Nephrin-Associated Molecule in Human Glomerular Diseases

Nephrin-associated molecules in human glomerular diseases

Pekka Ihalmo

Department of Bacteriology and Immunology Haartman Institute Faculty of Medicine

Division of Biochemistry Department of Biological and Environmental Sciences Faculty of Biosciences

University of Helsinki Finland

ACADEMIC DISSERTATION

To be publicly discussed with the permission of the Faculty of Biosciences, University of Helsinki, in Small Hall, University main building, Fabianinkatu 33 (4th ﬂ oor), Helsinki, on September 7th, 2007, at 12 noon.

Helsinki 2007 Supervised by Docent Harry Holthöfer, M.D., Ph.D. Department of Bacteriology and Immunology University of Helsinki Finland

Reviewed by Professor Markku Laakso, M.D., Ph.D. Department of Medicine University of Kuopio Finland

and

Docent Eero Lehtonen, M.D., Ph.D. Department of Pathology University of Helsinki Finland

Ofﬁ cial opponent Docent Marjo Kestilä, Ph.D. Department of Molecular Medicine National Public Health Institute Helsinki

ISBN 978-952-92-2472-2 (paperback) ISBN 978-952-10-4089-4 (PDF) http://ethesis.helsinki.ﬁ

Helsinki 2007

Contents

LIST OF ORIGINAL PUBLICATIONS ...... 6

ABBREVIATIONS ...... 7

1. ABSTRACT ...... 9

2. REVIEW OF THE LITERATURE...... 10 2.1. Glomerular capillary wall as the ﬁ ltration barrier in the kidney glomerulus ...... 10 2.2. The slit diaphragm is an adhesion and signalling complex in the podocytes ...... 12

Nephrin...... 13

Nephrin-like proteins NEPH1, NEPH2 and ﬁ ltrin ...... 14

Cadherin superfamily proteins...... 15

Intracellular scaffolding and signalling proteins...... 16

2.3. Development of the slit diaphragm from a tight junction . . . . 17

2.4. Proteinuria is a hallmark of glomerular injury...... 18

Minimal change disease ...... 18

Focal segmental glomerulosclerosis...... 19

Membranous glomerulopathy ...... 19

Hypertensive nephropathy ...... 20

Diabetic nephropathy ...... 20

2.5. Genetic basis of proteinuria ...... 21

3. AIMS OF THE STUDY ...... 25

4. MATERIALS AND METHODS ...... 26

4.1. Study subjects and clinical samples ...... 26

4.2. Animals...... 28

4.3. Cell culture ...... 28

4.4. Molecular cloning and mRNA expression analysis ...... 28

Complementary DNA clones...... 28

RNA isolation ...... 28

Reverse transcriptase-polymerase chain reaction (RT-PCR) . . . . 29

Northern Blotting ...... 30

4 RNA dot blot assay ...... 31

4.5. Immunochemical methods ...... 31

Antibodies ...... 31

Immunoblotting ...... 32

Immunoﬂ uorescence microscopy ...... 32

Immunoelectron microscopy ...... 32

4.6. Genotyping ...... 33

4.7. Databases, bioinformatics tools, and statistical methods . . . . . 33

5. RESULTS ...... 35 5.1. Filtrin, a novel nephrin-like protein, is expressed in glomerular podocytes (Studies I and III) ...... 35 5.2. Nphs1as mRNA is transcribed from the nephrin locus in a reverse orientation in the brain and lymphoid tissue (Study II) ...... 37 5.3. Glomerular expression of ﬁ ltrin in patients with proteinuric kidney diseases (Study III) ...... 40 5.4. Genetic association analysis of LRRC7, KIRREL, NPHS2, KIRREL2, and ACTN4 with diabetic nephropathy (Study IV) . . . . 43

6. DISCUSSION ...... 46

Filtrin emerges into the picture of the podocytes ...... 46

Function of ﬁ ltrin in other tissues...... 47

Expression of nephrin and novel regulatory aspects ...... 47 Chromosomal organisation of KIRREL2 and NPHS1 in the human genome ...... 49

Patients with proteinuria (Studies III and IV) ...... 50 Is ﬁ ltrin merely a by-stander in the podocyte or is it involved in podocyte injury? ...... 50 Can the genes behind monogenic proteinuric diseases contribute to the understanding of diabetic nephropathy? ...... 52

Concluding remarks ...... 55

7. ACKNOWLEDGEMENTS...... 57

8. APPENDIX...... 59 Members of the European Renal cDNA Bank at the time of the study...... 59

9. REFERENCES ...... 60

ORIGINAL PUBLICATIONS ...... 77

5 LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original articles, which are referred to by their Roman numerals in the text.

I Ihalmo, P., Palmén, T., Ahola, H., Valtonen, E., Holthöfer, H. Filtrin is a novel member of nephrin-like proteins. Bio- chemical Biophysical Research Communications. 2003, 300:364-70. II Ihalmo, P., Rinta-Valkama, J., Mai, P., Åstrom, E., Palmén, T., Pham, T.T., Floss, T., and Holthöfer, H. Molecular cloning and characterization of an endogenous antisense transcript of Nphs1. Genomics. 2004, 83:1134-40. III Ihalmo, P., Schmid, H., Rastaldi, M.P., Mattinzoli, D., Lang- ham, R.G., Luimula, P., Kilpikari, R., Lassila, M., Gilbert, R.E., Kerjaschki, D., Kretzler, M., and Holthöfer, H. Expression of ﬁ ltrin in human glomerular diseases. Nephrology Dialysis Transplantation. 2007, 22:1903-9. IV Ihalmo, P., Wessman, M., Kaunisto, M., Kilpikari, R., Parkko- nen, M., Forsblom, C., Holthöfer, H., and Groop, P.-H. for the FinnDiane Study Group. Association analysis of podocyte slit diaphragm genes as candidates for diabetic nephropathy (submitted).

The original articles have been reprinted with the permission of the pub- lishers.

6 ABBREVIATIONS

ACTN4 α-actinin-4 AER albumin excretion rate AMV avian myeloblastosis virus ANOVA analysis of variance BLAST basic local alignment search tool CD2AP CD2-associated protein CDH3 cadherin 3 (P-cadherin) CDH5 cadherin 5 (VE-cadherin) cDNA complementrary DNA CEPH Centre d’Etude du Polymorphisme Humain CI conﬁ dence interval CNF congenital nephrotic syndrome of the Finnish type COL4A3, -A4, -A5 α3-, α4-, α5(IV)collagen CON control group CpG CpG dinucleotide dCTP 2’-deoxycytidine 5’-triphosphate DN diabetic nephropathy Ena/VASP Enabled/vasodilator-stimulated phosphoprotein ERCB European Renal cDNA Bank ESRD end-stage renal disease EST expressed sequence tag FAT FAT tumor suppressor 1 (FAT1) FSGS focal segmental glomerulosclerosis GAPDH glyseraldehyde-3-phosphate dehydrogenase GBM glomerular basement membrane

HbA1c glycosylated hemoglobin HT hypertensive nephropathy HUGO Human Genome Organisation HWE Hardy-Weinberg equilibrium KIRREL kin of IRRE like (NEPH1) KIRREL2 kin of IRRE like 2 (ﬁ ltrin or NEPH3) KIRREL3 kin of IRRE like 3 (NEPH2) LAMB2 laminin β2 LD linkage disequilibrium LMX1B LIM homeobox transcription factor 1β LRRC7 leucine rich repeat containing 7 (densin-180) Macro macroalbuminuria MAF minor allele frequency MCD minimal change disease

7 MGN membranous glomerulopathy M-MLV moloney murine leukemia virus MYH9 myosin, heavy chain 9, non-muscle NCBI National Center for Biotechnology Information NEPH1-3 nephrin-like protein 1-3 Normo normoalbuminuria NPHS1 nephrosis 1 (nephrin) Nphs1as nephrosis 1 antisense NPHS2 nephrosis 2 (podocin) OR odds ratio PDZ PSD-95/disc large/ZO-1 PLCE1 phospholipase C ε1 RACE rapid ampliﬁ cation of cDNA ends SH2 Src homology 2 SNP single nucleotide polymorphism TJP1 tight junction protein 1 (ZO-1) TRPC6 transient receptor potential cation channel, subfamily C, member 6 US urinary space WT1 Wilm’s tumor 1 ZO-1 zonula occludens 1

8 1. ABSTRACT

The glomerular epithelial cells and their intercellular junctions, termed slit diaphragms, are essential components of the fi ltration barrier in the kidney glomerulus. Nephrin is a transmembrane adhesion protein of the slit diaphragm and a signalling molecule regulating podocyte physiol- ogy. In congenital nephrotic syndrome of the Finnish type, mutation of nephrin leads to disruption of the permeability barrier and leakage of plasma proteins into the urine. This doctoral thesis hypothesises that novel nephrin-associated molecules are involved in the function of the fi ltration barrier in health and disease. Bioinformatics tools were utilized to identify nephrin-like molecules in genomic databases, and their distribution in the kidney and other tissues was investigated. Filtrin, a novel nephrin homologue, is expressed in the glomerular podocytes and, according to immunoelectron microscopy, localizes at the slit diaphragm. Interestingly, the nephrin and fi ltrin genes, NPHS1 and KIRREL2, locate in a head-to-head orientation on chromosome 19q13.12. Another nephrin-like molecule, Nphs1as was cloned in the mouse, however, no expression was detected in the kidney but instead in the brain and lymphoid tissue. Notably, Nphs1as is transcribed from the nephrin locus in an antisense orientation. The glomerular mRNA and protein levels of fi ltrin were measured in kidney biopsies of patients with proteinuric diseases, and marked reduction of fi ltrin mRNA levels was detected in the proteinuric samples as compared to controls. In addition, altered distribution of fi ltrin in injured glomeruli was observed, with the most prominent decrease of the expression in focal segmental glomerulosclerosis. The role of the slit diaphragm-associated genes for the development of diabetic nephropathy was investigated by analysing single nucleotide polymorphisms. The genes encoding fi ltrin, densin- 180, NEPH1, podocin, and α-actinin-4 were analysed, and polymorphisms at the α-actinin-4 gene were associated with diabetic nephropathy in a gender-dependent manner. Filtrin is a novel podocyte-expressed protein with localization at the slit diaphragm, and the downregulation of fi ltrin seems to be characteristic for human proteinuric diseases. In the context of the crucial role of nephrin for the glomerular fi lter, fi ltrin appears to be a potential candidate molecule for proteinuria. Although not expressed in the kidney, the nephrin antisense Nphs1as may regulate the expression of nephrin in extrarenal tissues. The genetic association analysis suggested that the α-actinin-4 gene, encoding an actin-fi lament cross-linking protein of the podocytes, may contribute to susceptibility for diabetic nephropathy.

1 Abstract 9 2. REVIEW OF THE LITERATURE

2.1. Glomerular capillary wall as the ﬁ ltration barrier in the kidney glomerulus

The kidney is a vital organ responsible for secreting metabolic end-products into the urine. In addition, the kidney maintains body homeostasis by regulating the fl uid, acid-base and electrolyte balance. The nephron is the basic functional unit in the kidney, and primary urine is produced in the fi rst part of the nephron, the glomerulus, which is a capillary tuft surrounded by a urinary space and the Bowman’s capsule [1]. The mechanism of glomerular fi ltration has remained an enigma of nephrology for decades. Notably, about 180 liters of primary urine is fi l- tered through the glomerular capillary wall every day, which is essential for the effi cient excretion of metabolic end-products from the circulation. At the same time, this fi lter can retain albumin and other crucial plasma proteins in the circulation, but the fi lter itself does not clog in spite of the enormous load of 15 kg of protein that the kidneys handle every day [1]. The prevailing view is that the composite effect of all layers of the glomerular capillary wall contributes to the charge- and size selective properties of the fi ltration barrier (Figure 1). Blood enters the capillary tuft through the afferent arteriole, which branches into smaller capillaries [1]. The high capillary pressure (55 mmHg) forces water and small molecules through the capillary wall. It has been estimated that approximately 20% of the plasma volume is fi ltered through the capillary wall, the rest exits the glomerulus via the efferent arteriole [1]. The capillary endothelial cells are thin cells lining the luminal side of the glomerular basement membrane (GBM) (Figure 1) and they represent the initial barrier to the blood constituents. These cells have a porous structure with round to oval fenestrae of 70-100 nm in diameter [1]. The fenestrae and the whole luminal side of the cell are covered by a special glycocalyx with a negative charge due to sialic acid containing glycoproteins and lip- ids [2, 3]. The negative charge of the endothelial cells may account for the charge selectivity of the permeability barrier [4]. The mesangial cells support the glomerular structure on the endocap- illary side [5]. Similar to the smooth muscle cells, the mesangial cells con- tract in response to vasoactive agents and have thus been proposed to regulate glomerular fi ltration. These cells also have phagocytotic properties and secrete bioactive substances such as prostaglandins [6, 7].

10 2 Review of the literature Capillary lumen

Urinary space

Podocyte

Basement membrane

Endothelium

Mesangial cells

Figure 1. Schematic structure of the glomerulus. Primary urine is produced in the glomerulus, which is a capillary tuft surrounded by a urinary space and the Bowman’s capsule. Modiﬁ ed from [107].

