Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is an autosomal recessive disorder characterized by a nephrotic syndrome (NS) shortly after birth (1,2). For most patients with NPHS1, renal transplantation is the only effective treatment (2). Long-term graft function and survival in these patients is generally good (3). However, the recurrence of proteinuria in the renal graft is a major risk after transplantation. Seven years ago we described the recurrence of severe proteinuria in 7 of 29 nine transplanted grafts (4). Altogether, recurrence has so far been described in 10 patients with NPHS1, leading to a graft loss in 5 cases (4–8). The pathogenesis of this state has remained unknown.
Two years ago, mutations in NPHS1 were identified to be responsible for NPHS1 (9). NPHS1 is a novel 29 exon gene that codes for nephrin, a podocyte cell adhesion protein belonging to the immunoglobulin superfamily. This 1241 amino acid protein was recently localized to the slit diaphragm area of the kidney glomerulus (10–12). Two common mutations, named Fin-major and Fin-minor, were found in over 90% of Finnish patients with NPHS1 (9). Fin-major mutation is a 2-base pair deletion in exon 2, leading to a stop codon, and formation of only 18 N-terminal and 50 novel residues (due to frame shift). Fin-minor is a nonsense mutation in exon 26, leading to a translational stop and a truncated 1109-residue protein, which contains the extracellular part and one third of the intracellular part. Recently, we reported that both mutations lead to absence of nephrin and slit diaphragms in NPHS1 kidneys and to a clinically severe form of congenital nephrosis, CNF (13).
In the present study, we elucidated the role of nephrin in the pathogenesis of recurrent NS in patients with NPHS1. We analyzed the genetic and clinical data and we investigated renal pathology. We also studied the patients’ sera for circulating antibodies against nephrin and glomerular structures. The results show that the recurrence has occurred only in patients with the Fin-major/Fin-major genotype and that circulating anti-nephrin antibodies seem to be associated with this process.
This study included 45 patients with NPHS1 who received 51 kidney transplants at the Hospital for Children and Adolescents, University of Helsinki, between the years 1986 and 2000. The diagnosis of NPHS1 was based on the typical clinical picture of CNF (2) and the genetic analysis of NPHS1 mutations (9). Twenty-nine patients were homozygous for Fin-major mutation, four patients were homozygous for Fin-minor mutation, and eight patients were compound heterozygotes for these two mutations. Three patients had the Fin-major mutation in one and a missense mutation in the other allele, and finally, one patient had three missense mutations (Table 1).
The clinical data were collected from the patient records. The renal transplantation was performed at the mean age of 2.0 (range, 0.9–4.3) years. A mean of 2.1 mismatches was observed in the HLA-A, -B, and -DR loci. No significant differences were observed in the age at transplantation, donor age (mean 28 years), or HLA-AB/DR and cytomegalovirus mismatches between the patients with different genotypes (data not shown). After transplantation all children received standard triple-drug immunosuppression with cyclosporine, azathioprine, and methylprednisolone (4).
A total of 262 serum samples were used for analysis of anti-glomerular and anti-nephrin antibodies (see below). Ninety-eight samples were taken from nine NPHS1 patients with recurrent NS, 67 samples from 29 NPHS1 patients without recurrence, 52 samples from 31 non-NPHS1 children with kidney transplantation, and 47 samples from 47 children with liver or heart transplantation. The serum samples from patients with NPHS1 were collected between a few days and 12 years after kidney transplantation and were stored at −70°C. The samples in the other groups were collected 2–7 years after transplantation.
Renal tissue samples were obtained by percutaneous needle biopsy taken within the first 4 months after the onset of proteinuria. Routine histology and immunofluorescence studies for immunoglobulins and complement components were performed on all samples. Samples taken within 4 days from the onset of NS (episodes 2b, 4, 5a, 8, and 9 in Table 3) were also prepared for electron microscopy, immunoperoxidase staining, and in situ hybridization. In addition, we performed immunofluorescence and immunoelectron microscopy studies on two samples (episodes 2b and 4). As controls, we used routine biopsy samples taken 1.5–5 years after transplantation from NPHS1 patients with a well-functioning graft.
Fetal kidney samples used in the immunofluorescence studies were collected at autopsy from human fetuses at 22 and 23 weeks of gestation, obtained through prostaglandin-induced abortions due to anencephaly and gastroschisis.
This study was approved by the ethical committees of the Hospital for Children and Adolescents, and the Department of Obstetrics and Gynecology, University of Helsinki.
