Nephrin Redistribution on Podocytes Is a Potential Mechanism for Proteinuria in Patients with Primary Acquired Nephrotic Syndrome

Reagents

The mouse anti-nephrin antibody (IgG₁) is a mAb specific for the extracellular fibronectin type III-like motif of the recombinant human nephrin produced in A293 cells ( V. Ruotsalainen and K. Tryggvason, manuscript in preparation). In Western blots, nephrin antibody recognized the extracellular domain of recombinant human nephrin and a 180-kd protein in lysates of human glomeruli. 3 An irrelevant IgG₁ isotypic control antibody was purchased from Cedarlane (Hornby, Ontario Canada). Rabbit antiserum to human-IgG was purchased from Dade Behring (Marburg, Germany). Fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse IgG (adsorbed with human serum proteins); tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (adsorbed with human IgG); FITC-phalloidin; human IgG₄ kappa (IgG₄); human tumor necrosis factor-α (TNF-α); cytochalasin B; sodium azide; C5, C6, C7, C8, and C9 complement fragments; methionine; chloramine-T; and puromycin were obtained from Sigma Chemical Co. (Saint Louis, MO). Mouse mAb anti-human C9 neoantigen of the C5b-9 complex (MAC) was purchased from DAKO (Glostrup, Denmark). The antibody anti-E-selectin was obtained from Chemicon Int. (Temecula, CA). Vectashield mounting medium was purchased from Vector Laboratories, Inc. (Burlingame, CA).

Patients

The study included 30 proteinuric patients (27 of 30 with nephrotic syndrome) and six patients with minimal proteinuria. Nephrotic syndrome was defined as proteinuria ≥3.5 g/day and serum albumin ≤3.0 g/dL. The histological diagnosis of proteinuric patients was membranous GN in 13 cases, minimal change GN in 10 cases, and focal segmental glomerulosclerosis (FSGS) in seven cases. Patients with minimal proteinuria selected for the study presented IgA GN. None of the patients had evidence of systemic disease on a clinical or laboratory basis. Table 1 ▶ depicts some of the clinical features of the patients included in the study. As control, 10 specimens were obtained from normal kidney portions of patients undergoing surgery for cancer. Patients were selected for absence of proteinuria and lack of glomerular abnormalities detected by light and immunofluorescence microscopy.

For all patients, the protein content of 24-hour urinary samples was measured by the pyrogallol red method. The creatinine concentration in plasma was analyzed by kinetic Jaffé method with a Beckman Synchron CX3.

Culture of Glomerular Epithelial Cells

Decapsulated glomeruli were isolated by differential sieving from renal cortex fragments taken from surgically removed kidneys of five Caucasian patients. 9,10 Primary cultures of glomerular epithelial cells (GECs) were established as previously described. 11 GECs were obtained by plating at high density glomeruli untreated with collagenase. After a 10-day incubation in Dulbecco’s modified Eagle’s medium containing 20% fetal calf serum, the cultures were trypsinized and the outgrowing GECs were expanded. Phenotypic characterization was performed according to cell morphology (polyhedral cells with cobblestone-like appearance); positive staining for synaptopodin, podocalyxin, zonula occludens-1 (ZO-1), cytokeratin, vimentin, and laminin; negative staining for smooth muscle-type myosin, FVIIIr:Ag, and CD45; cytotoxicity in response to puromycin aminonucleoside (10 to 50 μg/ml). 10,12-14

Established lines of differentiated GECs were obtained by infection of pure primary cultures with a hybrid Adeno5/SV40 virus as previously described. 11,15 Individual foci of immortalized cells were subcultured and cloned. The selected clones were used between passages 25 and 40. The GEC line used in the present study was characterized as previously described according to the phenotype, following the criteria mentioned above. 11,13

Immunofluorescence Studies

Immunofluorescence (IF) studies were performed on kidney biopsies from the patients described above. The tissues were rapidly frozen in liquid nitrogen, and 2-μm-thick cryostat sections were fixed in 3.5% paraformaldehyde for 15 minutes and washed in phosphate-buffered saline (PBS). The sections were incubated with anti-nephrin mAb at a concentration of 10 μg/ml or with the irrelevant mouse IgG₁ isotypic control antibody, for 2 hours at room temperature, washed in PBS, and incubated with FITC-conjugated sheep anti-mouse IgG. Double staining was performed on biopsies from patients with membranous GN using a rabbit antiserum to human IgG and a TRITC-conjugated goat anti-rabbit IgG. A rabbit nonimmune serum was used as irrelevant control.