The GBM separates the endothelial cells from the podocytes (Figure 1). This membrane is a meshwork of glycoproteins (type IV collagen [α3α4α5], laminin-11 [α5β2γ1], and nidogen/entactin) and proteoglycans (agrin and perlecan) with a high water content (reviewed in [8, 9]). The GBM car- ries an anionic charge due to negatively charged glycosaminoglycan side chains of perlecan and agrin. Podocytes (Greek ‘pod’, foot) cover the GBM from the urinary side with cellular projections known as foot processes (Figure 1; reviewed in [10]). The podocytes are terminally differentiated epithelial cells with an arborized structure similar to neurons. These cells are highly polarized with their apical side and the cell body facing the urinary side of the Bowman’s space, and foot processes are anchored to the GBM on the basal side in an integrin- and dystroglycan-dependent fashion [11-13]. Bundles of actin ﬁ laments on the apical pole and the cortical actin net- work support the structure of the foot processes [14]. The interdigitating foot processes are separated by a slit of 30-40 nm and bridged by a unique cell junction termed the ‘slit diaphragm’ [15]. In the pioneering work by Rodewald and Karnovsky, the slit diaphragm was described as a zipper-like structure with a pore size corresponding to the dimensions of albumin [15]. Recent electron-tomographic data ac-

2 Review of the literature 11 cords with this view, revealing winding strands that form connections at the midline of the slit [16]. Conceptually, the slit diaphragm represents the border between the basal and apical domains of the podocyte [17, 18] and has been proposed to share properties of adherens and tight junctions [17, 19]. In the 1960s, tracer studies were launched to decipher the molecular barrier of the glomerular capillary wall. Experiments using ferritin and dextrans of different molecular weights as molecular markers indicated that the molecules of the size of albumin or larger cannot penetrate the GBM (reviewed in [20]). However, this approach neglected the infl uence of charge of the molecular components of the barrier on fi ltration. More conclusive results were obtained by using ferritins with anionic, neutral and cationic charge, and these experiments suggested that the GBM and endothelial cells prevent the passage of anionic molecules, whereas the fi ltration slit diaphragm appeared to retard the passage of molecules with positive charge [21]. However, this issue has remained a matter of debate. A gel permeation/diffusion hypothesis suggests that GBM is the size-selective barrier and the slit diaphragm only provides resistance to water fl ow [22], but completely contrasting views have also been proposed [23, 24]. A set of discoveries have brought the slit diaphragm into focus. In 1988, it was demonstrated that an injection of the monoclonal antibody 5-1-6, directed against the slit diaphragm, induced proteinuria in rats [25]. Later, it was ascertained that the antibody reacted with the slit diaphragm protein nephrin [26]. The discovery of the causative nephrin gene (NPHS1) for congenital nephrotic syndrome of the Finnish type in 1998 [27], and the elucidation of the genetic defects in other inherited nephropathies (reviewed in chapter 2.5) have launched a new era in nephrology.

2.2. The slit diaphragm is an adhesion and signalling complex in the podocytes

The slit diaphragm has been described as a ‘modifi ed adherens junction’ by Reiser and co-workers [19]. This cell-cell contact shares characteristics of the adherens junction (expression of P-cadherin [19]) and tight junction (zonula occludens-1 [17]), and the expression of slit diaphragm-specifi c proteins refl ects its unique properties (e.g. podocin [28]). The molecular composition of the slit diaphragm is reviewed in this chapter with special reference to the functions attributed to these proteins. Figure 2 summarizes the principal components of the slit diaphragm and the associated cytoplasmic adhesion complex. The slit diaphragm can be considered an epithelial cell adhesion junction and a fence between the apical and basal domains of the podocyte

12 2 Review of the literature Cadherin Catenins superfamily ZO-1 CD2AP Immunoglobulin superfamily Podocin

Podocyte

GBM Endothelium

Figure 2. The slit diaphragm is a unique cell-cell junction between the podocyte foot processes. Proteins of the immunoglobulin and cadherin superfamilies comprise the extracellular adhesion complex, while several adaptor and scaffolding proteins are located on the intracellular side. GBM, glomerular basement membrane. Modiﬁ ed from [61].

foot processes [17, 18]. Tight junctions have usually been acknowledged for their function as a selective ﬁ lter, but as illustrated by the permeability assays (reviewed in chapter 2.1) and the multitude of slit diaphragm encoding genes behind hereditary proteinuria (chapter 2.5), the slit diaphragm also seems to serve a ﬁ ltering function. Recently, the functions ascribed to the slit diaphragm have extended from a mere structural component to the regulator of transcriptional activity, apoptosis, and podocyte cytoskeleton (reviewed in [29-31]).

Nephrin Nephrin (the offi cial gene name by HUGO nomenclature NPHS1) is an integral adhesion molecule and a structural component of the slit diaphragm [32-34]. This protein belongs to the immunoglobulin superfamily with eight extracellular immunoglobulin-like domains followed by a fi bronectin type III domain, a single-span transmembrane region, and a C-terminal intracellular domain. A hypothetical model of the homophilic interaction of nephrin molecules was introduced already in the fi rst reports of nephrin [34], suggesting that nephrin molecules from adjacent foot processes would interact via their extracellular immunoglobulin-like domains and thus contribute to the fi lamentous diaphragm observed between foot processes. Subsequent experimental studies have provided support for this view [35-37].

2 Review of the literature 13 Nephrin resides in lipid rafts on the plasma membrane, and tyrosine residues of nephrin are phosphorylated upon foot process effacement [38, 39]. Furthermore, nephrin associates with and is phosphorylated by Src family kinase Fyn which stimulates intracellular signalling [40, 41]. Emphasizing the role of nephrin as a signaling protein, the intracellular tyrosine phosphorylated residues serve as potential docking sites for p85, the regulatory subunit of phosphoinositide 3-OH kinase [42], and for the adaptor protein Nck [39, 43], and these interactions appear to regulate the actin cytoskeleton and apoptosis in podocytes [39, 42, 43]. In addition, phosphorylation of nephrin seems to regulate gene expression in the podocytes, and this is facilitated by the interaction with another slit diaphragm protein, podocin [41, 44]. Similar to other plasma membrane receptors, nephrin is endocytosed by β-arrestin in a phosphorylation dependent manner [45]. Regulation of nephrin in acquired proteinuric diseases has been under intensive study, and altered protein and mRNA levels are found both in experimental animal models and human diseases (e.g. [46-48]). In particular, downregulation of nephrin in diabetic nephropathy is well established and, at least partially, mediated by angiotensin II and glycated albumin [49-51]. However, repression of nephrin expression might also be partially explained by the reduced number of podocytes per glomerulus [52].

Nephrin-like proteins NEPH1, NEPH2 and fi ltrin The cloning of NEPH1 (KIRREL) in 2001 led to the establishment of a protein family including two other closely related proteins, NEPH2 (KIRREL3) and fi ltrin (KIRREL2) [53-56]. Filtrin is also known as NEPH3. These proteins share a common domain architecture with fi ve extracellular immunoglobulin-like domains and a short intracellular domain of around 200 amino acids [57]. Within podocytes, NEPH1 and NEPH2 localize at the podocyte slit diaphragm [37, 54, 58] (Figure 2). In NEPH1-defi cient mice, the podocyte foot processes are effaced along the GBM, and the mice exhibit severe proteinuria [53]. Similarly to nephrin-defi cient mice, the pups die during the early postnatal period [53]. Soon after the cloning of nephrin-like proteins, the hypothesis of their potential interaction with nephrin was presented. Indeed, the extracellular domains of NEPH1 and NEPH2 appear to interact with nephrin, and the interactions are mediated by several extracellular immunoglobulin- like domains [35, 37, 54, 58]. NEPH2 has also been reported to form ho- modimers [54], whereas the data concerning the possible homodimerisa- tion of NEPH1 remains controversial [35, 37, 58]. Similarly to nephrin, NEPH1 resides in lipid rafts [37, 54]. The extracellular domain of NEPH2 is shedded from the plasma membrane by matrix metalloproteinases [54, 59], and seems to leak into the urine of both healthy individuals and proteinuric patients.

14 2 Review of the literature The physiological importance of NEPH1-nephrin interaction has been clarifi ed in vivo by injecting a combination of anti-NEPH1 and anti- nephrin antibodies into rats. The antibody mixture induced a transient pro teinuria in mice, whereas injection of anti-NEPH1 or anti-nephrin antibodies alone did not cause any changes. Even though the podocyte foot processes and slit diaphragms remained intact during this experiment, the expression of ZO-1, an intracellular binding partner of NEPH1, was considerably reduced [58]. In their intracellular part, NEPH1, NEPH2 and fi ltrin share a motif of nine conserved amino acids surrounding an SH2-binding site of growth factor receptor-bound protein 2 (Grb2) [57]. Sellin and colleagues hypoth- esized that this domain of nine amino acids could mediate interaction between the NEPH1 and podocin. They ascertained that both a single point mutation in the Grb2-binding site and tyrosine phosphorylation of this motif by Src kinases inhibited the interaction between the C-terminal domain of NEPH1 and podocin [57]. NEPH1 interaction via its intracellular domain with podocin was also verifi ed by immunoprecipitation [57]. Furthermore, co-expression of NEPH1 and the IL2-inducible T-cell kinase resulted in a marked activation of the transcription factor AP-1, arguing for a role of NEPH1 as a signalling molecule [57]. Since the C-terminal end of nephrin-like proteins contains a putative PDZ-binding motif, the interaction between them and a PDZ-domain- bearing protein ZO-1 has been assessed [60]. The interaction occurs both in vitro and in vivo and has been mapped to the fi rst PDZ domain of ZO-1 and the last three amino acids of the intracellular domain of NEPH1. This conclusion accords with the data presented by Liu and colleagues [58]. The interaction substantially increased signal transduction as measured by AP-1 activity and induced tyrosine phosphorylation of NEPH1 [60].

Cadherin superfamily proteins To date, only a few proteins in addition to nephrin and nephrin-like proteins have been localized to the extracellular adhesion junction of the slit diaphragm (Figure 2). These proteins include P-cadherin (CDH3) [19] and a recently reported VE-cadherin (CDH5) [61], originally attributed to its function in the vascular endothelium [62]. In a podocyte cell line, P-cadherin also recruites several catenins to the intracellular side [19]. Even though reduced levels of P-cadherin have been reported in diabetic nephropathy [63], the signifi cance of P-cadherin for maintaining the glomerular fi lter remains elusive, since the respective P-cadherin-defi cient mice show no kidney dysfunction [64]. In addition to classical cadherins, a large protocadherin FAT1 (FAT) with 34 extracellular cadherin repeats seems to be an essential component of the slit diaphragm, as refl ected by the lethal phenotype of Fat1-defi cient mice [65, 66]. These mice suffer from perinatal lethality and their foot processes are fused according to an electron microscopic examination

2 Review of the literature 15 [66]. FAT1 is acknowledged for its function as a regulator of actin polymerization (via its interaction with Ena/VASP proteins) and, in addition, in the establishment of cell polarity [67].

Intracellular scaffolding and signalling proteins A number of peripheral membrane proteins locate on the intracellular side of the slit diaphragm, where they link the immunoglobulin superfamily members and cadherins to the cortical actin cytoskeleton [68, 69]. Before nephrin, ZO-1 (TJP1) was the only identifi ed slit diaphragm-associated protein in the podocytes [17]. It is actually a typical tight junction protein, and the ZO-1 isoform without the motif-alpha is expressed in the slit diaphragms [70]. ZO-1 is localized on the intracellular side of the lateral foot process membranes, where it binds the nephrin-like proteins via its PDZ-domain [60]. Nephrin also provides a nexus for several binding partners on the intracellular side (Figure 2). Proteins including CD2-associated protein (CD2AP) [71, 72] and podocin [73] are located on the lateral foot process membrane and they are crucial for the proper fi ltration function. Podocin (NPHS2) is a member of the prohibitin-domain proteins and expressed al- most exclusively in the podocytes [28, 73]. Podocin forms homo-oligomers and is a constituent of lipid rafts [28, 74], where it binds cholesterol via its prohibitin-domain [75]. Podocin also functionally interacts with the slit diaphragm protein TRPC6 [76, 77], a non-selective cation channel, and regulates the activity of this channel in a cholesterol dependent fashion [75]. It has been speculated that podocin and TRPC6 could act as sensors for the intraglomerular pressure and/or glomerular fi ltration in the kidney [75]. Podocin associates via its C-terminal domain with nephrin and CD2AP [28, 44] and escorts nephrin to the lipid rafts [74]. CD2AP is an adaptor protein, originally recognized for its function in the immunological synapse [78]. CD2AP is an actin-associated protein in the podocytes [79, 80], but immunoelectron microscopic data and the reported interaction with nephrin suggest localization also at the slit diaphragm [72, 81]. In the close vicinity of the slit diaphragm resides an actin-bundling protein, α-actinin-4 (ACTN4) [82, 83]. Transgenic animal models of Actn4 have pinpointed a role for this molecule in cell motility and actin cytoskeleton regulation [84, 85] and also in the adhesion of podocytes to the GBM [86]. Densin-180 (LRRC7) is a founding member of a protein family containing several leucine-rich repeats and PDZ domains, and it had been originally identifi ed from the postsynaptic densities in the brain [87]. Recently, it has been reported as a novel nephrin-associated protein at the slit diaphragm [88] and as a potential interaction partner for α-actinin-4 [89].

16 2 Review of the literature 2.3. Development of the slit diaphragm from a tight junction

Development of the metanephric mammalian kidney is a process that involves reciprocal interaction of two tissue components, the mesenchymal blastema and the ureteric bud, originally the mesoderm-derived Wolf- fi an duct [90]. The glomerular development involves a series of events that initiates with the condensation of metanephric mesenchyme cells next to the tip of the ureteric bud [90]. These cells undergo mesenchyme- to-epithelium transition and form an epithelial vesicle, which gives rise to all the segments of the nephron, including the glomerulus and the proximal and distal tubules [90]. Glomerular anlagen are fi rst visible at the S-shape body stage, which has developed from the vesicle [91] (Figure 3). At this stage, the precursor cells of the endothelium and mesangium invade the proximal part of the S-shaped body, and the entity eventually forms a vascularized glomerulus [91, 92]. Visceral epithelial cells lining the presumptive Bowman’s space represent the future podocytes (Figure 3). At the early S-shape body stage, the cells are joined by ZO-1-positive apical tight junctions [17] and are fi rmly attached to the nascent basement membrane, a fence between the epithelial and capillary compartments [91]. During podocyte differentiation, the junctional complexes between the cell membranes migrate down towards the basal side. Eventually, a continuous intercellular space becomes visible between the cells, and ladder-like junctions are occasion- ally seen between the cells [91]. The layer of the future podocytes forms a cup around the developing capillary loops, and the formation and in- terdigitation of the foot processes begins (Figure 3). At the capillary loop stage, nephrin and ZO-1 localize at the cell-cell contacts between the developing podocytes [17, 93]. The foot processes surround the capillaries, and the tight junctions are gradually replaced with the mature slit diaphragms (Figure 3). [91]

S-shaped body Mature glomerulus

Figure 3. Development of the slit diaphragm. During the development the apical junctional complexes migrate towards the basal slit and ﬁ nally mature into slit diaphragms. Drawn after [235].

2 Review of the literature 17 In experimental nephrosis and human proteinuric diseases, foot process effacement is often observed [94, 95]. It has been postulated that the loss of the foot process architecture and appearance of tight junctions or ladder-like structures replacing the slit diaphragms corresponds to a reverse of the podocyte into more primitive developmental stages [91]. For example, in the puromycin aminonucleoside nephrosis of rats, tight junctions or ladder-like structures reappear in the intercellular space [94], and this condition may be reversible with the restoration of the foot process architecture. The elucidation of the morphological basis of proteinuria has lead to the view that podocytes are involved in the progression of proteinuric diseases (reviewed in [96]).