Immunofluorescence for anti-glomerular antibodies.
The presence of anti-glomerular antibodies in the patient sera was evaluated by indirect immunofluorescence using normal fetal kidney section as antigen. Fetal kidney was chosen because nephrin is strongly expressed in developing glomeruli (9,14). Unfixed 5-μm cryosections were air dried and sera were diluted in phophate-buffered saline (PBS) and incubated for 2–3 hr. After washes in PBS, the sections were incubated in cyanine-conjugated anti-human IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2–3 hr, washed, and mounted. The microscopy was performed independently by two observers. The presence of anti-glomerular antibodies in the patient sera was indicated when the glomerular reactivity was clearly detected by both observers (J.P. and H.J.).
ELISA for serum anti-nephrin antibodies.
Anti-nephrin antibodies were measured using microtiter plates coated with the extracellular part of nephrin. Generation of the nephrin antigen is described in detail elsewhere. 8 Briefly, the intracellular and transmembrane part of nephrin cDNA was replaced with a sequence of six histidines and a stop codon. cDNA was cloned into vector pcDNA3 (Invitrogen, Carlsbad, CA) and transfected into human embryonic kidney 293 cells. Recombinant protein, designated NphExHis, was purified from conditioned culture media of a stable cell lineage using anion exchange chromatography and metal affinity chromatography.
Microtiter plates (Nunc Maxisorp, Roskilde, Denmark) were coated overnight at 4°C with 50 μl of NphExHis at the concentration of 10 μg/ml in sodium carbonate buffer, pH 9.4. After blocking the wells with 1% bovine serum albumin for 30 min, the plates were washed with five cycles of 0.05% Tween-20 in PBS. Serum samples were diluted 1/100 in the assay buffer (1% bovine serum albumin and 0.05% Tween-20 in PBS), and 100 μL was applied to each well and incubated for 2 hr. The plates were washed three times, and 100 μL of peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories) was added. The plates were incubated for 30 min and washed, and peroxidase activity was visualized with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid). Absorbance at 410 nm was recorded.
After initial screening experiments, one positive serum sample (patient 1) was chosen to be used in the quantification of antibody titers. This sample was arbitrarily set to contain 500 U/mL. A twofold dilution series from 1/50 to 1/6400 was used in each plate together with 1/100 dilution of one positive (patient 1) and one negative control sample. All standards, controls, and samples were analyzed as duplicates.
A representative standard curve of the assay is shown in Figure 1A. For a positive control sample the intra-assay and interassay variances were 6.4% (n=6) and 6.5% (n=12), respectively. For a negative control sample, the intra-assay and interassay variances were 18% (n=6) and 28% (n=12), respectively. Analysis of 16 control samples collected from healthy volunteers gave a mean of 32.8±14 U/mL. Liver and heart transplant patients gave 33.4±2 U/mL (n=40) and 25.2±26 U/mL (n=16), respectively. All control groups together had a mean of 31.3±28 U/mL (n=16). Threshold for anti-nephrin antibody positivity was set to 105 U/mL, representing the 95th percentile of the controls.
On competitive assay, a serum dilution was chosen to give 0.3–0.5 absorbance units for each sample. Serum was mixed with NphExHis and preincubated for 30 min before applying to NphExHis-coated and blocked wells. Glutathione S-transferase was used as a negative control.
Light microscopy and immunofluorescence studies for immunoglobulins and complement components were performed in a standard fashion. For electron microscopy, the samples were fixed in 2.5% glutaraldehyde and embedded in Epon. Poststaining was performed with 1% uranyl acetate and lead. The sections were examined with a JEOL 1200EX electron microscope (JEOL USA, Inc., Peabody, MA) at 60 kV.
In situ hybridization for nephrin.
In situ hybridization was performed as described earlier (15). The probes for in situ studies were synthesized by subcloning a 287-base pair cDNA fragment corresponding to exon 10 in human NPHS1 into pBluescript (Stratagene, La Jolla, CA), and antisense and sense RNAs were produced using T3 and T7 RNA polymerase, respectively (9).
Immunofluorescence and immunoperoxidase staining protocols have been described earlier (13,15). We used monoclonal and polyclonal antibodies directed against the intracellular and extracellular parts of nephrin (14,15). Immunoelectron microscopic studies were performed using polyclonal antibodies directed against the extracellular (10) and intracellular part of nephrin (15). We compared the expression of nephrin to that of ZO-1 (a component of the cytoplasmic face of the slit diaphragm) (16). Antiserum against ZO-1 came from Zymed Laboratories Inc. (San Francisco, CA).
