No abstract

Two distinct signaling pathways, involving Wnt signaling and polycystin, have been found to be critical for normal kidney development. Renal tubulogenesis requires the presence of certain Wnt proteins, whereas mutations in polycystin impede the terminal differentiation of renal tubular epithelial cells, causing the development of large cystic kidneys that characterize autosomal dominant polycystic kidney disease. Polycystin is an integral membrane protein, consisting of several extracellular motifs indicative of cell-cell and cell-matrix interactions, coupled through multiple transmembrane domains to a functionally active cytoplasmic domain. We report here that expression of the C-terminal cytoplasmic domain of polycystin stabilizes soluble endogenous ␤-catenin and stimulates TCF-dependent gene transcription in human embryonic kidney cells. Microinjection of the polycystin C-terminal cytoplasmic domain induces dorsalization in zebrafish. Our findings suggest that polycystin has the capacity to modulate Wnt signaling during renal development.

Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking NEPH1 develop a nephrotic syndrome that resembles NPHS mutations, suggesting that all three proteins are essential for the integrity of glomerular podocytes. Podocin interacts with the C-terminal domain of nephrin and facilitates nephrin-dependent signaling. NEPH1, a member of the immunoglobulin superfamily, is structurally related to nephrin. We report now that NEPH1 belongs to a family of three closely related proteins that interact with the C-terminal domain of podocin. All three NEPH proteins share a conserved podocin-binding motif; mutation of a centrally located tyrosine residue dramatically lowers the affinity of NEPH1 for podocin. NEPH1 triggers AP-1 activation similarly to nephrin but requires the presence of Tec family kinases for efficient transactivation. We conclude that NEPH1 defines a new family of podocin-binding molecules that are potential candidates for hereditary nephrotic syndromes not linked to either NPHS1 or NPHS2.

Giant isoforms, encoded by Nesprin-1 (Syne1) and Nesprin-2 (Syne2), are multifunctional actin-binding and nuclear-envelope-associated proteins belonging to the spectrin superfamily. Here, we investigate the function of Nesprin-2 Giant (NUANCE) in skin by generating mice lacking the actin-binding domain of Nesprin-2 (Nesprin-2ΔABD). This loss results in a slight but significant thickening of the epidermis, which is a consequence of the increased epithelial nuclear size. Nonetheless, epidermal proliferation and differentiation appear normal in the knockout epidermis. Surprisingly, Nesprin-2 C-terminal-isoform expression and nuclear envelope localization were affected in certain tissues. Nuclei of primary dermal knockout fibroblasts and keratinocytes were heavily misshapen, displaying a striking similarity to nuclear deformations characteristic of laminopathies. Furthermore, emerin, the protein involved in the X-linked form of Emery-Dreifuss muscular dystrophy (EDMD), was unevenly distributed along the nuclear envelope in mutant fibroblasts, often forming aggregates in the deformed nuclear envelope areas. Thus, Nesprin-2 is an important scaffold protein implicated in the maintenance of nuclear envelope architecture. Aged knockout fibroblasts readily generated, by alternative splicing and alternative translation initiation, aberrant Nesprin-2 Giant isoforms that lacked an ABD but that were sufficient to restore nuclear shape and emerin localization; this suggests that other regions of Nesprin-2 Giant, potentially including its spectrin repeats, are crucial for these functions.

Primary focal segmental glomerulosclerosis (FSGS) is a disease with poor prognosis and high unmet therapeutic need. Here, we evaluated the safety and pharmacokinetics of single-dose infusions of fresolimumab, a human monoclonal antibody that inactivates all forms of transforming growth factor-β (TGF-β), in a phase I open-label, dose-ranging study. Patients with biopsy-confirmed, treatment-resistant, primary FSGS with a minimum estimated glomerular filtration rate (eGFR) of 25 ml/min per 1.73 m2, and a urine protein to creatinine ratio over 1.8 mg/mg were eligible. All 16 patients completed the study in which each received one of four single-dose levels of fresolimumab (up to 4 mg/kg) and was followed for 112 days. Fresolimumab was well tolerated with pustular rash the only adverse event in two patients. One patient was diagnosed with a histologically confirmed primitive neuroectodermal tumor 2 years after fresolimumab treatment. Consistent with treatment-resistant FSGS, there was a slight decline in eGFR (median decline baseline to final of 5.85 ml/min per 1.73 m2). Proteinuria fluctuated during the study with the median decline from baseline to final in urine protein to creatinine ratio of 1.2 mg/mg with all three Black patients having a mean decline of 3.6 mg/mg. The half-life of fresolimumab was ∼14 days, and the mean dose-normalized Cmax and area under the curve were independent of dose. Thus, single-dose fresolimumab was well tolerated in patients with primary resistant FSGS. Additional evaluation in a larger dose-ranging study is necessary.

