Use of iia compound in preparation of drugs for treating diseases related to kidney injury

Abstract

This invention belongs to the field of biomedical technology and relates to the application of IIA compounds in the preparation of drugs for the treatment of kidney injury-related diseases. Through cell experiments and animal experiments, it has been confirmed that IIA-12 can reduce apoptosis levels, regulate immune inflammatory responses, and improve renal function in in vivo and in vitro models of ischemia-reperfusion kidney injury. IIA-12 can reduce renal tubular damage caused by AKI, improve renal tubular epithelial cell apoptosis and inflammation, and promote renal cell function repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of IIA compounds in the preparation of drugs for the treatment of kidney injury-related diseases. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in kidney function over a short period. Its core diagnostic criteria include a rapid increase in serum creatinine levels and a significant decrease in urine output. In severe cases, it can lead to complete loss of kidney function, causing life-threatening complications such as uremia and multiple organ failure. The causes of AKI are extremely diverse, mainly divided into three categories: prerenal factors, such as dehydration, hypotension, and insufficient renal perfusion due to heart failure; renal factors, such as renal ischemia-reperfusion injury, drug / toxic kidney injury, glomerular diseases, and tubulointerstitial inflammation; and postrenal factors, such as urinary tract obstruction. Among these, renal ischemia-reperfusion injury and drug-induced kidney injury are the most common clinical precipitating factors.

[0003] As a highly prevalent clinical disease, AKI has an incidence rate of 10% to 20% among hospitalized patients, and the incidence rate in intensive care unit patients exceeds 50%. Moreover, the mortality rate remains high, with a short-term mortality rate of 30% to 50% for moderate to severe AKI patients. Survivors often suffer from chronic kidney disease, or even progress to end-stage renal disease. This not only seriously threatens the lives and health of patients, but also incurs huge medical expenses due to long-term dialysis and treatment of complications, placing a heavy economic burden on the social medical system and families.

[0004] From a pathophysiological perspective, renal tubular injury and repair are the core processes in the development of acute kidney injury (AKI). Renal tubular epithelial cells, as key cells in renal filtration and reabsorption, directly impair kidney function through necrosis, apoptosis, and shedding. The proliferation, differentiation, and tissue remodeling of these damaged epithelial cells determine the degree of renal function recovery. Immune inflammatory responses are crucial in the renal tubular injury and repair process in AKI. After injury, inflammatory cells such as macrophages and neutrophils infiltrate renal tissue in large numbers, releasing inflammatory factors such as interleukins and tumor necrosis factor. Excessive inflammation exacerbates tubular damage, while moderate inflammation can initiate tissue repair. Although research into the pathological mechanisms of AKI has deepened in recent years, clinical treatment remains primarily symptomatic and supportive, such as fluid resuscitation, correction of electrolyte imbalances, and renal replacement therapy. Currently, there is a lack of effective drugs that can specifically target renal tubular injury, regulate immune inflammatory responses, and promote renal function recovery. Developing highly effective and specific AKI treatments has become an urgent clinical need. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention modifies the structure of the IA series compounds. A total of 39 candidate compounds from the IA and IIA series were synthesized. Finally, the bioactivity of these candidate compounds was screened, and the efficiency of each compound in inhibiting lymphocyte proliferation in vitro was tested. Ultimately, IIA-12 was selected as the compound with the highest bioactivity. Cellular and animal experiments confirmed that IIA-12 can reduce apoptosis levels, regulate immune inflammatory responses, and improve renal function in in vitro and in vivo models of ischemia-reperfusion kidney injury. IIA-12 can alleviate renal tubular injury caused by AKI, improve renal tubular epithelial cell apoptosis and inflammation, and promote cell function repair.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] Application of IIA compounds in the preparation of drugs for treating kidney injury-related diseases, wherein the structural formula of the IIA compound is shown in Formula 1:

[0008]

[0009] Formula 1;

[0010] X is a flexible linker chain, including but not limited to one or more combinations of amino, C1-C6 straight-chain or branched alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, and C1-C6 alkoxyalkyl groups. Specifically, the C1-C6 straight-chain or branched alkyl groups include, but are not limited to, methyl (-CH3), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), isopropyl (-CH(CH3)2), n-butyl (-CH2CH2CH2CH3), isobutyl (-CH2CH(CH3)2), sec-butyl (-CH(CH3)CH2CH3), and tert-butyl (-C(CH3)3). ), n-pentyl (-CH2CH2CH2CH2CH3), isopentyl (-CH2CH2CH(CH3)2), neopentyl (-C(CH3)2CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), isohexyl (-CH2CH2CH2CH(CH3)2), etc.; the C1-C6 alkoxy groups include, but are not limited to, methoxy (-OCH3), ethoxy (-OCH2CH3), n-propoxy (-OCH2CH2CH3), isopropoxy (-OCH(CH3)2), n-butoxy (-OCH2CH2CH2CH3), isobutoxy (-O... CH2CH(CH3)2), n-pentoxy (-OCH2CH2CH2CH2CH3), n-hexyloxy (-OCH2CH2CH2CH2CH2CH3), etc.; the C1-C6 hydroxyalkyl group contains a hydroxyl (-OH) hydrogen bond donor structure including but not limited to hydroxymethyl (-CH2OH), hydroxyethyl (-CH2CH2OH), 1-hydroxypropyl (-CH(OH)CH2CH3), 2-hydroxypropyl (-CH2CH(OH)CH3), 1-hydroxybutyl (-CH(OH)CH2CH2CH3), 2-hydroxybutyl (-CH2CH(OH)CH2CH3), 3-hydroxybutyl (-CH2CH(OH)CH2CH3), etc. -CH2CH2CH(OH)CH3), hydroxypentyl, hydroxyhexyl, etc.; the C1-C6 alkoxyalkyl groups include, but are not limited to, methoxymethyl (-CH2OCH3), methoxyethyl (-CH2CH2OCH3), ethoxymethyl (-CH2OCH2CH3), ethoxyethyl (-CH2CH2OCH2CH3), propoxymethyl (-CH2OCH2CH2CH3), propoxypropyl (-CH2CH2CH2OCH2CH2CH3), butoxybutyl, pentoxypentyl, hexoxyhexyl, etc.; the above X groups can be used alone or can be combined through covalent bonds to form a composite flexible linker chain.

