CN111419860A

CN111419860A - Glomerular lobular nephropathy modeling method

Info

Publication number: CN111419860A
Application number: CN202010194856.2A
Authority: CN
Inventors: 姜洪波; 孙利伟; 田玉玲; 杜娟; 刘宇奇; 马英伟; 贺岩; 赵艳玲; 张亮
Original assignee: CHANGCHUN CHILDREN S HOSPITAL
Current assignee: CHANGCHUN CHILDREN S HOSPITAL
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-07-17

Abstract

The invention discloses a glomerular lobular nephropathy modeling method, which comprises the following steps: 6.5mg/kg of adriamycin is intravenously injected into a male Wistar rat body on the first day, and 30-70 mg/kg of ceftazidime is continuously injected for three days every day. The glomeruli of the obtained male Wistar rat kidney disease model appear to be lobulated. Provides a new animal kidney disease model for the research of pathophysiology mechanism, molecular cytology, molecular biology and the like of kidney diseases, and provides a basis for preventing and treating clinical patients earlier.

Description

Glomerular lobular nephropathy modeling method

Technical Field

The invention belongs to the technical field of animal model modeling, and particularly relates to a modeling method for glomerular lobular nephropathy.

Background

China is a large country with kidney diseases (nephropathy), the incidence rate of uremia is 100-. According to the disease progression rate, renal failure can be classified into Acute Renal Failure (ARF) and Chronic Renal Failure (CRF). The pathogenesis of renal failure is not clear, so no effective drug treatment exists. Research on pathogenesis of renal failure and development of safe and effective medicaments are always problems to be solved urgently in clinic. The preclinical research of drugs mainly takes the form of animal experiments of tested drugs, namely, animal models are established, and then pathogenesis or intervention (pharmaceutical) test research is carried out. For many years, renal disease researchers have developed animal models adapted to various renal diseases using rats as research subjects.

Acute renal failure refers to acute renal disease characterized by acute change of renal function within hours to days, resulting in loss of capability of kidney to discharge waste nitrogen and maintain stable function of water and electrolyte in vivo; they can be classified into pre-renal, neutral and post-renal. Prerenal refers to decreased perfusion of renal parenchyma due to various causes, renal indicates that renal parenchymal injury is present, with renal ischemia and Acute Tubular Necrosis (ATN) caused by nephrotoxic drugs or toxins being the most common, others include Acute Interstitial Nephritis (AIN), glomerular disease and renal vascular disease, etc., and obstruction can occur anywhere in the urinary tract from the renal pelvis to the urethra due to postrenal urinary tract infection.

Chronic renal failure is a long-time and progressive nephron injury, which causes dysfunction of renal excretion function, leads to retention of nitrogen metabolites caused by change of internal environment of an organism, and finally causes abnormal metabolism of the organism. The pathogenesis of the disease comprises: (1) nephron high perfusion, high filtration; (2) nephron hypermetabolism; (3) the effect of phenotypic transformation of epithelial cells of renal tissue; (4) the effects of cytokines and growth factors on promoting fibrosis; (5) and others.

Common molding methods for ARF and CRF are: physical method and chemical method, the ARF commonly used drugs include (1) glycerol type, (2) oleic acid type, (3) mercury chloride type, (4) aminoglycoside type, (5) snake venom induction, (6) sodium fluoride induction, (7) cisplatin induction, CRF commonly used drugs include adenine and adriamycin, and two drugs are used together to make animal model or two or more methods are used together. In recent years, the establishment methods of Acute Renal Failure (ARF) and Chronic Renal Failure (CRF) animal models are continuously improved, different animal models have respective characteristics and application ranges, each model cannot be completely matched with the characteristics of clinical diseases due to the influence of multiple factors, and the degree of renal injury is different. Therefore, the further exploration is more in line with the pathological change characteristics of human acute and chronic renal failure, and the search for a unified, stable and simple animal model is still a problem to be solved at present.

Adriamycin is a classic rodent nephritis injury inducer, can induce early stage focal segmental glomerulosclerosis, and can well simulate the chronic nephritis condition of a human body. Sternberg first reported in 1970 that anthracyclines could cause nephritis. The doxorubicin-induced rat nephritis model was first reported in 1976, after which the mouse doxorubicin nephritis model was reported in 1998. The doxorubicin nephritis rat model led the researchers to extensive and intensive studies to further reveal the pathological mechanism of kidney injury. Adriamycin (Adriamycin, ADR) has renal toxicity, can damage renal proximal convoluted tubule epithelial cells, and the exfoliated tubule epithelial cells, proteins and other cell components in a lumen form a tube shape to block the lumen, so that the intratubular pressure of the kidney is increased, the kidney injury is aggravated, and CRF is finally formed.

