NEPHRIN VESA Role in the renal ultrafilter and involvement in proteinuria RUOTSALAINEN
Biocenter Oulu and Department of Biochemistry, University of Oulu
OULU 2004
VESA RUOTSALAINEN
NEPHRIN Role in the renal ultrafilter and involvement in proteinuria
Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L10), Linnanmaa, on May 28th, 2004, at 9 a.m.
OULUN YLIOPISTO, OULU 2004 Copyright © 2004 University of Oulu, 2004
Reviewed by Professor Leena Ala-Kokko Associate Professor Annika Wernerson
ISBN 951-42-7348-6 (nid.) ISBN 951-42-7349-4 (PDF) http://herkules.oulu.fi/isbn9514273494/ ISSN 0355-3191 http://herkules.oulu.fi/issn03553191/
OULU UNIVERSITY PRESS OULU 2004 Ruotsalainen, Vesa, Nephrin. Role in the renal ultrafilter and involvement in proteinuria Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland, Department of Biochemistry, University of Oulu, P.O.Box 3000, FIN-90014 University of Oulu, Finland 2004 Oulu, Finland
Abstract Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessive disease that affects 1:8000 newborns in Finland. NPHS1 is characterised by heavy proteinuria already in utero and typical signs of nephrotic syndrome (NS) are present at or soon after birth. Due to the evident absence of extrarenal symptoms, NPHS1 has been considered a model disease for NS. In this study, the NPHS1 locus on chromosome 19q13.1 was sequenced and analysed with computer programs to identify new genes in the region. Genes were further characterised and sequenced from NPHS1 patient samples, as well as from controls. Analysis of the data resulted in the identification of the affected gene with two mutations that were found to explain 94% of the Finnish NPHS1 cases. The NPHS1 gene was found to encode a novel single-pass transmembrane protein, termed nephrin, which belongs to the immunoglobulin superfamily of cell adhesion molecules. The NPHS1 gene was cloned and recombinant nephrin fragments were produced in prokaryotic and eukaryotic expression systems. These fragments were used to raise antibodies that were utilized to characterise the spatial and temporal expression of nephrin in kidney glomeruli. Nephrin was localised by electron microscopy (EM) in ladder-like structures of the early junctional complexes of developing columnar podocytes at the capillary stage. In mature glomeruli, nephrin was localised to the slit diaphragm (SD) between adjacent glomerular podocyte foot processes. In order to investigate the more general involvement of nephrin in proteinuric disease, its expression was studied in primary acquired NS by immunofluorescence microscopy. The level of nephrin expression was found to be significantly reduced in membranous glomerulonephritis, minimal change disease and in focal segmental glomerulosclerosis. The known effects of nephrin mutations, together with the structure predicted from its sequence and localisation of the protein to the SD, emphasizes its indispensable role in maintaining the integrity of the glomerular filtration barrier. The glomerular basement membrane has long been considered to possess the size-selective filtration property of the filtration barrier. However, the identification of nephrin in the SD, as well as its alterations in proteinuria, has led us to reconsider SD as the final decisive size-selective filter.
Keywords: congenital nephrotic syndrome, Finnish disease heritage, glomerular filtration, molecular cloning, nephrin, ultrastructure
To Heli, Emmi and Olli
Acknowledgements
This work was carried out at the Biocenter Oulu and Department of Biochemistry, University of Oulu. First of all, I wish to express my gratitude to my supervisor, Professor Karl Tryggvason for engouragement and guidance during the years. I also wish to thank the staff at the Biochemistry Department, especially Professors Kalervo Hiltunen and Raili Myllylä, for creating inspirating athmosphere to the department. I am grateful to Professors Leena Ala-Kokko and Annika Wernerson for their valuable comments on the manuscript and to Dr. Deborah Kaska for the careful revision of the language of this thesis. I feel deep gratitude to all the co-authors and collaborators. Particularly, I wish to thank Professors Christer Holmberg and Jorma Wartiovaara and, especially, Docent Hannu Jalanko, Dr. Päivi Tissari and Dr. Jaakko Patrakka for their outstanding contribution to the project. I am in debt to the members of the TK-team and also to other colleagues at the department. Specially, I would like to thank the “CNF-subteam” members Marjo Kestilä, Minna Männikkö, Ulla Lenkkeri and Heli Putaala for the most productive teamwork. I would like to thank Paula Reponen, Reetta Vuolteenaho, Matti Höyhtyä, Pekka Kilpeläinen and Paula Martin, not only for their contribution and good advice during the years, but also especially for their friendship. I am in debt to Maire Jarva and Leena Ollitervo for their technical skills and for the good times in the laboratory. I express gratitude to my present colleagues at Orion Diagnostica for their support. My warmest thanks go to my parents for their support throughout my life. I owe to my sister and brother including their families. I would also like to thank my relatives-in-law for their support and relaxing moments over the weekends. Furthermore, I would like to thank all my friends, particularly Mika, Tuire, Tomi, Ulla and their families. Finally, I am in deepest gratitude to my family. Heli, your love has carried me through these tough years. Emmi and Olli, I wish I had your energy! This work was financially supported by several institutions listed in the original articles. Additionally, the work was supported by the Finnish Cultural Foundation.
Oulu, May 2004 Vesa Ruotsalainen
Abbreviations
AFP α-fetoprotein CD2AP CD2-associated protein ELISA enzyme-linked immunosorbent assay EM electron microscopy FSGS focal segmental glomerulosclerosis GBM glomerular basement membrane GN glomerulonephritis IF immunofluorescence mAb monoclonal antibody MC minimal change disease MW molecular weight NPHS1 congenital nephrotic syndrome of the Finnish type NS nephrotic syndrome SD slit diaphragm SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis ZO-1 zonula occludens –1
List of original articles
This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I Kestilä M*, Lenkkeri U*, Männikkö M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K (1998) Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital nephrotic syndrome. Mol Cell 1:575-82. II Ruotsalainen V*, Ljungberg P*, Wartiovaara J, Lenkkeri U, Kestilä M, Jalanko H, Holmberg C, Tryggvason K (1999) Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci U S A 96:7962-7. III Li C, Ruotsalainen V, Tryggvason K, Shaw A, Miner J (2000) CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere. Am J Physiol Renal Physiol 279: 785-92. IV Ruotsalainen V, Reponen P, Khoshnoodi J, Kilpeläinen, P, Tryggvason K (2004) Monoclonal antibodies to nephrin. Hybrid Hybridomics 23: 55-63. V Ruotsalainen V*, Patrakka J*, Tissari P, Reponen P, Hess M, Kestilä M, Holmberg C, Salonen R, Heikinheimo M, Wartiovaara J, Tryggvason K, Jalanko H (2000) Role of nephrin in cell junction formation in human nephrogenesis. Am J Pathol 157:1905- 16. VI Doublier S, Ruotsalainen V, Salvidio G, Lupia E, Biancone L, Conaldi P, Reponen P, Tryggvason K, Camussi G (2001) Nephrin redistribution on podocytes is a potential pathomechanism for proteinuria in patients with primary acquired nephrotic syndrome. Am J Pathol 152: 1723-31. *These authors contributed equally to these works. In addition, some unpublished observations are presented.
Contents
Acknowledgements Abbreviations List of original articles Contents 1 Introduction ...... 15 2 Review of the literature ...... 16 2.1 Renal glomerular filter...... 16 2.2 Structure of the glomerulus...... 16 2.2.1 Mesangium ...... 17 2.2.2 Endothelial cells...... 18 2.2.3 Glomerular basement membrane ...... 18 2.2.4 Podocytes...... 20 2.2.5 Slit diaphragm...... 21 2.3 Glomerulogenesis ...... 22 2.4 Glomerular ultrafiltration and proteinuria ...... 23 2.4.1 Alterations in the GBM...... 25 2.4.2 Alterations in podocytes...... 26 2.5 Congenital nephrotic syndrome of the Finnish type ...... 27 2.5.1 Clinical features...... 28 2.5.2 Diagnosis ...... 28 2.5.3 Treatment...... 29 2.5.4 Pathological findings ...... 29 2.5.5 Localisation of the NPHS1 gene to chromosome 19q13.1 ...... 30 3 Aims of the study...... 32 4 Materials and methods...... 33 4.1 Sequencing of cosmid clones and analysis of the sequence (I)...... 33 4.2 Characterisation of genomic DNA and cDNAs (I, II)...... 33 4.3 Mutation analysis (I)...... 34 4.4 Hybridisation techniques (I, V)...... 34 4.5 Recombinant protein expression and purification (II, IV, V)...... 34 4.6 Production of antibodies (II, III, V, IV) ...... 36
4.7 Protein electrophoresis and Western blot analysis (II, IV, VI)...... 36 4.8 Electron microscopy (II, V)...... 36 4.9 Immunohistochemistry (II, III, IV, V, VI)...... 37 4.10 Expression of nephrin in cultured podocytes (VI)...... 37 5 Results ...... 38 5.1 Identification of the NPHS1 gene on chromosome 19q13.1 (I)...... 38 5.2 Characterisation of the gene product (I) ...... 40 5.3 Protein expression and characterisation of antibodies (II, III, IV, V, VI)...... 41 5.4 Localisation of nephrin in the kidney glomerulus (II) ...... 41 5.5 Nephrin in developing normal and NPHS1 glomerulus (V)...... 42 5.6 Expression of nephrin and CD2AP in developing mouse and human glomeruli (III) ...... 43 5.7 Nephrin involvement in the pathogenesis of proteinuria (VI) ...... 43 6 Discussion...... 45 6.1 The NPHS1 mutations ...... 45 6.2 Nephrin – crucial component of the slit diaphragm...... 47 6.3 Nephrin in the developing glomerulus...... 48 6.4 Molecular structure of the slit diaphragm...... 49 6.5 Involvement of nephrin in the pathogenesis of proteinuria ...... 52 7 Conclusions ...... 55 References 1 Introduction
The formation of urine starts with the ultrafiltration of blood in the kidney glomerulus. The delicate glomerular filter consists of three layers: the fenestrated endothelium, glomerular basement membrane (GBM), and highly specialised epithelial cells, podocytes. Plasma flows through the pores in the endothelium, while blood cells are retained in the capillary lumen. The GBM is thought to act as a pre-filter, which prevents proteins from passing through the capillary wall. Finally, the filtrate is guided through a slit between two podocyte cell protrusions to the Bowman’s space and further to the tubular system of nephron, where it is concentrated to become the final urine. The filtration function of the kidney glomerulus is disturbed in numerous primary and secondary kidney diseases. This results in leakage of plasma proteins into the urine. Prolonged and extensive proteinuria can develop into a nephrotic syndrome (NS), and finally, to end stage renal disease. In the glomerular ultrafilter, prolonged proteinuria results in typical morphological changes on the epithelial podocytes. Congenital nephrotic syndrome of the Finnish type (NPHS1) is a rare autosomal recessive disease. It manifests with heavy proteinuria already in utero and the patients develop nephrosis soon after birth. Examination by electron microscopy (EM) shows massive effacement of the podocyte foot processes, a typical finding in NS from any cause. Because there are apparently no extra renal symptoms and the disease seem to affect only the filtration barrier, NPHS1 has been considered a model disease for NS. Prior to this study, the NPHS1 gene has been localised to chromosome 19q13 by genome wide screening of genetic markers utilising samples from Finnish NPHS1 families. In this study the critical region in chromosome 19q13 was further narrowed and genes from the critical region were characterised. Sequence analyses of NPHS1 patients resulted in the identification of the defected gene. Poly- and monoclonal antibodies were generated against this novel transmembrane protein, termed nephrin, and its location, developmental expression and alterations in acquired nephrotic syndromes were assessed by immunochemical methods. 2 Review of the literature
2.1 Renal glomerular filter
The kidney has an important function in maintaining the body homeostasis and one of the essential functions of the kidney is the ultrafiltration of blood (Briggs et al. 1998). The human kidney is a bean-shaped organ of about 150 grams lying in the retroperitoneal space. Each human kidney contains about one million filtration units, called nephrons. Each nephron is a self-functioning unit consisting of a renal corpuscle and a tubular system. The renal corpuscle consists of the glomerulus, a tuft of capillaries that is surrounded by Bowman's capsule. The epithelial layer of the Bowman’s capsule is impermeable to water. This capsule collects the primary urine and conducts it into the tubular system that consists of the proximal tubule, the loop of Henle, the distal tubule and the connecting segment. The nephron ends in a collecting system that directs the final urine to the renal pelvis, where the urine flows into the ureter and is collected in the bladder prior to its excretion from the body. The formation of urine starts with ultrafiltration of blood in the glomerulus (Tisher & Madsen 2000). Capillary plasma is filtered through the three layers of the capillary wall: the fenestrated endothelium, the GBM and a highly specialised epithelium. The plasma is believed to first flow through the pores in the endothelium, while the blood cells are retained in the capillary lumen. Next, the filtrate flows though the GMB, which is thought to possess both size and charge selective filtering properties that prevent the majority of proteins from passing through. Finally, the filtrate is guided through a narrow slit between two epithelial cell protrusions.
