Modeling method for constructing obesity exacerbation acute kidney injury animal model and application thereof

Abstract

The invention discloses a modeling method for constructing an obesity exacerbation acute kidney injury animal model and application of the modeling method, and belongs to the technical field of biomedical engineering and experimental zoology and metabolic disease and kidney disease science. According to the modeling method, after a mouse is induced to reach the obesity period by adopting a high-fat feed, kidney strike is performed by adopting a renal toxicity drug or an operation until an aggravated acute kidney injury phenotype is obtained and is difficult to relieve, and then the mouse obesity aggravated acute kidney injury animal model is successfully constructed. According to the invention, an ideal animal model can be provided for researching reasons and mechanisms for aggravation and difficulty in relieving of acute kidney injury of obese or overweight people, and the animal model is more in line with the disease development process of clinical obese patients suffering from acute kidney injury. The method can also be used for further researching the acute kidney injury mechanism of obese patients, and has huge potential application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedical engineering, experimental animal science, metabolic diseases and nephrology, and particularly relates to a method for constructing an animal model of obesity-aggravated acute kidney injury and its application. Background Art

[0002] Acute kidney injury (AKI) is a common complication and complication in hospitalized patients, closely associated with progression to chronic renal failure and increased mortality. The incidence of obesity has been increasing annually in recent years and is an independent risk factor for AKI. Studies have shown that obese patients are more susceptible to AKI and experience more severe deterioration in renal function. Treatments used in healthy individuals are ineffective, resulting in a significant economic burden and a key issue affecting patients' overall prognosis.

[0003] Previous studies have shown a significant interaction between metabolic and immune homeostasis. Metabolic disorders have been found to cause abnormal systemic or local inflammatory responses in both obese humans and mice fed a high-fat diet. This immune-metabolic interaction amplifies susceptibility to disease damage, leading to an increased risk of infection, exacerbated intestinal inflammatory responses, and exacerbated autoimmune and allergic reactions. Related studies in the kidney field have found that changes in renal tubular homeostasis in patients with diabetic nephropathy may increase the risk of AKI, and abnormal immune reprogramming may appear in the kidneys long before hyperglycemia occurs.

[0004] Obesity is a condition that affects the entire body. It can cause fat cells in the patient's body to enlarge, reduce the density of receptors on fat cells, and reduce the cells' sensitivity to insulin, which can easily lead to elevated blood sugar, diabetes, hyperlipidemia, atherosclerosis, coronary heart disease, and other diseases. The kidneys of obese patients are in a state of micro-inflammation and micro-damage, making them more susceptible to damage under various stressful conditions. The pathogenesis is not exactly the same as that of normal people. Therefore, clinically, obese patients have more severe symptoms, and treatments designed for normal people are ineffective. However, current research on the mechanisms and treatments of acute kidney injury mostly focuses on conditions that occur in healthy individuals. There is little research on the mechanisms that exacerbate acute kidney injury in obesity, and there is also a lack of corresponding animal models to study the formation process, causes, and other factors of acute kidney injury in obesity. Summary of the Invention

[0005] In order to overcome the problems and defects in the prior art, the present invention proposes a modeling method for constructing an animal model of obesity-aggravated acute kidney injury and its application.

[0006] The object of the present invention is to construct a method for establishing a model of obesity exacerbating acute kidney injury. The present invention induces an obese mouse model through a high-fat diet, with an obesity degree > 20% and showing the physical and chemical characteristics of obese mice. Then, through the said method, a nephrotoxic drug or ischemia-reperfusion surgery is used to construct an acute kidney injury animal model, so that the kidney shows an aggravated phenotype of acute kidney injury and is difficult to relieve, and thus a mouse model of obesity exacerbating acute kidney injury is successfully constructed. The present invention can provide an ideal animal model for studying the reasons and mechanisms for the exacerbation and intractability of acute kidney injury in obese or overweight people, and the said animal model is more in line with the disease development process of acute kidney injury in clinical obese patients. It can also be used to further study the mechanism of acute kidney injury in obese patients and has great potential application value.

[0007] The present invention also aims to provide a method for establishing a model of obesity exacerbating acute kidney injury in animals. First, a high-fat diet is used to induce mice to reach the obese stage, and then a nephrotoxic drug or surgery is used to strike the kidneys until an aggravated phenotype of acute kidney injury is obtained, that is, a mouse model of obesity exacerbating acute kidney injury is established.

[0008] The present invention also aims at acute kidney injury in obese mice prepared by the above method, which more mimics the microinflammatory state of the kidneys of clinical obese patients. There are immune cells, especially neutrophils and macrophages, in the isolated kidneys, indicating a pre-activated state of the kidneys.

[0009] Another object of the present invention is that the animal model prepared by the above method can not only provide a model for studying the exacerbation of acute kidney injury by animal obesity, etc., but also provide a model for poor drug response, difficult kidney recovery, aggravated kidney fibrosis, and lupus in the case of obesity, and can also provide a research tool for in-depth exploration of the regulatory role played by adipokines or related genes.

[0010] In order to achieve the above objects of the present invention, the following technical solutions are specifically adopted:

[0011] The present invention provides a method for establishing a model of obesity exacerbating acute kidney injury in animals. First, a high-fat diet is used to induce mice to reach the obese stage, and then a nephrotoxic drug or surgery is used to strike the kidneys until an aggravated phenotype of acute kidney injury is obtained.

[0012] Among them, the nephrotoxic drug includes one or more of cisplatin, folic acid, adenine, etc.; preferably cisplatin.

[0013] Among them, the surgery includes one or more of ischemia-reperfusion, unilateral ureteral obstruction, 5 / 6 nephrectomy, etc.; preferably ischemia-reperfusion.

[0014] Among them, the induction of mice by the high-fat diet lasts for 3 weeks.

[0015] Among them, the mice include wild-type mice, gene knockout mice, etc. Among them, the gene knockout mice include Rarres2-KO mice (strain number: T028752), Cmklr1-KO mice (strain number: T007406), etc.

[0016] In a preferred embodiment, the mice induced by high-fat diet are 6 weeks old, the induction of high-fat diet in mice lasts for 3 weeks, and the mice subjected to kidney injury by nephrotoxic drugs or surgery are 9 weeks old.

[0017] In a preferred embodiment, the mice induced by high-fat diet are C57BL / 6J.

[0018] Among them, the measurement standard for the obesity period is that the obesity degree is greater than 20%.

