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Nephrin Is Specifically Located at the Slit Diaphragm of Glomerular Podocytes

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Nephrin Is Specifically Located at the Slit Diaphragm of Glomerular Podocytes Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7962–7967, July 1999 Cell Biology Nephrin is specifically located at the slit diaphragm of glomerular podocytes VESA RUOTSALAINEN*†,PA¨IVI LJUNGBERG*‡§,JORMA WARTIOVAARA¶,ULLA LENKKERI†,MARJO KESTILA¨†, ʈ HANNU JALANKO‡,CHRISTER HOLMBERG‡, AND KARL TRYGGVASON† †Biocenter and Department of Biochemistry, University of Oulu, 90570 Oulu, Finland; ‡Hospital for Children and Adolescents, University Hospital of Helsinki, 00014 Helsinki, Finland; §Department of Bacteriology and Immunology, Haartman Institute and ¶Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland; and ʈDivision of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden Communicated by Peter A. Reichard, Karolinska Institute, Stockholm, Sweden, April 9, 1999 (Received for review February 12, 1999) ABSTRACT We describe here the size and location of tight junction protein ZO-1 (17) has been localized in the nephrin, the first protein to be identified at the glomerular glomerulus, predominantly to points where the slit diaphragm podocyte slit diaphragm. In Western blots, nephrin antibodies is inserted into the lateral cell membrane of the foot process generated against the two terminal extracellular Ig domains of (18). The ZO-1 protein possibly connects the slit diaphragm, recombinant human nephrin recognized a 180-kDa protein in directly or indirectly, to the cytoskeleton. lysates of human glomeruli and a 150-kDa protein in trans- In numerous primary and secondary diseases of the kidney, fected COS-7 cell lysates. In immunofluorescence, antibodies the filtration barrier is affected, resulting in proteinuria, i.e., to this transmembrane protein revealed reactivity in the leakage of albumin and larger plasma proteins into the urine, glomerular basement membrane region, whereas the podocyte with edema and nephrotic syndrome as a consequence. Many cell bodies remained negative. In immunogold-stained thin cases also include an immunological component in their sections, nephrin label was found at the slit between podocyte etiology which, however, is not the case in the congenital foot processes. The congenital nephrotic syndrome of the nephrotic syndrome of the Finnish type (NPHS1) (19). We Finnish type (NPHS1), a disease in which the nephrin gene is have recently identified the gene mutated in NPHS1 (20, 21). mutated, is characterized by massive proteinuria already in The disease specifically affects the kidney and is characterized utero and lack of slit diaphragm and foot processes. These by massive proteinuria already in utero. Electron microscopic features, together with the now demonstrated localization of examination of NPHS1 patient kidneys reveals thinner lamina nephrin to the slit diaphragm area, suggests an essential role densa of the GBM than in controls, but no structural abnor- for this protein in the normal glomerular filtration barrier. A mality of the GBM has been detected (22, 23). In kidneys of zipper-like model for nephrin assembly in the slit diaphragm patients with NPHS1, the podocyte foot processes are absent, is discussed, based on the present and previous data. and no slit diaphragms have been described in the podocyte cell–cell adhesions. This finding is typical for nephroses of any Ultrafiltration of blood during formation of the primary urine cause. in the glomerulus is one of the central functions of the human The gene mutated in NPHS1 codes for a putative trans- kidney (1). Structurally, the glomerulus is a tuft of anasto- membrane protein termed nephrin that belongs to the mosing capillary loops surrounded by the Bowman’s capsule Ig superfamily. It has an extracellular portion containing eight leading the primary urine to the tubular system. The glomer- Ig motifs and one type III fibronectin domain (20). These ular filtration barrier is formed by three layers: the innermost properties, together with the sequence of the intracellular fenestrated vascular endothelium, the glomerular basement domain that contains eight tyrosines, suggested that nephrin is membrane (GBM), and the podocyte layer. The podocytes a signaling adhesion molecule. By using in situ hybridization, form a tight web on top of the GBM with their interdigitating nephrin was shown to be exclusively expressed in glomerular foot processes joined by a slit diaphragm. podocytes (20). It is generally acknowledged that the molecules passing In the present study, we localized nephrin in immunofluo- through the glomerular filtration barrier are selected accord- rescence microscopy to the GBM region of newborn human ing to their size, charge, and shape (2–8). The exact locations glomeruli, using antibodies generated against recombinant of the various selective functions in the barrier are, however, antigen. Moreover, we demonstrated by immunoelectron mi- more controversial. The charge-selective filter has been croscopy that nephrin is located in