The episodes of recurrent NS.
During the years from 1986 to 2000, 51 renal transplantations were performed on 45 Finnish NPHS1 patients with a known genotype (Table 1). Patient and graft survival rates at 5 years after transplantation were 100% except in patients homozygous for the Fin-major mutation (graft survival 72%). Fifteen episodes of recurrent NS occurred in 13 grafts (25%) of 9 NPHS1 patients (20%). All nine recipients had the Fin-major/Fin-major genotype (Table 1). Recurrence occurred in 37% of the grafts of patients with this genotype. No significant differences were observed in donor type, number of acute rejection episodes, HLA-DR mismatches, or cytomegalovirus mismatches in these nine children as compared with patients with the Fin-major/Fin-major genotype and no recurrent NS (Table 2). Also, no delayed graft function was observed in the first grafts of any of the 9 patients as compared with 3 of 36 in those without recurrence. However, significantly more HLA-AB mismatches were found in patients with recurrence (P <0.01).
Recurrent NS was diagnosed from 5 days to 48 months after the transplantation (mean 12 months) (Table 3). The mean urinary protein concentration on admission was 24 (range, 3–59) g/L, and the serum creatinine value was normal or moderately elevated (mean 130 μmol/L). Thirteen of the 15 episodes were treated with oral cyclophosphamide (azathioprine stopped) and increased doses of oral methylprednisolone. In addition, plasmapheresis two to three times a week for 3 weeks was used as an adjunct in one episode appearing soon after transplantation (episode 2b in Table 3).
No response was achieved in eight episodes, and six grafts were lost (Table 3). Five grafts were removed because of renal failure, and one graft because of continuous heavy proteinuria. Seven episodes responded to therapy. In four of these, excretion of protein to urine decreased markedly within days from the start of treatment (episodes 5b, 6a, 6b, and 9 in Table 3), while three episodes responded to therapy only after several months (episodes 2b, 3b, 8 in Table 3).
Circulating anti-nephrin and anti-glomerular antibodies.
A total of 44 (45%) of the 98 samples from the patients with recurrent NS had circulating antibodies reacting against fetal kidney glomerulus, as studied by indirect immunofluorescence (Table 4). These were found in eight (89%) of the nine nephrotic patients, which was a significantly higher frequency than observed in other NPHS1 patients (41%) as well as in other control groups (Table 4). The staining pattern varied from linear staining along the glomerular basement membrane (GBM) to a more diffuse staining of the glomeruli (Fig. 2B). Also, the intensity of the immunofluorescence varied significantly. No constant difference in the staining pattern was noticed between the groups.
Specific anti-nephrin antibodies were studied by direct ELISA method using the extracellular part of nephrin as an antigen. High levels of anti-nephrin antibodies were detected in four (patients 1–4 in Table 3) of the nine patients with recurrent NS (maximal levels 378–968 U/mL) (Fig. 1B). The specificity of these antibodies was verified by inhibiting the binding by soluble nephrin in all four cases (Fig. 1C). So far, patients 1 and 3 have lost two grafts due to heavy proteinuria. These two patients showed first high titers of anti-nephrin antibodies at the time of primary graft loss (Fig. 2 for patient 1 in Table 3). Patient 2 lost the first graft due to NS, but the rescue therapy with cyclophosphamide and plasmapheresis saved the secondary graft despite the recurrent NS. Serum taken 2 days after the second transplantation contained high levels of anti-nephrin antibodies (946 U/mL). Patient 4 died of septic infection 6 days after the onset of NS in the first graft. High titer of anti-nephrin antibodies was detected in a serum sample taken at the beginning of proteinuria.
Anti-nephrin antibody levels above the 95th percentile of control samples (114 U/mL) were detected in two NPHS1 patients without recurrent NS and two non-NPHS1 patients with a renal graft (147–189 U/mL). This binding could be inhibited in only one of these patients (Table 4).
Light and electron microscopy of the proteinuric grafts.
At the onset of NS, glomerular morphology in light microscopy was quite normal. Interstitial cellular rejection was evident in two cases (episodes 4 and 5a in Table 3), but no glomerular lesions were seen. The routine immunofluorescence studies did not reveal the presence of immunoglobulin or complement components.
