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Tissue Expression of Nephrin in Human and Pig

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Tissue Expression of Nephrin in Human and Pig 0031-3998/04/5505-0774 PEDIATRIC RESEARCH Vol. 55, No. 5, 2004 Copyright © 2004 International Pediatric Research Foundation, Inc. Printed in U.S.A. Tissue Expression of Nephrin in Human and Pig ARVI-MATTI KUUSNIEMI, MARJO KESTILÄ, JAAKKO PATRAKKA, ANNE-TIINA LAHDENKARI, VESA RUOTSALAINEN, CHRISTER HOLMBERG, RIITTA KARIKOSKI, RIITTA SALONEN, KARL TRYGGVASON, AND HANNU JALANKO Hospital for Children and Adolescents and Biomedicum Helsinki [A.-M.K., J.P., A.-T.L., C.H., R.K., H.J.], University of Helsinki, 00290 Helsinki, Finland; Department of Molecular Medicine [M.K.], National Public Health Institute, 00290 Helsinki, Finland; Biocenter and Department of Biochemistry [V.R.], University of Oulu, 90571 Oulu, Finland; Department of Obstetrics and Gynecology [R.S.], University of Helsinki, 00290 Helsinki, Finland; and Division of Matrix Biology [J.P., K.T.], Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden ABSTRACT Nephrin is a major component of the glomerular filtration cine fetuses, newborns, and infants. Likewise, nephrin mRNA barrier. Mutations in the nephrin gene (NPHS1) are responsible expression was not observed outside kidney glomerulus in nor- for congenital nephrotic syndrome of the Finnish type (NPHS1). mal or NPHS1 children. The phenotype analysis of NPHS1 Nephrin was at first thought to be podocyte specific, but recent children with severe nephrin gene mutations supported the find- studies have suggested that nephrin is also expressed in nonrenal ings in the tissue expression studies and revealed no impairment tissues such as pancreas and CNS. We studied the expression of of the neurologic, testicular, or pancreatic function in a great nephrin in human and porcine tissues at different stages of majority of the patients. The studies suggest that nephrin has no development and correlated these findings to clinical character- major clinical significance outside the kidney. (Pediatr Res 55: istics of NPHS1 children. Immunofluorescence staining and 774–781, 2004) Western blotting were used to detect nephrin protein in frozen tissue samples. Polyclonal antibodies against the intracellular Abbreviations part of nephrin were used in these analyses. In situ hybridization NPHS1, congenital nephrotic syndrome of the Finnish type was used to detect nephrin mRNA in specimens from normal IF, immunofluorescence human subjects and patients with NPHS1. Nephrin protein was RT-PCR, reverse transcriptase–PCR not detected in nonrenal tissues obtained from human and por- EST, expressed sequence tag Nephrin is a cell adhesion protein produced in kidney by the 26 kb. Mutations in NPHS1 lead to congenital nephrotic syn- glomerular epithelial cells (1). It is located at the podocyte slit drome of the Finnish type (NPHS1), which is an autosomal diaphragm, which is a crucial component of the glomerular recessive disorder characterized by nephrotic syndrome soon filtration barrier (2–4). Nephrin has 1241 amino acid residues after birth (15). The disease is highly enriched in the Finnish and belongs to the immunoglobulin superfamily containing population, but cases of NPHS1 are found all over the world eight immunoglobulin-like domains and one fibronectin-type (16–18). The Finnish patients have two important mutations domain in the extracellular part. The intracellular part has no (Fin-major and Fin-minor), which both lead to a truncated homology with other known proteins. It contains tyrosine protein and total absence of nephrin in kidney glomerulus (19). residues, and nephrin was recently shown to take part in cell In electron microscopy, these kidneys also lack the filamentous signaling together with two other podocyte proteins, podocin image of slit diaphragm, supporting the central role of nephrin and CD2-associated protein (5–10). The extracellular part of in the architecture of the slit diaphragm (19). nephrin interacts with Neph1, and the two proteins probably The expression of nephrin was at first thought to be limited form the backbone of the podocyte slit diaphragm (11–13). to kidney (1). However, in rodents, activity of nephrin gene Nephrin is encoded by NPHS1 gene located on chromosome promoter was observed in some parts of the CNS, such as the 19q13.1 (14). This gene consists of 29 exons and has a size of ventricular zone of the fourth ventricle; the developing spinal cord, cerebellum, hippocampus, and olfactory bulb; and ␤ cells of the pancreas (20, 21). In addition, the expression of nephrin Received May 12, 2003; accepted November 11, 2003. Correspondence: Hannu Jalanko, M.D., Hospital for Children and Adolescents, Uni- in murine testis, spleen, and thymus has been suggested (22). versity of Helsinki, 00290 Helsinki, Finland; e-mail: hannu.jalanko@hus.fi In humans, nephrin has been located in pancreatic ␤ cells but DOI: 10.1203/01.PDR.0000117842.10241.2C not in the CNS (23). We studied the expression of nephrin in 774 TISSUE EXPRESSION OF NEPHRIN 775 human and porcine tissues using immunohistochemical, in situ Laemmli sample buffer, and the proteins were separated on a hybridization, and Western blotting techniques and analyzed gel (7%) and blotted onto an Immobilon-P polyvinylidene the clinical data of patients with NPHS1. fluoride membrane (Millipore, Bedford, MA, U.S.A.). After blocking with 5% nonfat dry milk in PBS, the membrane was METHODS stained with rabbit anti-nephrin antibodies followed by perox- idase-conjugated goat anti-rabbit IgG antibodies (Jackson Im- Tissue samples. Human fetal samples were collected at munoResearch). Bound antibodies were visualized using en- autopsy from fetuses at 14, 18, and 20 wk of gestation obtained hanced chemiluminescence (Amersham Biosciences, Uppsala, through prostaglandin-induced abortions as a result of trisomy Sweden). The same antibody preparation was used for Western 21 (Department of Obstetrics and Gynecology, University of blotting and IF. Helsinki). In this disorder, abnormalities have not been de- In situ Hybridization. Formalin-fixed, paraffin-embedded tected in chromosome 19, where the gene for nephrin is sections (10 ␮m) were deparaffinized in xylene, rehydrated in located. Tissue samples were also obtained at autopsies of a decreasing alcohol series, and treated with proteinase-K newborn (died in sepsis) and five infants. Two human testis (Sigma Chemical Co., St. Louis, MO, U.S.A.) before hybrid- samples came from orchiectomies of adult men (supplied by ization. Thereafter, the sections were subjected to in situ Dr. Leo Dunkel) and one testis sample from a preterm baby (24 hybridization as described previously (25), with some modifi- wk of gestation) who died of lung problems at the age of 2 wk. Two of the infants had NPHS1 (Fin-major/Fin-minor geno- cations. Briefly, the tissue sections were washed in PBS, acetylated and dehydrated, and then incubated with 1.2 ϫ 106 types) and had died after renal transplantation at the ages of 2 33 and 3 y. Three infants died of congenital heart failure at the P-labeled (1000 Ci/mmol; Amersham, Arlington Heights, IL, ages of 4 mo, 6 mo, and 3 y. Porcine fetal samples were U.S.A.) antisense and sense riboprobes in a total volume of 80 ␮ obtained from aborted pregnancies at days 55 and 80 of Lat60°C for 18 h. After washes with standard saline culture, gestation (the total length of gestation is 114 d). In addition, RNA digestion, and dehydration, the sections were dipped in samples from newborn and a 6-mo-old pigs were used. NTB2 nuclear emulsion (Kodak, Rochester, NY, U.S.A.) and The use of the tissue samples and the study protocol were exposed in the dark at 4°C for 1 or 2 wk. After developing, the approved by the ethical committees of the Department of sections were counterstained with hematoxylin and eosin. Mi- Obstetrics and Gynecology and the Hospital for Children and croscopy was carried out with a standard Leica DM RX light Adolescents of the University of Helsinki. All human studies microscope. were conducted with informed consent. The probes for in situ studies were synthesized by subclon- Immunofluorescence staining. Rabbit polyclonal antiserum ing a 287-bp cDNA fragment corresponding to exon 10 in directed against the intracellular part of nephrin molecule was human NPHS1 into pBluescript (Stratagene, La Jolla, CA, used for the immunohistochemistry of nephrin. This antiserum U.S.A.), and antisense and sense RNA were produced using T3 was prepared as described previously (24). It was completely and T7 RNA polymerase, respectively. specific for nephrin and did not give any immunofluorescence Clinical data on NPHS1 children. Totally 56 children with (IF) staining for nonglomerular structures even in low dilu- NPHS1 have received kidney transplants since 1987 in Fin- tions. One polyclonal antiserum and three MAb preparations land. The clinical records of these children were analyzed against the extracellular domains of nephrin (3, 24) were also retrospectively, and the data regarding the neurologic find- tested but not used in the IF. These gave a slight background ings and pancreatic function were recorded. For studying the staining on nonglomerular epithelial structures, suggesting function of pancreatic ␤ cells, an oral glucose tolerance test cross-reactivity with epitopes in other proteins. Polyclonal was performed on 36 NPHS1 patients and 25 other kidney guinea pig anti-insulin antiserum (A0564; Dako Corporation, transplant patients 1 to 5 y after renal transplantation. The Carpinteria, CA, U.S.A.) was used as a positive control in test was done according to standard protocols, and blood immunohistochemistry of human and porcine pancreas. glucose and serum insulin levels were recorded for2hat For the IF stainings, cryosections (5 ␮m) of the tissue 30-min intervals. samples were fixed with 3.5% paraformaldehyde and incubated For elucidating the testicular function, serum sex hormone overnight at 4°C with antibodies diluted in PBS. FITC- conjugated secondary antibodies (Jackson ImmunoResearch levels were measured from eight 15- to 17-y-old boys who had Laboratories, West Grove, PA, U.S.A.) were incubated for 1 h. NPHS1 and had received a kidney transplant in infancy.
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