BACKGROUND Anti-nephrin autoantibodies have emerged as a putative pathogenic driver in a subset of patients with podocytopathies, including those with posttransplant disease recurrence. METHODS We measured anti-nephrin autoantibodies in a cohort of 65 patients with podocytopathy associated with steroid-sensitive nephrotic syndrome ( n = 39) and steroid-resistant nephrotic syndrome ( n = 26) and in 34 patients with posttransplant podocytopathy recurrence. Fourteen patients with membranous nephropathy and 20 healthy volunteers served as controls. ELISA and immunoprecipitation assays were performed to detect anti-nephrin IgG using 2 different recombinant human nephrin proteins. Immunofluorescence analysis was performed to assess gG deposition and its colocalization with nephrin in renal biopsies. RESULTS When using an ELISA based on murine cell-derived human antigen, the highest positivity was found in healthy volunteers (55%), correlating with levels of circulating natural anti–α-galactose-α-1,3-galactose antibodies. This cross-reactivity was abrogated with recombinant human nephrin expressed in human cells. In this setting, very low prevalence (<5%) of anti-nephrin antibody-positive patients was found in steroid-sensitive and -resistant nephrotic syndrome cohorts and in patients with posttransplant disease recurrence. These frequencies were comparable to healthy volunteers. Using confocal and super-resolution microscopy, only trace amounts of IgM, but no IgG, were found in the glomeruli of analyzed biopsies, which did not colocalize with nephrin. CONCLUSION With the methodology presented here, anti-nephrin reactivity was extremely rare and occurred at comparably low frequencies in healthy controls, native-kidney podocytopathies, and posttransplant disease recurrence. This suggests that these autoantibodies are not inherently disease specific and may not serve as a broad biomarker across podocytopathies. TRIAL REGISTRATION ClinicalTrials.gov NCT06334692. FUNDING The Medici di Marignano family.

Characterizing kidney disease is challenged by marked cellular heterogeneity and limited tissue availability from renal biopsies. Conventional diagnostic workflows rely on multiple serial sections for parallel staining, increasing tissue consumption, sampling bias, and loss of spatial information, thereby constraining molecular characterization within intact tissue architecture. High-plex spatial proteomics may overcome these limitations by enabling comprehensive molecular profiling on a single section. Here, we present and evaluate a high-plex cyclic immunofluorescence imaging workflow (MACSima™, Miltenyi Biotec) applied to kidney transplant biopsies, including BK virus nephropathy (BKVN) and focal segmental glomerulosclerosis (FSGS), to characterize spatial immune organization with a focus on complement system components. Feasibility and subcellular resolution were first assessed in a lupus nephritis section, demonstrating compatibility with diagnostic immune panels and preservation of tissue morphology. A 48-marker multiplex panel interrogating immunity, oxidative stress, senescence, and fibrosis was then applied to BKVN samples, including paired pre- and post-treatment biopsies, revealing distinct proteomic patterns and dynamic changes following therapy. In FSGS, a glomerulus-focused panel identified spatially resolved innate and adaptive immune signatures, including complement-related patterns supporting exploratory analysis of glomerular immune architecture. Structural, nuclear, membrane, and phosphorylated signaling markers enabled precise delineation of renal compartments and assessment of cellular states such as proliferation, DNA damage, and pathway activation. The workflow also supported detection of extracellular vesicles in cultured renal cells, highlighting its versatility. Overall, this approach provides a robust, tissue-sparing platform for integrated spatial and molecular profiling of renal biopsies, reducing sampling bias while enabling discovery-level phenotyping from a single section. This unified strategy is particularly suited to kidney transplantation, where diagnosis, therapeutic decision-making, and longitudinal monitoring are closely interconnected.