the podocyte slit region. We thought to be located in the GBM, a crosslinked meshwork of propose that nephrin is most likely a component of the slit type IV collagen, laminin, nidogen, and proteoglycans (9, 10). diaphragm. The fact that the protein is mutated in NPHS1 The anionic charge of heparan sulfate side chains of proteo- further indicates an essential role for the slit diaphragm in the glycans is believed to hinder the traversal of anionic plasma maintenance and size selectivity of the glomerular filtration proteins (5, 11, 12). The location of the size-selective property barrier. of the filtration barrier has been attributed to the GBM alone or, alternatively, to the slit diaphragm (5, 13, 14). MATERIALS AND METHODS Concerning the molecular composition of the slit dia- phragm, monoclonal antibody 5–1-6 (15) that recognizes a Generation and Characterization of Antiserum. The N- 51-kDa protein has been shown in immunoelectron micros- terminal fragment of nephrin was produced in Escherichia coli copy to react exclusively with the slit diaphragm (16). However, by using the QiaExpressionist kit from Qiagen. The cDNA- the nature of this protein is still unknown. The ␣-isoform of the Abbreviation: GBM, glomerular basement membrane. The publication costs of this article were defrayed in part by page charge Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF035835). payment. This article must therefore be hereby marked ‘‘advertisement’’ in *These authors contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. ʈTo whom reprint requests should be addressed. e-mail: karl. PNAS is available online at www.pnas.org. [email protected]. 7962 Downloaded by guest on September 27, 2021 Cell Biology: Ruotsalainen et al. Proc. Natl. Acad. Sci. USA 96 (1999) 7963 encoding amino acids 22 to 240 (21) were amplified from fixed in ethanol for 10 min at room temperature and washed human fetal kidney 5ЈSTRETCH cDNA library (CLON- in PBS. Cultured cells were fixed with 4% paraformaldehyde TECH) by PCR. Synthetic oligonucleotides were used to for 3 min and then with ethanol for 10 min at room temper- generate additional restriction sites BglII (5Ј) and HindIII (3Ј) ature. To block nonspecific binding, the samples were incu- to the ends of the cDNA to facilitate cloning into BamHI͞ bated in 10% horse serum in PBS for 30 min at room HindIII-digested vector pQE-30. Production and initial puri- temperature. Rabbit primary antisera were used at dilutions fication of the six-histidine-tagged protein on Ni-NTA column 1:200 to 1:400 in 1% horse serum in PBS. Incubations were was carried out according to the manufacturer’s instructions. carried out for 2 hr at 37°C or overnight at 4°C.After washings Protein was purified under denaturing conditions in phosphate in PBS, the primary antibody was detected by using FITC- buffer containing 8 M urea. For further purification, the labeled anti-rabbit IgG antibody (Dako) at dilution 1:200 for protein was diluted 10-fold with an anion exchange chroma- 60 min at 37°C. After washings in PBS, the sections were tography buffer (8 M urea,͞0.05 M Tris͞1 mM dithiotreitol, analyzed with fluorescence microscopy. pH 8) and purified on Source Q column (Amersham Phar- Immunoelectron Microscopy. Kidney tissue from three macia Biotech) by using a linear NaCl-gradient. Protein was brain-dead intended donors (a 49-yr-old female unsuitable as precipitated by dialysis against PBS. The precipitate was donor because of being positive for hepatitis B surface antigen, solubilized in 1% SDS, diluted 10-fold with PBS, and used as a 30-yr-old female who was found to have variation in the an antigen to raise polyclonal antibodies in rabbits by using kidney vasculature, and a normal 17-yr-old male whose kidney standard procedures (SLA, Uppsala, Sweden). The antisera was biopsied) were processed for postembedding immunoelec- were characterized by using Western blotting, ELISA, and tron microscopy. The kidneys had been stored in ViaSpan immunofluorescence microscopy. solution (DuPont Merck) for 12, 15, and 24 hr, respectively, Purification of Antibodies. For immunolocalization studies, until processing. Cubicles (1 ϫ 1 ϫ 5 mm) were cut with a razor rabbit IgG was affinity purified with protein A Sepharose FF blade from the renal cortex in PBS. The samples were fixed in (Amersham Pharmacia Biotech). For Western blot analysis, 3.5% paraformaldehyde (Sigma) alone or combined with the antibody was purified by using nitrocellulose-bound re- 0.01% glutaraldehyde (Electron Microscopy Sciences, Fort combinant antigen (24). Briefly, 2 ␮g of antigen was separated Washington, PA) in 0.1 M phosphate buffer (pH 7.3) at room by preparative SDS gel electrophoresis and blotted onto temperature for 1, 1.5, and 19 hr, respectively. Fixed samples nitrocellulose membrane. The antigen-containing band was were washed in phosphate buffer, dehydrated in graded eth- cut into pieces and blocked with BSA. Antiserum was diluted anol, and embedded in LR white (London Resin, Basingstoke, 10-fold with TBS (0.05 M Tris͞0.15 M NaCl, pH 7.5) and U.K.) and Lowicryl K4M (Polysciences) resins.
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