Congenital nephrotic syndrome of the Finnish type is the most common single entity of congenital nephroses (1). The gene for this disease has recently been isolated and named NPHS1 (2). It codes for a podocyte-specific transmembrane cell adhesion protein, nephrin, which we and others located at the slit diaphragm area of the human kidney glomerulus (2,3,4,5). The two most common mutations in the NPHS1 gene, Fin-major and Fin-minor, lead to the severe Finnish type of congenital nephrosis characterized by lack of nephrin in the kidney glomerulus, proteinuria in utero, and nephrotic syndrome within weeks after birth (6). Other mutations in the NPHS1 gene have also been described, and some of them cause milder forms of proteinuric states (7,8,9). The findings indicate that nephrin and the slit diaphragm have a crucial role in the maintenance of normal glomerular filtration barrier.
The molecular composition of the slit diaphragm is still largely unrevealed (10). Besides nephrin, ZO-1 is known to be located at the slit diaphragm of human glomerular podocytes (11). The α-isoform of the tight junction protein ZO-1 is a component of the cytoplasmic face of the slit diaphragm where it probably connects the slit diaphragm to the cytoskeleton of the podocyte (11, 12). The presence of CD2-associated protein (CD2AP) in the podocyte foot processes has been reported (13). CD2AP probably anchors nephrin to podocytes. In addition, P-cadherin was recently localized at the slit diaphragm (14).
The podocytes are the target for injury in several renal diseases (15). Proteinuric diseases are associated with the fusion of the podocyte foot processes and detachment of podocytes from the underlying glomerular basement membrane (GBM) (16). Other changes in the podocytes include vacuolization, hypertrophy, cell body attenuation, and pseudocyst formation (17). These maladaptive changes have also been observed in various animal models (18, 19). Preliminary results of the downregulation of nephrin mRNA in puromycin nephrosis of the rat has also been reported (19). Recently, decreased expression of nephrin mRNA was described in three glomeruli from adult minimal change nephrosis (MCN) patients using PCR amplification technique (20).
In the present study, expression of nephrin was studied in renal biopsy samples from pediatric patients with acquired kidney disease using in situ hybridization and immunohistochemistry. The expression pattern was compared to that of ZO-1. With the use of these methods, no major alteration in the expression of nephrin in these diseases was observed.
Materials and Methods
Patients and Tissue Samples
Renal tissue samples were obtained by percutaneous needle biopsy taken on clinical indications from patients who were treated during the years 1989 to 1999 at the Hospital for Children and Adolescents, University of Helsinki. In addition, five samples from adult patients with membranous nephropathy (MN) from the Department of Internal Medicine, University of Helsinki, were studied. The diagnoses were based on typical clinical features, laboratory investigations, and kidney histology. The histologic diagnosis was established by a combination of light microscopy and immunofluorescence staining for immunoglobulins and complement components.
A total of 56 renal samples were included in this study (Table 1). Medical records of the patients were reviewed, especially concerning the medication before the time of biopsy. Data on proteinuria, hematuria, and serum creatinine levels at the time of renal biopsy were collected. Proteinuria was graded into three categories (<0.3 g/d, 0.3 to 3.0 g/d, and >3.0 g/d), and patients with more than five erythrocytes per high-power field were defined as hematuric. Fifteen patients had heavy proteinuria (>3.0 g/d) and 22 patients had moderate proteinuria (0.3 to 3.0 g/L) at the time of biopsy. Thirty-three patients were hematuric. Six patients had elevated serum creatinine levels and 19 patients had been treated with steroids or azathioprine before their renal biopsy.
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Table 1: Samples included in this study and clinical parameters at the time of biopsya
Immunohistochemical studies were performed on all samples. In situ hybridization for nephrin mRNA was performed on the following 17 samples: 6 steroid-resistant MCN, 2 focal segmental glomerulosclerosis (primary FSGS), 4 MN, 3 tubulointerstitial nephritis, 1 IgA nephropathy, and 1 with hematuria resulting from bladder injury. As controls for immunohistochemistry, we used cadaver kidneys that were unsuitable for transplantation because of vascular abnormalities (from the IV Department of Surgery, University of Helsinki).