IF on cultured GECs was performed as previously described. 9 Briefly, coverslip-attached GECs at subconfluent density were fixed in 3.5% paraformaldehyde containing 2% sucrose for 15 minutes at room temperature and washed in PBS. In experiments aimed to study intracellular localization of nephrin or cell cytoskeleton, cells were made permeable to large molecules by soaking coverslips for 5 minutes at 0°C in HEPES-Triton X-100 buffer (20 mmol/L HEPES, pH 7.4, 300 mmol/L sucrose, 50 mmol/L NaCl, 3 mmol/L MgCl₂, and 0.5 Triton X-100). Cells were then incubated with antibodies as described for the biopsies. The slides were then washed, mounted with Vectashield mounting medium, and examined.

Control experiments included incubation of sections or cells with nonimmune isotypic control antibodies or the omission of primary antibodies followed by the appropriate labeled secondary antibodies. The specificity of anti-nephrin mAb was tested by pre-adsorption of the antibody (10 μg/ml) with purified recombinant extracellular nephrin (30 μg/ml).

The number of glomeruli available on each biopsy for analysis of nephrin expression ranged between 3 to 7. Three nonsequential sections were examined for each specimen. Nephrin expression was analyzed semiquantitatively by measuring fluorescence intensity by digital image analysis (Windows MicroImage, version 3.4 CASTI Imaging, Venezia, Italy) of images obtained using a low-light video camera (Leica DC100, Wetzlar, Germany) on a 180-μm diameter field. The results were expressed as relative fluorescence intensity on a scale from 0 (fluorescence of background of tissue) to 255 (fluorescence of standard filter).

Detection of Nephrin mRNA Expression by Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR)

RT-PCR was performed using total RNA from GECs. Total RNA was extracted using Tri Reagent (Sigma) and precipitated with isopropanol. Complementary DNA was obtained by using oligo-p(dT)₁₅ primers (Boehringer Mannheim, Mannheim, Germany). Reverse transcription was performed at 42°C for 60 minutes; in addition to 1 μg of RNA, the reaction mixture (20 μl) contained 10 mmol/L Tris, 50 mmol/L KCl, pH 8.3, 5 mmol/L MgCl₂, 1 mmol/L dNTPs, 50 U RNase inhibitor and 20 U AMV reverse transcriptase (Boehringer Mannheim). For reverse transcriptase-negative controls, the enzyme was omitted. cDNA was then subjected to 35 cycles of amplification by the PCR in an automated DNA thermal cycler (Hybaid, Ashford, UK). For detection of human nephrin and human glyceraldehyde phosphate dehydrogenase (h-GAPDH), used as housekeeping gene, sequence-specific oligonucleotide primers (purchased from TIB Molbiol, Genova, Italy) were designed (nephrin: 3′reverse, TAC ACC AGA TGT CCC CTC AG; 5′ forward, TCT TAT TCC CGA GGT TTC AC; GAPDH: 3′reverse, TCT AGA CGG CAG GTC AGG TCC ACC; 5′ forward, CCA CCC ATG GCA AAT TCC ATG GCA). The PCR mixture (50 μl) contained 10 mmol/L Tris-HCl, pH 8.8, at 25°C, 50 mmol/L KCl, 0.1% Triton X-100, 1.25 mmol/L MgCl₂, 0.2 mmol/L dNTPs, 25 pmol of (+) and (−) primers, and 1.25 U thermostable DNA polymerase (Finnzymes). Times and temperatures for denaturation, annealing, and extension were 1 minute at 95°C, 1 minute at 60°C, and 3 minutes at 72°C, respectively. Amplification products (257 bp) for nephrin and (598 bp) for GAPDH were visualized by ethidium bromide staining after agarose gel electrophoresis.

Detection of Nephrin Expression by Western Blot Analysis

Protein concentration of GEC lysates obtained as previously described 16 was determined by the Bradford technique, and the protein content of the samples was normalized to 100 32 μg/sample by appropriate dilution with Laemmli buffer. Proteins were directly subjected to 8% SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose. The filters were incubated with blocking solution (10% low-fat milk in 20 mmol/L Tris/HCl, pH 7.6, and 17 mmol/L NaCl) for 60 minutes. The anti-nephrin mAb was then added at a concentration of 2.5 μg/ml, and the incubation was performed overnight at 4°C. For detection, the filters were washed four times (15 minutes each wash) with PBS and 0.5% Tween 20, and reacted for 60 minutes at room temperature with peroxidase-conjugated protein A (200 ng/ml; Amersham, Buckinghamshire, UK). The enzyme was removed by washing as above. The filters were incubated for 2 minutes with a chemiluminescence reagent (ECL, Amersham) and exposed to an autoradiography film for 1 to 5 minutes.

