The histopathological lesions, minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are entities without immune complex deposits which can cause podocyte injury, thus are... Show moreThe histopathological lesions, minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are entities without immune complex deposits which can cause podocyte injury, thus are frequently grouped under the umbrella of podocytopathies. Whether MCD and FSGS may represent a spectrum of the same disease remains a matter of conjecture. Both frequently require repeated high-dose glucocorticoid therapy with alternative immunosuppressive treatments reserved for relapsing or resistant cases and response rates are variable. There is an unmet need to identify patients who should receive immunosuppressive therapies as opposed to those who would benefit from supportive strategies. Therapeutic trials focusing on MCD are scarce, and the evidence used for the 2021 Kidney Disease: Improving Global Outcomes (KDIGO) guideline for the management of glomerular diseases largely stems from observational and pediatric trials. In FSGS, the differentiation between primary forms and those with underlying genetic variants or secondary forms further complicates trial design. This article provides a perspective of the Immunonephrology Working Group (IWG) of the European Renal Association (ERA) and discusses the KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases focusing on the management of MCD and primary forms of FSGS in the context of recently published evidence, with a special emphasis on the role of rituximab, cyclophosphamide, supportive treatment options and ongoing clinical trials in the field.10.1093/ndt/gfae025 Video Watch the video of this contribution at https://academic.oup.com/ndt/pages/author_videos gfae025Media1 6346336829112 Show less
Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of... Show moreHistorically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169(+) macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8(+) T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169(+) macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8(+) T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8(+) T cell activation during bacterial infection. However, after liposomal vaccination CD8(+) T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169(+) macrophages and cDC1 and emphasize the importance of platelets in induction of CD8(+) T cell responses in the context of liposomal vaccination. Show less
Objective:To provide mechanistic insight into key biological alterations in donation after circulatory death kidneys during continuous pefusion we performed mass spectrometry profiling of perfusate... Show moreObjective:To provide mechanistic insight into key biological alterations in donation after circulatory death kidneys during continuous pefusion we performed mass spectrometry profiling of perfusate samples collected during a phase 3 randomized double-blind paired clinical trial of hypothermic machine perfusion with and without oxygen (COMPARE).Background:Despite the clinical benefits of novel perfusion technologies aiming to better preserve donor organs, biological processes that may be altered during perfusion have remained largely unexplored. The collection of serial perfusate samples during the COMPARE clinical trial provided a unique resource to study perfusate proteomic profiles, with the hypothesis that in-depth profiling may reveal biologically meaningful information on how donor kidneys benefit from this intervention.Methods:Multiplexed liquid chromatography-tandem mass spectrometry was used to obtain a proteome profile of 210 perfusate samples. Partial least squares discriminant analysis and multivariate analysis involving clinical and perfusion parameters were used to identify associations between profiles and clinical outcomes.Results:Identification and quantitation of 1716 proteins indicated that proteins released during perfusion originate from the kidney tissue and blood, with blood-based proteins being the majority. Data show that the overall hypothermic machine perfusion duration is associated with increasing levels of a subgroup of proteins. Notably, high-density lipoprotein and complement cascade proteins are associated with 12-month outcomes, and blood-derived proteins are enriched in the perfusate of kidneys that developed acute rejection.Conclusions:Perfusate profiling by mass spectrometry was informative and revealed proteomic changes that are biologically meaningful and, in part, explain the clinical observations of the COMPARE trial. Show less
Hendriks, S.H.; Heidt, S.; Schulz, A.R.; Fijter, J.W. de; Reinders, M.E.J.; Koning, F.; Kooten, C. van 2023
