Human Molecular Genetics, 2001, Vol. 10, No. 1 1-8
© 2001 Oxford University Press
The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death
1Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden and 2Electron Microscopy Unit, Institute of Biotechnology, FIN-00014 University of Helsinki, Finland
Received 8 September 2000; Revised and Accepted 6 November 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter. Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h. The kidneys of null mice exhibited enlarged Bowman’s spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients. In addition to expression in glomerular podocytes, the reporter gene was expressed in the brain and pancreas of (+/–) and (–/–) mice. In the brain, expression was localized to the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb. In the cerebellum, the expression was seen in radial glial cells. Neither anatomical nor morphological abnormalities were observed in the brains of null mice.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Ultrafiltration of plasma is one of the major functions of the kidney. This function is affected in a variety of pathological conditions, such as diabetes, inflammatory diseases and poisoning complications, leading to proteinuria, i.e. excessive leakage of plasma proteins into the urine. This disorder constitutes a major health problem, but its molecular mechanisms are still poorly understood.
Blood filtration occurs in the renal glomeruli, which contain a tuft of capillaries located inside the Bowman’s capsule. The filtration barrier is composed of a fenestrated endothelial cell layer, a 300 nm thick glomerular basement membrane (GBM) and glomerular epithelial cells (podocytes) facing the urinary space of the glomerulus. The podocytes have a complex cellular organization with three distinct segments: the cell body, major primary processes and secondary foot processes. Hanging freely in the urinary space, the podocyte cell body sends out major processes which branch further into secondary foot processes, the basal surfaces of which are embedded in the GBM. Secondary foot processes from two podocytes form a characteristic interdigitating pattern on the outside of the capillary. These foot processes are separated by an 
40 nm wide slit, which contains an ultrathin membrane referred to as the slit diaphragm. The glomerular filtrate first traverses the fenestrated endothelium, then the GBM, a proposed pre-filter of large plasma proteins, and finally passes through the slit diaphragm, the ultimate size-selective macromolecular filter, which hinders proteins of the size of albumin or greater from passing into the urinary space.
The molecular composition of the slit diaphragm has long been a mystery, but recently a novel highly kidney-specific protein termed nephrin was shown to be located in the slit diaphragm (1,2). The nephrin gene is mutated in congenital nephrotic syndrome of the Finnish type (NPHS1), a human disease that leads to proteinuria in utero and to early death (3). Renal transplantation following intensive treatment, including nephrectomy, peritoneal dialysis and intravenous nutrition, is the only curative therapy for NPHS1 (4). Nephrin is a transmembrane protein that contains eight extracellular Ig-like motifs, a fibronectin type III-like repeat and a short intracellular domain. Nephrin molecules from two adjacent foot processes have been proposed to interact with each other in the center of the slit to form an isoporous zipper-like filter structure (2,5). Therefore, the renal failure in NPHS1 is believed to be caused by the absence or abnormal function of nephrin. Other proteins interacting with nephrin in the slit diaphragm have not been identified, but intracellular proteins, such as CD2AP (6) and podocin (7), that may connect nephrin to the cytoskeleton, have been reported. P-cadherin, an adherens junction protein present in many tissues, has been localized to the slit diaphragm region (8) but, since inactivation of its gene does not cause any renal phenotype (9), the protein is not likely to be important to the integrity and function of the slit diaphragm.
