Nicole C. A. J. van de Kar

C3 glomerulopathy (C3G) and immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) are severe complement-mediated kidney diseases. In a substantial proportion of these patients, C3 nephritic factors (C3NeFs) are detected; these autoantibodies stabilize the complement alternative pathway (AP) C3 convertase. Previous studies have investigated and distinguished properdin-dependent and properdin-independent C3NeFs. In this study, we investigated a distinct subset of C3NeFs that are exclusively active in the absence of properdin: properdin-inhibited C3NeFs. A two-step hemolytic AP convertase activity assay was employed to examine convertase activity in patient serum in the presence versus absence of properdin using the properdin inhibitor Salp20. In 7/16 patients and 1/23 healthy controls, who showed no convertase stabilization in full serum, convertase stabilization was observed upon properdin inhibition, indicating properdin-inhibited C3NeF activity. Consistent findings were obtained when purified patient Igs were added to properdin-depleted serum. Moreover, in an ELISA-based C3bBb binding assay, 6/7 patients with properdin-inhibited C3NeFs showed increased convertase binding, which decreased upon addition of properdin. Complement levels in patients with properdin-inhibited C3NeFs were not significantly different from those in the C3NeF-negative group, and properdin levels were generally within the refence range. In conclusion, our results support the existence of properdin-inhibited C3NeFs, thereby further expanding the functional heterogeneity of these autoantibodies. Although their functional relevance under physiological conditions remains unclear, these findings may have important implications for complement-targeted therapies, particularly strategies aimed at properdin inhibition, as the presence of these C3NeFs may predispose to unintended effects.

Key Points: Adolescents with C3 glomerulopathy/primary immune complex membranoproliferative GN received pegcetacoplan. Proteinuria reduction and stable eGFR in adolescents were consistent with the overall VALIANT population. Pegcetacoplan may resolve pathophysiology underlying C3 glomerulopathy/primary immune complex membranoproliferative GN and prevent long-term kidney damage in a key patient population. Background: In VALIANT (phase 3; NCT05067127), pegcetacoplan (C3/C3b inhibitor) led to significant proteinuria reduction (>68% versus placebo) and eGFR stabilization in native kidney and posttransplant recurrent C3 glomerulopathy and primary immune complex membranoproliferative GN. Here, we describe results for adolescents (12–17 years) in VALIANT. Methods: Patients (randomized 1:1) received pegcetacoplan twice weekly or placebo for 26 weeks. For overall population, the primary end point was baseline-to-week 26 change in log-transformed urine protein-to-creatinine ratio (UPCR) in pegcetacoplan versus placebo arms. Key secondary end points were patients achieving composite renal end point (≤15% eGFR reduction and ≥50% UPCR reduction), patients achieving ≥50% UPCR reduction, and eGFR change. Results: Twenty-eight adolescents received pegcetacoplan; 27 received placebo. Consistent with overall population, pegcetacoplan-treated adolescents achieved clinically meaningful UPCR reduction (relative reduction, 75% [95% confidence interval], 59 to 84; nominal P < 0.001). More adolescents in pegcetacoplan versus placebo arms achieved the composite end point (57% versus 4%; nominal P = 0.002) and ≥50% UPCR reduction (71% versus 4%; nominal P < 0.001). eGFR was stable for pegcetacoplan-treated adolescents (adjusted least squares mean difference [95% confidence interval] versus placebo, +9.7 [−0.026 to 19.382] ml/min per 1.73 m ² ; nominal P = 0.05). Three adolescents in each group experienced serious treatment-emergent adverse events (pyrexia in one pegcetacoplan-treated patient was considered treatment related). None experienced infection caused by encapsulated bacteria. Conclusions: Pegcetacoplan induced clinically meaningful proteinuria reduction and eGFR stabilization compared with placebo in adolescents with C3 glomerulopathy or primary immune complex membranoproliferative GN and was well tolerated. Clinical Trial registry name and registration number: ClincialTrials.gov, NCT05067127.

Thrombotic microangiopathy (TMA) in association with RNA exosome encoding mutations has only recently been recognized. Here, we present an infant (female) with an EXOSC5 mutation (c.230_232del p.Glu77del) associated with the clinical phenotype known as CABAC syndrome (cerebellar ataxia, brain abnormalities, and cardiac conduction defects), including pontocerebellar hypoplasia, who developed renal TMA. At the age of four months, she presented with signs of septic illness, after which she developed TMA. A stool culture showed rotavirus as a potential trigger. The patient received eculizumab once, alongside supportive treatment, while awaiting diagnostic analysis of TMA, including genetic complement analysis, all of which were negative. Eculizumab was withdrawn and the patient’s TMA recovered quickly. A review of the literature identified an additional four patients (age < 1 year) who developed TMA after a viral trigger in the presence of mutations in EXOSC3. The recurrence of TMA in one of these patients with an EXOSC3 mutation while on eculizumab treatment underscores the apparent lack of responsiveness to C5 inhibition. In conclusion, mutations in genes influencing the RNA exosome, like EXOSC3 and EXOSC5, characterized by neurodevelopment and neurodegenerative disorders could potentially lead to TMA in the absence of complement dysregulation. Hence, these patients were likely non-responsive to eculizumab.