[0011] The core functional fragment R is a functional fragment containing an aromatic conjugated system, including but not limited to one of the following: substituted or unsubstituted phenyl, substituted or unsubstituted phenoxyalkyl, substituted or unsubstituted five-membered aromatic heterocycle, substituted or unsubstituted six-membered aromatic heterocycle, substituted or unsubstituted fused-ring aromatic fragment, and complex active fragment; the substituted or unsubstituted phenyl is selected from phenyl, tolyl, ethylphenyl, 4-hydroxyphenyl, 3-methoxyphenyl, 4-aminophenyl, 3-amidophenyl, 4-trifluoromethylphenyl, 3,5-dihydroxyphenyl, and 4-ethoxyphenyl; the substituted or unsubstituted phenoxyalkyl is selected from phenoxymethyl, phenoxyethyl, and 4-hydroxyphenoxyethyl; the substituted or unsubstituted five-membered aromatic heterocycle is selected from furanyl, 3-ethoxyfuranyl, 2-hydroxymethylfuranyl, thiophenyl, 3-methylthiophenyl, 2-hydroxyethylthiophenyl, imidazolyl, 1-ethylimidazolyl, and 4-hydroxymethylimidazolyl. The substituted or unsubstituted six-membered aromatic heterocycle is selected from pyridyl, 3-hydroxypyridyl, 4-methoxypyridyl, 2-ethylpyridyl, 2-aminopyrimidinyl, 4-hydroxypyrimidinyl, 5-methylpyrimidinyl, pyrazinyl, 4-aminopyrazinyl, 3-hydroxypyridyl, morpholinylphenyl, and piperidinylpyridyl. The substituted or unsubstituted fused-ring aromatic fragments are selected from naphthyl, 1-methoxynaphthyl, 2-hydroxyethylnaphthyl, quinolinyl, 6-hydroxyquinolinyl, 3-propylisoquinolinyl, indoleyl, N-methylindoleyl, 3-indoleethyl, benzofuranyl, benzothiophenyl, and benzimidazolyl; the complex active fragments are selected from 4-ureidophenyl, 3-ureidopyridyl, 4-aminosulfonylphenyl, 3-sulfonamidethiophenyl, coumarinyl, and chromoneyl.

[0012] One end of the linker segment X is covalently connected to the compound nucleus, and the other end is covalently connected to the core functional segment R. The linker site is a substitution site on the aromatic ring or heterocycle of segment R, preferably para or meta. The linker bond between X and R is a C-C bond, a CO bond, or a CN bond.

[0013] Furthermore, X is amino or C1-C6, straight-chain or branched alkyl, alkoxy, hydroxyl, amino, amide, ester, hydroxyalkyl, alkoxyalkyl;

[0014] R is a substituted or unsubstituted five- or six-membered aromatic heterocycle, including: phenyl, naphthyl, quinolinyl, indolyl, furanyl, thiophene, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrroleyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.

[0015] The kidney injury-related diseases include, but are not limited to, acute kidney injury and inflammatory kidney injury. The acute kidney injury includes, but is not limited to, sepsis-induced acute kidney injury, ischemia-reperfusion kidney injury, drug-induced kidney injury, contrast agent-induced nephropathy, and folic acid-induced acute kidney injury.

[0016] The inflammatory kidney injury includes, but is not limited to, acute nephritis diseases such as glomerulonephritis and interstitial nephritis.

[0017] Preferably, the IIA compound of the present invention is IIA-12, and the structural formula of IIA-12 is shown in Formula 2:

[0018]

[0019] Formula 2;

[0020] A pharmaceutical formulation for treating kidney injury, comprising IIA-12 as the active ingredient and pharmaceutically acceptable excipients.

[0021] Furthermore, this invention protects the use of IIA-12 in the preparation of medicaments for the treatment of ischemia-reperfusion kidney injury.

[0022] Furthermore, the application is the use of IIA-12 in the preparation of drugs for the prevention, relief and / or treatment of ischemia-reperfusion kidney injury;

[0023] Furthermore, the application specifically refers to the use of a single IIA-12 formulation, or a drug prepared in combination with a pharmaceutically acceptable diluent or carrier, for the prevention, relief, and / or treatment of kidney injury and its directly related diseases. The kidney injury and its directly related diseases include, but are not limited to, acute kidney injury, chronic kidney disease, glomerulonephritis, tubulointerstitial injury, renal hypertension, renal anemia, nephrotic syndrome, uremia, as well as sepsis-induced kidney injury, drug-induced kidney injury, ischemic kidney injury, and various secondary kidney-related lesions caused by toxic kidney injury.

[0024] Furthermore, the present invention also provides a pharmaceutical composition (IIA-12 pharmaceutical preparation), comprising IIA-12 prepared as a single chemical component pharmaceutical preparation, or prepared in combination with other drugs to form a compound pharmaceutical preparation; the pharmaceutical preparation is formulated into various dosage forms for clinical application according to pharmaceutical requirements and clinical needs, and the dosage forms of the pharmaceutical preparation include, but are not limited to, tablets, capsules, injections, oral liquids, granules, etc.

[0025] The specific methods and requirements for the use of IIA-12 drug formulations for the prevention, relief, and / or treatment of kidney injury are as follows:

[0026] The IIA-12 formulation of the present invention for the prevention, relief and / or treatment of kidney injury can be used alone or in combination with other active ingredients, including products for the prevention, diagnosis, detection, protection, treatment and research of kidney injury and its directly related diseases, including drugs, reagents, food, etc., especially drugs.

[0027] In terms of specific applications, the IIA-12 for preparing the treatment of kidney injury described in this invention can be used alone or in combination with many other chemical substances. Regardless of whether these chemical substances are biologically active or have therapeutic functions, including adjuvant functions such as synergistic amplification, antagonism, or mitigation of side effects in preparing the IIA-12 for treating kidney injury, these chemical substances include one or more of pharmaceutically acceptable carriers, foods, natural products, chemically synthesized drugs, or human medicines; preferably, they include one or more of pharmaceutically acceptable carriers or foods; more preferably, pharmaceutically acceptable carriers.

[0028] As used herein, "pharmaceuticalally acceptable excipients" and "pharmaceuticalally acceptable diluents or carriers" include one or more of any and all physiologically suitable solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, or absorption delaying agents. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, glucose, glycerol, or ethanol, and combinations thereof. In many cases, it is preferable to include an isotonic agent in the composition, such as one or more of sugars, mannitol, sorbitol, polyols of sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may also contain small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which enhance the efficacy or potency of the preparation of IIA-12 for the treatment of kidney injury.

[0029] From a specific classification perspective, the pharmaceutically acceptable carriers refer to conventional drug carriers in the pharmaceutical field, including excipients such as starch or water (one or more); lubricants such as glycerin or magnesium stearate (one or more); disintegrants such as microcrystalline cellulose; fillers such as starch or lactose (one or more); binders such as pregelatinized starch, dextrin, cellulose derivatives, alginate, gelatin, or polyvinylpyrrolidone (one or more); osmotic pressure regulators such as glucose, sucrose, sorbitol, or mannitol (one or more); diluents such as water; disintegrants such as agar, calcium carbonate, or sodium bicarbonate (one or more); absorption enhancers such as quaternary ammonium compounds; surfactants such as hexadecyl alcohol; adsorbents such as kaolin or soap clay (one or more); lubricants such as talc, calcium stearate, magnesium stearate, or polyethylene glycol (one or more); and other excipients such as flavoring agents or sweeteners (one or more) may also be added to the composition.

[0030] For example, an injectable formulation can be prepared by dissolving, suspending, or emulsifying the active ingredient in a suitable aqueous solvent (e.g., one or more of distilled water, physiological saline, or gentian solution) or an oily solvent (e.g., one or more of vegetable oils such as olive oil, sesame oil, cottonseed oil, corn oil, or propylene glycol) to prepare an anti-ischemia-reperfusion renal injury formulation. The solvent may contain a dispersant (e.g., one or more of polysorbate 80, polyoxyethylene-cured castor oil 60, polyethylene glycol, benzyl alcohol, chlorobutanol, or phenol) and an osmotic pressure regulator (e.g., one or more of sodium chloride, glycerol, D9-mannose, D-sorbitol, or glucose). In this case, additives may be added if necessary, such as solubilizers (e.g., one or more of sodium salicylate or sodium acetate), stabilizers (e.g., human serum albumin), analgesics (e.g., benzyl alcohol), etc.