In order to examine the biological role of nephrin, as well as to determine its necessity for the development and integrity of the glomerular filtration barrier, we generated nephrin-deficient mice by targeting the nephrin gene in embryonic stem (ES) cells. The targeting construct contained the lacZ gene as a reporter for nephrin expression. The three mouse genotypes were born in expected Mendelian ratios, but the null allele mice died within 24 h after birth, exhibiting massive proteinuria, edema and absence of a slit diaphragm. These results demonstrate the essential role of nephrin for the development and maintenance of a functional kidney filter and confirm that congenital nephrotic syndrome in man is caused by the absence of functional nephrin. The lacZ gene was expressed under the nephrin gene promoter in the brain and pancreas, in addition to its strong expression in the kidney. In the cerebellum, nephrin was localized to radial glial cells by immunohistochemical stainings.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation and characterization of nephrin-deficient mice
The nephrin gene was disrupted in ES cells by homologous recombination using a targeting vector, in which exon 1 was fused in-frame to the Escherichia coli lacZ gene, located in a cassette with the PGK-neo selection gene transcribed in the opposite direction (Fig. 1a). Homologous recombination occurred in 30% of the ES cells that were selected for neo-resistance. Such relatively high recombination efficiencies have been reported for several other genes (10,11). As a possible explanation for the efficient homologous recombination was high activity of the locus in ES cells, we studied whether ß-galactosidase was expressed in the ES cells used for blastocyst injection. However, no expression was observed (data not shown), which precludes high expression of nephrin at the ES cell stage as the cause for the high rate of homologous recombination. Two ES cell clones were used to generate chimeric animals that passed the mutant allele to their offspring (Fig. 1b). Heterozygous mice, phenotypically indistinguishable from wild-type mice, were crossbred to generate nephrin null allele (–/–) mice, which were born alive at an expected frequency of 25%. Although the null allele pups were indistinguishable from (+/–) and (+/+) pups at birth and they started suckling, they soon became edemic and died within 24 h after birth. Western blot analyses demonstrated complete absence of nephrin protein in the (–/–) mice (Fig. 1c). Likewise, no nephrin mRNA was detected in (–/–) kidney mRNA using RT–PCR analysis, demonstrating a total inactivation of the nephrin gene (data not shown).
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Developmental expression of lacZ driven by the nephrin promoter
The lacZ gene inserted behind the nephrin gene promoter was used as a sensitive marker in studying temporal and spatial expression of Nphs1. The expression pattern was identical in heterozygous and homozygous nephrin null allele animals and no difference in the pattern of ß-galactosidase expression between mice originating from the two ES cell clones was observed. The highly restricted expression of lacZ seen in the kidney and brain (12) indicates that the expression of ß-galactosidase is a true reflection of nephrin promoter activity during both embryonic and postnatal developmental stages.
In the developing kidney, lacZ expression was identical to that found previously for nephrin by in situ hybridization and immunostaining (1,12,13). Thus, expression was first visible in the mesonephros (data not shown) and subsequently in the metanephros in late S-shaped bodies at embryonic day 13 (E13) (Fig. 2a). At this stage, the primitive cuboidal or columnar podocytes flatten and apically located tight junctions migrate along the lateral cell surface to the base of podocytes, where they are replaced later at the capillary loop stage by slit diaphragms (14). The expression persisted in the podocytes throughout embryonic development, as well as in adult heterozygous mice, indicating life-long turnover of nephrin in the slit diaphragm (Fig. 2b).
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Nephrin expression has previously been detected in the cerebellar anlage at E11 (12). In the present study, the brain expression was further studied by analyzing ß-galactosidase expression in the brains of (+/–) and (–/–) mice. Expression was first observed at E9.0 in the ventricular zone of the fourth ventricle and caudally along the walls of the neurocoele (Fig. 2c). At E12, strong expression was seen in the ventricular zone of the fourth ventricle (Fig. 2e) and along the entire spinal cord. In the spinal cord, the expression was detected in cells located at the boundary between ependymal and mantle layers, being also visible in the lateral white matter (Fig. 2d). The spinal cord was negative for ß-galactosidase expression after E12. At E13, the brain expression was completely restricted to the ventricular zone of the fourth ventricle in the developing cerebellar anlage, where it was seen throughout the remaining embryonic development. The ß-galactosidase expression pattern observed in the nervous system is in agreement with our previous in situ analysis of nephrin expression (12).
