Expression of nephrin in acquired human glomerular disease

Wooseong Huh1, Dae Joong Kim1,, Mi-Kyung Kim2, Yoon Goo Kim1, Ha-Young Oh1, Vesa Ruotsalainen3 and Karl Tryggvason4

1 Division of Nephrology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 2 Department of Diagnostic Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 3 Department of Biochemistry, University of Oulu, Oulu, Finland and 4 Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden



   Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Nephrin is a recently identified protein, which is synthesized in the podocytes and localized in the slit diaphragm area. Nephrin is a cell adhesion molecule of the immunoglobulin superfamily, and presumably is a part of the zipper-like structure of the slit membrane. As the mutation of the gene coding nephrin induces congenital nephrotic syndrome of Finnish type, which is a prototype of nephrotic syndrome, it has been suggested that nephrin also plays a role in acquired proteinuric kidney disease.

Methods. To address the above issue, the expression of nephrin in acquired human glomerular disease was studied by immunoelectron microscopy employing a polyclonal antibody against nephrin. Four normal human kidneys from nephrectomy specimens and eight kidney biopsy specimens from glomerular disease patients (one minimal change disease, one membranous glomerulonephritis (GN), one membranoproliferative GN, four IgA nephropathy, and one lupus nephritis) were studied. Proteinuria of the patients ranged from 448 to 11725 mg/day. Effacement of the foot processes was observed in all patients.

Results. The study demonstrated that the number and distribution of gold particles in the glomerular region, where the podocyte foot process was well preserved, were similar to that found in normal kidneys; however, gold particles were almost always absent in regions where the foot processes were effaced. The number of gold particles per foot process interspace was not different between normal controls and GN patients; however, the number of gold particles per defined length (1000 nm) of the glomerular basement membrane underlying the foot processes was significantly reduced in GN patients.

Conclusion. Using immunoelectron microscopy, we observed that the expression of nephrin in GN was lower in regions where the foot processes were effaced, and comparable with that of normal controls where the foot process interspaces were preserved. The significance of our observation in the context of proteinuria in acquired GN needs further clarification.

Keywords: nephrin; glomerulonephritis; podocyte; proteinuria; effacement



   Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Nephrin is a recently identified protein and is a putative cell adhesion molecule of the immunoglobulin superfamily. Mutation of the gene coding nephrin induces congenital nephrotic syndrome of Finnish type (NPHS1), which is characterized by heavy proteinuria that manifests as early as the fetal stage and the loss of the foot process of the glomerular epithelial cells. Nephrin is specifically expressed in podocytes. By immunoelectron microscopy employing a polyclonal antibody against nephrin, the epitope was shown to be located exclusively in the slit diaphragm. These findings, along with the presumed structure of the molecule, suggest that nephrin may assemble into the zipper-like isoporous filter of the slit membrane [1].

The barrier for the filtration of plasma during the formation of primary urine in the glomerulus consists of three layers; a fenestrated endothelium, the glomerular basement membrane, and the outermost epithelial podocyte foot processes with their interdigitating slit diaphragm. The ultimate barrier for proteins of the size of albumin seems to be the slit diaphragm [1].

Proteinuria is the hallmark of most, if not all, varieties of glomerular disease, such as glomerulonephritis (GN), diabetic nephropathy, and hypertensive nephrosclerosis, moreover, in these conditions it may also be considered as a prognostic indicator of the diseases [2]. Foot process effacement is a typical pathologic feature of a variety of proteinuric glomerular diseases in humans [3] and in animal models of glomerular injury [4]. This process is sometimes rapid and reversible, as demonstrated by steroid-responsive minimal change disease and puromycin-induced nephrosis [4]. Diffuse foot process effacement was also observed in NPHS1 and was the only pathologic change associated with the disease [5].

In view of the above, a role for nephrin has been suggested in a variety of acquired proteinuric kidney diseases [6]. Nephrin may be disrupted to induce effacement of foot processes and proteinuria as in NPHS1. Alternatively, the expression or localization of nephrin may be altered because of cytoskeletal rearrangements due to other factors.

In regard to the possibility of nephrin playing a role in acquired human glomerular disease, several studies have reported the expression of nephrin in the disease. However, all of the reported studies employed immunohistochemistry or mRNA quantification, which may not accurately reflect changes in nephrin expression or localization in the disease. To address this issue, we examined the expression and localization of nephrin in acquired human glomerular disease and compared this with that of normal human kidneys using immunoelectron microscopy.



   Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Kidney biopsy specimens from eight patients diagnosed with minimal change lesion (n=1), membranous GN (n=1), IgA nephropathy (n=4), membranoproliferative GN (n=1), or mixed diffuse proliferative and membranous lupus nephritis (n=1) were studied. For normal controls, normal portions of nephrectomy specimens (n=4) from patients with renal cell carcinoma were used. Clinical data and the pathological findings of the eight patients are summarized in Table 1Go. Proteinuria ranged from 448 to 11725 mg/day. The patient with minimal change disease went into remission with corticosteroid treatment, and the proteinuria of the patient with membranoproliferative GN decreased after immunosuppressive treatment, which included steroid and cyclosporin. The lupus patient progressed to chronic renal failure even with cyclophosphamide pulse therapy. The other patients did not receive immunosuppressive treatment. Ultrastructurally, foot process effacement of variable degree was noted in all patients.


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Table 1.  Clinical characteristics and pathologic diagnosis of the patients

 

Antibodies
A polyclonal antibody against the intracellular portion of the nephrin was prepared by immunizing the rabbit with the C-terminal fragments of nephrin produced in Escherichia coli by using the QiaExpressionist kit from Qiagen. The cDNA-encoding amino acids 1084–1241 for intracellular antigen were amplified from human fetal kidney 5'STRETCH cDNA library (Clontech) by PCR and cloned into vector pQE-30. Production and initial purification of the six-histidine-tagged protein on Ni–NTA column was carried out according to the manufacturer's instructions and used as an antigen to raise polyclonal antibodies in rabbits by using the standard procedures (SLA, Uppsala, Sweden). The antisera were characterized by western blotting, ELISA, and immunofluorescence microscopy. For immunolocalization studies, rabbit IgG was affinity purified with protein A–Sepharose FF (Amersham Pharmacia Biotech).

Western blotting
Glomerular proteins were extracted into sonication buffer (0.05 M sodium phosphate/0.15 M NaCl/1% Triton X-100/0.1 mM phenylmethylsulfonyl fluoride/10 mM ethylenediaminetetraacetic acid, pH 7.5). Samples were treated with or without 10 mM dithiotreitol in sonication buffer. After brief sonication, the samples were incubated on ice for 30 min and centrifuged to clear the supernatant. For SDS–PAGE, the lysates were diluted in Laemmli sample buffer, with or without 5% 2-mercaptoethanol, and run on 6% gel. COS-7 cells transiently expressing nephrin were treated similarly, except for the reduction, which was done only before the electrophoresis with mercaptoethanol.

SDS–PAGE and western blot analyses were performed according to the standard methods. Proteins were transferred onto poly(vinylidene difluoride) membrane and blocked with 1% BSA in Tris-buffered saline. After anti-nephrin or pre-immune IgG-incubation (0.1 µg/ml), primary antibodies were detected with alkaline phosphatase-conjugated anti-rabbit IgG (Dako) and chemiluminescent reagent (ECLplus, Amersham Pharmacia Biotech).

Indirect immunofluorescent microscopy
Cryosections 5–10 µm thick were placed on Silan-coated slides and dried at room temperature. Sections were fixed in a mixture of cold acetone and ethanol in a ratio of 4:1 for 10 min at room temperature and washed in phosphate-buffered saline (PBS). To block non-specific binding, the samples were incubated in 10% horse serum in PBS for 30 min at room temperature. Rabbit primary antisera were used at dilutions of 1:200 in 1% horse serum in PBS. Incubations were carried out for 1 hour at 37°C. After washing in PBS, the primary antibody was detected by using FITC-labeled anti-rabbit IgG antibody (Dako) at dilution of 1:200 for 60 min at 37°C. After washing in PBS, the sections were analysed with fluorescence microscopy.

Immunoelectron microscopy
Kidney tissues from eight patients with glomerular disease and four patients with renal cell carcinoma were processed for post-embedding immunoelectron microscopy. Cubicles, measuring 0.5x0.5x0.5 mm, were cut with a razor blade from the renal cortex in PBS. The samples were fixed in 3.5% paraformaldehyde (Sigma) combined with 0.01% glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA) in 0.1 M phosphate buffer, pH 7.4 at 4°C for 2 h. Fixed samples were rinsed in 0.1 M phosphate buffer pH 7.4 for 12 h, dehydrated from 10 to 100% in graded ethanol, infiltrated by 50% LR White (London Resin, Basingstoke, UK) for 2 h in 100% ethanol followed by LR White resin for 12 h. Gelatin capsules (Agar, Essex, UK) were filled to the brim with fresh LR White resin and closed. Gelatin capsules underwent UV polymerization at room temperature for 7 days or incubated at 45°C for 3 or 4 days. After the sections were cut on Leichert ultracut S (Wien, Austria), they were brought to the carbon-coated formval nickel grid as soon as it was possible.

