Altered ultrastructural distribution of nephrin in minimal change nephrotic syndrome

Annika Wernerson1,, Fredrik Dunér2, Erna Pettersson2, Silwa Mengarelli Widholm1, Ulla Berg3, Vesa Ruotsalainen4, Karl Tryggvason5, Kjell Hultenby1 and Magnus Söderberg1

1 Department of Pathology, 2 Department of Renal Medicine and 3 Department of Pediatrics, Karolinska Institutet, Huddinge University Hospital, Sweden, 4 Biocenter Oulu, Department of Biochemistry, University of Oulu, Finland and 5 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Nephrin is a cell-adhesion protein that is defective in congenital nephrotic syndrome of the Finnish type (CNF). Nephrin is synthesized by the podocytes and is localized to the slit membrane between individual foot processes of the podocytes. Although nephrin is apparently pivotal in the development of CNF, the role of nephrin in other causes of nephrotic syndrome is not fully understood.

Methods. Renal biopsy specimens from patients with minimal change nephrotic syndrome (MCNS) were investigated. Nephrin distribution was studied with immunohistochemical and semiquantitative immunoelectron microscopic techniques and the results were related to the degree of foot process effacement found under the electron microscope.

Results. In normal kidney, immunofluorescence revealed a linear staining along the capillary basement membranes, corresponding to the localization of nephrin in the slit membranes. In the biopsies from patients with MCNS, the nephrin pattern had become granular. The degree of granularization corresponded to the degree of foot process effacement. Ultrastructural semiquantification showed the amount of nephrin to be reduced both in areas with and without foot process effacement compared with the control specimens. The concentration of nephrin was lowest in the areas with foot process effacement and there was redistribution from the slits into the cytoplasm.

Conclusions. These findings demonstrate that nephrin expression is altered in MCNS. Whether this reflects a pathogenetic role for nephrin in MCNS or a phenomenon secondary to other causes of foot process effacement remains to be elucidated.

Keywords: glomerulus; nephrin; proteinuria; slit diaphragm; ultrastructure



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The production of primary urine involves large volumes of fluid and yet only small quantities of macromolecules escape from the circulation. The reason for this is the filtration barrier consisting of the endothelial cell layer, the glomerular basement membrane (GBM) and the visceral epithelial cells, the podocytes. The extent to which each of these structures restricts the filtration of proteins still remains to be determined.

Congenital nephrotic syndrome of the Finnish type (CNF) is a disorder characterized by massive proteinuria. Untreated patients usually die before the age of 2 years. The most remarkable finding is the complete effacement of foot processes found under the electron microscope, whereas only minor glomerular abnormalities are seen under the light microscope [1]. Recently, it was found that these patients have mutations in the gene for a previously unknown protein, nephrin [2]. Nephrin is a transmembrane protein of the immunoglobulin (Ig) superfamily; in the kidney it is specifically expressed in glomeruli and ultrastructurally it is localized to the slit membrane of adjacent foot processes of the podocytes [3,4]. Immunofluorescence (IFL) and immunoelectron microscopical studies have demonstrated almost complete loss of nephrin expression in CNF [5].

Foot process effacement is a common finding in several proteinuric disorders. Therefore, it is of interest to determine whether the nephrin pattern is altered in kidneys with causes of nephrotic syndrome other than CNF. Patrakka et al. [6] studied the expression of nephrin in various paediatric kidney diseases using light microscopy with immunohistochemistry and in situ hybridization. They observed no changes in nephrin expression in biopsies from patients with proteinuria compared with controls. In contrast, Furness et al. [7] found reduced amounts of nephrin mRNA in glomeruli isolated from patients with minimal change nephrotic syndrome (MCNS) and Doublier et al. [8], using IFL, reported loss of staining for nephrin in patients with primary nephrotic syndrome. However, none of these studies presented ultrastructural data. The aim of the present investigation was to semiquantitatively analyse the distribution of nephrin under the electron microscope and to correlate the findings with alterations of the foot process structure. Minimal change nephrotic syndrome was chosen because it is a common cause of nephrotic syndrome, and podocyte foot process effacement is the only morphological alteration found in this disease [9].