[0019] In a specific embodiment, the induction time of the high-fat diet is 3 weeks; by mass, the composition and content of the high-fat diet are: basal diet 52.2%, lard 15%, sucrose 20%, cholesterol 1.2%, sodium cholate 0.2%, casein 10%, calcium hydrogen phosphate 0.6%, stone powder 0.4%, premix 0.4%, etc.; by energy, the composition of the high-fat diet is: protein 20%, carbohydrate 35%, fat 45%, etc. Among them, the composition and content of the basal diet are: corn 48.37%, soybean meal 22%, wheat middlings 20%, fish meal 3.5%, calcium hydrogen phosphate 2%, soybean oil 1.8%, calcium carbonate 1.3%, additive 1%, choline 0.03%, etc.

[0020] Among them, the premix includes one or more of compound vitamins, compound minerals, etc.

[0021] Among them, the additive includes one or more of compound vitamins, compound minerals, choline bitartrate, FD&C blue dye, etc.

[0022] In a preferred embodiment, the nephrotoxic drug is cisplatin, and the induction dose of cisplatin is 15 mg / kg; the induction method of cisplatin is intraperitoneal injection.

[0023] In a preferred embodiment, the surgery is ischemia-reperfusion, and during the induction of ischemia-reperfusion, the bilateral renal pedicles of the mice that have completed diet induction are clamped for 30 minutes.

[0024] The present invention also provides the animal model of obesity-induced acute kidney injury constructed by the modeling method as described above.

[0025] The present invention also provides the application of the modeling method as described above, or the animal model of obesity-induced acute kidney injury constructed by the modeling method as described above, and the application is selected from any one or more of the following:

[0026] a. Screening / evaluating / preparing drugs for preventing / treating / diagnosing the diseases described above;

[0027] b. Screening / evaluating / preparing drugs that regulate, inhibit or antagonize the function of the chemerin-CMKLR1 axis;

[0028] c. Screening / evaluating / preparing drugs that regulate, inhibit, or antagonize the expression or function of chemerin and / or chemerin receptor protein CMKLR1;

[0029] d. Screening / evaluation / preparation of drugs that inhibit macrophage infiltration into kidneys with acute kidney injury;

[0030] e. Screening / evaluating / preparing drugs that reduce the dependence of chemerin on CMKLR1;

[0031] f. Preparation of diagnostic products for the diagnosis of the onset and / or cure and / or recurrence of the disease as described above;

[0032] g. Developing drug targets for the diseases described above;

[0033] h. Develop bioengineering products for detecting the diseases mentioned above;

[0034] i. Research on animal obesity-induced acute kidney injury, adverse drug reactions in obesity, difficulty in kidney recovery in obesity, renal fibrosis, lupus, and the regulatory effects of adipose factors or related genes.

[0035] Wherein, the chemerin-CMKLR1 axis includes chemerin and chemerin receptor protein CMKLR1;

[0036] Among them, the diseases include acute kidney injury, metabolic injury, metabolic syndrome, inflammation, obesity, diabetic nephropathy, etc.;

[0037] Among them, chemerin in the chemerin-CMKLR1 axis promotes macrophage infiltration into the kidneys of acute kidney injury in a CMKLR1-dependent manner, subsequently triggering a strong inflammatory response;

[0038] The drug has at least one of the following effects:

[0039] (1) Prevention / treatment / diagnosis of the diseases mentioned above;

[0040] (2) regulating, inhibiting, or antagonizing the function of the chemerin-CMKLR1 axis as described above;

[0041] (3) drugs that regulate, inhibit, or antagonize the expression or function of chemerin as described above;

[0042] (4) drugs that regulate, inhibit or antagonize the expression or function of the chemerin receptor protein CMKLR1 as described above;

[0043] (5) inhibiting macrophage infiltration into kidneys with acute kidney injury;

[0044] (6) Reduce chemerin's dependence on CMKLR1.

[0045] The present invention also provides a method for screening candidate drugs or studying the pathological mechanism of a disease, wherein the candidate drug is administered to the animal model of obesity-aggravated acute kidney injury obtained by the modeling method described above.

[0046] Specifically, candidate drugs are screened by comparing changes in an animal model of obesity-aggravated acute kidney injury before and after drug administration.

[0047] In the modeling method, application, and method of the present invention as described above, the mice induced by high-fat diet are 6 weeks old; and / or, the mice subjected to kidney damage by nephrotoxic drugs or surgery are 9 weeks old; and / or, the mice include wild-type mice and gene knockout mice; and / or, the mice induced by high-fat diet are C57BL / 6J; and / or, the drug is used to prevent / treat / diagnose acute kidney injury, preferably, the drug is used to prevent / treat / diagnose obesity-aggravated acute kidney injury.

[0048] The beneficial effects of the present invention include: the present invention provides a technology and method for aggravating acute kidney injury after diet-induced obesity; the acute kidney injury animal model achieved by the present invention using the above method more simulates the actual clinical situation, and the modeled mice are obese mice, which show an acute kidney injury phenotype that is aggravated compared with normal mice, which is of great significance for studying the occurrence of acute kidney injury under obesity and the interaction between various cells; the obesity-aggravated acute kidney injury technology prepared by the present invention using the above method provides an in vivo model for studying kidney health and nutritional regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0050] Figure 1High-fat diet increases susceptibility to cisplatin-induced acute kidney injury: (A) Mice were intraperitoneally injected with cisplatin or saline after 3 weeks of high-fat diet (HFD) or standard diet (STD) induction and sacrificed 3 days later. The weight changes of mice in the STD group and HFD group are shown below the experimental schematic diagram. (B) On the 3rd day after cisplatin modeling, the blood urea nitrogen (BUN) and serum creatinine (SCr) levels in the HFD group of mice were significantly increased. (C-D) Renal PAS staining showed more obvious tubular injury in the HFD group of mice after AKI. Scale bar = 100 μm. (E-F) Oil red O staining showed more significant lipid accumulation in the renal tubular epithelial cells of the HFD group of mice after cisplatin administration. Scale bar = 50 μm. (G-H) TUNEL staining showed a significant increase in renal cell apoptosis in the HFD group of mice after intraperitoneal injection of cisplatin. Scale bar = 50 μm.