In newborn mice, ß-galactosidase expression was observed in the cerebellum, along the midline of the mesencephalon, and in some glomeruli of the main olfactory bulb. Sagittal sections revealed that the cells expressing ß-galactosidase in the heterozygous cerebellum reside in the internal granular layer, occasional blue staining being visible in the external granular layer, as well as in the white matter of the central parts of the cerebellum (Fig. 3a). Most of the positive cells were aligned along the Purkinje cell layer. In null allele cerebellum, the ß-galactosidase-expressing cells appeared to be more scattered in the white matter than in wild-type cerebellum (Fig. 3b). This might be due to stronger ß-galactosidase expression in the (–/–) mice. Neither marked differences in expression patterns nor gross abnormalities in cerebellar foliation were observed in newborn null allele mice (Fig. 3a and b). Furthermore, cells attached to choroid plexus were positive both in (+/–) and (–/–) brains, the choroid plexus epithelium itself remaining negative throughout embryonic and postnatal development.
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At postnatal day 8 (P8), the ß-galactosidase-expressing cells in the cerebellum were located underneath the internal granular layer within the white matter central to each developing folium (Fig. 2f), a few positive cells being seen in the internal granular and Purkinje cell layers. The expression pattern of the ß-galactosidase gene at P16 was similar, but considerably weaker (data not shown). The cerebellum was virtually negative for ß-galactosidase staining in heterozygous young adult mice, the expression being visible only in some of the glomeruli of the main olfactory bulb and in hippocampus in the dentate gyrus molecular layer (Fig. 2g and h). In 1-year-old heterozygous mouse cerebellum, the ß-galactosidase expression was upregulated and seen in white matter central to each folium (data not shown). No expression was seen in mature Bergmann glial cells in adult cerebella of any age. During development, expression of the ß-galactosidase gene was also observed in the pancreas (Fig. 2i). The positive cells have been identified as insulin-producing ß islet cells (unpublished data).
Sequencing of RT–PCR products generated from isolated cerebellar poly(A) RNA, with primers specific to nephrin, showed no abnormally spliced nephrin variants (data not shown). Evidence for nephrin expression in the human brain was obtained using PCR of a human fetal brain (Quick-clone cDNA-mix; Clontech) using primers specific to human nephrin (data not shown).
Enlarged kidneys and massive proteinuria in nephrin-deficient mice
The kidneys of children with NPHS1 have been shown to be 2–3 times larger than normal (15,16). Therefore, the sizes of kidneys from (–/–), (+/–) and (+/+) animals of several litters were compared. Since the null mice died within 24 h after birth, the analysis could be carried out only on kidneys of newly born mice. The kidneys of newborn wild-type and null allele littermates were of comparable weight (data not shown), but comparison of kidneys from 24-h-old mice revealed an 12% increase in weight in (–/–) mice compared with (+/+) and (+/–) mice (P = 0.0039, t-test; Table 1).
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of urine from newborn nephrin-deficient mice demonstrated extensive non-selective proteinuria compared with heterozygous and wild-type littermates (Fig. 4a). Based on molecular size markers, most of the proteins in the urine of (–/–) mice are of the size of albumin and smaller proteins, a finding consistent with a similar type of non-selective proteinuria in the human NPHS1 disease, in which 90% of the urinary protein is albumin (4). However, some proteins larger than albumin can also be seen in the urine of nephrin-deficient mice (Fig. 4a).
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Histological and electron microscopic findings in kidneys of nephrin-deficient mice
The histological findings in (–/–) mouse kidneys were essentially identical to those observed in human NPHS1 patients (16–18), including an enlarged Bowman’s space, dilated proximal and distal tubules and mesangial cell hypercellularity, as well as microcysts in the cortex and medulla (Fig. 4b–e).
