Construction method and application of diabetic nephropathy animal model

Abstract

The invention belongs to the field of animal model building methods, and particularly discloses a diabetic nephropathy animal model building method and application thereof. The construction method comprises the following steps: (1) inducing hyperglycemia: injecting streptozotocin into a rat to induce hyperglycemia; and (2) saline feeding: after successful hyperglycemia induction, adopting a 0.3-2.0 wt% NaCl aqueous solution as drinking water for the rats, and continuously carrying out 6-16 weeks to obtain the diabetic nephropathy animal model. The diabetic nephropathy animal model obtained by the construction method can be used for development of medicines for preventing or treating nephropathy, medicines for reducing blood sugar and / or medicines for reducing blood pressure. The method provided by the invention is simple and convenient to operate and short in modeling time, the diabetic nephropathy animal model standards proposed by the American diabetic complication animal model association (Animal Models of Diabetic Complex Consortium, AMDCC) are met, various phenotypes are closer to clinical diabetic nephropathy characteristics, the scientific research requirements can be met, and the method has excellent technical effects.

Description

Technical Field

[0001] The present invention relates to the field of methods for establishing animal models, and in particular to a method for constructing a diabetic nephropathy animal model and its application. Background Art

[0002] Clinically, diabetic nephropathy is manifested by persistent large amounts of urinary albumin, at which time the glomerular filtration rate begins to decline rapidly, and glomerular mesangial nodules and renal interstitial fibrosis appear.

[0003] The Animal Models of Diabetic Complications Consortium (AMDCC) proposed the following standards for animal models of diabetic nephropathy: compared with the control group, the urinary albumin of the model animals increased by more than 10 times; the glomerular filtration rate of the model animals decreased by more than 50%; renal pathological changes included thickening of the glomerular basement membrane by more than 50% under electron microscopy, obvious mesangial matrix expansion with or without mesangial dissolution and nodular mesangial sclerosis, renal interstitial fibrosis, and varying degrees of arteriolar hyalinization.

[0004] Animal models of diabetic nephropathy can be divided into three categories according to the modeling method: inducible, spontaneous and transgenic. Among them, the latter two types of animal models are closer to human diabetic nephropathy, but their application is limited by factors such as difficulty in obtaining, high technical requirements, high prices, and slow disease progression. At present, most experiments use the inducible method to construct, especially the streptozotocin (STZ)-induced diabetes model is the most widely used. However, although the existing publicly reported STZ-induced diabetes model can cause a certain degree of increase in animal urinary albumin excretion, its renal lesions are relatively mild and cannot meet the diabetic nephropathy animal model standards and clinical diabetic nephropathy characteristics proposed by the American Association of Animal Models of Diabetes Complications (AMDCC).

[0005] Z. Qi et al. (Z. Qi, H. Fujita, J. Jin, LS Davis, Y. Wang, AB Fogo, MD Breyer, Characterization of Susceptibility of Inbred Mouse Strains to Diabetic Nephropathy, Diabetes, 54 (2005) 2628-2637.) used streptozotocin to induce C57BL / 6J, DBA / 2J and other mice, but the glomerular filtration rate (GFR) did not decrease, and the clinical characteristics of diabetic nephropathy could not be simulated.

[0006] F. Palm, H. et al. P. Hansell, P. Liss, P. -O. Carlsson, Differentiating between effects of streptozotocin per se and subsequent hyperglycemia on renal function and metabolism in the streptozotocin-diabetic rat model, Diabetes. Metab. Res. Rev, 20 (2004) 452-459.) After Wistar-Furth rats were induced with streptozotocin, the glomerular filtration rate (GFR) did not decrease, and there was no tubular atrophy, which could not simulate the clinical characteristics of diabetic nephropathy.