2.4. Proteinuria is a hallmark of glomerular injury

Minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and membranous glomerulopathy (MGN) are primary glomerular diseases manifesting as nephrotic syndrome [97]. These diseases are clini- cally characterized by heavy proteinuria (i.e. leakage of high molecular weight proteins into the blood ﬁ ltrate and urine), hypoalbuminemia, hyperlipidemia, and oedema [97]. Diabetic nephropathy and hypertensive nephropathy are secondary disorders that affect the glomerulus and present a more insidious pathogenetic process. The clinical sequelae of proteinuric diseases are well-described, but the pathogenetic mechanisms at the molecular level have remained elusive. The main question is, why the glomerulus becomes leaky under pathological conditions. It has become evident that a common denominator for these diseases is podocyte injury (reviewed in [98]).

Minimal change disease Minimal change disease is characterized by the nephrotic syndrome without glomerular lesions observed in light microscopy (ﬁ rst described by Munk in 1916 [99]). However, in electron microscopic examination, effacement of the podocyte foot processes is frequently encountered, while the structure of GBM seems unaltered [95]. This suggests that podocyte injury is involved in the pathogenesis of MCD [95]. MCD is the most common primary nephrotic syndrome in children, and it accounts for about 20% of primary glomerular disease in adults [100]. MCD can often be classiﬁ ed as ‘primary’, but secondary forms also exist in association with tumours, toxic or allergic reactions, infections, and autoimmune diseases [97]. Immune-complex deposition or complement activation in the glomerulus of the patients with MCD has not been reported. Instead, it has been postulated that MCD is a disorder of T cell function, where abnormal secretion of cytokines affecting the glomerular capillary permeability may be involved [101, 102]. MCD responds ef-

18 2 Review of the literature fectively to treatment with corticosteroids (prednisone) in children; in adults, steroid resistance and relapses are, on the other hand, common [103]. Gradual transition from MCD into focal segmental glomerulosclerosis has been reported making these diseases both diagnostically and therapeutically challenging [104]. The expression ratio of podocin to synaptopodin, two podocyte-expressed genes, has recently been suggested for disease stratiﬁ cation [47].

Focal segmental glomerulosclerosis The name ‘focal segmental glomerulosclerosis’ describes the lesions observed in the kidney in this disease: Scarring involves only some of the glomeruli (focal), and in the affected glomeruli, only a part of the glomerulus appears sclerotic (segmental) (ﬁ rst description published in [105]). Several variants of FSGS (e.g. collapsing, glomerular tip, or cellular variant) can be separated in histological examination [97]. Podocyte injury and dedifferentiation seems to be a primary cause of the disease [98, 106], and formation of scars may be a sign of a reparative process after podocyte injury [107]. FSGS is the cause of primary glomerular disease in about 15% of adults patients, but is less common in children [100]. FSGS can be considered either primary or secondary if associated with HIV, toxic compounds or drugs [97]. In some patients with primary FSGS, it has been speculated that a circulating factor underlies the glomerular permeability changes [108]. Interestingly, several familial forms of FSGS have been discovered with mutations in the podocyte-expressed genes (reviewed in chapter 2.5). FSGS responds poorly to corticosteroid treatment, and often progresses to chronic renal failure [103]. Anti-proliferative agents (e.g. cyclophosphamide) may be effective for the treatment of the steroid-resistant cases [103]. Notably, the prognosis of patients carrying mutations in genes linked to FSGS is poor.

Membranous glomerulopathy Membranous glomerulopathy is characterized by formation of subepi- thelial immune deposits of IgG and a diffusively thickened GBM [97]. Po- docyte foot process effacement is observed at the ultrastructural level [97, 109]. Even though MGN progresses slowly, it is a common cause of primary glomerular disease accounting for about 40% of cases in adults, but it is less common in children [100]. The formation of immune complexes against the podocyte-membrane proteins, with yet unidentiﬁ ed antigens, seems to trigger the membrane thickening (reviewed in [109]). In rare cases of neonatal membranous nephropathy, neutral endopeptidase in the podocyte membrane has been identiﬁ ed as a target for circulating antibodies [110]. In Heymann nephritis, a rat model of MGN, the antigen is the podocyte membrane protein megalin, which is a member of the LDL-receptor subfamily [111, 112]. Mechanistically, the immune complexes lead to complement activation

2 Review of the literature 19 and generation of C5b-9 membrane-attack complexes on the podocyte membranes, and formation of oxygen radicals by the injured podocytes seems to promote the glomerular damage [110, 113, 114]. Corticoster- oids alone have been reported to be ineffective in the therapy [103]; The combination of anti-proliferative agents with corticosteroids is considered a better approach [103].

Hypertensive nephropathy Hypertensive nephropathy is histologically characterized by the accumu- lation of hyaline in the walls of arteries and arterioles, collapse of the glomerular tuft, and glomerulosclerosis [115]. It has been estimated that hypertensive male patients suffer from manifold higher relative risk of developing renal failure than subjects with optimal blood pressure [116]. Malignant hypertension is recognized as a distinct cause of renal failure, while the link between essential hypertension and nephropathy is less well determined [117]. The podocytes are sensitive to injury, and animal models of hypertension suggest that podocyte failure in hypertension may promote the formation of glomerulosclerosis [118, 119]. It has been postulated that mechanical strain due to increased intraglomerular pressure is the underlying cause for podocyte injury [98, 120, 121]. Glomerular ischemia and/or hyperﬁ ltration have also been suggested to promote glomerular injury. Notably, systemic hypertension and/or elevated intraglomerular pressure associate with other types of nephropathy, e.g. diabetic nephropathy [115]. Control of hypertension with antihypertensive agents is essential for the treatment of the disease.

Diabetic nephropathy Diabetic nephropathy is the most common cause of nephrotic proteinuria [122]. It has been estimated that approximately one-third of patients with diabetes develop diabetic nephropathy [123, 124], which can be considered the most severe complication of diabetes due to the associated several fold increased risk of cardiovascular disease and mortality [125, 126]. During the course of diabetes, the incidence of diabetic nephropathy is not linear, but it increases until 15 years after the onset of diabetes and declines thereafter [123]. Microalbuminuria is the ﬁ rst sign of glomerular dysfunction in type 1 diabetes and is considered a powerful predictor of progression to overt nephropathy [127]. Microalbuminuria reﬂ ects the alterations in the glomerular capillary wall at the early stage of the disease: Increase in podocyte foot process width and reduced number of podocytes per glomerulus have been reported [128-130]. At later stages of the disease, nodular glomerulosclerosis with expansion of the mesangial matrix and thickened GBM can be observed [131]. It should be noted that diabetic nephropathy is part of a generalized microvascular damage occurring af-

20 2 Review of the literature ter prolonged hyperglycaemia [132]. During this process, involvement of the renin-angiotensin system, advanced glycosylation end-products and reactive oxygen species have been indicated as pathogenetic mechanisms (reviewed in [133]). Importantly, angiotensin II seems to have direct effects on the podocytes, causing e.g. podocyte apoptosis and cytoskeleton rearrangement [120, 134]. Hyperglycaemia is the most important risk factor for diabetic nephropathy both in type 1 and 2 diabetes [135, 136]. However, patients with poor glycaemic control do not necessarily develop nephropathy, indicating that other factors are involved. Additional risk factors include smoking [137], hyperlipidemia [138], and insulin resistance even in patients with type 1 diabetes [139]. The prevailing view is that the combination of environmental and genetic risk factors (reviewed in chapter 2.5) is the driving force for the development of diabetic nephropathy. Elimination of classical risk factors, improvement of glycaemic control, aggressive treatment of hypertension, and cessation of smoking are essential for the prevention of the development of diabetic nephropathy. Currently used therapies for diabetic nephropathy focus on blocking the renin-angiotensin system with angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists [140, 141].

2.5. Genetic basis of proteinuria

Milestone discoveries of the genetic basis of proteinuria have shed light on the molecular structure of glomerular capillary wall and have dis- closed mutations in genes encoding adhesion proteins, components of cell cytoskeleton, and mediators of signal transduction (reviewed in [142- 144]). Notably, most of these proteins were located in the glomerular podocytes. Therefore, renal research has in recent years adopted a new concept of a ‘podocentric view of nephrology’ [145]. According to the classiﬁ cation by Wilhelm Kriz [142], hereditary diseases affecting the podocytes can be divided into two groups based on their age of onset. First, genetic defects manifesting at birth or soon after birth (‘early onset’) are characterized by the lack of or deformed slit diaphragms and/or nephrotic range proteinuria [142] (Table 1). Second, proteinuric disorders occurring from childhood to late adulthood can be regarded as a consequence of the reduced ability of the podocyte to respond to physiological challenge, accounting for the later age of onset [142]. Typically, these diseases segregate in an autosomal-dominant pattern in families with variable penetrance (Table 1). The gene encoding the slitdiaphragm protein nephrin, NPHS1, is mutated in congenital nephropathy of the Finnish type (CNF) [27]. This disorder was ﬁ rst characterized by Hallman and co-workers in 1956 [146], and it belongs to the Finnish disease heritage [147], a term used to describe

2 Review of the literature 21 Table 1. Hereditary proteinuric diseases classiﬁ ed according to age at onset. Modiﬁ ed from [142] and [143].

Chromosomal Mode of Disease Gene (protein) region inheritance References

Early onset

Congenital nephrotic syndrome of NPHS1 (nephrin) 19q13.12 recessive [27] the Finnish type

Denys-Drash syndrome WT1 (WT1) 11p13 dominant [161]

Pierson’s syndrome LAMB2 (laminin β2 3p21.31 recessive [152] chain)

Nephrotic syndrome type 3 (NPHS3) PLCE1 (phospholipase 10q23.33 recessive [151] C ε1

Steroid-resistant nephrotic syndrome NPHS2 (podocin) 1q25.2 recessive [73]

Late onset

Alport syndrome COL4A3 (α3(IV) 2q36.3 recessive / [228] [229] collagen) dominant [230]

COL4A4 (α4(IV) 2q36.3 collagen)

COL4A5 (α5(IV) Xq22.3 X-linked [231] collagen)

Epstein and Fechtner syndromes MYH9 (non-muscle 22q12.3 dominant [155] myosin heavy chain 9)

Focal segmental glomerulosclerosis ACTN4 (α-actinin-4) 19q13.2 dominant [83]

Focal segmental glomerulosclerosis TRPC6 (TRPC6) 11q22.1 dominant [77] [76]

Frasier’s syndrome WT1 (WT1) 11p13 dominant [162]

Nail-patella syndrome LMX1B (LMX1B) 9q33.3 dominant [160]

The chromosomal regions are derived from the UCSC Genome Browser (March 2006 Assembly).

22 2 Review of the literature diseases that occur more frequently in Finland than elsewhere in the Eu- rope. In CNF, the podocyte foot processes are fl attened and effaced, and the slit diaphragms are missing [148, 149]. Affected babies suffer from massive treatment-resistant proteinuria already in utero [146, 150]. The NPHS2 gene encoding podocin is mutated in steroid-resistant nephrotic syndrome [73]. Recently, a third nephrosis gene, namely phospholipase C ε1 (PLCE1), was discovered, with expression in the developing and mature podocytes [151]. Interestingly, some patients with mutations in PLCE1 were steroid-sensitive, indicating that patients carrying certain mutations in PLCE1 could be cured. Laminin β2-chain is a component of mature glomerular basement membranes, and certain mutations in the human LAMB2 gene account for Pierson’s syndrome [152]. Pierson’s syndrome is characterized by congenital nephrosis with mesangial sclerosis and ocular defects [152]. Familial forms of focal segmental glomerulosclerosis (FSGS) with defects in the non-selective cation channel TRPC6 [76, 77] and the actin-fi lament cross-linking protein α-actinin-4 (ACTN4) [83] have been characterized. Interestingly, mutations of NPHS2 with later onset have also been reported in familial FSGS [153], and NPHS2 has been suggested to associate with microalbuminuria in the general population [154]. The importance of the podocyte cytoskeleton was further emphasized by the May- Hegglin anomaly, and Sebastian, Epstein and Fechtner syndromes that involve mutations in the gene encoding the non-muscle myosin heavy chain 9 (MYH9) expressed in the podocytes [155]. In these syndromes, the patients suffer from macrothrombocytopenia and variably from other symptoms including proteinuria [156]. Alport syndrome involves mutations in the genes encoding the constituents of type IV collagen (α3α4α5) of the GBM and presents with hematuria and progressive glomerular disease, hearing loss and ocular lesions (reviewed in [157]). Transcription factors LMX1B and WT1 regulate several podocyte-expressed genes and are involved in the early phases of the kidney and podocyte differentiation [158, 159]. Mutations in the LMX1B gene have been mapped in patients with nail-patella syndrome, a dominantly inherited disorder with skeletal malformations and, in some patients, renal dysfunction due to glomerular lesions [160]. Variants in the Wilms tumor 1 gene WT1 have variable phenotypes: The mutation spectrum of Denys- Drash syndrome is broad covering the whole gene locus resulting in an early onset of nephropathy [161]. Patients with Denys-Drash syndrome suffer from generalized urogenital developmental abnormalities [161]. In Frasier syndrome, characterized by male pseudohermaphrodism and progressive glomerular disease, proteinuria develops later due to mutations in the donor splice site of intron 9 of the WT1 gene [162]. Kriz classifi es nail-patella syndrome and Frasier syndrome in the late-onset group based on their clinical features, but emphasizes that the disease mechanisms are not well understood [142].