Tacrolimus is the backbone of immunosuppressive agents to prevent transplant rejection. Paradoxically, tacrolimus is nephrotoxic, causing irreversible tubulointerstitial damage. Therefore, infusion... Show moreTacrolimus is the backbone of immunosuppressive agents to prevent transplant rejection. Paradoxically, tacrolimus is nephrotoxic, causing irreversible tubulointerstitial damage. Therefore, infusion of mesenchymal stromal cells (MSC) 6 and 7 weeks post-transplantation was assessed to facilitate withdrawal of tacrolimus in the randomized phase II TRITON trial. Here, we performed detailed analysis of the peripheral blood immune composition using mass cytometry to assess potential effects of MSC therapy on the immune system. We developed two metal-conjugated antibody panels containing 40 antibodies each. PBMC samples from 21 MSC-treated patients and 13 controls, obtained pre-transplant and at 24 and 52 weeks post-transplantation, were analyzed. In the MSC group at 24 weeks, 17 CD4(+) T cell clusters were increased of which 14 Th2-like clusters and three Th1/Th2-like clusters, as well as CD4+FoxP3+ Tregs. Additionally, five B cell clusters were increased, representing either class switched memory B cells or proliferating B cells. At 52 weeks, CCR7(+)CD38(+) mature B cells were decreased. Finally, eight Tc1 (effector) memory cytotoxic T cell clusters were increased. Our work provides a comprehensive account of the peripheral blood immune cell composition in kidney transplant recipients after MSC therapy and tacrolimus withdrawal. These results may help improving therapeutic strategies using MSCs with the aim to reduce the use of calcineurin inhibitors. Show less
Essen, M.F. van; Schlagwein, N.; Hoven, E.M.P. van den; Gijlswijk-Janssen, D.J. van; Lubbers, R.; Bos, R.M. van den; ... ; COMBAT Consortium 2023
Properdin, the only known positive regulator of the complement system, stabilizes the C3 convertase, thereby increasing its half-life. In contrast to most other complement factors, properdin is... Show moreProperdin, the only known positive regulator of the complement system, stabilizes the C3 convertase, thereby increasing its half-life. In contrast to most other complement factors, properdin is mainly produced extrahepatically by myeloid cells. Recent data suggest a role for properdin as a pattern recognition molecule. Here, we confirmed previous findings of properdin binding to different necrotic cells including Jurkat T cells. Binding can occur independent of C3, as demonstrated by HAP-1 C3 KO cells, excluding a role for endogenous C3. In view of the cellular source of properdin, interaction with myeloid cells was examined. Properdin bound to the surface of viable monocyte-derived pro- and anti-inflammatory macrophages, but not to DCs. Binding was demonstrated for purified properdin as well as fractionated P2, P3, and P4 properdin oligomers. Binding contributed to local complement activation as determined by C3 and C5b-9 deposition on the cell surfaces and seems a prerequisite for alternative pathway activation. Interaction of properdin with cell surfaces could be inhibited with the tick protein Salp20 and by different polysaccharides, depending on sulfation and chain length. These data identify properdin as a factor interacting with different cell surfaces, being either dead or alive, contributing to the local stimulation of complement activation. Show less
Hilhorst, M.; Bemelman, F.J.; Bruchfeld, A.; Fernandez-Juarez, G.M.; Floege, J.; Frangou, E.; ... ; Immunonephrol Working Grp European 2023
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic led to rapid vaccine development and large global vaccination schemes. However, patients with immune-mediated kidney... Show moreThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic led to rapid vaccine development and large global vaccination schemes. However, patients with immune-mediated kidney disease, chronic kidney diseases and kidney transplant recipients show high non-response rates to vaccination despite more than three vaccinations and, consequently, reduced viral clearance capacity when infected while receiving certain immunosuppressants, carrying an elevated risk for coronavirus disease 2019 (COVID-19)-related morbidity and mortality. SARS-CoV-2 evolution has been characterized by the emergence of novel variants and spike mutations contributing to waning efficacy of neutralizing antibodies. To this end, the therapeutic field expands from vaccination towards a combined approach of immunization, pre-exposure prophylaxis and early post-exposure treatment using direct-acting antivirals and neutralizing monoclonal antibodies to treat early in the disease course and avoid hospitalization. This expert opinion paper from the Immunonephrology Working Group of the European Renal Association (ERA-IWG) summarizes available prophylactic and/or early treatment options (i.e. neutralizing monoclonal antibodies and direct-acting antivirals) of SARS-CoV-2-infected patients with immune-mediated kidney disease, chronic kidney disease and kidney transplant recipients. Show less
Gaykema, L.H.; Nieuwland, R.Y. van; Dekkers, M.C.; Essen, M.F. van; Heidt, S.; Zaldumbide, A.; ... ; Kooten, C. van 2023