In Situ Hybridization
Formalin-fixed, paraffin-embedded sections (10 μm) were deparaffinized in xylene, rehydrated in decreasing alcohol series, and treated with proteinase-K (Sigma Chemical Co., St. Louis, MO) before hybridization. Thereafter, the sections were subjected to in situ hybridization as described previously (21), with some modifications. Briefly, the tissue sections were washed in phosphate buffered saline (PBS), acetylated and dehydrated, and then incubated with 1.2 × 10633P-labeled (1000 Ci/mmol; Amersham, Arlington Heights, IL) antisense and sense riboprobes in a total volume of 80 μl at 60°C for 18 h. After washes with SSC, RNA digestion, and dehydration, the sections were dipped in NTB2 nuclear emulsion (Kodak, Rochester, NY) and exposed in the dark at 4°C for 1 or 2 wk. After developing, the sections were counterstained with hematoxylin and eosin. Microscopy was performed with a standard Leica DM RX light microscope.
The probes for in situ studies were synthesized by subcloning a 287-bp cDNA fragment corresponding to exon 10 in human NPHS1 into pBluescript (Stratagene, La Jolla, CA), and antisense and sense RNA were produced using T3 and T7 RNA polymerase, respectively.
Antibodies
For the immunohistochemistry of nephrin, we used rabbit polyclonal antibodies directed against the intracellular part of nephrin (see below) and a pool of three mouse monoclonal antibodies directed against the extracellular part of nephrin (Ruotsalainen et al., manuscript in preparation). Rabbit polyclonal antibody, directed against the cytoplasmic slit diaphragm protein ZO-1, was purchased from Zymed Laboratories Inc. (San Francisco, CA).
The antigen for the polyclonal anti-nephrin antibodies was prepared as follows. The intracellular portion of nephrin was expressed as six-histidine tagged mouse dihydrofolate reductase fusion protein in Escherichia coli using Qiaexpresionist-expression system (Qiagen, Hilden, Germany). A DNA fragment coding for amino acids 1084 to 1241 of human nephrin was amplified by PCR and cloned into vector PUC18 with sureclone ligation kit (Pharmacia, Uppsala, Sweden). The fragment was cleaved from the vector using cleavage sites for BgIII and HindIII synthesized into PCR primers. The cleaved fragment was ligated into BgIII/HindIII-digested vector pQE-40. This expression plasmid was transformed into E. Coli strain XL-1 blue. The fusion protein was expressed at high levels only as insoluble inclusion bodies. Protein purification was initiated by lysing bacterial cells by brief sonication and pelleting the inclusion bodies at 10,000 × g for 30 min. The pellet was washed with 0.2% sodium deoxycholate in Tris-buffered saline and recentrifuged. Insoluble proteins were solubilized, and the fusion protein was purified in 8 M urea using affinity chromatography. The purified intracellular nephrin was used to raise polyclonal antibodies in three NZW rabbits using standard protocols (SVA, Uppsala, Sweden). Antisera titers were measured in enzyme-linked immunosorbent assay using the antigen as capture reagent.
Western Blot Analysis
The isolation of normal kidney glomeruli was performed by sieving as described before (22). The glomerular proteins were extracted using 1% Triton X-100 in PBS. Ten μg of total protein was diluted in Laemmli sample buffer, and the proteins were separated on a gradient gel (4 to 15%) and blotted onto a polyvinyl difluoride membrane. After blocking with 1% bovine serum albumin in Tris-buffered saline, the membrane was stained with protein-A—purified immune and preimmune IgG followed by alkaline phosphatase—conjugated coat anti-rabbit IgG and anti-mouse IgG antibodies (Jackson Immuno Research, West Grove, PA).
Immunohistochemistry
The sections (3 μm) of formalin-fixed, paraffin-embedded renal samples were deparaffinized with xylene and then rehydrated. Endogenous peroxidase activity was quenched with 3% hydrogen peroxidase for 5 min, and to improve antibody penetration, microwave treatment in 10 mM citric acid for 10 min was performed. Blocking was achieved by incubating the samples in a solution of 1.5% goat serum in 0.1% tween-PBS for 30 min. The polyclonal anti-nephrin antibodies were diluted to 0.1% tween-PBS (1:1000) and incubated with the sections for 1 h at room temperature. The monoclonal anti-nephrin antibodies were diluted to PBS (1 μg/ml) and incubated with the tissue sections overnight at 4°C. Amplification of the primary antibody reaction was achieved by incubating the sections with biotinylated anti-rabbit IgG and anti-mouse IgG (Vector Laboratories Inc, Burlingame, CA) for 30 min, respectively. This was followed by a complex of avidin and biotinylated peroxidase (Vector laboratories Inc.) for another 30 min. Each incubation was followed by three 5-min washes in PBS. The binding was visualized using diaminobenzidine substrate (Dako Corporation, Carpinteria, CA), and the tissues were counterstained with Harris's hematoxylin. Microscopic observations were carried out with a standard Leica DM RX light microscope.