[0051] Figure 2 Rarres2 deficiency alleviates cisplatin-induced acute kidney injury in HFD-fed mice. WT mice and Rarres2 - / - mice were given cisplatin to induce AKI after 3 weeks of STD or HFD diet. (A) There was no significant difference in the weight changes of WT and Rarres2 - / - mice fed with HFD. (B) After HFD diet induction, compared with Rarres2 - / - mice, the survival rate of WT mice was significantly lower 3 - 7 days after AKI, indicating persistent and unrelieved kidney injury. (C) In the HFD group, the BUN and SCr levels in Rarres2 - / - mice were significantly lower than those in WT mice after cisplatin administration. (D) PAS staining showed less severe proximal tubular injury in Rarres2 - / - mice after cisplatin modeling. Scale bar = 100 μm. (E) qPCR showed a lower expression level of renal Ngal in Rarres2 - / - mice than in WT mice, where Ngal is an early marker of tubular injury. (F-G) Renal oil red O staining and TUNEL staining showed less lipid accumulation and less apoptosis in the kidneys of Rarres2 - / - mice. Scale bar = 50 μm.

[0052] Figure 3In kidney immune reprogramming, chemerin promotes macrophage chemotaxis in a Cmklr1-dependent manner. (A) Flow cytometry analysis showed that compared with STD mice, the number of immune-inflammatory cells in the kidneys of HFD mice was significantly increased after AKI, including total CD45+ leukocytes, Ly6g+ neutrophils, F4 / 80+ macrophages, and Ly6C+ monocytes. In addition, Rarres deficiency could reduce kidney immune cell infiltration in STD-AKI mice and HFD-AKI mice. (B) The G protein-coupled receptor CMKLR1 is an important membrane receptor through which chemerin mediates chemotaxis. ScRNA-seq (sequencing) was used to study the expression level of Cmklr1 in different kidney cell populations after AKI and found that Cmklr1 was mainly present in kidney mononuclear phagocytes (MPCs), with macrophages being the most prominent (see red circle). (C) Flow cytometry further confirmed that after AKI, CMKLR1 was mainly expressed in macrophages rather than other leukocytes or epithelial cells. (D) Schematic of the experiment: designed to verify the mechanism by which tubular epithelial cells (TECs) interact with macrophages through the chemerin-CMKLR1 axis, and study the primary WT / Rarres2 - / - TECs and WT / Cmklr1 - / - Bone marrow-derived macrophages (BMDMs) were subjected to migration assays. (E) After cisplatin stimulation, the chemotaxis of BMDMs towards WT TECs was significantly increased. The migration of BMDMs towards Rarres2 - / - TECs was reduced, and this phenomenon was reversed to a certain extent after exogenous supplementation of recombinant chemerin. (F) There was no significant difference in the number of migrated Cmklr1 - / - BMDMs between WT and Rarres2 - / - TECs groups, suggesting that the chemotactic ability of Cmklr1 - / - BMDMs towards WT and Rarres2 - / - TECs was impaired. (G) The study further constructed an adoptive transfer experiment. WT / Cmklr1 - / - BDMDs were transferred into AKI mice, and the kidneys were collected after 20 hours to measure the number of infiltrated macrophages. The results showed that the proportion of kidney macrophages among total CD45+ leukocytes and the proportion of CMTMR-labeled BMDMs among macrophages in the WT group were significantly higher than those in the Cmklr1 - / - group, verifying that the chemotactic activity of Cmklr1-deficient BDMDs was impaired in vivo.

[0053] Figure 4 For chemerin in a Cmklr1 hiMacrophage-dependent manner promotes immune reprogramming and tissue damage in AKI. (A) GO analysis showed that Cmklr1 hi IMs-2 and Cmklr1 hi KRMs-2 cell populations exhibited similar enhanced functions in the positive regulation of inflammatory responses and cell migration. (B) Tnfα and Il6, classical inflammatory genes associated with macrophages, were expressed in Cmklr1 hi (C) qPCR showed that the expression levels of pro-inflammatory genes Tnfα and Il6 in the kidneys of mice induced by HFD diet after AKI were significantly increased compared with those in the STD group. - / - -The expression levels of pro-inflammatory genes Tnfα and Il6 in the kidneys of AKI mice were significantly reduced in both STD and HFD groups. (D) To further confirm that the damaging effect of chemerin in AKI is due to its amplifying effect on the inflammatory response, we investigated the effect of chemerin on the expression of Rarres2 during AKI. - / - Mice were injected with exogenous recombinant TNF-α and IL-6. The results showed that after supplementation with TNF-α and IL-6, Rarres2 - / - The renal damage of mice was reversed, as shown by increased levels of BUN and SCr. (EG) PAS staining showed that Rarres2 - / - The renal tubular injury scores of mice were significantly increased after supplementation with exogenous recombinant TNF-α and IL-6. Scale bar = 100 μm. DETAILED DESCRIPTION

[0054] The present invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0055] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention, its application, or use. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0056] In all examples shown and discussed herein, any specific values should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0057] The object of the present invention also lies in that obesity is one of the known risk factors for acute kidney injury, but the exact mechanism behind it has not been fully elucidated. The present invention demonstrates that short-term high-fat diet (HFD) exacerbates kidney injury in AKI mice, which may be due to HFD-induced immune reprogramming. Through omics analysis, the present invention identifies a unique kidney injury-related adipokine, chemerin (encoded by the Rarres2 gene), which is enriched in AKI tubular epithelial cells, especially after exposure to a high-fat diet. Detection of serum and kidney tissues from obese AKI patients by the present invention reveals increased chemerin expression levels, which are negatively correlated with renal function. Lack of Rarres2 in mice protects them from HFD-induced AKI susceptibility and AKI caused by various reasons, suggesting that Rarres2 has a direct effect on AKI independent of metabolic factors. The present invention also proposes that the chemerin receptor (CMKLR1, chemerin chemokine-like receptor 1) is expressed on renal mononuclear phagocytes (MPCs). Single-cell RNA sequencing (scRNA-seq) analysis reveals the presence of a unique Cmklr1 hi cluster in AKI kidney MPCs, which exhibits stronger chemotaxis and pro-inflammatory ability. Functionally, chemerin acts as a chemokine, enhancing the chemotaxis of Cmklr1hi-MPCs. The present invention proposes that chemerin is a unique kidney injury-related adipokine that can enhance Cmklr1 hi chemotaxis of mononuclear phagocytes (MPCs) and further exacerbate tissue damage. Finally, the present invention proposes that administration of cytokines secreted by MPCs eliminates the protective effect of Rarres2 deficiency on kidney injury, demonstrating the role of the chemerin-CMKLR1 axis in promoting inflammation and kidney injury. In summary, the present invention identifies chemerin as a unique inflammation and metabolism target that exerts its effect through Cmklr1 hi macrophages with highly efficient pro-inflammatory effects. The present invention shows that targeting the chemerin-CMKLR1 axis is expected to become a new treatment strategy for AKI patients, especially those with metabolic syndrome.