End stage renal disease is an increasing problem worldwide driven by aging of the population and increased prevalence of metabolic disorders and cardiovascular disease. Currently, kidney... Show moreEnd stage renal disease is an increasing problem worldwide driven by aging of the population and increased prevalence of metabolic disorders and cardiovascular disease. Currently, kidney transplantation is the only curative option, but donor organ shortages greatly limit its application. Regenerative medicine has the potential to solve the shortage by using stem cells to grow the desired tissues, like kidney tissue. Immune rejection poses a great threat towards the implementation of stem cell derived tissues and various strategies have been explored to limit the immune response towards these tissues. However, these studies are limited by targeting mainly T cell mediated immune rejection while the rejection process also involves innate and humoral immunity. In this study we investigate whether inhibition of the complement system in human induced pluripotent stem cells (iPSC) could provide protection from such immune injury. To this end we created knock-in iPSC lines of the membrane bound complement inhibitor CD55 to create a transplant-specific protection towards complement activation. CD55 inhibits the central driver of the complement cascade, C3 convertase, and we show that overexpression is able to decrease complement activation on both iPSCs as well as differentiated kidney organoids upon stimulation with anti-HLA antibodies to mimic the mechanism of humoral rejection. Show less
Alem, C.M.A. van; Bank, J.R.; Vries, D.K. de; Bajema, I.M.; Mallat, M.J.K.; Fijter, J.W. de; ... ; Kooten, C. van 2022
Acute cellular rejection (ACR) occurs in 10% of renal allograft recipients and is characterized by leukocyte infiltration as observed in needle biopsies. ACR onset is subject to several risk... Show moreAcute cellular rejection (ACR) occurs in 10% of renal allograft recipients and is characterized by leukocyte infiltration as observed in needle biopsies. ACR onset is subject to several risk factors, including delayed graft function (DGF). As the impact of DGF on the etiology of ACR remains unclear, this study analyzed the association between presence of leukocyte subsets and ACR onset, in DCD kidney biopsies with extensive DGF following transplantation. Immunohistochemical analysis of protocol biopsies taken 10 days after kidney transplantation revealed that patients with high levels of renal CD163+ macrophages have a decreased risk (OR = 0.021, P = 0.008) for ACR in the first 6 months after transplantation. In pre-transplant biopsies of a comparable DCD cohort, with >80% DGF, presence of donor CD163+ macrophages showed no effect on ACR risk. Therefore, leukocyte infiltrate present during the inflammatory response at the time of DGF may contain anti-inflammatory macrophages that exert a protective effect against ACR development. Show less
Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic... Show moreComplement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/ dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney. Show less
Schaik, M. van; Arends, E.J.; Soonawala, D.; Ommen, E. van; Leeuw, K. de; Limper, M.; ... ; Teng, Y.K.O. 2022
Background: Belimumab, an anti-B-cell activating factor antibody, is approved for the treatment of auto-antibody positive systemic lupus erythematosus with a high degree of disease activity. Anti... Show moreBackground: Belimumab, an anti-B-cell activating factor antibody, is approved for the treatment of auto-antibody positive systemic lupus erythematosus with a high degree of disease activity. Anti-CD20 B cell depletion with rituximab is used in refractory SLE as well, although with variable responses. We hypothesized that incomplete B cell depletion, related to a surge in BAFF levels following rituximab treatment, can cause ongoing disease activity and flares. The Synbiose 1 study primarily focused on immunological effects and shows the preliminary clinical benefit of combined rituximab and belimumab in SLE. The Synbiose 2 study will evaluate the clinical efficacy of combining belimumab with rituximab in patients with severe SLE, allowing the tapering of prednisolone and mycophenolate. Methods: Synbiose 2 is a phase 3, multicenter, randomized, controlled, open-label 2-year clinical trial. Seventy adults with severe SLE including lupus nephritis will be randomized 1:1 to receive either standard of care consisting of prednisolone and mycophenolate as induction and maintenance treatment, or belimumab and rituximab combined with standard of care as induction treatment, followed by prednisolone and belimumab as maintenance treatment. The primary objective is to assess whether combined B cell therapy will lead to a reduction of treatment failure. Secondary endpoints are complete and partial clinical and renal response and the improvement of SLE-specific autoimmune phenomena. Safety endpoints include the incidence of adverse events, with a special interest in infections. Discussion: The Synbiose 2 trial is the first multicenter phase 3 clinical trial investigating combined B cell targeted therapy in SLE, including lupus nephritis. The outcome of this study will provide further evidence for the clinical efficacy of this new treatment strategy in severe SLE. Show less