Transmission electron microscopic analysis of glomeruli in (+/+) mice revealed a variable width of podocyte foot processes along the GBM (Fig. 5a) with uniformly wide slits and an apparent diaphragm structure (Fig. 5b). In contrast, the glomeruli of (–/–) mice had partial effacement of podocyte foot processes, which were abnormally low in number, the normal slit diaphragm being completely absent (Fig. 5c). High magnification revealed foot processes in closer apposition, with intervening cellular junctions devoid of slit diaphragms (Fig. 5d). Similar findings have been described in the glomeruli of rats with puromycin aminonucleoside (PAN)-induced nephrosis (19). Histological and ultrastructural analyses revealed no differences in the kidneys of heterozygous and wild-type animals (data not shown). Together, these results, which are similar to those found in the glomeruli of patients with NPHS1 (20,21), confirm that we have generated a mouse model for NPHS1.
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Immunohistochemical analysis of the brain
The nephrin promoter-driven ß-galactosidase expression pattern in the brains of (+/–) and (–/–) mice found in this study, as well as in previous in situ hybridization studies (12), indicate that nephrin has a role in the development of certain regions of the brain. However, histological analysis did not reveal any apparent morphological changes in the cerebellum of (–/–) mice (Fig. 3a and b).
In order to determine which brain cell types produce nephrin, we carried out double immunofluorescence stainings on newborn cerebella using antibodies against nephrin (Fig. 3c) and against vimentin (Fig. 3d) that detect the fibers of radial glial cells (22,23). Merged images of the two immunostains showed complete co-distribution of the two proteins (Fig. 3e). Consequently, it can be concluded that the cerebellar cells expressing nephrin in newborn mice are radial glial cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present work we generated a mouse model for NPHS1 by inactivating the gene for nephrin, a central component of the kidney ultrafilter—the podocyte slit diaphragm. Although the nephrin-deficient mice were born alive and seemingly normal, they immediately developed massive proteinuria and died within 24 h of birth. The null mice were edemic, and electron microscopic analyses revealed effacement of podocyte foot processes in the glomeruli and absence of the slit diaphragm. The massive loss of plasma proteins manifested by non-selective proteinuria is likely to be the cause of death of null mice.
An interesting question is also how nephrin expression is correlated to the development of the slit diaphragm. We have recently shown by immunoelectron microscopy that nephrin is first expressed in ladder-like junctions appearing between developing human podocytes in capillary loop stage glomeruli (24). These ladder-like structures, as well as the slit diaphragm of mature stage glomeruli, were missing in human NPHS1 patients where the nephrin gene is mutated and the protein is not expressed. Essentially the same results were obtained in the nephrin-deficient mice, which verifies this association.
The phenotype of nephrin-deficient mice is essentially the same as that observed in human patients with NPHS1. The human NPHS1 patients develop proteinuria in utero and usually develop nephrotic syndrome, with massive non-selective proteinuria and edema, within the first week of life. The human patients die soon after birth without intensive care, mainly involving dialysis and intravenous nutrition. To date, the only curative treatment for NPHS1 is kidney transplantation, after which the patients usually develop normally, at least until adolescence. As described above, the histopathological and electron microscopic findings in the kidneys of null mice were similar to those found in human NPHS1 patients. As in human patients, electron microscopy showed effacement of foot process structures with occasional normal foot process-like structures in the null mice. These processes were, however, in closer apposition than in wild-type glomeruli, and they had cellular junctions that appear similar to those described in the PAN nephrosis animal model (19). In this model, the junctions display the tight junction protein ZO-1 at the inner surface of the podocyte cell membrane, possibly representing a bona fide tight junction (19). The lack of a slit diaphragm in the Nphs1 null mice probably leads to the formation of abnormal cell junctions similar to those in PAN nephrosis.