[0058] To achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0059] The present invention also provides the application of the chemerin-CMKLR1 axis as a drug target in the preparation of drugs for preventing / treating diseases, wherein the chemerin-CMKLR1 axis includes chemerin and the chemerin receptor protein CMKLR1;

[0060] The diseases include acute kidney injury, metabolic injury, metabolic syndrome, inflammation, obesity, and diabetic nephropathy.

[0061] Specifically, the drug can regulate, inhibit or antagonize the function of the chemerin-CMKLR1 axis as described above, and / or the expression or function of chemerin as described above, and / or the expression or function of the chemerin receptor protein CMKLR1 as described above.

[0062] Specifically, the chemerin-CMKLR1 axis promotes AKI immune reprogramming and kidney injury. In the chemerin-CMKLR1 axis, chemerin promotes macrophage infiltration into the kidneys of acute kidney injury in a CMKLR1-dependent manner, subsequently triggering a strong inflammatory response. Chemerin is a unique adipokine associated with kidney injury and is enriched in the kidneys of AKI induced by HFD (high-fat diet). Serum and kidney chemerin levels are elevated in obese AKI patients and are negatively correlated with renal function. Cmklr1 hi As a kidney injury-related subset, macrophages have enhanced chemotaxis and a powerful inflammatory amplification ability; regardless of the dietary pattern, chemerin deficiency exhibits a protective effect in reducing kidney injury and inflammation.

[0063] The present invention also provides an inhibitor / antagonist / regulator that can regulate, inhibit or antagonize the function of the chemerin-CMKLR1 axis as described above, and / or the expression or function of chemerin as described above, and / or the expression or function of the chemerin receptor protein CMKLR1 as described above.

[0064] Specifically, the inhibitor / antagonist / regulator includes one or more of nucleic acid molecules, carbohydrates, lipids, small molecules, antibodies, polypeptides, proteins, viruses, cells, etc. Among them, the nucleic acid molecules include one or more of siRNA, shRNA, etc.

[0065] The present invention also provides a drug / drug composition that contains the inhibitor / antagonist / regulator as described above; further, it also contains a pharmaceutically acceptable carrier.

[0066] Specifically, the drug / drug composition is administered orally, by injection, intranasally, transdermally or mucosally, etc. Specifically, the drug / drug composition includes liquid dosage forms, gas dosage forms, solid dosage forms and semi-solid dosage forms, etc.

[0067] Preferably, the pharmaceutically acceptable carrier means that when the drug is appropriately administered to animals or humans, they do not produce adverse, allergic or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: saccharides such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffer solutions, etc. These substances are used as needed to help the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0068] Specifically, the drug / drug composition can be made into injections, sterile powders for injection, tablets, pills, capsules, lozenges, spirits, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalants, or suppositories, etc. The drug / drug composition of the above various dosage forms can be prepared according to the conventional methods in the pharmaceutical field. Specifically, in the drug / drug composition, physiologically compatible excipients can also be included, and the excipients include buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc.

[0069] Specifically, the drug / drug composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal dropping, eye dropping, penetration, absorption, physical or chemical mediated methods; or be introduced into the body after being mixed or encapsulated with other substances. Preferably, it is administered by injection. The drug / drug composition can also be used in combination with other treatment means, and the other means include surgery, radiotherapy, chemotherapy, targeted therapy.

[0070] The dosage level of the drug / drug composition of the present invention can be adjusted according to the amount of the composition required to achieve the desired diagnostic or therapeutic result. The administration regimen can also be a single injection or multiple injections, or be adjusted. The selected dosage level and regimen are reasonably adjusted depending on various factors including the activity and stability (i.e., half-life) of the cell drug / drug composition, the formulation, the administration route, the combination with other drugs or treatments, the disease or disorder to be detected and / or treated, and the health status and previous medical history of the subject to be treated, etc.

[0071] The therapeutically effective dose for the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and / or primates. Animal models can also be used to measure suitable administration concentration ranges and approaches. Subsequently, it can be used to determine the useful dose and approach used in humans. Generally, the determination and adjustment of an effective dose or dosage and the assessment of when and how to perform such adjustments are known to those skilled in the art.

[0072] For additional guidance on formulation, dosage, administration schedules, and measurable therapeutic outcomes, see, among others, Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; and Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0073] Furthermore, the drug / drug composition is used alone and / or in combination with other drugs.

[0074] The present invention also provides a reagent / kit for detecting the function of the chemerin-CMKLR1 axis as described above, and / or the expression or function of the chemerin as described above, and / or the expression or function of the chemerin receptor protein CMKLR1 as described above.

[0075] The present invention also provides a use of the above-mentioned inhibitor / antagonist / modulator, or the above-mentioned drug / pharmaceutical composition, or the above-mentioned reagent / kit, wherein the use is selected from any one or more of the following:

[0076] a. Screening / evaluating / preparing drugs for preventing / treating / diagnosing the diseases described above;

[0077] b. Screening / evaluating / preparing drugs that regulate, inhibit or antagonize the function of the chemerin-CMKLR1 axis as described above;

[0078] c. Screening / evaluating / preparing drugs that regulate, inhibit, or antagonize the expression or function of chemerin and / or the chemerin receptor protein CMKLR1 as described above;

[0079] d. Screening / evaluation / preparation of drugs that inhibit macrophage infiltration into kidneys with acute kidney injury;

[0080] e. Screening / evaluating / preparing drugs that reduce the dependence of chemerin on CMKLR1;

[0081] f. Preparation of diagnostic products for the diagnosis of the onset and / or cure and / or recurrence of the disease as described above;

[0082] g. Developing drug targets for the diseases described above;

[0083] h. Develop bioengineering products for detecting the diseases mentioned above;

[0084] i. Research on animal obesity-induced acute kidney injury, adverse drug reactions in obesity, difficulty in kidney recovery in obesity, renal fibrosis, lupus, and the regulatory effects of adipose factors or related genes.