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patient selection
Patients who fulfilled the criteria for MCNS clinically and histopathologically were chosen for the study [9]. Nephrosis was defined as proteinuria exceeding 3.0 g/24 h in adults and 1.0 g/m2 of body area/24 h in children. Clinical data are shown in Table 1Go. At the time of biopsy, one adult and all children but one were on steroid treatment and their proteinuria had diminished. Most children were biopsied due to poor response to steroid treatment. Both treated and untreated patients were included in the present study in order to elucidate whether there is any correlation between the degree of proteinuria, the extent of foot process effacement and the nephrin pattern. In total, biopsies from 13 patients were investigated by light microscopy and IFL. All of the procedures were approved by the local ethical committee.


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Table 1.  Data of patients included in the study and nephrin pattern observed on immunofluorescence

 

Tissue specimens
Control kidney specimens were obtained from macroscopically normal portions of kidneys resected because of localized neoplasms. Routine renal biopsy specimens from patients fulfilling the criteria for MCNS were used for the special stainings of the study. Biopsy specimens for routine light microscopy had been fixed in buffered 4% paraformaldehyde, whereas those used for electron microscopy were fixed with an additional 2% of glutaraldehyde (GA). Samples for IFL were snap frozen in liquid nitrogen and stored at -70°C until analysis. Tissues used for immunoelectron microscopy were fixed in 1% paraformaldehyde and 0.5% GA, in a 0.1 M phosphate buffer.

Antibodies
Polyclonal rabbit anti-nephrin antibodies against the extracellular and intracellular domains of nephrin were used. Production and characterization of these antibodies has been described in detail elsewhere [3,6]. Characterization of the antibodies has included demonstration of reactivity against the used antigen in ELISA, reactivity against nephrin cDNA-transfected mammalian cell lines measured by IFL, lack of reactivity against vector-transfected mammalian cell lines measured by IFL, reactivity against human glomeruli using IFL, reactivity in western blot analysis against transfected cell line lysate proteins and human glomerular extract, with a lack of reactivity when pre-immune antisera/IgG was used instead. In western blot analysis of glomerular lysates, a stronger upper band and an occasional weaker lower band are obtained, using both antibodies.

Immunofluorescence
Cryostat sections, 5 µm thick, incubated overnight at 4°C with anti-nephrin antibodies (1.2 mg/ml, diluted 1:50) were rinsed three times in PBS and incubated with FITC-conjugated swine anti-rabbit IgG (1:50 dilution) (DAKO, Copenhagen, Denmark) for 1 h at 37°C. Stained sections were imaged with a CCD camera mounted on a Leica DM RXA epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) and processed using Photoshop software (Adobe Systems, San Jose, USA). The staining pattern for nephrin was assessed semiquantitatively and described as linear, mostly linear, mostly granular or granular.

Immunoelectron microscopy
Low-temperature dehydration, followed by embedding in Lowicryl resin at low temperature, is known to preserve the antigenic sites better, thereby improving the immunohistochemical results [10]. Tissues were dehydrated with methanol and embedded in Lowicryl K11M (Chemische Werke Lowi GmbH, Waldkreiburg, Germany). The specimens were cut into ultrathin sections (40 nm) with a diamond knife and mounted on carbon/formvar nickel grids. To minimize non-specific labelling, grids were incubated in 10% bovine serum albumin (BSA) in 0.1 M phosphate buffer at pH 7.4 for 2 h at room temperature followed by incubation overnight with anti-nephrin antibodies, diluted 1:500, at 4°C. Control sections were incubated with normal rat serum diluted 1:500. After rinsing, protein A conjugated with 10 nm colloidal gold (Amersham Pharmacia Biotech, Little Chalfont, UK) diluted 1:100 was added and incubated for 2 h at room temperature. The grids were then rinsed in 0.1 M phosphate buffer with 0.1% BSA and post-fixation was performed in 2% GA in 0.1 M cacodylate buffer with 0.1 M sucrose for 20 min. The specimens were contrasted with 4% uranyl acetate followed by Reynold's lead citrate. Sections were examined in a LEO 906 transmission microscope (LEO Electron Microscopy Ltd, Cambridge, UK) at 80 kV. On the basis of a pilot study made to determine the number of profiles needed for an appropriate sample [11], three glomeruli were examined in each specimen. In the MCNS cases, five evenly distributed locations per glomerulus were randomly selected along the peripheral glomerular capillary membranes. Micrographs were taken from each location, both from areas with foot process effacement and areas with normal foot processes. Controls were examined similarly and micrographs were taken from each of the five randomly selected areas in each of the three glomeruli.