The phenotypical similarities of nephrin-deficient mice and human NPHS1 patients demonstrate nephrin’s crucial role in the development and function of the renal filtration barrier. Nephrin has been proposed to form quite uniform 40 nm-wide zipper-like isoporous structures in the slit between two adjacent podocyte foot processes, lack of nephrin leading to complete absence of a normal slit diaphragm (2,5). This hypothesis was confirmed by the present study, since no slit diaphragm could be seen between neighboring podocytes in nephrin-deficient mouse kidneys. To date, no other extracellular proteins essential to the slit diaphragm structure have been identified. P-cadherin, a widely distributed cell adherens junction protein, has been localized to the slit diaphragm (8), but since p-cadherin null mice develop neither a renal nor any other known phenotype (9), this protein cannot be of importance to the development or maintenance of the renal ultrafilter. In contrast, two intracellular proteins that are potentially important for connecting the intracellular portion of nephrin to the cytoskeleton have been described. First, Shih et al. (6) reported that the CD2 adaptor protein (CD2AP), a widely distributed membrane-associated protein, is present in podocyte foot processes, where it probably binds to the intracellular domain of nephrin. The importance of CD2AP to the integrity of the filtration barrier became evident from gene targeting experiments, in which CD2AP-deficient mice developed proteinuria soon after birth and died from nephrotic syndrome at the age of 6–7 weeks (6). Second, Boute et al. (7) recently isolated the gene mutated in late-onset steroid-resistant congenital nephrotic syndrome (NPHS2). The NPHS2 gene encodes a novel plasma membrane protein, termed podocin, which also seems to contribute to the maintenance of the slit diaphragm. However, as both CD2AP-deficient mice and podocin-deficient NPHS2 patients develop nephrotic syndrome much later than nephrin-deficient mice or NPHS1 patients, the function of CD2AP and podocin might be compensated for at some stage by other proteins. Therefore, several proteins are likely to be involved in the intracellular interactions with nephrin.
Initial northern blot analyses showed evidence for nephrin mRNA being exclusively transcribed in the kidney and not in other organs, including the brain (1). However, our previous in situ hybridization studies (12), the present lacZ expression analyses and nephrin immunostaining studies clearly demonstrated that nephrin is also expressed during brain development. Therefore, it was of interest to determine which brain cells express nephrin, to gain insight into its potential role in the development or function of the brain.
The present results showed lacZ reporter gene expression in several cerebral regions, including the cerebellum, glomeruli of the main olfactory bulb and the hippocampal dentate gyrus, as well as transiently in the spinal cord during embryonic development. In the newborn mouse cerebellum, nephrin expression was localized to radial glial cells. These cells are transiently present during short periods of development in most regions of the brain where they provide a scaffold for directional migration of neurons. They also have a role in the formation of diverse glial cell lineages (for a review see ref. 25). During cerebellar development, the granular cells, which are situated externally to the Purkinje cell layer, migrate, with the help of the radial fibers, through the Purkinje cell layer to form an internal granular layer. Furthermore, the radial glia have a role in Purkinje cell migration (for reviews see refs 26,27). In mice, the migration of granular cells in the cerebellum is usually completed at P15, after which the transient radial glial cells have differentiated to Bergmann glia through less mature transitional forms (28). The localization of nephrin to radial glial cells in the cerebellum indicates that the cells expressing ß-galactosidase in the main olfactory bulb glomeruli and in the dentate gyrus molecular layer are also of the glial lineage. Whether nephrin has a significant biological role in these regions remains to be determined. Furthermore, the potential role of nephrin in pancreatic ß islet cells is unknown.
Unfortunately, nephrin null mice die during their first day of life, which hampers studies on the potential effects of nephrin loss on brain development and function. As demonstrated by the apparent normal development of NPHS1 patients after kidney transplantation (29), nephrin may not be very critical for brain development. The mild ataxia occasionally observed in NPHS1 patients might be due to the absence of nephrin in the cerebellum, but it may also be a complication of the congenital nephrotic syndrome itself. Several cell adhesion molecules of the immunoglobulin superfamily are expressed in the nervous system (for reviews see refs 30–32), some of them possibly compensating for the absence of nephrin in the brain.