Introduction:Approximately 30% of patients with primary membranous nephropathy (MN) and nephrotic syndrome (NS) fail rituximab treatment through mechanisms that could be overcome by the human IgG1 monoclonal anti-CD38 antibody, felzartamab. Methods:In this prospective, single-arm, single-center, open-label trial, 10 consenting Caucasian adult patients with MN, rituximab-resistant NS, and estimated glomerular filtration rate (GFR) > 30 ml/min per 1.73 m2 received a 5-month, 9-dose course of 16 mg/kg felzartamab infusions at the Nephrology Unit of Bergamo Hospital, Italy between November 9, 2021 and February 1, 2023 and were followed up with for 24 months. Clinical and laboratory parameters were evaluated at baseline, 1, 2, 5, 6, 9, 18, and 24 months, whereas GFR as well as albumin and IgG fractional clearances were measured at baseline and at 6, 9, 12, 18, and 24 months posttreatment. The primary outcome was 24-hour proteinuria (median of 3 consecutive measurements) at 12 months. Results:Twelve-month 24-hour proteinuria was similar to baseline. Linear-mixed model analyses showed no significant time-dependent changes in 24-hour proteinuria and albuminuria; serum total-protein, albumin, creatinine and lipid levels, GFR and albumin fractional clearances. Circulating anti-phospholipase A2 receptor (PLA2R) antibodies transiently decreased but were never depleted. All considered Igs transiently decreased up to month 12, and recovered to baseline thereafter. Felzartamab deeply and persistently decreased natural killer (NK), B cells, and antigen-inexperienced transitional B cells; however, it did not affect CD20-expressing memory B cells, plasmablasts, and plasma cells. Treatment was safe and well-tolerated. Conclusion:One course of felzartamab was safe and well-tolerated, but ineffective in patients with MN and rituximab-resistant NS, possibly because of the inability to persistently deplete nephritogenic autoantibody-producing CD38-expressing B cells.

Pathogenic antibodies produced by alloreactive B cells mediate antibody-mediated rejection after kidney transplantation, but the mechanisms remain poorly understood. Follicular regulatory T (Tfr) cells modulate follicular helper T cell-mediated B cell responses, but the functions of Tfr in controlling alloreactive antibody are unknown. Here we study the developmental signals and functions of Tfr cells in mouse allogeneic kidney transplantation models, and show that costimulatory blockade alters the development of Tfr cells disproportionately by decreasing germinal center (GC)-like Tfr cells but increasing follicular-like Tfr cells. Functionally, global Tfr cell deletion results in accelerated graft rejection and increases in donor-specific B cells in both draining lymph nodes and kidney allografts. Mechanistically, Tfr cell deletion increases GC B cell expression of pro-inflammatory cytokines such as IL-15, while neutralization of IL-15 compensates for the loss of Tfr cells and prolongs the survival of mice receiving kidney transplants. Together our preclinical mouse data demonstrate how Tfr restrains kidney allograft rejection by limiting alloreactive B cell responses.

Introduction Approximately 30% of patients with primary membranous nephropathy (MN) and persistent nephrotic syndrome (NS) fail rituximab therapy through mechanisms that could be overcome by obinutuzumab. Methods In this prospective, single-arm, single-center, open-label trial, twenty consenting adults with MN and rituximab-resistant NS received three 1000 mg obinutuzumab infusions at the Bergamo Nephrology Unit (Italy) between March 2022 and February 2024 and were monitored for at least 12 months. The primary outcome was a composite endpoint of normo-albuminemia and complete (proteinuria <0.3 g/day) or partial (proteinuria <3.5 g/day with ≥50% reduction from baseline) NS remission at 12-month follow-up. Twenty-four-hour proteinuria and GFR were evaluated at baseline and at 3, 6, 9, 12, 18, and 24 months post-treatment. Results At 12 months, 16 patients met the combined endpoint, 4 with complete remission. No patient relapsed after remission. Median [IQR] 24-hour proteinuria decreased from 5.7 [4.7-8.1] to 1.3 [0.5-2.7] g/24-hour. Albumin and IgG fractional clearances also decreased, whereas serum albumin increased from 2.9±0.6 g/dl to 3.9±0.3 g/dl (p<0.0001 for all changes). Dyslipidemia, hypocalcemia and hypo-gammaglobulinemia improved significantly and GFR stabilized. At 12 months, total B-cells and circulating anti-PLA2R antibodies were depleted. Similar findings were observed in the cohort of ten patients who completed 24 months of follow-up. However, total B-cells re-emerged in the circulation without an increase in anti-PLA2R antibodies and proteinuria, or relapses. Treatment was safe and well tolerated. Conclusions Obinutuzumab treatment is extremely effective and safe in patients with MN and rituximab-resistant NS and can achieve persistent remission in this population.