The mechanism of the antibiotic-induced kidney damage has two aspects, namely, the direct cytotoxicity effect is realized, the toxic effect is directly generated by the chemical structure of the medicine, the toxic kidney damage is caused, and the body has anaphylactic reaction to the medicine, such as acute interstitial nephritis, and the medicine is used as an antigen, so that the immune reaction is activated, and the kidney is influenced. Ceftiofur causes renal damage. It causes kidney damage parallel to the dose administered, which can cause toxic kidney damage. The drug can be injected into experimental animals through intramuscular, intravenous or intraperitoneal injection, and finally accumulated in renal tubular epithelial cells through a series of metabolism in the bodies. Due to its cytotoxic effects, it eventually leads to tubular epithelial cell injury and necrosis, inducing ARF production.

Disclosure of Invention

The invention aims to provide a glomerular lobular nephropathy modeling method.

Application of adriamycin and ceftazidime in preparation of glomerular lobular kidney disease animal model

A method for modeling glomerular lobular nephropathy, comprising: injecting adriamycin into the animal body 1); 2) injecting ceftiofur;

the injection in the step 1) is intravenous injection; the injection in the step 2) is intramuscular injection;

the animal is a rat;

the animal is a male Wistar rat;

the step 1) injecting adriamycin on the first day; 2) starting to inject ceftiofur on the next day, and continuously injecting for three days;

the adriamycin injection amount on the first day is 1.5-7.5 mg/kg; the ceftiofur is 30-70 mg/kg per day;

the adriamycin is 6.5mg/kg, and the cefotaxime is 50-70 mg/kg per day.

The invention provides a glomerular lobular nephropathy modeling method, which comprises the following steps: 6.5mg/kg of adriamycin is intravenously injected into a male Wistar rat body on the first day, and 50-70 mg/kg of ceftiofur is continuously injected for three days every day. The glomeruli of the obtained male Wistar rat kidney disease model appear to be lobulated. Provides a new animal kidney disease model for the research of pathophysiology mechanism, molecular cytology, molecular biology and the like of kidney diseases, and provides a basis for preventing and treating clinical patients earlier.

Drawings

FIG. 1 is a diagram of the steps of animal experiment; 1. weighing; 2. anaesthetizing; 3. disinfecting the skin at the tail part; 4. tail vein injection; 5. sterilizing; 6. intramuscular injection; 7. cutting open the abdominal cavity; 8. isolating the abdominal aorta; 9. taking blood from abdominal aorta; 10. taking out the kidney;

FIG. 2A-C are standard curves of phosphorus content;

FIG. 3D-F sets of standard curves for phosphorus content;

FIG. 4 microscopic observation of rat kidney section results; a-1, magnifying by 100 times and observing a normal control group; a-2, magnifying by 400 times and observing a normal control group; b-1, observing a doxorubicin 6.5mg/kg dose group under 100-fold magnification; b-2, observing a doxorubicin 6.5mg/kg dose group under 400-fold magnification; c-1, a group with 100 times of magnification of 60mg/kg of ceftazidime; c-2, a 400-fold magnification ceftazidime 60mg/kg dose group;

FIG. 5 microscopic observation of rat kidney section results; d-1, observing a dosage group of 6.5mg/kg of adriamycin and 30mg/kg of ceftazidime under the magnification of 100 times; d-2, observing a dosage group of 6.5mg/kg of adriamycin and 30mg/kg of ceftazidime under 400 times of magnification; e-1, observing a dosage group of 6.5mg/kg of adriamycin and 50mg/kg of ceftazidime under the magnification of 100 times; e-2, observing a dosage group of 6.5mg/kg of adriamycin and 50mg/kg of ceftazidime under 400-fold magnification; f-1, observing a dosage group of 6.5mg/kg of adriamycin and 70mg/kg of ceftazidime under the magnification of 100 times; f-2, observing the adriamycin 6.5mg/kg and ceftazidime 70mg/kg dosage group under 400-fold magnification.

Detailed Description

Example 1 Adriamycin in combination with ceftiofur in establishing rat model of chronic renal failure

1. Animal selection and grouping

Rats were selected because most species of rats were very sensitive to doxorubicin kidney damage, male Wistar rats required an doxorubicin dose in the range of 1.5-7.5 mg/kg.ba L B/c mice of 9.8-10.4 mg/kg, male BA L B/c SCID mice required only 5.3 mg/kg.c57b L/c mice highly resistant to doxorubicin-induced kidney damage, but high doses were able to induce kidney damage.

