Circulating levels of endogenous complement inhibitors correlate inversely with complement consumption in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is marked by excessive complement activation, contributing to tissue damage. Complement activation can be detected in many organs including the skin, kidney, and brain. The involvement of the central nervous system is particularly relevant to understanding neuropsychiatric SLE (NPSLE), one of the poorest understood manifestations of SLE for which no biomarkers are available. We studied the levels of complement inhibitors in SLE in relation to disease activity and as possible biomarkers to identify NPSLE. Serum levels of complement inhibitors C1-inhibitor (C1-INH), C4b-binding protein (C4BP), Factor I, and Factor H were measured in 345 SLE patients (including 102 with NPSLE) and 108 healthy controls. Compared with controls, SLE patients had higher C1-INH and C4BP but lower Factor I and H levels. All inhibitors positively correlated with total C3 and C4 levels. While correlating with the SLE Disease Activity Index (SLEDAI), no distinction in inhibitor levels was found between SLE and NPSLE patients. Over time, C1-INH and Factor H levels normalized, but no significant changes were observed for C4BP and Factor I. In SLE the levels of circulating complement inhibitors are inversely correlated to complement consumption but do not serve as biomarkers for NPSLE.

Application of spatial-omics to the classification of kidney biopsy samples in transplantation.

Improvement of long-term outcomes through targeted treatment is a primary concern in kidney transplant medicine. Currently, the validation of a rejection diagnosis and subsequent treatment depends on the histological assessment of allograft biopsy samples, according to the Banff classification system. However, the lack of (early) disease-specific tissue markers hinders accurate diagnosis and thus timely intervention. This challenge mainly results from an incomplete understanding of the pathophysiological processes underlying late allograft failure. Integration of large-scale multimodal approaches for investigating allograft biopsy samples might offer new insights into this pathophysiology, which are necessary for the identification of novel therapeutic targets and the development of tailored immunotherapeutic interventions. Several omics technologies - including transcriptomic, proteomic, lipidomic and metabolomic tools (and multimodal data analysis strategies) - can be applied to allograft biopsy investigation. However, despite their successful application in research settings and their potential clinical value, several barriers limit the broad implementation of many of these tools into clinical practice. Among spatial-omics technologies, mass spectrometry imaging, which is under-represented in the transplant field, has the potential to enable multi-omics investigations that might expand the insights gained with current clinical analysis technologies.

IDO+ Endothelial Cells in Glomeruli of Kidney Transplantation Patients With Glomerulitis.

Background: Kidney transplantation is the preferred treatment option for patients with end-stage renal disease. However, long-term graft survival remains a challenge. The enzyme indoleamine 2,3 dioxygenase (IDO) has been reported to have immunomodulatory effects with IDO transcripts being elevated in both antibody-mediated rejection and T cell-mediated rejection. Methods: A metal-conjugated antibody panel for the staining of kidney biopsies was developed, allowing the visualization of 41 structural and immune markers on a single tissue slide to gain in-depth insight into the composition and localization of the immune cell compartment. Staining was applied to week 4 and 24 protocol biopsies of 49 patients as well as on 15 indication biopsies of the TRITON study and 4 additional transplantation biopsies with glomerulitis. Results: A highly distinctive and specific glomerular IDO expression was observed in biopsies from 3 of 49 patients in imaging mass cytometry. Immunohistochemistry confirmed IDO expression in glomeruli of 10 of 10 cases with glomerulitis. IDO was found to be expressed by CD31+ glomerular endothelial cells, accompanied by the presence of granzyme-B+Tbet+CD7+CD45RA+ natural killer cells and CD68+ macrophages. Furthermore, a proportion of both the immune cells and endothelial cells expressed Ki-67, indicative of cell proliferation, which was not observed in control glomeruli. Conclusions: Our results show glomerular IDO expression in transplanted kidneys with glomerulitis, which is accompanied by increased numbers of natural killer cells and macrophages and likely reflects local immune activation.

Urinary beta-2 microglobulin increases whereas TIMP-2 and IGFBP7 decline after unilateral nephrectomy in healthy kidney donors.

