1 Graduate Institutes of Medical Sciences, 2 School of Pharmacy, National Defense Medical Center, 3 Division of Nephrology, Department of Internal Medicine, 4 Department of Pathology, Tri-Service General Hospital and5 National Health Research Institutes, Taipei, Taiwan
Correspondence and offprint requests to: Yuh-Feng Lin, MD, Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital 325, Sec. 2, Cheng-Kung Road, Neihu 114, Taipei, Taiwan, Republic of China. Email: linyf{at}ndmctsgh.edu.tw
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Abstract |
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Methods. cBSA in an optimum dose was given intravenously to 8-week-old female ICR, BALB/c and C57BL/6 mice for 4 weeks. The disease state was verified by renal histopathology as well as by serum and urine metabolic profiles. Serum concentrations of anti-cBSA immunoglobulins (Igs) and circulating immune complex (CIC) were assayed to study the mechanisms of initiation and progression. T-helper (Th) cell subsets in peripheral blood were examined using flow cytometry, and the Th1/Th2 subset distribution was determined by comparing the serum concentrations of IgG1 and IgG2a, using quantitative heterologous interpolation enzyme-linked immunosorbent assays.
Results. Only ICR and BALB/c mice developed the typical clinical and pathological patterns of MN in response to an optimum dose of cBSA. Disease induction was dose related and strain specific. The serum concentrations of anti-cBSA were significantly higher in the strains that developed MN, but there were no differences in CIC concentrations. This suggests that in situ immune-complex glomerulonephritis may be involved in the development of MN. The Th2 type immune response may predominate in the ICR and BALB/c mice models, as the serum concentration of IgG1 was higher than that of IgG2a; moreover Th2 type strain specificity was necessary for the development of MN.
Conclusions. This improved mouse model of MN induced by cBSA more closely duplicates human MN than the other available models. Disease generation is antigen dose related and strain specific.
Keywords: cationic bovine serum albumin; circulating immune complex; membranous nephropathy; strain difference; T-helper lymphocytes
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Introduction |
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One of the most frequently used models of human MN is Heymann's nephritis, using the rat [1,4]. Alternative models are induced by repeated doses of cationic bovine serum albumin (cBSA) in the dog [5], cat [6], rabbit [79] and rat [10,11] (Table 1). However, there are few well-characterized mouse models for the study of human MN although mice offer the advantage of being cheaper, easier to manipulate, and suitable for ample experimental applications. Here we have developed a mouse model of MN using cBSA, and have characterized it clinically, pathologically and genetically. We also evaluated the susceptibility of various strains of mice in response to different doses of cBSA.
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Materials and methods |
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Preparation of cBSA
The cBSA was prepared as previously described [5] with slight modification. Briefly, 200 g of crystallized bovine serum albumin (Sigma, St Louis, MO) was dissolved in 1.2 ml of distilled water. Anhydrous ethylenediamine (EDA) (Sigma) was prepared by mixing 2.68 ml of EDA and 20 ml of distilled water. The pH was adjusted to 4.75 with 6 N HCl and the solution cooled to 25°C. This EDA solution was added slowly to the BSA solution followed by 150 mg of 1-ethyl-3-[(3-dimethylaminopropyl)-carbodiimide hydrochloride] (EDC) (Sigma) with continuous stirring for 4 h. The product was purified using the pH-dependent binding technique [12]. The reaction solution was applied to a 1 ml Econo Pac® High S cationic ion-exchange column (BioRad, Hercules, CA). After binding of BSA to the ion-exchange beads, the column was washed with 10 ml of pH 6.8 phosphate buffer (40 mM) to remove the non-reacted BSA. Subsequently, the modified BSA was eluted with 5 ml of 40 mM phosphate buffer (pH 10.6). All collections were then pooled, concentrated and reconstituted with saline. Prior to use, the final protein concentration was measured using UV absorbance at 280 nm.
Experimental design
To determine the optimum dose of cBSA for MN induction, ICR mice (10 in each group) received the antigen intravenously at 1, 3, 5, 7 and 9 mg/kg, three times per week every other day (tiw), two weeks after immunization by the same route with 0.2 mg of cBSA emulsified in an equal volume of complete Freund's adjuvant. The severity of proteinuria was measured using urine strips (Bayer Diagnostics, Bridgend, South Wales, UK). When all members of a group developed grade 4 proteinuria, that minimum dose was defined as the optimum. BALB/c and C57BL/6 mice received the same protocol to define the ideal dose for those strains.