Grouping: a: a normal control group; b: doxorubicin at 6.5mg/kg dose group; c: ceftiofur 60mg/kg dose group; d: a 6.5mg/kg doxorubicin + 30mg/kg ceftiofur dose group; e: doxorubicin at 6.5mg/kg + ceftiofur 50 mg/kg; f: 6.5mg/kg of adriamycin and 70mg/kg of ceftazidime; there were 6 groups of 3, 18 each.

2. Drug selection

Doxorubicin is an anthracycline antibiotic, which is rapidly cleared from the plasma and significantly associated with tissue, primarily metabolized by the liver, after entering the body. 4 to 5 percent of adriamycin can be excreted by human body through urine 5d, and 40 to 50 percent of the administration amount can be excreted through bile 7 d. After intravenous administration in rats and mice, doxorubicin is rapidly cleared from plasma, deposited in tissues, and slowly excreted via urine and bile. Doxorubicin is slowly metabolized and accumulates primarily in the kidney. Ceftiofur is a first-generation cephalosporin, is a pyridine derivative of cephalothin, has stronger effect on gram-positive bacteria than the former and also has stronger effect on escherichia coli, is mainly used for respiratory tract infection, skin and soft tissue infection, urinary tract infection, biliary tract infection and thoracic cavity and abdominal cavity infection caused by sensitive bacteria, and can also be used for meningitis, septicemia and pleurisy. After injection, the blood concentration is higher than that of cephalothin, and the excretion is slower. There was significant nephrotoxicity, which parallels the dose administered to the kidney. Doxorubicin and ceftiofur have certain complementary action in vivo metabolism, can both generate residual renal toxicity, are combined to prepare the renal failure animal model, have definite and stable effect, can be said to be short of the time of simple doxorubicin magic property, and are reported at home and abroad.

3. Animal modeling

The animal rooms alternate day and night for 12h to 12h, the animals are kept drinking water and eating freely at the temperature of 23 ℃ to 25 ℃, the animals are raised in a metabolism cage, and the experimental animals enter the animal rooms for adaptive feeding for one week and then enter the experiment; the rats were weighed and anesthetized, placed flat on a laboratory bench, with the left thumb and index finger pinching the two sides of the tail to fill the vein, the middle finger was used to hold the tail from below, the ring finger was used to clamp the tip of the tail, and the right tail vein was selected for injection. B. D, E, F group is administered by tail vein injection with adriamycin once at a dose of 6.5 mg/kg; the experimental mouse is fixed the next day after tail vein injection, one hind limb is selected to be placed at the muscle part at the outer side of thigh, the skin surface is disinfected by 75% alcohol, the right hand-held injector forms 60 degrees with muscle, muscle is quickly punctured, and muscle is pumped back to carry out intramuscular injection after no backflow matter exists. C. D, E, F the ceftiofur is injected into the groups according to the dosage of 60mg/kg, 30mg/kg, 50mg/kg and 70mg/kg respectively for three days; normal group a rats were given equal volumes of saline tail vein or intramuscular injection.

Collecting a specimen: collecting urine 3 days, 5 days, 7 days and 2 weeks after intramuscular injection, calculating urine volume, and detecting urine protein volume; the rats were drunk with 28 Gastrodia elata on 14 days, and the femoral artery was sampled (operation process).

Example 2 detection of urine protein content

Taking urine: feeding mice in a metabolism cage, normally providing water and feed, collecting and calculating 24h urine of each rat on the 3 rd day, the 7 th day and the second week after intramuscular injection, and collecting the urine for biochemical detection; the grouping is as follows:

1) normal control group: a3, A7, A14

2) Doxorubicin 6.5mg/kg group: b3, B7, B14

3) Ceftiofur 60mg/kg group: c3, C7, C14

4) Doxorubicin 6.5mg/kg + ceftiofur 30mg/kg group: d3, D7, D14

5) Doxorubicin 6.5mg/kg + ceftiofur 50mg/kg group: e3, E7, E14

6) Doxorubicin 6.5mg/kg + ceftiofur 70mg/kg group: f3, F7, F14

3. 7, 14 represent 3 day, 7 day, 14 day time points, respectively; as a result, it was found (Table 1) that the urine protein content of the group containing doxorubicin at 6.5mg/kg and ceftiofur at 60mg/kg increased as the drug action time extended, as compared with the normal control group; compared with the group with single action of adriamycin and ceftazidime, the group with combined action of adriamycin and ceftazidime has increased urine protein content.