[0007] KA, J et al. (KA, JG, EA C, AA, Rapid development of glomerulosclerosis in diabetic Dahl salt-sensitive rats, Diabetologia, 40 (1997) 367-373.) used streptozotocin to induce glomerulosclerosis in Dahl salt-sensitive rats, but found that the glomerulosclerosis was not obvious and could not simulate the clinical characteristics of diabetic nephropathy.

[0008] Therefore, there is still an urgent need for a method for constructing an animal model of diabetic nephropathy that is simple to operate, takes a short time to build a model, meets the standards for animal models of diabetic nephropathy proposed by the American Animal Models of Diabetes Complications Consortium (AMDCC), has various phenotypes that are closer to the clinical manifestations of diabetic nephropathy, can meet scientific research needs, and has excellent technical effects. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides a method for constructing an animal model of diabetic nephropathy.

[0011] A method for constructing an animal model of diabetic nephropathy comprises the following steps:

[0012] (1) Inducing hyperglycemia: Rats were injected with streptozotocin (STZ) to induce hyperglycemia;

[0013] (2) Saline feeding: After successful induction of hyperglycemia, 0.3 wt%-2.0 wt% NaCl aqueous solution was used as drinking water for 6 to 16 weeks to obtain an animal model of diabetic nephropathy.

[0014] In some embodiments, the rat is a Dahl salt sensitive (DS) rat.

[0015] In some embodiments, the step (2) comprises: after the hyperglycemia is successfully induced, a 0.3wt%-2.0wt% NaCl aqueous solution is used as the drinking water of the rats for 11 to 13 weeks (e.g., 11 weeks, 12 weeks, or 13 weeks) to obtain a diabetic nephropathy animal model. In some embodiments, the step (2) comprises: after the hyperglycemia is successfully induced, a 0.3wt%-2.0wt% NaCl aqueous solution is used as the drinking water of the rats for 12 weeks to obtain a diabetic nephropathy animal model.

[0016] In some embodiments, the injection in step (1) is intraperitoneal injection.

[0017] In some embodiments, the step of inducing hyperglycemia comprises: intraperitoneally injecting 30mg-60mg or 40mg-50mg of streptozotocin per 1kg of rat body weight to induce hyperglycemia. In some embodiments, the step of inducing hyperglycemia comprises: intraperitoneally injecting 30mg, 35mg, 40mg, 45mg, 50mg, 55mg or 60mg of streptozotocin per 1kg of rat body weight to induce hyperglycemia.

[0018] In some embodiments, the step of inducing hyperglycemia comprises: intraperitoneally injecting 45 mg of streptozotocin per 1 kg of rat body weight to induce hyperglycemia.

[0019] In some embodiments, the successful induction of hyperglycemia means that blood glucose is ≥16.7 mM for two consecutive days after randomly testing blood glucose for 7 days after injection of streptozotocin.

[0020] In some embodiments, the rats are fasted for 8 hours to 12 hours before the injection of streptozotocin, but are not deprived of water.

[0021] In some embodiments, the streptozotocin is dissolved into a solution of 7mg / ml-12mg / ml or 9mg / ml with a sodium citrate buffer solution before intraperitoneal injection. In some embodiments, the citrate concentration in the sodium citrate buffer solution is 0.05mol / L-0.20mol / L. In some embodiments, the citrate concentration in the sodium citrate buffer solution is 0.05mol / L, 0.06mol / L, 0.07mol / L, 0.08mol / L, 0.09mol / L, 0.1mol / L, 0.11mol / L, 0.12mol / L, 0.13mol / L, 0.14mol / L, 0.15mol / L or 0.2mol / L. In some embodiments, the citrate concentration in the sodium citrate buffer solution is 0.1mol / L.