[0031] The IIA-12 formulation for preparing ischemia-reperfusion kidney injury described in this invention can also be used in combination with other chemical substances, such as drugs, used to treat animals, especially mammals, including humans or other animals, or similar compositions. These mammals include one or more of humans, mice, rats, sheep, monkeys, cattle, pigs, horses, rabbits, dogs, chimpanzees, baboons, marmosets, macaques, or rhesus monkeys, preferably one or more of humans, mice, rats, monkeys, pigs, rabbits, or dogs, and more preferably one or more of humans, rats, or monkeys. For example, the IIA-12 formulation for preparing ischemia-reperfusion kidney injury described in this invention can be added to a pharmaceutical composition suitable for administration to a patient. Typically, this pharmaceutical composition comprises the IIA-12 formulation for preparing ischemia-reperfusion kidney injury described in this invention and a pharmaceutically acceptable carrier.

[0032] IIA-12 formulations for treating ischemia-reperfusion kidney injury can be prepared into various dosage forms using conventional manufacturing methods known in the art, such as mixing the active ingredient with one or more carriers and then preparing it into the desired dosage form.

[0033] When used on patients, the dosage of the IIA-12 formulation for treating kidney injury described in this invention is 5 to 100 mg / kg. This dosage or amount is usually determined based on the patient's or user's age and weight, as well as their physical condition or the severity of their symptoms.

[0034] The beneficial effects of this invention are as follows: This application has discovered a novel compound, IIA-12, that can treat ischemia-reperfusion kidney injury. Through cell experiments and animal experiments, it has been confirmed that IIA-12 can reduce apoptosis levels, regulate immune inflammatory responses, and improve renal function in in vivo and in vitro models of ischemia-reperfusion kidney injury. IIA-12 can reduce renal tubular damage caused by AKI, improve renal tubular epithelial cell apoptosis and inflammation, and promote cell function repair. Drug formulations with IIA-12 as the main component have great potential in the treatment of kidney injury. Attached Figure Description

[0035] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0036] Figure 1 Results of experiments on IIA-12 alleviating renal function damage in IRI-AKI model mice;

[0037] In this study, AB: the kit was used to detect serum creatinine and blood urea nitrogen levels in each group of mice; C: the metabolic cage was used to collect and count the urine volume (ml / kg / 12h) of each group of mice after IRI modeling or Sham treatment; D: HE staining was used to clarify the pathological changes in the morphology and structure of the kidney tissue in each group of mice, with black scale bar = 100μm and red scale bar = 50μm; E: semi-quantitative analysis of the renal tubular injury index in each group in HE staining.

[0038] Figure 2 The results of mouse experiments showed that compound IIA-12 could improve renal tubular injury caused by IRI;

[0039] In this study, A: PAS staining and KIM-1 immunohistochemical staining were performed on kidney sections from mice in each group. B: Semi-quantitative analysis of the renal tubular injury index in each group based on PAS staining. C: Semi-quantitative analysis of the positive area of ​​KIM-1 immunohistochemical staining in each group. D: Western blot detection of the expression levels of KIM-1, AQP-2, and NGAL proteins in the kidney tissues of mice in each group. EG: Grayscale values ​​were statistically analyzed to semi-quantitatively determine the expression levels of KIM-1, AQP-2, and NGAL proteins in the kidney tissues of mice in each group. Black bar = 100 μm; Red bar = 50 μm;

[0040] Figure 3 Cellular experimental results showing that compound IIA-12 alleviates IRI-induced apoptosis in renal tubular epithelial cells;

[0041] A: TUNEL staining analysis of apoptosis levels in kidney tissues of mice in the Sham, IRI, and IRI+IIA-12 (30 mg / kg) groups. B: Statistical analysis of TUNEL staining results. C: Western blot detection of expression levels of apoptosis-related proteins BCL-2 and BAX in kidney tissues of mice in each group. D: Semi-quantitative analysis of WB results. E: Transmission electron microscopy showing the ultrastructure of kidney tissues of mice in each group. TUNEL staining scale bar = 100 μm; electron microscopy scale bar = 0.5 μm.

[0042] Figure 4 Cellular experimental results showing that compound IIA-12 improves IRI-induced inflammatory response in mouse kidney tissue;

[0043] In this study, A: IHC staining analysis revealed the expression levels of TNF-α and IL-6 molecules in the kidney tissues of mice in each group. BC: Semi-quantitative analysis showed the positive area of ​​TNF-α and IL-6 immunohistochemical staining. D: Western blot analysis detected the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the kidney tissues of mice in each group. EG: Statistical analysis of the WB results. Scale bar = 50 μm.

[0044] Figure 5 Cellular experimental results showing that compound IIA-12 alleviates H / R-induced damage and apoptosis in HK-2 cells;

[0045] A: CCK-8 assay to detect the effect of different concentrations of IIA-12 stimulation on HK-2 cell viability for 12h and 24h. B: Cell experiments were divided into four groups: Control group, IIA-12 (5μM) group, H / R group, and H / R+IIA-12 group. Western blot was used to detect the expression levels of KIM-1, BCL-2, and BAX proteins in each group. C: Semi-quantitative analysis of WB results. D: Flow cytometry to detect the proportion of apoptotic cells in each group. E: Statistical analysis of the flow cytometry apoptosis detection results.

[0046] Figure 6 Cellular experimental results showing that compound IIA-12 alleviates H / R-induced inflammatory response in HK-2 cells;

[0047] In this study, A: Western blot was used to detect the expression levels of AHR, FOXO3, IL-1β, and TNF-α proteins in each group of cells. BE: The gray values ​​of each group in the WB results were statistically analyzed and semi-quantitatively analyzed. F: RT-qPCR was used to analyze the differences in IL-6 gene expression levels among the Control group, IIA-12 group, H / R group, and H / R+IIA-12 group. Detailed Implementation

[0048] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0049] The experimental materials and methods involved in the embodiments of this application are as follows:

[0050] 1. Experimental Materials

[0051] 1.1 Experimental Animals: The C57BL / 6J mice (male, 6-8 weeks old, 18-20g) used in the experiment were purchased from Beijing Vital River Biotechnology Co., Ltd. All mice were housed at the Model Animal Research Center of Shandong University. All animal husbandry and experiments strictly adhered to the guidelines of the Animal Experiment Ethics Committee of Shandong University.

[0052] 1.2 Experimental Cells: Human renal tubular epithelial cell line (HK-2 cells) was obtained from the National Model and Characteristic Experimental Cell Resource Bank of the Chinese Academy of Sciences. It was cultured in DMEM complete medium (containing 10% FBS + 1% streptomycin / penicillin) in a 37℃ constant temperature cell incubator.

[0053] 1.3 Clinical samples: Paraffin-embedded kidney tissue sections were obtained from Qilu Hospital of Shandong University, from patients pathologically diagnosed with acute tubular injury and minimal change disease. Detailed clinical baseline information is provided in the appendix. Patient privacy was strictly protected in accordance with the ethical principles outlined in the Declaration of Helsinki, and the use of kidney biopsy samples followed the guidelines of the Ethics Committee of Qilu Hospital of Shandong University.