Introduction: Both rituximab (RTX) and cyclophosphamide (CYC) are effectively used in combination with steroids as remission induction therapy for patients with antineutrophil cytoplasmic antibody ... Show moreIntroduction: Both rituximab (RTX) and cyclophosphamide (CYC) are effectively used in combination with steroids as remission induction therapy for patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Several studies have shown that the effect on achieving (clinical) remission, frequency and severity of relapses is equivalent for both therapies, but there is accumulating data that the long-term safety profile of RTX might outperform CYC. Combination of RTX with low-dose CYC (LD-CYC) has been investigated in only a few uncontrolled cohort studies, in which clinical remission and a favourable immunological state with low relapse rates was quickly achieved. In this randomised controlled trial, we aim to investigate whether the combination treatment (RTX+LD CYC) is superior in comparison to standard care with RTX only. Methods and analysis: This study is an open-label, multicentre, 1:1 randomised, prospective study for patients with AAV with generalised disease, defined as involvement of major organs, that is, kidneys, lungs, heart and nervous system. In total, 100 patients will be randomised 1:1 to receive either remission induction therapy with standard of care (RTX) or combination treatment (RTX+LD CYC) in addition to steroids and both arms are followed by maintenance with RTX retreatments (tailored to B-cell and ANCA status). Our primary outcome is the number of retreatments needed to maintain clinical remission over 2 years. Secondary outcomes are relevant clinical endpoints, safety, quality of life and immunological responses. Ethics and dissemination: This study has received approval of the Medical Ethics Committee of the Leiden University Medical Center (P18.216, NL67515.058.18, date: 7 March 2019). The results of this trial (positive and negative) will be submitted for publication in relevant peer-reviewed publications and the key findings presented at national and international conferences. Show less
Seidel, F.; Kleemann, R.; Duyvenvoorde, W. van; Trigt, N. van; Keijzer, N.; Kooij, S. van der; ... ; Morrison, M.C. 2022
Background: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in... Show moreBackground: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear. Methods: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots. Results: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF). Conclusion: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression. Show less
(1) Background: Soluble Fms-like tyrosine kinase 1 (sFLT1) is an endogenous VEGF inhibitor. sFLT1 has been described as an anti-inflammatory treatment for diabetic nephropathy and heart fibrosis.... Show more(1) Background: Soluble Fms-like tyrosine kinase 1 (sFLT1) is an endogenous VEGF inhibitor. sFLT1 has been described as an anti-inflammatory treatment for diabetic nephropathy and heart fibrosis. However, sFLT1 has also been related to peritubular capillary (PTC) loss, which promotes fibrogenesis. Here, we studied whether transfection with sFlt1 aggravates experimental AKI-to-CKD transition and whether sFLT1 is increased in human kidney fibrosis. (2) Methods: Mice were transfected via electroporation with sFlt1. After confirming transfection efficacy, mice underwent unilateral ischemia/reperfusion injury (IRI) and were sacrificed 28 days later. Kidney histology and RNA were analyzed to study renal fibrosis, PTC damage and inflammation. Renal sFLT1 mRNA expression was measured in CKD biopsies and control kidney tissue. (3) Results: sFlt1 transfection did not aggravate renal fibrosis, PTC loss or macrophage recruitment in IRI mice. In contrast, higher transfection efficiency was correlated with reduced expression of pro-fibrotic and pro-inflammatory markers. In the human samples, sFLT1 mRNA levels were similar in CKD and control kidneys and were not correlated with interstitial fibrosis or PTC loss. (4) Conclusion: As we previously found that sFLT1 has therapeutic potential in diabetic nephropathy, our findings indicate that sFLT1 can be administered at a dose that is therapeutically effective in reducing inflammation, without promoting maladaptive kidney damage. Show less
Essen, M.F. van; Schlagwein, N.; Gijlswijk-Janssen, D.J. van; Ruben, J.M.; Kooten, C. van; COMBAT Consortium 2022