As negative control for the expression of nephrin, an immortalized cell line of human renal tubular epithelial cells was used. 15

Experimental Design

The expression of nephrin by GECs was evaluated by indirect IF on cells fixed with paraformaldehyde followed or not by permeabilization with Triton X-100. In some experiments, living (unfixed) GECs were incubated at 37°C or 4°C for 60 minutes with anti-nephrin mAb to evaluate antibody-induced antigen redistribution. 9 To study in vitro the role of immune complexes on the surface expression and redistribution of nephrin, GECs were incubated with human aggregated IgG₄ (agIgG₄) (1 μg/ml) in Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum for 60 minutes at 37°C before fixation and staining with anti-nephrin mAb and rabbit anti-human-IgG. AgIgG₄ was obtained by heating at 63°C for 30 minutes, as described. 17 As control, cells were incubated with human disaggregated IgG₄ in Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum for 60 minutes at 37°C before staining with antibodies. Human IgG₄ were disaggregated after centrifugation for 4 hours at 100,000 × g at 4°C. 18

Actin microfilament alterations in agIgG₄-stimulated GECs were evaluated by FITC-phalloidin staining after permeabilization of the cells. Other stimuli, such as TNF-α, puromycin, or MAC, known to be involved in the pathogenesis of GN and to affect the cell cytoskeleton, were used to evaluate the role of actin microfilament alterations on the expression and redistribution of nephrin. Cells were incubated with human TNF-α (10 ng/ml) or puromycin (5 μg/ml) in Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum for 1 hour at 37°C before staining with anti-nephrin mAb. Actin microfilament alterations in TNF-α or puromycin-stimulated GECs were evaluated as FITC-phalloidin staining. Assembly of the MAC on GECs was performed as previously described. 19 Briefly, nonenzymatic formation of a C5b-like C5C6 complex was obtained by incubation of 10 μg of C5 in 10 μl of veronal buffer with 10 μl of 0.32 mmol/L chloramine-T for 10 minutes at room temperature. After inactivation of chloramine-T with 10 μl methionine (1 mmol/L), purified C6 (20 μg) was added in 300 μl of serum-free medium and incubated for 24 hours at 37°C. The assembly of the MAC into cell plasma membrane was obtained by 15 minutes of pre-incubation (37°C) of C7 (10 μg/ml) with the C5b-C6 complex (5 μg/ml). After two washings with serum-free medium, C8 (10 μg/ml) and C9 (10 μg/ml) complement components were added and incubated for 30 minutes at 37°C. In selected experiments heat inactivated (100°C for 30 minutes) or polymyxin B-treated (5 to 50 μg/ml) complement components were used as control. After cell fixation, the expression of nephrin was evaluated by IF with anti-nephrin mAb. In parallel experiments the insertion of MAC into GEC plasma membrane was assessed by IF using a mAb anti-human C5b-9 reacting with a neoepitope exposed only in activated C9 in the solid-phase membrane form and in the fluid form of MAC, but not in native C9. 20

In some experiments, before incubation with the various stimuli, the GECs were pre-incubated for 15 minutes with drugs that interfere with cytoskeletal function such as cytochalasin B (5 and 10 μg/ml) or with cell metabolism such as sodium azide (10⁻³ to 10⁻¹ mol/L). The cell viability was assessed by trypan blue exclusion.

Statistical Analysis

All results are given as mean values ± SD. Differences between multiple groups were analyzed by one-way analysis of variance in combination with Tukey’s multiple comparison test. Linear regression analysis was performed between relative fluorescence intensity for nephrin and extent of proteinuria. A P value of <0.05 was considered significant.

Expression of Nephrin in Primary Acquired Glomerular Diseases

The expression of nephrin was evaluated by indirect IF in 30 proteinuric patients (27 with nephrotic syndrome) including 13 membranous GN, 10 minimal change GN, and seven FSGS, and in six patients with IgA GN and minimal proteinuria (Table 1) ▶ using mAb specific for the extracellular domain of nephrin. As control, 10 specimens of normal kidney portions obtained from patients undergoing surgery for renal tumors were used.

In normal controls, nephrin exhibited a glomerular epithelial pattern with a punctate/linear distribution along the peripheral capillary loops (Figure 1A) ▶ . In glomeruli of patients with membranous GN, a more granular pattern or a loss of staining of nephrin was observed (Figure 1B) ▶ . Figure 1, C and D ▶ , shows the co-localization of nephrin and IgG evaluated by double IF in the same glomerulus of a patient with membranous GN. In Figure 1C ▶ , the green fluorescence depicts the distribution of nephrin. The red fluorescence observed in Figure 1D ▶ shows the granular distribution of IgG along the glomerular basement membrane. The overlap of staining for nephrin (green) and IgG (red) results in a yellow staining of granular deposits in the merge (Figure 1D) ▶ . A granular pattern with aspects of apparent plasmalemmal dislocation from the normal expression sites of nephrin and a loss of staining were also observed in patients with minimal change GN (Figure 2, A and B) ▶ and FSGS (Figure 2C) ▶ . In IgA GN, the staining pattern of nephrin showed an epithelial distribution, similar to that of controls (Figure 2D) ▶ . As shown in Figure 3 ▶ , relative fluorescence intensity was significantly reduced in all proteinuric patients irrespective of the primary disease. In contrast, the fluorescence intensity in glomeruli of patients with IgA GN with minimal proteinuria did not significantly differ from that of controls (Figure 3) ▶ . The average variability in nephrin staining pattern from one glomerulus to the next within individual patients and controls was 20.6 ± 16 and 14.4 ± 4, respectively.

