#2553 Circulating anti-slit diaphragm NEPHRIN and KIRREL-2 antibodies associate with nephrotic syndrome

Abstract Background and Aims Pathogenesis of Idiopathic Nephrotic Syndrome (INS) is still largely unknown. Recently, circulating anti-NEPHRIN antibodies have been proposed as responsible of disarrangement of the slit diaphragm architecture upon glomerular deposition. However, previous experience referred to limited cases and controls with different ELISA assays for antibodies detection. We compared the performance of three different ELISA assays quantifying circulating anti-NEPHRIN antibodies and we developed ELISA assays to quantify circulating antibodies against two proteins homologous of NEPHRIN, the KIRREL.1 and KIRREL-2. Method We compared the performance of three different ELISA assays, two against the extracellular (aa 1-1055NEPHRIN-A and aa 23-1029 NEPHRIN-B) and one against the intracytoplasmic (aa 1160-1241 NEPHRIN-C) domain of NEPHRIN. We tested a large cohort of 166 patients (396 serum samples) with INS, 32 focal segmental glomerulosclerosis kidney transplant recipients, 143 healthy controls and 40 lupus nephritis and membranous nephropathy, respectively. Furthermore, to investigate sensitivity and specificity of circulating anti-NEPHRIN antibodies, we developed ELISA assays to quantify circulating antibodies against two proteins homologous of NEPHRIN, the KIRREL-1 and KIRREL-2. We tested NEPHRIN and KIRREL-2 gene expression among glomeruli in kidney biopsies from 4 FSGS patient and 4 controls. Results Serum levels of anti-NEPHRIN antibodies against the two extracellular domains were highly correlated, but differed significantly to the levels detected using the intracytoplasmic domain of NEPHRIN. Neither the circulating anti-NEPHRIN targeting the extracellular nor the intracellular domains of the protein could differentiate INS patients overall from healthy controls (Fig. 1A). However, after age stratification, we found that circulating anti-NEPHRIN antibodies against the extracellular domain were significatively higher in INS than in healthy controls >6 years old (Fig. 1B).Circulating anti-NEPHRIN antibodies against extracellular domain appeared more specific for INS than anti-NEPHRIN antibodies against cytoplasmic domain. Circulating anti-NEPHRIN antibodies did not correlate with disease activity nor FSGS recurrence after kidney transplant (Fig. 1C). Of note, circulating anti-KIRREL-2 antibodies could differentiate INS and healthy controls more effectively than anti-NEPHRIN antibodies (Fig. 1D). We than confirmed that KIRREL-2 is transcript in glomeruli (Fig. 1E) and, in line with circulating anti-KIRREL-2 antibodies, spatial transcriptomic analyses showed that KIRREL-2 is increased in FSGS than in controls. Conclusion In a large cohort of patients, we showed that circulating anti-NEPHRIN antibodies are positive in a subset of INS and FSGS recurrence. However, they are not associated with disease activity and the circulating levels appeared as age related. Circulating anti-KIRREL-2 antibodies are more strongly associated with INS. Therefore, based on our findings, several podocyte antigens may be supposed target for specific autoantibodies in INS.