[0085] The present invention also provides a method for screening candidate drugs for preventing / inhibiting / alleviating / reducing / treating the diseases as described above, the method comprising detecting the effect of the candidate drug on the expression or function of the chemerin-CMKLR1 axis as described above, and / or the expression or function of chemerin as described above, and / or the expression or function of the chemerin receptor protein CMKLR1 as described above in a subject or a sample obtained from the subject.

[0086] Among them, after use of the candidate drug, the expression or function of the chemerin-CMKLR1 axis as described above, and / or the expression or function of chemerin as described above, and / or the expression or function of the chemerin receptor protein CMKLR1 as described above can be inhibited, indicating that the candidate drug has the effect of preventing / inhibiting / alleviating / reducing / treating the diseases as described above.

[0087] The present invention also provides a method for preventing / inhibiting / alleviating / reducing / treating the diseases described above, comprising administering the inhibitor / antagonist / modulator described above, or the drug / drug composition described above to a subject.

[0088] The present invention also provides a biomarker for predicting drug resistance and / or prognosis and / or treatment effect of the above-mentioned disease, wherein the biomarker is the chemerin-CMKLR1 axis as described above.

[0089] The present invention also provides a method for predicting the therapeutic effect / drug resistance / prognosis of the disease as described above, the method comprising: determining the expression or function of the chemerin-CMKLR1 axis as described above, or the expression or function of chemerin as described above, or the expression or function of the chemerin receptor protein CMKLR1 as described above, in a sample from a patient with the disease as described above, and predicting whether the patient with the disease as described above has the therapeutic effect / drug resistance / prognosis of the disease as described above based on the level.

[0090] In the present invention, in the application, inhibitor / antagonist / modulator, drug / drug composition, reagent / kits, method, biomarker as described above, the chemerin-CMKLR1 axis includes chemerin and the chemerin receptor protein CMKLR1;

[0091] The disease includes acute kidney injury, metabolic injury, metabolic syndrome, inflammation, obesity, diabetic nephropathy, etc.;

[0092] In the chemerin-CMKLR1 axis, chemerin promotes macrophage infiltration into the kidney of acute kidney injury in a CMKLR1-dependent manner, and subsequently triggers a strong inflammatory response;

[0093] The drug has at least one of the following effects:

[0094] (1) Prevent / treat / diagnose the disease as described above;

[0095] (2) Regulate or inhibit or antagonize the function of the chemerin-CMKLR1 axis as described above;

[0096] (3) A drug that regulates or inhibits or antagonizes the expression or function of chemerin as described above;

[0097] (4) A drug that regulates or inhibits or antagonizes the expression or function of the chemerin receptor protein CMKLR1 as described above;

[0098] (5) Inhibit macrophage infiltration into the kidney of acute kidney injury;

[0099] (6) Reduce the dependence of chemerin on CMKLR1.

[0100] The present invention also provides the application of the Rarres2 gene as a drug target in the preparation of a drug for preventing / treating a disease, wherein the Rarres2 gene encodes chemerin;

[0101] The disease includes acute kidney injury, metabolic injury, metabolic syndrome, inflammation, obesity, diabetic nephropathy, etc.

[0102] Specifically, the lack of Rarres2 exhibits a protective effect in alleviating kidney injury and inflammation.

[0103] The present invention also provides a biomarker for predicting drug resistance and / or prognosis and / or treatment effect of the disease as described above, and the biomarker is the Rarres2 gene as described above, and the NCBI sequence number of its nucleotide sequence is NM_002889.4 (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_002889.4).

[0104] The present invention also provides a method for screening a drug target / biomarker for acute kidney injury, and the screening method includes the following steps:

[0105] 1) Select GSE 144838 from the GEO database as a high-fat diet model data set, and sequence mice treated with cisplatin or normal saline to obtain cisplatin model data;

[0106] 2) Identify differentially expressed genes with co-upregulated expression by analyzing the upregulated genes in the kidney in the acute kidney injury and high-fat diet model data sets through a Venn diagram;

[0107] 3) Screen out the Rarres2 gene as a drug target / biomarker for acute kidney injury;

[0108] 4) Verify the expression level of chemerin (the protein encoded by Rarres2) in kidney tissue homogenate by ELISA detection.

[0109] The beneficial effects of the present invention also include: the present invention clarifies the mechanism of kidney immune reprogramming in the presence of metabolic injury, and determines the key molecule chemerin and its receptor CMKLR1. Cmklr1 hi Macrophages, as a kidney injury-related subset, have enhanced chemotaxis and a powerful inflammation amplification ability. Chemerin promotes macrophage infiltration into the AKI kidney in a CMKLR1-dependent manner, subsequently triggering a strong inflammatory response. Therefore, targeting the chemerin-CMKLR axis may be a new and promising strategy for the treatment of clinical AKI (especially in combination with abnormal metabolic status).

[0110] The test materials used in the examples are all conventional biochemical reagents unless otherwise specified.

[0111] Examples

[0112] 1. Materials and Methods

[0113] 1.1 Animals and Human Samples

[0114] Rarres2-KO mice (strain number: T028752), Cmklr1-KO mice (strain number: T007406), and WT mice were purchased from Model Animal Research Center of Nanjing University (China). All animal experiments were conducted in accordance with the protocols approved by the IACUC of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Human kidney tissue and serum samples were obtained from the kidney histopathology library of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, and experiments were conducted in accordance with the regulations of the histopathology library management and approved by the ethics committee.

[0115] 2. Methods

[0116] 2.1 Animal model construction

[0117] 2.1.1 Diet induction: Six-week-old male mice were randomly assigned to a normal diet group or a high-fat diet group (60% kcal fat; #D12492, Research Diet InC., USA) for 3 weeks.

[0118] 2.1.2 Cisplatin-induced AKI model: After diet induction, male mice were intraperitoneally injected with cisplatin (Sigma, St Louis, MO) at a dose of 15 mg / kg, and the control group was injected with an equal volume of normal saline (0.9%).

[0119] 2.1.3 Ischemia-reperfusion (IR)-induced AKI model: After diet induction, the bilateral renal pedicles of male mice were clamped for 30 minutes, and the control group received sham surgery.

[0120] 2.1.4 TNF-α and IL-6 recombinant protein intervention: WT and Rarres2 - / - mice were randomly assigned to a control group and an intervention group. For the intervention group, mice were intravenously injected with TNF-α and IL-6 (5 mg / kg each) (both from eBioscience, USA) daily from the start of the model until the mice were sacrificed.