Allergic diseases are common and affect a large proportion of the population. Interleukin-13 (IL-13)-expressing follicular helper T (TFH13) cells are a newly identified population of TFH cells that have been associated with high-affinity IgE responses. However, the origins, developmental signals, transcriptional programming and precise functions of TFH13 cells are unknown. Here, we examined the developmental signals for TFH13 cells and found a direct and progressive differentiation pathway marked by the production of IL-21. These two pathways differed in kinetics and extrinsic requirements. However, both pathways converged, forming transcriptionally similar TFH13 cells that express the transcription factor JunB as a critical stabilizing factor. Using an intersectional genetics-based TFH13-diphtheria toxin receptor model to perturb these cells, we found that TFH13 cells were essential to drive broad germinal center responses and allergen-specific IgG and IgE. Moreover, we found that IL-21 is a broad positive regulator of allergen germinal center B cells and synergizes with IL-13 produced by TFH13 cells to amplify allergic responses. Thus, TFH13 cells orchestrate multiple features of allergic inflammation. TFH cells that express IL-13 are associated with high-affinity IgE responses, but factors controlling their development, transcriptional programming and exact function have remained unclear. Here, Chandrakar et al. find that the transcription factor JunB is required for TFH13 cell maintenance and that TFH13 cells producing IL-21 drive broad germinal center responses to allergen-specific IgG and IgE.

Introduction:Approximately 30% of patients with primary membranous nephropathy (MN) and persistent nephrotic syndrome (NS) fail rituximab therapy through mechanisms that could be overcome by obinutuzumab. Methods:In this prospective, single-arm, single-center, open-label trial, 20 consenting adults with MN and rituximab-resistant NS received three 1000 mg obinutuzumab infusions at the Bergamo Nephrology Unit (Italy) between March 2022 and February 2024 and were monitored for ≤ 12 months. The primary outcome was a composite end point of normo-albuminemia and complete (proteinuria < 0.3 g/d) or partial (proteinuria < 3.5 g/d with ≥ 50% reduction from baseline) NS remission at 12-month follow-up. Twenty-four-hour proteinuria and glomerular filtration rate (GFR) were evaluated at baseline and at 3, 6, 9, 12, 18, and 24 months posttreatment. Results:At 12 months, 16 patients met the combined end point, 4 with complete remission. No patient relapsed after remission. Median (interquartile range) 24-h proteinuria decreased from 5.7 (4.7-8.1) to 1.3 (0.5-2.7) g/24 h. Albumin and IgG fractional clearances also decreased, whereas serum albumin increased from 2.9 ± 0.6 g/dl to 3.9 ± 0.3 g/dl (P < 0.0001 for all changes). Dyslipidemia, hypocalcemia, and hypo-gammaglobulinemia improved significantly and GFR stabilized. At 12 months, total B cells and circulating anti-phospholipase A2 receptor (PLA2R) antibodies were depleted. Similar findings were observed in the cohort of 10 patients who completed 24 months of follow-up. However, total B cells reemerged in the circulation without an increase in anti-PLA2R antibodies and proteinuria, or relapses. Treatment was safe and well-tolerated. Conclusion:Obinutuzumab treatment is extremely effective and safe in patients with MN and rituximab-resistant NS and can achieve persistent remission in this population.

Humoral immunity is orchestrated by follicular helper T (Tfh) cells, which promote cognate B cells to produce high-affinity, protective antibodies. In aged individuals, humoral immunity after vaccination is diminished despite the presence of Tfh cells, suggesting defects after initial Tfh cell formation. In this study, we utilized both murine and human systems to investigate how aging alters Tfh cell differentiation after influenza vaccination. We found that young Tfh cells underwent progressive differentiation after influenza vaccination, culminating in clonal expansion of effector-like cells in both draining lymph nodes and blood. In aging, early stages of Tfh cell development occurred normally. However, aging rewired the later stages of development in Tfh cells, resulting in a transcriptional program reflective of cellular senescence, sustained pro-inflammatory cytokine production, and metabolic reprogramming. We investigated the extent to which this rewiring of aged Tfh cells is due to the age-associated inflammatory (“inflammaging”) microenvironment and found that this setting was sufficient to both block the transition of Tfh cells to a post-effector resting state and skew Tfh cells toward the age-rewired state. Together, these data suggest that aging dampens humoral immunity by cytokine-mediated rewiring of late effector Tfh cell differentiation into an activated, yet less functional, cellular state.