2.2 Structure of the glomerulus
The glomerulus is a ball of capillaries surrounded by the Bowman’s capsule (Kanwar 1984, Tisher & Madsen 2000). The glomerulus contains three cell types and two types of extracellular matrix: mesangial matrix and the GBM. The luminal side of the capillary 17
GBM is covered by the endothelium and the outer side by a highly specialised epithelium. The capillary tuft is supported by the centrally located mesangium, which is composed of mesangial cells and the mesangial matrix. Endothelial and epithelial cells synthesize and maintain the GBM that is the major physical support of the glomerular capillary wall. The structure of the glomerulus is depicted in Fig. 1.
EFFERENT MACULA DENSA ARTERIO LE B A AFFERENT ARTERIO LE MESANGIUM
C APILLARY LUM EN PA RI ETAL EPITHELIUM ENDO THELIUM
Water flow C APILLARY LUMEN GBM
EPITHELIAL PODOC YTE PO DO C YTE FOOT PRO C ESS
PRO XIM AL TUBULE URINARY SPAC E SLIT DIAPHRAGM URINARY SPACE
Fig. 1. A. Anatomy of the glomerulus. B. Structure of the glomerular capillary ultrafilter containing the tri-laminar basement membrane. Note that the filtration route is extracellular as both the endothelium and epithelium are fenestrated.
2.2.1 Mesangium
The mesangium makes an important contribution to the mechanical stability of the glomerulus (for review, see Kriz et al. 1995). It is situated in the intracapillary region of the glomerulus and it is composed of mesangial cells and mesangial matrix. The mesangial cells contain long processes that extent and are connected to the GBM. The cytoplasm of mesangial cells and especially the processes, contain bundles of microfilaments similar to those is vascular smooth muscle cells (see Kriz et al. 1990). The GBM does not completely encircle the capillary tube; the juxtamesangial part of the capillary is devoid of GBM. Especially in this interface, microfilaments of the mesangial cell processes interconnect the opposing mesangial angles of GBM and restrict the expansive forces generated by the pressure gradient between the capillary lumen and the Bowman’s space. The mesangial cells produce the mesangial matrix and are embedded therein. Major constituents of the mesangial matrix are type IV collagen α1(IV) and α2(IV) chains, laminins-1, -2 and -10, entactin, perlecan and bamacan (for references, see Miner 1999). The appearance of the mesangial matrix in electron micrographs resembles that of basement membranes, except that it is less compact and contains microfibrils (Farquhar & Palade 1962, Kriz et al. 1990). The juxtamesangial part of the capillary is leaky and 18 loose. Tracer studies (e.g., Farquhar & Palade 1962) have demonstrated that the plasma apparently has an alternative route to the urinary space through the mesangium and the perimesangial GBM. These studies also show that mesangial cells have a capability for phagocytosis and they are therefore able to clean the mesangial matrix from trapped particulate material. The contractile property of the mesangial cells indicates that they are able to regulate the microcirculation and ultrafiltration inside the glomerulus. It has been demonstrated that mesangial cells contract in response to various vasoactive compounds, e.g., angiotensin II (Ausiello et al. 1980). This fact, together with the observation that at some parts of the glomerulus mesangial cells surround the circumference of capillaries (Inkyo- Hayasaka et al. 1996), supports the hypothesis that mesangial cells would be able to regulate glomerular hemodynamics. On the other hand, it has been suggested that the contractile system might have a role in the static support of the glomerular structure rather than a dynamic role in regulating the hemodynamics (Kriz et al. 1990).
2.2.2 Endothelial cells
The luminal surface of the glomerular capillary is covered by an endothelium (Tisher & Madsen 2000). The bulky, nucleus-containing part of the endothelial cell lies in close proximity to the mesangium and the thinner part of the cell covers the GBM. The glomerular capillary endothelium is fenestrated with pores of 70-100 nm in diameter. These fenestrae are small enough to restrict blood cells and platelets, while allowing plasma to reach the GBM and, thus, the fenestrated endothelium forms the initial barrier of the glomerular filter. The fenestrated endothelium has been estimated to cover one-half of the total surface area of capillary wall (Bulger et al. 1983). The highly arranged cytoskeleton of the endothelial cells organizes the endothelium into a regularly shaped net over the GBM and both the pores and the endothelial processes are nearly round in shape (Lea et al. 1989). Thin diaphragms are observed in these fenestrae during glomerulogenesis, but they disappear at the capillary stage. The endothelial cell membrane is negatively charged with podocalyxin (Kerjaschki et al. 1984), podoendin (Huang & Langlois 1985) and proteoglycans (Simionescu et al. 1981). This negative charge contributes to the overall charge-selectivity of the glomerular filtration barrier.
2.2.3 Glomerular basement membrane
Basement membranes are special extracellular structures that have a sheet-like form. Several excellent reviews on basement membranes are available; see (Timpl 1996, Timpl & Brown 1996, Erickson & Couchman 2000) and references therein. The GBM separates two cell layers: endothelial and epithelial cells. It originates from a fusion of two BMs, one of epithelial and another of endothelial origin (Martinez-Hernandez & Amenta 1983). 19
The special function of the GBM, the ultrafiltration of blood, is reflected by its exceptional thickness. This 350 nm thick membrane can be seen in EM as a tri-laminar sandwich as opposed to the common two-layered sheet. The inner, more electron dense layer, called the lamina densa is between two electron lucent layers designated the lamina rara interna and lamina rara externa. As these layers are not always seen, depending on the fixation, it has been suggested that they represent a fixation artefact (Goldberg & Escaig-Haye 1986). The main components of basement membranes are type IV collagen and laminin. These proteins form two independent networks which self-assemble from soluble monomeric components and form heterogeneous polymeric insoluble matrices. The type IV collagen monomer is a triple-helical molecule that consists of three α-chains (for review, see Hudson et al. 2003). In humans, six different chains, α1(IV) through α6(IV), are known and they have different tissue distributions. The α1(IV) and α2(IV) chains assemble into the classical α1·α2 network in a 2:1 ratio. This network is ubiquitously found in all basement membranes. During glomerular development, a transition occurs. Immature GBM contains only the α1·α2-network whereas the mature, adult type GBM mainly contains an α3·α4·α5 -network, composed of α3(IV), α4(IV) and α5(IV) chains in a 1:1:1 ratio (Gunwar et al. 1998, Boutaud et al. 2000). Laminins are large heterotrimeric proteins with a molecular weight (MW) of 5x105-1x106 Thus far, at least 12 different trimeric laminin molecules have been identified (Patarroyo et al. 2002). Laminin-11 (comprised of α5-, β2- and γ1-chains) is the major laminin in the GBM (Virtanen et al. 1995, Miner 1998). However, the immature GBM in the vascular cleft of the developing glomerulus contains laminin-1 (α1β1γ1) and laminin-8 (α4β1γ1)(Durbeej et al. 1996, Miner et al. 1997, Sorokin et al. 1997). During the S-shaped phase and capillary loop phase the expression of α5- and β2-chains appear and eventually laminin- 11 displaces laminins-1 and –8 (Miner et al. 1998). The two networks of type IV collagen and laminin are connected to each other via nidogen/entactin (Aumailley et al. 1989). Nidogen/entactin is a glycoprotein with a MW of 150 000 first isolated from murine EHS tumour and cell cultures (Carlin et al. 1981, Timpl et al. 1983). Recently, nidogen-2 has also been described and, furthermore, a nidogen-2 variant called osteonidogen has been identified (Kimura et al. 1998, Kohfeldt et al. 1998). The multiple binding properties of the nidogens imply that they stabilize the laminin and collagen IV networks in basement membranes and also integrate the proteoglycan perlecan into the scaffold. All basement membranes contain proteoglycans that consist of a protein core with covalently attached polysaccharide glycosaminoglycan. Heparan sulphate is the most abundant glycosaminoglycan in the GBM (Kanwar et al. 1981) and perlecan was the first heparan sulphate proteoglycan isolated from basement membranes (Hassell et al. 1980). Perlecan is widely expressed in basement membranes (Murdoch et al. 1994), but in the mature GBM it is restricted to the subendothelial aspect of the GBM while another heparan sulphate proteoglycan, agrin, seems to be present throughout the width of the GBM (Groffen et al. 1998). Both perlecan and agrin bind to other basement membrane components and cell surface receptors and thus strengthen the GBM and anchor the negative charge to the GBM. The negative charge has been thought to be important for the charge-selective ultrafiltration properties of the GBM, but a recent report on perlecan deficient mice suggest that the negative charge of perlecan is not essential for a functional glomerular filtration barrier (Rossi et al. 2003). 20
2.2.4 Podocytes
Visceral epithelial cells, podocytes, cover the outer aspect of the glomerular capillary (for recent reviews on podocyte biology, see Smoyer & Mundel 1998, Endlich et al. 2001, Pavenstadt et al. 2003). The podocyte has a complex cellular organization and the cell can be divided into three distinct parts: the cell body, primary major processes and secondary foot processes. The major processes protrude from the cell body towards the GBM and divide into numerous foot processes. Only the foot processes are attached to the GBM, leaving the rest of the cell hanging freely in the urinary space of the Bowman’s capsule. At the GBM level, the foot processes of neighbouring cells interdigitate and cover the GBM. Between the interdigitating foot processes there are a narrow slit and an electron dense slit diaphragm (SD) extending from one process to another. Podocytes have several important functions in the kidney ultrafilter. They produce and maintain the GBM. For this purpose podocytes have a well-developed endoplasmic reticulum and a Golgi apparatus. Podocytes also support the glomerular capillary. The architecture of the podocyte cell layer and the highly developed cytoskeleton enables the cells to restrict the expanding forces produced by the transcapillary pressure gradient. Podocytes also have a highly negative coating on the cell surface, which allows them to prevent the fusion of the capillary tuft to parietal epithelium and keep the filtration slit between two foot processes open.
2.2.4.1 Podocyte cytoskeleton
The complex cellular organization of podocytes is formed and maintained by a highly organised cytoskeleton (Andrews 1981, Drenckhahn & Franke 1988). The podocyte cell body contains mainly intermediate type filaments consisting of vimentin (Bachmann et al. 1983, Holthöfer et al. 1984, Oosterwijk et al. 1990) and desmin (Yaoita et al. 1990). To some degree, the cell body also contains microtubules and the cytosolic side of the entire podocyte plasma membrane is covered with microfilaments. Major processes are filled with microtubules of a mixed polarity and the motor protein CHO1/MKLP1 of the kinesin superfamily is associated with them (Kobayashi et al. 1998). The nonuniform polarity formed by the CHO1/MKLP1 appears to be necessary for the process formation. The foot processes branching from the major processes contain an actin-based cytoskeleton with a complete contractile apparatus. Actin filaments run along the longitudinal axis of the foot process as loops beginning from the tip of one process, bend in the major process, and end in the tip of a second process. The filaments are probably connected to the microtubular skeleton of the major processes (Mundel & Kriz 1995). Actin filaments are composed of actin, myosin II and α-actinin (Drenckhahn & Franke 1988). The podocytes have been shown to specifically express α-actinin-4, which is also involved in cell motility of non-renal cells (Honda et al. 1998, Kaplan et al. 2000). Cytoskeletal filaments are connected to the GBM at the sole of the foot process via integrin and dystroglycan complexes. Integrin complexes are composed of α3β1-integrin (Korhonen et al. 1990), vinculin, talin (Drenckhahn & Franke 1988), paxillin (Turner et 21 al. 1990) and probably of some other molecules generally linked to integrin signalling, like integrin-linked kinase (Haltia et al. 1999, Kretzler et al. 2001), focal adhesion kinase and p130cas (reviewed in Giancotti & Ruoslahti 1999). Dystroglycan complexes consist of utrophin and β- and α-dystroglycans that binds to laminin-11 and agrin (Durbeej et al. 1998, Raats et al. 2000, Regele et al. 2000). The α3β1 integrin has been shown to bind with high affinity to the α5-chain of laminin-11 (Delwel et al. 1994) and with lower affinity to other GBM components (for references, see Kreidberg & Symons 2000).
2.2.4.2 Glycocalyx
The podocyte cell membrane is divided into basal and apical cell membrane at the SD level. The apical cell membrane of podocytes has a negative charge, which is thought to prevent the collapse of the slit and fusion of podocytes to the parietal epithelium. It also contributes to the overall negative charge of the filtration barrier. Carbohydrates contribute to the negative charge of the podocyte cell membrane and this coating is usually referred as the glycocalyx. In particular, the glycocalyx is rich in sialoproteins podocalyxin, podoendin and SGP-115/107 (Kerjaschki et al. 1984, Huang & Langlois 1985, Mendrick & Rennke 1988). Podocalyxin, the major sialoprotein on the apical surface of the podocytes, contributes approximately 80 % of the negative charge of the podocyte glycocalyx. This protein with a MW of 150 000 is also expressed on the vascular endothelium (Kerjaschki et al. 1984, Horvat et al. 1986) and recently it was found to be expressed in hematopoietic stem cells, megakaryocytes and thrombocytes (McNagny et al. 1997). The extracellular portion of this single-pass transmembrane protein is rich in serine, threonine and proline and is heavily sialylated and O-glycosylated. Podocalyxin is connected to the cytoskeleton via interaction of its cytoplasmic tail with ezrin (Orlando et al. 2001).