Ultrastructural studies of the graft from four patients with recurrent NS showed extensive effacement of podocyte foot processes (Fig. 3A). From each capillary loop analyzed, only a few quite normal looking foot processes and slit pores were present. Interestingly, the filamentous image of the slit diaphragm was found in only 35% (179/513) of studied filtration slits in the proteinuric grafts (Table 5). This was significantly less (P =0.002) than in control kidneys, where the slit diaphragm was detected in over 68% (167/245) of podocyte slit areas (Table 5). In addition, junctions with ladder-like structures (17) were observed in three proteinuric grafts. The GBM* were normal.
Expression of nephrin in the proteinuric grafts.
Using in situ hybridization, kidney glomeruli were strongly positive for nephrin mRNA, whereas only slight background signal was seen outside the glomeruli (Fig. 4). The expression level of nephrin mRNA was evaluated by counting the grains and correlating this number to the area of glomerular cross-section (Table 5). With this semiquantitative method, we found a significant decrease in the expression level of nephrin mRNA in two proteinuric grafts, as compared with control glomeruli (Table 5 and Fig. 4). In three cases, no significant alterations in the expression levels of nephrin mRNA were found.
Immunofluorescence and immunoperoxidase stainings for nephrin gave a linear pattern along the GBM in the control grafts (Fig. 5). The staining for nephrin had shifted to a discontinuous coarse granular pattern in both proteinuric grafts studied by immunofluorescence. In immunoperoxidase staining, similar granularity (mostly inside the podocytes) was seen in three proteinuric grafts, whereas the staining appeared normal in two cases (Table 5). The distribution of ZO-1 in the glomerular capillary tufts was comparable to that of nephrin in control and proteinuric grafts (Fig. 5).
Immunoelectron microscopy of two nephrotic grafts revealed gold labeling for nephrin mostly at the slit pores (Fig. 3C). Occasionally, nephrin labeling was found on the urinary surface and inside the podocyte foot processes (Fig. 3D). Labeling for ZO-1 was comparable to that of nephrin.
In this work we studied the pathophysiology of proteinuria in kidney grafts of children with congenital nephrosis (NPHS1). This “model” serves as an opportunity to study the role of the slit diaphragm and nephrin in the development of proteinuria. The results suggest that an immune reactivity against this crucial slit diaphragm component can cause massive proteinuria.
We first reported the recurrence of severe proteinuria in kidney grafts of Finnish children with NPHS1 in 1993 (4). At that time, recurrence had occurred in 7 (24%) of 29 grafts, leading to a graft loss in three cases. As shown here, the frequency has remained the same during the last years. Fifteen episodes have appeared in 51 grafts (29%) transplanted to Finnish patients with NPHS1. Although remission has been achieved in half of the episodes with cyclophosphamide and steroids, six grafts have finally been lost.
A novel finding in the present study was that recurrent NS appeared only in patients with the Fin-major/Fin-major genotype. This severe mutation leads to a peptide of only 90 amino acid residues (many of them being novel due to frame shift) and the complete absence of nephrin in the native kidney (9,13). Fetuses with this genotype do not encounter nephrin during normal maturation of their immunological system and do not develop tolerance to nephrin. When nephrin is exposed in the kidney graft after transplantation, immunization against this “novel” antigen may occur. This is analogous to that observed in Alport’s syndrome, which is caused by mutations in the gene(s) coding for the type IV collagen (18). It forms the backbone of the GBM, and immune response against the previously unseen collagen epitopes in the kidney graft is responsible for the well-characterized de novo anti-GBM disease in patients with Alport’s syndrome (18,19).
Recurrence of NS in non-Finnish patients with NPHS1 is rare (5–8). This is most probably explained by the difference in mutations observed in the Finnish and non-Finnish patients with NPHS1 (20,21). Although a wide variety of missense mutations, insertions, and deletions in the NPHS1 gene has been described in non-Finnish children, the severe Fin-major mutation is very rare outside Finland. Thus, it seems possible that, in most children with NPHS1, a natural tolerance to nephrin molecule develops even though the molecule is defective.
What is the pathophysiology of this process? We found that four of the nine children with recurrent NS had high levels of anti-nephrin antibodies demonstrable by the ELISA method. Whether these antibodies were responsible for proteinuria remains to be seen. However, their pathogenic role is supported by two observations: (1) Three of the children had two episodes of NS, and the latter episode occurred soon (5–31 days) after retransplantation, suggesting that the preformed antibodies might have had a pathogenetic role. (2) In rats, injection of monoclonal antibody 5-1-6, directed against the extracellular domain of rat nephrin, causes severe proteinuria within days after the injection (22,23). In this model the antibodies obviously penetrate the GBM and reach the nephrin molecule. In the present work, we did not see immunoglobulin or complement deposits in the proteinuric grafts, which would speak against the role of these antibodies in the development of proteinuria. However, the amount of the antibodies involved in the process may be small and, perhaps, the antibodies are lost in the urine.