Results
In Situ Hybridization for Nephrin mRNA
Kidney glomeruli gave an intense signal for nephrin mRNA, whereas only a weak signal was observed outside the glomeruli (Figure 1A). A weak background signal was also seen with the nephrin sense riboprobe (Figure 1B). While the grains were scattered over the capillary tufts, clustering over glomerular podocytes was often seen with high magnification (Figure 1, C and D).
Figure 1: In situ hybridization for nephrin mRNA. (A) Darkfield view of the kidney biopsy from a patient with normal-appearing glomeruli. Glomeruli show strong positive hybridization with the nephrin antisense riboprobe. Faint background signal is observed. (B) Using the control nephrin sense riboprobe, only background signal is seen in control kidneys. (C, D) High-magnification bright-field hematoxylineosin and respective darkfield views of a normal-appearing glomerulus. Grains for nephrin mRNA are clustered around podocytes (arrows) in glomeruli, whereas no positive hybridization is seen in mesangial areas of capillary tuft. Note that because of shorter exposing time (1 wk) to NTB2 emulsion, signal intensity in this glomerulus is reduced as compared with glomeruli in other figures. (E) Glomerulus from a patient with hematuria resulting from bladder injury. (F) Minimal change nephrosis (MCN) patient with a proteinuria of 6.3 g/d. (G) Focal segmental glomerulosclerosis (FSGS) patient with a proteinuria of 1.0 g/d and (H) Membranous nephropathy (MN) patient with a proteinuria of 4.6 g/d. No major difference in the expression of nephrin mRNA between the samples is seen. Magnifications: ×100 in A and B; ×1250 in C and D; ×400 in E through H.
The expression of nephrin mRNA was studied in the non-proliferative kidney diseases by evaluating the signal intensity visually (grading from 0 to 3) and by counting the grains in separate glomeruli (Table 2). The number of grains varied from 638 to 3050/glomerulus. Because the area of the glomerular section varied (Figure 1, E through H), the number of grains was correlated to this area in the final analysis. No major difference was seen in the visual signal intensity or in the amount of grains for nephrin mRNA in the samples from MCN, FSGS, MN, tubulointerstitial nephritis, and a patient with hematuria resulting from bladder injury (Table 2, Figure 1, E through H).
Table 2: In situ hybridization for nephrin mRNA in 239 glomeruli of 17 renal samplesa
Immunoperoxidase Staining for Nephrin
Both polyclonal antibody against the intracellular part and monoclonal antibodies against the extracellular part and of nephrin gave an immunoreactive band at 180 kD in Western blot analysis of glomerular extracts (Figure 2). In immunoperoxidase staining of a normal kidney, a similar staining pattern was also observed with the two antisera (Figure 3, A and B). Nephrin was found along the glomerular capillary wall. A linear staining along the GBM was obvious (Figure 3C), while also faint staining in the podocytes was seen.
Figure 2: Western blot analysis of normal human glomerular extract using the rabbit polyclonal antibodies directed against the intracellular (lane 1) and the pool of monoclonal antibodies directed against the extracellular part of nephrin (lane 3). A strong band at 180 kD can be seen by both antibodies. Corresponding preimmune IgG (lanes 2 and 4) did not show any immunoreactivity.
Figure 3: Immunoperoxidase staining for nephrin and ZO-1 in biopsy samples. (A through C) Nephrin in normal kidney glomeruli using antibodies against the extracellular (A) and intracellular (B, C) parts of the molecule. Both antibodies give similar staining pattern, a linear reactivity along the capillary wall, and faint staining in the podocytes. (D through F) Glomeruli in MCN kidney stained with antibodies against the extracellular (D) and intracellular (E, F) parts of nephrin. No difference in the staining patterns is seen as compared with normal glomeruli. Slight variation in the staining intensity and in the amount of intracellular staining is observed at higher magnification. (G) Nephrin in FSGS, the sclerotic lesions show no staining. (H, I) Nephrin in MN, normal staining pattern (H) and mesangial expansion with scattered and sparse staining (I). Nephrin in MPGN (J) and Henoch-Scho[Combining Diaeresis]nlein nephritis (K), glomerular crescents and sclerotic lesions are negative for nephrin and mesangial expansion led to scattered and sparse staining. (L) ZO-1 staining in normal kidney glomeruli showing immunoreactivity along the capillary wall. Magnifications: ×400 in A, B, D, E, and G through L; ×1250 in C and F.