Printed copies at a final magnification of x30 000 were then examined in a blinded fashion by another investigator. On each micrograph, a parallel line was drawn 3 cm (corresponding to 1 µm) from the basement membrane on the epithelial side. The area of the foot processes present within this defined zone was measured by using a Zeiss Videoplan semiautomatic interactive image analyser. The following calculations were made (Table 2Go). (i) Slits/µm GBM. On the same micrographs, the total length of the basement membrane was measured and the number of slits were counted and expressed as slits per micrometre. (ii) Au/µm2. The total number of gold markers in the foot processes were counted and expressed as number of gold particles per square micrometre (Au/µm2). (iii) Au/slit. To quantify the amount of nephrin in and around the slits, a 1-cm square on a transparent film was placed centrally on each slit. Gold markers in the defined square (corresponding to 0.1 µm2) were counted and expressed as Au per slit. (iv) Percentage of Au on slits. To discern whether redistribution of nephrin within the podocytes occurs, the number of gold markers located in the vicinity of the slits was divided by the total number of gold particles in the foot process area described above.


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Table 2.  Immunoelectron microscopic findings

 

Statistical analysis
The statistical analyses presented in Table 2Go were performed using Prism software. Comparison of the groups was performed using one-way ANOVA. Differences between groups were calculated using Tukey's multiple-comparison test. Spearman rank correlation and linear regression analysis were also used.



   Results
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 Subjects and methods
 Results
 Discussion
 References
 
Routine evaluation of specimens
All kidney specimens were re-evaluated. The control tissue was essentially normal in all respects, and the diagnosis of MCNS was confirmed by the lack of major histological changes on light microscopy, lack of immune complex depositions on IFL and presence of foot process effacement without any other morphological alterations on electron microscopy.

Immunofluorescence with anti-nephrin antibodies
Similar results were obtained using antibodies directed against both the extra- and intracellular domains of nephrin. Normal kidneys showed a consistently linear staining for nephrin along the outsides of the GBM (Figure 1AGo). No other renal structures stained positively. In glomeruli from patients with MCNS, the nephrin pattern was variable. In patients with severe proteinuria, especially with recent onset, the linear nephrin pattern was virtually abolished, being replaced by a granular pattern, less clearly localized along the GBM aspect of the epithelial cells (Figure 1BGo). In patients with milder proteinuria and patients undergoing treatment, there was a mix of linear and granular patterns. Ultrastructural findings revealed that biopsies with more extensive foot process effacement showed the most pronounced granular nephrin pattern (Table 1Go). There was a direct correlation between the extent of foot process effacement and nephrin granularity ({rho}=0.755, Spearman rank correlation). Furthermore, there was an inverse correlation between serum albumin concentration and the extent of foot process effacement (r=0.688, P=0.0093). There was also a direct correlation between the extent of foot process effacement and urine albumin excretion, which did not reach statistical significance (r=0.582, P=0.0606), however. Patients with the highest albumin excretion rate were those with a granular nephrin pattern.



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Fig. 1.  Nephrin pattern of glomeruli from a control patient (A) and a patient with MCNS (B). The figures are immunofluorescence micrographs (original magnifications x500), in which fresh frozen human kidney was incubated with polyclonal rabbit anti-nephrin antibodies. The linear staining pattern for nephrin in the normal glomerulus (A) is lost in MCNS (B), which characteristically displays a granular staining pattern. The rabbit anti-human nephrin antibodies were detected with FITC-conjugated swine anti-rabbit IgG.

 

Immunoelectron microscopy
As in IFL, similar results were obtained using antibodies directed against both the extra- and intracellular domains of nephrin. In the glomerulus, gold particles were mainly found in the slit diaphragms and in the cytoplasm of the foot processes in the close vicinity of the slits. There was no labelling in specific compartments, such as vesicles or granules (Figure 2AGo). Very low labelling was found in other glomerular structures and in basement membranes, reflecting a low background level. Control sections incubated with normal rabbit serum also showed negligible amounts of labelling. The results of the semiquantitative evaluations of nephrin expression are given in Table 2Go. As expected, in areas with foot process effacement the number of slits was markedly reduced and the amount of labelled nephrin per square micrometre of total podocyte area was significantly reduced both in areas with and without foot process effacement. In areas without foot process effacement from the patients, the number of slits per micrometre GBM was unchanged compared with controls. Looking at individual slits, the amounts of Au-labelled nephrin (Au/slit) were comparable in the control and MCNS specimens. The percentage of Au on slits was altered in MCNS, with proportionally more nephrin found in the cytoplasm of the podocytes than in the slits (Table 2Go).



