title: Podocin localizes in the kidney to the slit diaphragm area.
abstract: We recently cloned a novel gene, NPHS2, involved in autosomal recessive steroid-resistant nephrotic syndrome. This gene encodes a novel podocyte protein, podocin. Given its similarity with the stomatin family proteins, podocin is predicted to be an integral membrane protein with a single membrane domain forming a hairpin-like structure placing both N- and C-termini in the cytosol. Here, we show by in situ hybridization, that during development, the NPHS2 transcript is first expressed in mesonephric podocytes from the S-shaped body and, later, in the metanephric kidney, in the future podocytes at the late S-shaped body stage. In the mature kidney, NPHS2 is exclusively expressed in the podocytes of mature glomeruli. We generated rabbit polyclonal antibodies against fusion proteins derived from the N- and the C-terminal regions of podocin which detected a single band of 49-kd in transfected HEK293 cell lysates by immunoprecipitation and Western blotting. By immunohistology, podocin was detected in podocytes from the early capillary loop stage in the developing nephrons, and at the basal pole, along the GBM, in mature glomeruli. By electron microscopy, we demonstrate that podocin is facing the slit diaphragm with its two ends in the cytoplasm of the foot processes, in agreement with its predicted structure. Our results suggest that podocin could serve to anchor directly or indirectly components of the slit diaphragm to the cytoskeleton.
authors.affiliation: Inserm U423, Hôpital Necker-Enfants Malades, Paris, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Roselli
authors.forename: Olivier
authors.initials: O
authors.lastname: Gribouval
authors.forename: Nicolas
authors.initials: N
authors.lastname: Boute
authors.forename: Mireille
authors.initials: M
authors.lastname: Sich
authors.forename: France
authors.initials: F
authors.lastname: Benessy
authors.forename: Tania
authors.initials: T
authors.lastname: Attié
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Gubler
authors.forename: Corinne
authors.initials: C
authors.lastname: Antignac
mesh_terms: D002460:Cell Line; D005333:Fetus; D005455:Fluorescent Antibody Technique; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism* / Q000648:ultrastructure; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D012333:RNA, Messenger / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein; D012333:RNA, Messenger