[0121] 2.2 Renal injury assessment

[0122] Blood urea nitrogen and creatinine levels were measured using the DIUR-100 QuantiChrom TM BUN assay kit and the DICT-500 QuantiChrom TMThe SCr detection kit (Bioassay System, USA) was used for detection according to the reagent instructions. Kidney tissues were collected after perfusion, fixed with 4% paraformaldehyde, and stained with PAS. Then, 10 non-overlapping fields of view were randomly selected from each sample under the microscope for scoring to evaluate the degree of diseased renal tubular injury. Briefly, renal tubular injury was defined as tubular necrosis, cast formation, loss of brush border, and accumulation of cell debris. The renal tubular injury score was determined according to the percentage of damaged renal tubules: 0 (no injury), 1 (<25% injury), 2 (25 - 50% injury), 3 (50 - 75% injury), 4 (>75% injury).

[0123] 2.3 Flow cytometry

[0124] Briefly, the collected kidneys were removed from the capsule, chopped, and digested with 1 mg / ml type IV collagenase and DNase I (Sigma, USA) at 37 °C for 40 minutes. After digestion, the samples were filtered through a 70-μm cell strainer to remove aggregated cells. Subsequently, the cells were resuspended and lysed for red blood cells in RBC lysis buffer (Biolegend, USA). Then, the cells were resuspended and filtered to obtain a single-cell suspension for subsequent flow cytometry analysis or single-cell sequencing. Flow cytometry analysis was performed on a CyAn instrument (Beckman Coulter, USA), and the analysis was carried out using FlowJo V10 software (Tree Star, Washington State, USA). Single cells were incubated with fluorescently labeled antibodies and 7-AAD. The percentages of CD45+CD11b+Ly6G+, CD45+CD11b+Ly6C+F4 / 80+, and CD45+Ly6g-CD11bhiF4 / 80int cells in total kidney cells were calculated respectively to determine the percentages of neutrophils, monocytes, and macrophages in total kidney cells.

[0125] 2.4 Single-cell RNA sequencing analysis

[0126] 2.4.1 Single-cell RNA sequencing data processing

[0127] Dead cell removal beads (Miltenyi Biotec, Germany) were used to enrich single-cell suspensions. Single cells were captured in droplets and reverse transcription was performed. The amplified cDNA was used for 3'-gene expression library construction, followed by sequencing on a Nova seq 6000 instrument (Illumina, USA) using 150 bp paired-end runs. Single-cell RNA sequencing data were normalized using the Seurat package (version 3.0) in R (version 3.5.3). In this study, the Seurat package (https: / / satijalab.org / seurat / ) was used for cell normalization and cell filtering, considering metrics such as the MT percentage, minimum gene count, and maximum gene count. In quality control, genes expressed in fewer than three cells were excluded, and cells expressing fewer than 200 genes or with a mitochondrial gene percentage exceeding 50% were also excluded. The DoubletFinder method was used to remove doublets. The batch effect was removed using the standard harmony integration process. Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) methods were used to clarify the relationships between single cells. Cell clustering was performed using Graphcluster and K-means, while marker gene analysis was performed using the Wilcoxon rank-sum test. The clustering resolution was set to 0.8.

[0128] 2.4.2 QuSAGE analysis

[0129] QuSAGE analysis (http: / / clip.med.yale.edu / qusage / ) eliminates false positive errors caused by correlations between different genes within a gene population through the variance inflation factor. QuSAGE analysis was used to evaluate the activation levels of different MPC gene sets.

[0130] 2.4.3 SCENIC analysis

[0131] To evaluate the effectiveness of transcription factor regulation, the study adopted the workflow of single-cell regulatory network inference and clustering (pySCENIC, version 0.9.5, https: / / aertslab.org / #scenic). This analysis involved using a 20,000-motif database obtained from RcisTarget and GRNboost. Transcription factors with significant regulatory strength and central roles were selected and the results were displayed in a heatmap.

[0132] 2.4.4 Cell communication analysis

[0133] Cell-cell communication analysis was performed based on CellPhoneDB (version 4.1.0, https: / / www.cellphonedb.org), a publicly available database containing curated receptors, ligands, and their interactions. This database helps predict rich cell interactions between two cell types from single-cell transcriptome data. The average cell communication significance (P<0.05) between ligands and receptors was calculated based on the normalized cell matrix generated by Seurat normalization.

[0134] 2.4.5 Gene clustering based on pseudotime expression patterns:

[0135] Differentiation trajectory analysis at the single-cell level was performed using the reverse graph embedding method in the Monocle2 package (version 2.22.0, https: / / cole-trapnell-lab.github.io / monocle-release / ). Cells were projected into a low-dimensional space while learning a smooth tree-like manifold, and cells were assigned on the manifold. Dynamic genes along the pseudotime were clustered and visualized on a heatmap using the "plot_pseudotime_heatmap" function of the Monocle package. The obtained gene modules were further analyzed by GOBP comparison.

[0136] 2.5 Transwell migration assay

[0137] Bone marrow-derived macrophages (BMDMs) were isolated from the femurs of WT or Cmklr1 - / - mice and cultured in RPMI 1640 medium containing 20 ng / ml M-CSF (R&D Systems) for 6 days. Tubular epithelial cells (TECs) were isolated from the kidneys of WT or Rarres2 - / - mice and seeded in the lower layer. Then the TECs were stimulated with cisplatin (10 μM) for 24 hours. 200 μL of WT or Cmklr1 - / - BMDMs (2×10 5 / mL) were transferred to the upper layer (5 μm pore size; Corning, USA), and WT or Rarres2 - / - mTECs were placed in the bottom layer. A vehicle control was added to the bottom chamber, with or without chemerin (3 nM). After incubation at 37 °C for 4 hours, non-migrating cells on the filter membrane were removed, and the cells in the bottom layer were fixed with 4% formaldehyde. Then an inverted microscope (Nikon, Japan) was used to determine the number of migrating BMDMs. At least 5 random images were selected for analysis in each well and averaged.

[0138] 2.6 Statistical analysis

[0139] Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups were performed using the unpaired two-tailed Student's t-test. Comparisons between multiple groups were performed using one-way ANOVA with Tukey's multiple comparisons. Statistical analysis was performed using GraphPad Prism (version 8.0). P < 0.05 was considered statistically significant. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0140] Other experiments in the embodiments of the present invention: PCR, WB (Western blotting), IF (immunofluorescence), ELISA (enzyme-linked immunosorbent assay), Oil Red O staining, PAS (peroxy acid Schiff) staining, conventional cell experiments, etc. are not particularly described and are common techniques in the art.