[0054] 2. Experimental Methods

[0055] 2.1 Animal Experiments All animal experiments were conducted at the Model Animal Research Center of Shandong University. Mice were fed according to SPF-grade animal husbandry conditions, housed in independent ventilated cages, with no more than 5 mice per cage, and provided with sterilized feed, ample drinking water, and a 12h-12h circadian light cycle. Mouse experimental designs adhered to the "3R principle." All animal experiments followed the guidelines of the Animal Experiment Ethics Committee of Shandong University.

[0056] 2.1.1 Construction of a mouse model of ischemia-reperfusion kidney injury

[0057] (1) Preparation of materials: Drug preparation: 1.25% tribromoethanol (Avorin), 75% ethanol solution, povidone-iodine solution, 0.9% NaCl solution. Equipment preparation: Mouse weighing scale (0.01g), medical cotton swabs, 1ml syringe, scalpel blade, medical tape, medical gauze, heat lamp. Instrument preparation: Artery clamp, ophthalmic scissors, forceps, needle holder, surgical sutures, suture needles, tissue scissors.

[0058] (2) Experimental steps: First, the experimental table and materials were irradiated with ultraviolet light for 30 minutes. The weight of the mice was weighed and recorded. The abdominal skin of the mice was disinfected with 75% ethanol solution. The mice were anesthetized by intraperitoneal injection of aphthylamine solution at a dose of 30 μl / g. After 5 minutes, the mice were fixed in a supine position on a foam board. The abdominal skin was prepared with a scalpel and disinfected with povidone-iodine. Next, a midline abdominal incision was made. The skin was lifted with forceps to expose the surgical field. The contents of the mouse's abdominal cavity were pushed to one side with a medical cotton swab to expose the contralateral renal pedicle. The renal artery was bluntly dissected with toothless forceps. Further, the renal arteries of both sides were clamped one after another with arterial clamps. After observing that the mouse kidneys changed from bright red to purplish red, sterile gauze was covered at the incision site and a heat lamp was turned on to prevent the mice from hypothermia. The arterial clamps were removed after 30 minutes. The mice in the Sham group only had a surgical incision made without clamping the renal arteries. Finally, the abdominal muscle layer and abdominal skin were sutured one after another. Disinfect the surgical incision, add 0.5 ml of 0.9% NaCl solution, and wait for the mouse to wake up. Keep the mouse warm throughout the experiment.

[0059] 2.1.2 Collection of mouse tissue and urine samples

[0060] (1) Collection of mouse urine samples: First, the weight of the mice was routinely weighed and recorded before collecting urine. The urine of the mice was collected for 12 hours using a metabolic cage, and the urine volume was recorded. Second, the collected urine was placed in a 5 ml centrifuge tube and centrifuged at 3000 rpm for 10 min at 4℃. Finally, the supernatant was placed in a sterile EP tube, labeled, and stored at -20℃ for biochemical detection.

[0061] (2) Collection of mouse serum samples: First, after anesthetizing the mice, blood was drawn through the retro-orbital vein using a sterile capillary glass tube. After drawing 2-3 drops (approximately 50 μl) of blood, hemostasis was achieved by applying pressure. Second, the collected blood samples were numbered and recorded, and after standing for 60 min, they were centrifuged at 8000 rpm for 15 min at 4℃. Finally, after centrifugation, the supernatant was transferred to a new EP tube, numbered, and stored at -20℃ for subsequent biochemical detection experiments.

[0062] (3) Mouse kidney tissue collection: First, 24 hours after ischemia-reperfusion modeling, mice were anesthetized by intraperitoneal injection of afodin, and the mice were fixed in a supine position on the operating table with medical tape. Second, the chest wall was opened using a scalpel and tissue scissors, and pre-cooled physiological saline was drawn into a 10ml syringe and perfused along the apex of the heart. Finally, after the blood was drained, the kidney tissue was excised and the renal fascia layer was removed. A longitudinal section was made in the renal cortex, and half of the tissue was fixed in 4% paraformaldehyde for histological staining. The remaining kidney tissue was immediately cryopreserved in liquid nitrogen for molecular biological detection.

[0063] 2.2 Biochemical Detection Experiment

[0064] 2.2.1 Detection of serum creatinine levels in mice

[0065] (1) Mouse serum samples were collected and numbered. The creatinine level in mouse blood was determined using the sarcosine oxidase method. Enzyme solution A, enzyme solution B and standard were obtained from creatinine detection kit (Nanjing Jiancheng).

[0066] (2) Prepare a 96-well plate, divided into wells for assay, blank control, and standard. Add 6 μl of the test sample, ddw, and standard to each well. Then add 180 μl of enzyme solution A to each well.

[0067] (3) React in an incubator at 37°C for 5 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance OD value A1 at a wavelength of 546 nm.

[0068] (4) After measuring A1, add 60 μl of enzyme solution B to each well, place it in an incubator at 37°C for 5 min, and measure the absorbance OD value A2 at 546 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader.

[0069] Note: The concentration of the creatinine standard is 442 μmol / L; dilution factor K = 0.756.

[0070] 2.2.2 Detection of blood urea nitrogen levels in mice

[0071] (1) Collect mouse serum samples and record them. First, dilute each serum sample 20 times with ddw. Prepare enzyme solution, phenol chromogenic reagent, alkaline sodium hypochlorite and standard, all of which are from urea nitrogen detection kit (Nanjing Jiancheng). Use urease method to detect the urea nitrogen level in the diluted mouse serum.

[0072] (2) Prepare buffer enzyme solution: Prepare buffer enzyme solution according to the ratio of enzyme stock solution to enzyme dilution solution of 3:1000.

[0073] 2.3 Cell Experiment Techniques

[0074] 2.3.1 Cell culture medium change: Disinfect the laboratory bench and supplies with ultraviolet light for 30 minutes. First, prepare complete culture medium: Add fetal bovine serum (final concentration 10%) and penicillin / streptomycin (final concentration 1%) to DMEM medium. Next, discard the old culture medium with a Pasteur pipette, wash three times with 1X PBS, and then add freshly prepared culture medium. Generally, 5 ml of complete culture medium is added to each T25 cell culture flask.

[0075] 2.3.2 Cell passage

[0076] (1) Observe the cell density in the T25 culture flask under a microscope. When the cell density is about 60%-80%, the cells can be passaged. Disinfect the experimental table and experimental supplies with ultraviolet light for 30 min. Use a Pasteur pipette to discard the old culture medium. Wash three times with 1XPBS and add 1 ml of trypsin. Place the flask in a 37℃ cell culture incubator for 1 min to digest.

[0077] (2) Observe the cell morphology under a microscope. If the adherent cells become round and a glowing outline appears around them, stop digestion. Add complete culture medium. Use a sterile pipette tip to draw up the complete culture medium and gently blow it into the bottom of the vial. Divide the vial into four quadrants and blow into each quadrant three times.

[0078] (3) Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 8000 rpm for 5 min. Discard the supernatant, resuspend the cells in complete culture medium, transfer them to a new culture flask, mark the passage time, and then transfer them to a 37°C cell incubator for culture.