To block non-specific binding, grids were incubated with protein blocking agent (Immunotech, Glostrup, Denmark) in PGB (0.1 M glycine and 1% bovine serum albumin in PBS) at room temperature for 10 min twice. Grids were incubated for 1 h at 37°C in the first antibody, which had been diluted in PGB (1:100 for rabbit anti-nephrin antibody). After rinsing with PGB for 5 min six times, grids were labelled with immunogold conjugate EM goat anti-rabbit IgG (British BioCell International, Cardiff, UK) in PGB at 37°C for 30 min. After rinsing with PGB for 5 min three times and rinsing with 0.1 M phosphate buffer pH 7.4, for 5 min three times, the grids were fixed with 0.1% glutaraldehyde in 0.1 M phosphate buffer for 10 min. After rinsing with 0.1 M phophate buffer for 1 min three times and rinsing with distilled water for 1 min six times, the grids were dried at room temperature. Grids were post-stained with 4% uranyl acetate in 60% ethanol at room temperature for 20 min. After rinsing in distilled water for 1 min six times, the grids were stained with lead citrate for 3 min. The sections were examined under a Hitachi 7100 electron microscope at 75 kV accelerating voltage.

Morphometric evaluation
Gold particles from the anti-nephrin antibody were counted from eight medium-power immunoelectron micrographs from each patient and normal control, which were systemically obtained and printed at a final magnification of x15000. The curved length of the peripheral capillary basement membrane (BML) was measured after the micrographs were digitized using a flat scanner and transferred to the computer-based morphometric system. The number of slits overlying the capillary basement membrane was counted and the average foot process width (Wp) was calculated with the following formula [7]:


(001)
where the {sum}slits is the number of slits counted.

Statistical analysis
All results are expressed as mean±SD. The difference between the mean of each group was assessed by the Mann–Whitney test and the correlation between the variables were expressed with Spearmann's r. The proportions of gold particles located other than in the slit diaphragm area were compared using the {chi}2 test.



   Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
A polyclonal antibody against the intracellular portion of nephrin reacted with a 180-kDa protein present in Triton X-100 extracts of human glomeruli. The antibody was also reactive against COS-7 cells transfected with full-length nephrin cDNA. Full-length recombinant nephrin produced in COS-7 cells has a smaller size of 150 kDa. Mock-transfected cells did not yield immunoreactivity (Figure 1Go). Immunofluorescent staining of a normal kidney showed finely dotted linear reactivity, giving a preferentially epithelial-like staining pattern as has been reported previously (Figure 2Go) [8].



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Fig. 1.  Western blot analysis using rabbit polyclonal antibody against the intracellular domain of nephrin. Lysate of normal human glomeruli (lane 1), NPHS1 glomeruli of FIN major-homozygote (lane 2), transfected COS-7 cells (lane 3) and cells transfected with vehicle (lane 4) were separated on 6% SDS–PAGE and transferred onto PVDF-membrane to be analysed with antisera. Alkaline phosphatase-conjugated anti-rabbit antibody and BCIP/NBT was used to visualize rabbit IgG.

 


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Fig. 2.  Immunofluorescent staining with antibody against intracellular portion of nephrin showed pseudolinear granular pattern in the glomerulus of normal kidney. Magnification x400.

 
In normal kidneys, gold particles, corresponding to nephrin, were usually observed within the podocyte cytoplasm, near the cell membrane that faces podocyte foot process interspaces (Figure 3AGo) as shown in previous reports [8]. The number and distribution of the gold particles in the glomerulus of patients with glomerular diseases was dependent upon the preservation of podocyte foot processes. Areas with intact foot processes showed similar numbers and distributions of gold particles as observed in the normal kidney (Figure 3BGo and D), whereas areas with effaced podocyte foot processes almost always showed no gold particles (Figure 3CGo and E). Some gold particles were also observed on the surface of podocytes and on the base of the foot processes, both in normal controls and in the patients with glomerular disease. However, the proportions of such particles were similar in normal individuals and GN patients (16.0 vs 17.3%, P>0.5).



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Fig. 3.  Gold particles for antibody against intracellular portion of nephrin were easily detected in normal kidney, especially around foot process interspaces (A). Whereas the particles were detected in the glomerulus of the patients where the foot processes were relatively preserved (B and D), it was invariably absent in the area where the foot processes were effaced (C and E). US, urinary space; GBM, glomerular basement membrane. Immunoelectron microscopy, magnification x20000 (A and C), x15000 (B, D, and E). Case 3 shown in (B) and (C) and case 8 in (D) and (E).