2.2.5 Slit diaphragm
The slit diaphragm (SD) is a sheet-like structure that connects two adjacent foot processes originating from neighbouring podocyte cell bodies. In EM it is seen as a thin line extending from one podocyte foot process cell membrane to the other. There is evidence that passage of molecules of the size of albumin and larger are restricted by the SD (Latta 1970) and also studies by Deen and co-workers (Drumond & Deen 1994, Edwards et al. 1999, Deen et al. 2001) support the view that the SD accounts for the size- selectivity of the glomerular filter. The width of the slit is estimated to be from 20 to 50 nm, depending on the fixation method (Furukawa et al. 1991), and it has been proposed that the total area covered by the SD in the glomerulus might increase in response to increased intraglomerular pressure (Kriz et al. 1996, Yu et al. 1997). Based on transmission EM analysis of sections cut in parallel with the GBM plane Rodevald and Karnovsky (1974) presented a zipper-like model of the SD. In this model, 22 alternating bridges extend from a podocyte foot process plasma membrane towards a central filament, which runs in parallel to the cell membranes. The validity of the model has been questioned as rather harsh fixation methods were used. A milder freeze-fracture method did not produce a porous structure. Instead a more sheet-like structure was proposed (Hora et al. 1990, Furukawa et al. 1991). Only limited knowledge about the molecular structure of the SD has been available. Until recently, the only component of the SD region identified has been zonula occludens -1 (ZO-1). The α- isoform of this protein has been localised in podocytes to the vicinity of the SD (Schnabel et al. 1990, Kurihara et al. 1992). ZO-1 has MW of 225 000, and it is a ubiquitously expressed tight-junction associated protein that belongs to the MAGUK family of proteins. MAGUK members are modular proteins containing several domains that participate in protein-protein interaction (for reviews, see Gonzalez- Mariscal et al. 2000, Hung & Sheng 2002). ZO-1 has three PDZ domains, one SH3 domain and one guanylate kinase domain through which it binds to other proteins in the tight junction. In the podocytes the α- isoform of ZO-1 lies in the intracellular site where the SD is inserted into the plasma membrane and it has been proposed that the SD is a modified tight junction, where ZO-1 connects the SD to the cytoskeleton (Schnabel et al. 1990). In addition to ZO-1 an unidentified antigen with a MW of 50 000, has been localised to the SD. This rat antigen is recognised by mouse a monoclonal antibody (mAb) 5-1-6, which was generated by immunisation of mice with a preparation of rat glomeruli (Orikasa et al. 1988). Injection of this antibody into rats causes proteinuria and the antigen has been localised exclusively to the SD (Kawachi et al. 1995). Attempts to identify this SD specific antigen were unsuccessful for a decade (Topham et al. 1999). Recently, several molecules in the SD region have been identified. More discussion about the molecular structure of the SD can be found in the discussion section of this thesis.
2.3 Glomerulogenesis
Development of the metanephric kidney begins after pro- and mesonepheric stages (for review, see Saxen 1987). In mammals and birds the pro- and mesonephros are transient structures and considered to reflect stages in kidney evolution, while the metanephros is the permanent functional kidney. Development of the metanephros begins at the fifth week of gestation during human development, when the nephritic duct has fused to the cloaca. A bud emerges from the nephritic duct and grows towards the metanephric mesenchyme. The ureteric bud enters the metanephric mesenchyme and starts to branch dichotomously. The tip of the branching ureteric bud transmits signals that induce the surrounding mesenchymal cells to condense and generate pretubular aggregates that initiate nephrogenesis. The ureter continues to branch and induce new nephrons until a total of about one million nephrons are formed in the human kidney. The glomerular development has been divided into several stages: vesicle, comma and S-shaped stages, capillary and maturing stages (see Abrahamson 1991, Horster et al. 1999). The ureteric bud induced aggregates of mesenchymal cells assume epithelial-like morphology and form the vesicle stage nephron from which the cells subsequently 23 differentiate into several epithelial phenotypes of the mature nephron. The differentiation begins with the formation of two clefts in the vesicle, which at first is comma-shaped and soon thereafter becomes the S-shaped form. The lower vascular cleft separates the S- shaped body into upper and lower limbs. The upper limb, that is closer to the ureter, finally fuses to it and forms the tubular system, while the lower part will form the glomerular tuft. Microvessels can be shown to invade the vascular cleft and the cells of these vessels are thought to be the origin of the endothelial cells of the mature glomeruli. The two epithelial cell layers beneath the vascular cleft are separated by a narrow space, which will become the Bowman’s space. The epithelial cells above the Bowman’s space will become podocytes and the cells below will form the parietal epithelium that covers the inside of the Bowman’s space. At the capillary loop stage, the basement membrane and the epithelial and endothelial cells begin to adopt their normal function and location. The forming glomerulus contains more vascular elements along with the afferent and efferent arterioles. Cells that appear to be mesangial cells can be observed and a matrix around these cells eventually begins to form. The basement membrane produced by the endothelial cells and the one produced by the presumptive podocytes fuse together and form the GBM. As the glomeruli develop further, they start to resemble mature glomeruli except that they are smaller. The phases of glomerular development are depicted in Fig. 2. Podocytes are first identified in the S-shaped stage. These presumptive podocytes are seen as a typical columnar epithelial cell layer with tight and adherens junctions at the apical-lateral cell membrane. As development proceeds, the junctions migrate down to the basal cell surface where the GBM is forming (Fig. 3). At the capillary loop stage, the basal membranes of the podocytes begin to interdigitate and the normal tight and adherens junctions are replaced by SDs. Before the SDs are formed, ladder-like structures are observed (Reeves et al. 1980). These structures disappear before the appearance of the SDs, but they have also been found in animal models of nephrotic syndrome (Ryan et al. 1975, Schneeberger & Grupe 1976).
2.4 Glomerular ultrafiltration and proteinuria
Proteins passing through the glomerular filter are selected according to their size, shape and charge. These distinct selective properties have been extensively examined by clearance and tracer studies using protein and dextran markers (for review, see Kanwar et al. 1991). Generally, proteins larger than albumin (MW 70 000) are almost completely restricted from passing through the ultrafilter, while low molecular weight proteins pass almost freely through the filter to be reabsorbed in the tubular system. The location of the size selective filter has been attributed to the GBM or alternatively to the SD (Latta 1970). A charge selective property has been demonstrated by using charged dextran molecules (Bohrer et al. 1978, Brenner et al. 1978). The behaviour of these molecules in the glomerular filter depends upon their charge. Cationic and neutral dextran molecules freely pass through the filter in contrast to anionic dextran molecules that are more restricted. A similar dependency on charge has been presented with globular proteins (Rennke et al. 1975, Rennke et al. 1978). The charge selectivity has been attributed to proteoglycans in the GBM. The anionic heparan sulphate side chains of the proteoglycans 24 are thought to hinder the movement of anionic plasma proteins (Caulfield & Farquhar 1978, Kanwar & Farquhar 1979). However, the recent results showing no proteinuria in mice with heparan sulphate deficient perlecan casts some doubt on the significance of glycosaminoglycans in macromolecular filtration (Rossi et al. 2003). In addition to the intrinsic properties of the glomerular filter, the transcapillary passage of water and proteins is also regulated by the glomerular plasma flow rate, transcapillary hydraulic pressure difference and intraglomerular hemodynamics (Tisher & Madsen 2000).
Fig. 2. Development of the nephron. A. Mesenchymal condensate. Cells of the metanephric mesenchyme aggregate around the branching ureteric tube. B. Vesicle stage. The mesenchymal cells acquire epithelial morphology C. Comma-shaped stage. The vesicle polarizes and the first cleft appears. D. S-shaped stage. At this stage the segments of the developing nephron can be observed for the first time. E. Capillary stage glomerulus. The upper limb of the S-shaped stage has fused to the ureter and microvessels have invaded the lower cleft. F. Maturing stage glomerulus. More capillaries form and finally fill the Bowman’s space. Illustration from (Saxen 1987), copyright Cambridge University Press. 25
Fig. 3. Development of the slit diaphragms from apical junctional complexes. In the late S- shaped stage the junctional complexes of the presumptive podocytes migrate along the lateral cell membrane towards the basal surface where the GBM is forming. The ladder-like structures transiently appear before the junctions mature to form slit diaphragms. Illustration adapted from (Schnabel et al. 1989) with permission, copyright Wissenschaftliche Verlagsgesellschaft mbH.
2.4.1 Alterations in the GBM
Alterations associated with proteinuria in the GBM have been extensively studied with experimental animals and recently the molecular cloning of GBM components has further revealed the essential role of the GBM for glomerular ultrafiltration. Farquhar and Palade (1962) demonstrated alterations in the negative charge of the GBM in puromycin aminonucleoside induced proteinuric rats. They showed that an anionic ferritin tracer penetrated deeper into the GBM of the proteinuric animal than in controls. It has been shown by several other studies that manipulation of the negative charge, which is contributed by heparan sulphate side chains, causes proteinuria (Kelley & Cavallo 1978, Kanwar et al. 1980, van den Born et al. 1992). Moreover, the concentration of heparan sulphate has been reported to be reduced in human glomerular disease as well (Vernier et al. 1983). However, deficiency of heparan sulphate side chains in perlecan does not lead to proteinuria in mice (Rossi et al. 2003). Importance of the type IV collagen α3·α4·α5 –network for the proper function of glomerular ultrafilter is emphasized by the effect of mutations in the genes coding for these chains (for review, see Hudson et al. 2003). A mutation in one of the three genes, COL4A3, COL4A4 or COL4A5, in most cases causes absence of all the chains from the GBM and only the α1·α2 –network persists. Mutations in the α5(IV)-chain coding gene, COL4A5, are causative for X-linked Alport syndrome, which is a progressive hereditary kidney disease characterised by hematuria, sensorineural hearing loss, ocular lesions and structural defects in the GBM (Barker et al. 1990). Mutations of COL4A3 and COL4A4 have been reported both in autosomal-recessive and autosomal-dominant forms of Alport syndrome, as well as in benign familial hematuria (Lemmink et al. 1994, Mochizuki et al. 1994, Lemmink et al. 1996, van der Loop et al. 2000, Ciccarese et al. 2001). 26
Furthermore, the α3(IV) chain NC1-domain is the target for autoantibodies in Goodpasture syndrome, which is characterised by glomerulonephritis (GN) and pulmonary haemorrhage (Saus et al. 1988). These data suggest a major structural role for type IV collagen in the GBM, while its function in macromolecular filtration is probably insignificant. In addition to type IV collagen genes, defective laminin genes are also associated with glomerular defects in experimental animal models. In mice lacking the laminin β2-chain, expression of the β1-chain persists in the GBM. Although, the ultrastructure of the GBM seems normal, podocytes are abnormally broadened, and animals develop severe proteinuria and die at 3-5 weeks of age (Noakes et al. 1995). Mice deficient of the laminin α5-chain die between embryonic day 14-17 with defects in neural tube closure, digit septation and placentation (Miner et al. 1998). Analysis of the embryonic metanephric kidneys revealed defective glomerulogenesis from the capillary stage onwards. Displacement of endothelial and mesangial cells resulted in avascular glomeruli (Miner & Li 2000).
2.4.2 Alterations in podocytes
Morphological changes in the podocytes are associated with NS (reviewed by Smoyer & Mundel 1998). The most prominent structural alteration is the effacement of podocyte foot processes. The processes broaden and finally a continuous cytoplasmic sheet covers the GBM. The most severe change is the detachment of the podocyte from the GBM, resulting in leakage of protein across the bare GBM (Kanwar & Rosenzweig 1982, Messina et al. 1987, Whiteside et al. 1989). These changes in the cellular architecture imply an indispensable role for podocytes in the glomerular filter. Complex cellular morphology is reflected in the organization of the podocyte cytoskeleton. Actin filaments have an abnormal distribution in effaced podocytes (Hoffmann 1982, Whiteside et al. 1993) and a redistribution of actin and α-actinin in the podocytes of puromycin aminonucleoside induced nephrotic rats is observed (Lachapelle & Bendayan 1991). Recently, a dominant mutation in the gene for α-actinin-4 has been detected (Kaplan et al. 2000). The mutated protein binds more strongly to actin and is suggested to cause an altered regulation of the cytoskeleton and as a consequence glomerular disease. In order to restrict the expansive forces of capillary pressure, the podocyte cytoskeleton is connected to GBM and to neighbouring foot processes. The receptors that have been identified at the podocyte-GBM interface are α3β1-integrin (Kerjaschki et al. 1989), dystroglycan (Durbeej et al. 1995) and megalin/gp330 (Kerjaschki & Farquhar 1983). Integrin α3β1 is essential for the maturation of podocytes as foot processes do not develop normally in α3-integrin deficient mice (Kreidberg et al. 1996). Megalin is a component of clathrin coated pits present both at the apical and basolateral membrane of the podocytes and has been identified as the pathogenic antigen of Heyman nephritis, a model of membranous glomerulonephritis (GN) (Kerjaschki et al. 1987, Saito et al. 1994). Expression of dystroglycan has been reported to be reduced in minimal change disease (MC) and in passive Heyman nephritis (Raats et al. 2000, Regele et al. 2000). 27
The connection of the cytoskeleton to neighbouring foot process is via the SD. In proteinuria, the SD is displaced apically and replaced by occluding type junctions. A decrease in the filtration slit frequency, narrowing of the slit and development of tight junctions between foot processes has been reported (Kurihara et al. 1992). Injection of an SD specific antibody into rats causes massive proteinuria with no apparent morphological changes (Orikasa et al. 1988). Later this antibody has been shown to be directed against nephrin (Topham et al. 1999). The negative charge of the apical membrane of podocytes seems to have a critical role in the foot process structure. Neutralization of the negative charge by perfusion of polycations or by enzymatic removal of sialic acid residues in vivo causes foot process effacement (Seiler et al. 1975, Seiler et al. 1977) and tyrosine phosphorylation of ZO-1 (Kurihara et al. 1995). The negative charge is mainly contributed by podocalyxin (Kerjaschki et al. 1984, Kershaw et al. 1997) and it has recently been demonstrated that neutralization of the negative charge dissociates the podocalyxin from the NHERF- 2/Ezrin complex that connects the podocalyxin to the cytoskeleton (Takeda et al. 2001). Deletion of the podocalyxin gene from mice leads to neonatal lethality and failure to form foot processes. Slit diaphragms of the homozygote mice are replaced by tight and adherens junctions resulting in anuria and death within 24 hours of birth (Doyonnas et al. 2001). The morphological changes of podocytes observed in proteinuric stages emphasize the importance of podocytes for normal ultrafiltration. Podocytes have a highly complex cellular architecture, and probably due to this complexity they are not able to proliferate in response to damage (Pabst & Sterzel 1983, Fries et al. 1989, Nagata et al. 1995).