The expression of nephrin was normal in glomeruli from patients with MCN (Figure 3, D through F). Nephrin was not absent focally or segmentally in the 11 samples evaluated. The staining intensity varied somewhat along the capillary wall and in the podocytes (Figure 3F), but this was the case also in normal glomeruli. In FSGS, sclerotic areas were negative for nephrin (Figure 3G), whereas glomeruli without these lesions showed no changes in the expression of nephrin. Mild to moderate mesangial expansion was seen in MN glomeruli, and the staining for nephrin was concentrated to the peripheral capillary loops (Figure 3I). Also, normal-looking glomeruli were observed in MN samples, and these showed no changes in the staining (Figure 3H).
In the proliferative forms of glomerulonephritides, glomerular crescents and sclerotic areas were negative for nephrin (Figure 3, J and K). In membranoproliferative glomerulonephritis, the glomerular tufts were enlarged and lobulated and nephrin was mainly found at the peripheral capillary loops (Figure 3J). Mesangial proliferation of various amounts lead to scattered and sparse staining patterns (Figure 3K). In kidneys with acute tubulointerstitial nephritis and symptomless hematuria, no changes in the glomerular staining for nephrin were observed.
Comparison of the Expression of Nephrin and ZO-1
The expression of nephrin and ZO-1 were compared by staining the subsequent sections with the two anti-nephrin and one anti—ZO-1 antisera. In normal glomeruli, ZO-1—specific peroxidase was detected in a similar pattern along glomerular capillary loops as nephrin (Figure 3L). No difference was seen between the expression of nephrin and ZO-1 in glomerular capillary tufts in MCN, FSGS, MN, or other diseases. ZO-1 was also detected as dots or short strings between the epithelial cells in Bowman's capsule and tubuli representing the tight junctions between these cells.
Discussion
The characterization of nephrin has opened new possibilities for studying the pathogenesis of proteinuria (2, 23, 24). Mutations in the nephrin gene lead to heavy proteinuria in congenital nephrosis (NPHS1), suggesting a central role for this molecule in the glomerular filtration barrier. This is the first report in which the expression of nephrin has been systematically studied in acquired kidney diseases.
The basic mechanisms that lead to proteinuria in most renal diseases are not known (25). The decrease of negative charge in the GBM and the detachment of podocyte foot processes from the GBM have been shown in some clinical entities as well as in experimental models (26, 27). The localization of nephrin to the slit diaphragm, however, favors an idea that structural and functional defects in this poorly known structure may have a crucial role in the development of proteinuria. This is supported by the fact that human nephroses, including NPHS1, are associated with ultrastructural changes and fusion of podocyte foot processes (15, 28). In NPHS1, the absence of nephrin leads to disappearance of the slit diaphragm as studied by electron microscopy (6).
In this work, we especially studied the expression of nephrin in MCN, FSGS, and MN. These diseases typically manifest as nephrosis with little hematuria or azotemia. Their pathogenesis is not known. MCN is associated with abnormalities in humoral and cellular immunity, and the patients usually respond to immunosuppressive therapy (29, 30). This is also seen in some cases of FSGS (31). MN is associated with autoimmune diseases, and subepithelial immune deposits in the glomeruli are diagnostic for it (32). While these findings suggest an immunological basis for these diseases, neither the target structures nor molecules in the glomerular filtration barrier has been revealed.
Our results showed no major alterations in the glomerular expression of nephrin in MCN, FSGS, MN or the proliferative glomerulonephritides studied. In MCN, this finding is different from that recently reported by Furness et al. (20). Using a PCR amplification technique, they noticed decreased amount of nephrin mRNA in three glomeruli from three adult patients with MCN as compared with six controls. In our study, the signal for nephrin mRNA using in situ hybridization did not show a major difference as compared with controls. The number of grains in 16 MCN glomeruli was similar to that of 26 control glomeruli. Although in situ hybridization is only a semiquantitative method, the counting of grains still most likely would reveal a major difference between the levels of nephrin mRNA in capillary tufts. Also, immunohistochemistry for nephrin gave a similar staining pattern in 11 samples from MCN patients as in controls. All of these patients were proteinuric at the time of biopsy, although most of them had received steroids or other immunosuppressive drugs.
