1 Department of Cell Biology, Institute of Nephrology, 2 Department of Internal Medicine (II), Niigata University School of Medicine, Niigata and 3 Department of Medicine, Teikyo University School of Medicine, Tokyo, Japan
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Abstract |
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Methods. After treatment to induce immune tolerance to mouse immunoglobulin, 20 rats were injected with anti-rat slit diaphragm monoclonal antibody (mAb) 5-1-6 twice a week for 6 months and were then sacrificed.
Results. mAb 5-1-6 induced massive proteinuria in 11 rats. In nine rats with mild proteinuria, no histological alteration could be detected with light microscopy and immunofluorescence. In nephrotic rats, light microscopy showed minor glomerular abnormalities, with interstitial oedema, tubular epithelial cell degeneration and interstitial cell infiltration. Immunofluorescence revealed increased expression of vimentin and an increased number of OX1-, OX19- and ED1-positive cells. However, we could not detect any accumulation of type I and IV collagen or laminin in the tubulointerstitium. RTPCR showed that the expression of mRNA for type I collagen was not increased, compared with that in control rats.
Conclusions. We succeeded in developing a model of persistent nephrosis without severe glomerular abnormalities, nephrectomy or other manoeuvres known to induce disturbed haemodynamics, using an agent without tubulointerstitial toxicity, and considered it to be suitable for investigating the direct toxicity of proteinuria. In this model, isolated massive proteinuria induced interstitial injury. However, the degree of injury was suggested to be much less than that observed in other previously developed models.
Keywords: monoclonal antibody 5-1-6; sustained proteinuria; tubulointerstitial injury
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Introduction |
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We have established a monoclonal antibody (mAb), 5-1-6, which binds specifically to the p51 antigen located on the slit diaphragm of rat glomerular epithelial cells and causes severe proteinuria when injected into rats [7]. mAb 5-1-6 does not appear to bind to antigens on tubular epithelial cells or in the interstitial area. The proteinuria induced by mAb 5-1-6 is independent of complement and inflammatory cells [7,8]. Histological alterations can hardly be detected, after a single injection of mAb 5-1-6. Even the foot processes of the podocytes remain well preserved except for focal areas of effacement [7]. Since mAb 5-1-6 can therefore be considered to exert no direct toxicity on the tubulointerstitium and does not cause severe glomerular abnormalities, we sought to establish an animal model of persistent nephrosis using multiple injections of mAb 5-1-6.
In the present study, we succeeded in producing a rat model of continuous proteinuria using multiple injections of mAb 5-1-6 after treatment to induce immunotolerance to mouse immunoglobulin (Ig). We then applied this model to investigate the toxic effects of enhanced and sustained excretion of host protein on the tubulointerstitium, without interference caused by other toxic agents. During the 6-month observation period, tubular epithelial cell injury and interstitial mononuclear cell infiltration were observed but interstitial fibrosis was not.
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Materials and methods |
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Induction of immunotolerance to mouse -globulin (MGG)
To prevent the production of antibodies to injected mAb 5-1-6, immunotolerance to mouse Ig was induced. We prepared MGG from the sera of normal mice by precipitation with 50% saturated ammonium sulfate, followed by dialysis against phosphate-buffered saline (PBS). The MGG was centrifuged at 17 000 g for 30 min and the supernatant, designated soluble MGG (sMGG), was collected. As a preliminary to confirm that a tolerant state was induced in rats by an intravenous injection of 5 mg of sMGG, 2 weeks after this initial injection, we challenged three sMGG-treated and three untreated control rats with 1 mg of MGG administered subcutaneously with Freund's complete adjuvant. Two weeks after the challenge injection, their sera were sampled and the titre of antibody to mouse Ig was examined by indirect enzyme-linked immunosorbent assay (ELISA) as described in Estimation of serum antibody titre. The titre of anti-mouse Ig in the sMGG-treated group was less than one-tenth of that in the untreated group, showing that tolerance had been induced successfully. For further experiments, each rat was therefore injected with 5 mg of sMGG to induce immunotolerance.
Preparation of mAb
mAb 5-1-6 was prepared as described previously [7]. The hybridoma was cultured in serum-free medium (protein-free hybridoma medium II, Gibco-BRL, Rockville, MD) and the supernatant was collected. Ammonium sulfate (350 g/l) was added immediately to the supernatant, which was then stored at 4°C. The preparation subsequently was centrifuged at 10 000 r.p.m. for 30 min. The sediment was suspended in PBS, dialysed extensively against PBS and stored at -20°C. Just before injection, the mAb was diluted to 500 µg or 250 µg/ml with PBS. Only traces of proteins other than IgG were detected by silver staining after (SDSPAGE). As a control, murine IgG1 mAb, RVG1 (against rotavirus, demonstrated not to react with rat kidneys) was used.
Experimental design
For the first time, we used an mAb derived from the supernatant of a culture in serum-free medium. In a preliminary study, we confirmed that the initial injection of 500 µg, followed by the twice weekly injections of 250 µg, was enough to induce sustained proteinuria.
As shown in Figure 1, we first injected 5 mg of sMGG intravenously into 24 rats to induce immunotolerance to mouse IgG. Two weeks later, 500 µg of mAb 5-1-6 was injected into 20 rats. From the next injection onwards, 250 µg mAb was injected twice a week (every Monday and Friday). Four rats were injected with the same dose of mAb RVG1 as negative controls. Twenty-four hour urine was collected on MondayTuesday and ThursdayFriday, and sera were sampled every 4 weeks. Twenty-five weeks after the first injection, the rats were killed under anaesthesia with ether. After cardiac puncture for blood sampling, the left renal artery and vein were clamped and the left kidney was removed and weighed. The left kidney was then used for extraction of RNA. The right kidney was perfused with PBS via the heart and was then removed for light, electron and immunofluorescence (IF) microscopy.
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Characterization of proteinuria
Twenty-four hour urinary specimens were collected from the rats using metabolic cages. The urine was centrifuged at 1000 g for 10 min, and the protein content was determined by the biuret method using bovine serum albumin (BSA) as the standard.
Urine and serum were sampled at 4, 16 and 25 weeks from rats with massive proteinuria, in order to calculate the selectivity index. The samples were heated for 2 min at 100°C in SDSPAGE sample buffer (10% sucrose, 6.25 mM Tris-HCl (pH 6.8), 2% SDS, 10 mM dithiothreitol, and 0.0025% bromophenol blue). Urinary protein (about 20 µg) and serum protein (20 µl of serum diluted 1 : 50) in sample buffer were applied to a 7.5% gel. SDSPAGE was performed as previously described [7]. The albumin, IgG and other bands were stained with brilliant blue R (Sigma, St Louis, MO) and quantified by densitometry (ATTO, Tokyo, Japan). The amount of protein which was greater than albumin in urinary total protein was calculated. From urinary albumin (Ualb), urinary IgG (UIgG), serum albumin (Salb) and serum IgG (SIgG) levels thus measured, the selectivity of urinary protein (selectivity index; SI) was also calculated using the following formula [9]: SI=(UIgGxSalb)/(UalbxSIgG)
Laboratory investigations
Serum creatinine, serum blood urea nitrogen (BUN), serum total cholesterol and albumin, serum N-acetyl glucosaminase (NAG) activity and complement (CH50), urinary NAG activity and urinary creatinine levels were measured. From the data on serum creatinine (S), urine creatinine (U), 24 h urine volume (V) and body weight (BW) at sacrifice, the 24 h endogenous creatinine clearance (Ccr) was calculated using following formula.
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BS=body surface area of rats calculated using the formula.
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The occult blood and glucose contents of the urine at sacrifice were examined semiquantitatively with MultistixTM (Bayer-Sankyo Co., Tokyo, Japan).
Morphological and immunohistological study
For light microscopy, parts of the kidney were fixed with 10% neutral formalin and embedded in paraffin. Sections (23-µm thick) were stained with periodic acidSchiff (PAS) and periodic acidmethenamin silver (PAM). A semiquantitative morphological study of the glomerular lesions was carried out in a blinded manner, as described by Raij et al. [10]. A total of 50 glomeruli were analysed in each specimen. Severity of glomerular injury was expressed as matrix score.
For IF microscopy, the renal tissues were quick-frozen in n-hexane cooled to -70°C and 4-µm thick sections were cut with a cryostat. For the indirect method, the sections were incubated with the anti-rat leukocyte common antigen mAb, OX1 (Serotec, Oxford, UK), the anti-rat pan T-cell mAb, OX19 (Serotec), the anti-rat pan macrophage/monocyte mAb, ED1 (Biogene, Sundown, NH), the anti-rat neutrophil mAb, RP3 (kindly provided by Dr Sendo, Yamagata University, Yamagata, Japan), rabbit anti-rat transforming growth factor-ß1 (TGF-ß1) polyclonal antibody (kindly provided by Dr Muramatsu, Research Center, Mitsubishi Kasei, Yokohama, Japan), rabbit anti-vimentin polyclonal antibody (Bio-science products AG, Emmenbrucke, Switzerland), rabbit anti-rat type-I collagen polyclonal antibody (Chemicon International Inc., Temecula, CA), rabbit anti-rat type IV collagen polyclonal antibody (LSL, Tokyo, Japan), rabbit anti-laminin polyclonal antibody or anti--smooth muscle actin (SMA) mAb (Sigma) at 37°C for 30 min, then washed with PBS for 10 min at room temperature. The sections first incubated with rabbit polyclonal antibodies were then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit Ig (Dako, Glostrup, Denmark), while those treated with OX1, OX19, ED1 or RP3 were incubated with FITC-conjugated anti-mouse Igs (Dako) and those treated with anti-
-SMA were incubated with FITC-conjugated anti-mouse IgG2a (Southern Biotechnology Associates Inc., Birmingham, AL). For the direct method, the sections were incubated with FITC-conjugated anti-mouse Igs, anti-rat Igs (Dako) or anti-rat C3 antibody (Cappel, Durham, NC) at 37°C for 30 min, then washed with PBS for 10 min at room temperature. The FITC-conjugated anti-rabbit and anti-mouse Igs were absorbed with rat whole serum. The sections were observed under an immunofluorescent microscope (Olympus Vanox AHBS3, Tokyo, Japan). More than 30 glomeruli from each rat were analysed and the severity of the lesions was graded in a blinded manner on the scale of 04+ according to the percentage of glomerular
-SMA involvement. The IF score was obtained by multiplying the degree of damage (04+) by the percentage of glomeruli (0, no staining; 1+, <25%; 2+, 2550%; 3+, 5075%, 4+, >75%) with the same degree of injury. In the interstitium, 50 randomly selected high power fields, each 0.5 mm in diameter (i.e. covering a total area of about 10 mm2), were evaluated. The number of OX1-, OX19-, ED1- or RP3-positive cells was counted and the accumulation of matrix proteins was assessed.
For electron microscopy, a part of the renal cortex from each rat was cut into small pieces and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for several days at 4°C. After washing in PB and post-fixing in 1% OsO4 for 2 h, the fixed material was dehydrated through an ethanolpropylene oxide series and embedded in Araldite M. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined under a JEOL 1200EX electron microscope (Tokyo, Japan).
Reverse transcriptasepolymerase chain reaction (RTPCR)
Total RNA was extracted from the kidney cortex with Trizol (Gibco-BRL) following the standard protocol. The final product was air dried, dissolved in diethyl pyrocarbonate (DEPC)-treated water, and stored at -80°C. First strand complementary DNA (cDNA) was synthesized using the SuperScript Preamplification System (Gibco-BRL), following the standard protocol. Amplification was carried out using a PC-800 programmable temperature control system (Astec, Fukuoka, Japan) through 2040 cycles of denaturation at 95°C for 30 s, annealing at individual temperatures for 30 s and extension at 72°C for 10 min. The optimal cycle numbers were determined in a preliminary trial to be in the linear phase of amplification. Products were analysed by molecular weight on 1.5% agarose gels, and stained with ethidium bromide. mRNA for collagen type I and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by densitometry (ATTO), and collagen type I/GAPDH was calculated. The following rat sequences were used as primers: (i) rat collagen type I (sense 5'-CCCACGTAGGTGTCCTAAAGT-3', antisense 5'-CCGTGGTGCTAAAATAATAAA-3'), (ii) rat GAPDH (sense 5'-GGATGACCTTGCCCACAGC-3', antisense 5'-CTCTACCCACGGCAAGTT CAA-3').
Estimation of serum antibody titre
The rat anti-mouse IgG antibody titre was measured by an indirect ELISA. Microtitre plates were coated with 100 µl of a 50 µg/ml solution of 5-1-6 in 0.05 M TrisHCl buffer (pH 7.4) for 2 h at 37°C. After extensive washing, the plate was blocked with 0.5% BSA for 2 h at 37°C. The following reagents were added sequentially, washing after each addition: 100 µl of test serum (1:1000 dilution), 100 µl of peroxidase-conjugated anti-rat IgG (1:200 000 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and 200 µl of substrate solution (0.05 M sodium citrate, pH 5.0, containing 0.4 mg/ml o-phenylendiamine and 0.4 mg/ml urea hydrogen peroxide). The reaction was stopped by adding 50 µl of 6M H2SO4 and the absorbance was measured at 490 nm with an ELISA autoreader (Bio-Rad, Boston, MA). The reagent blanks and normal rat serum were used as negative controls. Rat sera which had been sampled 2 weeks after the injection of 1 mg of 5-1-6 with Freund's complete adjuvant were used as positive controls. A standard curve was drawn from the optical densities (ODs) of the positive control sera diluted 1 : 100, 1 : 300, 1 : 1000, 1 : 3000, 1 : 10 000, 1 : 30 000, 1 : 100 000 and 1 : 300 000 with 0.05 M TrisHCl buffer (pH 7.4), and the titre for each test serum was calculated by comparison with the standard curve to determine the percentage titre relative to the positive control sera. The rats used as positive controls were immunized with mouse Ig at the pre-treatment dose for inducing immune complex glomerulonephritis by heterogeneous IgG [11].
Statistical analysis
Statistical analysis was performed using Student's or Welch's t-test or MannWhitney's U-test as appropriate. A P value <0.05 was considered significant.
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Results |
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Body weight and kidney weight
Body weight and kidney weight differed significantly between group 1 and group 2 (Table 1). A positive correlation of kidney weight with proteinuria (Figure 3A
) was observed.
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Laboratory investigations
Serum albumin, total cholesterol, CH50 levels and urinary NAG activity differed significantly between group 1 and group 2 (Table 1), whereas BUN and creatinine clearance (Ccr) did not differ significantly (Table 1
). Serum albumin (Figure 3B
) and total cholesterol (Figure 3C
) correlated significantly with the severity of proteinuria. Glucose and occult blood were not detected in the urine of any of the rats at sacrifice.
Titre of anti-mouse IgG antibody
The anti-mouse IgG antibody titre increased from 4 weeks in all groups of rats. The serum titre in group 2 was 25 times greater than that in group 1 at all times except on day 0. The serum titres of eight of the rats in group 2 and all of the rats in group 1 was >10% of the values recorded for the positive control.
Morphological and immunohistological study
Light microscopy.
No global glomerulosclerosis was observed. Segmental matrix expansion was observed in some glomeruli from rats in all groups but, in each, the area of expansion was <25% of the mesangial area (Figure 4A and B). Capsular adhesions were observed in some glomeruli from some rats of group 1, but the renal interstitium of these rats was not particularly damaged. Matrix scores are shown in Table 1
.
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Immunofluorescence microscopy.
On direct IF, mouse IgG was detected in the glomeruli from rats in both group 1 and group 2 but was not seen in the tubulointerstitial area (Figure 5A and B
). A granular pattern could be seen more clearly in group 1, while a pseudo-linear pattern was observed in group 2. In the control group, neither the glomeruli nor the tubulointerstitial area were stained with anti-mouse IgG. No significant deposits of rat Ig occurred in the glomeruli or the interstitium in any group. Rat C3 was not deposited in the glomeruli in any group, but was observed in the intratubular space in the rats in group 1 (Figure 5C
).
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Electron microscopy.
No mesangial cell proliferation, matrix expansion nor subepithelial electron-dense deposits were observed in the glomeruli of rats from any of the groups. Partial effacement of the foot processes of the epithelial cells was found occasionally, especially in group 1 (Figure 10).
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Expression of mRNA for type I collagen
Expression of mRNA for type I collagen was not increased in group 1 (three rats with the heaviest proteinuria) when compared with the controls after 35 PCR cycles (Figure 11).
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Discussion |
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Previous in vivo studies to evaluate the injurious effects of proteinuria have used models such as PAN nephrosis [3,5], protein-overload proteinuria [4], adriamycin nephropathy [6] or passive Heymann nephritis [13], in which many factors may be involved in the pathogenesis. PAN or adriamycin may be injurious to tubular epithelial cells [3,6]. In the chronic PAN nephritis model in the unilaterally nephrectomized rat, the obliteration of the post-glomerular capillaries induced by glomerulosclerosis may lead to severe interstitial injury following tubulointerstitial ischaemia [14,15]. In addition, the artificial modification of haemodynamics may cause the progression of tubular damage. Haemodynamics are also greatly modified by nephrectomy and hyperalbuminaemia in the protein-overload proteinuria model in unilaterally nephrectomized rats. The main urinary protein component in this model is heterologous albumin [4]. In passive Heymann nephritis, the anti-Fx1A antibodies filtered into the urine might bind to the antigen in the brush border and be harmful to tubular epithelial cells. Therefore, the possibility that factors other than proteinuria contribute to progressive tubulointerstitial injury cannot be ruled out for any of these models, and it remains unclear whether proteinuria alone harms the tubulointerstitium, promoting severe interstial injury and the progression of chronic renal failure. Taking all these factors into consideration, it is evident that an animal model of persistent nephrosis, which involves no severe glomerular abnormalities or artificially induced haemodynamic alterations, and can be induced by an agent which is not directly toxic to the tubulointerstitium, needs to be developed in order to investigate whether tubulointerstitial injuries would be caused directly by continuous proteinuria.
In this study, no abnormal findings were detected in either group 2 or the control group (Figures 49, Table 1
). This suggests that 5-1-6 has no direct toxic effect on the tubulointerstitium. We prepared mAb 5-1-6 from the supernatant of a culture in serum-free medium and induced immunotolerance to mouse Ig for this study. These procedures were considered to prevent immune complex formation leading to glomerulonephritis or interstitial damage. In group 1, massive proteinuria, hypoalbuminaemia and hypercholesterolaemia were observed (Figure 3C
and D
, Table 1
), and almost all of the urinary proteins were derived from host serum proteins (the average amount of urinary protein excreted was about 3 g/week and the mAb dose was 500 µg/week in group 1). Therefore, this model was similar to the nephrotic state in humans. In addition, severe glomerular injury was not induced (Figure 4
, Table 1
) and the animals were not manipulated with nephrectomy in the present model. Thus, we achieved our aim of developing a model of persistent nephrosis with no manoeuvres known to induce artificial haemodynamics and no severe glomerular abnormalities using an agent without direct tubular toxicity.
Using the model established in the present study, we examined the relationships between severity of proteinuria and several other factors. Interstitial oedema, cell infiltration and degeneration of the tubular epithelial cells were observed in the interstitium in nephrotic rats (Figure 4C). The kidney weight was positively correlated with the severity of proteinuria (Figure 3B
), and the increase in kidney weight might have been derived mainly from the interstitial oedema. No systemic oedema, massive ascites or pleural effusion was observed at sacrifice. The expression of vimentin indicated that tubular epithelial cell injury had occurred in group 1 (Figure 6A
). Similar findings have been made in PAN nephritis [3] and protein-overload proteinuria [4,16]. The number of OX1-, OX19- and ED1-positive cells that had infiltrated into the interstitium (Figure 3H
, Table 1
) was related to the severity of proteinuria. These inflammatory cell infiltrations are considered to be mediated by the release of chemoattractants, including MCP-1, RANTES, lipid-derived factors, etc. and by the up-regulation of adhesion molecules expression, as suggested by Eddy et al. [17]. In some rats with relatively high-grade proteinuria in group 1, the expression of TGF-ß on the tubular epithelial cells and the number of interstitial
-SMA-expressing cells were increased (Figure 9A
and C). Sustained proteinuria was suggested to induce these tubulointerstitial alterations dose dependently. However, renal function, which was evaluated by BUN and creatinine clearance, was not related to the severity of proteinuria (Figure 3E
and F
).
Progressive interstitial fibrosis was reported to be induced by sustained proteinuria in chronic PAN [5] and protein-overload proteinuria [4]. Jones et al. reported that in six unilaterally nephrectomized and PAN-injected rats which secreted daily urinary albumin of 130150 mg/100 g BW, interstitial fibrosis and increase of mRNA for collagen type I were induced by 6 weeks [5]. Although proteinuria was not examined selectivity, the amount of urinary daily total protein was estimated to be 200230 mg/100 g BW (if protein selectivity was entirely lost, content of urinary protein was equal to that of serum protein; albumin is about 65% of total protein; protein whose molecular weight is higher than albumin is about 30%). In the present model, the average amount of proteinuria was 208 mg/100 g BW in six rats with the heaviest proteinuria, and protein whose molecular weight was greater than that of albumin was >10% of total urinary protein in three rats with the heaviest proteinuria. Our observation period, 25 weeks, was 4-fold longer than the 6 weeks in their model. Thus, the tubulointerstitium in the present model is suggested to be exposed to 4-fold higher amounts of total protein and 1.3-fold more protein which was larger than albumin, even if the proteinuria in chronic PAN nephritis would be estimated at maximum. In their model, increases of matrix proteins in the interstitium were assessed semiquantitatively, and a relative intensity score (1 represented a normal scoring pattern; 2, slightly increased ECM staining; and 3, markedly increased ECM staining) was calculated [5]. The scores for collagen types I, III and IV, fibronectin and laminin ranged from 1.8 to 2.3 at 6 weeks [5]. In our model, according to the calculation method described by Jones et al. [5], the relative intensity scores for collagen types I and IV and laminin remained close to 1. Furthermore, interstitial fibrosis was reported to be induced within 3 weeks in the rat model of protein-overload proteinuria. The average amount of urinary protein was about 80 mg/100 g BW in this model. Thus, tubular cells in our model are considered to be totally exposed to a greater amount of urinary protein than those in previously applied models. The possibility that the interstitial fibrosis would be induced also in the present model by exposure to protein for a longer period could not be ruled out because interstitial cell infiltration, interstitial oedema, tubular epithelial cell injury and an increase of TGF-ß and -SMA staining were observed in the present model. However, the tubulointerstitial injury evidently was mild. The reason why the lesser degree of interstitial injury was induced in this study was considered to be that tubulotoxic and fibrogenic factors other than proteinuria were excluded. In order to induce rapidly progressive interstitial fibrosis, tubulotoxic or fibrogenic factors other than proteinuria may be essential.
In conclusion, we developed a novel rat model of nephrosis, which involved no manoeuvres known to induce artificial haemodynamics and no severe glomerular abnormalities. Isolated proteinuria induced the tubulointerstitial lesions, such as oedema, tubular epithelial cell injury and cell infiltrations, but the degree of the injury was much less than that in other previously applied models.
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Acknowledgments |
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Notes |
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References |
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