Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (CNF or NPHS1) is an autosomal recessive kidney disorder resulting in severe proteinurea and renal dysfunction. Although the disease occurs predominantly in the Finnish population, many cases in other populations have also been reported. The disease gene (NPHS1) encodes nephrin, a podocyte transmembrane protein that is an essential component of the podocyte slit diaphragm, the renal ultrafilter. Since the discovery of the gene, many mutations have been reported in the NPHS1 gene in patients with diverse ethnic background. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, we have investigated the fate of 21 such mutations hitherto identified in NPHS1 patients. Immunostaining of stable transfected cells expressing the nephrin mutants demonstrated that most of the mutants showed only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly showed an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, our data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1.

Molecular basis of glomerular permselectivity.

Recent discoveries in kidney research have given new insights into the molecular make-up of the glomerular filter and mechanisms of permselectivity. The identification of mutations in the genes for glomerular basement membrane type IV collagen has thus demonstrated the central role of the glomerular basement membrane as the structural skeleton of the glomerular capillary. Regional deterioration of this framework not only leads to proteinuria, but also to significant leakage of red blood cells into the urinary space. Tracer studies and the characterization of other glomerular basement membrane components, such as proteoglycans, have also emphasized the role of the glomerular basement membrane in the permselectivity process. However, more recent studies on nephrin, a key component of the slit diaphragm, as well as the podocyte and slit diaphragm-associated intracellular proteins, CD2-associated protein, podocin and alpha-actinin-4, have emphasized the role of the slit diaphragm as a central size-selective filtration barrier. These data have provided a completely new understanding of the mechanisms of proteinuria, both in inherited and acquired diseases. In this review, we present the recent progress made in the characterization of proteins that are important for glomerular permselectivity.

The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death.

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.

Role of nephrin in cell junction formation in human nephrogenesis.

Nephrin is a cell adhesion protein located at the slit diaphragm area of glomerular podocytes. Mutations in nephrin-coding gene (NPHS1) cause congenital nephrotic syndrome (NPHS1). We studied the developmental expression of nephrin, ZO-1 and P-cadherin in normal fetal kidneys and in NPHS1 kidneys. We used in situ hybridization and immunohistochemistry at light and electron microscopic levels. Nephrin and zonula occludens-1 (ZO-1) were first expressed in late S-shaped bodies. During capillary loop stage, nephrin and ZO-1 localized at the basal margin and in the cell-cell adhesion sites between developing podocytes, especially in junctions with ladder-like structures. In mature glomeruli, nephrin and ZO-1 concentrated at the slit diaphragm area. P-cadherin was first detected in ureteric buds, tubules, and vesicle stage glomeruli. Later, P-cadherin was seen at the basal margin of developing podocytes. Fetal NPHS1 kidneys with Fin-major/Fin-major genotype did not express nephrin, whereas the expression of ZO-1 and P-cadherin was comparable to that of control kidneys. Although early junctional complexes proved structurally normal, junctions with ladder-like structures and slit diaphragms were completely missing. The results indicate that nephrin is dispensable for early development of podocyte junctional complexes. However, nephrin appears to be essential for formation of junctions with ladder-like structures and slit diaphragms.

Congenital nephrotic syndrome (NPHS1): features resulting from different mutations in Finnish patients.

BACKGROUND: Congenital nephrotic syndrome (NPHS1) is a rare disease inherited as an autosomally recessive trait. The NPHS1 gene mutated in NPHS1 children has recently been identified. The gene codes for nephrin, a cell-surface protein of podocytes. Two mutations, named Fin-major and Fin-minor, have been found in over 90% of the Finnish patients. In this study, we correlated the NPHS1 gene mutations to the clinical features and renal findings in 46 Finnish NPHS1 children. METHODS: Clinical data were collected from patient files, and kidney histology and electron microscopy samples were re-evaluated. The expression of nephrin was studied using immunohistochemistry, Western blotting, and in situ hybridization. RESULTS: Nephrotic syndrome was detected in most patients within days after birth regardless of the genotype detected. No difference could be found in neonatal, renal, cardiac, or neurological features in patients with different mutations. Nephrin was not expressed in kidneys with Fin-major or Fin-minor mutations, while another slit diaphragm-associated protein, ZO-1, stained normally. In electron microscopy, podocyte fusion and podocyte filtration slits of various sizes were detected. The slit diaphragms, however, were missing. In contrast to this, a nephrotic infant with Fin-major/R743C genotype expressed nephrin in kidney had normal slit diaphragms and responded to therapy with an angiotensin-converting enzyme inhibitor and indomethacin. CONCLUSIONS: The most common NPHS1 gene mutations, Fin-major and Fin-minor, both lead to an absence of nephrin and podocyte slit diaphragms, as well as a clinically severe form of NPHS1, the Finnish type of congenital nephrotic syndrome.

