Identification of antibodies against phospholipase A2 receptor peptides in PLA2R-associated membranous nephropathy with negative circulating anti-PLA2R antibodies

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.