Zhao Cui

Background: About 30% of patients with phospholipase A2 receptor (PLA2R) associated membranous nephropathy (MN) have negative circulating anti-PLA2R antibodies. We investigated whether these patients harbor antibodies against linear PLA2R peptides, with the aim of improving serological detection. Methods: We synthesized 123 sequential overlapping linear peptides spanning the extracellular region of PLA2R across ten domains. Circulating IgG antibodies to these peptides were measured by ELISA in 42 biopsy-proven PLA2R-associated MN patients with negative circulating anti-PLA2R antibodies. Results: Twenty-six of 42 (61.9%) anti-PLA2R-negative MN patients reacted with at least one PLA2R peptide. Overall, 68 of 123 (55.3%) peptides were recognized, with the highest recognition frequencies for CysR-11 (21.4%), CTLD7-1 (14.3%) and CTLD7-2 (16.7%). Reactivity to these peptides was rare in patients with minimal change disease, focal segmental glomerulosclerosis, IgA nephropathy, hepatitis B virus-associated MN, or tumor-associated MN. Combined any positivity of antibodies to the three peptides yielded a sensitivity of 43.7% and a specificity of 81.3% for identifying PLA2R-associated MN in the absence of circulating anti-PLA2R antibodies. In inhibition assays, none of the three peptides detectably blocked binding of circulating anti-PLA2R antibodies to full-length PLA2R under the experimental conditions used. Among 32 anti-PLA2R-positive MN patients, 28 (87.5%) also reacted with PLA2R peptides, with frequent recognition of CysR-5, CTLD1-7, CTLD4-7, CTLD5-6, CTLD5-9, CTLD6-2, and CTLD7-2. Conclusions: Antibodies to linear PLA2R peptides are detectable in a substantial proportion of PLA2R-associated MN patients who are seronegative by conventional anti-PLA2R assays. CysR-11, CTLD7-1, and CTLD7–2 may serve as exploratory candidate biomarkers, and their pathogenic relevance warrants further study.

The non-collagenous domain 1 of the α3 chain of type IV collagen (α3(IV)NC1) is the primary autoantigen in anti-glomerular basement membrane (anti-GBM) disease. We previously developed a modified antigen-specific peptide, m-P14, derived from the nephritogenic epitope α3_127–148, which ameliorated experimental anti-GBM nephritis. However, its short half-life limits clinical translation. This study evaluated a butyrate-conjugated derivative (m-P14-BA) to improve pharmacokinetic properties while preserving therapeutic efficacy. M-P14-BA and m-P14 were administered to α3_127–148 immunized Wistar Kyoto rats in early and late treatment settings. Renal injury parameters and intrarenal inflammation were assessed, and pharmacokinetic profiles were evaluated following intraperitoneal administration in beagle dogs. M-P14-BA reduced proteinuria, crescent formation, glomerular IgG deposition, complement activation, and inflammatory cell infiltration, with overall efficacy comparable to m-P14 in early treatment settings. In late treatment settings, m-P14-BA was associated with a significant improvement in blood urea nitrogen levels and modest reductions in proteinuria and histopathological injury. Butyrate conjugation markedly improved pharmacokinetics, prolonging plasma elimination half-life by approximately 2.8-fold and increasing systemic exposure nearly fourfold. These pharmacokinetic improvements were associated with maintained therapeutic efficacy at a reduced dose, with 10 mg/kg m-P14-BA achieving effects broadly similar to those observed with 30 mg/kg m-P14. In summary, butyrate conjugation improves the pharmacokinetic profile of an antigen-specific therapeutic peptide while preserving therapeutic activity, suggesting a potential strategy to enhance the translational feasibility of peptide-based immunotherapy in anti-GBM disease.

Rationale: Regulatory T (Treg) cells suppress autoimmunity and restrain inflammatory responses, showing promising potential in autoimmune glomerulonephritis (GN) therapy with minimizing nonspecific immunosuppression. Although low-dose interleukin-2 (IL-2) has been shown to promote Treg expansion, its clinical utility is constrained by its short half-life and concurrent effector T cell activation.

Calcineurin inhibitors, such as cyclosporine, are primary treatments for membranous nephropathy (MN). Optimizing this regimen is crucial to reduce nephrotoxicity and enhance immunological remission. Sirolimus, when combined with cyclosporine, may offer non-inferior clinical remission to cyclosporine monotherapy, while improving kidney function preservation and antibody clearance. This single-center, randomized controlled, phase 2 clinical trial involved 74 patients with biopsy-proven primary MN and persistent proteinuria > 3.5 g/d, despite 6 months of supportive care. Participants were randomly assigned (1:1) to receive either sirolimus plus cyclosporine or cyclosporine monotherapy for 12 months. At 12 months, composite remission was achieved in 26 of 36 patients (72%) receiving combination therapy and in 24 of 36 (67%) receiving cyclosporine alone (95% confidence interval for noninferiority: 0.48—3.56). Immunological remission, defined as anti-phospholipase A2 receptor antibody conversion from positive to negative, was significantly higher in the combination group (70% vs. 20%, P < 0.001) at 6 months. Moreover, eGFR decline was significantly attenuated in the combination group (ΔeGFR: -7.1 vs. -21.3 ml/min/1.73m2, P < 0.001) at 12 months. One serious adverse event occurred in the combination group, compared to none in the monotherapy group (P = 0.317). Sirolimus combined with cyclosporine was noninferior to cyclosporine monotherapy in achieving clinical remission in MN and demonstrated superior benefits in immunological remission and kidney function preservation.

Relapse of membranous nephropathy (MN) and other glomerular diseases has been observed after COVID-19 infection. The risk factors contributing to disease progression in MN patients after contracting COVID-19 remain unclear. This retrospective study included 656 consecutive patients with biopsy-proven primary MN who received treatment and were regularly followed up for over six months. Logistic regression analyses were conducted to identify risk factors for disease progression. Among the cohort, 555 patients (84.6%) contracted COVID-19 from November 11th, 2022, to February 22nd, 2023. Of them, 112 patients (20.2%) experienced a > 50% increase in proteinuria, including 30 patients (5.4%) who experienced nephrotic syndrome relapse. Sixteen patients (2.9%) showed immune aggravation with elevated anti-PLA2R antibody levels, and five patients (0.9%) had immune relapse characterized by antibody reoccurrence. Kidney dysfunction, defined as an eGFR reduction > 30% from baseline, occurred in 10 patients (1.8%), with two patients (0.4%) progressing to ESKD. Four patients (0.7%) died of respiratory failure. Overall, 132 patients (24.0%) experienced disease progression after COVID-19 infection. Multivariate logistic regression identified longer fever duration (OR 1.118, 95% CI 1.029–1.356, P = 0.018), withdrawal of immunosuppressants and/or steroids (OR 2.571, 95% CI 1.377–4.799, P = 0.003) and extended drug cessation (OR 1.113, 95% CI 1.045–1.186, P = 0.001) as independent risk factors for MN progression. These findings suggest prompt antiviral treatment and minimizing the duration of immunosuppressant withdrawal to optimize kidney outcomes in MN patients with COVID-19.