The complement system does not only play an important role in the defence against microorganism and pathogens, but also contributes to the regulation of innate and adaptive immunity. Especially... Show moreThe complement system does not only play an important role in the defence against microorganism and pathogens, but also contributes to the regulation of innate and adaptive immunity. Especially activation fragments C3a and C5a and complement activation at the interface of antigen presenting cell (APC) and T cell, were shown to have a role in T cell activation and proliferation. Whereas most complement factors are produced by the liver, properdin, a positive regulator of the C3 convertase, is mainly produced by myeloid cells. Here we show that properdin can be detected in myeloid cell infiltrate during human renal allograft rejection. In vitro, properdin is produced and secreted by human immature dendritic cells (iDCs), which is further increased by CD40-L-matured DCs (mDCs). Transfection with a specific properdin siRNA reduced properdin secretion by iDCs and mDCs, without affecting the expression of co-stimulatory markers CD80 and CD86. Co-culture of properdin siRNA-transfected iDCs and mDCs with human allogeneic T cells resulted in reduced T cell proliferation, especially under lower DC-T cell ratio's (1:30 and 1:90 ratio). In addition, T cell cytokines were altered, including a reduced TNF-alpha and IL-17 secretion by T cells co-cultured with properdin siRNA-transfected iDCs. Taken together, these results indicate a local role for properdin during the interaction of DCs and allogeneic T cells, contributing to the shaping of T cell proliferation and activation. Show less
Jager, N.M.; Venema, L.H.; Arykbaeva, A.S.; Meter-Arkema, A.H.; Ottens, P.J.; Kooten, C. van; ... ; PROPER study consortium 2022
Background: The gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with... Show moreBackground: The gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP. Methods: NMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition. Results: Complement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death. Conclusion: Normothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation. Show less
Duijl, T.T. van; Ruhaak, L.R.; Kooten, C. van; Fijter, J.W. de; Cobbaert, C.M. 2022
Background: LC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we... Show moreBackground: LC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we examined the correlation and agreement between immunoassay and LC-MS/MS in the quantitation of kidney injury biomarkers and evaluated the application of technical LC-MS/MS meta-data assessment to ensure test result validity. Methods: NGAL, IGFBP7, TIMP2, and KIM-1 were quantified in 345 urine samples with one multiplex labdeveloped test that combines immunocapture with mass spectrometry read-out and 4 singleplex sandwich-type immunoassays. Assay performance and imprecision were monitored by 2 urine-based quality controls. Ion ratios, signal intensity, and retention time were monitored over all study samples. Results: The LC-MS/MS retention time drift was <= 1.2%, ion ratios were within 20% of the target values at concentrations of >100 pmol/L, and peptides originating from the same protein were in agreement (slopes between 1.03 and 1.41). The interassay CV was between 9.3% and 19.1% for LC-MS/MS analysis and between 4.2% and 10.9% for immunoassay. Direct LC-MS/MS analysis was correlated with immunoassay in the quantitation of NGAL (r = 0.93; range: 0.01-37 nmol/L), IGFBP7 (r = 0.80; range: 0.01-2.6 nmol/L), TIMP2 (r = 0.85; range: 0.01-6.3 nmol/L), and KIM-1 (r = 0.70; range 0.01-0.4 nmol/L), but the analytical methodologies differed in measurands and calibration strategies. Conclusions: LC-MS/MS is explored as a next-generation technology for multiplex urinary protein measurement. It has great potential to overcome nonselectivity and lack of standardization because of its capability of directly measuring well-defined molecular proteins. Show less
Duijl, T.T. van; Ruhaak, L.R.; Kooten, C. van; Fijter, J.W. de; Cobbaert, C.M. 2022
BackgroundLC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we... Show moreBackgroundLC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we examined the correlation and agreement between immunoassay and LC-MS/MS in the quantitation of kidney injury biomarkers and evaluated the application of technical LC-MS/MS meta-data assessment to ensure test result validity.MethodsNGAL, IGFBP7, TIMP2, and KIM-1 were quantified in 345 urine samples with one multiplex lab-developed test that combines immunocapture with mass spectrometry read-out and 4 singleplex sandwich-type immunoassays. Assay performance and imprecision were monitored by 2 urine-based quality controls. Ion ratios, signal intensity, and retention time were monitored over all study samples.ResultsThe LC-MS/MS retention time drift was ≤1.2%, ion ratios were within 20% of the target values at concentrations of >100 pmol/L, and peptides originating from the same protein were in agreement (slopes between 1.03 and 1.41). The interassay CV was between 9.3% and 19.1% for LC-MS/MS analysis and between 4.2% and 10.9% for immunoassay. Direct LC-MS/MS analysis was correlated with immunoassay in the quantitation of NGAL (r = 0.93; range: 0.01–37 nmol/L), IGFBP7 (r = 0.80; range: 0.01–2.6 nmol/L), TIMP2 (r = 0.85; range: 0.01–6.3 nmol/L), and KIM-1 (r = 0.70; range 0.01–0.4 nmol/L), but the analytical methodologies differed in measurands and calibration strategies.ConclusionsLC-MS/MS is explored as a next-generation technology for multiplex urinary protein measurement. It has great potential to overcome nonselectivity and lack of standardization because of its capability of directly measuring well-defined molecular proteins. Show less