[0079] 2.3.3 Seeding Cells: Observe the cell density in the T25 culture flask under a microscope. When the cell density reaches approximately 60%-80%, transfer the cells to cell culture plates. Pre-sterilize laboratory equipment and the worktable with UV light for 30 minutes beforehand. Remove the culture flask, discard the old culture medium, wash three times with 1X PBS, and then digest the cells with trypsin. After digestion, discard the trypsin, resuspend the cells in complete culture medium, rinse the bottom of the flask, centrifuge at 800 rpm for 5 minutes, discard the supernatant, resuspend the cells in complete culture medium, and transfer the resuspended cell culture plate. Add 2 ml of complete culture medium to each well of a 6-well cell culture plate; add 1 ml of complete culture medium to each well of a 12-well plate; and add 0.5 ml of complete culture medium to each well of a 24-well plate. After marking the seeding time, transfer the cell culture plate to a cell culture incubator for further culture.

[0080] 2.3.4 After seeding small interfering cells into a cell culture plate and culturing them in an incubator for a period of time, small interfering RNA transfection can be performed when the cell density reaches approximately 50%-70% under a microscope. The experimental setup and equipment are sterilized by UV irradiation for 30 minutes beforehand. First, the siRNA and transfection reagent are diluted 1:50 with buffer solution to prepare the transfection system. The mixture is gently pipetted and allowed to stand for 10 minutes. The 6-well plate is removed from the cell incubator and washed twice with 1X PBS solution in a clean bench. Then, 1.8 ml of culture medium and 200 μl of the transfection system are added to each well. The cells are incubated in the cell incubator for 6 hours, then replaced with complete culture medium at normal serum concentration. PCR is used to detect changes in the target gene RNA expression level after 48 hours, and Western blotting is used to detect changes in the target gene protein expression level after 72 hours.

[0081] 2.3.5 Cell cryopreservation: Observe the cell density in the T25 culture flask under a microscope. Cells are ready for cryopreservation when the density reaches approximately 70%-90%. Beforehand, irradiate the cell culture bench and equipment with ultraviolet light for 30 minutes, disinfect the surfaces with 75% ethanol, discard the old culture medium, digest with trypsin, centrifuge at 800 rpm for 5 minutes, and discard the supernatant. Add 2 ml of cell cryopreservation solution, gently pipette to resuspend the cells, and transfer them into cryovials. Use a gradient cryopreservation method: first, transfer the cryovials to a -20°C freezer for 2 hours, then to a -80°C freezer for short-term storage for 12 hours, and finally transfer them to a liquid nitrogen tank for long-term storage.

[0082] 2.3.6 Cell Thawing: Preheat a 37°C water bath. Remove the cryovials from the -80°C freezer and thaw them in the water bath with stirring for 1 min. Transfer the thawed cell suspension to a sterile centrifuge tube and centrifuge at 800 rpm for 5 min. Discard the supernatant, resuspend the cells in complete culture medium, seed the resuspended cells in vials or well plates, label the thawing time, and place in a 37°C cell culture incubator.

[0083] 2.3.7 Constructing a cellular hypoxia / reoxygenation (H / R) model

[0084] (1) After the cells were grouped, when the density of adherent cells in the cell culture plate was 60%-80%, the cells were washed three times with 1XPBS. The control group cells were given DMEM complete medium, while the modeling group was given DMEM / F12=1 low glucose serum-free medium.

[0085] (2) The cells of the modeling group were placed in a hypoxic incubator and incubated for 2 hours in a hypoxic incubator containing (1% oxygen, 5% carbon dioxide, 94% nitrogen). The cell culture plates were then removed and replaced with DMEM complete medium. The cells of the control group were cultured in a regular incubator.

[0086] (3) After the cell culture medium of the modeling group was changed, the cells were transferred to a regular incubator and the timing was started. Cell viability was detected after 24 hours. Cells were collected and cell proteins or total RNA were extracted for further molecular biological detection.

[0087] 2.4 Flow cytometry detection of apoptosis

[0088] (1) Cell collection: After the cell experiment reaches the stimulation time, remove the culture plate, collect the supernatant in the flow cytometer according to the experimental group, add trypsin without EDTA for digestion, stop digestion after the cells become round and gently resuspend the cells, mix the resuspended solution with the supernatant, centrifuge at 18000rpm for 5min at 4℃, discard the supernatant, and resuspend the cells in 1XPBS.

[0089] (2) Washing cells: Centrifugation → Resuspension: Centrifuge at 18000rpm for 5min at 4℃ and gently resuspend the cells in 1XPBS. Repeat twice.

[0090] (3) Add binding buffer: Add 100 μl binding buffer and gently mix until a single cell suspension is formed.

[0091] (4) Staining: Protect from light, add 5 μl APC and 5 μl PI, mix gently by blowing, protect from light, and incubate at room temperature for 10 min.

[0092] (5) Dilution: Protect from light, add 400 μl binding buffer, shake to mix, and use a flow cytometer to detect the sample within 1 hour.

[0093] (6) Flow cytometry analysis and results analysis: Each sample tube was numbered and recorded, and apoptosis data were collected using a flow cytometer. The proportion of various cell types in the data was analyzed using FlowJo (v.10) software.

[0094] 2.5 Western blot assay for cell proteins

[0095] (1) Cell protein extraction: Prepare protein lysis buffer and ice pack. Wash well plates with 1XPBS, digest cells with trypsin according to experimental groups, and centrifuge. Discard the supernatant, add protein lysis buffer to the precipitate, and lyse on ice for 30 min.

[0096] (2) Gel electrophoresis → protein transfer → immunoblotting → development, the experimental steps are the same as those for tissue protein immunoblotting.

[0097] 2.6 Real-time quantitative PCR experiment for cells

[0098] 2.6.1 Trizol method for extracting cellular RNA

[0099] (1) Lysis: After the stimulation time is reached in the cell experiment, wash twice with 1XPBS and aspirate any residual liquid. Follow the 1ml / 10 6 Add one cell to Trizol solution, mix well, and let stand at room temperature for 5 minutes to lyse the cells.

[0100] (2) Phase separation: Add 200 μl of chloroform to each EP tube, shake to mix, and let stand at room temperature for 10 min. Then centrifuge at 12000 rpm for 15 min at 4℃. The liquid separates into three layers: the upper aqueous phase contains RNA, the middle layer is protein, and the lower layer is organic phase.

[0101] (3) Precipitation: Gently aspirate the supernatant (avoid contact with the middle layer), transfer it to a new EP tube, add an equal volume of isopropanol, and gently invert to mix. After standing for 10 min, centrifuge at 12000 rpm for 15 min at 4℃. The white precipitate at the bottom of the EP tube is RNA.

[0102] (4) Washing: Discard the supernatant, add 1 ml of pre-cooled 75% ethanol, mix well, and centrifuge at 12000 rpm for 10 min at 4℃. After centrifugation, discard the supernatant, add 20 μl of DEPC water, and take 1 μl to measure RNA concentration and purity.

[0103] 2.7 CCK8 cell viability assay

[0104] (1) Cell seeding: Select a 96-well plate, digest the cells according to the group under sterile conditions and seed the plate. Add 100 μl of culture medium to each well and pre-culture the cells in a 37°C incubator for 24 h.

[0105] (2) Add different concentrations of the test drug to the 96-well plate and incubate the plate in an incubator for 12h or 24h.

[0106] (3) Slowly add 10 μl of CCK-8 solution to each well to avoid the generation of bubbles.