Abstract. Nephrin and NEPH1, the gene products of NPHS1 and NEPH1, are podocyte membrane proteins of the Ig superfamily. Similar to the nephrin knockout, mice lacking NEPH1 show severe proteinuria leading to perinatal death. To identify the ligand of NEPH1, the extracellular domain of NEPH1 was fused to human IgG. This NEPH1-Ig fusion protein labeled the glomerular capillary wall of mouse kidneys in a staining pattern identical to NEPH1 and nephrin, prompting speculation that that NEPH1 might form homodimers and/or heterodimers with nephrin. In coimmunoprecipitation and pull-down assays, the NEPH1-Ig fusion protein precipitated wild-type NEPH1 from overexpressing HEK 293T cells. Truncational analysis revealed that the adhesive properties were not confined to a single Ig domain of NEPH1. Fusion proteins containing two Ig domains of NEPH1 were sufficient to immobilize NEPH1, but they failed to interact with control protein containing the phylogenetically related PKD repeats of polycystin-1. NEPH1 also precipitated nephrin, a protein with eight Ig domains and a fibronectin-like domain. Truncational analysis of nephrin revealed a very similar mode of interaction, i.e., two nephrin Ig domains fused to human IgG precipitated either nephrin or NEPH1, but not the control protein. Both NEPH1 and nephrin interactions were strictly dependent upon posttranslational glycosylation, and bacterially expressed protein failed to bind NEPH1. These findings demonstrate that the Ig domains of NEPH1 and nephrin form promiscuous homodimeric and heterodimeric interactions that may facilitate cis-and trans-homodimerizations and heterodimerizations of these molecules at the glomerular slit diaphragm.Renal filtration of small solutes and water without loss of larger molecules is intimately linked to the glomerular basement membrane and the slit diaphragm between interdigitating podocytes. Alterations of these structures, either acquired or hereditary, commonly lead to proteinuria.Hereditary nephrotic syndromes are a heterogeneous group, displaying severe proteinuria and renal failure. Best-characterized is the congenital nephrotic syndrome of the Finnish type, caused by mutations in NPHS1, the gene encoding nephrin. Affected individuals exhibit massive proteinuria in utero and nephrosis at birth (1). Nephrin is an integral membrane protein located at adjacent sites of secondary foot processes of podocytes, a specialized epithelial cell that ensures size and charge selective ultrafiltration (reviewed in reference 2). The precise function of nephrin is unknown; however, it appears to form a zipper-like filter structure within the approximately 40-nmwide slits between two foot processes (3).In mice, the deletion of NEPH1 causes severe proteinuria and perinatal death (4). Like nephrin, NEPH1 is a transmembrane protein of the Ig superfamily expressed by podocytes and localizes to the slit diaphragm by electron microscopy (5). The extracellular domain of NEPH1 contains five Ig domains and the integrin recognition motif RGD. The third Ig domain ...

␤-Arrestins mediate internalization of plasma membrane receptors. Nephrin, a structural component of the glomerular slit diaphragm, is a single transmembrane spanning receptor and belongs to the family of adhesion molecules. Its mutation causes a hereditary nephrotic syndrome. We report the previously undescribed interaction of ␤-arrestin2 with the nephrin C terminus. The phosphorylation status of nephrin Y1193 regulates inversely the binding of ␤-arrestin2 and podocin. The Src-family member Yes, known to enhance podocin-nephrin interaction by nephrin phosphorylation, diminishes ␤-arrestin2-nephrin interaction. ␤-Arrestin2 induces nephrin endocytosis and attenuates nephrin signaling. This finding suggests that nephrin Y1193 serves as a molecular switch that determines the integrity of the slit diaphragm by functional competition between ␤-arrestin2 and podocin. This concept offers a molecular pathomechanism of slit diaphragm distortion and opens therapeutic avenues for glomerular diseases. glomerular slit diaphragm ͉ signaling ͉ podocin ͉ podocyte

The PSD95/Dlg/ZO-1 (PDZ) domain-containing protein zonula occludens-1 (ZO-1) selectively localizes to the cytoplasmic basis of the slit diaphragm, a specialized cellcell contact in between glomerular podocytes necessary to prevent the loss of protein in the urine. However, the function of ZO-1 at the slit diaphragm has remained elusive. Deletion of Neph1, a slit diaphragm protein of the immunoglobulin superfamily with a cytoplasmic PDZ binding site, causes proteinuria in mice. We demonstrate now that Neph1 binds ZO-1. This interaction was mediated by the first PDZ domain of ZO-1 and involved the conserved PDZ domain binding motif present in the carboxyl terminus of the three known Neph family members. Furthermore, Neph1 co-immunoprecipitates with ZO-1 from lysates of mouse kidneys, demonstrating that this interaction occurs in vivo. Both deletion of the PDZ binding motif of Neph1 as well as threonine-to-glutamate mutation of the threonine within the binding motif abrogated binding of ZO-1, suggesting that phosphorylation may regulate this interaction. ZO-1 binding was associated with a strong increase in tyrosine phosphorylation of the cytoplasmic tail of Neph1 and dramatically accelerated the ability of Neph1 to induce signal transduction. Thus, our data suggest that ZO-1 may organize Neph proteins and recruit signal transduction components to the slit diaphragm of podocytes.