Based on these titration results, we designed a study to clarify the initiation and progression of cBSA-induced MN. Eight-week-old ICR mice were divided into four groups (10 mice in each group): control (C); immunization (I); low dose (L); and high dose (H). Groups I, L and H were immunized as described. Two weeks later, groups L and H were injected intravenously with 1 and 7 mg/kg of cBSA, tiw, respectively. Group I received a saline injection after the immunization. Group C did not receive immunization but mice were given saline injections on the same schedule as group H. For the BALB/c and C57BL/6 strains, only the groups with successfully induced MN and controls were investigated. After 4 weeks, the animals were killed, and we collected blood, urine and kidney samples for analysis.
Blood and urine metabolic data
Urine samples were collected in a metabolic cage 1 day before death, microcentrifuged and stored at 70°C until assayed. Before death, blood samples were taken from the retro-orbital venous plexus, microcentrifuged, and stored at 70°C until assayed.
Proteinuria was measured as the ratio of urinary protein (mg/ml) to urine creatinine (Cr) (mg/dl) (Up/UCr). Urinary protein was quantitatively determined using a BCA protein assay kit (Pierce, Rockford, IL). Urine Cr was measured using a creatinine assay kit (Sigma). Concentrations of blood urea nitrogen (BUN), Cr, albumin and total cholesterol (T. chol) were determined with kits from Sigma. All assays were performed in duplicate according to the manufacturer's instructions.
Histopathology of renal tissues
Renal tissues were snap frozen and fixed in CarsonMillonig's solution for fluorescence and electron microscopy (EM) and 10% formalin for light microscopy. Formalin-fixed tissues were dehydrated in a graded ethanol series and embedded in paraffin wax. Sections (3 µm) were stained with haematoxylin and eosin (H&E). Frozen tissues were sectioned, dried in air, fixed in acetone for 10 min at room temperature, and washed with PBS prior to incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (Ig) A, IgG, IgM and C3 (Capple, Durham, NC). The sections were observed using a fluorescence photomicroscope (Olympus, Tokyo, Japan). Semi-quantitative evaluation of fluorescence was performed as previously described [13]. For EM examination, small sections of fixed renal tissue were washed in CarsonMillonig's solution, post-fixed for 1 h in 1% aqueous osmium tetroxide, dehydrated in a graded alcohol series, and embedded in Epon resin. Ultrathin sections were cut using diamond knives, double stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope (JEM-1230; JEOL, Peabody, MA).
Study of glomerular polyanions
Glomerular polyanions were investigated after staining according to Mowry [13,14] using colloidal iron staining for light microscopy. Renal samples were processed as described above for H&E staining. Deparaffinized sections were stained for 2 h in colloidal iron solution, followed by three rinses with 30% acetic acid (pH 1.5). The specimens were incubated for 20 min in a freshly prepared solution of 1% hydrochloric acid and 1% potassium ferrocyanide. Sections were then observed using an optical photomicroscope (Olympus). Semi-quantitative evaluation of glomerular staining was performed as previously reported [13]. The values ranged from 0 to a maximum of 300.
Serum concentrations of anti-cBSA Igs, IgG1 and IgG2a antibody, and circulating immune complex
The serum concentrations of the anti-cBSA Igs, IgG1, and IgG2a, were measured using an enzyme-linked immunosorbent assay (ELISA), which was modified from a previous method [11,15]. In brief, 96 well polyvinyl chloride microtitration plates (FALCON; Becton Dickinson and Co., Franklin Lakes, NJ) were coated with cBSA, 0.5 µg/well, in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 10 mM MgCl2, pH 9.6), incubated overnight at 4°C, and then washed five times with washing buffer (150 mM NaCl, 1/1000 Tween-20, pH 7.4). The plates were blocked with 300 µl of 1% goat serum albumin for 30 min at room temperature, followed by five washes with washing buffer. The serum samples, diluted in ELISA buffer (150 mM NaCl, 10 mM Na2HPO4, 1 mM KH2PO4, 2.6 mM KCl, 0.5% BSA, 1/1000 Tween-20, pH 7.4), were assayed at different dilution for Igs, IgG1 and IgG2a (Igs, 1/10 000; IgG1, 1/10 0001/640000; IgG2a, 11/640 000). After 1 h incubation at room temperature, the plates were washed five times with washing buffer. Then, 100 µl aliquots of horseradish peroxidase-conjugated goat anti-mouse Igs (DAKO EnVisionTM Doublestain system; DAKO, Carpinteria, CA), IgG1 and IgG2a (mouse IgG1 and IgG2a quantitation kit; Bethyl Laboratories Inc., Montgomery, TX), each diluted 1/1000, 1/5000 and 1/5000 with ELISA buffer, were added. After 1 h of incubation at room temperature, the plates were washed again five times, 100 µl of 3,3',5,5'-tetramethybenzidine (Chemicon International Inc.; Temecula, CA) substrate solution was added into each well for 15 min at room temperature, and the reaction was stopped with 50 µl of 2 N H2SO4. The optical density (OD) was read at 450 nm on an automated microplate reader (MRX; Dynex Technology, Chantilly, VA). The absorbance of the test sample blank was subtracted from all data for each plate. Data were expressed as OD at 450 nm. The IgG1 and IgG2a mouse reference sera (mouse IgG1 and IgG2a quantitation kits) were used to construct a standard curve according to the manufacturer's instructions.