 
The podocyte foot processes in patients were effaced, to a variable extent, in almost all the patients and, accordingly, the average foot process width in patients was far greater than that of normal kidneys (955.1±259.8 vs 460.8±64.8 nm, Figure 4AGo). However, the average number of gold particles per foot process interspace was not significantly different between patients and normal controls (1.04±0.29 vs 1.24±0.23, Figure 4BGo), and the average number of gold particles per unit length of capillary basement membrane underlying the foot process (1000 nm) was far lower in patients than in controls (1.15±0.42 vs 2.62±0.40, P<0.01, Figure 4CGo). The correlation between the number of foot process interspaces and the number of the gold particles in each patient is shown in Figure 4DGo.



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Fig. 4.  Comparison of the foot process width and the number of gold particles between normal and GN patients. As the foot process effacement of variable degree was noted in all the GN patients, average width of the foot process of the GN patients was far greater than that of the normal control (A). Whereas the number of gold particles per foot process interspaces was not different between normal control and GN patients (B), the number of gold particles per defined length (1000 nm) of GBM underlying the foot process was significantly lesser in the GN patients (C). The correlation between the number of gold particles and foot process interspaces in GN patients is shown in bottom right panel (Spearman's r=0.61, P=0.1, D). Patients with IgA nephropathy ({blacksquare}), minimal change disease ({blacklozenge}), lupus nephritis ({blacktriangledown}), MPGN (•), membranous GN ({blacktriangleup}), and normal control ({square}).

 



   Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
A single gene mutation of nephrin, such as that found in congenital nephrotic syndrome of Finnish type, causes effacement of the podocyte foot processes and proteinuria [5]. Foot process effacement and proteinuria of variable degrees are also observed in a wide variety of acquired human glomerular diseases and in several animal models of GN. Immunoelecton microscopy with anti-nephrin antibodies in human glomeruli demonstrated that the epitope is localized exclusively in the slit membrane region, which is a key component of the filtration barrier of the glomerulus, presumably forming an interdigitating zipper-like isoporous filter structure [1] similar to that presented by Rodewald and Karnovsky [9].

In view of the above findings, nephrin is suggested to play a role not only in congenital kidney disease but also in the pathogenesis of proteinuria in acquired human glomerular disease. Several reports have investigated the expression of nephrin in acquired human glomerular disease, which were not always consistent. Furness et al. [6] compared the expression of nephrin in samples of six normal kidneys and four specimens of human nephrotic syndrome using the reverse transcription polymerase chain reaction (RT–PCR) and found that PCR amplification of nephrin cDNA corrected for GAPDH decreased in nephrotic cases. Decreased expression of nephrin was also reported in animal models of nephrotic syndrome [10]. In contrast, Patrakka et al. [11] did not observe any major alterations in the expression of nephrin in proteinuric kidney disease, using immunohistochemistry and in situ hybridization. Our results may reconcile the above difference, by demonstrating that the expression of nephrin is comparable with that of normal where the foot process interspaces are preserved, and that the expression of nephrin decreases in areas where the foot processes are effaced. As foot process effacement is not homogeneous in GN, it is difficult to observe differences in nephrin expression between normal and glomerulonephritic kidneys before the foot processes are severely effaced. Furthermore, we would argue that PCR and immunohistochemistry, as used in the reports referred to, are not the methods of choice for detecting such a difference.

With this in mind, it is tempting to say that the expression of nephrin is altered in acquired glomerular disease and consequently, that the slit membrane, which is presumably formed by nephrin, is disrupted and podocyte foot processes are effaced, as in congenital nephrotic syndrome of Finnish type. However, it is also possible that the altered cytoskeleton of podocytes with effaced foot processes can change or suppress the expression of nephrin. The recent identification of mutation of podocin [12] and {alpha}-actinin-4 [14] in familial idiopathic nephritic syndromes and congenital nephrotic syndrome in mice lacking CD2-associated protein (CD2AP) [14] are the examples of the latter possibility.

Nephrin redistribution on podocyte has been suggested as a potential mechanism of proteinuria in patients with primary acquired nephrotic syndrome [15]. However, comparisons of the distribution of gold particles in GN cases and controls in this experiment were not consistent with the above suggestion. As nephrin expression and localizatin can be different according to the pathogenetic mechanisms underlying each type of GN, this hypothesis needs to be tested with more homogeneous samples.

In conclusion, we found that the expression of nephrin in GN was decreased in the areas where the foot processes were effaced; however, its expression was comparable with that of normal kidneys where the foot process interspaces were preserved. The significance of our observations, in the context of proteinuria in acquired GN, needs further clarification.



   Notes
 
Correspondence and offprint requests to: Dae Joong Kim, MD, Division of Nephrology, Samsung Medical Center, 50 Ilwondong, Kangnamgoo, Seoul, Korea 135-710. Email: kimdjmed{at}dreamwiz.com Back



   References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 15. 5.01
Accepted in revised form: 11.10.01