[0022] In some embodiments, the pH of the sodium citrate buffer solution is 4.0-5.0. In some embodiments, the pH of the sodium citrate buffer solution is 4.0, 4.1, 4.2, 4.3, .4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0023] In some preferred embodiments, the drinking water in step (2) is a 0.3wt%-1.5wt% NaCl aqueous solution. In some embodiments, the drinking water in step (2) is a 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt% or 1.5wt% NaCl aqueous solution. In some more preferred embodiments, the drinking water in step (2) is a 0.3wt%-0.9wt% NaCl aqueous solution. In some most preferred embodiments, the drinking water in step (2) is a 0.8wt%-0.9wt% NaCl aqueous solution. In some embodiments, the drinking water in step (2) is most preferably a 0.9wt% NaCl aqueous solution, which is more conducive to obtaining a model that meets the diabetic nephropathy model standards proposed by AMDCC and clinical diabetic kidney characteristics, including increased urinary albumin, a decrease in glomerular filtration rate of more than 50%, thickening of the glomerular basement membrane, expansion of the mesangial matrix accompanied by tuberous sclerosis, renal interstitial fibrosis and arteriolar hyalinization.

[0024] In some embodiments, the step (1) of injecting streptozotocin uses 6-7 week old DS rats.

[0025] In some embodiments, the rat is male.

[0026] In a second aspect, the present invention provides an application of the aforementioned construction method.

[0027] A use of the diabetic nephropathy animal model constructed by the construction method of the first aspect in the development of drugs for preventing or treating nephropathy, drugs for lowering blood sugar and / or drugs for lowering blood pressure.

[0028] In some embodiments, the drug for preventing or treating kidney disease includes: a drug for preventing or treating chronic kidney disease and / or a drug for preventing or treating diabetic nephropathy.

[0029] Beneficial Effects

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The method provided by the present invention is easy to operate, takes a short time to establish the model, meets the animal model standards for diabetic nephropathy proposed by AMDCC, and has various phenotypes that are closer to the clinical conditions of diabetic nephropathy, can meet the needs of scientific research, and has excellent technical effects.

[0032] (2) Compared with other concentrations of NaCl aqueous solution, the present invention preferably uses 0.8wt%-0.9wt% NaCl aqueous solution, and more preferably uses 0.9wt% NaCl aqueous solution as drinking water for rats after successful STZ-induced diabetes, which is conducive to obtaining a model that meets the diabetic nephropathy model standards proposed by AMDCC and clinical diabetic nephropathy characteristics, including an increase of more than 10 times in urine albumin, a decrease of more than 50% in glomerular filtration rate, thickening of glomerular basement membrane, expansion of mesangial matrix accompanied by tuberous sclerosis, renal interstitial fibrosis and arteriolar hyalinization, etc.

[0033] Terminology

[0034] In the description of the present invention, “plurality” means two or more than two, unless otherwise clearly and specifically defined.

[0035] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0036] In the following, all numbers disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number with a value of N is disclosed, any number with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15% or N+ / -20% will be explicitly disclosed, where "+ / -" means plus or minus.

[0037] The term "wt%" means percent by mass.

[0038] The term "% vol" means volume percentage.

[0039] The term "P<0.001" indicates that the result has extremely significant or extremely highly significant statistical significance, indicating that there is a probability of more than 99.9% that the difference or relationship reflected in the sample data is almost impossible to be generated by random, and is very reliable and significant.

[0040] The term "P<0.01" indicates that the result has a highly significant statistical significance, which means that there is a probability of more than 99% that the differences or relationships reflected in the sample data are not caused by random factors, and the reliability of the result is high.

[0041] The term "P<0.05" indicates that the result is statistically significant, indicating that the differences or relationships reflected in the sample data are unlikely to be caused by random errors, and there is a probability of more than 95% that there are real differences or relationships. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a statistical graph showing the effects of streptozotocin and saline on body weight, cumulative water intake, random blood glucose and animal survival rate in Example 1.

[0043] Figure 2 The statistical graphs show the effects of streptozotocin and saline induction on urine albumin and urine albumin to creatinine ratio (UACR) in Example 1.

[0044] Figure 3 This is a statistical chart showing the effects of streptozotocin and saline on blood pressure and eGFR (eGFR) in Example 1.

