A method for constructing a folate-induced rabbit AKI model based on a single intravenous injection

Abstract

The application discloses a method for constructing a folate-induced rabbit AKI model based on single intravenous injection and belongs to the technical field of biomedical experimental models. The application solves the contradiction between folate solubility and the volume limitation of intravenous injection of rabbits, first establishes a standardized method suitable for a folate-induced acute kidney injury model of rabbits, and fills the blank of the folate-induced acute kidney injury model of medium-sized animals.

Description

Technical Field

[0001] This invention belongs to the field of biomedical experimental model technology, specifically relating to a method for constructing a folic acid-induced rabbit AKI model based on a single intravenous injection. Background Technology

[0002] Acute kidney injury (AKI) is a common critical illness among hospitalized patients, with an incidence rate as high as 10-15%, and is closely related to high mortality and the progression of chronic kidney disease (CKD). AKI is classified into prerenal, renal, and postrenal types, with renal AKI primarily caused by acute tubular necrosis (ATN). Currently, research on the pathological mechanisms of AKI and drug development heavily rely on animal models, but existing models have significant limitations: surgical models (such as ischemia-reperfusion) are complex to perform and have a mortality rate of 20-30%, and are prone to complications such as infection and multi-organ damage; nephrotoxic drug models (such as cisplatin and gentamicin) have multi-organ toxicity (such as liver damage) and interfere with research on kidney-specific mechanisms; sepsis models (such as LPS injection) exhibit strong systemic inflammatory responses and poor model stability.

[0003] Folic acid (FA), as vitamin B9, is highly expressed by folate receptors in the renal tubules. After glomerular filtration, its reabsorption rate is nearly 100%, and it accumulates in the proximal convoluted tubule. High doses can selectively induce kidney damage, leading to acute tubular necrosis (AKI) through mechanisms such as activating oxidative stress. This model is simple to operate, primarily targets the kidneys, has minimal impact on other organs, requires no surgery, and can successfully induce AKI within 24–48 hours. It is currently well-established and applied in rodents. In rodents, the commonly used dose of folic acid for inducing AKI is 250 mg / kg (intraperitoneal injection), and the folic acid concentration should not exceed 12.5 mg / mL, as excessively high concentrations increase the risk of animal mortality.

[0004] Rabbits, as medium-sized laboratory animals, possess multi-papillary kidney structure and a GFR range similar to humans. Their moderate size facilitates multiple blood draws and delicate procedures such as renal artery cannulation, making them a more ideal model for AKI research. However, applying folic acid models to rabbits faces unique challenges: folic acid has poor solubility. To meet the limitation of a single intravenous injection volume in rabbits (usually ≤10 mL / kg), using the conventional concentration (not exceeding 12.5 mg / mL) for rodent models would result in an excessive injection volume, potentially inducing acute heart failure. If fractionated injections are used, pharmacokinetic changes may affect the model's uniformity and stability. Preparing low-concentration solutions leads to excessively large injection volumes, easily inducing acute heart failure and significantly increasing the risk of animal mortality; fractionated injections may also affect the model's uniformity and stability due to changes in pharmacokinetic metabolism. Conversely, preparing excessively high-concentration folic acid solutions presents technical bottlenecks such as dissolution difficulties. The lack of systematically validated effective parameters for folic acid-induced AKI models in rabbits has long limited the application of medium-sized animal models in AKI research. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention provides a method for constructing a folic acid-induced acute kidney injury (AKI) model in rabbits based on a single intravenous injection. By resolving the contradiction between folic acid solubility and the limitation of intravenous injection volume in rabbits, this invention establishes for the first time a standardized method suitable for rabbits to develop a folic acid-induced acute kidney injury model, including specific dose gradients, safe concentration ranges, and optimal modeling window time, filling the gap in folic acid-induced acute kidney injury models for medium-sized animals.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a method for constructing a folic acid-induced rabbit AKI model based on a single intravenous injection, which includes the following steps: (1) Prepare a folic acid solution with a concentration of 25 mg / mL; (2) Calculate the injection volume of folic acid solution in step (1) based on the folic acid dose required for the AKI model and the weight of the rabbit, so that it is ≤10 mL / kg; (3) Based on the injection volume determined in step (2), a folic acid-induced rabbit AKI model is obtained by a single injection via the marginal ear vein.