Example 3 serum marker detection

1. Grouping of rats

1) Normal control group: a14, A28

2) Doxorubicin 6.5mg/kg group: b14 and B28

3) Ceftiofur 60mg/kg group: c14, C28

4) Doxorubicin 6.5mg/kg + ceftiofur 30mg/kg group: d14 and D28

5) Doxorubicin 6.5mg/kg + ceftiofur 50mg/kg group: e14, E28

6) Doxorubicin 6.5mg/kg + ceftiofur 70mg/kg group: f14, F28;

14. 28 represent 14 day, 28 day time points, respectively;

2. serum creatinine assay

Under alkaline conditions, creatinine (Cr) in serum reacts with picric acid to produce an orange-colored picric acid creatinine complex. Detecting the change of absorbance at the wavelength of 505nm to obtain the concentration of creatinine in the detected serum; creatinine (CR) assay kit (Nanjing institute) was used; adding 0.2ml of serum into 2ml of tungstic acid protein testing agent, fully and uniformly mixing, rotating/min at 3500 rpm, centrifuging for 10 minutes, taking supernatant, and testing according to the following table;

mixing, water bathing at 37 deg.C for 10min, cooling with running water, and measuring absorbance at 510nm with enzyme-labeling instrument;

calculating the formula:

3. urea nitrogen detection

Urea is hydrolyzed under the action of urease to generate ammonia ions and carbon dioxide, the ammonia ions and a phenol color developing agent generate a blue substance in an alkaline medium, the generation amount of the substance is in direct proportion to the content of the urea, and the color comparison is carried out at the wavelength of 640 nm.

The operation steps are as follows:

mixing, water bathing at 37 deg.C for 10min, wavelength of 640nm, light path of 1cm, adjusting to zero with double distilled water, and measuring absorbance

Calculating the formula:

4. serum potassium detection

In an alkaline medium, reacting potassium ions in the sample treated by the protein precipitator with NA-TPB to generate turbid and stable suspension; the turbidity is in direct proportion to the concentration of potassium ions in the sample; detecting with potassium (K) test kit (built in Nanjing); taking 20ul serum and 180ul precipitator (prepared in situ), fully and uniformly mixing, 3500 rpm/min, centrifuging for 10min, and taking 50ul supernatant for determination; the operation steps are as follows:

mixing, standing for 5min at wavelength of 460nm and light path of 1cm, adjusting to zero with double distilled water, and measuring absorbance of each tube, wherein the calculation formula is as follows:

5. serum calcium detection

Calcium ions in the sample are combined with Methyl Thymol Blue (MTB) in an alkaline solution to generate a blue complex; comparing the color with a calcium standard treated in the same way to calculate the content of calcium in the sample; detecting with calcium content determination kit (Nanjing kit); serum procedure is as follows:

uniformly mixing, standing for 5 minutes, carrying out color comparison by an enzyme-labeling instrument at the wavelength of 610nm, and measuring the OD value of each hole; calculating the formula:

6. serum phosphorus detection

Inorganic phosphorus in the sample reacts with molybdic acid to generate phosphomolybdic acid, the phosphomolybdic acid is reduced to molybdenum blue, a maximum absorption peak is formed at 660nm, and the content of the inorganic phosphorus can be calculated through colorimetry; detecting with phosphorus content determination kit (Nanjing kit); taking 0.1ml of serum and 0.4ml of precipitator, fully and uniformly mixing, carrying out 3500 rpm centrifugation for 10 minutes, and taking the supernatant to be tested; the operation steps are as follows:

mixing, water bathing at 37 deg.C for 30 min, cooling to room temperature, wavelength of 660nm, optical path of 1cm, adjusting to zero with double distilled water, and measuring absorbance of each tube;

preparing and calculating standard curve by diluting 10 mmol/L phosphorus standard stock solution with deionized water to different concentrations (0.0625, 0.125, 0.25, 0.5, 1, 2 mmol/L), preparing standard curve according to operation table, drawing standard curve with the concentration of standard substance as abscissa and OD value as ordinate, and calculating linear regression equation y =0.4475x +0.006 (R) of standard curve by using the concentration of standard substance and OD value²= 0.9945), the OD value of the sample is substituted into the equation to calculate the concentration of the sample, and then the sample concentration is multiplied by the dilution factor to obtain the actual concentration of the sample, the OD value of the sample is measured, and the content of phosphorus (mmol/L) in the sample is calculated according to a standard curve, wherein the standard curve of the phosphorus content is shown in figure 2 and figure 3;

7. results

The experimental results are shown in Table 3, and it can be seen from the table that, compared with the normal control group, the serum creatinine, urea nitrogen, blood potassium and phosphorus contents of the adriamycin group of 6.5mg/kg and the ceftiofur group of 60mg/kg are increased, and the calcium content is reduced; the content of serum creatinine, urea nitrogen, blood potassium and phosphorus is continuously increased along with the prolonging of the action time of the medicine, and the content of calcium is reduced; compared with the group with simple action of the adriamycin and the ceftazidime, the group with combined action of the adriamycin and the ceftazidime has the advantages that the serum creatinine, the urea nitrogen, the serum potassium and the serum phosphorus content are increased, the calcium content is reduced, and the index values of the group with 6.5mg/kg of the adriamycin and 70mg/kg of the ceftazidime are increased or reduced the most.