2.5 Congenital nephrotic syndrome of the Finnish type
Nephrotic syndrome (NS) rarely manifests during the first year of life (Holmberg et al. 1999). The two major forms of congenital nephrotic syndromes are congenital nephrosis of the Finnish type (NPHS1, formerly CNF) and diffuse mesangial sclerosis (DMS). The latter can be isolated or associated with a variety of malformations, e.g., Denys-Drash and Mowat-Galloway syndromes. Also, familial forms of focal segmental glomerulosclerosis (FSGS) sometimes manifest in the newborn period. Secondary forms of NS in infancy can be caused by infections (syphilis, cytomegalovirus, toxoplasmosis). NPHS1 is an autosomal and recessively inherited disease first described by Hallman and co-workers in 1956 (Hallman et al. 1956). This deleterious disease belongs to a group of inherited diseases known as the Finnish disease heritage (Norio et al. 1973). There are about 35 diseases that are more common in Finland than anywhere else in the world (for review, see Peltonen et al. 1999). In contrast, common genetic diseases like cystic fibrosis or phenylketouria are practically nonexistent in Finland. NPHS1 affects about 1:8400 newborns in Finland (Huttunen 1976), while outside Finland the incidence is considerably lower, e.g., in North America the NPHS1 incidence has been estimated to be 1:50 000 (Albright et al. 1990). Although, the incidence outside Finland is lower, 28
NPHS1 cases have been described worldwide (Mahan et al. 1984, Grahame-Smith et al. 1988, Bucciarelli et al. 1989, Fuchshuber et al. 1996).
2.5.1 Clinical features
NPHS1 children are usually born prematurely at gestation week 36 on average. Typically, the first sign of NPHS1 is an enlarged placenta (Hallman et al. 1967). The proteinuria starts already in utero and symptoms of NS invariably develop during the first three months of life. Edema and abdominal distension is seen immediately at birth in one fourth of the cases and over 90 % develop NS within the first week (Huttunen 1976). Without nutritional support and albumin substitution the typical appearance of the NPHS1 child develops: widened cranial sutures, small low-bridged nose, wide-set eyes and low ears, abdominal distension, ascites, umbilical hernias, opistotonic position and generalized edema (Hallman et al. 1956, Holmberg et al. 1999). Before the development of renal transplantation techniques, NPHS1 children usually died within the first 6 months of life. At the beginning the proteinuria is highly selective, with 90 % of the lost protein being albumin (Huttunen 1976), but other proteins are also lost into the urine. The serum levels of these proteins and their ligands are low, which leads to secondary metabolic disturbances. Urinary loss of gammaglobulin and complement factors makes NPHS1 children susceptible to infections (Huttunen 1976, Mahan et al. 1984). Urinary excretion of plasminogen and antithrombin III results in decreased levels of these coagulation factors in serum and compensatory proteins synthesis may lead to increased levels of other coagulation factors and to severe coagulation problems (Llach et al. 1980, Panicucci et al. 1983, Mahan et al. 1984).
2.5.2 Diagnosis
Diagnosis of NPHS1 was previously based on a positive family history, severe proteinuria of intrauterine onset, large placenta, normal glomerular filtration rate and exclusion of other causes of congenital NS (Holmberg et al. 1995). Prenatal diagnosis of NPHS1 has earlier been based on elevated maternal serum or amniotic fluid α-fetoprotein (AFP) concentration (Seppälä et al. 1976). AFP is a normal fetal serum protein synthesized in the liver, yolk sac and gastrointestinal canal (Gitlin et al. 1972). Leakage of the protein to the amniotic fluid has been demonstrated in neural tube defects as well as in other pathological conditions (Brock & Sutcliffe 1972, Ryynänen et al. 1983). In NPHS1, the presence of AFP in the amniotic fluid indicates intrauterine proteinuria and the source of maternal serum AFP is the amniotic fluid. Elevated amniotic fluid and maternal serum concentrations of AFP in NPHS1 are observed especially in the second trimester of gestation (Seppälä et al. 1967, Seppälä et al. 1976, Aula et al. 1978). A maternal serum determination between gestation weeks 14 29 and 18 has been used in screening programmes (Ryynänen et al. 1983, Albright et al. 1990, Heinonen et al. 1996). However, serum AFP concentrations are elevated on carrier pregnancies as well and are therefore unreliable (Männikkö et al. 1997, Patrakka et al. 2002a). Diagnosis of the disease has dramatically improved due to a recent process that is based on the results of this thesis study (see below).
2.5.3 Treatment
Renal transplantation is the only life-saving treatment for NPHS1 patients. Before the children reach adequate weight and size for transplantation, intensive medical attention including bilateral nephrectomy and dialysis is needed (Holmberg et al. 1995). Before nephrectomy, aggressive protein substitution and high-calorie diet are used to prevent malnutrition and reduced growth caused by urinary loss of protein. Patients are susceptible to septic infections and require prompt antibiotic therapy. Medication is used to treat hypothyroxinemia and to reduce the susceptibility to thrombosis. Despite this aggressive treatment, the children are still malnourished and hypoproteinemic (Antikainen et al. 1992). Bilateral nephrectomy and dialysis ends the proteinuria and corrects protein and lipid status prior renal transplantation. The patients’ nutritional state improves and transplantation can be performed after three months on dialysis (Holmberg et al. 1995).
2.5.4 Pathological findings
NPHS1 kidneys are pale, soft and 2-3 times larger than normal at autopsy (Huttunen et al. 1980, Holmberg et al. 1999). The size may also be close to normal due to possible atrophy and scarring of the renal parenchyma in older patients. At the light microscopy level, the most characteristic feature is tubular dilations and microcysts that are filled with colloidal material (Autio-Harmainen & Rapola 1981, Rapola et al. 1984). The number of glomeruli is abnormally high, the mean volume of the glomeruli being larger resulting in a marked increase in the total glomerular volume (Tryggvason & Kouvalainen 1975, Tryggvason 1978). At the EM level, the most striking feature is the fusion of podocyte foot processes, which is evident already at second trimester of fetal life (Autio-Harmainen & Rapola 1983). The pathological changes are considered to be secondary to proteinuria and are progressive with age (Holmberg et al. 1999). Kidney weight and size, as well as the number of glomeruli are normal at mid-pregnancy and the overall picture of the organogenesis and nephrogenesis of the affected fetuses seem normal. However, the proportion of the mature glomeruli is increased and percentage of podocytes is lower, which suggests that glomerular maturation is accelerated (Autio-Harmainen & Rapola 1981). Occasional dilated tubuli filled with eosinophilic material can be seen close to the corticomedullary border and podocyte effacement is already evident in electron 30 micrographs. At birth, the changes are still subtle, but become more pronounced during the first months of life. At this stage, dilation of the tubuli is more evident, the glomeruli are enlarged and the Bowman’s space is dilated. Progressively with time, the degree of degenerative changes, such as interstitial fibrosis and inflammation, shrinking of the glomerular tuft, fibrotic thickening of the Bowman capsule and glomerular sclerosis increases.
2.5.5 Localisation of the NPHS1 gene to chromosome 19q13.1
Since its discovery in 1956, the cause of NPHS1 has been investigated using biochemical, histological and immunological methods. Generally, massive proteinuria indicates a defect in glomerular ultrafiltration. Thus defects in the components of the GBM were considered as possible causes of NPHS1. However, no major changes in the composition of the GBM were found (Van den Heuvel et al. 1992, Ljungberg et al. 1993, Ljungberg et al. 1995). Although the molecular mechanism behind an inherited disease is unknown, the gene causing the disease can still be cloned by positional candidate or by positional cloning methods (Collins 1992). These cloning methods are based on the knowledge that when the disease gene locus and a genetic marker locus are close to each other on the same chromosome, they are inherited together. The positional cloning method usually requires a random analysis of numerous markers spanning the entire genome, while in the positional candidate cloning method markers close to a selected candidate gene are used. A prerequisite for a positional cloning project is a good sample population material from affected families. A search for the NPHS1 gene was initiated at the end of 1980’s by collecting samples from 29 Finnish NPHS1 families. Out of these families, 17 were large enough to be used in linkage analyses. First, the positional candidate cloning method was applied. By using restriction fragment length polymorphism markers (RFLP) and single strand conformation polymorphism analyses (SSCP) the genes coding for all the known basement membrane genes were excluded as causative for NPHS1 (Kestilä et al. 1994b). These genes were those coding for α1(IV), α2(IV), α3(IV) and α4(IV) chains of type IV collagen, β1, γ1 and γ2 chains of laminin and the gene coding for perlecan. A dominant gain of function mutation of PAX-2 gene has been shown to cause congenital NS in mice (Dressler et al. 1993). Deregulation of this DNA-binding protein causes development of tubular microcysts, loss of foot processes and proteinuria. PAX-2 has been localised to 10q24.3- q25.1 (Narahara et al. 1997) and analysis of 7 microsatellite markers from chromosome 10q demonstrated that PAX-2 is not causative for NPHS1 (Kestilä et al. 1994c). Since none of the known GBM components could be linked to the disease, random linkage analysis spanning the entire genome was initiated. After analysis of 90 markers a significant linkage to markers on the long arm of chromosome 19 was observed (Kestilä et al. 1994a). The critical region was about 3 cM in 19q12-13.1 close to the markers D19S224 and D19S220. To narrow the critical region, new markers from the region were established (Männikkö et al. 1995). Analysis revealed that the disease locus was in the 31 vicinity of marker D19S610. Overlapping cosmid clones from this 1-Mb region were isolated (Männikkö et al. 1995, Olsen et al. 1996) and the NPHS1 gene was fine-mapped to a region of 150-Mb (I). This region was sequenced and computer programs were used to analyse for exons. These analyses predicted the existence of at least 11 genes in the region. Comparison of the predicted exons against sequence databanks revealed that only one of the genes was previously known. This gene, APLP1, codes for amyloid precursor- like protein (Lenkkeri et al. 1998) and was excluded as causative for NPHS1 by sequencing and comparing patient and control DNA. 3 Aims of the study
The unique feature of the NPHS1 patients, the development of a progressive nephrotic syndrome soon after birth and the lack of any extra renal symptoms, make it a model disease to study the pathomechanisms of inherited proteinuria. Identification of the original molecular defect underlying NPHS1 could help to reveal the molecular pathomechanism and development of this severe syndrome, and would definitively increase our knowledge on the physiology of glomerular ultrafiltration. Since its first description in 1956, attempts to find the biochemical defect behind NPHS1 have failed. The development of molecular biology techniques and molecular genetics in the 1980’s made a positional cloning approach feasible. Prior this thesis study an extensive amount of work had been carried out in collecting samples and analysing genetic markers to map the gene locus to chromosome 19q13.1 (Kestilä et al. 1994a, Männikkö et al. 1995).
The primary aim of this study was to extend this work, the specific tasks being to:
1. Characterise genes from the 150-kb NPHS1 locus in order to find the gene mutated in the disease.
2. Generate and characterise poly and monoclonal antibodies against the protein (nephrin) defective in NPHS1.
3. Determine the precise location of nephrin in the renal ultrafilter and to study the expression of nephrin during development.
4. Analyse the expression of nephrin in renal biopsies from patients with primary acquired NS (MC, membranous GN and FSGS).
4 Materials and methods
Detailed description of the methods used can be found in the original articles.
4.1 Sequencing of cosmid clones and analysis of the sequence (I)
DNA of cosmid clones F191541, R33502, F15549, R28051, F19399, R31158 and R31874 (Olsen et al. 1996) spanning the critical NPHS1 region between markers D19S208 and D19S608 on chromosome 19q13.1 were mechanically sheared by nebulization. Fragments of 1000-2000 bp were isolated and subcloned into M13 phage and sequenced using automatic sequencers. Homology searches were performed with BLASTX and BLASTN programs (Altschul et al. 1990) against GenBank sequence databases. The nucleotide sequence was filtered using CENSOR (Jurka et al. 1996) to mask out repeat regions prior to BLASTN analysis. The programs GRAIL (Uberbacher & Mural 1991), GENSCAN (Burge & Karlin 1997), FGENEH and HEXON (Solovyev et al. 1994) were used to predict exon sequences. Mutation searches were performed with the FASTA program of the GCG package (Genetics Computer Group 1996). Protein sequence analysis was conducted with the BLASTP-program and the EXPASY molecular biology server (Appel et al. 1994).