[0141] Table 1: Summary of primers involved in qPCR

[0142]

[0143] 3. Results

[0144] The experimental results of the present invention show that a high-fat diet increases the susceptibility of cisplatin-induced acute kidney injury (see Figure 1 ),in Figure 1 A shows mice that were fed a high-fat diet (HFD) or standard diet (STD) for 3 weeks and then intraperitoneally injected with cisplatin or saline. The mice were sacrificed 3 days later. Body weight changes in the STD and HFD groups are shown below the schematic diagram. Figure 1 B shows that on the 3rd day after cisplatin modeling, the blood urea nitrogen (BUN) and serum creatinine (SCr) levels of mice in the HFD group were significantly increased. Figure 1 C- Figure 1 D shows PAS staining of the kidneys, which revealed more pronounced renal tubular damage in HFD-treated mice after AKI. Scale bar = 100 μm. Figure 1 E- Figure 1 F shows that oil red O staining showed that lipid accumulation in renal tubular epithelial cells was more pronounced in HFD mice after cisplatin administration. Scale bar = 50 μm. Figure 1 G- Figure 1 H shows TUNEL staining showing a significant increase in renal cell apoptosis in HFD mice after intraperitoneal injection of cisplatin. Scale bar = 50 μm.

[0145] The experimental results of the present invention show that Rarres2 deficiency alleviates HFD mice from cisplatin-induced acute kidney injury (see Figure 2 ). WT mice and Rarres2 - / -Mice were given cisplatin to induce AKI after 3 weeks of STD or HFD diet. Among them, Figure 2 A shows that there was no significant difference in the weight change of WT and Rarres2 - / - mice after being given HFD diet. Figure 2 B shows that after induction with HFD diet, compared with Rarres2 - / - mice, the survival rate of WT mice was significantly reduced 3 - 7 days after AKI, suggesting that kidney injury persisted and was not alleviated. Figure 2 C shows that in the HFD group, the levels of BUN and SCr in Rarres2 - / - mice were significantly lower than those in WT mice after cisplatin administration. Figure 2 D shows that PAS staining revealed less severe proximal tubular injury in Rarres2 - / - mice after cisplatin-induced AKI. Scale bar = 100 μm. Figure 2 In E, qPCR showed that the expression level of Ngal in the kidneys of Rarres2 - / - mice was lower than that in WT mice, where Ngal is an early marker of tubular injury. Figure 2 F - Figure 2 G shows that kidney Oil Red O staining and TUNEL staining revealed less lipid accumulation and less apoptosis in the kidneys of Rarres2 - / - mice. Scale bar = 50 μm.

[0146] The experimental results of the present invention show that in kidney immune reprogramming, chemerin promotes macrophage chemotaxis in a Cmklr1-dependent manner (see Figure 3 ). Among them, Figure 3 A shows that flow cytometry analysis revealed a significant increase in immune-inflammatory cells in the kidneys of HFD mice after AKI compared with STD mice, including total CD45+ leukocytes, Ly6g+ neutrophils, F4 / 80+ macrophages, and Ly6C+ monocytes. In addition, Rarres deficiency was able to reduce kidney immune cell infiltration in STD-AKI mice and HFD-AKI mice. Figure 3 B shows that the G protein-coupled receptor CMKLR1 is an important membrane receptor through which chemerin mediates chemotaxis. ScRNA-seq (sequencing) studied the expression level of Cmklr1 in different kidney cell populations after AKI and found that Cmklr1 was mainly present in kidney mononuclear phagocytes (MPCs), with macrophages being the most prominent (see the red circle). Figure 3 C Flow cytometry further confirmed that after AKI, CMKLR1 was mainly expressed in macrophages rather than other leukocytes or epithelial cells. Figure 3D is the experimental diagram: the purpose is to verify the mechanism of interaction between tubular epithelial cells (TECs) and macrophages through the chemerin-CMKLR1 axis, and to study the effect of primary WT / Rarres2 - / - TECs and WT / Cmklr1 - / - Bone marrow-derived macrophages (BMDMs) were used for migration assay. Figure 3 E shows that after cisplatin stimulation, the chemotaxis of BMDMs to WT TECs increased significantly. - / - TECs migration was reduced, and this phenomenon was reversed to some extent after exogenous supplementation of recombinant chemerin. Figure 3 F indicates the difference between WT and Rarres2 - / - Among TECs groups, Cmklr1 - / - There was no significant difference in the number of BMDMs migrating, suggesting that Cmklr1 - / - BMDMs were sensitive to WT and Rarres2 - / - The chemotactic ability of TECs was impaired. Figure 3 G shows that the study further constructed adoptive transfer experiments to transfer WT / Cmklr1 - / - BDMDs were transferred into AKI mice, and the kidneys were collected 20 hours later to determine the number of infiltrating macrophages. The results showed that the proportion of renal macrophages to total CD45+ leukocytes and the proportion of CMTMR-labeled BMDM to macrophages in the WT group were significantly higher than those in the Cmklr1 group. - / - The results showed that the chemotactic activity of BDMDs lacking Cmklr1 was impaired in vivo.

[0147] The experimental results of the present invention show that chemerin is expressed as Cmklr1 hi Macrophage-dependent mechanisms promote immune reprogramming and tissue damage in AKI (see Figure 4 ).in, Figure 4 A is the GO analysis showed that Cmklr1 hi IMs-2 and Cmklr1 hi The KRMs-2 cell population exhibited similar enhanced functions in the positive regulation of inflammatory responses and cell migration. Figure 4 B shows that the classical inflammatory genes Tnfα and Il6 associated with macrophages are expressed in Cmklr1 hi Highly enriched and activated in subpopulations. Figure 4 qPCR in C showed that the expression levels of pro-inflammatory genes Tnfα and Il6 in the kidneys of mice induced by HFD diet after AKI were significantly increased compared with those in the STD group. - / --The expression levels of pro-inflammatory genes Tnfα and Il6 in the kidneys of AKI mice were significantly reduced in both the STD and HFD groups. Figure 4 D is to further confirm that the damaging effect of chemerin in AKI is due to its amplifying effect on inflammatory response. - / - Mice were injected with exogenous recombinant TNF-α and IL-6. The results showed that after supplementation with TNF-α and IL-6, Rarres2 - / - The kidney damage in mice was reversed, as evidenced by increased BUN and SCr levels. Figure 4 E- Figure 4 PAS staining in G showed that Rarres2 - / - The renal tubular injury scores of mice were significantly increased after supplementation with exogenous recombinant TNF-α and IL-6. Scale bar = 100 μm.