2 Review of the literature 23 In diabetic nephropathy, familial clustering has been reported in several studies indicating the existence of a genetic component in the λ disease [163-166]. In the Finnish population, the sibling recurrent risk s is 2.3, which indicates the risk that diabetic siblings of a nephropathic proband have compared to siblings of a proband without diabetic neph- ropahty [163]. Furthermore, the albumin excretion rate and the extent of glomerular lesions seem to be partially heritable and genetically determined in families with patients with diabetes [167, 168]. Diabetic nephropathy can be considered a typical complex disease with multiple genetic loci that contribute to the disease development in combination with other biological effects (e.g. hyperglycaemia) [169]. Genome-wide screens using sib-pairs or case-control setting have revealed potential susceptibility loci for diabetic nephropathy on chromo- somes 10q and 3q [170-172]. Candidate gene studies have focused on the known biological pathways involved in the pathogenesis of diabetic nephropathy. These pathways include the genes of the renin-angiotensin system, and a recent meta-analysis of several studies support the involvement of angiotensin I converting enzyme insertion/deletion polymorphism in the development of diabetic nephropathy [173]. However, most attempts to determine the effect of a genetic variant in a particular gene on the development of diabetic nephropathy have suffered from the lack of suffi cient statistical power (discussed in [174]). Transgenic animal models have provided new insights in the genetics of proteinuria. Defi ciency of the slit diaphragm proteins NEPH1, a nephrin-homologue, and FAT1, a member of the cadherin superfamily, was discovered to cause massive proteinuria and lethality in mouse [53, 66]. Inactivation of the gene encoding CD2-associated protein (Cd2ap) in mice results in congenital nephrotic syndrome and death at 6 to 7 weeks of age [71]. Interestingly, haploinsuffi ciency of CD2AP may also be involved in human glomerular disease [175]. To my knowledge, no genetic defect has been mapped to the human homologues of NEPH1 and FAT1 in proteinuria.

24 2 Review of the literature 3. AIMS OF THE STUDY

Since the discovery of the causative gene for the congenital nephrotic syndrome of the Finnish type, NPHS1, and the identiﬁ cation of the gene product nephrin as a constituent of the slit diaphragm [27, 32, 34], the prevailing view has been that the slit diaphragms are of pivotal importance for the molecular sieve in the kidney glomerulus. This thesis hy- pothesized that additional nephrin-associated molecules participate in maintaining the function of the ﬁ ltration barrier in health and disease.

The speciﬁ c aims of this thesis were to evaluate the following: 1. To determine the expression and localization of ﬁ ltrin, a novel nephrin-like protein, in the kidney.

2. To clone and characterize the nephrin antisense transcript Nphs1as in mouse.

3. To elucidate the expression pattern and distribution of ﬁ ltrin in the kidneys of patients with proteinuric diseases.

4. To investigate the genetic association of the nephrin-associated genes densin-180 (LRRC7), NEPH1 (KIRREL), podocin (NPHS2), ﬁ ltrin (KIR- REL2), and α-actinin-4 (ACTN4) with diabetic nephropathy in type 1 diabetes.

3 Aims of the study 25 4. MATERIALS AND METHODS

4.1. Study subjects and clinical samples

Samples of normal human kidney were obtained from cadaver donors inappropriate for transplantation due to vascular anatomic reasons (De- partment of Surgery, Helsinki University Central Hospital), and glomeruli were extracted from the cortical kidney tissue using the graded sieving method [176] (Study I). Primary cultures of human podocytes (chapter 4.3) were established from kidney specimens obtained from renal carci- noma patients undergoing nephrectomy. Two sets of patients were selected for quantitative RT-PCR analysis of fi ltrin (KIRREL2), nephrin (NPHS1), and podocin (NPHS2) mRNAs (Study III). The fi rst set of 69 kidney biopsies was collected within the European Renal cDNA Consortium (members listed in Appendix I), and the biopsies were divided into the following groups according to histological diagnosis (Table 2). In these samples, the glomerular compartment was microdissected and processed for RNA isolation as described previously [177]. The second set of samples was collected at the St. Vincent’s Hospital (Fitzroy, Australia; Table 2), and total RNA was extracted from whole biopsies. Nonaffected parts of tumor nephrectomies served as a control group. For immunohistochemical examination of fi ltrin in injured glomeruli, a total of 15 biopsies obtained for routine diagnostic purposes at the San Carlo Borromeo Hospital (Milan, Italy) were used (Study III). The diagnostic groups of the biopsies were as follows: minimal change disease (n = 4), membranous glomerulopathy (n = 6), and focal segmental glomerulosclerosis (n = 5). In addition, renal samples of the tumor-free part of nephrectomy specimen were used as controls (n = 3). Two groups of Finnish patients with type 1 diabetes with and without diabetic nephropathy were selected for the genetic association analysis of densin-180 (LRRC7), NEPH1 (KIRREL), podocin (NPHS2), fi ltrin (KIRREL2), and α-actinin-4 (ACTN4). The patients were part of the Finnish Diabetic Nephropathy (FinnDiane) Study (Study IV). First, the extensive screening of single nucleotide polymorphisms (SNPs) was performed in 1103 patients. Second, an additional set of 1025 patients was analysed in order to confi rm the positive fi ndings (Table 2). The renal status of the patients was based on the albumin excretion rate (AER). Normoalbuminuria was defi ned as AER below 30 mg/24h (< 20 μg/min in an overnight urine col- lection), microalbuminuria as AER of 30-300 mg/24h (20-200 μg/min), and

26 4 Materials and methods Table 2. Clinical characteristics of analyzed subjects.

Age Serum creatinine Diagnostic group n (years) (µmol/l) Proteinuria (g/24h)

European Renal cDNA Consortium

Control subjects 7 64±6 NA NA

Minimal change disease 13 36±16 101±44 5.7 (0.2 - 14.8

Hypertensive nephropathy 16 56±10 172±151 1.0 (0 - 2.6)

Membranous glomerulopathy 31 55±23 130±108 4.9 (0.4 - 17.0)

Focal segmental glomerulosclerosis 9 47±16 128±45 2.3 (0.2 - 10.4)

St. Vincent’s Hospital

Control subjects 6 NA NA NA

Membranous glomerulonephropathy 4 65±4 125±50 4.1 (1.9 - 8.0)

Diabetic nephropathy 7 51±19 173±80 1.2 (0.6 - 21.2)

Minimal change disease 5 31±12 86±21 8.2 (6.3 - 14)

Focal segmental glomerulosclerosis 11 56±19 145±42 4.0 (1.0 - 14.0)

FinnDiane - 1st set of patients AER (mg/24h)

Normoalbuminuria 459 43±10 85±15 7 (1 - 85)

Microalbuminuria 276 37±11 90±19 59 (2 - 613)

Macroalbuminuria 368 39±9 165±125 588 (4 - 8348)

FinnDiane - 2nd set of patients

Normoalbuminuria 607 40±12 83±17 8 (1 - 101)

Macroalbuminuria 158 45±10 163±114 426 (11 - 4609)

End-stage renal disease 260 44±8 - -

Data are summarized as mean ± standard deviation or median (range). AER, albumin excretion rate; NA, not available.

4 Materials and methods 27 macroalbuminuria as AER over 300 mg/24h (> 200 μg/min) in two out of three consecutive urine collections [178]. Patients with normoalbuminuria were required to have duration of diabetes over 15 years. Patients on dialysis or with a kidney transplant were considered to have an end-stage renal disease (ESRD). Informed consent was obtained from all subjects. The study protocols followed the principles of the Declaration of Helsinki and were approved by local ethics committees.

4.2. Animals

Normal rat kidneys (Sprague-Dawley) were used to explore the expression and localization of ﬁ ltrin (Studies I and III). Glomeruli were isolated from the kidney tissue using the sieving method as previously described for the rat tissue [176]. Tissues (brain, kidney, pancreas, mesenteric and peripheral lymph nodes, spleen, and thymus) of adult male mice (strain NMRI) were prepared for the expression analyses of the Nphs1 and Nphs1as mRNAs (Study II). In addition, femoral and tibial bone marrow were harvested for preparing of dendritic cells [179]. Macrophages were obtained from peritoneal cavity after stimulation with intraperitoneally injected fetal calf serum. Macrophages were collected from the peritoneal cavity with RPMI 1640 medium (BioWhittaker Europe, Verviers, Bel- gium). The local research ethics committees approved all experiments.

4.3. Cell culture

Various cell lines and primary cell cultures were employed to investigate the expression pattern of ﬁ ltrin, nephrin and Nphs1as (Studies I, II and III; Table 3)

4.4. Molecular cloning and mRNA expression analysis

Complementary DNA clones The cDNA clones UI-M-BH2.1-apt-g-03-0-U (mouse Nphs1as cDNA) and DKFZp564A1164 (human ﬁ ltrin cDNA) were purchased from Research Genetics (Invitrogen, Paisley, UK) and RZPD Resource Center (Berlin, Ger- many) for further experiments.

RNA isolation Total RNA was isolated from tissues and cultured cells using chloroform- phenol extraction in the presence of Trizol reagent (Life Technologies,

28 4 Materials and methods Table 3. Summary of cell culture experiments.

Cell culture Culture media Reference Study

Human podocytes DMEM containing 10% fetal calf serum, 2.5 mM [232] I glutamine, 0.1 mM sodium pyruvate, 5 mM HEPES buff er, insulin, transferrin, and 5 mM sodium selenite.

Mouse dendritic cells RPMI 1640 medium with 10% fetal calf serum [179] II supplemented with 10 ng/ml murine recombinant GM-CSF.

Mouse embryonic stem cell DMEM with 20% fetal calf serum containing 1000 [233] II line 129/Sv units/ml of leukemia inhibitory factor.

Mouse epithelial cortical 1:1 mix of Ham F12 medium and DMEM containing [81] II collecting duct cell line M-1 5% fetal bovine serum and 5 μM dexamethasone.

Mouse insulinoma DMEM with GlutaMax-1 supplemented with [234] II cell line MIN6 15% fetal calf serum, 1 mM sodium pyruvate, and 0.05 mM β-mercaptoethanol.

Gibco BRL, Paisley, UK) or with an RNA isolation kit (Atlas Glass Total RNA Isolation kit [Clontech, Carlsbad, CA, USA] and RNeasy kit [Qiagen, Hilden, Germany]) according to the manufacturers’ instructions. To re- move traces of genomic DNA, isolated RNA was incubated with RNase- free DNaseI (Promega, Madison, WI, USA) for 30 minutes together with human placental RNase inhibitor (Promega).

Reverse transcriptase-polymerase chain reaction (RT-PCR) Several applications of reverse transcriptase-polymerase chain reaction (RT-PCR) were employed in gene expression analysis at the mRNA level. The methods included qualitative, (semi)quantitative, and orientation- speciﬁ c RT-PCR analysis, and, for mRNA cloning, rapid ampliﬁ cation of cDNA ends (RACE). Complementary DNA (cDNA) was synthesized from RNA using the M- MLV (Promega) or AMV (Roche Diagnostic GmbH, Mannheim, Germany)

reverse transcriptase enzymes using either oligo-dT15 primers or random hexamers (Roche Diagnostic GmbH). In orientation-specifi c RT-PCR analysis of the Nphs1 and Nphs1 as mRNAs, gene-specifi c oligonucleotides were used for priming the cDNA synthesis (Study II). Reactions without the reverse transcriptase enzyme served as negative controls. Thereafter, the cDNA was amplifi ed using gene-specifi c primer pairs (Studies I and II) with AmpliTaq Gold DNA polymerase (Applied Biosys- tems, Foster City, CA). In semiquantitative RT-PCR of Nphs1 and Nphs1as

4 Materials and methods 29 (Study II), serial dilutions (1:5 - 1:125) of cDNA samples were amplifi ed within the linear range of the PCR reaction, and the mRNA levels were normalized to the house-keeping gene cyclophilin A (Ppia, peptidylprolyl isomerase A). Cyclophilin A can be considered a suitable internal reference gene for comparing the expression of a target gene between different tissues, since the expression of cyclophilin A is not restricted to a specifi c tissue or cell type [180]. The RT-PCR products were analyzed electrophoretically and visualized using ethidium bromide. Appropriate controls without the cDNA template were included in each experiment. The amplifi ed PCR products were confi rmed with DNA sequencing using an ABI 373 sequencer (Applied Biosystems, Foster City, CA, USA) and ABI Prism Dye Terminator kit (Applied Biosystems). For quantitative real-time RT-PCR analysis of RNA isolated from human kidney biopsies, the gene expression assays were ordered from Applied Biosystems. Pre-developed assay reagents were used for fi ltrin (Assay ID: Hs00375638_m1) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Hs99999905_m1). According to a paper reporting the validation of internal reference genes for microdissected biopsies [181], the cyclophilin A mRNA was expressed at low levels in isolated glomeruli and was thus not suitable as a reference gene. Instead, GAPDH can be regarded as a potential housekeeping gene in microdissected glomeruli [181, 182]. For nephrin and podocin, previously developed forward and reverse primers and probes were used [47]. The gene expression measurement was based on the TaqMan technology which utilizes the 5’-nuclease activity of the AmpliTaq Gold DNA polymerase and fl uorescently labeled gene-specifi c probe containing a reporter dye at the 5’-end of the probe and a quencher at the 3’-end of the probe. Each sample was measured in duplicate, and the PCR amplifi cation was performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems) equipped with in- strumentation for fl uorescence detection. The normalized expression of ΔCt Δ the analysed gene was calculated with the 2 method [177], where Ct was defi ned as Ct(gene) - Ct(GAPDH), Ct denoting the threshold cycle for each measurement. For cloning the antisense transcript of the mouse nephrin gene (Nphs1as), RACE [183] was performed with the GeneRacer kit (Invitrogen) according to the manufacturer’s instructions using the gene-specifi c 5’- and 3’-RACE primers (Study II). The nested PCR products were subcloned into a pCR4-TOPO vector (Invitrogen) and sequenced as described above.

Northern Blotting The molecular weight of the Nphs1as mRNA was determined using the Northern blotting analysis [184]. RNA isolated from ES-cells (strain 129/ Sv) was separated with gel-electrophoresis and blotted onto a nylon membrane. The ﬁ lter was hybridized with a [α−32P]UTP-labeled riboprobe. This strand-speciﬁ c probe was synthesized with a T7 polymerase

30 4 Materials and methods (Promega) from the pCR4-TOPO vector containing the 3’-RACE fragment of the Nphs1as cDNA.

RNA dot blot assay In order to get insight into the fi ltrin mRNA expression pattern in human tissues, an RNA dot blot technique was chosen, and Human Multiple Tis- sue Expression (MTE™) Array (Clontech Laboratories) containing poly(A) mRNAs from 76 tissues was selected for the expression profi ling. Briefl y, [α−32P]dCTP labeled cDNA probe was synthesized with Rediprime II random prime labeling system (Amersham Pharmacia Biotech, Uppsala, Swe- den) using the insert of nucleotides 496-2273 from the DKFZp564A1164 clone as a template. The MTE blot was hybridized overnight with the purifi ed (NICK column; Amersham Pharmacia Biotech) probe in a hybridi- zation oven, and analyzed after an overnight exposure to a phosphoim- ager plate at -75°C with a Bio-imaging analyzer (Fuji Photo Film co, Kana- kawa, Japan).