[0107] (4) After incubating the culture plate in an incubator for 1 hour, measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0108] (5) Prepare a standard curve: Dilute the cells in a proportional manner with culture medium to form a concentration gradient. After inoculation, culture for 2 hours to allow the cells to adhere to the wall. Then, add 10 μl of CCK-8 reagent to every 100 μl of culture medium and culture for a certain period of time before measuring the OD value. Prepare a standard curve with the number of cells as the x-axis and the OD value as the y-axis.

[0109] (6) Calculate the activity of each group of cells based on the standard curve and absorbance.

[0110] 2.8 Western Blot (WB) Assay

[0111] 2.8.1 Kidney tissue protein extraction

[0112] (1) First, prepare tissue lysis buffer according to the ratio of PMSF:RIPA lysis buffer = 1:100 and place it on ice for later use. Take mouse kidney tissue samples from an 80°C freezer and cut 3mm sections with a scalpel. 3 The kidney tissue was placed in new EP tubes and numbered. The remaining tissue was returned to storage at -80°C.

[0113] (2) Add 50 μl of tissue lysis buffer to the EP tube, place it in an ice box, grind the kidney tissue into a homogenate for 1 min using a tissue homogenizer, add 150 μl of tissue lysis buffer to the EP tube after grinding, shake and centrifuge at low speed to mix the tissue and lysis buffer thoroughly, and place the EP tube in an ice box for lysis for 30 min.

[0114] (3) The tissue lysis products were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected. The protein concentration was measured and recorded using an ultra-micro spectrophotometer.

[0115] 2.8.2 Tissue Protein Denaturation: Add 1 / 4 volume of loading buffer to the protein lysis buffer extracted in step 2.8.1, vortex for 1 min, centrifuge at low speed to mix thoroughly, then heat in a 100℃ metal bath for 10 min. After removal, cool on ice and use directly or store at -20℃ for a short period.

[0116] 2.8.3 SDS-PAGE gel electrophoresis

[0117] (1) Install the glue-making plate: Clean the glass plate with a DDW and dry the surface of the glass plate in a 55℃ constant temperature oven. Install the glass plate on the glue-making frame, aligning the ends of the thick and thin glass plates and fixing them with plastic clips. After installation, use a DDW to test for leaks. After confirming that there is no leakage, pour out the liquid and remove the moisture with absorbent paper.

[0118] (2) Preparation of separating gel (bottom layer gel): Select the appropriate concentration of separating gel according to the molecular weight of the target protein. Proteins with a molecular weight of 30-100 kDa typically use a 10% separating gel, proteins with a molecular weight of 20-30 kDa use a 12% bottom layer gel, and proteins with a molecular weight below 20 kDa require a 15% separating gel. The corresponding concentrations of separating gel solution, separating gel buffer, and modified coagulant are all from the gel preparation kit (Yamei Biotechnology), prepared in a ratio of 1:1:0.02. Approximately 8 ml of bottom layer gel is needed per plate.

[0119] (3) Flattening the separating gel: Add 1 ml of isopropanol to the top of the separating gel in the glass plate. After observing that the liquid level is horizontal, let it stand for 10 minutes. When a clear boundary appears between the isopropanol and the separating gel, it proves that the separating gel has solidified. Discard the isopropanol and use absorbent paper to dry the liquid.

[0120] (4) Preparation of stacking gel (top layer gel): Prepare a 5% stacking gel. Take an equal volume of top layer gel solution and stacking gel buffer, mix well, then add 1 / 50 volume of modified coagulant and mix well. Pour the mixture into the gel casting plate and insert the comb. Each plate requires approximately 4 ml of stacking gel. The stacking gel will solidify after standing for 15 minutes.

[0121] (5) After the concentrated gel solidifies, remove the gel preparation plate, soak it in ddw, and store it in a refrigerator at 4°C for later use.

[0122] (6) Prepare the SDS gel electrophoresis buffer, install the electrophoresis apparatus, and completely immerse the glass plate in the electrophoresis buffer. Sample loading: Remove the comb, add 3-6 μl of protein ladder to both sides of the sample well, and calculate the required loading volume for 30 μg of total protein based on the protein concentration in step 2.8.1. Electrophoresis: Maintain a constant voltage of 80V for approximately 30 minutes. When the molecular weights in the ladder separate, adjust the voltage to 120V. Stop electrophoresis when the bromophenol blue reaches the bottom of the glass plate.

[0123] 2.8.4 Protein Transfer

[0124] (1) Transfer preparation: First, prepare the transfer buffer and store it in a 4°C refrigerator for later use. Take out the glass plate from the electrophoresis buffer and cut it to an appropriate size according to the molecular weight of the target protein, then cut the appropriate PVDF membrane according to the size of the gel. For target proteins with a molecular weight greater than 50 kDa, select a PVDF membrane with a pore size of 0.45 μm. If the molecular weight of the target protein is less than 50 kDa, a PVDF membrane with a pore size of 0.2 μm is required. First, activate the PVDF membrane by placing it in methanol for 1 min. Open the transfer sandwich clamp, immerse it in the transfer buffer, and then place the sponge, filter paper, gel, and activated PVDF membrane on it in sequence. Use a roller to remove air bubbles between the gel and the membrane, and always keep the gel and PVDF membrane moist. Close and press the transfer clamp tightly and place it in the transfer rack.

[0125] (2) Protein transfer: Install the transfer rack into the transfer tank, pour in the transfer solution to soak the transfer rack, and then place the transfer tank in a pre-prepared ice box. Place the PVDF membrane at the positive electrode and the gel at the negative electrode. The constant current is 245mA. The transfer time is calculated to be 1.5xmin based on the protein molecular weight xkd at a ratio of 1:1.5.

[0126] 2.8.5 Immunoblotting

[0127] (1) Blocking: Preparation of blocking solution: Weigh BSA or skim milk powder and dissolve it in 1XTBST solution to prepare a 5% protein blocking solution. Place the PVDF membrane with the side closest to the gel facing up in the blocking solution and block it at room temperature for 2 hours on a shaker at 40 rpm. After blocking, remove the PVDF membrane and wash it three times with 1XTBST at 80 rpm for 5 minutes each time.

[0128] (2) Incubation with primary antibody: Prepare the primary antibody working solution according to the dilution ratio in the instructions using the primary antibody diluent. Immerse the PVDF membrane in the primary antibody working solution and incubate overnight at 50 rpm on a shaker at 4°C. (3) Incubation with secondary antibody: Remove the PVDF membrane from the primary antibody, add 1XTBST, and wash 4 times at 80 rpm on a shaker for 5 min each time. The secondary antibody working solution for goat anti-mouse or goat anti-rabbit is prepared by mixing 5% BSA at a ratio of 1:8000. Immerse the PVDF membrane after washing with the primary antibody in the secondary antibody working solution and incubate at room temperature at 60 rpm on a shaker for 1 h. Remove the PVDF membrane again and wash 4 times with 1XTBST for 5 min each time. Keep the PVDF membrane moist during the process.

[0129] (4) Protein Development: Mix developer A and developer B in equal proportions, prepare fresh, and store in a light-proof EP tube for later use. Before development, remove moisture from the PVDF membrane with absorbent paper, add developer, and expose the membrane in the developing instrument to obtain the image. Save and record the image. (5) Immunoblot Image Analysis: The raw image data comes from the gel imaging system (Tanon4800). The gray values ​​of the target protein bands are analyzed using ImageJ (v.1.54) software, and semi-quantitative calculations are performed. The immunoblotting detection of the target protein in each independent experiment is repeated at least 3 times.