We could not demonstrate anti-nephrin antibodies by the ELISA method in five of the patients with recurrent NS. The extracellular part of nephrin molecule was used as an antigen in the assay, and it is possible that the method did not detect all anti-nephrin antibodies in these patients. Interestingly, four of the five patients with negative ELISA results had antibodies against fetal glomerulus in the immunofluorescence. The specificity of these antibodies remains to be solved. It is, of course, possible that these antibodies were totally unrelated to nephrin and other podocyte structures and played no role in the recurrence of proteinuria. Development of antibodies against alloantigens is common in organ transplant recipients (24), which was evident also in our patients. Approximately 25% of the patients in each transplant group (kidney, liver, and heart) had antibodies against endothelial or epithelial structures of fetal glomerulus, as shown by the indirect immunofluorescence.
The role of T cell-mediated response in the development of recurrent NS remains to be solved (especially in patients with no detectable anti-nephrin antibodies). Although no inflammatory reaction was seen in the proteinuric grafts, cellular immunity against nephrin in the patients with NPHS1 seems possible. Regulatory T cells might also control the immune responsiveness to nephrin and this might explain why recurrence of NS occur in only some patients with the Fin-major/Fin-major genotype. This situation is somewhat similar to that seen in focal segmental glomerulosclerosis, in which recurrent NS but no inflammation is often seen in some transplanted grafts (25).
How is the glomerular filtration barrier affected in the recurrent NS? As reported previously (4), the glomeruli looked quite normal at light microscopy in biopsy specimens taken within a few days after the start of proteinuria. At the ultrastructural level, effacement of the podocyte foot processes was evident, as is the case in many nephrotic processes (26). Also, quite normal foot processes were seen. An interesting finding was that the frequency of the slit pores, devoid of filamentous image of the slit diaphragm in electron microscopy, was clearly increased. In addition, junctions with the ladder-like structures (17) were occasionally observed between the podocytes. These suggest that disruption of the slit diaphragms takes place, and possibly leads to foot process fusion. Junctions with the ladder-like structures could be a result of the disruption and represent a fragmented form of the slit diaphragm.
At the molecular level, our results of the nephrin expression were somewhat equivocal. Immunofluorescence staining was altered to a coarse granular pattern in both cryosections gained from the proteinuric grafts. Although immunoperoxidase staining detected nephrin mostly inside the podocytes in three proteinuric grafts, staining was mainly distributed along the GBM in two samples (as in controls). In immunoelectron microscopy, nephrin labeling was mostly found at the slit pore. However, labeling was also occasionally present on the urinary surface and inside the foot processes. Labeling for ZO-1 (the cytoplasmic component of the slit diaphragm) in glomerular capillary tufts was comparable to distribution of nephrin in both control and proteinuric grafts. Further, using in situ hybridization, we found marked reduction in the expression level of nephrin mRNA in two of five proteinuric grafts analyzed. Similar findings have been reported in rats (27). In puromycin aminonucleoside nephropathy and in nephrotic rats induced by the injection of monoclonal antibody 5-1-6, there was a significant decrease of the expression of nephrin mRNA, and the staining pattern for nephrin shifted from a linear to a granular pattern.
In conclusion, the present study describes 15 episodes of the recurrent NS in kidney grafts of Finnish patients with NPHS1. The recurrence has occurred only in patients homozygous for the severe Fin-major mutation. The immune reaction against nephrin seems to be involved in the pathogenesis of this recurrence, leading perhaps to disruption of the normal slit diaphragm structure and protein leakage. Based on these findings we have started a follow-up of anti-nephrin antibodies in the NPHS1 patients with kidney grafts. Children with the Fin-major/Fin-major genotype, and especially those with anti-nephrin antibodies or a history of recurrent NS, have a high risk of graft loss. These patients are evaluated carefully, and increased immunosuppression has generally been used. So far, our experience is limited and the optimal management remains to be solved.
The skillful technical assistance of Maire Jarva, Leena Ollitervo, and Mirka Parkkinen is gratefully acknowledged.
8 Ruotsalainen et al. Manuscript in preparation.
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