Randomized clinical trials are underway to evaluate the efficacy of novel agents targeting the alternative complement pathway in patients with C3 glomerulopathy (C3G), a rare glomerular disease. The Kidney Health Initiative convened a panel of experts in C3G to (1) assess the data supporting the use of the prespecified trial end points as measures of clinical benefit and (2) opine on efficacy findings they would consider compelling as treatment(s) of C3G in native kidneys. Two subpanels of the C3G Trial Endpoints Work Group reviewed the available evidence and uncertainties for the association between the three prespecified end points—(1) proteinuria, (2) eGFR, and (3) histopathology—and anticipated outcomes. The full work group provided feedback on the summaries provided by the subpanels and on what potential treatment effects on the proposed end points they would consider compelling to support evidence of an investigational product's effectiveness for treating C3G. Members of the full work group agreed with the characterization of the data, evidence, and uncertainties, supporting the end points. Given the limitations of the available data, the work group was unable to define a minimum threshold for change in any of the end points that might be considered clinically meaningful. The work group concluded that a favorable treatment effect on all three end points would provide convincing evidence of efficacy in the setting of a therapy that targeted the complement pathway. A therapy might be considered effective in the absence of complete alignment in all three end points if there was meaningful lowering of proteinuria and stabilization or improvement in eGFR. The panel unanimously supported efforts to foster data sharing between academic and industry partners to address the gaps in the current knowledge identified by the review of the end points in the aforementioned trials.

In this issue of Blood, Brocklebank et al1 present the largest single-center cohort with suspected complement-associated atypical hemolytic uremic syndrome (CaH

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is the most common cause of hemolytic uremic syndrome (HUS), one of the main causes of acute kidney injury in children. Stx plays an important role in endothelium damage and pathogenesis of STEC-HUS. However, the effects of Stx on neutrophils and neutrophil extracellular trap (NET) formation are not well understood. In this study, we investigated how Stx2a affects NET formation and NETotic pathways (NADPH or NOX-dependent and -independent) using neutrophils isolated from healthy donors and patients with STEC-HUS, during the acute and recovery phase of the disease. Stx2a dose-dependently induced NETosis in neutrophils isolated from both healthy controls and STEC-HUS patients. NETosis kinetics and mechanistic data with pathway-specific inhibitors including diphenyleneiodonium (DPI)-, ERK-, and P38-inhibitors showed that Stx2a-induced NETosis via the NOX-dependent pathway. Neutrophils from STEC-HUS patients in the acute phase showed less ROS and NETs formation compared to neutrophils of the recovery phase of the disease and in healthy controls. NETs induced by Stx2a may lead to the activation of endothelial cells, which might contribute to the manifestation of thrombotic microangiopathy in STEC-HUS.

The complement system is an important part of innate immunity. Complement activation leads to formation of convertase enzymes, switch of their specificity from C3 to C5 cleavage, and generation of lytic membrane attack complexes (C5b-9) on surfaces of pathogens. Most C5 cleavage occurs via the complement alternative pathway (AP). The regulator properdin promotes generation and stabilization of AP convertases. However, its role in C5 activation is not yet understood. In this work, we showed that serum properdin is essential for LPS- and zymosan-induced C5b-9 generation and C5b-9–mediated lysis of rabbit erythrocytes. Furthermore, we demonstrated its essential role in C5 cleavage by AP convertases. To this end, we developed a hemolytic assay in which AP convertases were generated on rabbit erythrocytes by using properdin-depleted serum in the presence of C5 inhibitor (step 1), followed by washing and addition of purified C5–C9 components to allow C5b-9 formation (step 2). In this assay, addition of purified properdin to properdin-depleted serum during convertase formation (step 1) was required to restore C5 cleavage and C5b-9–mediated hemolysis. Importantly, C5 convertase activity was also fully restored when properdin was added together with C5b-9 components (step 2), thus after convertase formation. Moreover, with C3-depleted serum, not capable of forming new convertases but containing properdin, in step 2 of the assay, again full C5b-9 formation was observed and blocked by addition of properdin inhibitor Salp20. Thus, properdin is essential for the convertase specificity switch toward C5, and this function is independent of properdin’s role in new convertase formation.