Sclerotic lesions in FSGS glomeruli were negative for nephrin, and in MN glomeruli with mesangial expansion, the staining was found at the peripheral capillary loops. Otherwise, these samples did not show significant changes in the glomerular expression of nephrin or nephrin mRNA. None of these patients had been given immunosuppressive drugs before biopsy, and all patients were proteinuric at the time of biopsy. Proliferative lesions, as well as glomerular crescents, were observed in other glomerulonephritides, such as Henoch-Scho[Combining Diaeresis]nlein nephritis, IgA nephropathy, lupus nephritis, and postinfectious and membranoproliferative glomerulonephritis. In these states, sclerotic lesions and glomerular crescents were negative for nephrin. Whether these areas contribute to proteinuria remains to be seen. In general, epithelial crescents and sclerosis are not associated with proteinuria, and we believe that the lack of nephrin in these lesions is secondary, representing the distortion of normal structures.
Although our study showed little changes in the glomerular expression of nephrin at light microscopy, ultrastructural changes cannot be excluded. In rats, monoclonal antibody 5-1-6 detects an antigen located at the slit diaphragm (33). This antigen, p51, was recently shown to be an epitope of the extracellular part of nephrin (34). A single injection of 5-1-6 antibody induces nephrosis in rats, which is associated with a relocalization of p51 antigen. In immunoelectron microscopy, p51 is moved to the urinary surface of podocyte foot processes and later into the lysosomal compartment of the epithelial cells. At the same time, a progressive decline in stainable ZO-1 antigen is noticed (17, 18). One can speculate that similar alterations at the ultrastructural level might occur in human nephrotic states. Our preliminary results using immunoelectron microscopy indicate that nephrin and ZO-1 label can be seen at slits between some of the podocyte foot processes in MCN glomeruli (Patrakka et al., unpublished results). However, the interpretation of these results is difficult because the gold particles are evident in only a minority of the slits even in normal kidney glomeruli (3). In contrast to the rat model, the expression of ZO-1 seemed normal with light microscopy. This is in agreement with the findings of Bains et al. (35). In their quantitative immunohistochemical study, the glomerular expression of ZO-1 in proteinuric states (MCN and MN) did not show changes in the staining intensity or distribution.
Nephrin is a cell adhesion protein that most likely has both extra- and intracellular interactions with other molecules (13), and it most probably has a role in signal transduction. It is obvious that pathologic processes that interfere with the function of nephrin and other slit diaphragm components may cause proteinuria even if the proteins are present in the podocyte slits (23). In NPHS1, a single amino acid change in the nephrin molecule can cause profound proteinuria (2), suggesting that minor defects in the nephrin molecule may have major functional consequences.
Thus, it is obvious that immunohistochemistry and in situ hybridization are not optimal methods for studying the role of nephrin in proteinuric renal diseases. Both methods may fail to acknowledge modest alterations in the amount or distribution of nephrin in capillary tufts. Also, no molecular data are obtained. The research material naturally limits the possible methodology. The amount of kidney tissue obtained from a clinical biopsy sample is small, and glomeruli represent only a small fraction of this material. So, quantitative assays of nephrin mRNA or protein in the biopsy material is more difficult than in animal models, in which larger amounts of tissue is available. However, the use of single glomeruli, as described by Furness et al. (20), is problematic because the glomeruli are not evenly affected in acquired renal diseases.
In conclusion, we have documented the expression of the slit diaphragm—specific protein nephrin in several acquired kidney diseases using immunohistochemistry and in situ hybridization. No constant changes in the staining intensity or distribution of the molecule were observed, suggesting that the grave reduction of nephrin in capillary tufts is not the underlying mechanism of proteinuria in these disorders.
This work was supported by The Sigrid Juselius Foundation, The Ulla Hjelt Fund of The Foundation for Pediatric Research, The Swedish Medical Research Council, Novo Nordisk Foundation, NIH grant DK 54724, and The Helsinki University Central Hospital Research Fund. The authors thank Sinikka Heikkila[Combining Diaeresis] and Mirka Parkkinen for excellent technical assistance, and Dr. Kristiina Bogulowsky for many helpful advices. We are also grateful to Dr. Helena Isoniemi for providing us with control kidney material.
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