergents, including Rapigest, PPS, and ProteaseMax. Methods were applied on 4 and 15 mu m thick FF tissues, and 4 mu m thick FFPE tissues. We evaluated sensitivity and repeatability of the methods and found that the protocol containing Rapigest enabled detection of 630 proteins from FF tissue of 1 mm(2) and 15 mu m thick, whereas 498 and 297 proteins were detected with the protocols containing ProteaseMax and PPS, respectively. Surprisingly, PPS-containing buffer showed good extraction of the proteins from 4 mu m thick FFPE tissue with the average of 270 protein identifications (1 mm(2)), similar to the results on 4 mu m thick FF. Moreover, we found that temperature increases during incubation with urea on 4 mu m thick FF tissue revealed a decrease in the number of identified proteins and increase in the number of the carbamylated peptides. 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
Hermsen, M.; Volk, V.; Braesen, J.H.; Geijs, D.J.; Gwinner, W.; Kers, J.; ... ; Laak, J.A.W.M. van der 2021
Delayed graft function (DGF) is a strong risk factor for development of interstitial fibrosis and tubular atrophy (IFTA) in kidney transplants. Quantitative assessment of inflammatory infiltrates... Show moreDelayed graft function (DGF) is a strong risk factor for development of interstitial fibrosis and tubular atrophy (IFTA) in kidney transplants. Quantitative assessment of inflammatory infiltrates in kidney biopsies of DGF patients can reveal predictive markers for IFTA development. In this study, we combined multiplex tyramide signal amplification (mTSA) and convolutional neural networks (CNNs) to assess the inflammatory microenvironment in kidney biopsies of DGF patients (n = 22) taken at 6 weeks post-transplantation. Patients were stratified for IFTA development (<10% versus >= 10%) from 6 weeks to 6 months post-transplantation, based on histopathological assessment by three kidney pathologists. One mTSA panel was developed for visualization of capillaries, T- and B-lymphocytes and macrophages and a second mTSA panel for T-helper cell and macrophage subsets. The slides were multi spectrally imaged and custom-made python scripts enabled conversion to artificial brightfield whole-slide images (WSI). We used an existing CNN for the detection of lymphocytes with cytoplasmatic staining patterns in immunohistochemistry and developed two new CNNs for the detection of macrophages and nuclear-stained lymphocytes. F1-scores were 0.77 (nuclear-stained lymphocytes), 0.81 (cytoplasmatic-stained lymphocytes), and 0.82 (macrophages) on a test set of artificial brightfield WSI. The CNNs were used to detect inflammatory cells, after which we assessed the peritubular capillary extent, cell density, cell ratios, and cell distance in the two patient groups. In this cohort, distance of macrophages to other immune cells and peritubular capillary extent did not vary significantly at 6 weeks post-transplantation between patient groups. CD163(+) cell density was higher in patients with >= 10% IFTA development 6 months post-transplantation (p < 0.05). CD3(+)CD8(-)/CD3(+)CD8(+) ratios were higher in patients with <10% IFTA development (p < 0.05). We observed a high correlation between CD163(+) and CD4(+)GATA3(+) cell density (R = 0.74, p < 0.001). Our study demonstrates that CNNs can be used to leverage reliable, quantitative results from mTSA-stained, multi spectrally imaged slides of kidney transplant biopsies.This study describes a methodology to assess the microenvironment in sparse tissue samples. Deep learning, multiplex immunohistochemistry, and mathematical image processing techniques were incorporated to quantify lymphocytes, macrophages, and capillaries in kidney transplant biopsies of delayed graft function patients. The quantitative results were used to assess correlations with development of interstitial fibrosis and tubular atrophy. Show less
Reinders, M.E.J.; Groeneweg, K.E.; Hendriks, S.H.; Bank, J.R.; Dreyer, G.J.; Vries, A.P.J. de; ... ; Fijter, J.W. de 2021
After renal transplantation, there is a need for immunosuppressive regimens which effectively prevent allograft rejection, while preserving renal function and minimizing side effects. From this... Show moreAfter renal transplantation, there is a need for immunosuppressive regimens which effectively prevent allograft rejection, while preserving renal function and minimizing side effects. From this perspective, mesenchymal stromal cell (MSC) therapy is of interest. In this randomized prospective, single-center, open-label trial, we compared MSCs infused 6 and 7 weeks after renal transplantation and early tacrolimus withdrawal with a control tacrolimus group. Primary end point was quantitative evaluation of interstitial fibrosis in protocol biopsies at 4 and 24 weeks posttransplant. Secondary end points included acute rejection, graft loss, death, renal function, adverse events, and immunological responses. Seventy patients were randomly assigned of which 57 patients were included in the final analysis (29 MSC; 28 controls). Quantitative progression of fibrosis failed to show benefit in the MSC group and GFR remained stable in both groups. One acute rejection was documented (MSC group), while subclinical rejection in week 24 protocol biopsies occurred in seven patients (four MSC; three controls). In the MSC group, regulatory T cell numbers were significantly higher compared to controls (p = .014, week 24). In conclusion, early tacrolimus withdrawal with MSC therapy was safe and feasible without increased rejection and with preserved renal function. MSC therapy is a potentially useful approach after renal transplantation. Show less
Putten, C. van der; Remmerswaal, E.B.M.; Terpstra, M.L.; Bom, N.D. van der; Kers, J.; Berge, I.J.M. ten; ... ; Aalderen, M.C. van 2021
Background: At border sites, and in internal organs, tissue resident memory T cells (T-RM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However,... Show moreBackground: At border sites, and in internal organs, tissue resident memory T cells (T-RM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However, information on the presence and function of these cells in the human kidney is scant. In order to better understand the T cell-mediated immunological defense in this organ, we aimed to determine phenotypic and functional aspects of CD8 and CD4 T cells present in healthy and allograft kidney tissue. Methods: Using multichannel flow cytometry, we assessed the phenotype and function of T cells in healthy renal tissue samples (n = 5) and kidney allograft tissue (n = 7) and compared these aspects to T cells in peripheral blood from healthy controls (n = 13). Results: Kidney tissue samples contained substantial amounts of CD8 and CD4 T cells. In contrast to the circulating cells, kidney T cells frequently expressed CD69 and CD103, and were more often actively cycling. Furthermore, nearly all kidney T cells expressed CXCR3, and often expressed CXCR6 compared to T cells in the circulation. Markedly, kidney T cells produced greater quantities of IFN gamma than circulating cells and were frequently polyfunctional. Conclusion: Functional T cells with the characteristic traits of T-RM reside in human kidney tissues. These cells are more often actively cycling and frequently express CXCR3 and CXCR6. 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