[0007] Furthermore, the preparation process of the folic acid solution in step (1) is as follows: Prepare a weak alkaline solution with a pH of 8-9 and a concentration of 0.3 mM using ultrapure water. Then add folic acid powder and stir in a 37°C water bath until completely dissolved to obtain a folic acid solution with a pH of 7-7.5 and a final concentration of 25 mg / mL.

[0008] Furthermore, the weak alkaline solution is a sodium bicarbonate solution.

[0009] Furthermore, the folic acid dose used to construct the AKI model was 100-150 mg / kg.

[0010] Furthermore, the folic acid dose for the mild to moderate AKI model was 100 mg / kg, and the folic acid dose for the severe AKI model was 150 mg / kg.

[0011] Furthermore, in step (3), the injection rate for a single injection is 3~5 mL / min.

[0012] The beneficial effects of this invention are: 1. Addressing the core contradiction of excessive liquid load and difficulty in dissolving high concentrations of folic acid when extending the folic acid-induced AKI model from rodents to rabbits, this invention, through a systematic dose-concentration effect study, has for the first time verified that a single intravenous injection of folic acid solution in the concentration range of 25 mg / mL can successfully construct a rabbit AKI model while ensuring animal safety. This fills the gap in medium-sized animal models in this field and breaks through the technical bottleneck of species expansion.

[0013] 2. Studies have found that while using the conventional upper concentration limit (12.5 mg / mL) in rodent models can induce kidney injury in rabbits, the large injection volume easily leads to the risk of acute heart failure (prodromal symptoms such as shortness of breath during injection), resulting in poor model safety. This invention, by increasing the folic acid concentration to 25 mg / mL, did not observe any increase in vascular stimulation or systemic toxicity in the rabbit model. Instead, by reducing the injection volume, it achieved precise grading and modeling of the degree of kidney injury while ensuring animal safety.

[0014] 3. This invention successfully constructed rabbit models simulating mild-to-moderate and severe clinical AKI by establishing two dosage gradients of 100 mg / kg and 150 mg / kg, respectively. Verification through multiple dimensions, including serum biochemistry (BUN, Scr), urinary urinary cytokine response (UACR), pathological scores, and electron microscopy ultrastructure, confirmed that there were significant gradient differences in the degree of damage between the different dosage groups, and the models demonstrated good discriminative power. Attached Figure Description

[0015] Figure 1 A folic acid solution with a concentration of 25 mg / mL was prepared for this invention; Figure 2 Changes in BUN and Scr 24 hours after folic acid injection in different experimental groups; Figure 3 Changes in BUN and Scr at different time points after folic acid injection in Group 4; Figure 4UACR test results for different experimental groups after folic acid injection; Figure 5 Kidney morphology after folic acid injection in different experimental groups; Figure 6 Pathological staining results of kidneys after folic acid injection in different experimental groups; Figure 7 Acute kidney injury scores were measured in different experimental groups after folic acid injection. Figure 8 Transmission electron micrographs of kidney tissue after folic acid injection in different experimental groups. Detailed Implementation

[0016] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0017] Example 1 1. Preparation of folic acid solution Because folic acid (F8758, Sigma) has low solubility in water, a weakly alkaline solvent is required to aid dissolution. The specific preparation process is as follows: A 0.3 mM sodium bicarbonate solution (NaHCO3, S5761, Sigma) with pH 8.40 was prepared using ultrapure water. Folic acid powder was added to the above sodium bicarbonate solution and stirred in a 37 °C water bath until completely dissolved, forming a homogeneous, transparent, amber-colored solution. The final folic acid concentration was 25 mg / mL, and the pH was 7.34 (see [link to relevant documentation]). Figure 1 Store away from light.