Example 4 routine pathological examination of rat kidney

Anesthetizing rats in each group (A-F) on day 28, cutting off abdominal cavities, taking kidney tissues of the rats, fixing and embedding the kidney tissues, cutting the kidney tissues into paraffin sections with the thickness of 3 mu m, staining the kidney tissues by hematoxylin-eosin (HE), and observing pathological morphological changes of glomeruli and tubulointerstitial tissues under a light microscope; the method comprises the following specific steps:

1. tissue paraffin preparation

1) After the material was taken, the material was washed with PBS and fixed with 4% paraformaldehyde.

2) And (3) dehydrating: soaking the tissue in 75% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I and 100% ethanol II for 10min each;

3) and (3) transparency: soaking the dehydrated tissue in 1/2 xylene, xylene I and xylene II for 10 min;

4) wax penetration: soaking the transparent treated tissue in melted paraffin for 3 h;

5) embedding;

6) slicing: slicing the tissue in the paraffin block into thin slices with the thickness of 5um by using a slicing machine, and flatly paving the thin slices on an anti-falling glass sheet;

7) baking slices: the slices were placed on a 55 ℃ slide-baking machine to allow the tissue pieces to cling to the anti-detachment slides.

2. Paraffin section dewaxing rehydration

1) Dewaxing: soaking the paraffin sections in xylene I, xylene II and 1/2 xylene respectively for 10 min;

2) rehydration: soaking the dewaxed paraffin section in 100% ethanol I, 100% ethanol II, 95% ethanol I, 95% ethanol II, 85% ethanol, and 75% ethanol for 5min respectively;

3) cleaning with double distilled water for 2min for 3 times;

3. HE staining

1) Dyeing with hematoxylin dye liquor for 10 min;

2) washing the redundant dyeing solution with tap water for about 5 min; washing with distilled water again (for several seconds);

3) performing microscopic examination, and decolorizing with 1% hydrochloric acid alcohol if hematoxylin is stained too deeply, and removing excessive hematoxylin staining solution in cytoplasm;

4) dyeing with eosin dye liquor for 30 s;

5) washing the excessive dye liquor with tap water for about 3 min; washing with distilled water again (i.e. microscopic examination);

4. dehydration seal

Dehydrating with 95% ethanol for 2min, replacing with fresh 95% ethanol, and dehydrating for 2 min; the xylene is transparent for 5min, fresh xylene is used for replacement, and the xylene is transparent for 5min again; sealing the neutral resin; microscopic examination, the nucleus turns blue, and the cytoplasm turns red or pink;

the experimental result shows that the kidney shape of the normal control group is normal, the kidney structure is clear, and the glomerulus is regular; compared with a normal control group, glomerular mesangial cells of an adriamycin group of 6.5mg/kg and a ceftazidime group of 60mg/kg are proliferated, and a basement membrane is thickened; part of glomeruli of the adriamycin and ceftiofur combined action group are lobulated, plasma proteins in renal capsules exude, and lesions have certain changes along with the increase of the concentration of the ceftiofur.

Claims

1. Application of adriamycin and ceftazidime in preparation of glomerular lobular kidney disease animal models.

2. A method for modeling glomerular lobular nephropathy, comprising: injecting adriamycin into the animal body 1); 2) ceftiofur is injected.

3. The method of claim 1, wherein the method comprises: the injection in the step 1) is intravenous injection; the injection in the step 2) is intramuscular injection.

4. The method of claim 3, wherein the method comprises: the animal is a rat.

5. The method of claim 3 or 4, wherein the method comprises: the animal is a male Wistar rat.

6. The method of claim 5, wherein the method comprises: the step 1) injecting adriamycin on the first day; 2) ceftiofur was injected starting the next day for three consecutive days.

7. The method of claim 6, wherein the method comprises: the adriamycin injection amount on the first day is 1.5-7.5 mg/kg; the ceftiofur is 30-70 mg/kg per day.

8. The method of claim 7, wherein the method comprises: the adriamycin is 6.5mg/kg, and the cefotaxime is 50-70 mg/kg per day.