[0045] Figure 4 These are H&E, periodic acid-Schiff (PAS), Masson staining images and histopathological scoring statistics of kidney sections in Example 1.

[0046] In the figure, * indicates that compared with the DS rat group, P<0.05; ** indicates that compared with the DS rat group, P<0.01; *** indicates that compared with the DS rat group, P<0.001. # indicates that compared with the STZ-DS rat group, P<0.05; ## indicates that compared with the STZ-DS rat group, P<0.01; ### indicates that compared with the STZ-DS rat group, P<0.001. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further describe the present invention in detail.

[0048] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0049] The content of the NaCl aqueous solution in the following examples is in mass percentage, such as 0.3% NaCl aqueous solution means 0.3wt% NaCl aqueous solution; 0.9% NaCl aqueous solution means 0.9wt% NaCl aqueous solution; 2.0% NaCl aqueous solution means 2.0wt% NaCl aqueous solution.

[0050] Sodium citrate buffer solution: citrate 0.1 mol / L, pH 4.5.

[0051] Detection methods for each detection item of the present invention:

[0052] 1. Urine albumin to creatinine ratio (UACR) test

[0053] Urinary albumin concentration was measured using a rat microalbumin ELISA kit (Ethos Biosciences, product number NR002), and urine creatinine concentration was measured using an automatic biochemical analyzer (Roche, Cobas c311). The urine albumin-creatinine ratio (UACR) was calculated as urine albumin (μg) / urine creatinine (g).

[0054] 2. Glomerular filtration rate (eGFR)

[0055] Rats were anesthetized with isoflurane (1.5% vol, flow rate 0.5 L / min) and then placed on a warming table to maintain body temperature at 37°C. Afterwards, 0.9 wt% NaCl aqueous solution containing 2% bovine serum albumin and 2 mg / ml fluorescein isothiocyanate (FITC) marker was intravenously infused into the rats to measure glomerular filtration rate. At 1 minute, 2 minutes, 3 minutes, 5 minutes, 7 minutes, 15 minutes, 35 minutes, 55 minutes and 75 minutes after injection, 5 μl of blood was collected from the rat orbit using a disposable micro-blood collection tube, and the centrifuge tube containing anticoagulant (5 μl heparin sodium solution) was stored in the dark. After centrifugation at 3000 rpm for 10 minutes, 1 μl of supernatant (plasma) was diluted with 40 μl 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer (pH = 7.4) in a 96-well plate. The plasma inulin concentration was measured using a fluorescence microplate reader (PHERAstar FS, BMG Labtech, Germany) and the glomerular filtration rate was calculated.

[0056] 3. Histopathological Scoring

[0057] Glomerular injury and renal interstitial fibrosis were scored in a blinded manner. Glomerular injury was scored according to the thickening of the basement membrane, expansion of the mesangial matrix with or without mesangial dissolution and nodular mesangial sclerosis, and renal interstitial fibrosis was scored according to the number, morphology and distribution of fibers. The score ranged from 0 to 4 points. Among them, 0 points represented normal; 1 point represented 25% of the damaged area; 2 points represented 50% of the damaged area; 3 points represented 75% of the damaged area; 4 points represented more than 75% of the damaged area.

[0058] Tubular injury was also scored in a blinded manner, including epithelial flattening, dilation, necrosis, inflammatory infiltration, and protein casts. The score range is 0-5 points. 0 points represent normal; 1 point represents 11% of the damaged area; 2 points represent 25% of the damaged area; 3 points represent 50% of the damaged area; 4 points represent 75% of the damaged area; 5 points represent more than 75% of the damaged area.

[0059] All morphological measurements were performed by two examiners who were blinded to the treatment regimen.

[0060] Statistical Analysis

[0061] Data for pharmacological experiments are presented as mean ± standard error of the mean (SEM). Differences among groups were analyzed by one-way or two-way analysis of variance (ANOVA) using GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were considered statistically significant. P values ​​< 0.05, < 0.01, and < 0.001 were reported in the text.

