Kidney filtration barrier components nephrin and Neph3: transcriptional regulation and role in cell adhesion Mervi Ristola Department of Pathology, Haartman Institute and Helsinki Biomedical Graduate Program Faculty of Medicine University of Helsinki Finland ACADEMIC DISSERTATION To be publicly discussed, with the permission of the Faculty of Medicine of the University of Helsinki, in the Auditorium of Arppeanum, Helsinki University Museum, Snellmaninkatu 3, Helsinki, on 8th of June 2012, at 12 noon. Helsinki 2012 1 Supervised by Sanna Lehtonen, Ph.D. Satu Juhila née Arpiainen, Ph.D. Assistant Professor R&D, In Vitro Biology Department of Pathology Orion Corporation, Orion Pharma University of Helsinki Espoo, Finland Helsinki, Finland Reviewed by Marjo Kestilä, Ph.D. Jorma Palvimo, Ph.D. Docent Professor Department of Chronic Disease Prevention Institute of Biomedicine National Institute for Health and Welfare (THL) University of Eastern Finland Helsinki, Finland Kuopio, Finland Official opponent Seppo Vainio, Ph.D. Professor Department of Medical Biochemistry and Molecular Biology Institute of Biomedicine Oulu Center For Cell Matrix Research Biocenter Oulu University of Oulu Oulu, Finland Helsinki University Biomedical Dissertations No. 165 ISSN 1457-8433 ISBN 978-952-10-8030-2 (paperback) ISBN 978-952-10-8031-9 (PDF) http://ethesis.helsinki.fi Unigrafia Oy Helsinki 2012 2 “The first step in the journey is to lose your way.” - Galway Kinnell 3 TABLE OF CONTENTS LIST OF ORIGINAL PUBLICATIONS ........................................................................ 7 ABBREVIATIONS ......................................................................................................... 8 ABSTRACT .................................................................................................................... 9 1 REVIEW OF THE LITERATURE .......................................................................... 10 1.1 Kidney – anatomy and function ......................................................................... 10 1.2 Glomerular filtration barrier ............................................................................. 10 1.2.1 Podocytes and the slit diaphragm (SD) ........................................................ 10 1.2.1.1 Composition and functions of the SD.............................................. 12 1.2.1.2 Podocyte cytoskeleton .................................................................... 13 1.2.1.3 Nephrogenesis and development of podocytes and the SD .............. 14 1.2.2 Glomerular basement membrane ................................................................. 14 1.2.3 Endothelial cells ......................................................................................... 15 1.2.4 Mesangium ................................................................................................. 15 1.2.5 Glomerular filtration ................................................................................... 15 1.3 Nephrin and Neph-protein family ...................................................................... 16 1.3.1 Nephrin ...................................................................................................... 16 1.3.1.1 Congenital nephrotic syndrome of the Finnish type (CNF) ............. 16 1.3.1.2 Structure and functions of nephrin protein ..................................... 17 1.3.1.3 Nephrin expression and its splicing variants .................................. 18 1.3.2 Neph-protein family.................................................................................... 19 1.3.3 Orthologues of nephrin and Neph-family members in Drosophila and Caenorhabditis elegans ............................................................................. 20 1.4 Transcriptional regulation in podocytes ............................................................ 21 1.4.1 General aspects of transcriptional regulation................................................ 21 1.4.1.1 Transcription initiation complex .................................................... 22 1.4.1.2 Controlling the assembly and regulation of chromatin.................... 23 1.4.1.3 Co-regulators ................................................................................ 26 1.4.2 Transcription factors in podocytes ............................................................... 26 1.4.2.1 WT1 .............................................................................................. 26 1.4.2.2 NF-țB ........................................................................................... 28 1.4.2.3 Sp1 ................................................................................................ 29 1.4.2.4 Other transcription factors ............................................................ 30 1.4.3 Transcriptional regulation of nephrin and Neph3 genes................................ 31 1.4.3.1 Tissue-specific regulatory elements in nephrin promoter ................ 31 4 1.4.3.2 Transcription factors and agents regulating nephrin gene in podocytes ..................................................................................... 32 1.4.3.3 Transcription factors regulating nephrin and Neph3 genes in other tissues .................................................................................. 34 1.4.4 Summary of transcription factors and agents of interest ............................... 34 2 AIMS OF THE STUDY ............................................................................................ 36 3 MATERIALS AND METHODS............................................................................... 37 3.1 DNA constructs .................................................................................................. 37 3.1.1 Expression vectors (I-IV) ............................................................................ 37 3.1.2 Luciferase reporter gene constructs (II-IV) .................................................. 38 3.2 Cell culture (I-IV)............................................................................................... 38 3.3 Animals............................................................................................................... 39 3.3.1 Nephrin-deficient mouse line (I).................................................................. 39 3.3.2 Sprague-Dawley rats (IV) ........................................................................... 40 3.4 Antibodies (I-IV) ................................................................................................ 40 3.5 Cell assays........................................................................................................... 41 3.5.1 Hanging drop aggregation assay (I) ............................................................. 41 3.5.2 Reporter gene assay (II-IV) ......................................................................... 41 3.5.3 Transcription factor overexpression studies (II, III) ..................................... 41 3.5.4 TNF-Į, BAY 11-7082 and 5-aza-2’-deoxycytidine treatments (III) .............. 41 3.6 Protein studies .................................................................................................... 42 3.6.1 Preparation of E17 kidney lysates and glomerular and tubular lysates (I, IV) ......................................................................................................... 42 3.6.2 Preparation of total cell lysates (I, IV) ......................................................... 42 3.6.3 Preparation of nuclear extracts (IV) ............................................................. 42 3.6.4 Immunoblotting (I, III, IV) .......................................................................... 43 3.6.5 Immunofluorescence microscopy ................................................................ 43 3.6.5.1 Immunofluorescence microscopy of kidney tissue (I, IV) ................. 43 3.6.5.2 Immunofluorescence microscopy of cultured cells (I, III, IV) .......... 43 3.6.5.3 Surface staining (I) ........................................................................ 44 3.6.6 Protein interaction studies ........................................................................... 44 3.6.6.1 Co-immunoprecipitation assay (I, III) ............................................ 44 3.6.6.2 Pull-down assays with the extracellular domain of Neph3 (I) ......... 44 3.7 Computer-based promoter analysis ................................................................... 45 3.7.1 Promoter alignment and identification of transcription factor binding sites (II-IV) ................................................................................................ 45 3.7.2 Identification of Alu elements and CpG islands (II, III) ............................... 45 3.8 Chromatin immunoprecipitation (II-IV) ........................................................... 45 3.9 mRNA expression analysis ................................................................................. 46 5 3.9.1 RNA preparation (II, III) ............................................................................. 46 3.9.2 Conventional reverse transcription-PCR (II) ................................................ 46 3.9.3 Quantitative reverse transcription-PCR (II, III) ............................................ 46 3.10 Statistical analyses ............................................................................................ 46 4 RESULTS .................................................................................................................