4.5. Immunochemical methods

Antibodies Polyclonal anti-fi ltrin antibodies were generated in rabbits, since fi ltrin was a novel and previously uncharacterized protein. Two hydrophilic oli- gopeptides were selected from the fi ltrin protein sequence: The extracellular peptide GPS PHF LQQ PED LVV LLG EE(C) represented the amino acids 21-40 of fi ltrin and was highly conserved in the rat and mouse. An additional cysteine (C) was added for coupling the peptide to a carrier protein. The second peptide consists of the amino acids 533-548, CWR HSK ASA SFS EQK N, in the intracellular domain of fi ltrin. The peptides were synthesized, purifi ed, and coupled to the carrier protein keyhole limpet hemocyanin by Alpha Diagnostics International Inc. (San Antonio, TX, USA). Two rabbits (NZW strain) were immunized with each peptide according to standard protocols. Prior to immunizations, appropriate control serum samples were collected. The antiserum against the extracellular peptide was immuno-purifi ed with the corresponding peptide coupled to Epoxy-Activated Sepharose CL-6B Resin (Sigma-Aldrich, St. Louis, MO, USA) to yield peptide-specifi c fractions, and antiserum against the intracellular peptide with the protein-A-sepharose (Amersham Bio- sciences), respectively, following the manufacturers’ instructions. The polyclonal anti-nephrin antibody was raised in rabbits immunized with a recombinant intracellular part of rat nephrin [88]. The antiserum was purifi ed with a protein-A-column to isolate IgG fractions. The monoclonal anti-synaptopodin antibody from Progen (Goettingen, Germany) was used as a podocyte-specifi c marker.

4 Materials and methods 31 Immunoblotting The molecular weight and expression of ﬁ ltrin and nephrin were determined by immunoblotting in the kidney and other tissues. Tissue samples were homogenized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS and 0.5% sodiumdeoxycholate, pH 8.0) in the presence of a protease inhibitor cocktail (Complete Mini, EDTA-free, Roche Diagnostics). The detergent extracts were suspended in the Laemmli sample buffer [185], boiled for 5 minutes, separated with SDS-PAGE, and electrotransferred to nitrocellulose membranes (Amersham Life Sciences, Buckinghamshire, UK). Proteins were detected either with the intracellular anti-ﬁ ltrin or with the anti-nephrin antibody followed by HRP- conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) using the ECL method (SuperSignal West Pico Chemiluminescent Substrate kit; Pierce, Rockford, IL, USA).

Immunofl uorescence microscopy The distribution of fi ltrin was investigated in the cortical kidney tissue from cadavers and in kidney biopsies from patients with proteinuric diseases using immunofl uorescence microscopy (Studies I and III). Five μm thick frozen sections were fi xed with either ice cold-acetone or 3.5% paraformaldehyde in PBS followed by permeabilization with 0.1% Tri- ton X-100 in PBS, and detected with the indirect immunofl uorescence method. Briefl y, the intra- or extracellular anti-fi ltrin antibodies were incubated on the sections followed by appropriate fl uorescently-labelled secondary antibodies (FITC goat anti-rabbit IgG (H+L) [Jackson Immu- noResearch Laboratories] or Alexa Fluor 546 goat anti-rabbit IgG [Molec- ular Probes, Invitrogen]). In dual labeling experiments, the sections were incubated with the anti-synaptopodin antibody and Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) as a secondary antibody prior to staining with the anti-fi ltrin antibody. The sections were embedded in an anti-fading aqueous medium and examined under a fl uorescence microscope equipped with appropriate fi lters. As a secondary antibody control, the primary antibodies were omitted or replaced with irrelevant IgGs. Additionally, the intracellular anti-fi ltrin was immunodepleted by preincubation with the antigenic peptide to confi rm the specifi city in immunofl uorescence analysis.

Immunoelectron microscopy Subcellular localization of fi ltrin in podocytes was explored with indirect immunoelectron microscopy (Study III). Rat kidneys were perfusion fi xed with 4% freshly prepared paraformaldehyde in PBS. Ultrathin sections were incubated with the extracellular anti-fi ltrin antibody, followed by incubation with 10 nm gold-conjugated goat anti-rabbit IgG (1:50; Am- ersham Biosciences).

32 4 Materials and methods 4.6. Genotyping

In Study IV, the genetic association of LRRC7, KIRREL, NPHS2, KIRREL2, and ACTN4 with diabetic nephropathy was investigated using a compre- hensive set of bi-allelic single nucleotide polymorphisms (SNPs). The SNPs were selected from public SNP databases (Table 4). The Tagger program [186] was employed to select tag SNPs for ACTN4 and NPHS2 with pair- wise r2 > 0.8. In LRRC7, KIRREL, KIRREL2 evenly distributed SNPs covering the gene loci were chosen. DNA was extracted from peripheral blood collected from the patients. Genotyping was performed using the Homog- enous MassExtend MassArray System (Sequenom, San Diego, CA, USA) based on the MALDI-TOF determination of primer extension products [187] or with the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) utilizing allele-speciﬁ c TaqMan probes [188]. To assure genotyping accuracy, internal controls were included in each analysis.

4.7. Databases, bioinformatics tools, and statistical methods

Publicly available databases and bioinformatics search engines were utilized in searching for novel nephrin homologues (including fi ltrin [KIRREL2] and Nphs1as) and in gaining information about their gene structure and genomic localization, expression profi les and protein architecture (Table 4). Genomic databases were employed for the selection of SNPs, and appropriate tools were selected to estimate the power of statistical tests and haplotype and linkage disequilibrium (LD) values of the analyzed genes (Table 4). Continuous variables (clinical variables and gene expression data) are summarized as mean value ± standard deviation or median value (range). Differences between multiple groups for normally distributed variables were evaluated with ANOVA or the Dunnett 2-sided test, non-normally distributed variables were either log10 transformed prior to analysis or assessed with the non-parametric Kruskal-Wallis test. Pearson correlation coeffi cients were calculated and their signifi cances were tested to evaluate correlation between two variables. Hardy-Weinberg calcula- tions were performed to test genotype equilibrium for each SNP, and the genotype data were compared with the χ2-test. Logistic regression model with the nephropathy status as the dependent variable and risk factors as covariates was used to estimate the independent association of SNPs. Statistical analysis was performed with SPSS 12.0.1 for Windows software (SPSS Inc., Chicago, IL, USA).

4 Materials and methods 33 Table 4. Summary of bioinformatic databases and computational tools.

Database / Tool Description URL Study

AUG_EVALUATOR Prediction of translation start codons http://www.itba.mi.cnr.it/webgene/ I

BLAST Basic local alignment search tool http://www.ncbi.nlm.nih.gov/BLAST/ I, II

ClustalW Multiple sequence alignment tool http://www.ebi.ac.uk/clustalw/ I

CpGPlot Detection of http://www.ebi.ac.uk/emboss/cpgplot I, II CpG-rich regions in genomic DNA dbSNP Database of single nucleotide http://www.ncbi.nlm.nih.gov/SNP/ IV polymorphisms

GenBank Database of genetic sequences http://www.ncbi.nlm.nih.gov/Genbank/ I, II

Genetic Power Power analysis of genetic http://pngu.mgh.harvard.edu/~purcell/gpc/ IV Calculator association tests

Genome Browser Database of genetic information http://genome.ucsc.edu/ IV

Genscan Prediction of gene structures in http://genes.mit.edu/GENSCAN.html I genomic DNA

Haploview Tool for haplotype and linkage http://www.broad.mit.edu/mpg/haploview/ IV disequilibrium analysis

HapMap Database of human genetic variation http://www.hapmap.org/ IV

HCtata Prediction of TATA-signals in http://www.itba.mi.cnr.it/webgene/ II eukaryotic genes

InterPro Database of protein families, http://www.ebi.ac.uk/interpro/ I domains and functional sites

NetOGlyc Prediction of mucin type GalNAc http://www.cbs.dtu.dk/services/NetOGlyc/ I O-glycosylation sites in proteins

Primer3 Tool for designing primers for http://frodo.wi.mit.edu/cgi-bin/primer3/ I, II exprssion analysis primer3_www.cgi

Proscan Prediction of promoter regions http://bimas.dcrt.nih.gov/molbio/proscan/ I

Prosite Database of protein families, http://www.expasy.ch/tools/scanprosite/ I domains and functional sites

SignalP Prediction of signal peptide cleavage http://www.cbs.dtu.dk/services/SignalP/ I sites in proteins

Tagger Tool for the selection and evaluation http://www.broad.mit.edu/mpg/tagger/ IV of tag SNPs from genotype data

TMHMM Prediction of transmembrane helices http://www.cbs.dtu.dk/services/TMHMM- I in proteins 2.0/

UniGene Database of gene transtcripts http://www.ncbi.nlm.nih.gov/entrez/ I, II

34 4 Materials and methods 5. RESULTS

5.1. Filtrin, a novel nephrin-like protein, is expressed in glomerular podocytes (Studies I and III)

The NCBI BLAST algorithm was used to search for uncharacterized cDNA clones and expressed sequence tags (ESTs) homologous to human nephrin. The cDNA clone DKFZp564A1164, originally isolated from the human fetal brain, was chosen as the candidate molecule for detailed analysis. This molecule was named ‘fi ltrin’, and the offi cial gene name provided by the HUGO Gene Nomenclature Committee is ‘kin of irre like 2’ (KIRREL2) due to its similarity with D. melanogaster irre. The sequence analysis revealed that fi ltrin is a type I transmembrane protein of the immunoglobulin superfamily of adhesion proteins. It is composed of a cleavable N-terminal signal sequence, fi ve extracellular immunoglobulin-like domains followed by an acid alpha-helical transmembrane region, and a cytoplasmic carboxy-terminal domain with a proline-rich region (Study I; Figure 4). Several putative N- and O-glycosylation sites were detected at the extracellular side, and the cytoplasmic domain has potential phosphorylation sites at serine, threonine or tyrosine residues. The full-length form of fi ltrin consists of 708 amino acids. In addition to sequence homology to human nephrin (30%), fi ltrin shows considerable homology and structural similarity to the nephrin-like proteins NEPH1 (44%) and NEPH2 (41%) (Table 5).

TM PDZK1

lg Grb2-SH2

Figure 4. Schematic domain composition of the nephrin-like proteins. The proteins consist of ﬁ ve extracellular immunoglobulin-like domains (Ig) followed by a transmembrane domain (TM) and a cytoplasmic domain with the Grb2-SH2 and PDZK1 binding sites. The nephrin-like proteins interact with their cytoplasmic Grb2-SH2 and PDZK1 binding sites with podocin and ZO-1, respectively. SP, signal peptide. Drawn after [57].

5 Results 35 Table 5. Nephrin and the nephrin-like proteins, the genes encoding them and molecular characteristics.

Chromosomal location Protein

Protein Gene Human Mouse Size (aa) Molecular weigth (kDa)

Nephrin NPHS1 19q13.12 7qB1 1241 180

NEPH1 KIRREL 1q23.1 3qF1 757 90-110

NEPH2 KIRREL3 11q24.2 9qA4 778 100-125

Filtrin/NEPH3 KIRREL2 19q13.12 7qB1 708 107

The size of the protein is given in the number amino acids, and the apparent molecular weight represents the full-length isoform. The chromosomal regions are from the UCSC Genome Browser (March 2006 Assembly for human; February 2006 for mouse). aa, amino acid; kDa, kilo Dalton.

The human fi ltrin gene KIRREL2 resides on chromosome 19q13.12 adjacent to NPHS1 encoding nephrin (Study I). It is of particular note that NPHS1 and KIRREL2 are transcribed in the opposite directions, and the distance between the transcription start sites is approximately 5 kb in humans and 3 kb in the mouse and rat. This chromosomal arrangement is conserved among the species (Figure 5). In addition to the full-length form of fi ltrin, alternatively spliced variants of the fi ltrin mRNA were discovered. The alpha-form lacks exon 4, potentially deleting a part of the extracellular immunoglobulin-like domains of the translated protein, and the beta-forms represents a variant consisting only of the sequence coding for the signal peptide fused to the carboxy-terminal intracellular part of the protein (Study I). As reported by Sun and colleagues [56], the spectrum of alternatively spliced variants was broadened with the cloning of a mRNA variant, which lacks exon 2. Two independent methods were employed to detect the expression of fi ltrin in the kidney. First, the fi ltrin mRNA was successfully amplifi ed from the isolated human and rat glomeruli and from the cultured human podocytes with RT-PCR (Study I). Second, anti-fi ltrin antibodies were raised in rabbits to explore fi ltrin at the protein level, and the antibodies were affi nity-purifi ed to reach maximal specifi city. The immunoblotting studies of the glomerular lysates with the extracellular anti-fi ltrin antibody showed the presence of a protein band with an apparent molecular weight of 107 kDa (Study I). In addition, the cultured human podocytes expressed protein isoforms of varying molecular weight. The difference between the theoretical molecular weight of 75.1 kDa and the experi- mentally verifi ed size of 107 kDa may be explained by post-translational modifi cation by N- or O-glycosylation or other covalent modifi cations.

36 5 Results ~5 kb

NPHS1 KIRREL2

Figure 5. The genomic organization and exon-intron structure of the human NPHS1 and KIRREL2 genes in 19q13.12. Their transcription initiation sites are separated by a stretch of 5 kb, possibly sharing common regulatory elements. The arrows indicate the 5’-3’ direction of the mRNA transcription (Study I).

In order to obtain an insight into the global expression pattern of fi ltrin in human tissues, both bioinformatics and experimental methods were utilized. First, the expression of the fi ltrin-specifi c mRNA was explored with the EST sequence analysis: The UniGene cluster Hs.145729 contained EST sequences isolated from the lung, eye, lymphatic tissue, pancreas, brain, kidney, and pituitary gland (Study I). Second, the expression pattern was deciphered with hybridizing the radioactively labelled fi ltrin-specifi c probe to the nylon fi lter containing poly(A) mRNAs from 72 human tissues. According to the RNA dot blot analysis, fi ltrin mRNA is primarily expressed in the lymph nodes and the pancreas (Study I). The distribution of fi ltrin in the kidney was examined with immuno- histological methods. Immunofl uorescence staining with the extracellular anti-fi ltrin antibody revealed expression in the glomerulus and to some extent in proximal and distal tubules (Study I). According to the staining with the intracellular antibody, fi ltrin is expressed in an epithelial cell-like pattern in the glomerulus (Figure 6A; Study III). Most of the fi ltrin expression within the glomerulus was confi ned to podocytes as indicated by immunofl uorescent double labeling with the podocyte marker synaptopodin (Study III). The expression of fi ltrin was also examined at the ultrastructural level in podocytes, and the results indicate that fi ltrin localizes at the slit diaphragm of the podocyte foot processes (Figure 7; Study III).