[0062] Embodiment 1:

[0063] 1. Methods and Materials

[0064] 1.1 Animals / Ethics

[0065] Animal experiments were performed according to protocols approved by the Animal Care and Use Committee of the Laboratory Animal Center of Guangdong East Sunshine Pharmaceutical Co., Ltd. All animals were euthanized by carbon dioxide inhalation to ensure minimal suffering to the animals.

[0066] DS rats (Dahl salt-sensitive rats) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Unless otherwise stated, all animals were housed in a temperature- and humidity-controlled environment with a 12-h light / dark cycle and free access to food and water. Animals were acclimated to the study conditions for at least three days before the first dose.

[0067] 1.2 Construction of diabetic nephropathy model

[0068] (1) Inducing hyperglycemia: 6-7 week old male DS rats were intraperitoneally injected with 45 mg STZ per kg of rat body weight (using 9 mg / ml STZ sodium citrate buffer solution for intraperitoneal injection) to induce hyperglycemia; another 6 6-7 week old male DS rats were intraperitoneally injected with 5 ml sodium citrate buffer solution per kg of rat body weight to serve as DS rats without STZ induction;

[0069] (2) Water or saline feeding: 7 days after injection of STZ, DS rats (diabetes induced successfully) with blood glucose ≥16.7 mM for two consecutive days were selected and grouped according to Table 1 for subsequent operations. DS rats without STZ induction and DS rats after STZ induction were fed with water, 0.3 wt% NaCl aqueous solution, 0.9 wt% NaCl aqueous solution, and 2.0 wt% NaCl aqueous solution as drinking water (see Table 1), and were fed with conventional animal feed for 12 consecutive weeks to obtain diabetic nephropathy animal models. During the 12 consecutive weeks, the amount of water consumed was measured every day, the random blood glucose and body weight were measured every week, the blood pressure was measured in the 8th week, and the glomerular filtration rate was measured in the 10th week. During the entire study period, urine was collected every 4 weeks to measure the urine albumin and creatinine in the urine, and then the urine albumin to creatinine ratio (UACR) was calculated. At the end of the study, the kidneys were collected and fixed in 10% buffered formalin solution. Paraffin sections were prepared and stained with H&E, periodic acid-Schiff (PAS), and Masson to assess the extent of tubular, glomerular, and interstitial damage.

[0070] Table 1: Grouping

[0071]

[0072] 1.3 Results

[0073] 1.3.1 Effects of streptozotocin and saline induction on body weight, cumulative water intake, random blood glucose levels, and animal survival rate

[0074] Weight trends Figure 1 Figure A in the middle; the trend of cumulative water intake is shown in Figure 1 Figure B; random blood sugar trend is shown in Figure 1 Middle C; survival rate see Figure 1 Figure D.

[0075] Depend on Figure 1 As shown in Figure A, compared with the DS group, the body weight of the STZ-induced DS rats (STZ-DS group, STZ-DS+0.3% NaCl group, STZ-DS+0.9% NaCl group) decreased significantly.

[0076] Depend on Figure 1 As shown in Figure B, compared with the DS group, the cumulative water intake of the STZ-DS group was significantly increased; when NaCl was added to the drinking water (STZ-DS + 0.3% NaCl group, STZ-DS + 0.9% NaCl group), the water intake of DS rats further increased, among which the increase in the STZ-DS + 0.9% NaCl group was more significant.

[0077] Depend on Figure 1 As shown in Figure C, compared with the DS group, the random blood glucose of STZ-induced DS rats (STZ-DS group, STZ-DS+0.3% NaCl group, STZ-DS+0.9% NaCl group) was significantly increased, indicating that the diabetes model was successfully established.