Discovery of the congenital nephrotic syndrome gene discloses the structure of the mysterious molecular sieve of the kidney.

The molecular nature of the glomerular slit diaphragm, the site of renal ultrafiltration, has until recently remained a mystery. However, the identification of the gene affected in congenital nephrotic syndrome has revealed the presence of a novel protein, possibly specific for the slit diaphragm. This protein, which has been termed nephrin, is a transmembrane protein that probably forms the main building block of an isoporous zipper-like slit diaphragm filter structure. Defects in nephrin lead to abnormal or absent slit diaphragm leading to massive proteinuria and renal failure. The discovery of nephrin sheds new light on the glomerular filtration barrier, provides new insight into the pathomechanisms of proteinuria, and even opens up possibilities for the development of novel therapies for this common and severe kidney complication.

Developmental regulation and ultrastructure of glycogen deposits during murine tooth morphogenesis.

The distribution and ultrastructure of glycogen deposits were investigated in the murine tooth germ by histochemical periodic acid-Schiff (PAS) staining and transmission electron microscopy. Lower and upper first molars were examined in mouse embryos at embryonic days 11.5-17 (E11.5-E17) and in 2-day-old postnatal (P2) mice. The oral and dental epithelia and the mesenchymal cells were generally PAS-positive during tooth morphogenesis. PAS-negative cells were present at E13 in the distal tip of the tooth bud epithelium and in the contacting mesenchyme, and this complete lack of PAS reactivity continued in the dental papilla mesenchyme and inner enamel epithelium during the cap and bell stages. The lack of glycogen deposits in the interacting epithelium and mesenchyme during early morphogenesis may be associated with their demonstrated high signaling activities. Mesenchymal cells in the dental follicle consistently possessed small clusters or large pools of glycogen, which disappeared by P2. Since an intense PAS reaction was seen in mesenchymal cells at future bone sites, the glycogen in the dental follicle cells may be associated with their development into hard-tissue-forming cells. Ultrastructural observation of the enamel organ cells from the cap to early bell stages (E14-E15) revealed the occurrence of glycogen pools, which were associated with the Golgi apparatus and with vesicles having amorphous contents. Glycogen particles were also occasionally present inside vesicles or in the extracellular matrix. These may be associated with the exocytosis of glycosaminoglycan components into extracellular spaces and the formation of the stellate reticulum.

Nephrin is specifically located at the slit diaphragm of glomerular podocytes.

We describe here the size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognized a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein revealed reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remained negative. In immunogold-stained thin sections, nephrin label was found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggests an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data.

Ultrastructural localization of beta-actin and amphoterin mRNA in cultured cells: application of tyramide signal amplification and comparison of detection methods.

We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding beta-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold enhanced by silver and gold toning, and fluorescently labeled tyramides. Diaminobenzidine and Nanogold can be used in both light and electron microscopy. The nickel-enhanced diaminobenzidine was the most sensitive visualization method. It is easy to accomplish but a drawback is poor spatial resolution, which restricts its use at high magnifications. Nanogold visualization has considerably better spatial resolution and is therefore recommended for electron microscopy. Fluorescent tyramides, especially TRITC-tyramide, offer a good detection method for fluorescence and confocal microscopy. The methods were used to localize amphoterin and beta-actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, beta-actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, beta-actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen. (J Histochem Cytochem 47:99-112, 1999)

Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium.