Abstract Background and Aims Systemic lupus erythematosus (SLE) is an autoimmune disorder in which autoantibody formation can lead to severe kidney pathology. Interleukin (IL)17-producing T cells have been suggested to play an important role in the pathogenesis of SLE, and IL-17 inhibition was shown to ameliorate SLE pathology in lupus-prone mice. Moreover, the frequency of circulating follicular helper T (Tfh) cells, a T cell subset that orchestrates the maturation of high-affinity antibodies in the germinal center of secondary lymphoid organs, is associated with disease activity in SLE. Increased frequencies of IL17-producing Tfh have been reported in SLE patients compared to controls, but their role in SLE pathogenesis is still unknown. Method By leveraging the B6.Sle1NZM2410/AegYaa strain, we generated lupus-prone mice in which IL17-producing Tfh cells could be either fate-mapped (SLE Tfh17-FM, Il17aCRERosaLoxSTOPLox-YFPSle1+/+Yaa+) or fate-mapped and selectively depleted by intraperitoneal diphtheria toxin (DT) administration (SLE Tfh17-DTR, Il17aCRERosaLoxSTOPLox-YFPCxcr5LoxSTOPLox-DTRSle1+/+Yaa+). Tfh17-FM and Tfh17-DTR mice (both Sle1−/−Yaa−) were used as controls. Results Tfh17 comprised approximately 0.4% of total splenic Tfh in SLE mice at 6 weeks of life, and DT administration caused potent and selective depletion of these cells in the SLE Tfh17-DTR strain (Fig. 1A). Early Tfh17 deletion from week 4 to week 9 did not modify the increased Tfh frequency observed at 20 weeks in SLE mice, but resulted in long-term reduction of splenic germinal center (GC) B cells and plasmacells (Fig. 1B-C). IL17 receptor A was overexpressed in non-GC B cells and plasmacells of SLE mice compared to controls, suggesting a possible increased susceptibility of these subsets to IL17 (Fig. 1D). SLE Tfh17-DTR mice treated with DT displayed a trend towards decreased anti-DNA levels compared to SLE Tfh17-FM (Fig. 1E), but both glomerular immunocomplex deposition and renal pathology were not significantly impacted (Fig. 1F-G). Conclusion The germinal center reaction is affected by selective depletion of IL17-producing Tfh cells in SLE mice, with reduced differentiation of GC B cells and plasmacells. The modest effect of Tfh17 depletion on autoantibody levels and lupus nephritis pathology suggests that other Tfh subsets may play a role in GC-derived autoantibody generation. In addition, extrafollicular sources may also contribute to autoantibody production in SLE.

Rationale & Objective: Rituximab is the first- choice therapy for patients with primary membranous nephropathy (MN) and nephrotic syndrome. However, approximately 30% of patients are treatment-resistant or become treatment-intolerant with hypersensitivity reactions upon repeated drug exposures. We aimed to assess whether ofatumumab, a fully human second-generation anti-CD20 antibody, could be a valuable alternative to rituximab in this population. Study Design: Case series. Setting & Participants: 7 rituximab-intolerant and 10 rituximab-resistant patients with MN who consented to receive ofatumumab (50-300 mg, single intravenous infusion) and were followed at the nephrology unit of Azienda SocioSanitaria Territoriale Papa Giovanni XXIII (Bergamo, Italy) between September 2015 and January 2019. Findings: Over a median (IQR) follow-up of 5.0 (3.0-9.8) months, all 7 rituximab-intolerant and 3 of the 10 rituximab-resistant patients exhibited complete (proteinuria <0.3 g/d) or partial (proteinuria <3.5 g/d with >= 50% reduction vs baseline) remission of nephrotic syndrome. Circulating B cells were similarly depleted in all patients by 1 week, and serum anti-phospholipase A2 receptor antibody concentrations decreased to <2.7 relative units/ mL in 3 of 4 rituximab-intolerant and 4 of 8 rituximab-resistant patients with phospholipase A2 receptor-related disease. Ofatumumab significantly reduced 24-hour urinary protein and immunoglobulin G excretion and increased serum albumin and immunoglobulin G levels. These effects were greater in rituximabintolerant than in rituximab-resistant patients. Measured glomerular filtration rate significantly increased by an average of 13.4% at 24 months compared with baseline (P= 0.036) among all patients in the series. There were 14 nonserious infusion-related adverse events in 9 patients that recovered with temporary infusion interruption. Limitations: Retrospective design, limited number of patients. Conclusions: Ofatumumab may represent an effective and safe treatment for rituximabintolerant cases of MN. Larger prospective studies will be needed to validate these preliminary findings and explore the effectiveness of other second-generation antiCD20 antibodies in this clinical setting.