[0078] Depend on Figure 1 As shown in Figure D, all rats in the STZ-DS+2.0% NaCl group died on the 7th day after saline feeding, and no organ abnormalities were found in the autopsy of the animals. On the 63rd day of saline feeding, one animal in the STZ-DS+0.9% NaCl group died. On the 67th day of saline feeding, one animal in the STZ-DS group died. Therefore, the maximum concentration of NaCl should not exceed 2%.

[0079] 1.3.2 Effects of streptozotocin and saline induction on urine albumin and urine albumin:creatinine ratio

[0080] Urine albumin results are shown in Figure 2 Figure A, urine albumin-to-creatinine ratio (UACR) results are shown in Figure 2 Middle Figure B.

[0081] Depend on Figure 2As shown in Figure A, compared with the DS group, the urine albumin levels of STZ-induced DS rats (STZ-DS group, STZ-DS + 0.3% NaCl group, STZ-DS + 0.9% NaCl group) were significantly increased. At the 12th week of modeling, the urine albumin level of the STZ-DS + 0.9% NaCl group (2,174.2 ± 307.3) was about 12 times that of the DS group (175.2 ± 42.6), and the urine albumin level of the STZ-DS group (872.6 ± 223.1) was about 5 times that of the DS group. Compared with the STZ-DS group, the urine albumin level of the STZ-DS + 0.9% NaCl group increased more significantly.

[0082] UACR is used to assess kidney health and detect potential kidney disease. When UACR is significantly elevated, it indicates that the kidneys may be damaged. Figure 2 As shown in Figure B, the UACR of each modeling group showed a gradual upward trend during the feeding period, indicating that renal damage gradually worsened with the passage of modeling time. The UACR of STZ-induced DS rats (STZ-DS group, STZ-DS + 0.3% NaCl group, STZ-DS + 0.9% NaCl group) increased faster. At the 12th week of modeling, compared with the STZ-DS group, the STZ-DS + 0.3% NaCl group and the STZ-DS + 0.9% NaCl group were significantly increased.

[0083] In summary, the urine albumin in the STZ-DS+0.9% NaCl group was more consistent with the animal model standard of diabetic nephropathy proposed by AMDCC.

[0084] 1.3.3 Effects of streptozotocin and saline induction on blood pressure and estimated glomerular filtration rate (eGFR)

[0085] Blood pressure results at week 8 Figure 3 Figure A, the results of eGFR at week 12 are shown in Figure 3 Middle Figure B.

[0086] Depend on Figure 3 As shown in Figure A, compared with the DS group, the blood pressure of STZ-induced DS rats (STZ-DS group, STZ-DS+0.3% NaCl group, STZ-DS+0.9% NaCl group) was significantly increased, among which the blood pressure of the STZ-DS+0.9% NaCl group was even higher.

[0087] Depend on Figure 3 As shown in Figure B, at the 12th week of modeling, the eGFR of the STZ-DS group + 0.9% NaCl group (138.9±14.35) was reduced by 50% compared with the DS group (281.9±12.80), and the eGFR of the other groups decreased slightly.

[0088] Therefore, the blood pressure and eGFR of the STZ-DS+0.9% NaCl group were more consistent with the animal model standard of diabetic nephropathy proposed by AMDCC.

[0089] 1.3.4 Effects of streptozotocin and saline on renal pathology

[0090] At the end of the experiment, kidneys were collected and sectioned for staining to evaluate kidney damage. The experimental results are shown in Figure 4 shown.

[0091] Depend on Figure 4 As shown in Figures A, B, and C, the kidneys of the STZ-DS+0.9% NaCl group showed severe renal damage, including thickening of the glomerular basement membrane, expansion of the mesangial matrix with tuberous sclerosis, renal interstitial fibrosis, tubular necrosis, and arteriolar hyalinization. Figure 4 Middle A, blue arrows indicate tubular necrosis; Figure 4 Middle A, green arrows indicate renal interstitial inflammation; Figure 4 Middle B: Yellow arrows show expansion of mesangial matrix with tuberous sclerosis. Figure 4 Middle B, black arrows indicate glomerular nodule formation; Figure 4 (B, red arrows indicate hyalinosis of small arteries).