[0018] 2. Construction of a rabbit acute kidney injury model (1) Twenty-five healthy adult New Zealand white rabbits (weighing 2.5–3.0 kg) were selected and acclimatized for ≥1 week under standard laboratory animal conditions, with free access to water and food. They were fasted for 12 hours before the experiment, but water was allowed. They were then divided into Groups 1–5 for the experiment, as follows: Group 1: Control group (physiological saline 12 mL / kg); Group 2: Low-dose folic acid solution at standard concentrations (100 mg / kg, 12.5 mg / mL). Group 3: Medium-dose folic acid solution at standard concentrations (150 mg / kg, 12.5 mg / mL). Group 4: Low-dose folic acid solution concentration group of the present invention (100 mg / kg, 25 mg / mL). Group 5: Medium-dose folic acid solution concentration group of the present invention (150 mg / kg, 25 mg / mL).

[0019] (2) Calculation of administration volume: Administration volume (mL) = body weight (kg) × dose (mg / kg) / solution concentration (mg / mL).

[0020] (3) After weighing, the experimental rabbits were fixed in a special rabbit rack. The marginal ear vein was selected, and after routine disinfection, a 24G indwelling needle was inserted into the ear vein. The injection was slowly administered in a single dose through the indwelling needle at a rate of 3-5 ml / min. The control group was given an equal volume of physiological saline. After the injection, the rabbits were returned to their cages, and their condition was closely observed. They were allowed free access to food and water.

[0021] 3. Model Validation and Evaluation (1) Comparison of serum biochemical indicators Serum urea nitrogen and creatinine levels were measured in each group 24 hours after drug administration. The results are shown in Table 1 and [Table data would be inserted here]. Figure 2 As shown: Table 1. Changes in BUN and Scr 24 hours after folic acid injection in different groups

[0022] Note: BUN-Pre blood urea nitrogen before folic acid injection, BUN-Pro blood urea nitrogen 24 hours after folic acid injection, Scr-Pre blood creatinine before folic acid injection, Scr-Pro blood creatinine 24 hours after folic acid injection; * P<0.05, *** P<0.001.

[0023] Group 1: BUN and Scr remained stable over 24 hours, at 8.30±0.65 mmol / L and 102.50±5.93 μmol / L, respectively, with no significant changes compared to baseline.

[0024] Group 2: BUN and Scr increased slightly over 24 hours, to 10.85±4.43 mmol / L and 113.24±13.00 μmol / L, respectively, but this change did not reach statistical significance.

[0025] Group 3: BUN and Scr were significantly elevated within 24 hours, at 13.38±4.87 mmol / L (P<0.05) and 152.66±12.90 μmol / L (P<0.001), respectively.

[0026] Group 4: BUN and Scr were significantly elevated, at 11.75±1.34 mmol / L (P<0.001) and 194.14±9.93 μmol / L (P<0.001), respectively.

[0027] Group 5: BUN and Scr were significantly elevated, at 28.30±3.16 mmol / L (P<0.001) and 625.06±92.4 μmol / L (P<0.001), respectively, showing the largest increase. No rabbit deaths were observed in any group during the experiment, but rabbits in Group 3 exhibited significantly rapid breathing during injection.

[0028] The above results demonstrate that the concentration of this invention (25 mg / mL) can achieve dose-dependent renal function injury while ensuring animal safety, and successfully constructs mild to moderate (Group 4) and severe (Group 5) folic acid-induced AKI models.

[0029] (2) Dynamic changes in renal function BUN and Scr levels were measured in Group 4 before drug administration and at 12h, 24h, and 48h after drug administration. The results are shown in the table below. Figure 3 .

[0030] like Figure 3 As shown, BUN and Scr began to rise at 12h, peaked at 24h, and partially declined at 48h but remained above baseline, indicating that folic acid-induced renal function damage is time-dependent, with 24 hours being the optimal assessment window for model formation.