5.2. Nphs1as mRNA is transcribed from the nephrin locus in a reverse orientation in the brain and lymphoid tissue (Study II)

The characterization of the nephrin-deﬁ cient mouse model generated by random insertional mutagenesis revealed that the gene-trap vector PT1βgeo, containing an Engrailed splice-acceptor site fused in-frame to the LacZ/neomycin fusion gene [189], was integrated in an inverted orientation in exon 8 of the Nphs1 gene [190] (Figure 8B). Notably, the

5 Results 37 AB

Figure 6. Distribution of fi ltrin in the kidney glomerulus as shown by immunofl uorescence microscopy. (A) Filtrin is expressed in an epithelial cell-like pattern in the normal glomerulus. (B) In primary FSGS, a decrease in the fi ltrin staining can be observed, ranging from segmental to global.

Figure 7. (A and B) On immunoelectron microscopical examination, gold particles were detected at the slit diaphragm area suggesting that ﬁ ltrin localizes at the slit diaphragm. US, urinary space; GBM, glomerular basement membrane (Study III).

38 5 Results trapped ES-cells were successfully screened by the selection marker gene and 5’-RACE, indicating the presence of an actively transcribed gene in an antisense orientation to nephrin. To elucidate this issue, Nphs1-like expressed sequence tags (ESTs) were searched from dbEST using the BLAST tool at NCBI. Among several positive hits, the clone UI-M-BH2.1-apt-g- 03-0-UI, originally isolated and sequenced from the mouse brain (strain C57BL/6J), showed exact complementarity, however, in an antisense orientation (Figure 8A; Study II). An orientation-specifi c RT-PCR technique was chosen to exclude possible sequence artifacts and to confi rm that the clone represents a true mRNA transcribed from the Nphs1 locus. Several primer pairs were de- signed with the help of the cDNA clone UI-M-BH2.1-apt-g-03-0-UI and the Nphs1 cDNA. This analysis confi rmed the presence of the Nphs1as mRNA in the mouse brain (strain NMRI) and embryonic stem (ES) cells (strain 129/Sv). No PCR product could be amplifi ed from cDNAs prepared from the kidney and pancreas, suggesting that these tissues do not express Nphs1as. All tissues examined and the ES cells expressed the Nphs1 mRNA (Study II). Notably, the amplifi cation of Nphs1as was only successful with the primer pair crossing the intron 7 of Nphs1 (Figure 8A), which is in agreement with the alignment of the EST clone UI-M-BH2.1-apt-g- 03-0-UI with the genomic sequence. More extensive screening of mouse lymphoid tissues indicated that the Nphs1as mRNA is transcribed in the thymus, peripheral lymph nodes and dendritic cells, whereas no expression was observed in the mesenteric lymph nodes, spleen and macrophages (Study II).

Figure 8. Nphs1as is transcribed from the nephrin encoding locus in a reverse orientation. (A) Genomic location and exon–intron structure of Nphs1 and Nphs1as mRNAs. Locations of the primers 2s/2as and 2Bs/2Bas used to amplify Nphs1as in Study II are indicated. (B) Targeting vector PT1βgeo disrupts exon 8 of Nphs1 in the nephrin- deﬁ cient mouse model. Notably, the vector is integrated in an inverted orientation. CpG, CpG island (Study II).

5 Results 39 Mounting evidence of the existence of the nephrin antisense transcript encouraged us to proceed to cloning the full-length cDNA. A technique known as the Rapid amplifi cation of cDNA ends (RACE) [183] was utilized to clone both the 5’- and 3’-ends of the transcript from the wild type murine brain RNA. The assembly of the isolated RACE-fragments with the sequence of the clone UI-M-BH2.1-apt-g-03-0-UI revealed that Nphs1as is a continuous 1339 bp mRNA (Study II). Northern blot analysis was used to further examine the molecular weight of Nphs1as mRNA. An RNA blot from embryonic stem cells was hybridized with a strand-specifi c 3’-RACE riboprobe. The size of the single transcript of approximately 1.5 kb detected in the analysis accords with the length of the Nphs1as cDNA isolated with RACE (Study II). Based on the information of the Nphs1 exon-intron structure [191], the transcription of Nphs1as initiates from the Nphs1 exon 12 and ex- tends until intron 6 (Figure 8A). Nphs1as is composed of three exons with consensus GT-AG splice sequences: the nucleotides 1-143 form exon 1, 144-558 exon 2, and 558-1339 exon 3. It is likely that the isolated sequence contains a genuine 3’end since poly(A)-tail was located at the end and multiple poly(A) signal sites (nucleotide positions 1125-1137) were present near the A-rich region. These sites were not completely identical with the conserved AAUAAA or AUUAAA eukaryotic signal sequences [192], possibly reducing the accuracy or effi ciency of poly(A) tail formation. A translated protein is not to be expected, because the sequence analysis did not revealed an open reading frame. To throw light on the possible correlation between the Nphs1 and Nphs1as levels and nephrin protein expression, semiquantitative RT-PCR of the sense and antisense transcripts and immunoblotting with nephrin- specifi c antibody were employed. Immunoblotting analysis identifi ed a double band of 185 kDa and 165 kDa in the kidney lysates, and a protein of 165 kDa in the lysates from the pancreas and brain (Study II). The intensity of amplifi ed RT-PCR products in dilution series of the cDNAs (from 1:5 to 1:125) was evaluated visually after gel-electrophoresis. In tissues expressing the sense and antisense transcripts, the level of the Nphs1as mRNA was remarkably higher in the brain than in peripheral lymph nodes and thymus. The Nphs1 mRNA was present in similar levels in the brain and peripheral lymph nodes, whereas the thymus expressed only a fraction of the other two (Study II).

5.3. Glomerular expression of ﬁ ltrin in patients with proteinuric kidney diseases (Study III)

The expression of ﬁ ltrin was examined in patients with proteinuric kidney diseases. First, ﬁ ltrin mRNA expression was analyzed in isolated glomeruli from patients with focal segmental glomerulosclerosis (FSGS), minimal

40 5 Results A B

0.9 3.0 0.8 2.5 0.7 0.6 2.0 0.5 1.5 0.4 0.3 1.0

Filtrin/GAPDH 0.2 Filtrin/Nephrin 0.5 0.1 0.0 0.0 CON MCDHT MGN FSGS CON MCDHT MGN FSGS

Figure 9. (A) Expression of the ﬁ ltrin mRNA in glomerular diseases in the ERCB samples. The results are normalized to the GAPDH gene. (B) An alternative normalization method was applied, where ﬁ ltrin mRNA levels were adjusted to the nephrin mRNA levels in each sample. CON, control group (non-affected parts of tumour nephrectomies); MCD, minimal change disease; HT, hypertensive nephropathy; MGN, membranous glomerulopathy; FSGS, focal segmental glomerulosclerosis (Study III).

change disease (MCD), hypertensive nephropathy (HT), and membranous glomerulonephropathy (MGN). Control samples were obtained from the healthy kidney poles of individuals who underwent tumor nephrectomies. By real-time quantitative RT-PCR, the ratio of fi ltrin mRNA to GAPDH mRNA was determined. In addition, the levels of nephrin and podocin mRNA were measured from the same samples. Compared with control kidneys (n = 7), statistically signifi cant under-expression of fi ltrin was observed in all diagnostic groups, with the highest repression in patients with FSGS (n = 9, p = 4.5·10-6) and MGN (n = 31, p = 0.3·10-6; Figure 9A). Second, expression ratios of fi ltrin/nephrin and fi ltrin/podocin were calculated in order to adjust the levels of fi ltrin to the podocyte marker genes (Figure 9B). This confi rmed the reduced expression of fi ltrin mRNA in relation to the podocyte markers in proteinuric diseases, thus over- coming the possibility that the observed decrease was due to a reduced number of podocytes per glomerulus. In addition, a positive correlation was observed in the glomerular fi ltrin/GAPDH and nephrin/GAPDH values (n = 63, r = 0.72, p = 2.9·10-11). Third, replication of the mRNA results was performed in a second set of samples, where the cDNAs were prepared from whole biopsies. The results were in agreement with the previous analysis in the case of FSGS (n = 11, p = 1.6·10-3) and also revealed a decrease in patients with diabetic nephropathy (n = 7, p = 5.1·10-3) compared to controls (n = 6). In MGN and MCD, the decrease in fi ltrin mRNA did not reach statistical signifi - cance. In spite of the general decrease in fi ltrin mRNA levels in proteinuric diseases, no correlation was observed with the expression of fi ltrin and the levels of proteinuria or serum creatinine at the time of biopsy.

5 Results 41 Figure 10. The exon-intron structure of the ACTN4 gene, with the chromosomal location of the genotyped SNPs. The marker-marker linkage disequilibrium (LD) plots are presented with r2 values (white = 0, 0 < grey < 1, black =1). Haplotype blocks of strong LD are indicated. LD, linkage disequilibrium (Study IV).

Collectively, these results indicate that the mRNA expression of fi ltrin is decreased in kidneys of patients with proteinuric kidney diseases. A moderate staining intensity of fi ltrin was observed in the glomerulus of patients with MCD (n = 4) and MGN (n = 6) as compared to controls (n = 3) in immunohistochemical examination. In MCD, fi ltrin was also detected in the mesangium, whereas in MGN, the glomerular staining of fi ltrin appeared granular. A marked decrease in the glomerular staining of fi ltrin characterized patients with FSGS (n = 5), ranging from segmental to global (Figure 6).

42 5 Results 5.4. Genetic association analysis of LRRC7, KIRREL, NPHS2, KIRREL2, and ACTN4 with diabetic nephropathy (Study IV)

To investigate the genetic association of selected nephrin-associated genes with nephropathy in type 1 diabetes, a genetic association analysis was performed in a cross-sectional case-control study setting. A set of 1103 Finnish type 1 diabetic patients was included into the fi rst analysis, and the patients were classifi ed as having normoalbuminuria (n = 459), microalbuminuria (n = 276), or macroalbuminuria (n = 368). In order to confi rm the results of the fi rst analysis, an analysis of patients with normoalbuminuria (n = 607) and macroalbuminuria (n = 158) was performed for the selected SNPs at the second stage of analysis. In addition, a set of patients with ESRD (n = 260) was included. In the fi rst screening, a total of 45 SNPs in the LRRC7, KIRREL, NPHS2, KIRREL2, and ACTN4 genes were genotyped for the association analysis (Study IV). Genotype distributions were in Hardy-Weinberg equilibrium (HWE) in all SNPs except in the LRRC7 SNP rs984950 (HWE p-value in patients with normoalbuminuria 2.2·10-6), indicating that the results of this SNP should be interpreted with caution. Statistical analysis revealed that no differences were evident in genotype, allele, or haplotype frequencies between patients with normal albumin excretion rate and diabetic nephropathy. The independent association of the SNPs was evaluated by adjusting the genetic effect with duration of type 1 diabetes, systolic and diastolic blood pressure, and HbA1c. However, this did not change the results. Neither were the polymorphisms associated with the albumin excretion rate, serum creatinine level nor time from the onset of type 1 diabetes to the development of diabetic nephropathy, when these variables were used as quantitative variables in the analysis instead of the categorical nephropathy status. Notably, this analysis included also patients with microalbuminuria. Since male gender has been considered as a risk factor for diabetic nephropathy in type 1 diabetes [124], males and females were also analyzed separately. The gender-specifi c analysis yielded modest genotype and allelic associations with diabetic nephropathy for the SNPs rs979972 (p = 0.053), rs6508813 (p = 0.025), rs749701 (p = 0.008) and rs1060186 (p = 0.025) in the ACTN4 gene in females. Figure 10 describes the exon-intron structure of the ACTN4 SNPs with the location of the genotyped SNPs. In- terestingly, high pair-wise linkage disequilibrium (r2 > 0.8) was measured for the ACTN4 SNPs rs6508813-rs1060186. The four SNPs in the ACTN4 gene reaching the statistical signifi cance level 0.05 were selected for the second stage of analysis. At the second stage, a joint analysis of the genotypes from the fi rst and second set of patients was performed. The effect of stratifi cation by gender was investigated, since gender-specifi c effects were observed

5 Results 43 already in the ﬁ rst sample set. The SNPs rs979972 and rs749701 were associated with diabetic nephropathy and ESRD in females (Table 6). In this analysis, the genetic effect was adjusted for duration of type 1 diabetes, systolic and diastolic blood pressure, and HbA1c. For the SNPs rs979972 and rs749701, p-values below 0.05 were detected in the Hardy-Weinberg equilibrium test in macroalbuminuric patients (Table 6). Interestingly, in the joint analysis of patients, the SNPs rs979972 and rs749701 yielded nominally signiﬁ cant association with ESRD also in males, when the patients with normoalbuminuria and ESRD were compared (Table 6).

44 5 Results Table 6. Four ACTN4 SNPs with evidence of an association with diabetic nephropathy. The patients of the ﬁ rst and second stage of analysis were combined, minor allele frequencies are presented separately for male and female patients with the corresponding adjusted odds ratios (OR) and p-values.