Early kidney injury may be detected by urinary markers, such as beta-2 microglobulin (B2M), tissue inhibitor of metalloproteinases-2 (TIMP-2), insulin-like growth factor-binding protein 7 (IGFBP7), kidney injury molecule-1 (KIM-1) and/or neutrophil gelatinase-associated lipocalin (NGAL). Of these biomarkers information on pathophysiology and reference ranges in both healthy and diseased populations are scarce. Differences in urinary levels of B2M, TIMP-2, IGFBP7, KIM-1 and NGAL were compared 24 h before and after nephrectomy in 38 living kidney donors from the REnal Protection Against Ischaemia-Reperfusion in transplantation study. Linear regression was used to assess the relation between baseline biomarker concentration and kidney function 1 year after nephrectomy. Median levels of urinary creatinine and creatinine standardized B2M, TIMP-2, IGFBP7, KIM-1, NGAL, and albumin 24 h before nephrectomy in donors were 9.4 mmol/L, 14 µg/mmol, 16 pmol/mmol, 99 pmol/mmol, 63 ng/mmol, 1390 ng/mmol and 0.7 mg/mmol, with median differences 24 h after nephrectomy of - 0.9, + 1906, - 7.1, - 38.3, - 6.9, + 2378 and + 1.2, respectively. The change of donor eGFR after 12 months per SD increment at baseline of B2M, TIMP-2, IGFBP7, KIM-1 and NGAL was: - 1.1, - 2.3, - 0.7, - 1.6 and - 2.8, respectively. Urinary TIMP-2 and IGFBP7 excretion halved after nephrectomy, similar to urinary creatinine, suggesting these markers predominantly reflect glomerular filtration. B2M and NGAL excretion increased significantly, similar to albumin, indicating decreased proximal tubular reabsorption following nephrectomy. KIM-1 did not change considerably after nephrectomy. Even though none of these biomarkers showed a strong relation with long-term donor eGFR, these results provide valuable insight into the pathophysiology of these urinary biomarkers.

A cell-free nutrient-supplemented perfusate allows four-day ex vivo metabolic preservation of human kidneys.

The growing disparity between the demand for transplants and the available donor supply, coupled with an aging donor population and increasing prevalence of chronic diseases, highlights the urgent need for the development of platforms enabling reconditioning, repair, and regeneration of deceased donor organs. This necessitates the ability to preserve metabolically active kidneys ex vivo for days. However, current kidney normothermic machine perfusion (NMP) approaches allow metabolic preservation only for hours. Here we show that human kidneys discarded for transplantation can be preserved in a metabolically active state up to 4 days when perfused with a cell-free perfusate supplemented with TCA cycle intermediates at subnormothermia (25 °C). Using spatially resolved isotope tracing we demonstrate preserved metabolic fluxes in the kidney microenvironment up to Day 4 of perfusion. Beyond Day 4, significant changes were observed in renal cell populations through spatial lipidomics, and increases in injury markers such as LDH, NGAL and oxidized lipids. Finally, we demonstrate that perfused kidneys maintain functional parameters up to Day 4. Collectively, these findings provide evidence that this approach enables metabolic and functional preservation of human kidneys over multiple days, establishing a solid foundation for future clinical investigations.

Disruption of memory B-cell trafficking by belimumab in patients with systemic lupus erythematosus.

