REGULATORY ROLE AND MECHANISM OF FNBP1L/FNBP1 ON CDC42 IN GLOMERULAR PODOCYTES

Podocytes are terminally differentiated cells with a distinctive cellular morphology. Their function is largely dependent on a highly dynamic cytoskeletal network, which is crucial for maintaining the health and integrity of the glomerulus. Kidney diseases resulting from podocyte injury (podocytopathies) represent a significant public health burden. However, the mechanisms underlying podocyte injury and repair remain incompletely understood. The Rho GTPase molecule CDC42 functions as a "molecular switch" and serves as a master regulator of the podocyte cytoskeletal network, playing an essential role in preserving podocyte structural and functional integrity. Genetic deletion of CDC42 leads to podocyte injury and loss, ultimately resulting in mortality in mice. Nevertheless, the regulation of CDC42 expression and function in podocytes remains poorly understood. Our preliminary studies have identified FNBP1L, a member of the F-BAR family, as an essential gene in podocytes. This study aims to investigate the regulatory roles and mechanisms of FNBP1L and FNBP1 on CDC42, with the goal of identifying novel therapeutic targets for podocytopathies.

We examined the expression and alterations of FNBP1L in podocytes using human podocyte cell lines, model organisms, and renal biopsy samples from patients. Subsequently, we generated conventional FNBP1L knockout mice and podocyte-specific FNBP1 knockout mice. Through breeding, we obtained podocyte-specific FNBP1L & FNBP1 double-knockout mice (Fnbp1l-dKO), and then established podocyte injury models in these mice, including lipopolysaccharide (LPS), nephrotoxic serum (NTS), and diabetic nephropathy (STZ) models. In FNBP1L & FNBP1 double-knockout human podocyte cell lines, we assessed various cellular functions—such as endocytosis, migration, cytoskeletal organization, apoptosis, autophagy, and cell adhesion—using endocytosis assays, scratch wound healing assays, phalloidin staining, TUNEL staining, and Western blotting. Furthermore, we investigated the impact of FNBP1L deficiency on CDC42 through both in vivo and in vitro experiments, as well as in patient biopsy samples.

We found that both FNBP1 and FNBP1L are expressed in normal human and mouse podocytes(Fig A,B), but their expression is downregulated in renal biopsy samples from patients with FSGS and Diabetic Nephropathy. Furthermore, their expression in podocytes was decreased in both in vivo and in vitro models induced by Adriamycin (ADR), Lipopolysaccharide (LPS), and Puromycin Aminonucleoside (PAN). Subsequently, we observed that neither Fnbp1l knockout (KO) mice nor podocyte-specific Fnbp1 conditional KO (cKO) mice developed spontaneous proteinuria. However, upon challenge with NTS, STZ, or LPS, these mice exhibited exacerbated podocyte injury and proteinuria. Interestingly, the double-knockout mice (Fnbp-dKO) were developmentally delayed (Fig C) and developed massive proteinuria approximately 3 weeks after birth and succumbed to renal failure around 5 weeks of age(Fig D,E). Pathological examination of Fnbp-dKO mice revealed foot process effacement(Fig F), glomerulosclerosis, and impaired endocytosis—a phenotype entirely consistent with previously reported podocyte-specific Cdc42 knockout mice. We also discovered that knockout of FNBP1L/FNBP1 severely reduced CDC42 protein levels and disrupted its co-localization with effector proteins (N-WASP, ARP2/3, INF2, mDia1), suggesting that FNBP proteins function to stabilize CDC42 and mediate its interactions with effectors. In human podocyte cell lines (HPC), knockdown of FNBP1/FNBP1L impaired cell viability, migration, endocytosis, autophagy, and G-actin polymerization capacity, accompanied by reductions in lamellipodia, filopodia, and focal adhesions—phenotypes similar to those induced by CDC42 knockdown. Conversely, podocyte-specific FNBP1L transgenic mice exhibited significant protection against kidney injury in disease models and maintained CDC42 stability in podocytes.

FNBP1 and FNBP1L maintain normal podocyte structure and function by stabilizing CDC42 protein and mediating its interactions with effector molecules. In glomerular diseases, the downregulation of FNBP1 and FNBP1L leads to loss of CDC42 expression and function, resulting in podocyte injury. Therefore, these proteins represent potential therapeutic targets for podocytopathies.