ILDR2 is a conserved podocyte stress marker uncoupled from glomerular barrier function and local immune modulation​

Background ILDR2 (angulin-3) is a tricellular tight junction protein selectively expressed by podocytes within the kidney and consistently upregulated in glomerular disease. Whether this induction contributes to glomerular barrier function or represents a local immunoregulatory response remains unknown. We investigated whether ILDR2 is required for podocyte development, maintenance, and injury responses and whether its cell-specific upregulation reflects an immunoregulatory function that can be mimicked by exogenous ILDR2 substitution. Methods Single-cell transcriptomics was used to characterize glomerular cell-specific ILDR2 regulation in experimental podocytopathies, and ILDR2 immunofluorescence was quantified in human kidney biopsies. Germline and F0 ildr2 knockout zebrafish were generated using CRISPR/Cas9 and challenged by selective pharmacogenetic podocyte injury. Angulin family expression was assessed for compensatory regulation. Global proteomic profiling of freshly isolated and cultured Ildr2 knockout and wild-type mouse glomeruli was performed by LC-MS/MS. Recombinant ILDR2-mFc was administered during murine nephrotoxic serum nephritis (NTS-N), followed by assessment of proteinuria, histological injury, immune cell infiltration, fibrosis, and inflammatory gene expression. Results Single-cell transcriptomics revealed selective upregulation of ILDR2 in injured podocytes but not other glomerular cell populations. Consistently, ILDR2 was increased in primary but not secondary podocytopathies in human biopsies, supporting its potential to discriminate between these disease entities. Genetic loss of ILDR2 did not induce compensatory upregulation of other angulin family members and did not impair glomerular development, filtration barrier integrity, or susceptibility to podocyte injury. Despite extensive proteomic remodeling during ex vivo glomerular dedifferentiation, ILDR2 knockout and wild-type glomeruli showed nearly identical responses, with only seven proteins differentially regulated between genotypes. Moreover, ILDR2-mFc treatment failed to reduce proteinuria, histological injury, inflammation, or fibrosis in NTS-N. Conclusions ILDR2 is a highly conserved, podocyte-specific injury-responsive protein with potential to discriminate primary from secondary podocytopathies. Despite its robust disease-associated upregulation, ILDR2 is dispensable for glomerular development, filtration barrier maintenance, and podocyte injury responses, while exogenous ILDR2-mFc does not ameliorate immune-mediated glomerulonephritis. Thus, ILDR2 primarily represents a podocyte stress signature rather than a functional determinant of glomerular barrier integrity or local immune regulation.