OBJECTIVES: Autoreactive memory B cells contribute to chronic and progressive courses in autoimmune diseases like systemic lupus erythematosus (SLE). The efficacy of belimumab (BEL), the first approved biologic treatment for SLE and lupus nephritis (LN), is generally attributed to depletion of activated naïve B cells and inhibition of B cell activation. BEL's effect on memory B cells (MBCs) is currently unexplained. We performed an in-depth cellular and transcriptomic analysis of BEL's impact on the blood MBC compartment in patients with SLE. METHODS: A retrospective meta-analysis was conducted, pooling flow cytometry data from four randomized trials involving 1245 patients with SLE treated with intravenous BEL or placebo. Then, extensive MBC phenotyping was performed using high-sensitivity flow cytometry in patients with mild/moderate SLE and severe SLE/LN treated with subcutaneous BEL. Finally, transcriptomic characterization of surging MBCs was performed by single-cell RNA sequencing. RESULTS: In BEL-treated patients, a significant increase in circulating MBCs, in a broad range of MBC subsets, was established at week 2, gradually returning to baseline by week 52. The increase was most prominent in patients with higher SLE disease activity, serologically active patients, and patients aged ≤18 years. MBCs had a non-proliferating phenotype with a prominent decrease in activation status and downregulation of numerous migration genes. CONCLUSION: Upon BEL initiation, an increase of MBCs was firmly established. In the small cohort investigated, circulating MBCs were de-activated, non-proliferative, and demonstrated characteristics of disrupted lymphocyte trafficking, expanding on our understanding of the therapeutic mechanism of B cell-activating factor inhibition by BEL. TRIAL REGISTRATION: ClinicalTrials.gov NCT00071487, NCT00410384, NCT01632241, NCT01649765, NCT03312907, NCT03747159.

Management of adult patients with podocytopathies: an update from the ERA Immunonephrology Working Group.

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 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.

Red blood cells as oxygen carrier during normothermic machine perfusion of kidney grafts: Friend or foe?

Renal ex vivo normothermic machine perfusion (NMP) is under development as an assessment tool for high-risk kidney grafts and as a means of achieving more physiologically accurate organ preservation. On-going hemolysis has been reported during NMP, as this technique relies on red blood cells for oxygen delivery. In this study, we confirm the occurrence of progressive hemolysis during 6-hour kidney NMP. NMP-associated erythrostasis in the glomeruli and in peri-glomerular vascular networks points to an interaction between the red blood cells and the graft. Continuous hemolysis resulted in prooxidative changes in the perfusate, which could be quenched by addition of fresh frozen plasma. In a cell-based system, this hemolysis induced redox stress and exhibited toxic effects at high concentrations. These findings highlight the need for a more refined oxygen carrier in the context of renal NMP.

Voclosporin and the Antiviral Effect Against SARS-CoV-2 in Immunocompromised Kidney Patients.

Introduction: Immunocompromised kidney patients are at increased risk of prolonged SARS-CoV-2 infection and related complications. Preclinical evidence demonstrates a more potent inhibitory effect of voclosporin on SARS-CoV-2 replication than tacrolimus in vitro. We investigated the potential antiviral effects of voclosporin on SARS-CoV-2 in immunocompromised patients. Methods: First, we conducted a prospective, randomized, open-label, proof-of-concept study in 20 kidney transplant recipients (KTRs) on tacrolimus-based immunosuppression who contracted mild to moderate SARS-CoV-2 infection. Patients were randomized to continue tacrolimus or switch to voclosporin. Second, we performed a post hoc analysis on SARS-CoV-2 infections in 216 patients with lupus nephritis (LN) on standard immunosuppression who were randomly exposed to voclosporin or placebo as part of a clinical trial that was conducted during the worldwide COVID-19 pandemic. Results: The primary end point was clearance of SARS-CoV-2 viral load and that did not differ between voclosporin-treated KTRs (median 12 days, interquartile range [IQR] 8-28) and tacrolimus-treated KTRs (median 12 days, IQR 4-16) nor was there a difference in clinical recovery. Pharmacokinetic analyses demonstrated that, when voclosporin trough levels were on-target, SARS-CoV-2 viral load dropped significantly more (ΔCt 7.7 [3.4-10.7]) compared to tacrolimus-treated KTRs (ΔCt 2.7 [2.0-4.3]; P = 0.035). In voclosporin-exposed patients with LN, SARS-CoV-2 infection was detected in 6% (7/116) compared to 12% (12/100) in placebo-exposed patients (relative risk [RR] 1.4 [0.97-2.06]). Notably, no voclosporin-exposed patients with LN died from severe SARS-CoV-2 infection compared to 3% (3/100) in placebo-exposed patients (RR 2.2 [1.90-2.54]). Conclusion: This proof-of-concept study shows a potential positive risk-benefit profile for voclosporin in immunocompromised patients with SARS-CoV-2 infection. These results warrant further investigations on voclosporin to establish an equipoise between infection and maintenance immunosuppression.