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MATERIALS AND METHODS |
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Gene targeting in ES cells and generation of mice
Isolation of a genomic bacterial artificial chromosome (BAC) clone containing the entire murine nephrin gene has been described previously (12). The targeting vector was generated by isolating an
6.5 kb SpeI–SalI fragment containing the 5' region, exon 1 and part of exon 2. An artificial HindIII site was created with PCR into exon 1 using the primer 5'-AGT TCT GTG CAC TAA GCT TGC CC-3' and ligated into the HpaI site of the SpeI–SalI fragment. For the downstream short arm, a 1.2 kb HindIII–SpeI fragment containing exons 1–4 was made using PCR with a forward primer from the vector and a primer (5'-GGG CAA GCT TAG TGC ACA GAA CT-3') that creates an artificial HindIII site into exon 1. An EcoRI fragment containing a plasmid described by Putaala et al. (12) was used as a template. The long and short arms were ligated together at their HindIII sites, after which a lacZ/neo cassette was inserted into the same HindIII site. In the cassette, the direction of PGK-neomycin transcription was opposite to that of lacZ. The resulting plasmid contained a targeting construct consisting of exon 1 from the nephrin gene fused in a reading frame with the lacZ gene, as was confirmed by the sequenced clone. The plasmid was cut with NotI located in the 3' end of the short arm in the vector polylinker and 25 µg of the targeting construct was electroporated into RW4 ES cells, followed by selection with 400 µg/ml G418 (Gibco). Of 254 ES clones, 78 were shown to be positive by Southern blotting using a 3' external probe (Fig. 1a). Two of those were used in C57BL/6 blastocyst injections; one chimeric male was derived from each cell line, giving germline transmission when mated with C57BL/6 females. The F1 and F2 progenies in mixed C57BL/6 x 129/SV background were analyzed in this study.
Genotyping
Tail biopsies from F1 animals were genotyped by PCR, the results of which were confirmed in some cases by Southern blot analysis. F2 animals were mostly analyzed by PCR. The presence of the mutant allele was detected using primers neo-1n, 5'-TGT CAT CTC ACC TTG CTC CTG-3', and neo-2n, 5'-TCA AGA AGG CGA TAG AAG GCG-3', resulting in a 490 bp PCR product. The wild-type allele was identified using primers w1 from mouse nephrin exon 1, 5'-GAC AGC AAC AAA CAA GCT GCT GG-3', and m1b from mouse nephrin exon 2, 5'-CAG AAG CAG CCC ATC CTT AGC-3', resulting in a 360 bp product.
Histological and electron microscopic analyses
For analysis of lacZ expression in embryonic tissues, whole-mount 5-bromo-4-chloro-3-indolylbeta-D-galactosidase staining was carried out as described by Gossler and Zachgo (33) and the samples were post-fixed overnight in 10% formaline in phosphate-buffered saline (PBS) pH 7.2 at 4°C, dehydrated and embedded in paraffin. Sections of 8 and 12 µm were made from kidneys and whole embryos, as well as from brains, respectively. The sections were then rehydrated and counterstained with either hematoxylin–eosin or safranine.
For analyses of tissues from developmental stages P8 and older, the animals were first perfused intracardially with PBS pH 7.2 and then with 2.5% glutaraldehyde, 1% paraformaldehyde in PBS pH 7.2. The brains were additionally post-fixed in the same fixation solution for 2 h and cut in sagittal 1 mm sections with a mouse braintree mold. The sections were washed extensively with the wash solution and stained to detect ß-galactosidase activity as reported by Gossler and Zachgo (33).
For electron microscopy, standard procedures were used. Fixed samples were dehydrated in graded ethanol and embedded in epoxy resin. Thin sections were post-stained with uranyl acetate and lead citrate. Sections were examined under a Jeol 1200 EX electron microscope at 60 kV accelerating voltage.