[0130] 2.9 Kidney tissue section staining experiment

[0131] 2.9.1 Hematoxylin-eosin (HE) staining

[0132] (1) Dehydration of tissue embedded in paraffin: The kidney tissue was placed in dehydration boxes and numbered. All dehydration boxes were placed in a dehydrator for sequential dehydration with ethanol. 75% ethanol 4h → 85% ethanol 2h → 90% ethanol 2h → 95% ethanol 1h → anhydrous ethanol I 30min → anhydrous ethanol II 30min → paraffin I 1h → paraffin II 1h → paraffin III 1h. Embedding: The paraffin-soaked tissue was embedded in an embedding machine. First, the melted wax was placed in the embedding frame. The kidney tissue was taken out of the dehydration box before the paraffin solidified, with the cross-section facing up, and placed in the embedding frame and labeled accordingly. Fixation: Cooled in an ice-water mixture for 10min. After the paraffin solidified, the wax block was removed and the edges of the wax block were trimmed with a scraper.

[0133] (2) Tissue sectioning: Fix the trimmed wax block onto the paraffin microtome and section it to a thickness of 4 μm. Float the tissue section on the surface of water at 37°C in the slide spreader until the tissue is flattened. Use the corresponding numbered glass slide to lift the tissue section horizontally and place it in an oven at 60°C to dry the moisture. Then collect the tissue slide and store it in a slide box at room temperature.

[0134] (3) Dewaxing the sections: Place the sections to be dewaxed in a 65°C incubator for 1 hour, and dewax them with a gradient of environmentally friendly dewaxing agents and ethanol solutions to water: environmentally friendly dewaxing agent I 10 min → environmentally friendly dewaxing agent II 10 min → 90% ethanol 5 min → 80% ethanol 5 min → 70% ethanol 5 min → 1XPBS shaker at 80 rpm twice, 5 min each time.

[0135] (4) Hematoxylin-Eosin Staining: Hematoxylin staining: First, outline the tissue with a tissue pen. Add 50 μl of hematoxylin staining solution to each tissue, ensuring the staining solution covers the tissue. After staining for 2 min, wash and rinse three times with 1XPBS at 80 rpm for 5 min each time. Differentiation: Immerse in 5% hydrochloric acid alcohol differentiation solution for 30 sec, rinse with tap water for 5 min to return to blue. Eosin staining: Place the sections in eosin solution for staining for 3 min, then wash and rinse three times with 1XPBS at 80 rpm for 5 min each time. Dehydration: Immerse the sections in the following solutions in sequence: 70% ethanol → 80% ethanol → 90% ethanol → anhydrous ethanol, for 1, 2, 3, and 4 min respectively. Clearing: Place in environmentally friendly dewaxing agent I for 5 min → environmentally friendly dewaxing agent II for 5 min, and air dry. Mounting: Add 20 μl of neutral resin to each tissue and then mount, avoiding the formation of air bubbles during mounting. (5) Collect images and score: At least 3 stained sections of each kidney tissue, and at least 3 high-power fields of view for each section. Renal tubular damage is counted and scored as follows: Percentage of renal tubules showing cell atrophy and necrosis, tubular dilation, cast formation, brush border loss, or immune cell infiltration: 0 points: none; 1 point: none.

[0136] 2.9.3 Immunohistochemical staining (IHC)

[0137] (1) Dewaxing of sections: The procedure is the same as that for HE staining.

[0138] (2) Antigen retrieval: Prepare 0.01M citrate buffer (pH=6.0) antigen retrieval solution, immerse the slide in the retrieval solution, place it in a microwave oven and heat on high (100%) for 3 min, then turn to low (40%) for 15 min, cool naturally to room temperature and wash, wash 3 times with 1XPBS on a shaker at 80 rpm for 5 min each time.

[0139] (3) Cell permeation: Prepare a cell permeation solution, namely a 3% hydrogen peroxide aqueous solution. Immerse the slides in the permeation solution and permeate at room temperature for 20 min. Wash the slides and wash them three times with 1XPBS at 80 rpm for 5 min each time.

[0140] (4) Antigen blocking: Prepare blocking solution by selecting host serum from the same source as the secondary antibody and diluting it 4 times with 1XPBS. Use a tissue pen to outline the area to be stained, add 50 μl of blocking solution to each tissue to cover the tissue, and place it in an incubator at 37°C for 30 min.

[0141] (5) Add primary antibody: Prepare the primary antibody working solution according to the antibody instructions. After removing the blocking solution with absorbent paper, add 20 μl of primary antibody to each tissue to cover the tissue, place it in a humidified box, and incubate it in a 37°C incubator for 1 h.

[0142] (6) Adding secondary antibody: Recover the primary antibody into an EP tube, wash the slides in 1XPBS, and wash three times at 80 rpm for 5 min each time. After adding the enhancer, incubate at 37℃ for 20 min, and wash three times at 80 rpm with 1XPBS solution. Add 50 μl of goat anti-mouse or rabbit antibody to each tissue according to the host source of the primary antibody, and incubate at 37℃ for 30 min; wash three times at 80 rpm with 1XPBS solution for 5 min each time.

[0143] (7) DAB staining: Prepare DAB staining solution at a ratio of 1:20, using freshly prepared solution. Remove surface moisture from the sections with absorbent paper and observe under a microscope, adjusting the focus to concentrate on the tissue plane. Add 50 μl of DAB staining solution to each tissue and start timing. Staining is complete when a brownish-yellow color change and appropriate color depth are observed under the microscope. After staining, place the sections in PBS to stop staining and record the staining time. The staining time must be consistent for the same target protein in different groups within the same batch of experiments.

[0144] (8) Staining and mounting: After DAB staining, add 50 μl of hematoxylin staining solution to each tissue sample to cover the tissue. After staining for 2 min, rinse with running water for 5 min, immerse in differentiation solution for 2 sec, and continue rinsing with running tap water for 5 min to return to blue. Dehydration: Place the sections in 70% ethanol → 80% ethanol → 90% ethanol → anhydrous ethanol in sequence for 1, 2, 3, and 4 min respectively. Clearing: Place in environmentally friendly dewaxing agent I for 5 min → environmentally friendly dewaxing agent II for 5 min, and air dry at room temperature. Mounting: Add 20 μl of neutral resin to each tissue and then mount, avoiding the formation of air bubbles during mounting. (9) Image acquisition and analysis: After mounting, place the sections in a machine with an automatic image acquisition system to observe and acquire images at 200x and 400x magnification. At least 3 high-power fields of view are acquired for each section. Use ImageJ (v.1.54) software to identify the area of ​​immunohistochemically positive staining regions, calculate the positive area ratio, and then perform semi-quantitative analysis. 2.9.4 Immunofluorescence staining (IF)

[0145] (1) Tissue embedding and dewaxing of sections: same as HE staining.

[0146] (2) Antigen retrieval: Same as IHC staining.

[0147] (3) Antigen blocking: Circle the outline of the stained area with a tissue pen, add 50 μl of serum blocking solution to each tissue, and block for 30 min at room temperature.