4.2 Characterisation of genomic DNA and cDNAs (I, II)
To characterise potential novel genes in the NPHS1 region, PCR-primers were selected from predicted exon sequences to amplify cDNA fragments from kidney poly(A) RNA. These fragments were sequenced, labelled with 32P and utilised as probes to screen cDNA-libraries using standard techniques (Ausubel et al. 1989). Marathon ready cDNA kits (Clontech) were used to amplify the 5’ and 3’ ends. cDNAs were cloned into the pUC general cloning vector, sequenced and the sequence compared with the genomic sequence 34 to establish gene structures. A full-length nephrin cDNA lambda clone was isolated from a human fetal kidney cDNA library (Clontech, 5'-STRETCH).
4.3 Mutation analysis (I)
Southern analysis was used to detect possible genomic rearrangements in NPHS1 patients. For these analyses, 10 µg of genomic DNA from patients and controls were digested with EcoRI and BamHI restriction enzymes and electrophoresed on 1 % agarose gels. DNA was transferred onto nylon membranes and hybridised with 32P-labelled cDNA probes. A total of 49 Finnish NPHS1 patients, their parents and siblings together with 83 control individuals were analysed for mutations in the NPHS1 gene. All 29 exons were amplified from genomic DNA by PCR (Perkin Elmer Cetus 9600) using AmpliTaq Gold DNA Polymerase (Perkin Elmer) and specific primers. The PCR products were purified using Qiaex II gel extraction kit (Qiagen) and sequenced using termination sequencing chemistry on ABI377 and ABI310 sequencers (Perkin Elmer).
4.4 Hybridisation techniques (I, V)
Northern filters containing poly(A) RNA from eight adult and four fetal tissues (Clontech) were probed with 1.4 kb NPHS1 cDNA (exons 1-10) made by RT-PCR from fetal kidney poly(A) RNA. The hybridisations were carried out overnight at 65°C using Express Hyb hybridisation buffer (Clontech). The probe for in situ hybridizations was a 287 bp cDNA fragment from NPHS1 exon 10 corresponding to the fourth immunoglobulin like motif in the nephrin molecule. Both a digoxigenin labelled DNA probe (I) and 32P –labelled RNA sense and antisense probes (V) were used. The digoxigenin labelled probe was cut to ~150 bp fragments by alkaline hydrolysis. Tissue sections were treated proteinase K and hybridised with the probe at 62°C for 16 hr. After rinsing in 50 % formamide and standard sodium citrate, the probe was immunologically detected with anti-digoxigenin conjugated to alkaline phosphatase (Roche). The 32P-labelled probes were used as described (Wilkinson 1992) with some modifications (V).
4.5 Recombinant protein expression and purification (II, IV, V)
For prokaryotic expression of nephrin fragments in E. coli, vectors pQE-30 and pQE-40 from Qiagen were used. Eukaryotic expression in HEK293 and COS-7 cells was achieved by using vectors pcDNA3 and pCR3 (Invitrogen). The pCR3 contained a cDNA 35
Table 1. Recombinant nephrin proteins.
Protein Amino Domains1 Vector Host Fusion Purification acids partner AG-12 1084-1241 Intracellular pQE-40 E.coli His-DHFR3 + AG-2 23-240 Ig1-2 pQE-30 E.coli His4 + AG-3 1-1059 Extracellular pCR3 HEK293 Fc5 + Nph1-142 1-142 Ig1 pcDNA3 COS-7 - - Nph1-243 1-243 Ig1-2 pcDNA3 COS-7 - - Nph1-344 1-344 Ig1-3 pcDNA3 COS-7 - - Nph1-450 1-450 Ig1-4 pcDNA3 COS-7 - - Nph1-558 1-558 Ig1-5 pcDNA3 COS-7 - - Nph1-646 1-646 Ig1-6 pcDNA3 COS-7 - - Nph1-748 1-748 Ig1-SP pcDNA3 COS-7 - - Nph1-845 1-845 Ig1-7 pcDNA3 COS-7 - - Nph1-954 1-954 Ig1-8 pcDNA3 COS-7 - - NphExHis 1-1059 Extracellular pcDNA3 HEK293 His6 + Full-length 1-1241 Full-length pcDNA3 COS-7 - -
1 Ig1 through Ig1-8 stand for extracellular immunoglobulin-like domains in nephrin. 2 AG-1 = antigen 1, 3Amino-terminal sequence of six histidines and mouse dihydrofolate reductase (DHFR). 4 Amino-terminal sequence of six histidines for immobilized metal affinity chromatography. 5 Fc portion of human IgG. 6 Carboxy-terminal sequence of six histidines.
cassette encoding the Fc part of human IgG1 (Schneider et al. 1997). The nephrin lambda cDNA clone was subcloned as two separate EcoRI fragments into pBluescript (Stratagene). Synthetic oligonucleotides and PCR were used to introduce new restriction sites to the cDNA in order to facilitate ligation into expression vectors. These modified fragments and the cDNA clones were utilised in engineering the expression plasmids presented in Table 1. Constructs were sequenced by using termination sequencing chemistry on ABI377 and ABI310 sequencers (Perkin Elmer). Recombinant proteins AG-1 and AG-2 were solubilized from E. coli inclusion particles in 8 M urea and purified with immobilized metal affinity chromatography on a Ni-NTA column (Qiagen) and with anion exchange chromatography on a Q-sepharose column (Amersham). The fragment AG-3 containing the human IgG Fc-portion was purified from cell culture media by utilising a HiTrap protein A column (Amersham). The fragment NphExHis was purified with a Q-Sepharose column (Amersham) and a TALON affinity resin (Clontech). 36
4.6 Production of antibodies (II, III, V, IV)
Polyclonal antibodies (PoAb) were raised in rabbits using standard procedures (SLA, Uppsala, Sweden). The antisera were characterised by using Western blotting, enzyme- linked immunosorbent assay (ELISA) and IF-microscopy by using preimmune sera as a negative control. Rabbit IgG was purified from serum using protein A-chromatography (Amersham Pharmacia Biotech). Immunisation of mice and production of monoclonal antibodies (mAb) was done following standard protocols (Ausubel et al. 1989). The protocol and use of animals was accepted by the animal care and use committee of the University of Oulu. Antiserum titres were determined with ELISA using antigen-coated plates. Growing hybridomas were screened with the ELISA and IF-microscopy using nephrin expressing COS-7 cells and cryosections of human kidney. Cell lines were cloned by the limiting dilution method and IgG-subclasses were determined. Cultures were expanded and IgG was purified from conditioned media using protein G-column chromatography (Amersham Pharmacia Biotech).
4.7 Protein electrophoresis and Western blot analysis (II, IV, VI)
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses were performed according to standard methods (Laemmli 1970, Towbin et al. 1979). Proteins were transferred onto a polyvinylene difluoride membrane. The membrane was blocked with bovine albumin or low-fat milk and probed with diluted antiserum or purified IgG. After washings, the bound immunoglobulins were detected with enzyme conjugated secondary antibody (Jackson ImmunoResearch) or protein A (Amersham). Peroxidase enzyme was detected either with diaminobenzedine or a chemiluminescent reagent (ECL, Amersham Pharmacia biotech) and alkaline phosphatase with BCIP/NBT.
4.8 Electron microscopy (II, V)
EM was performed according to standard procedures (fixation in 2.5 % glutaraldehyde, followed by OsO4 and Epon embedding). Thin sections were post-stained with 1 % uranyl acetate and lead. For immuno-EM, kidneys were stored in ViaSpan solution (DuPont Merck) for 12-24 hr until processing. Tissue cubes (1 x 1 x 5mm) were fixed in 3.5 % paraformaldehyde (Sigma) with or without 0.01 % glutaraldehyde (Electron Microscopy Sciences) in 0.1 M phosphate buffer (pH 7.3) at room temperature. Fixed samples were washed in phosphate buffer, dehydrated in graded ethanol and embedded in LR white (London Resins) and Lowicryl K4M (Polysciences) resins. Thin sections were placed on Pioloform (Agar Scientific) and carbon-coated nickel grids. Grids were 37 incubated for 60 min in the first antibody diluted in 3 % bovine albumin. After incubation in gold conjugated secondary antibodies, the sections were post-stained in 1 % uranyl acetate. Double labelling was performed sequentially on different sides of thin resin sections, as described earlier (Bendayan 1982). The sections were examined with a Jeol 1200 EX electron microscope at 60 kV.
4.9 Immunohistochemistry (II, III, IV, V, VI)
Immunohistochemistry was accessed by indirect methods. Samples for immuno- peroxidase staining were fixed in 10 % formalin or 4 % paraformaldehyde in phosphate buffer. Paraffin sections were deparaffinized and incubated with 3 % peroxide to quench endogenous peroxidase. Slides were heat-treated in citrate buffer to unmask antibody epitopes. Primary antibodies were detected with biotin-labelled secondary antibody and peroxidase-avidin complex (ABC-reagent, Vector Laboratories) and visualized with diaminobenzidine. For immunofluorescence (IF) microscopy, cryosections and cultured cells were fixed in ethanol or phosphate buffered 2-4 % paraformaldehyde. Free aldehyde groups generated by formaldyhyde were blocked with glycine and nonspecific binding was quenched with bovine albumin in phosphate buffer. The primary antibody was detected with FITC, Cy2, Cy3 or TRICT labelled secondary antibody and the slides were examined with fluorescence microscope. Control experiments were performed with preimmune antisera or by omitting the primary antibody.
4.10 Expression of nephrin in cultured podocytes (VI)
Primary cultures of human podocytes were established as previously described (Camussi et al. 1985, Striker & Striker 1985, Conaldi et al. 1997). Established lines of differentiated podocytes were obtained by infection of pure primary cultures with a hybrid Adeno5/SV40 virus (Conaldi et al. 1998). The expression of nephrin in cultured podocytes was evaluated by indirect IF-microscopy on paraformaldehyde fixed cells. To access intracellular staining, cells were permeabilized with Triton X-100. Actin microfilament alterations were evaluated with FITC-phalloidin staining after permeabilization of the cells. To study in vitro the role of immune complexes in nephrin expression and distribution, cells were incubated with aggregated human IgG prior to fixation and detection with anti-nephrin. Dissaggregated human IgG was used in control experiments. The effect of the cell cytoskeleton on nephrin redistribution was evaluated by incubating podocytes with TNF-α, puromycin, cytochalasin B, sodium azide and the membrane attack complex of complement. 5 Results
5.1 Identification of the NPHS1 gene on chromosome 19q13.1 (I)
Mapping of the NPHS1 locus to chromosomal region 19q13.1 has been described earlier (Kestilä et al. 1994a, Männikkö et al. 1995). Overlapping cosmids clones spanning 920 kb of the critical region between markers D19S208 and D19224 were isolated and new microsatellite markers were identified from the region and used in fine-mapping the NPHS1 gene. Significant linkage disequilibrium was observed with markers D19S608 and D19S610 as well as with new markers (D19S1173, D19S1175and D19S1176) and the gene was mapped to a 150 kb region between markers D19S1175 and D19S608. Southern analysis was used determine possible genomic rearrangements in the region, but no variation was observed. The 150 kb region was sequenced at the Lawrence Livermore National Library and analysed with several programs to predict exon sequences. The sequence was found to contain over 100 putative exons and EST-hits were found to at least eight genes. At least 11 genes could be predicted in the 150 kb region. We determined the gene structure for five genes in the locus. Northern analysis could not exclude any of the genes studied as causative for NPHS1, as they were all expressed in the kidney. The locus contained only one gene that was previously known. This gene, APLP1, encodes the amyloid precursor-like protein (Lenkkeri et al. 1998). Another gene (gene B) that was characterised and shown to be highly expressed in peripheral leukocytes (Fig. 4) has later turned out to encode TYROBP (formerly DAP12), a protein of significance in immune response (Lanier et al. 1998). Interestingly, we found this gene to be deleted in another disease of the Finnish disease heritage, PLOSL, characterised by early dementia and bone cysts (Paloneva et al. 2000). The structure of APLP1 and novel genes A, B (TYROBP) and C were determined and excluded as the NPHS1 gene by sequencing all the exons from patient and control DNAs. One gene with a transcript of about 4.3-kb was found to be strongly expressed in human embryonic and adult kidney, with no detectable signal in the other tissues studied (I, Fig. 2). The cDNA was constructed using PCR primers based on predicted exon sequences. cDNA fragments were amplified from poly(A)mRNA, sequenced and 39 compared to genomic sequence to determine the gene structure. The gene, NPHS1, contained 29 exons spread over 26 kb of genomic DNA. Sequencing of the exons from patient DNA revealed two major mutations in over 90% of NPHS1 chromosomes. Of the 49 Finnish NPHS1 patients studied, 32 were homozygous for a 2-bp deletion in exon 2. This deletion, nt121(del2), termed Fin-major, explains the majority of Finnish cases. The Fin-major mutation leads to a frame shift and a premature stop codon. The second mutation, termed Fin-minor, is a nonsense mutation nt3325(C→T) in exon 26 in which an arginine codon is replaced with a stop codon. The Fin-minor mutation was homozygous in 4 patients, while 8 patients were compound heterozygotes for the Fin-major/Fin-minor alleles. Four patients had the Fin-major in one allele and a completely different mutation in the other allele. One patient had a mutation different from the Fin-major or minor in both chromosomes. None of the 108 parents and 54 healthy siblings was a homozygote or a compound heterozygote for the mutations. Furthermore, only one heterozygous Fin-major mutation was found in 83 healthy controls.
