Animal model of chronic aristolochic acid nephropathy
Aristolochia plants can cause kidney damage. It has been found that the weight-loss drug "Slimming Pill", which contains Chinese herbs, can cause chronic renal failure, and its pathological change is "rapidly progressive renal interstitial fibrosis", and this kind of renal damage has been named as "Chinese herhsnephrology" (中草药肾病). Over the past few years, the problem of kidney damage caused by Chinese herbs has attracted extensive attention from scholars at home and abroad. It has been found that the main component of Chinese herbs causing such renal damage is aristolochic acid, and the renal damage caused by Chinese herbs is called "aristolochic acid nephropathy" (aristolochic arid nephrology). In recent years, acute kidney injury in rats caused by the application of the traditional Chinese medicines Aristolochic acid and Guanmutong has been reported in the domestic literature, but chronic aristolochic acid nephropathy has not been confirmed by animal experiments. Therefore, it is of great theoretical and practical significance to establish an animal morlels ofchronic aristolochic aecid nephrology in order to further study the mechanism of aristolochic acid-induced renal damage and its prevention and treatment measures.
Principle
Depending on the experimental method, the corresponding principles differ:
The basic principle of the animal model of chronic aristolochic acid nephropathy:
Mechanisms of renal injury induced by aristolochic acid include the ability to induce renal tubular epithelial cell enlargement, vacuolar degeneration, necrosis, and detachment, and prolonged use of the drug can lead to renal tubular atrophy, tubular transdifferentiation, and renal interstitial fibrosis.
Appliance
The commonly used application areas of chronic aristolochic acid nephropathy animal model are as follows: chronic aristolochic acid nephropathy animal model can be used to study the nephrotoxicity of aristolochic acid and its toxicity to other organs, and can also be used to study the mechanism of the occurrence of chronic interstitial fibrosis, which can help to evaluate the safety of the relevant Chinese herbal medicines.
Operation method
Animal model of chronic aristolochic acid nephropathy
Principle
Mechanisms of renal injury induced by aristolochic acid include induction of renal tubular epithelial cell enlargement, vacuolar degeneration, necrosis, and detachment, and prolonged use of the drug can lead to renal tubular atrophy, tubular transdifferentiation, and renal interstitial fibrosis.
Materials and Instruments
Subjects: Move The basic process of the animal model of chronic aristolochic acid nephropathy can be divided into the following steps: (2) Renal histomorphometry: Renal tissue specimens were fixed in 4% neutral buffered formaldehyde solution. Paraffin embedded specimens were made into 2 um thick sections, stained with HEPAS, PASM and Masson staining, and examined by light microscopy. The paraffin-embedded specimens were cut into 4 um thick sections, digested and examined by immunofluorescence. Renal cortex specimens were fixed with 3% glutaraldehyde for electron microscopy. Renal pathology image analysis method: CMIAS true color pathology image analysis system (developed by Beijing University of Aeronautics and Astronautics) was used to take images through an optical microscope magnified 100~400 times and input them into the image analysis system for pathology image analysis. Measurement of renal tubular area: 10 fields of view were randomly selected from each specimen under a 100× light microscope, and 5 proximal tubules were measured in each field of view, and the parameters of measurement were renal tubular area, tubular lumen area, tubular wall area, tubular lumen area/tubular area ratio, and tubular wall area/tubular area ratio. Interstitial area was measured by Masson staining at 400× light microscopy. Ten fields of view were randomly selected for each specimen, and the relative area of the renal interstitium, i.e., the ratio of the area of the renal interstitium to the area of the statistical field, was measured separately for each field of view. Caveat 1. Changes in blood and urine indicators. In the experimental group, the quantitative amount of 24-hour urinary protein increased slightly at different time points after treatment with aristolochic acid. Blood urea nitrogen and blood creatinine of rats in the aristolochic acid group increased significantly at 16 weeks after treatment. After stopping the drug, these two indexes did not decrease significantly, but continued to increase. 2. Observation of kidney specimens and pathological examination results. The renal surface of the rats in both groups was smooth, and no unevenness was observed. In the aristolochic acid group, the renal cortex became thinner after 16 weeks of drug administration, and the weight of the kidney was significantly reduced.Light microscopic examination showed that: at 8 weeks after the administration of aristolochic acid, there was no obvious damage to the kidneys, and only a few tubular epithelial cells had vacuolar degeneration; at 12 weeks after the administration of aristolochic acid, the number of tubular epithelial cells with vacuolar degeneration was increased, and the nuclei of some of the tubular epithelial cells were detached; at 16 weeks after the administration of aristolochic acid, the proximal tubular epithelial cells of the kidneys were swollen and the lumen of the tubes was narrowed, and some of the tubular epithelial cells were necrotic and apoptotic. At 20 weeks, the swollen renal tubular epithelial cells gradually shrunk, and some distal tubules were dilated, but no interstitial fibrosis was seen; at 24 weeks, more renal tubules were atrophied, and the renal interstitium showed multifocal fibrosis. The glomeruli were basically normal at 8 and 12 weeks, and there was a mild increase in glomerular mesangial cells at 16 weeks; the renal vasculature remained unchanged.Immunofluorescence: Aristolochic acid was negative for lgG, IgA, lgM, C3, and Clq, and fibrinogen was weakly positive at 16, 20, and 24 weeks (±).Electron microscopy showed that the primary lysosomes of rats in the aristolochic acid group increased slightly compared with those of the control group at 8 and 12 weeks, and a small amount of secondary lysosomes could be seen; at 16 weeks, the primary lysosomes and secondary lysosomes increased significantly, and the brush border of some renal tubular epithelial cells disappeared; at 20 and 24 weeks, a lot of secondary lysosomes could be seen in the cytoplasm, and medullary bodies could be seen, and the brush border of some renal tubular epithelial cells disappeared. For more product details, please visit Aladdin Scientific website.
① Rats.
Experimental reagents:
① 0.5 mg/mL aristolochic acid preparation;
② 4% neutral buffered formaldehyde solution.
A. Experimental animals and preparations.
(1) Experimental animals: female Wistar rats, 20~24 weeks old, weighing 190-210 g, provided by the Animal Center of Chinese Academy of Military Medical Sciences. During the experimental period, the rats were free to drink and eat, and the indoor temperature was controlled at 20~24 ℃ with good ventilation and humidity.
(2) Experimental reagents: Aristolochic acid standard (provided by the National Institute for the Control of Pharmaceutical and Biological Products of the National Health and Family Planning Commission) was used to formulate 0.5 mg/mL aristolochic acid preparation, which was autoclaved and stored in the refrigerator at 4 ℃ for spare use.
B. Modeling methods and detection indexes. Forty-two Wistar rats were selected, and each rat was injected intraperitoneally with aristolochic acid at a dose of 5 mg/(kg-d) for 16 experiments. Six rats were killed at the 8th, 12th, 16th, 20th and 24th weeks after the start of drug administration. The weight of the rats was measured at the time of execution, and blood, urine and kidney specimens were collected and stored in a refrigerator at -70 ℃ for biochemical and pathological examinations.
(1) Blood and urine specimens: blood urea nitrogen, serum creatinine (automatic biochemical analyzer), 24-hour urine protein quantification (turbidimetric method with trichloroacetic acid), and urinary β2-microglobulin (γ-radioimmunoassay).