SDS–PAGE and western blotting analyses
In order to study the presence of protein in the urine of newborn mice, the animals were sacrificed by decapitation and the bodies were fixed with needles on a styrofoam pad. The urinary bladder was pressed with forceps to release the urine, which was then collected with a pipette. The urine samples were analyzed for proteins by mixing 3 µl of unconcentrated urine with SDS–sample buffer and subjecting the sample to electrophoresis on 7% SDS–polyacrylamide gel under non-reducing conditions. After electrophoresis, proteins were visualized by silver staining.
To examine for the presence of nephrin in kidney tissues of (+/+), (+/–) and (–/–) mice, proteins were extracted from newborn kidneys into an extraction buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 1 mM PMSF, 1% Triton and supplemented with Complete EDTA-free protease inhibitor cocktail (Boehringer Mannheim). Lysates were treated with 10 mM dithiotreitol, briefly sonicated and incubated on ice for 30 min. The lysates were clarified by centrifugation for 20 min at 13 500 g at +4°C and 7 µl of each lysate was mixed with SDS sample buffer and electrophoresed on a 7% SDS–polyacrylamide gel. The proteins were then transferred to poly(vinylidene difluoride) membrane and immunostained with an affinity-purified rabbit anti-mouse nephrin antibody according to established procedures. Immunostaining was visualized using the Renaissance chemiluminescent reagent (NEN Life Science Products).
Immunohistochemical analysis
For the generation of antiserum against mouse nephrin, a glutathione S-transferase (GST) fusion protein containing intracellular residues 1102–1256 was generated in E.coli BL21 strain using the pGEX–2TK vector (Amersham Pharmacia Biotech) and standard procedures recommended by the manufacturer. Purified fusion protein was used as antigen to raise polyclonal antibodies in rabbits according to standard procedures (Statens Veterinärmedicinska anstalt). Rabbit IgG was affinity-purified using protein A–Sepharose FF (Amersham Pharmacia Biotech) and an affinity matrix prepared by coupling E.coli BL21 lysate containing GST protein to CNBr-activated Sepharose 4FF (Amersham Pharmacia Biotech). Antiserum and purified IgG were characterized by western blotting, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence microscopy.
For immunostaining of P0-stage brain tissues, the brains were excised, embedded in Tissue-Tek OTC (Sakura) and frozen fresh in cold 2-butanol cooled in liquid nitrogen. Cryosections of 12–15 µm thickness were fixed in acetone at +4°C for 10 min, washed several times with PBS pH 7.2 and blocked with 20% donkey normal serum (Jackson Immunoresearch). The primary antibodies were diluted in 5% donkey normal serum 1:1000 and incubated in a humid chamber at 4°C overnight. The sections were washed repeatedly in PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody in 1:100 dilution (Jackson Immunoresearch) for 30 min. The sections were washed several times in PBS and double-stained with goat anti-vimentin (Santa Cruz) 1:450 dilution for 1 h and then with RRX-labeled donkey anti-goat antibody (Jackson Immunoresearch) in 1:100 dilution.
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ACKNOWLEDGEMENTS |
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We are grateful to Johanna Kela for assistance with statistical analyses. We thank Johanna Räisänen, Maria Laisi, Johanna Isopahkala and Ulla Mikkonen for technical help and Miikka Putaala for computer work. This work was supported in part by NIH grant no. DK 54724, as well as grants from the Novo Nordisk Foundation, the Sigrid Juselius Foundation and the Swedish Medical Research Council.
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FOOTNOTES |
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+ Present address: Biocenter Oulu, Department of Medical Biochemistry, 90014 University of Oulu, Finland
§ To whom correspondence should be addressed. Tel: +46 8 728 7720; Fax: +46 8 316 165; Email: karl.tryggvason@mbb.ki.se
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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