The shapes of different organs can be explained largely by two fundamental characteristics of their epithelial rudiments - the pattern of branching and the rate of proliferation. Glial-cell-line-derived neurotrophic factor (GDNF) has recently been implicated in the development of metanephric ureteric epithelium (Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A.-C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B.J., Sariola, H. and Westphal, H. (1996). Nature 382, 73-76; Sánchez, M.P., Silos-Santiago, I., Frisén, J., He, B., Lira, S.A. and Barbacid, M. (1996). Nature 382, 70-73; Vega, Q.C., Worby, C.A., Lechner, M.S., Dixon, J.E. and Dressler, G.R. (1996). Proc. Nat. Acad. Sci. USA 93, 10657-10661). We have analysed the target cells of GDNF and the manner in which it controls ureteric development, and have compared it with other growth factors that have been associated with the regulation of branching morphogenesis, namely hepatocyte growth factor (HGF) and transforming growth factor-beta1 (TGFbeta1). We show that GDNF binds directly to the tips of ureteric bud branches, and that it has the ability to promote primary ureteric buds from various segments of Wolffian duct and to attract ureteric branches towards the source of GDNF. It increases cell adhesion, but is not obviously mitogenic for ureteric cells. The data indicate that GDNF is required primarily for bud initiation. Comparison of GDNF, HGF and TGFbeta1 suggests that the latter act later than GDNF, and may represent a partially redundant set of mesenchyme-derived growth factors that control ureteric development. Thus, GDNF is the first defined inducer in the embryonic metanephric kidney.

Variations in the formation of the human caudal spinal cord.

Collection of 15 human embryos between 4-8 developmental weeks was used to histologically investigate variations in the development of the caudal part of the spinal cord and the neighboring axial organs (notochord and vertebral column). In the 4-week embryo, two types of neurulation were parallelly observed along the anteroposterior body axis: primary in the areas cranial to the neuroporus caudalis and secondary in the more caudal tail regions. In the 5-week embryos, both parts of the neural tube fused, forming only one continuous lumen in the developing spinal cord. In the three examined embryos we found anomalous pattern of spinal cord formation. Caudal parts of these spinal cords displayed division of their central canal into two or three separate lumina, each surrounded by neuroepithelial layer. In the caudal area of the spinal cord, derived by secondary neurulation, formation of separate lumina was neither connected to any anomalous notochord or vertebral column formation, nor the appearance of any major axial disturbances. We suggest that development of the caudal part of the spinal cord differs from its cranial region not only in the type of neurulation, but also in the destiny of its derivatives and possible modes of abnormality formation.

Morphological and immunohistochemical characteristics of axial structures in the transitory human tail.

Ultrastructural relationships between the notochord and neighboring spinal cord were examined during the regression of the human tail. Also, the presence of certain extracellular matrix components in the notochord was immuno-histochemically analysed in the 4th to 12th week old embryos. At the early stages, a close apposition of the notochord to the spinal cord exists in the entire tail region. The external surface of both structures is covered with a continuous basal lamina. The narrow tissue interspace contains interdigitating cell processes and both amorphous and fibrillar extracellular matrix material. With advancing embryonic age, separation of the two structures occurs in craniocaudal direction and the widening interspace becomes occupied by mesenchymal cells. During tail regression and spinal cord retraction, the appearance of large intercellular spaces and cell degeneration takes place in both tissues. With age, the extracellular matrix of the notochord, predominantly the perinotochordal sheath, increases in amount and antigenic complexity. While the intensity of laminin, collagen type IV and type III expression rises continuously during the period examined, the expression of fibronectin begins first at later stages, after the separation of the notochord from the spinal cord. The possible developmental significance of the described phenomena in the regression of the posterior end of the human tail remains to be elucidated.

Contractile proteins and nonerythroid spectrin in oogenesis of Xenopus laevis.