[0148] (4) Antibody incubation: Remove serum with absorbent paper, dilute the primary antibody according to the antibody instructions, add 50 μl of primary antibody to each tissue, and place the slides in a humidified chamber and incubate at 37°C for 1 h. After recovering the primary antibody, wash the slides three times with 1XPBS solution at 80 rpm for 5 min each time. Protect from light, add fluorescent secondary antibody working solution, and incubate at 37°C for 1 h. Wash three times with 1XPBS solution at 80 rpm for 5 min each time.

[0149] (5) Nucleus staining and mounting: Protect from light and mount with anti-fluorescence quenching mounting medium containing DAPI.

[0150] (6) Image acquisition and analysis: Use an Olympus fluorescence microscope to observe and acquire fluorescence images. At least 3 high-power fields of view are acquired for each slide. Use ImageJ (v.1.54) software to identify the proportion of immunofluorescence staining positive areas and perform semi-quantitative analysis. 2.9.5 TUNEL staining of tissue (1) Tissue embedding and dewaxing of slides: The steps are the same as HE staining. (2) Adding proteinase K: Place the slides in a humidified box, circle the outline of the staining area with a tissue pen, add 20 μg / ml proteinase K without DNase to each tissue, incubate at 37℃ for 30 min, and then wash. Wash 4 times with 1XPBS solution at 80 rpm, 5 min each time. (3) Preparation of TUNEL staining solution: Prepare according to the ratio of TdT enzyme: fluorescent labeling solution: TUNEL detection solution equal to 1:9:10, and use immediately after preparation. Add 50 μl of staining solution to each tissue sample, protect from light, incubate at 37℃ for 1 h, wash, and wash 3 times with 1XPBS solution at 80 rpm, 5 min each time. (4) Nuclear staining and mounting: Protect from light and mount with a DAPI-containing anti-fluorescence quenching mounting medium. (5) Image acquisition and analysis: Observe and acquire staining results under a fluorescence microscope. The excitation wavelength of Cy3 is 550 nm and the emission wavelength is 570 nm. At least 3 slides should be acquired for each group, and at least 3 high-power fields should be acquired for each slide. Use ImageJ (v.1.54) software to identify the proportion of TUNEL-positive cells and perform semi-quantitative analysis.

[0151] 2.9.6 Electron microscopy: After sampling mouse kidney tissue, it was immediately cut into pieces of approximately 1 mm. 3 The samples were prepared by forming cubes of the desired size and then transferring them into an electron microscopy fixative. The ultrastructure of renal tubular epithelial cells in the control, model, and treatment groups was observed and imaged using transmission electron microscopy (TEM). Electron microscopy equipment and technical support were provided by Wuhan Sewell Biotechnology Co., Ltd.

[0152] Example 1

[0153] Compound IIA-12 alleviates renal function damage in the IRI-AKI model.

[0154] To further explore the role of IIA-12 in ischemia-reperfusion kidney injury, we designed the following animal experiments using 6-8 week old C57BL / 6J mice, which were divided into four groups:

[0155] 1. Sham group: sham surgery was performed, and 0.1 ml of PBS was administered twice a day before modeling.

[0156] 2. IRI group: Mice were modeled with IRI-AKI. Before modeling, they were given 0.1 ml of PBS twice a day.

[0157] 3. IRI+IIA-12L group: Mice were modeled with IRI-AKI, and were given 15 mg / kg of IIA-12 by gavage twice a day before modeling.

[0158] 4. IRI+IIA-12H group: Mice were modeled with IRI-AKI. Before modeling, they were given 30 mg / kg of IIA12 by gavage twice a day.

[0159] Twenty-four hours after IRI modeling, indicators of renal function impairment in mice of each group were examined. First, serum creatinine and blood urea nitrogen levels were measured in each group. Results showed that compared to the Sham group, serum creatinine and blood urea nitrogen levels were significantly increased in the IRI group, indicating successful establishment of the IRI acute kidney injury model. Serum creatinine and blood urea nitrogen levels in the IRI+IIA-12L group were lower than those in the IRI group, and serum creatinine and blood urea nitrogen levels in the IRI+IIA-12H group were significantly lower than those in the IRI group, showing a dose-dependent relationship with IIA-12. Figure 1 (AB). Statistical results of the urine-to-body weight ratio of mice in each group showed that the urine volume of mice in the IRI group after IRI modeling was significantly higher than that in the Sham group, while the urine volume of mice in the IRI+IIA-12L and IRI+IIA-12H groups after IRI modeling was significantly lower than that in the IRI group. Figure 1 (C) Further hematologic staining of kidney tissue sections was performed, and the renal tubular injury score was calculated. The results showed that the degree of renal tubular injury in the IRI group was significantly higher than that in the Sham group. The renal tubular injury score in the IRI+IIA-12L group was lower than that in the IRI group, and the renal tubular injury score in the IRI+IIA-12H group was significantly lower than that in the IRI group. The effect of IIA-12 was dose-dependent. Figure 1 (DE). Histological staining results were consistent with the results of renal function index testing.

[0160] Example 2

[0161] Compound IIA-12 can improve renal tubular damage caused by IRI.

[0162] To further investigate the effect of IIA-12 on renal tubular injury caused by ischemia-reperfusion injury, we first stained renal tissue sections from mice in each group with PAS. The results showed that the renal tubules in the Sham group exhibited normal histological morphology, while the renal tubules in the IRI group showed brush border loss of renal tubular epithelial cells, vacuolar degeneration, nuclear pyknosis, tubular swelling, and cast formation within the lumen. After adding IIA-12 treatment to the IRI model, the pathological changes in the renal kidneys induced by IRI were alleviated with increasing IIA-12 concentration, and this effect was dose-dependent on IIA-12. Figure 2(AB). Secondly, we performed immunohistochemical staining on KIM-1, which showed that KIM-1 protein expression was significantly higher in the IRI group than in the Sham group, and decreased in a dose-dependent manner in the IIA-12 treatment group (AB). Figure 2 The results of Western blot analysis of KIM-1 protein were consistent with those of immunohistochemical staining (C). Figure 2 (DE). Meanwhile, Western blot analysis of NGAL and AQP-2 showed that, compared to the Sham group, the IRI group exhibited significantly decreased AQP-2 expression and significantly increased NGAL expression. However, in the IIA-12 treatment group, the expression levels of both proteins showed a dose-dependent recovery with IIA-12. Figure 2 (FG). Consistent with the WB detection results of KIM-1 protein.

[0163] Example 3

[0164] Compound IIA-12 reduces renal tubular epithelial cell apoptosis induced by IRI.

[0165] We used TUNEL staining to evaluate renal tissue cell apoptosis in each group. Analysis of TUNEL-positive cell counts revealed that the level of renal tubular epithelial cell apoptosis was significantly higher in the IRI group than in the Sham group, while the number of apoptotic cells was significantly lower in the IRI+IIA-12 group. Figure 3 Western blot analysis (WB) revealed that BCL-2 protein expression in the IRI group was significantly lower than that in the Sham group, and BCL-2 protein expression in the IIA-12 treatment group increased in a dose-dependent manner. Compared with the Sham group, BAX protein expression in the IRI group was significantly increased, and BCL-2 protein expression in the IIA-12 treatment group decreased in a dose-dependent manner. Figure 3 (CD). