|Year : 2015 | Volume
| Issue : 1 | Page : 35-37
Establishment and study of passive heymann nephritis model
Xiaofan Cai, Yueyi Deng, Yifei Zhong
Division of Nephrology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
|Date of Web Publication||23-Jan-2015|
No. 725 South Wanping Road, Shanghai 200032
Source of Support: None, Conflict of Interest: None
Objective: To discuss the establishment and stability of passive Heymann nephritis (PHN) in order to provide help for experimental study of membranous nephropathy (MN). Materials and Methods: We used 20 male rats of Sprague-Dawley weighing 120 to 150 g to prepare renal antigen Fx1A based on hypotonic lysis, Ca 2+ aggregation of contaminants and differential centrifugation and used six male New Zealand white rabbits for preparing anti-serum which was injected to SD rats to establish the model in different dosages (1 mL/100 g and 0.5 mL/100 g). The proteinuria of six model making rats were tested after 1st Week and then sacrificed and studied by direct immunofluorescence (IF) for deposition of rat IgG and electron microscopy (EM) in different time (1 st week and 7 th week ) with or without a booster injection. Results: After 7 days (1 st week) of model making, all six rats showed proteinuria (higher than 100 mg/dL). IF and EM study showed a typical pathological features of PHN (MN) but the intensity of IF intensity and electron dense deposits decreased in the rats without a booster injection. Conclusions: A PHN model could be successfully prepared according to the method we reported while a spontaneous remission may appear several weeks after the model making.
Keywords: Animal model, Heymann nephritis, human membranous nephropathy
|How to cite this article:|
Cai X, Deng Y, Zhong Y. Establishment and study of passive heymann nephritis model. J Integr Nephrol Androl 2015;2:35-7
| Introduction|| |
Heymann nephritis (HN) is an experimental model of human membranous nephropathy (MN) in the rat, which can cause proteinuria ascribed to in situ formation of immune complexes and podocyte injury. It can be divided into two types, active Heymann nephritis (AHN) and passive Heymann nephritis (PHN). The active model utilizes the production of autologous antibodies to renal antigens, such as megalin and receptor-associated protein (RAP) by the rats' own immune system, while the passive one utilizes the antiserum to such antigens. [1 ] In PHN model, anti-serum(often another species including rabbits, goats, or sheep) to renal tubular border, that is, anti-Fx1A serum, which mainly contains anti-megalin and anti-RAP antibody is injected to rats for inducing the extremely similar pathological changes to MN. At present, PHN has already become a proverbial model for study of MN. In this article, we will discuss the establishment and stability of PHN in order to provide help for experimental study of MN.
| Materials and methods|| |
Preparation of renal tubular epithelial fractions (Fx1A)
The extraction of Fx1A were performed in accordance with the rapid isolation of kidney brush-border membranes.  We used 20 male rats of Sprague-Dawley weighing 120 to 150 g, whose kidneys were perfused in vivo with heparin saline (1000 mL physiological saline plus heparin 12,500 U, pH 7.0). Their cortices were separated and cut into paste. After adding 30 volume (volume/weight: mL/g) 50 mmol/L mannitol-2 mmol/L Tris-HC1 (4°C, pH 7.0) and 1 mol/L CaCl 2 solution to a final concentration of 10 mmol/L, the paste was homogenized for 5 min at the speed of 40 r/min and filtered by a 100-mesh stainless steel sieve. Stirred in ice bath for 5-10 min the homogenate was then centrifuged at 5000 r/min for 15 min by Hitachi Himac CR21G II. The sediment was removed and the clear supernatant extract was carefully decanted and centrifuged at 18,900 r/min for 20 min. The sediment, representing the subcellular brush-border membranes, was washed with distilled water and centrifuged at 18,900 r/min × g for 20 min again. The final sediment was designated to be Fx1A. It was resuspended in physiological saline (pH 7.0) to a concentration of 20 mg/mL and divided into 1.5 mL eppendorf tubules for 1 mL each.
Preparation of anti-sera to Fx1A
Six adult male New Zealand White rabbits weighing 2 to 2.5 kg were used. Anti-Fx1A sera were prepared by giving intermittent injections of Fx1A-Freund's complete adjuvant (CFA) mixture. 20 mg/mL Fx1A solution was mixed with the same volume of CFA by a self-made antigen emulsifying device for 2 min at the speed of 10,000 r/min to make it be an "oil in water" emulsion. Subcutaneous injections were given to each rabbits with the same dosage (20 mg Fx1A/rabbit) at intervals of 10 to 14 days over a period of nearly 2 months. The rabbits were drawn blood after injection 3 times and tested by ELISA-method.
Detection of anti-sera's titer by ELISA-method
20 mg/mL Fx1A antigen solution 50 μL was added into 0.01 mmol/L phosphate buffer saline (pH 7.4) to a final volume of 10 mL. After 4°C overnight, the coated antigen was added to 96-well plates for 100 μL/well. The anti-sera were successively diluted by five times following the initial titer 1:500 and were required to achieve 1:32,000 or higher as successful preparation.  Once again, 20 mg/mL Fx1A antigen solution was added into each well (50 μL/well) and incubated with the anti-serum at 37°C for 2 hours. After being washed by 0.01 mmol/L PBS-0.5% Tween (pH 7.4) for five times, horse radish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:10,000) was added (100 μL/well) and incubated at 37°C for 45 min following another five times washed as above-mentioned. Finally, the reaction liquid was colored by TMB and OD value measured.
Preparation and verification of Rats PHN model
Six rats of Sprague-Dawley (male/female: 3/3) weighing 150 to 180 g were divided into three groups (one male and one female each). All the rats were given two intraperitoneal injections of anti-Fx1A serum at intervals of 5 days while the dosages were different. Groups A and B were injected 1 mL/100 g body weight, while group C was given only half of that (0.5 mL/100 g). All the rats underwent proteinuria detection by test paper after 7 days of the second injection. Then group A was sacrificed and studied by direct immunofluorescence (IF) for deposition of rat IgG and electron microscopy (EM). Groups B and C were continuously fed to the sixth week and the male rats of the two groups were given another intraperitoneal injection of anti-serum with the same dosage before. A week later, the remaining rats were also killed and underwent IF and EM tests.
| Results|| |
Titer of anti-sera
After the fourth injection, the anti-sera of the remaining five rabbits (one rabbits died 10 days after the first injection) have already exceeded the titer 1:32,000. The graph charts of each anti-sera are shown in [Figure 1].
|Figure 1: Anti-sera's titer. All the titer demonstrated a significantly declined between 1:62,500 and 1:312,500|
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Proteinuria and pathological changes of PHN model
After 7 days of the second injection, urine protein test paper demonstrated that proteinuria of all six rats showed positive and higher than 100 mg/dL. The determinate results are shown in [Figure 2]. The kidneys of group A studied by IF had 2 to 3+ finely granular deposits of rat IgG. Electron dense deposits can be found in the subepithelial space [Figure 3] and [Figure 4].
|Figure 2: Proteinuria test by test paper method. Compared with the standard color version above, all the samples were positive (the test paper on the left-most is an unused one)|
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|Figure 3: Direct IF on group A rat kidneys stained for rat IgG. Strongly positive granular staining along the glomerular capillary wall is present (×400)|
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|Figure 4: Electron micrograph of group A. Electron dense deposits are located in subepithelial side near or in the GBM (×7000)|
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The kidneys of remaining rats in group B and C (the female rat of group B died on 5 th week) were also studied by the IF and EM after 7th week. In group B (the male one), after a booster injection the IF intensity was still 2+ and the electron dense deposits could be clearly found. In group C, the IF intensity of the male one which had a booster injection of anti-serum was 1 to 2+ while its electron dense deposits was basically the same with the group B. Instead, the female one which did not have a booster injection performed a decreased IF intensity (1+) and electron dense deposits [Figure 5] and [Figure 6].
|Figure 5: Direct IF on group B and C rat kidneys stained for rat IgG. The IF intensity of the one (group C female) without booster injection decreased|
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|Figure 6: Electron micrograph of group B and C. The amount and intensity of the electron dense deposits declined in the one (group C female) without booster injection|
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| Discussion|| |
In 1967, Edgington TS and Glassock RJ  reported a method for isolation of specific renal tubular epithelial antigens. It is now the most common method for the preparation of Fx1A antigen of Heymann nephritis. In our present study, we used a method for extracting brush-border membranes which is different from the one we mentioned above. It is a feasible method as the main target antigen of HN, megalin, is highly expressed in proximal tubule brush border. Simple steps and relative low speed of centrifuge are the advantages of this method. A higher dosage of anti-serum is needed as we did not choose tail vein injection but intraperitoneal one in order to prevent the death after injection. From the results of proteinuria test, IF and EM, we could confirm the model had been successfully made.
From the study on the stability of the model, we found that after 7 weeks, there is an alleviation of pathological changes in the model without a booster injection, mainly manifested as a decreased IF intensity and electron dense deposits. It is evitable that the titer and dosage of the anti-serum play an important role in the stability on the model. The higher titer and the greater dosage of anti-serum can make the stable time loger while the mortality may also be increased. But as the animal model of MN, PHN may also have a tendency of spontaneous remission. Moreover, female gender is associated with better probability of spontaneous remission and slower rate of progression in MN.  This may be the another reason to explain why the pathological lesions of the female rat without a booster injection reduced. Therefore, the result may be related to a variety of reasons.
In conclusion, PHN model could be successfully prepared according to the method we reported while a spontaneous remission may appear 6 weeks after model making.
| References|| |
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Edgington TS, Glassock RJ, Watson JI, Dixon FJ. Characterization and isolation of specific renal tubular epithelial antigens. J Immunol 1967;99:1199-210.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]