The distribution of contractile proteins, actin and myosin, and an actin-binding protein, spectrin, was studied in oogenesis of Xenopus laevis. These proteins are present in oocytes already at the previtellogenic stages, which are characterized by their diffuse distribution. The localization of proteins changed with the beginning of vitellogenesis. At all vitellogenic stages, including the fully grown oocyte, animal-vegetal differences were noted in localization of actin and myosin: in the animal hemisphere they appear as fibrillar-like structures, while in the vegetal one they are localized around the yolk platelets. By the end of the oocyte's growth, a cortical gradient appeared: predominant localization of actin and myosin in the cortical area. As the oocyte maturation proceeded, the distribution of actin and myosin again became diffuse and nonuniform, so that a cortical gradient appears. At the beginning of vitellogenesis spectrin is distributed as a network all over the ooplasm, while in the fully grown oocyte it is localized mostly in the subcortical area of the animal hemisphere and, as individual inclusions, in other regions of the oocyte. No spectrin is found by the end of maturation. Actin, myosin, and spectrin are also present in the oocyte's nuclei. Changes in the distribution of contractile proteins and spectrin during oocyte maturation are discussed with respect to the development of cortical contractility, as well as to the changes in spatial distribution of yolk platelets and regional sensitivity of the maturing oocyte to cytochalasin B.

[A morphological study of the keratin cytoskeleton of the oocyte from the clawed toad using heterologous monoclonal antibodies]. / Izuchenie morfologii keratinovogo tsitoskeleta ootsita shportsevoi liagushki s pomoshch'iu geterologicheskikh monoklonal'nykh antitel.

Heterologous monoclonal antibodies E2 (against rat cytokeratin 8) and OSC-1 (against cytoskeletal preparations of mouse oocytes) were used to study the presence and distribution of cytokeratins in Xenopus oocytes during their maturation and growth. To improve visualization of cytokeratins, more adequate methods of oocyte fixation and processing were developed. The results on distribution of cytokeratins obtained with the above mentioned antibodies were compared with one another and with published data. It was found that data obtained by different authors, who used different types of antibodies and different methods for fixation and visualization of Xenopus oocyte cytokeratins, are often contradictory and inconsistent. Along with that, common features of cytokeratin distribution are revealed that have been noticed by every author: animal-vegetal asymmetry of cytokeratin distribution and the existence of two keratin domains, cortical and ooplasmic ones. Specific for the cortical domain of the animal hemisphere is the arrangement of filaments in parallels to the surface, whereas filaments of the ooplasmic domain are oriented radially. The vegetal hemisphere is characterized by the presence of a network formed by filaments with different orientation. We also describe certain peculiarities of cytokeratin distribution in previtellogenic oocytes, as well as disintegration of the keratin system by the end of oocyte maturation.

Cell contacts in early human pituitary development.

The ultrastructure of cell and tissue contacts in the developing human pituitary gland were studied in embryos form 3 to 5 weeks of age. At the early stage, the pituitary anlage, Rathke's pouch (RP) of the pharyngeal gut wall, had no direct contact with the diencephalon. At later stages, several close cell-to-cell contact sites were observed between the two tissues, on the upper anterior aspect of RP. However, even at these contact sites a thin layer of extracellular material always remained between the tissues, elsewhere covered by a well-developed basement membrane. RP was, in addition, surrounded by close-coming mesenchymal cells during the time studied. The findings are discussed in reference to different tissue interaction models.

Spinal cord-notochord relationship in normal human embryos and in a human embryo with double spinal cord.

Spinal cord-notochord relationship was analyzed histologically and immunohistochemically in normal human conceptuses between the 4-8 developmental weeks and in a 8-week embryo with double spinal cord. In the early 4-week embryo, the gradual closure of the neural tube along the cranio-caudal body axis was paralleled by the differentiation of the median hindge point cells at the ventral midline of the tube and by its temporary close association with the notochord. During the 5th-8th developmental weeks, the neuroepithelium differentiating into three distinct layers was accompanied by a solid, ventromedially positioned notochord. In the abnormal 8-week embryo, the additional spinal cord was located ventrolaterally from the vertebral column. Both spinal cords appeared bilaterally asymmetric, with their floor and roof plates irregularly formed. An abnormally enhanced pattern of neuroepithelial differentiation characterized their dorsal parts. Furthermore, additional spinal nerves and ganglia and an abnormal bony structure were associated with the spinal cord positioned outside the vertebral column. The underlying vertebral bodies were misshaped and contained scattered supernumerary groups of notochord cells. Our investigation underlines the importance of the notochord-neural tube relationship in the morphogenesis of the spinal cord. We suggest that the double spinal cord was induced by the split notochord.