For CIC detection, plates coated with C1q to detect CIC (Alpha Diagnostic International Inc., San Antonio, TX) were used with sera diluted 1/10, 1/20, 1/40, 1/80, 1/160 and 1/320 in the sample buffer. Subsequent procedures were performed according to the manufacturer's instructions. Data were read and expressed as described above.
Preparation of peripheral blood and flow cytometry
Before death, blood samples were taken from the retro-orbital venous plexus, 500 µl being collected in an EDTA tube and mixed by inversion. Subsequently, 10 ml of 1 N NH4Cl was added to whole blood, and the mixture was incubated for 10 min at room temperature before centrifugation at 750 g at 4°C for 6 min. Cells were washed twice with 0.2% BSA in 1 M PBS. More than 90% cell viability was verified using the Trypan Blue exclusion method, and the cell concentration was determined.
A sample of 1.02.0 x 105 cells was incubated for 30 min on ice with cell surface monoclonal antibodies (MoAbs) against T cells. Dual colour immunofluorescence (IF) was performed using phycoerythrin (PE)-conjugated rat anti-mouse CD3 (PharMingen, San Diego, CA) and FITC-conjugated rat anti-mouse CD4 and CD8 (Serotec, Kidlington, Oxford, UK). The stained cells were washed with 1 M PBS and then analysed by fluorescence-activated cell sorting (FACS) using a FACScan flow cytometer (Becton-Dickinson). A minimum of 10 000 events was acquired, and the data were analysed using CellQuest software (version 3.3).
Statistical analyses
Results are expressed as mean±SEM. Statistical significance was calculated by one-way ANOVA for multiple comparisons and Student's t-test for unpaired data. P < 0.05 was regarded as significant.
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Results |
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Light microscopy
Thickness of the GBM for groups I and L was the same as for group C (data not shown), but group H exhibited the typical MN morphologic pattern, namely, diffuse thickening of the GBM and no significant mesangial proliferation in comparison with group C (Figure 1A).
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Electron microscopy
Ultrastructurally, in group H kidneys the basement membrane showed severe irregular thickening of the laminae rarae, interna and externa, and subepithelial deposits were layered and more confluent (Figure 1C), suggesting glomerular size selectivity defect [16]. GBM spikes were observed extending between the deposits and there was loss of podocyte foot processes.
Colloidal iron staining
There was a reduced intensity of blue staining along the GCW, indicating the loss of glomerular anions. Staining was moderately attenuated in group H kidneys (Figure 2A) compared with group C (Figure 2B), and the total mean intensity of staining was decreased to approximately 120 on a semi-quantitative scale [13].
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Polarization of Th cell subsets
Two strategies were used to evaluate the polarization of Th subsets in our MN mouse models. First, the two inbred strains, BALB/c and C57BL/6, each with a distinct genetic background, were studied. BALB/c is a strain carrying the Th2 subset; however, C57 carries Th1 [17]. We found that MN could be induced in BALB/c, but not in C57 mice. The Th2 subset response may thus play an important role in the development of MN. Secondly, the functionally distinct Th1 and Th2 subsets can be differentiated according to individual cytokine production or subsequent cytokine effects. Th1 regulates Ig switching to the IgG2a type, whereas Th2 enhances IgG1 secretion [15]. We therefore assessed Th functions in our successful MN mouse modelsthe ICR and BALB/c strainsby measuring the serum concentrations of IgG1 for Th2 and of IgG2a for Th1. Using quantitative heterologous interpolation ELISA [18], we found that the reference curves constructed with IgG1 and IgG2a were similar; the data subsequently were applied to interpolate test samples. As shown in Figures 4A and B, no matter what the sample dilution, the slope, intercept and R2 of the regression lines for IgG1 were significantly higher than those of IgG2a in ICR and BALB/c mice. Thus, the MN mouse model appeared attributable to a Th2 response.
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Discussion |
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The cBSA antigen has been utilized to induce MN in various animals, as summarized and compared with our model in Table 1. For all models, both initial antigen stimulation and continuous boosting doses are required for disease induction. Among these models, all animals develop proteinuria except the dog. Our mice also demonstrated hypoalbuminaemia and hypercholesterolaemia, suggesting that this model exhibits clinical manifestations similar to those of human MN with nephrotic syndrome. Histopathologically, all animal models display GBM thickening, IgG granular deposition and subepithelial deposits. However, variants of MN are sometimes observed, including inflammatory-cell infiltration, segmental endocapillary proliferation, and mesangial cells proliferation. Our mice did not develop these variants to any significant extent. Such variants of MN might reflect the purity of the cBSA antigen. The heterogeneity of the antigenic charge distinctly affects its ability to cause damage: the more cationic the immunogen, the more nephritogenic it is and the greater its tendency to produce a typical MN pattern [8,19]. The cBSA preparations previously used were only characterized by polyacrylamide gel flat bed electrophoresis and fast protein liquid chromatography [5,8,9], which could not exclude the presence of native anionic and slightly cationic BSA. Because we used a more homogenous cBSA preparation purified by the pH-dependent binding technique [12], our model disclosed greater consistency.
Antigen quantity also seems to be a cofactor influencing MN induction and a threshold exposure to antigen is needed for induction [20]. Our experience of induction was consistent with this. In preliminary studies, we found that ICR mice given a low dose of cBSA (3 mg/kg) could recover from proteinuria if administration was stopped (data not shown). Human MN has a variable course, and 1560% of patients exhibit spontaneous remission and complete recovery without treatment [21]. Although the mechanisms responsible for this variable course remain unclear, our results suggest that lower antigen exposure might increase the chance of spontaneous remission. We speculate that the antigen source, dose and exposure duration are factors in the pathological and clinical diversity of MN.
The antigen-associated renal injury mostly involves two mechanisms: initiation and progression. Initiation is strongly suggested to be a consequence of circulating autoantibody against antigen located on the epithelial side of the glomerular capillaries [22], indicating in situ immune-complex GN. Consistent with this idea, the mechanism of initiation in this MN mouse model is proposed to be deposition of cBSA antigen on the subepithelial side, followed by specific antibody aggregation leading to in situ immune-complex GN. With regard to MN progression, it is proposed that immune complexes, once deposited in the glomerulus, generate a cascade of local injury events that contribute to reduced glomerular size and charge selectivity [22,23] and excessive protein leakage. In our model, combined impairment of charge and size selectivity was responsible for proteinuria, as was also demonstrated by colloidal iron staining and ultrastructural studies. Another mechanism of accelerated MN progression is a T-cell-related systemic immune response. The Th2 type, a predominantly humoral immune response is strongly suggested [3]. In our study, MN was successfully induced in the strain of mice carrying Th2, but not in the Th1-carrying strain. This suggests that a genetic background with a predisposition for Th2 cells may determine the successful induction and progression of MN. Moreover, when analysing IgG1 and IgG2a, in our model, the IgG isotype profile showed a bias for IgG1. Polarization to a Th2 type immune response in the initiation and progression of our MN mouse model is thus likely. The CD4 and CD8 T-cell phenotypes showed no statistically significant changes between baseline and after MN induction. Various studies of the changes in T-cell subsets in MN have not shown consistent results [3,24,25], for unknown reasons.
The Heymann's nephritis rat is a generalized model with morphological and functional aspects similar to those of human MN [4]. However, it has rarely been applied to the mouse. Our MN model induced by cBSA not only provides a new model to study the process of MN, but also shows strain-specific difference in the establishment of disease. These models, including inbred and outbred strains of mice, offer promise for studies of the initiation, progression and treatment of human MN. Moreover, as we found a dose-related effect on induction, this model may be suitable for investigation of the varied course of MN.
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Acknowledgments |
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Conflict of interest statement. None.
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References |
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