SNP HWE MAFa Diabetic nephropathy ESRD

p-value ORb (95% CI) p-value ORb (95% CI) p-value

Males rs979972 Normo 0.82 0.52 0.74 (0.48 - 1.15) 0.183 0.42 (0.21 - 0.85) 0.015 Macro 0.96 0.51 ESRD 0.09 0.47

rs6508813 Normo 0.55 0.27 0.64 (0.34 - 1.18) 0.153 0.60 (0.23 - 1.45) 0.244 Macro 0.70 0.24 ESRD 0.33 0.27

rs749701 Normo 0.44 0.44 0.65 (0.41 - 1.01) 0.057 0.43 (0.21 - 0.88) 0.021 Macro 0.69 0.40 ESRD 0.11 0.40

rs1060186 Normo 0.46 0.26 0.63 (0.34 - 1.19) 0.157 0.59 (0.24 - 1.47) 0.258 Macro 0.60 0.24 ESRD 0.45 0.26

Females rs979972 Normo 0.56 0.51 1.81 (1.18 - 2.79) 0.007 2.26 (1.14 - 4.48) 0.020 Macro 0.03 0.56 ESRD 0.08 0.60

rs6508813 Normo 0.91 0.27 1.70 (0.98 - 2.94) 0.058 0.98 (0.37 - 2.59) 0.961 Macro 0.35 0.34 ESRD 0.47 0.25

rs749701 Normo 0.67 0.42 1.93 (1.25 - 2.96) 0.003 2.12 (1.05 - 4.28) 0.036 Macro 0.02 0.49 ESRD 0.63 0.48

rs1060186 Normo 0.54 0.26 1.73 (1.00 - 3.00) 0.052 0.86 (0.31 - 2.39) 0.766 Macro 0.33 0.34 ESRD 0.72 0.25

aMinor allele is deﬁ ned as in Study IV. bThe ORs were adjusted for duration of type 1 diabetes, systolic and diastolic blood pressure, and HbA1c. Abbreviations: CI, conﬁ dence interval; ESRD, end- stage renal disease; HWE, Hardy-Weinberg equilibrium; Macro, macroalbuminuria; MAF, minor allele frequency; Normo, normoalbuminuria; OR, odds ratio.

5 Results 45 6. DISCUSSION

The hypothesis of this thesis stated that novel, yet unknown, nephrin- associated molecules participate in maintaining the slit diaphragm function with nephrin. This has been proved to be at least partially true. Novel nephrin homologues NEPH1 [53], NEPH2 [54], and ﬁ ltrin (Studies I and III) have been identiﬁ ed and localized at the slit diaphragm. In addition, an endogenous nephrin antisense transcript Nphs1as was cloned in mouse (Study II), but to date no expression of Nphs1as has been detected in the kidney.

Filtrin emerges into the picture of the podocytes

Filtrin is a novel nephrin-like protein of the immunoglobulin superfamily (Study I) [56, 57]. This is the fi rst study to determine the endogenous fi ltrin at the protein level in the kidney. The colocalization of fi ltrin with synaptopodin, an established podocyte marker, in dual immunofl uorescence microscopy of human kidney cortex together with the immunoblotting and RT-PCR data of isolated glomeruli support the expression of fi ltrin in the podocytes (Studies I and III). Nephrin is a crucial protein of the slit diaphragm [32-34]. Therefore, the question arises, which data support the association of fi ltrin with the slit diaphragm complex? At the ultrastructural level, immunoelectron microscopy using fi ltrin-specifi c antibodies indicates that fi ltrin localizes at the slit diaphragm area (Study III). In addition, fi ltrin, similar to the other nephrin-like molecules, interacts with the slit diaphragm proteins podocin and ZO-1 [57, 60]. Collectively, these data suggest that fi ltrin may belong, together with nephrin, to the cell adhesion junction between the podocyte foot processes. The nephrin-like proteins NEPH1, NEPH2 and fi ltrin all localize in the slit diaphragm, and experimental data suggest that NEPH1 and NEPH2 form heterodimers with nephrin [35, 37, 54]. However, the dimensions of the extracellular domain of fi ve tandem immunoglobulin like repeats of the nephrin-like proteins might not allow direct interactions extending over the slit between the lateral foot process membranes. Instead, they may interact with the proximal part of the nephrin and, thus, stabilize the slit diaphragm structure as suggested by Tryggvason and colleagues [143]. Two other slit diaphragm proteins, nephrin and FAT1, are likely to be the ones that extend and form trans-interactions [34, 65]. Importantly, information on the signalling function for NEPH1 is emerging, involving

46 6 Discussion dynamic interactions with podocin and ZO-1, and NEPH1 seems to regulate podocyte gene expression [29].

Function of ﬁ ltrin in other tissues

The expression profi ling of human tissues suggested that fi ltrin-specifi c mRNA is predominantly expressed in the pancreas and lymph nodes (Study I). The expression in the pancreas has been replicated in later studies, and immunohistochemical data suggest localization in the β-cells of the islets of Langerhans [56, 193]. Recently published data on patients with type 1 diabetes indicates that anti-fi ltrin autoantibodies can be detected in the serum in approximately 10% of the patients during a follow-up time of 10 years after the diagnosis of type 1 diabetes [194]. This suggests that fi ltrin is involved in the autoimmune destruction of pancreatic β-cell. In the case of nephrin and NEPH1, intravenous injection of the antibodies in experimental models results in proteinuria [25, 58], and it is tempting to speculate that fi ltrin in the podocytes could act as an autoantigen in patients with type 1 diabetes and ultimately predispose to diabetic nephropathy. However, no evidence on the association of anti-fi ltrin autoantibodies with diabetic nephropathy was observed in the clinical study by Rinta-Valkama and colleagues [194]. In the brain, fi ltrin (NEPH3) appears transiently in the post-mitotic neural precursor cells, where it accumulates at the cellular processes and ZO-1 positive adherens junctions [195]. Confi rming the observation, fi ltrin localizes at the cell contacts in an adherens junction model in vitro. No- tably, fi ltrin proteins were shown to form homophilic trans-interactions with each other [195]. In olfactory sensory neurons, fi ltrin and its homologue NEPH2 act as homophilic adhesive molecules, and, interestingly, these proteins are expressed in a mutually exclusive pattern in the bun- dle of axons [196]. In these cells, fi ltrin presumably participates in axon guidance [196]. The podocytes have been described as neuron-like cells due to their characteristic morphology, and these cell types share some similarities at the molecular level [10]. However, it should be noted that the properties, especially the gap of 25 nm [197], of a typical adherens junction are not similar to the slit diaphragm. Thus the above mentioned results cannot be directly extrapolated to the mature slit diaphragm, where the lateral juxtaposed foot process membranes are separated by a distance of 30-40 nm [15].

Expression of nephrin and novel regulatory aspects

Expression of nephrin outside the kidney has been reported. In humans, nephrin has been associated with the islets of Langerhans in the pancreas

6 Discussion 47 [193, 198] and the lymphoid tissue [199], but the fi ndings remain contra- dictory (as reported in [200]) and may suffer from methodological limita- tions. In the mouse, the expression pattern is also promiscuous including the pancreas and distinct parts of the brain [201]. In line with the expression profi le in humans, the nephrin mRNA was also successfully amplifi ed in the lymphoid tissue in mice (Study II). Distinct promoter regions and alternatively used upstream exons may partly explain the tissue- and cell type-specifi c expression of nephrin [202-205]. Mechanistically, the transcription factors WT1, PPAR-gamma, and retinoic acid receptors participate in the regulation of nephrin at the promoter level [206, 207]. In addition, an alternatively spliced variant of NPHS1 mRNA has been isolated from the kidney [32]. The cloning of the endogenous antisense transcript of nephrin, Nphs1as, revealed yet another regulatory aspect of nephrin. Nphs1as is expressed in the brain, lymphoid tissue, and embryonic stem cells in the mouse (Study II), but, in contrast to the hypothesis, Nphs1as does not seem to be transcribed in the kidney and thus does not participate in the regulation of the slit diaphragm in the podocytes. It is reasonable to question, if Nphs1as mRNA is truly transcribed from the nephrin encoding locus in the mouse. How- ever, sequence alignments of the isolated cDNA with the genomic DNA sequence (Study II) and the incorporation of the TRAP vector in exon 8 in the nephrin-defi cient mouse model [190] indicate that Nphs1as is transcribed from the nephrin locus. The expression of nephrin mRNA in embryonic stem cells has recently been confi rmed by independent research groups [208, 209]. The results presented here do not allow direct conclusions about the function of Nphs1as, but speculations can be presented based on previous knowledge of endogenous antisense transcripts: A number of functions, including transcriptional interference, RNA masking and double-stranded RNA-dependent mechanisms, has been attributed to endogenously expressed antisense transcripts (reviewed in [210]). As discussed by Lapidot and colleagues [210], the relationship between the expression pattern of Nphs1as and its target Nphs1 might give certain indications regarding the mode of action. Nephrin is expressed in the brain in a cell-type specifi c manner [191, 201], and both the Nphs1as and Nphs1 mRNA are expressed in reasonable amounts (Study II). Thus, it can be proposed that the function of Nphs1as in the brain is to stabilize the Nphs1 mRNA transcript levels, but, of course, other mechanisms cannot be excluded. To my knowledge, the human counterpart of the nephrin antisense transcript has not been isolated.

48 6 Discussion Chromosomal organisation of KIRREL2 and NPHS1 in the human genome

The human fi ltrin gene KIRREL2 resides on chromosome 19q13.12 adjacent to NPHS1 which encodes nephrin, and the distance between the transcription initiation sites is approximately 5 kb in human (Study I; Ta- ble 5) and 3 kb in mouse and rat (data not shown). The inserted repeti- tive DNA elements in the human DNA sequence may explain the difference in the distance of the intervening region. It has been demonstrated that the regulatory elements required for the endogenous Nphs1 expression pattern in the mouse are located 5.4 kb or even 8.3 kb upstream of the transcription initiation site [202]. Moreover, the region 1.25 kb upstream is shown to direct podocyte-specifi c expression both in human and mouse [203, 204]. Overlapping expression sites of nephrin and fi ltrin exist, particularly in the podocytes and the pancreatic beta-cells. Thus, the regulatory regions of NPHS1 and KIRREL2, although read in opposite directions, may overlap, suggesting the possibility of transcriptional co- regulation. The head-to-head orientation of genes is a rather common phenome- non, but the mechanisms of regulation of bi-directional genes are poorly known [211]. Systematic analysis of gene expression databases has revealed that many bi-directional genes are coregulated but some, instead, are antiregulated [211]. In addition, transient transfection assays for a randomly chosen set of genes suggested that most bi-directional promoters share gene regulatory elements that control both genes [211]. The TATA-element of the proximal promoter region is suggested to contribute to the directionality of transcription and, therefore, bi-directional promoters contain very rarely TATA-boxes [211]. Interestingly, neither the NPHS1 nor KIRREL2 promoter has a TATA-box in their proximal promoter area (Study I). Gene pairs coding for homologous proteins are uncommon. An example of such a case is provided by the genes SAFB1 and SAFB2, which is a homolous gene pair arranged in a bi-directional divergent confi guration in 19p13.3 [212]. The genes are separated by a rather short intervening region of 500 bp and share similar functions as oestrogen receptors. They are widely co-expressed in human tissues, but the translated proteins have somewhat differing subcellular localizations [212]. On human chromosome 19, a conserved gene arrangement of orthol- ogous genes in mouse and human has been described [213, 214]. Nota- bly, one third of genes on chromosome 19 has been observed to belong to clustered gene families, possibly due to duplication of ancestral genes [214]. It appears that the nephrin and the nephrin-like genes are not confi ned to a single mammalian species, suggesting that chromosomal arrangements or duplications have occurred prior to the divergence of mammalian ancestors.

6 Discussion 49 Patients with proteinuria (Studies III and IV)

This study utilized several patient groups in elucidating the pathogenetic mechanism in proteinuria. Two sample sets were collected using a multi- central approach. First, the Finnish FinnDiane cohort focusing on complications in type 1 diabetes, particularly diabetic nephropathy, was utilized for the genetic association analysis of the nephrin-associated genes (Study IV). Second, the European Renal cDNA bank, a multinational ini- tiative to investigate the functional genetics of proteinuric renal diseases (participating centres listed in Appendix) was used for gene expression analysis of fi ltrin and the podocyte marker genes (Study III). The other two sample collections were derived from a single hospital in Fitzroy, Australia and in Milan, Italy, and were examined in Study III. All clinical data were analysed in a cross-sectional setting (studies III and IV), which is a powerful study setting, when the phenotypes are well- defi ned and the number of samples provides suffi cient statistical power. Ideally, the study setting would be a prospective one having follow-up data available after the time of collecting the samples. It is also note- worthy that kidney biopsies usually represent a rather established stage of the disease, and may thus provide limited information on the early phases of the disease. Therefore, experimental animal models will be crucial to gain insight into the time perspective of the variation in the expression of fi ltrin and in order to exclude the effect of different environmental effects.

Is ﬁ ltrin merely a by-stander in the podocyte or is it involved in podocyte injury?

Mutations in the genes encoding nephrin (NPHS1) and NEPH1 (KIRREL) seem to trigger congenital nephrosis, and these molecules are indispensa- ble for the maintenance of the slit diaphragm. We tested the hypothesis, whether fi ltrin is involved in the primary glomerular diseases including minimal change disease, focal segmental glomerulosclerosis and membranous glomerulopathy (Study III). The mRNA levels of fi ltrin, nephrin, podocin and GAPDH were measured in kidney biopsies. GAPDH was used for standard normalization and nephrin and podocin were considered established podocyte marker genes. Two set of patients (ERCB and the St. Vincent Hospital) were analyzed. Collectively, the results indicate that fi ltrin is down-regulated in patients with primary glomerular disease in relation to GAPDH and also to the podocyte marker gene nephrin and podocin. The sample size was limited but still suffi cient to detect a statistically signifi cant difference in expression values in all diagnostic groups in the ERCB samples, whereas in the second set of samples from the St. Vincent’s Hospital the low number of patients per diagnostic group and

50 6 Discussion high variability in the expression values did not allow drawing defi nitive conclusions in all disease categories (Study III). A statistically signifi cant, positive correlation (p ≈ 2.9·10-11) was observed in the expression values of fi ltrin and nephrin. This result gives additional support for the view that the gene expression in the podocyte is orchestrated potentially via common regulatory pathways. In addition, it is particularly interesting due to the intriguing chromosomal arrangement of the nephrin and fi l- trin genes NPHS1 and KIRREL2. Altered distribution of fi ltrin in the glomerulus was observed in patients with glomerular diseases, especially in FSGS (Study III), which supports the results of the gene expression analysis. Can the nephrin-like proteins mutually compensate each other’s function? It is known that the nephrin-like proteins are only homologous in their extracellular domains but not in the intracellular domain (except the podocin-interacting domain) [57], and these structural differences may indicate that these proteins do not have identical signal transductions mechanisms. The measurement of mRNA levels in kidney samples does not perfectly represent the gene expression activity. It rather refl ects the steady-state level of mRNA turnover, where degradation, modifi cations and transcription have all their own effects. As for the podocytes, altered podocyte number due to their detachment from the basement membrane or possibly via apoptotic mechanisms may reduce the proportion of podocytes per glomerulus as reported in FSGS and diabetic nephropathy [129, 215]. Another concern is the number of glomeruli per one kidney biopsy. These facts may cause a bias in the expression analysis. In this study, the concept of ‘in silico’ normalization according to the podocyte marker genes nephrin and podocin, originally introduced by the group of Dr. Matthias Kretzler [47], was successfully adapted for the expression analysis of fi ltrin (Study III). The results indicated that fi ltrin mRNA is down-regulated beyond the regulation of nephrin and podocin mRNAs, consistent with an mRNA repression per podocyte and not only a consequence of podocyte loss. This allowed us to overcome - at least partially - the concerns mentioned above. Furthermore, the effect of dis- secting the kidney biopsies into distinct glomerular and tubulointerstitial compartments (described in [177]) was also evaluated in this study. The expression of fi ltrin in microdissected glomeruli was compared to that measured from whole biopsies (Study III). It appeared that a trend of decreased expression was observed in both sample sets, irrespective of the mode of the preparation of the samples (Study III). Based on the knowledge of the pathogenesis of ‘podocyte diseases’, additional means of normalization should be taken into use (including the ‘in silico’ normalization) beyond the normalization in the house-keeping genes like GAPDH, which has been a golden standard in renal research.