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Fig. 2.  (A) Labelling of nephrin in a normal glomerulus. Gold particles (arrowheads) are found predominantly in the slits between adjoining foot processes. (B) Labelling of nephrin in MCNS shows reduced amounts of gold particles in the foot processes. The bar represents 0.5 µm. BM, basement membrane.

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The mechanisms leading to the structural abnormalities in the filtration barrier associated with nephrotic syndrome are still poorly understood. Apparently, a number of different structural components are necessary to maintain the integrity of the filtration barrier [12]. In addition to nephrin, several different membrane and intracellular proteins have recently been found to be associated with the development of proteinuric states. Podocin, a podocyte-specific membrane protein, is mutated in some patients with steroid-resistant familial nephrotic syndrome [13]. Mice lacking CD2-associated protein (CD2AP) develop proteinuria at ~2 weeks of age and die from renal failure 4–5 weeks later [14]. Although CD2AP is associated with nephrin [15], the role it plays in human nephrotic disease is still unknown. Alpha-actinin-4, a component of the cytoskeleton of podocytes, has been found to be mutated in some patients with an autosomal dominant form of focal segmental glomerulosclerosis [16]. Thus, these observations have opened an exciting new field of investigation in this important group of renal disorders.

The lack of, or abnormal function of, nephrin in CNF is apparently crucial for the development of this disease. Its absence leads to massive proteinuria in humans as early as in utero [17], whereas mice with an inactivated nephrin gene exhibit extensive protein leakage and die on their first day of life [18]. Some studies of nephrin expression in experimental animal models of the nephrotic syndrome have been published recently. A lowered synthesis of nephrin was reported, as well as a redistribution of nephrin at the ultrastructural level [19,20]. The extent to which the findings in these experimental systems are applicable to human nephrotic disorders is unclear.

The characteristic morphological finding in CNF and in nephrin knockout mice is pronounced foot process effacement [18]. An analogous morphological finding is the characteristic of MCNS as well. But is nephrin involved in this disease? Our IFL studies show that nephrin loses its linear pattern along the basement membranes and is found instead in a granular fashion. These results are in line with those of Doublier et al. [8], who recently reported similar findings in patients with primary acquired nephrotic syndrome, including MCNS. On the other hand, Patrakka et al. [6] found an unchanged linear staining pattern for nephrin in MCNS. The conflicting results could, at least in part, be explained by differences in methodology; Patrakka et al. used immunohistochemistry, whereas we and Doublier et al. used IFL. In addition, neither Patrakka et al. nor Doublier et al. did any ultrastructural investigation and, consequently, there is no information about the degree of foot process effacement in the cases they studied. One of the major problems when studying patients with MCNS is that the degree of foot process effacement varies considerably, not only between patients but also between glomeruli and even between segments of individual glomeruli. These segmental differences are further augmented by steroid treatment, a regimen to which most patients have been subjected at the time of biopsy. Therefore, when studying the role of nephrin in the pathogenesis of proteinuria in MCNS it is important to correlate any findings of altered nephrin expression with the actual ultrastructural state of the filtration barrier, i.e. the extent of foot process effacement. In contrast to previous studies, we have correlated our IFL results with the ultrastructural findings and have found that the degree of granularity on IFL correlated well with the degree of foot process effacement. We believe that the granular staining pattern reflects the uneven distribution of foot process effacement. The IFL technique visualizes a complex of fluorescence markers as a spot in the microscope. In control tissue, where there is a short distance between the slits, these spots will not be separated, since the distance between individual antibody complexes is too short. This will result in a linear staining pattern. However, in MCNS, segments with foot process effacement will appear as dark areas, while segments without will show labelling. This is visualized as a ‘granular’ staining pattern.