[0092] Depend on Figure 4 As shown in Figures D, E, and F, compared with the DS group, the renal tubular injury index ( Figure 4 Middle D), glomerular sclerosis index ( Figure 4 E) and renal interstitial fibrosis score ( Figure 4 The levels of leucocytes in the STZ-DS+0.9% NaCl group were increased, and the increase was most significant in the STZ-DS+0.9% NaCl group.

[0093] 1.4 Conclusion

[0094] Compared with DS rats fed with water after STZ induction (STZ-DS group) and DS rats fed with 0.3wt% NaCl aqueous solution after STZ induction (STZ-DS+0.3% NaCl group), DS rats fed with 0.9wt% NaCl aqueous solution after STZ induction (STZ-DS+0.9% NaCl group) are more in line with the diabetic nephropathy model standard proposed by AMDCC and clinical diabetic kidney characteristics, including more than 10-fold increase in urinary albumin, more than 50% decrease in glomerular filtration rate, thickening of glomerular basement membrane, expansion of mesangial matrix with nodular mesangial sclerosis, renal interstitial fibrosis and arteriolar hyalinization. Unexpected technical effects were achieved.

[0095] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for constructing an animal model of diabetic nephropathy, characterized in that: The following steps are involved: (1) Inducing hyperglycemia: Rats were injected with streptozotocin to induce hyperglycemia; (2) Saline feeding: After successful induction of hyperglycemia, 0.3 wt% to 2.0 wt% NaCl aqueous solution was used as drinking water for 6 to 16 weeks to obtain an animal model of diabetic nephropathy.

2. The construction method according to claim 1, characterized in that: The rats are Dahl salt-sensitive rats; and / or The step (2) comprises: after successful induction of hyperglycemia, using 0.3wt%-2.0wt% NaCl aqueous solution as drinking water for the rats for 11 weeks to 13 weeks or 12 weeks to obtain an animal model of diabetic nephropathy.

3. According to the construction method according to any one of claims 1-2, the injection in step (1) is intraperitoneal injection.

4. The construction method according to any one of claims 1 to 3, characterized in that: The step of inducing hyperglycemia comprises: intraperitoneally injecting 30 mg-60 mg or 40 mg-50 mg of streptozotocin per kg of rat body weight to induce hyperglycemia; or The step of inducing hyperglycemia comprises: intraperitoneally injecting 45 mg of streptozotocin per 1 kg of rat body weight to induce hyperglycemia.

5. The construction method according to any one of claims 1 to 4, characterized in that: The rats were fasted for 8-12 hours before injection of streptozotocin, but were not allowed to drink water.

6. The construction method according to any one of claims 1 to 5, characterized in that: The streptozotocin is first dissolved in sodium citrate buffer solution into a solution of 7 mg / ml-12 mg / ml or 9 mg / ml before intraperitoneal injection.

7. The construction method according to any one of claims 1 to 6, characterized in that: The drinking water in step (2) is a 0.3wt%-1.5wt% or 0.3wt%-0.9wt% NaCl aqueous solution.

8. The construction method according to any one of claims 1 to 7, characterized in that: The drinking water in step (2) is a 0.8wt%-0.9wt% NaCl aqueous solution.

9. The construction method according to any one of claims 1 to 8, characterized in that: The drinking water in step (2) is a 0.9 wt % NaCl aqueous solution.

10. Use of the diabetic nephropathy animal model constructed by the construction method according to any one of claims 1 to 9 in the development of drugs for preventing or treating nephropathy, drugs for lowering blood sugar and / or drugs for lowering blood pressure; Optionally, the drug for preventing or treating kidney disease comprises: Drugs for the prevention or treatment of chronic kidney disease and / or drugs for the prevention or treatment of diabetic nephropathy.

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