Fig. 4. Northern blot analysis of gene B (TYROBP) expression with mRNA from human embryonic and adult tissues. Multiple tissue northern blots (Clontech) containing 2 µg of poly(A)RNA per lane were hybridised with a 0.8 kb cDNA probe. The transcript of about 0.8 kb (arrow) can be observed in all the tissues studied. However, the strongest expression is in peripheral blood leukocytes. On the left are the RNA size markers (kb). 40
5.2 Characterisation of the gene product (I)
Sequence analysis of the cDNA-deduced translation product revealed a 1241 residue protein with a putative 22 amino acid N-terminal signal sequence and another 28 residue hydrophobic region that was predicted to be a transmembrane domain. These findings indicated that the protein would be a type I single-pass transmembrane protein. The computer analyses and data base comparisons revealed that the predicted 1036 amino acid extracellular domain contains eight immunoglobulin modules and one fibronectin type III module, while the 155 amino acid intracellular domain had no homology to other proteins. The putative extracellular domain contains ten NXT and NXS sequences known as sites for N-glycosylation and seven SG doublets that are potential heparan sulphate attachment sites. The carboxyterminal intracellular domain contains 9 tyrosine residues, which are possible targets of phosphorylation. The sequence predicted structure of the protein is shown in Fig. 5. Northern hybridisation analysis revealed expression of the mRNA in both embryonic and adult kidney. No detectable expression was observed in the other tissues studied. In situ hybridisation was carried out on a kidney sample from a 23-week-old human embryo. Expression of the mRNA was found in both developing and mature-stage glomeruli. Localisation of the signal to the periphery of the glomeruli leaving the central mesangial regions negative indicated that the positive cells are podocytes. As the gene was expressed only in the nephron with no apparent extrarenal expression, the gene product was termed nephrin.
polyclonal antibodies PoAb2 (antigen AG-2)
12345 6 7 8FnTm domain C structure N
32A7 41F2 48E3 43H7 3D11 32B1 43C7 49H12 47B8 7C1 50A9 47H12 49E9 46G6 48E11 48H1
Fig. 5. Domain structure of nephrin and location of anti-nephrin antibody epitopes. Nephrin is a single-pass transmembrane protein of the immunoglobulin superfamily. The extracellular domain of nephrin consists of eight Ig-modules depicted with semicircles (1-8) and one fibronectin like module (Fn). The C-terminal intracellular domain is separated from the extracellular domain by a short transmembrane domain (Tm). Locations of epitopes for polyclonal and monoclonal antibodies are depicted with brackets. 41
5.3 Protein expression and characterisation of antibodies (II, III, IV, V, V I )
In order to obtain antigens for raising anti-nephrin antibodies, various fragments of nephrin were produced as recombinant protein in prokaryotic and eukaryotic expression systems. SDS-PAGE and Western blot analyses were conducted to access the purity and size of the antigens. Antigens AG-1 and AG-1 migrated with the expected sizes (~48 000 and ~29 000, respectively) under both non-reducing and reducing conditions. Antigen NphExHis had a MW of ~140 000 under reducing conditions, but migrated slightly faster under non-reducing conditions. Antigen AG-3 migrated with a MW of ~280 000 under non-reducing conditions, while upon reduction of the Fc-disulphide with dithiotreitol the antigen migrated with a MW of ~150 000. Nested nephrin fragments were analysed from cell lysates and conditioned cell culture media. Analysis with a mAb recognising the first Ig-like module revealed varying sized nephrin fragments that migrated in accordance with their calculated MW. The antigens AG-1 and AG-2 were used to raise rabbit polyclonal antibodies and antigens AG-1, AG-2 and AG-3 were used to immunise mice for mAb production. Antibodies were characterised using Western blot analysis, ELISA, microscopy and immunoprecipitation techniques. Epitopes for the mAbs against extracellular domain were determined by Western blot analysis of nested nephrin fragments and by sandwich ELISA using NphExHis as antigen. The location of the antibody epitopes is summarised in Fig. 5. The antibodies recognised a protein with a MW of 180 000 in Western analysis from extracted human glomeruli (IV, Fig. 1C, NHG), while the full-length recombinant nephrin had a MW of about 150 000 (IV, Fig. 1C, Full-length). Analysis of mock- transfected cells and NPHS1 glomeruli did not reveal immunoreactive bands (Patrakka et al. 2000), nor did an extract of normal glomeruli or recombinant nephrin when analysed with preimmune antisera (Patrakka et al. 2001).
5.4 Localisation of nephrin in the kidney glomerulus (II)
Using indirect IF-microscopy (PoAb1) on cryosections of human kidney nephrin immunoreactivity was observed along the GBM while podocyte cell bodies were negative. This immunostaining pattern, the in situ hybridisation result (I) and the sequence predicted structure suggested that nephrin would be expressed by podocytes and transported to the cell membrane in the podocyte-GBM interface or to the podocyte- podocyte junction. To localise nephrin more precisely, post-embedding immuno-EM was used utilising the rabbit anti-nephrin antibody (PoAb1). Bound rabbit IgG was detected with a goat anti-rabbit antibody that was labelled with gold particles. Although the labelling efficiency was rather low, practically all the label was concentrated to the SD between podocyte foot processes. One or two gold particles were seen in approximately every fifth to tenth foot process interspace. No labelling was observed in endothelial or 42 mesangial cells and no label was present in the podocyte-GBM interspace. Some gold particles were detected in the cytoplasm of podocyte foot processes.
5.5 Nephrin in developing normal and NPHS1 glomerulus (V)
The expression of nephrin mRNA was studied by the in situ hybridisation technique using fetal kidney samples. Early stages of glomerulogenesis remained negative for nephrin mRNA, and no signal was obtained from vesicle, comma-shaped and early S- shaped stages. Synthesis of nephrin messenger began in the late S-shaped glomeruli and was most intense at the beginning of the capillary loop stage. The signal was located near the area of the vascular cleft. With IF and immunoperoxidase staining of fetal kidney with anti-nephrin antibodies (PoAb2 and the pool of mAbs 43C7, 50A9 and 48E11) the staining was observed in the late S-shape stage. A faint staining was observed in the columnar epithelial cells near the vascular cleft. At the capillary loop stage, staining was observed in the basal margin of the epithelial cells and also between the developing podocytes. Strongly staining dots were present between developing podocytes in 36% of the 70 capillary stage glomeruli analysed. In the maturing glomeruli the stain appeared in a strong linear pattern in the basal margin of the podocytes. Using immuno-EM (PoAb1 and PoAb2), nephrin was localised to ladder-like junctions of the columnar epithelial cells of the capillary loop stage glomeruli. In maturing glomeruli, nephrin staining was concentrated into the SD area. Analysis of the two NPHS1 kidneys carrying homozygous Fin-major mutation showed a normal level of nephrin mRNA signal based on the in situ hybridisation result, but IF staining failed to detect any nephrin. By EM, the typical fusion and irregularity of the podocyte foot processes was observed in the maturing glomeruli of these patients. The SD was completely absent and, moreover, ladder-like junctions were not observed. The tight junction protein ZO-1 co-localised spatially and temporally with nephrin in normal glomeruli. In addition to podocyte staining, ZO-1 was also found in parietal and tubular epithelium. ZO-1 was first detected in S-shaped stage basal margin and also between presumptive podocytes. Moreover, ZO-1 was also seen as intensively stained dots between developing podocytes in the capillary loop stage. In immunoelectron microscopy ZO-1 co-localised with nephrin to the ladder-like structures of developing capillary stage podocytes. Staining for P-cadherin was first seen in the apical margin of the vesicle stage glomeruli. Signal in the glomerulus was weaker compared to a strong expression in ureteric buds and developing tubuli. In the late S-shaped stage, P-cadherin co-localised with nephrin and ZO-1 to the basal margin of the epithelial cells. Dotted staining between podocytes in the capillary stage was not detected. Staining for ZO-1 and P-cadherin was indistinguishable from normal in NPHS1 kidneys. 43
5.6 Expression of nephrin and CD2AP in developing mouse and human glomeruli (III)
CD2AP is a protein interacting with the T-cell receptor CD2 (Dustin et al. 1998). Surprisingly, CD2AP deficient mice develop nephrotic syndrome and CD2AP was shown to interact with intracellular domain of nephrin in transfected cells (Shih et al. 1999). Therefore, we decided to compare the expression of nephrin and CD2AP in developing mouse and human kidneys. Patterns of expression of nephrin and CD2AP were determined and compared by IF- microscopy. In newborn normal and Cd2ap +/- mice expression of CD2AP and nephrin (PoAb2) first appeared in the capillary loop stage. The staining of both proteins in the glomerulus was concentrated in the basal margin of podocytes, with some diffuse staining in podocyte cytoplasm. Newborn Cd2ap -/- mice were negative for CD2AP, but the expression of nephrin was unaltered. In 2-week old Cd2ap +/- mice, both proteins were detected basally adjacent to the GBM, with some degree of diffuse staining of the podocyte cytoplasm. In Cd2ap -/- mice the expression of nephrin was normal, while CD2AP staining was absent. In adult Cd2ap +/- glomeruli (7-week), the CD2AP was less diffuse and more basally concentrated, whereas nephrin maintained a diffuse pattern restricted to podocytes. In Cd2ap -/- glomeruli, nephrin staining was undetectable in the great majority of glomeruli. At this stage, mice were severely damaged by mesangial matrix deposition, chronic NS and widespread foot process effacement. However, in some occasional glomeruli, there was segmental immunoreactivity for nephrin. In fetal human kidney, nephrin became detectable slightly earlier than CD2AP. The difference was minimal and both proteins were first detected at the capillary loop stage. In maturing glomeruli, both proteins co-localised to the basal margin of podocytes.
5.7 Nephrin involvement in the pathogenesis of proteinuria (VI)
Indirect IF-microscopy utilising monoclonal anti-nephrin antibody (48E11) was used to evaluate expression of nephrin in proteinuric patients. Nephrin expression was analysed semiquantitatively by measuring fluorescence intensity of images obtained using a low- light video camera and image-analysis software. A total of 30 samples from patients suffering from membranous GN (13), MC (10) and FSGS (7) were studied together with 10 control specimens. Out of the 30 patients, 27 had developed NS. Also 6 patients having IgA GN with minimal proteinuria were studied. In normal controls, staining for nephrin was in a finely punctuated to linear pattern along the GBM indicating glomerular epithelial staining. In glomeruli of patients with membranous GN, nephrin was in a more granular pattern and significant reduction in fluorescence was observed. Staining for nephrin co-localised with the staining for human IgG in membranous GN patients. A change from a linear to a more granular pattern and reduction in fluorescence intensity was also observed with patients suffering from MC and FSGS. In samples from patients with IgA GN, the staining pattern and fluorescence intensity was similar to controls. 44
In cultured glomerular epithelial cells nephrin had a finely granular distribution on the cell surface. Incubation of the cells prior to fixation with anti-nephrin mAb (48E11) caused conversion of nephrin staining from a fine granular to a patchy pattern. Incubation of the cells with aggregated human IgG4 caused a redistribution of the nephrin from an even to a focal distribution and a disruption of the normal cellular organization of F-actin. The overall intensity of the staining was reduced and nephrin co-localised with aggregated human IgG. Disaggregated human IgG did not induce alterations in nephrin and F-actin staining. The membrane attack complex of complement, TNF-α and puromycin are known to affect the cytoskeleton of podocytes (Camussi et al. 1987, Camussi et al. 1993, Coers et al. 1994). Introduction of these factors into cultured podocytes induced a significant reduction of IF-staining and a redistribution of nephrin. This effect was reversible since 24-hours after removal of TNF-α from the culture medium, nephrin expression was back to the normal level. Sodium azide and cytochalasin B prevented the redistribution and reduction in expression of nephrin. 6 Discussion
The major result of this thesis study is the identification of the NPHS1 gene and localisation of the gene product – nephrin – to the podocyte SD. Identification of the disease gene not only provides valuable tools for diagnosing the disease, but it also improves our understanding the pathogenesis of this serious disease and possibly serves as a guide to better treatment of NPHS1 patients. For example, we have been able to develop an assay and identify circulating anti-nephrin antibodies in NPHS1 patients suffering from recurrent nephrosis (Patrakka et al. 2002b). The assay can be used to monitor the NPHS1 patients in order to identify those at risk for recurrent proteinuria in the kidney graft. The SD has been an enigmatic structure for decades and nephrin, being the first molecule to be identified to the SD itself, has drawn interest to this practically uncharacterised cell junction. New molecules to the SD region have subsequently been identified and the molecular nature of this mysterious structure has begun to emerge. Furthermore, the spatial and temporal location of nephrin in developing normal and diseased kidneys was analysed and compared to the expression of three other SD components. In addition to a role for nephrin as a structural component of the SD, these studies indicate a specific role for nephrin in the development of the SD. Finally, the observation of reduced nephrin expression in patients suffering from acquired kidney diseases emphasizes the importance of nephrin and the integrity of the SD not only in NPHS1, but also more generally in normal and disturbed filtration process.