[0148] 4. Conclusion

[0149] This study reveals the mechanism of renal immune reprogramming in abnormal metabolism and AKI, and identifies chemerin, an important adipokine associated with renal injury. Key findings from this study include:

[0150] (1) HFD increases the risk of AKI occurrence and aggravation, which may be due to renal immune reprogramming.

[0151] (2) Chemerin is a unique kidney injury-related adipokine that is enriched in the kidneys of AKI patients induced by a HFD diet. Serum and kidney chemerin levels are elevated in obese patients with AKI and are negatively correlated with renal function.

[0152] (3) Regardless of the dietary pattern (STD or HFD), Rarres2 deficiency exhibited a protective effect in alleviating renal injury and inflammation.

[0153] (4) ScRNA-seq analysis identified a unique Cmklr1 in renal MPCs after AKI hi subpopulations, which exhibit enhanced chemotactic and pro-inflammatory capabilities.

[0154] (5) In vivo and in vitro experiments demonstrated the role of the chemerin-CMKLR1 axis in promoting AKI immune reprogramming and renal injury.

[0155] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0156] As used in the present invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the content specified in the present invention but do not exclude other aspects.

[0157] As used in the present invention, the term "and / or" includes any and all combinations of one or more of the related listed items.

[0158] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are used as the protection scope.

Claims

1. A method for establishing an animal model of obesity - aggravated acute kidney injury, characterized in that, After first inducing mice to reach the obese stage with a high-fat diet, kidney injury is then induced using a nephrotoxic drug or surgery until a phenotype of aggravated acute kidney injury is obtained.

2. The modeling method according to claim 1, wherein The criterion for measuring the obese stage is that the obesity degree is greater than 20%.

3. The modeling method according to claim 1, characterized in that, The nephrotoxic drugs include one or more of cisplatin, folic acid, and adenine; and / or, the surgery includes one or more of ischemia-reperfusion, unilateral ureteral obstruction, and 5 / 6 nephrectomy.

4. The modeling method according to claim 3, wherein, The dosage of cisplatin induction is 15 mg / kg; and / or, the way of cisplatin induction is intraperitoneal injection.

5. The modeling method according to claim 3, wherein, During the ischemia-reperfusion induction, the bilateral renal pedicles of the mice that have completed diet induction are clamped for 30 minutes.

6. The modeling method according to claim 1, wherein The mice induced by the high-fat diet are 6 weeks old; and / or, the mice subjected to kidney injury induction using a nephrotoxic drug or surgery are 9 weeks old.

7. The modeling method according to claim 1, wherein By mass, the high-fat diet contains: 52.2% basal diet, 15% lard, 20% sucrose, 1.2% cholesterol, 0.2% sodium cholate, 10% casein, 0.6% calcium hydrogen phosphate, 0.4% stone powder, 0.4% premix; and / or, by energy, the high-fat diet contains: 20% protein, 35% carbohydrate, 45% fat; Among them, the premix includes one or two of compound vitamins and compound minerals.

8. The modeling method according to claim 7, characterized in that, The basal diet contains: 48.37% corn, 22% soybean meal, 20% wheat middlings, 3.5% fish meal, 2% calcium hydrogen phosphate, 1.8% soybean oil, 1.3% calcium carbonate, 1% additive, 0.03% choline.

9. The modeling method according to claim 8, characterized in that, The additive includes one or more of compound vitamins, compound minerals, choline bitartrate, and FD&C blue dye.

10. The modeling method according to claim 1, characterized in that, The induction of the high-fat diet in mice lasts for 3 weeks.

11. The modeling method according to claim 1, characterized in that, The mice include wild-type mice and gene knockout mice.

12. The modeling method according to claim 10, characterized in that, The gene knockout mice include Rarres2-KO mice and Cmklr1-KO mice.

13. The modeling method according to claim 1, wherein, The mice are C57BL / 6J.

14. The modeling method according to any one of claims 1-13, or the application of an animal model of obesity-induced acute kidney injury constructed by the modeling method according to any one of claims 1-13, characterized in that, The applications are selected from any one or more of the following: a. Screening / evaluating / preparing drugs for preventing / treating / diagnosing diseases; b. Screening / evaluating / preparing drugs for regulating or inhibiting or antagonizing the function of the chemerin-CMKLR1 axis; c. Screening / evaluating / preparing drugs for regulating or inhibiting or antagonizing the expression or function of chemerin and / or the chemerin receptor protein CMKLR1; d. Screening / evaluating / preparing drugs for inhibiting macrophage infiltration into the kidneys of acute kidney injury; e. Screening / evaluating / preparing drugs for reducing chemerin dependence on CMKLR1; f. Preparing diagnostic products for diagnosing the onset and / or cure and / or recurrence of diseases; g. Developing drug targets for diseases; h. Developing related bioengineering products for detecting diseases; i. Research on the aggravation of acute kidney injury by animal obesity, research on poor drug response in obesity, research on the difficulty of kidney recovery in obesity, research on kidney fibrosis, research on lupus, research on the regulatory role of adipokines or related genes.

15. The application according to claim 14, wherein The chemerin-CMKLR1 axis includes chemerin and the chemerin receptor protein CMKLR1; and / or, The diseases include acute kidney injury, metabolic injury, metabolic syndrome, inflammation, obesity, and diabetic nephropathy; and / or, In the chemerin-CMKLR1 axis, chemerin promotes macrophage infiltration into the kidneys of acute kidney injury in a CMKLR1-dependent manner, subsequently triggering a strong inflammatory response.

16. A method for screening candidate drugs or for studying the pathological mechanism of a disease, characterized in that, The method administers a candidate drug to an animal model of obesity-induced exacerbation of acute kidney injury constructed by the modeling method described in any one of claims 1-13.

17. The method according to claim 16, wherein By comparing the changes in the animal model of obesity-induced exacerbation of acute kidney injury before and after drug administration, candidate drugs are screened out.

18. The modeling method, application, method according to any one of claims 1-17, characterized in that, The mice induced by the high-fat diet are 6 weeks old; and / or, the mice subjected to kidney injury by nephrotoxic drugs or surgery are 9 weeks old; and / or, the mice include wild-type mice and gene knockout mice; and / or, the mice are C57BL / 6J; and / or, the drug is used for preventing / treating acute kidney injury.

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