6 Discussion 51 Can the genes behind monogenic proteinuric diseases contribute to the understanding of diabetic nephropathy?

Most of the current understanding of common diseases derives from the elucidation of the molecular background of rare familial forms of these common traits (discussed in [216]). The genes underlying the hereditary proteinuric diseases are expressed in the podocytes and include several slit diaphragm associated genes (chapter 2.5). In this study, we tested the hypothesis, whether the genes encoding fi ltrin, NEPH1, podocin, α-actinin-4 and densin-180 act as susceptibility genes for diabetic nephropathy (Study IV). The kidney complication of diabetes manifests fi rst with microalbuminuria [127], which suggests that the glomerular capillary wall, responsible for retaining albumin within circulation, is affected. The selection of the population is critical for the validity of the results of association analysis in complex diseases. The homogeneity and structure of the Finnish population is defi nitely an asset for performing genetic studies (reviewed in [217]). The Finnish population has a characteristic disease heritage, and many of these diseases have a Mendelian pattern of inheritance. Several causative genes have been successfully identifi ed, including NPHS1 [27]. The population has also proved useful for complex disease genetic mapping, as illustrated by the mapping of susceptibility gene for asthma [218]. A recent population based study supports the involvement of a genetic component in diabetic nephropathy in the Finnish population [163]. In addition, Finland has the highest incidence rate of type 1 diabetes in the world [219]. For the genes ACTN4 and NPHS2, tag SNPs were selected based on the HapMap data in the CEPH (Centre d’Etude Polymorphism Humain) population. This approach assumes that the information content of the tag SNPs in the CEPH population is transferable to the Finnish population. The issue of tag transferability was recently examined by de Bakker and colleagues [220]. They estimated the correlation (r2) in the genotype data of a set of candidate genes between the CEPH population and the Botnia sample from Finland, and the results indicate that it is justifi ed to utilize the HapMap data for selection of tag SNPs for genotyping in the Finnish population without compromising the statistical power. A spacing approach (with an interval of 6-14 kb) was employed in the selection of the genotyped SNPs for KIRREL and KIRREL2. The HapMap data were utilized also in this selection, but not all tag SNPs were included in genotyping due to the large number of SNPs that the complete covering of these relative small genes would have required. In the case of LRRC7, only a few SNPs were genotyped, so it can only be considered as a pilot study. The results of the genetic association analysis suggest that polymorphisms in the ACTN4 gene, encoding α-actinin-4, may be involved

52 6 Discussion in susceptibility to diabetic nephropathy in type 1 diabetes (Study IV). However, the link between ACTN4 and diabetic nephropathy was not straightforward, but a genetic interaction of the gender and the ACTN4 polymorphisms was observed. The results were supported by the fi nding that the same variants also seemed to associate with cardiovascular disease (Study IV). Furthermore, the genotype distribution of the polymorphisms showed a deviation from the Hardy-Weinberg equilibrium in the female patients with macroalbuminuria (Study IV), which may indicate a true genetic association as proposed by Nielsen and colleagues [221]. α-actinin-4 is an actin-fi lament cross-linking protein located in the proximity of the slit diaphragm in podocyte foot processes [82, 83]. The structure and function of the cytoskeleton are of pivotal importance for maintaining the cell shape of the podocytes, and disturbances of the podocyte cytoskeleton have been detected in the presence of high glucose both in vitro and in vivo [222]. Interestingly, α-actinin-4 is down-regulated in kidney biopsies of diabetic patients with microalbuminuria as compared to controls [223], and its expression is also disturbed in cultured cells by diabetes-related pathogenetic factors including advanced glycation end-products [224]. The connection between ACTN4 and nephropathy was fi rst discovered in patients with familial focal segmental glomerulosclerosis (FSGS) carrying mutations in the ACTN4 gene [83]. This disease belongs to the ‘late-onset’ group according to the classifi cation by Kriz [142]. Regarding diabetic nephropathy, the complication develops gradually and the incidence is at its highest after about 15 years’ duration of type 1 diabetes [124]. The results support the hypothesis that a gene previously linked to a rare monogenic type of nephropathy is also a good candidate for diabetic nephropathy, a more common type of nephropathy. The statistical power was estimated for the genetic association analysis [225]. While the fi rst set of patients alone remained underpowered to detect association with markers with low minor allele frequencies, the additional genotyping and the joint analysis of the patients at the second stage of analysis increased the statistical power remarkably (Study IV). Low minor allele frequency (MAF) for certain genotyped SNPs may limit the interpretation of the results. Especially in KIRREL2, all SNPs had MAF under 0.05 in patients with a normal albumin excretion rate (Study IV). Therefore, direct sequencing of the KIRREL2 locus on chromosome 19q13.12 to detect population-specifi c polymorphisms may be required. Furthermore, we cannot exclude the existence of rare variants in some cases of diabetic nephropathy. It can be speculated that the mutation phenotype of the genes of monogenic proteinuria with a congenital onset of the disease, e.g. NPHS1 and NPHS2, would not perfectly fi t with the insidious and slow pathogenesis of diabetic nephropathy. On the other hand, the negative results of the association analysis might also arise from to the fact that the

6 Discussion 53 genotyped SNPs covered only the coding region and the close vicinity of the genes under analysis. This approach assumed that causative mutations locate in the coding, splicing site and/or regulatory regions in the proximity of the analysed gene. The causative variants may also reside over long distances from the coding region as exemplifi ed by the case of adult-type hypolactasia [226], and this possibility has to be taken into account when interpreting these results. Even if the genotyped SNPs in the fi ltrin gene KIRREL2 were not associated with diabetic nephropathy, it appears that fi ltrin is down-regulated at the mRNA level in kidney biopsies of patients with diabetic nephropathy (Study III). This seems to be the case with the nephrin gene NPHS1: NPHS1 has been excluded as a susceptibility gene for diabetic nephropathy [227], but nephrin still seems to contribute to the pathogenesis of diabetic nephropathy. Decreased levels of nephrin are often observed in the glomerulus, and this seems to be mediated in an angiotensin II dependent fashion [49, 51]. In addition, a similar down-regulation of fi ltrin mRNA was detected in another secondary glomerular disease hypertensive nephropathy (Study III). Interestingly, diabetic and hypertensive nephropathy may have common predisposing factors and pathogenetic mechanisms [98].

54 6 Discussion Concluding remarks

Since the cloning of the nephrin gene NPHS1, novel nephrin-associated molecules of pivotal importance for the slit diaphragm have been discovered. This thesis adds two potentially interesting molecules to this list, and addresses their involvement in human glomerular diseases. It is of note that the term ‘nephrin-associated’ was interpreted in a quite broad sense in this thesis, including both nephrin-like and nephrin-interacting molecules. This thesis employed a bioinformatics approach for fi shing novel homologous genes and proceeded to assess the signifi cance of the molecules in pathological conditions by analysing human sample collections. This approach proved successful. Two novel candidates molecules, fi ltrin and Nphs1as, were discovered, and moreover, they are both transcribed from the nephrin locus or its proximity. Filtrin is a novel podocyte-expressed protein with localization at the slit diaphragm, and the expression of fi ltrin is down-regulated in human proteinuric diseases. However, several aspects of fi ltrin still deserve to be explored. Molecules of the immunoglobulin superfamily tend to form both homo- and heterodimers, and, therefore, it will be interesting to defi ne the molecular interactions of fi ltrin at the slit diaphragm - especially with nephrin and the nephrin-like proteins NEPH1 and NEPH2. Regarding the fi ltrin gene KIRREL2, an intriguing question is also the potential transcriptional co-regulation of KIRREL2 and NPHS1 in physiological and pathological conditions. Whether or not these genes share common regulatory elements in their intergenic region remains to be the elucidated. This thesis reports the discovery of Nphs1as, an endogenous antisense transcript of the nephrin gene, but this line of research was not continued due to the apparent lack of expression in the mouse kidney. Nevertheless, this does not rule out the possibility that Nphs1as could be biologically important. The possible human orthologue has not been cloned, and it is also tempting to speculate that de novo expression of the antisense transcript would emerge in the kidney under proteinuric conditions, which would provide an unprecedented view in the role of nephrin in glomerular diseases. It will be important to determine at which level (transcription, pre-mRNA splicing, or translation) the possible interaction between Nphs1 and Nphs1as mRNAs occurs. These experiments may indicate novel regulatory functions of Nphs1as. A candidate gene approach was selected to evaluate the genetic factors in diabetic nephropathy. It seems that common variants in ACTN4 contribute to genetic susceptibility for diabetic nephropathy in type 1 diabetes. The results suggest that a gene previously linked to a monogenic focal segmental glomerulosclerosis (FSGS) may confer susceptibility to a secondary form of nephropathy, namely diabetic nephropathy. Replica- tion of the fi nding in other populations and fi ne mapping of the gene

6 Discussion 55 locus will be required. It is also worth examining the role of two other nephrin-associated genes TRPC6 and CD2AP in diabetic nephropathy. No- tably, these genes have previously been linked to FSGS. In conclusion, it seems that novel nephrin-like and nephrin-associated molecules are crucial for maintaining the function of the glomerular ﬁ ltration barrier. The ﬁ ndings presented in this thesis provide novel insights into the molecular basis of human glomerular diseases and extend the understanding of the molecular anatomy of the slit diaphragm.

56 6 Discussion 7. ACKNOWLEDGEMENTS

This work was carried out at the Department of Bacteriology and Im- munology, University of Helsinki and at the Folkhälsan Research Center in Biomedicum Helsinki during the years 2001 – 2007. I am grateful to the previous and present heads of both the institutes, Professors Risto Renkonen, Seppo Meri, Per-Henrik Groop and Anna-Elina Lehesjoki, for providing excellent research facilities. I wish to thank Professor Carl G. Gahmberg for guiding me through the ﬁ nal metres of my Ph.D. studies and for accepting the role of custos in my defence. Professor Markku Laakso and Docent Eero Lehtonen are warmly acknowledged for reviewing this thesis and for their constructive criticism. I want to express my gratitude to Docent Harry Holthöfer for intro- ducing me to the fascinating world of podocyte research. His encourage- ment during the thesis work and never-ending enthusiasm will not be forgotten. I am most grateful to Docent Per-Henrik Groop. He has taught me the essence of science and created a pleasant and supportive atmos- phere to work at. My special thanks belong to Docent Maija Wessman. I am grateful to Maija for all for her time and effort that she has unself- ishly devoted to my research and this Ph.D. thesis. My collaborators and co-authors have been numerous. It has been a pleasure to work with Tuula Palmén, Heikki Ahola, Johanna Rinta- Valkama, Petra Mai, Eva Åström, Thu Trang Pham, Thomas Floss, Holger Schmid, Maria Pia Rastaldi, Deborah Mattinzoli, Robyn G. Langham, Pauliina Luimula, Riika Kilpikari, Markus Lassila, Richard E. Gilbert, Dontscho Kerjaschki, Matthias Kretzler, Mari Kaunisto, Riika Kilpikari, Maija Parkkonen, and Carol Forsblom. Their contribution has made the publications and this thesis possible. Thank you! It has been a privilege to participate in the work in two research groups. All the previous and present members of the Glomerular Biology Study Group and the FinnDiane Study Group are acknowledged for their help and support. We have shared the excitement of discovery and some- times depressing moments together. Especially, I thank three excellent scientists, Docent Markus Lassila, Sanna Lehtonen, and Carol Forsblom, for always ﬁ nding time for inspiring discussions about science and for their valuable advice regarding practical problems. This work would not have been possible without the FinnDiane patients and other people who have participated in the various studies. Their involvement in the projects – without expecting any personal gain – is acknowledged.

7 Acknowledgements 57 Sami Ruotsalainen is thanked for his skilful help with the image processing of this thesis. I want to thank all my friends for all their support and for keeping me in touch with the world surrounding Biomedicum. I owe my warm- est thanks my family, my parents Tuula and Erkki Ihalmo, my sister Teija Sirko, brother Matti Ihalmo, and their families, for their loving care and endless support. This work was ﬁ nancially supported by the European Commission and numerous private foundations. The personal grants from the Finnish Cultural Foundation, the Federation of European Biochemical Societies (FEBS), the Kyllikki and Uolevi Lehikoinen Foundation, and the Finnish Kidney Foundation are gratefully acknowledged.

Helsinki, July 2007 Pekka Ihalmo

58 7 Acknowledgements 8. APPENDIX

Members of the European Renal cDNA Bank at the time of the study:

C. Cohen, H. Schmid, M. Kretzler, and D. Schlöndorff, Munich, Germany; P. Ronco and J.D. Sraer, Paris, France; M.P. Rastaldi and G. D’Amico, Mi- lan, Italy; F. Mampaso, Madrid, Spain; P. Doran and H.R. Brady, Dublin, Ireland; D. Mönks and C. Wanner, Würzburg, Germany; A.J. Rees and P. Brown, Aberdeen, Scotland; F. Strutz and G. Müller, Göttingen, Germany; P. Mertens and J. Floege, Aachen, Germany; N. Braun and T. Risler, Tü- bingen, Germany; L. Gesualdo and F.P. Schena, Bari, Italy; J. Gerth and G. Wolf, Jena, Germany; R. Oberbauer and D. Kerjaschki, Vienna, Austria; B. Banas and B.K. Krämer, Regensburg, Germany; W. Samtleben, Munich, Germany; H. Peters and H.H. Neumayer, Berlin, Germany; K. Ivens and B. Grabensee, Düsseldorf, Germany; and V. Tesar, Prague, Italy.

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