6.1 The NPHS1 mutations
This thesis study led to identification of the gene causing the congenital nephrotic syndrome of the Finnish type. The critical NPHS1 region on chromosome 19q13.1 was narrowed further to a 150-kb region and the cosmid clones spanning the area were sequenced. As initial analysis did not reveal larger genomic rearrangements and all the genes studied were expressed in the kidney, there were no candidate genes in the region. However, by systematic analysis of the computer predicted exon sequences the disease- 46 causing gene was identified. Following sequencing of five genes, two mutations were found in Finnish patients in a novel gene, now known as NPHS1. The two mutations, termed Fin-major and Fin-minor, were found to cause over 94% of the Finnish NPHS1 cases. Typically, in diseases belonging to the Finnish disease heritage, one mutation is carried by practically all the affected patients (Peltonen & Uusitalo 1997), meaning that one founder introduced the mutation into the population. Observation of two separate mutations indicates that there have been two founders for the NPHS1 in the Finnish population history. Both of the Finnish mutations lead to equally severe nephrotic syndrome and no differences between the patient-groups have been observed (Patrakka et al. 2000). These mutations also lead to total absence of nephrin and SDs in the kidney. Interestingly, transcription of the NPHS1 mRNA was detected in NPHS1 kidneys as accessed by in situ hybridisation technique (Patrakka et al. 2000). Usually mRNAs containing premature stop codons are strongly downregulated (Wagner & Lykke-Andersen 2002). In contrast to a typical Finnish NPHS1 patient, analysis of a biopsy specimen from a heterozygous NPHS1 patient with Fin-major/R743C alleles revealed expression of the protein and visible slit diaphragms. Furthermore, unlike typical Finnish NPHS1 patients this patient responded to therapy that reduces glomerular filtration pressure, thereby avoiding transplantation (Patrakka et al. 2000). This is an important observation that may affect the treatment and outcome of patients, especially of non-Finnish origin (see below). Already the identification of the NPHS1 locus enabled prenatal diagnosis on Finnish families with NPHS1 history (Männikkö et al. 1997). Now after identification and characterization of the gene, the diagnosis is more accurate and can be now performed on non-Finnish families as well. Historically, the prenatal diagnosis of NPHS1 has been based on elevated maternal serum or amniotic fluid AFP concentration and exclusion of other reasons of elevated AFP by ultrasonography. After termination of the pregnancy the diagnosis has been confirmed by EM microscopy of the fetal kidneys. The source for the amniotic fluid and maternal serum AFP is the fetal serum and in NPHS1 it indicates prenatal proteinuria. However, it is now evident that NPHS1 carrier fetuses have temporal proteinuria in utero and therefore the diagnosis based on AFP measurement may lead to unjustified termination of the pregnancy (Patrakka et al. 2002a). To date, more than 60 different mutations have been discovered in the nephrin molecule in patients with different ethnic backgrounds (I, Bolk et al. 1999, Lenkkeri et al. 1999, Aya et al. 2000, Beltcheva et al. 2001, Gigante et al. 2002, Koziell et al. 2002). These mutations include deletions, insertions, nonsense, missense, splice site and promoter mutations. The mutations seem to span almost the entire length of the nephrin molecule. Surprisingly many of the mutations are missense mutations resulting in single amino acid substitutions. Liu and co-workers (2001b) studied the fate of 21 missense mutations on transfected cells. Over 75 % of the mutated molecules were not transported to the cell surface. Instead, they were trapped in the endoplasmic reticulum probably due to misfolding of the molecule. Although NPHS1 is most abundant in Finland, the numerous non-Finnish-type mutations reported in other countries demonstrate that the disease is not restricted to Finland. It is now also evident that milder forms of the disease exist (e.g., the Fin- major/R743C case). As discussed above, the Fin-major and Fin-minor mutations lead to a severe nephrotic syndrome. However, there have been some reports of congenital 47 nephrotic syndrome patients that have responded to angiotensin converting enzyme inhibitor therapy (Guez et al. 1998, Patrakka et al. 2000). In a recent report, Koziel and co-workers (Koziell et al. 2002) reported that six out of 16 patients with an R1160X mutation have such an atypically mild disease. The report also describes how mutations in another SD protein gene, NPHS2, can cause the classical severe type seen in NPHS1. NPHS2 encodes for podocin (see below) and has been reported to be mutated in an early onset autosomal recessive form of steroid-resistant NS (Boute et al. 2000). Furthermore, patients possessing mutations simultaneously in both NPHS1 and NPHS2 genes have a phenotype of congenital FSGS. These observations widen the spectrum of both the genetics and phenotype of NPHS1 (Koziell et al. 2002).
6.2 Nephrin – crucial component of the slit diaphragm
A significant result of this thesis work is the localisation of nephrin to the SD between two podocyte foot processes. Two other research groups have confirmed this observation (Holthöfer et al. 1999, Holzman et al. 1999). The SD has been a rather mysterious structure for decades and no molecule apart from ZO-1 has been associated with it prior to nephrin. Subsequently, other molecules have been localised to the SD region and the molecular structure of the SD has begun to emerge as discussed below. Identification of nephrin in the SD has given us new insights into the glomerular filter and led us to consider the SD the as the final decisive size-selective barrier. Structurally, nephrin belongs to the immunoglobulin superfamily of cell adhesion molecules. Nephrin has three distinct domains: the large extracellular domain, single-pass transmembrane domain and the intracellular domain. The extracellular domain contains eight immunoglobulin modules and one fibronectin type III module. The extracellular domain is highly glycosylated and the glycosylation is critical for the plasma membrane location of the protein (Yan et al. 2002). The intracellular domain contains several tyrosine residues that are possible targets for phosphorylation. In fact, nephrin has been demonstrated to be phosphorylated (Simons et al. 2001). The structure of nephrin and its location to the SD led to the hypothesis that nephrin would interact in a homophilic manner over the filtration slit between two opposing podocyte foot processes thus forming the SD (II, Fig. 5). There is recent evidence supporting this hypothesis (Gerke et al. 2003). Nephrin is indispensable for proper glomerular filtration. As already discussed, most of the mutations in the nephrin molecule cause complete lack of functional nephrin in podocytes. More evidence on the important role of nephrin in controlling the glomerular filtration comes from the studies with rat nephrin. Injection of monoclonal anti-nephrin antibody (at that time the antibody was known as mAb 5-1-6) induces rapid and reversible proteinuria (Orikasa et al. 1988). Interestingly, the anti-nephrin injection causes a decrease in ZO-1 expression (Kawachi et al. 1997). This is in contrast to results with NPHS1 patients possessing the Fin-major and Fin-minor mutations. The ZO-1 expression is normal in the patients as assessed by IF-microscopy and immunoblotting (Patrakka et al. 2000). The proteinuria caused by 5-1-6 is independent of the 48 inflammatory cells and complement and no histological abnormalities can be detected at the light microscopy level (Kawachi et al. 2002a). By day 5 after the injection, when proteinuria is most severe, only partial retraction of the podocyte foot processes can be observed on EM. These observations together with the observation that nephrin dislocates from the filtration slit while the SD remains visible by EM caused Kawachi and co- workers (Kawachi et al. 2000) to suspect the structural role of nephrin in the SD. However, the phenotype of NPHS1 patients, the location of nephrin in SD, and the studies with mAb 5-1-6 emphasize the role for nephrin in the regulation of glomerular filtration. A transgenic model of NPHS1 has been generated (Putaala et al. 2001, Rantanen et al. 2002). Nephrin deficient homozygous mice are born alive but rapidly develop edema and die within 24 hours after birth. Analysis of the urine revealed heavy proteinuria, with serum albumin as the major protein. Histological examination of the null allele pups revealed findings typical for human NPHS1 kidneys. Therefore, the transgenic model of NPHS1 confirms the observations made for human disease.
6.3 Nephrin in the developing glomerulus
Developmental expression of nephrin in rat and mouse has been reported (Kawachi et al. 1995, Holzman et al. 1999). The expression has also been studied with in situ hybridisation techniques and promoter analyses utilising reporter gene techniques in transgenic mice (Moeller et al. 2000, Putaala et al. 2000, Wong et al. 2000, Putaala et al. 2001, Beltcheva et al. 2003). In the developing rat kidney, nephrin was first detected (by using the mAb 5-1-6) at the S-shaped body stage of glomerular development (Kawachi et al. 1995). Nephrin was seen below the tight junctions on the basal and lateral cell membranes of the capillary loop stage glomeruli and was later concentrated at the filtration slits. At the S-shaped body stage, ZO-1 was localised to the intracellular site of junctional complexes and migrated down to the basal level along with the junctional complexes. In mouse, nephrin was absent in the S-shape body stage, but it was localised to the vicinity of the intercellular junctions of capillary loop stage glomeruli (Holzman et al. 1999). In this thesis work, the expression of nephrin in human and mouse glomerular development was studied. The expression of nephrin was compared to three other molecules of the SD-region: ZO-1, P-cadherin and CD2AP. In addition to normal glomerular development, expression of nephrin was studied in human NPHS1 kidneys and in kidneys of CD2AP deficient mice. Expression of nephrin, ZO-1 and CD2AP appeared simultaneously during the late S-shaped stage or in the capillary stage of glomerular development depending on the species and possibly on the sensitivity of the detection method used. In contrast to these three proteins, the expression of P-cadherin started already at the vesicle stage. However, at the capillary loop stage the expression of P-cadherin was co-localised with the other proteins to the basal and lateral surfaces of the developing podocytes. At the human capillary loop stage glomerulus, nephrin and ZO-1 were found as intensively staining dots at the lateral surface between developing 49 columnar podocytes and to some extent at the basal margin of podocytes. The observation that the temporal and spatial expression of nephrin, ZO-1 and CD2AP overlaps indicates that these proteins may interact during glomerular development. Perhaps the most interesting observation of these developmental studies is the localisation of nephrin and ZO-1 to the ladder-like structures. These structures are seen between developing podocytes in the capillary loop stage glomeruli and they have also been reported in animal models of nephrotic syndrome (Ryan et al. 1975, Schneeberger & Grupe 1976). In NPHS1 kidneys these ladder-like structures and mature SDs were completely missing, but the early junctions between columnar podocytes were present and ZO-1 was normally localised in these complexes. These results indicate that nephrin is not essential for the early junctional complexes, but it is essential for the development of the ladder-like structures and the SDs. However, the relationship between these two structures remains unclear. Interestingly, in newborn CD2AP deficient mice the expression of nephrin was comparable to controls. This observation is in line with the fact that these mice are not nephrotic at birth. Instead, they begin to develop proteinuria at 2 weeks of age and die at 6-7 weeks of age. Histological examination of the kidneys at 7 weeks of age reveals global glomerulosclerosis and extracellular matrix depositions in the mesangium (Shih et al. 2001). At this stage the expression of nephrin is severely altered with only segmental expression in occasional glomeruli. Although the CD2AP does not seem to be crucial for the podocyte and SD development, it is evidently important for the maintenance of the filtration barrier. Expression of nephrin was initially thought to be limited to the kidney (I). However in rodents, nephrin mRNA expression has been subsequently observed in certain regions of the central nervous system, pancreas, spleen, thymus and testis (Putaala et al. 2000, Liu et al. 2001a, Putaala et al. 2001). This tissue and cell lineage specific expression is achieved by alternatively used promoters (Beltcheva et al. 2003). In addition to this complex pattern of expression, different splice variants have been reported (Holthöfer et al. 1999, Luimula et al. 2000a). In man the nephrin expression in pancreatic β-cells has been reported, but no expression in the central nervous system was observed (Palmen et al. 2001). The significance of the extrarenal expression of nephrin remains to be investigated and interestingly, a recent study reports no extrarenal expression in man and swine (Kuusniemi et al. 2004).