The functional implications of the staining pattern is emphasized by the finding that the degree of granularity correlates with the amount of proteinuria. The significant inverse correlation between serum albumin concentration and the extent of foot process effacement is in agreement with a previous study in MCNS children [21].

This is the first report where nephrin expression is studied in a group of MCNS patients, using semiquantitative immunoelectron microscopy. A preliminary report has been published on the expression of nephrin at the ultrastructural level in acquired human glomerular disorders [22]. When those disorders were studied as a group, nephrin expression was generally reduced. Only one patient with MCNS was included, however, and the authors suggested that further studies should be made on each different diagnosis group, since the pathogenesis leading to protein leakage is likely to differ.

The strength of the present study is the use of semiquantitative immunoelectron microscopy, where bound antibodies were detected by gold-conjugated protein A, which only bind one IgG molecule (primary antibody) [23]. Under these conditions, the relation between bound antibodies and gold particles is almost 1:1, which is a prerequisite for reliable semiquantification. Areas with and without foot process effacement have been analysed in the same glomerulus and compared regarding the expression of nephrin. In areas with foot process effacement, the amounts of nephrin are reduced. We also compared areas that appeared normal, i.e. without foot process effacement, to corresponding areas in biopsies from control patients. Interestingly, the nephrin expression per total podocyte area is reduced in MCNS patients, also in areas without foot process effacement. This novel finding suggests that the reduction of nephrin is not a phenomenon that is merely secondary to the effacement of foot processes.

These results may indicate a lowered synthesis of nephrin in podocytes displaying foot process effacement. This is supported by Furness et al. [7], who found decreased expression of glomerular nephrin mRNA in patients with MCNS, but contrasts with the findings of Patrakka et al. [6], who reported unchanged expression of nephrin in MCNS using in situ hybridization. A pivotal role for nephrin in the development of different kinds of acquired nephrotic syndrome has been suggested by Doublier et al. [8]. Their in vitro studies show that nephrin redistribution, with dislocation from the plasma membrane, is a common response to several stimuli known to be involved in the pathogenesis of proteinuric states.

We also found that the amount of nephrin is unchanged in the vicinity of the slits that are still present. Possibly, a certain concentration of nephrin is necessary for the formation of slit membranes. Furthermore, we found a redistribution of nephrin in areas with foot process effacement; the percentage of nephrin present in the vicinity of slits was lowered when compared with that in control specimens. In areas without foot process effacement in the MCNS patients, this relation is unchanged. A possible explanation for our findings could be that when the amount of nephrin is locally reduced below a certain pivotal level, the slit membranes dissolve and the nephrin molecules become dispersed in the plasma membrane of the podocytes and may be recirculated intracellularly. Alternatively, nephrin is lost in the urine and the observed granular staining on IFL represents newly synthesized nephrin. If conditions, including the amounts of nephrin, are returned to normal, it is possible for slits to reform rapidly. This notion is supported by the rapid recovery of foot processes demonstrated in experimental conditions when the cause of foot process effacement is eliminated [24]. It is well known that MCNS is usually a reversible condition that recovers spontaneously or responds positively to steroid treatment. Ultrastructural examinations reveal that the recovery also involves the podocytes. Biopsies obtained during clinical remission show complete reinstatement of the foot processes [25].

In summary, our semiquantitative ultrastructural studies show a reduced concentration of nephrin in foot processes of glomerular podocytes in MCNS, especially in segments with foot process effacement. Further studies are needed to elucidate whether this phenomenon is of importance for the pathogenesis of MCNS.



   Acknowledgments
 
We thank Mrs Anneli Hansson and Ms Maria Jepsen for skilful technical assistance. This study was supported in part by grants from Karolinska Institutet, the Swedish Medical Research Council, the Novo Nordisk Foundation and by NIH Grant DK-54724. Parts of the results have been presented in abstract form at Nordiska njurdagar, Odense, May 2001 and at ERA-EDTA, Vienna, June 2001. A young investigators award was granted to F.D. in Vienna.



   Notes
 
Correspondence and offprint requests to: Annika Wernerson, Department of Pathology, F46, Huddinge University Hospital, S-141 86 Stockholm, Sweden. Email: Annika.Wernerson{at}pathlab.hs.sll.se Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 7. 2.02
Revision received 20. 8.02.