[0031] (3) Comparison of urinary index (UACR) The UACR values ​​of rabbits in Group 1, Group 4, and Group 5 were measured, and the results are as follows: Figure 4 As shown, the UACR in Groups 4 and 5 was significantly higher than that in Group 1, with a larger increase in Group 5 (P<0.001) (see...). Figure 4 The results were consistent with changes in serum biochemical indicators, indicating that mild to moderate (Group 4) and severe (Group 5) folic acid-induced AKI models were successfully constructed.

[0032] (4) Morphological observation of the kidney The kidney morphology of rabbits in Groups 1, 4, and 5 was observed, and the results are as follows: Figure 5 As shown, Figure 5 From left to right, the measurements are as follows: Group 5, kidney length diameter 3.7cm; Group 4, kidney length diameter 4.4cm; Group 1, kidney length diameter 3.4cm; the unit of measurement is cm.

[0033] like Figure 5 As shown, the kidneys in Group 1 are normal in shape and rosy in color; the kidneys in Group 5 are most swollen and pale in color; the kidneys in Group 4 are between the two in terms of swelling and color, which is consistent with the degree of damage.

[0034] (5) Renal histopathological evaluation Rabbits in Groups 1, 4, and 5 underwent HE, PAS, and Masson staining to observe the pathological staining results of renal tissue. A semi-quantitative area score based on PAS staining was used to assess the degree of acute tubular injury. The results are shown in [Table / Reference]. Figure 6 and Figure 7 .

[0035] like Figure 6 and Figure 7 As shown, the kidney tissue structure of rabbits in Group 1 was intact, the renal tubular epithelial cells were tightly arranged, the brush border was clear, and the median acute tubular score was 1 (range 0-1); the renal tubular epithelial cells of rabbits in Group 4 were mildly swollen, the lumen was dilated, and the median semi-quantitative area score of PAS staining was 2 (range 1-2); the renal tubules of rabbits in Group 5 were extensively necrotic and sloughed off, the lumen was significantly dilated, accompanied by interstitial inflammatory cell infiltration, and the median semi-quantitative area score of PAS staining was 3 (range 2-4).

[0036] (6) Observation of ultrastructure Electron microscopy revealed the following: Group 1: Normal cellular structure in the renal tubular region, with no obvious abnormalities in the morphology of organelles such as the nucleus, mitochondria, endoplasmic reticulum, and lysosomes. Group 4: Mild swelling of mitochondria and mild dilation of the endoplasmic reticulum in the renal tubular region. Group 5: Significant swelling of mitochondria, marked dilation of the endoplasmic reticulum with vacuolar changes, and a significant increase in lysosomes in the renal tubular region (see [link to article]). Figure 8 This indicates that increased folic acid dosage exacerbates ultrastructural damage to renal tubular epithelial cells, which is positively correlated with the severity of AKI.

[0037] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing a folic acid-induced rabbit AKI model based on a single intravenous injection, characterized in that, Includes the following steps: (1) Prepare a folic acid solution with a concentration of 25 mg / mL; (2) Calculate the injection volume of folic acid solution in step (1) based on the folic acid dose required for the AKI model and the weight of the rabbit, so that it is ≤10 mL / kg; (3) Based on the injection volume determined in step (2), a folic acid-induced rabbit AKI model is obtained by a single injection via the marginal ear vein.

2. The method according to claim 1, characterized in that, The preparation process of folic acid solution in step (1) is as follows: Prepare a weak alkaline solution with a pH of 8-9 and a concentration of 0.3 mM using ultrapure water. Then add folic acid powder and stir in a 37 ℃ water bath until completely dissolved to obtain a folic acid solution with a pH of 7-7.5 and a final concentration of 25 mg / mL.

3. The method according to claim 2, characterized in that, The weak alkaline solution is a sodium bicarbonate solution.

4. The method according to claim 1, characterized in that, The folic acid dosage for constructing the AKI model is 100-150 mg / kg.

5. The method according to claim 4, characterized in that, The folic acid dose for mild to moderate AKI models was 100 mg / kg, and the folic acid dose for severe AKI models was 150 mg / kg.

6. The method according to claim 1, characterized in that, In step (3), the injection rate for a single injection is 3~5 mL / min.

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