6.4 Molecular structure of the slit diaphragm
The composition of the SD has been a mystery for decades. Previously the only molecules associated with this structure have been ZO-1 and the unknown antigen for mAb 5-1-6 (Orikasa et al. 1988, Schnabel et al. 1990). Localisation of nephrin to the SD made it the first known protein to be identified in this structure. Subsequently, antibody 5-1-6 has been shown to react with rat nephrin (Topham et al. 1999). After identification of nephrin as a component of the SD several other proteins have been localised to this structure. Some of these proteins have structures similar to cell adhesion receptors with 50 extra and intracellular domains, while others are integral membrane proteins or intracellular adapter proteins that organize and connect the receptors to the cytoskeleton. A second receptor-type protein is P-cadherin. Reiser and co-workers (Reiser et al. 2000) demonstrated that P-cadherin co-localised with the adaptor proteins ZO-1 and α-, β- and γ-catenins on cultured podocytes and P-cadherin was also shown to be present in the SD of rat kidney glomeruli. It was suggested that P-cadherin forms the backbone of the SD, while nephrin might be responsible for the size-selective property of the slit. A similar suggestion was made based on the observation that anti-nephrin (mAb 5-1-6) injection to rats dislocates nephrin from the slit to apical plasma membrane of podocyte foot process, while the visual SD seems to remain intact (Fujigaki et al. 1996, Kawachi et al. 1997, Kawachi et al. 2000). However, this model is in contrast with the observed effects of the nephrin mutations both in human and mice. Furthermore, patients with a mutation in the P-cadherin gene lack nephrotic phenotype (Sprecher et al. 2001) and P- cadherin deficient mice do not have kidney defects either (Radice et al. 1997). These observations imply that the role of P-cadherin in glomerular filtration is dispensable. The third receptor type protein of the SD is FAT, a mammalian homolog for the Drosophila tumour suppressor gene fat (Ponassi et al. 1999). FAT is a large (MW >500 000) protein and the sequence predicted structure suggests that it is an adhesion molecule of the cadherin superfamily. It contains 34 consecutive cadherin repeats in the extracellular domain, a transmembrane region and an intracellular domain that contains a potential β-catenin binding region. FAT is widely expressed in fetal tissues, while in the adult its expression is low. In the podocytes FAT was localised to the SD (Inoue et al. 2001). The sequence predicted structure and the localisation of this huge protein in the podocytes indicates that it is a structural component of the SD. Recently, podocyte effacement and the perinatal death in FAT deficient mice has been reported (Ciani et al. 2003). The fourth receptor type protein identified in the SD is Neph1. A retrovirus mediated gene-trap method was used to identify this novel murine transmembrane protein (Donoviel et al. 2001). Based on the cDNA sequence, Neph1 is a putative transmembrane protein with five extracellular Ig-like domains and it has a MW of 67 000. It has some degree of sequence homology (18% identity) to nephrin. Neph1 deficient mice suffer from heavy proteinuria without edema and most of the pups die before the age of 12 days. Reporter gene analysis of Neph1 +/- newborn mice and adult kidney confirmed the expression of the protein in glomerular epithelium, although expression was not restricted to podocytes and extra-renal expression was also observed. Two recent papers report identification of two other Neph1-like proteins (Ihalmo et al. 2003, Sellin et al. 2003). Neph1 has recently been localised in the podocytes to the SD, and Neph1 directly interacts with nephrin (Barletta et al. 2003, Gerke et al. 2003) and ZO-1 (Huber et al. 2003). The interaction of nephrin and Neph1 was detected between both the extracellular and intracellular domains of the proteins. Furthermore, the studies show that the nephrin extracellular domain is capable of homophilic interaction. These results indicate that Neph1 and nephrin can form homo- and heterodimers/oligomers and may interconnect between opposing podocyte foot processes over the filtration slit. CD2AP is a mouse adaptor protein that has been localised to the SD region. CD2AP is ubiquitously expressed in the body. This protein with a MW of 80 000 was first shown to interact with the intracellular domain of the T-cell receptor CD2 (Dustin et al. 1998). 51
CD2AP has three consecutive SH3 domains at the amino terminus, a proline-rich region and C-terminal coiled coil region. SH3 domains are conserved protein modules that mediate specific protein-protein interactions. In addition to CD2, CD2AP has been shown to interact with other receptor molecules (see Shaw & Miner 2001) and the human homologue for CD2AP is CMS, which is identified as a protein associated with the focal adhesion protein p130Cas (Kirsch et al. 1999). CD2AP is critical in stabilizing the contact between a T-cell and the antigen presenting cell, and CD2AP knock out mice have a compromised immune function (Shih et al. 1999). Interestingly, CD2AP deficient mice develop NS and die at 6-7 weeks of age. In EM examination of the kidneys, effacement of podocyte foot processes is evident. In the kidney CD2AP has been shown to interact with polycystin-2 (Lehtonen et al. 2000), nephrin (Shih et al. 1999, Shih et al. 2001, Palmen et al. 2002) and actin (Lehtonen et al. 2002). Localisation of this protein in the SD region (Shih et al. 2001) and the interaction of this protein with both nephrin and actin suggests that it stabilizes the SD by connecting it to the podocyte cytoskeleton. Recently, mutations in CD2AP/CMS have also been linked to human disease (Kim et al. 2003). Kim and co-workers found that two of the 45 analysed FSGS patients were heterozygotes for a mutation that was predicted to ablate expression of one allele. The result indicates that haploinsufficiency of CD2AP leads to increased susceptibility to glomerular disease. Podocin is an integral membrane protein that is mutated in the early onset autosomal recessive form of steroid-resistant NS. The gene for podocin, NPHS2, was localised to chromosome 1q25-q31 and cloned (Boute et al. 2000). Podocin is a 384-amino acid protein with a MW of 42 000 that is exclusively expressed in podocytes. By similarity, it belongs to proteins of the band-7-stomatin family. Homology to human stomatin predicts that podocin forms a hairpin-like structure with one transmembrane region. Podocin has been localised to the podocyte SD area (Roselli et al. 2002) and it associates with lipid rafts and interacts with nephrin (Huber et al. 2001, Schwarz et al. 2001) and Neph1 (Sellin et al. 2003), and potentially with CD2AP (Schwarz et al. 2001). Although podocin mutations were first described in familial NS (Boute et al. 2000), mutations have been observed in sporadic cases as well (Caridi et al. 2001, Karle et al. 2002, Caridi et al. 2003a, Caridi et al. 2003b). The gene for the autosomal dominant form of FSGS was localized to the vicinity of the nephrin locus (Mathis et al. 1998). Sequence analysis of the nephrin gene from affected patients did not reveal any alterations. However, analysis of other candidate genes from 19q13 identified ACTN4 as the affected gene (Kaplan et al. 2000). ACTN4 encodes for α-actinin-4, which is an actin cross-linking protein (Honda et al. 1998) Mouse models with mutated α-actinin-4 have been generated and these studies further confirm the linkage of the gene to the disease (Kos et al. 2003, Michaud et al. 2003). The effects of α-actinin-4 mutations in familial inherited FSGS emphasizes the important role of podocyte cytoskeleton in the integrity of the glomerular filtration barrier. Discovery of these SD components enables us to establish a model of the filtration slit at the molecular level. Nephrin, podocin and Neph-1 most probably organize into oligomeric complexes and interact over the filtration slit (Schwarz et al. 2001, Gerke et al. 2003). This complex is connected to filamentous actin via CD2AP and ZO-1. P- cadherin and FAT may connect to the cytoskeleton via catenins and ZO-1. Like other junctions, assembly and maintenance of the SD requires outside-in and inside-out 52 signalling. In fact, nephrin has been shown to be able to activate mitogen activated protein kinases (Huber et al. 2001). This activation is enhanced by podocin interaction. Furthermore, injection of antibody recognising a podocyte-specific 9-O-acetylated GD2 ganglioside causes tyrosine phosphorylation of nephrin (Simons et al. 2001) and binding of antibody to extracellular domain of nephrin induces clustering and tyrosine phosphorylation of nephrin on the intracellular domain by Fyn kinase of the src family (Lahdenperä et al. 2003). Fyn deficient mice also have somewhat altered kidney morphology on EM examination (Verma et al. 2003). Fig. 6 summarises the recently discovered structural building blocks of the SD and their possible organization. Most probably still more components will be found in the SD-region. In fact, very recently a new protein, densin, has been localised to the SD region (Ahola et al. 2003).
Fig. 6. Molecular structure of the podocyte slit diaphragm.
6.5 Involvement of nephrin in the pathogenesis of proteinuria
The crucial role of nephrin in the development and maintenance of the SD is demonstrated by the adverse effects of NPHS1 mutations. In addition to its role in NPHS1, nephrin may have a more general role in the pathogenesis of proteinuria. Therefore, the expression of nephrin was studied in acquired NS by IF-microscopy of kidney biopsy specimens. A significant reduction in the staining intensity and a change in staining pattern was observed in patients suffering from membranous GN, MC and FSGS. It is notable, that the change in the nephrin expression was more related to the rate of proteinuria rather 53 than to any of the specific diseases studied. To date, substantial amount of data have accumulated on membranous GN, MC and FSGS (Furness et al. 1999, Patrakka et al. 2001, Huh et al. 2002, Kim et al. 2002, Koop et al. 2003, Wernerson et al. 2003) as well as IgA GN (Huh et al. 2002, Koop et al. 2003) and diabetic nephropathy (Langham et al. 2002, Doublier et al. 2003, Koop et al. 2003). Although, some discrepancies are evident due to semi-quantitative nature of the used techniques, these studies confirm that nephrin protein levels are decreased in human proteinuric disease and nephrin location shifts from finely granular staining to more granular pattern. On the mRNA-level the results are more controversial than on the protein level. For example, Furness and co-workers (1999) reported a downregulation in the nephrin mRNA in patients with NS, while a recent report from Koop and co-workers (2003) describe an upregulation of nephrin mRNA. In addition to studies with human biopsies, numerous studies involving animal models of human proteinuric diseases have been conducted and confirm the inverse correlation of nephrin levels and proteinuria. These studies include the passive Heymann nephritis model of the membranous GN (Benigni et al. 2001, Kawachi et al. 2002b, Yuan et al. 2002b), puromycin aminonucleoside induced nephrosis model of MC (Ahola et al. 1999, Luimula et al. 2000a, Luimula et al. 2000b, Kawachi et al. 2002b), animal models of diabetic nephropathy (Aaltonen et al. 2001, Bonnet et al. 2001, Bonnet et al. 2002, Forbes et al. 2002, Davis et al. 2003) and anti-nephrin induced nephrosis (Orikasa et al. 1988, Kawachi et al. 2000, Kawachi et al. 2002b). It is evident from all this data that in addition to its role in the rare inherited NPHS1 disease, nephrin is also critical for the integrity of glomerular filter in acquired kidney diseases. The possible mechanism behind the reduced nephrin expression was studied by using cultured podocytes. Aggregated IgG4, which is known to be the major immunoglobulin subclass in membranous GN (Oliveira 1998), induced focal redistribution of nephrin in the cultured cells, but disaggregated IgG had no effect. Result indicates that the antibody aggregate may interact with a neonatal Fc-receptor present on the podocyte plasma membrane (Haymann et al. 2000) and this causes the observed cytoskeletal rearrangements. As nephrin is connected to actin filaments (Saleem et al. 2002, Yuan et al. 2002a) the consequence would be a redistribution of nephrin in the cell membrane. The finding was confirmed by incubation of the cells with the membrane attack complex of complement, puromycin and TNF-α that are known to affect the cytoskeletal organisation. These findings indicate that the reduced nephrin expression observed in acquired proteinuric disease may be the consequence of a podocyte injury triggered by different stimuli. As discussed above, a number of molecules besides nephrin have been localised to the SD and linked to proteinuria (Table 2). These recent advancements underscore the importance of the SD for the filtration barrier integrity. Challenges for the future research will be not only in identifying all the components of the SD, but in identifying the interplay between these components and the downstream and upstream signalling events in normal and disease states. Unravelling of these interactions will not only increase our understanding of the basis of proteinuric disease, but it will most probably also benefit patients in a form of new treatment possibilities. 54
Table 2. SD components that link to a proteinuric disease.
Protein Human gene Human disease Animal model
Name Locus Name Reference
Nephrin NPHS1 19q13.1 Congenital nephrotic (I) Putaala et al. 2001 syndrome of the Finnish type (NPHS1, CNF)
Podocin NPHS2 1q25-32 Steroid-resistant nephrotic Boute et al. 2000 - syndrome (SRNS)
CMS/CD2AP CMS 6p12 Focal segmental Kim et al. 2003 Shih et al. 1999 glomerular sclerosis (FSGS3)
FAT FAT 4q34-q35 - - Ciani et al. 2003
Neph1 NEPH1 1q21-25 - - Doviel et al. 2001
7 Conclusions
In this study, the gene defective in NPHS1 was identified in the chromosomal region of 19q13.1. The NPHS1-gene was shown to encode for a single-pass transmembrane protein of the immunoglobulin superfamily. As the gene expression was originally observed only in the nephron, the gene product was termed nephrin. Importantly, in the kidney nephrin was localised to glomerular podocytes, more precisely to the filtration slit between two podocyte foot processes. Developmentally, nephrin was detected prior to or at the capillary loop stage, when the adherens and occluding type junctions of the presumptive podocytes begin to develop into the SD. Furthermore, nephrin expression was studied and also found to be reduced in acquired nephrotic syndromes. The observed changes in the localisation of nephrin in cultured podocytes upon induction of cytoskeletal changes indicate that nephrin is connected to the actin-based cytoskeleton of podocytes. Nephrin is the first identified component of the podocyte SD. It evidently has an eminent role in the filtration barrier, as both the human mutations and the inactivation of nephrin gene in mouse lead to NS already in utero. The reduced expression observed in the acquired NS indicates that nephrin has a more general role in the integrity of the filtration barrier. Furthermore, identification of nephrin as a structural component of the SD has lead to the discovery of other molecules of this mysterious structure. Consequently, this study can also be seen as an example of how studies on a rare genetic disease can provide valuable new information on a normal physiological process. In this case the results have improved our understanding of the glomerular ultrafiltration and the molecular structure of the SD. References
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