30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth. Amino acid sequence and localization in the filopodia of the advancing plasma membrane.

A cDNA library constructed from mRNA of rat brain was used to clone the cDNA that encodes the 30-kDa heparin-binding protein (amphoterin) that is developmentally regulated in brain and enhances neurite outgrowth in cerebral neurons. cDNA and peptide sequencing identified a dipolar sequence that has been previously found in studies of high mobility group 1 protein: the 184-amino acid cationic region is followed by a cluster of 30 anionic residues. The mRNA encoding amphoterin is also developmentally regulated; it is strongly reduced in quantity after the rapid perinatal growth phase of the rat brain. Anti-synthetic peptide antibodies raised according to the sequence of amphoterin were shown to bind specifically to the protein isolated from brain, and were used to detect amphoterin in subcellular fractions and in immunostaining of cells. Amphoterin was found in the cytoplasm of the cell soma, in the cell processes, and the substrate-attached material. In cells that are at an active stage of spreading and extending their cytoplasmic processes amphoterin was especially associated with plasma membrane filopodia. The distinct localization to the filopodia of the advancing plasma membrane suggests that endogenous amphoterin has a role in the extension of neurite-type cytoplasmic processes in developing cells. This inference is further supported by the finding that both anti-amphoterin and the anti-synthetic peptide antibodies in the culture media strongly inhibit the outgrowth of cytoplasmic processes.

Basement membrane matrices in mouse embryogenesis, teratocarcinoma differentiation and in neuromuscular maturation.

In this paper we discuss studies on basement membrane and interstitial matrix molecules in early development and teratocarcinoma differentiation. In the early embryo a compartmentalization of newly formed cell types takes place immediately by formation of basement membranes. The stage-specific developmental appearance of extracellular matrix molecules such as type IV collagen, laminin, entactin, fibronectin and proteoglycans seems to reflect a diversified role of extracellular matrices already in the earliest stages of development. In teratocarcinoma cultures the appearance and composition of extracellular matrices during the differentiation of endoderm cells closely resembles that found in the early embryo. Also in this respect the teratocarcinoma system can be used as a model for studies on early development. In later developmental phenomena other matrix molecules can also be of importance. Merosin, a novel tissue-specific basement membrane-associated protein that appears during muscle and nerve maturation is an example of such molecules.

Ultrastructure of oral leukoplakia and lichen planus. II. A correlated scanning and transmission electron microscopic study of epithelial surface cells.

Nine cases of homogenous leukoplakia and 21 cases of lichen planus (11 reticular and 10 erosive) were studied under the scanning and transmission electron microscopes. Characteristic differences between leukoplakia and lichen planus in surface patterns of the epithelial surface cells were noted. Microridges in leukoplakia form parallel and anastomosing rows whereas the picture in lichen planus is irregular with microridges varying in width. The transmission electron microscopic findings confirm and correlate with these findings, the microridges representing invaginations of the plasma membrane. The keratin patterns exhibited by the surface cells were also characteristic regarding type and site of lesion. Scanning electron microscopy can be used as an additional diagnostic aid in differentiating these lesions.

Ultrastructure of oral leukoplakia and lichen planus. I. Basal region and inflammatory cells.

Nine cases of homogenous leukoplakia and 21 cases of lichen planus (11 reticular and 10 erosive) were studied under the electron microscope. The changes found in leukoplakia were limited to occasional breaks in the basal lamina and modest changes in the cytoplasm of the basal cells. The basal lamina in lichen planus was found to exhibit 3 distinctly different pictures reflecting the clinical types of lichen planus. The structural abnormalities in the basal cells increased with increasing severity of the lesions. The inflammatory infiltrate in lichen planus was found to contain mainly small-to-medium-sized lymphocytes but plasma-cells with widely dilated rough endoplasmic reticulum containing a granular substance were frequently seen. Similar changes are seen in other disorders and, therefore, specific diagnostic criteria cannot be established on the basis of the present material.