Hypothermic machine perfusion (HMP) has become the new gold standard in clinical donor kidney preservation and a promising novel strategy in higher risk donor livers in several countries. As shown... Show moreHypothermic machine perfusion (HMP) has become the new gold standard in clinical donor kidney preservation and a promising novel strategy in higher risk donor livers in several countries. As shown by meta-analysis for the kidney, HMP decreases the risk of delayed graft function (DGF) and improves graft survival. For the liver, HMP immediately prior to transplantation may reduce the chance of early allograft dysfunction (EAD) and reduce ischemic sequelae in the biliary tract. Ischemia-reperfusion injury (IRI), unavoidable during transplantation, can lead to massive cell death and is one of the main causes for DGF, EAD or longer term impact. Molecular mechanisms that are affected in IRI include levels of hypoxia inducible factor (HIF), induction of cell death, endothelial dysfunction and immune responses. In this review we have summarized and discussed mechanisms on how HMP can ameliorate IRI. Better insight into how HMP influences IRI in kidney and liver transplantation may lead to new therapies and improved transplant outcomes. Show less
In recent decades, several important advances have taken place in the understanding of the pathogenesis underlying membranous nephropathy (MN) that have sparked renewed interest in its management.... Show moreIn recent decades, several important advances have taken place in the understanding of the pathogenesis underlying membranous nephropathy (MN) that have sparked renewed interest in its management. Four landmark trials in MN and a fifth clinical trial-which was a pilot study-have been published in recent years. The results from some of these trials have had a significant impact on the recommendations included in the 2021 Kidney Disease: Improving Global Outcomes (KDIGO) Guideline for the Management of Glomerular Diseases, representing a significant step forward compared with the previous guideline in several aspects, including diagnosis, disease monitoring and treatment strategies. However, considering the rapidly evolving advances in the knowledge of MN and the recent publication of the STARMEN and RI-CYCLO trials, several recommendations contained in the guideline warrant updates. This article provides a perspective of the Immunonephrology Working Group of the European Renal Association regarding the management of MN in native kidneys of adult patients. Show less
Patients with immune-mediated kidney diseases are at increased risk of severe coronavirus disease 2019 (COVID-19). The international rollout of COVID-19 vaccines has provided varying degrees of... Show morePatients with immune-mediated kidney diseases are at increased risk of severe coronavirus disease 2019 (COVID-19). The international rollout of COVID-19 vaccines has provided varying degrees of protection and enabled the understanding of vaccine efficacy and safety. The immune response to COVID-19 vaccines is lower in most patients with immune-mediated kidney diseases; either related to immunosuppression or comorbidities and complications caused by the underlying disease. Humoral vaccine response, measured by the presence of antibodies, is impaired or absent in patients receiving rituximab, mycophenolate mofetil (MMF), higher doses of glucocorticoids and likely other immunosuppressants, such as cyclophosphamide. The timing between the use of these agents and administration of vaccines is associated with the level of immune response: with rituximab, vaccine response can only be expected once B cells start to recover and patients with transient discontinuation of MMF mount a humoral response more frequently. The emergence of new COVID-19 variants and waning of vaccine-induced immunity highlight the value of a booster dose and the need to develop mutant-proof vaccines. COVID-19 vaccines are safe, exhibiting a very low risk of de novo or relapsing immune-mediated kidney disease. Population-based studies will determine whether this is causal or coincidental. Such cases respond to standard management, including the use of immunosuppression. The Immunonephrology Working Group and European Vasculitis Society recommend that patients with immune-mediated kidney diseases follow national guidance on vaccination. Booster doses based on antibody measurements could be considered. Show less