As shown in Figure 4 ▶ an inverse correlation between relative fluorescence intensity and extent of proteinuria was detected when all patients with GN were analyzed. In contrast, when linear regression analysis was performed only on patients with nephrotic syndrome, no significant correlation was observed.

The staining for nephrin was completely abrogated by pre-adsorption of the antibody with the purified human recombinant extracellular nephrin, indicating staining specificity. Moreover, sections incubated with the nonimmune isotypic control antibodies or with the appropriate labeled secondary antibodies without the primary antibody were always negative (data not shown).

Expression of Nephrin by GECs

Primary and immortalized GECs showed expression of the nephrin mRNA and of the protein by RT-PCR (Figure 5A) ▶ and Western blot analysis (Figure 5B) ▶ , respectively. When tested by indirect IF, antibody specific for the extracellular domain of nephrin bound to GECs unfixed or fixed with paraformaldehyde with a punctate granular pattern, suggesting a surface expression of nephrin (Figure 6A) ▶ . In paraformaldehyde-fixed and permeabilized GECs a combination of fine granular and diffuse staining was observed suggesting the presence of an intracellular pool of nephrin. In permeabilized cells the staining was more evident around the nuclei (data not shown).

Modulation of Nephrin Expression on GECs

Experiments of nephrin redistribution were performed on immortalized GECs. When living (unfixed) GECs were incubated with anti-nephrin mAb for 60 minutes at 37°C, but not at 4°C, the binding was rapidly converted from fine granular into a patchy pattern, suggesting antibody-induced antigen redistribution (data not shown). Therefore, we tested whether agIgG₄ were able to change the surface expression of nephrin. Incubation of living GECs at 4°C for 60 minutes with agIgG₄ resulted in diffuse surface binding of IgG₄ to GECs (data not shown). When incubated at 37°C for 60 minutes, agIgG₄ appeared redistributed in one or multiple aggregates (capping) (data not shown). When cells were fixed with paraformaldehyde after 60 minutes incubation with agIgG₄, nephrin appeared focally redistributed on the cell surface, leaving variable parts of the cell surface devoid of antigen (Figure 6B) ▶ . Double staining of nephrin and IgG showed focal co-localization on the cell surface (Figure 6B ▶ , inset). In most GECs the disappearance of nephrin from the surface was predominant, as indicated by a significant decrease of IF staining (Figures 6C and 7A) ▶ ▶ , suggesting shedding of the protein from the cell surface. AgIgG₄ induced disruption of normal cellular organization of F-actin (Figure 6D) ▶ in GECs, with loss of stress fibers and peripheral actin localization (Figure 6, E and F) ▶ . In contrast, disaggregated IgG₄ did not induce nephrin redistribution and disappearance (Figure 6G) ▶ or cytoskeletal changes (data not shown). We therefore investigated whether the observed nephrin redistribution could be ascribed to cytoskeletal reorganization induced by agIgG₄. Plasma membrane insertion of MAC, TNF-α, or puromycin, stimuli known to affect the cytoskeleton of GECs, 14,21,22 also induced a significant reduction of IF intensity (Figure 7A) ▶ and a staining pattern compatible with surface patching and shedding of nephrin (Figure 6, H and I) ▶ . Within 24 hours of removal of TNF-α from the culture medium, nephrin was fully re-expressed on GECs, indicating that the effect of this cytokine was not to be ascribed to a cytotoxic effect (Figure 6J) ▶ . Moreover, the process of nephrin redistribution was an active energy-requiring process, because sodium azide, an inhibitor of oxidative phosphorylation and of glycolysis, prevented patching and shedding induced by agIgG₄, TNF-α, and puromycin (Figures 6K and 7B) ▶ ▶ . Similar results were obtained with cytochalasin B, a compound that affects the microfilaments of the microtubular system, 23 which prevented reduction of fluorescence intensity and morphological evidences of nephrin redistribution (Figures 6L and 7C) ▶ ▶ .

In all of the experiments of cytoskeleton activation, the reduction in cell viability ranged from 6 to 11%.

In control experiments, the staining for nephrin was completely abrogated by pre-adsorption of the antibody with the purified human recombinant extracellular nephrin. When the relevant antibodies were substituted with the nonimmune isotypic control antibodies or with the appropriate labeled secondary antibodies without the primary antibody the immunofluorescence was always negative (data not shown).

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