INTRODUCTION:Rejection remains a major obstacle to successful kidney transplantation. The complex pathophysiology of rejection depends on a fine-tuned interplay between the innate and adaptive immune systems. AREAS COVERED:This review provides a comprehensive analysis of the pathophysiology of rejection of kidney grafts, performed through careful selection of most relevant papers available on the topic in the PubMed database. The two types of rejection usually observed at the kidney biopsy, i.e. cellular and humoral rejection, are described with an accurate outline of the biological processes that lead to their development. EXPERT OPINION:The incidence of T-cell-mediated rejection is decreasing, and most cases promptly respond to appropriate immunosuppression. However, late diagnosis or incomplete response to treatment may have deleterious consequences in the long term. The main issue is represented by antibody-mediated rejection, which unsatisfactorily responds to aggressive immunosuppression, especially when diagnosed late. Prevention of acute ABMR rests on HLA-specific antibody detection prior to transplantation, adequate immunosuppression, and optimal patients' compliance. Late diagnosis and poor response to treatment inevitably lead to chronic ABMR, for which no therapies are currently available.

The deposition of antipodocyte autoantibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.

Purpose of reviewAntibody-mediated rejection (AMR) after solid organ transplantation remains an unsolved problem and leads to poor early and late patient outcomes. The complement system is a well recognized pathogenic mediator of AMR. Herein, we review the known molecular mechanisms of disease and results from ongoing clinical testing of complement inhibitors after solid organ transplant.Recent findingsActivation and regulation of the complement cascade is critical not only for the terminal effector function of donor-specific antibodies, but also for the regulation of T and B cell subsets to generate the antidonor humoral response. Donor-specific antibodies (DSA) have heterogenous features, as are their interactions with the complement system. Clinical testing of complement inhibitors in transplant patients have shown good safety profiles but mixed efficacy to date.SummaryThe complement cascade is a critical mediator of AMR and clinical trials have shown early promising results. With the steady emergence of novel complement inhibitors and our greater understanding of the molecular mechanisms linking complement and AMR, there is greater optimism now for new prognostic and therapeutic tools to deploy in transplant patients with AMR.

Results from recent randomized controlled trials on inhibitors of the sodium-glucose cotransporter 2 (SGLT2) have determined a paradigm shift in the treatment of patients with type 2 diabetes mellitus. These agents have been shown not only to ameliorate metabolic control, but also to independently protect from cardiovascular events and to reduce the progression of chronic kidney disease (CKD) in these patients. The magnitude of the nephroprotective effect observed in these studies is likely to make SGLT2 inhibitors the most impactful drug class for the treatment of diabetic patients with CKD since the discovery of renin-angiotensin system inhibitors. Even more surprisingly, SGLT2 inhibitors have also been shown to slow CKD progression in non-diabetic individuals with varying degrees of proteinuria, suggesting that activation of SGLT2 is involved in the pathogenesis of CKD independent of its etiology. As indications continue to expand, it is still unclear whether the observed benefits of SGLT2 inhibitors may extend to CKD patients at lower risk of progression and if their association with other agents may confer additional protection.

Follicular helper T (Tfh) cells have been implicated in controlling rejection after allogeneic kidney transplantation, but the precise subsets, origins, and functions of Tfh cells in this process have not been fully characterized. Here we show that a subset of effector Tfh cells marked by previous IL-21 production is potently induced during allogeneic kidney transplantation and is inhibited by immunosuppressive agents. Single-cell RNA-Seq revealed that these lymph node (LN) effector Tfh cells have transcriptional and clonal overlap with IL-21-producing kidney-infiltrating Tfh cells, implicating common origins and developmental trajectories. To investigate the precise functions of IL-21-producing effector Tfh cells in LNs and allografts, we used a mouse model to selectively eliminate these cells and assessed allogeneic B cell clonal dynamics using a single B cell culture system. We found that IL-21-producing effector Tfh cells were essential for transplant rejection by regulating donor-specific germinal center B cell clonal dynamics both systemically in the draining LN and locally within kidney grafts. Thus, IL-21-producing effector Tfh cells have multifaceted roles in Ab-mediated rejection after kidney transplantation by promoting B cell alloimmunity.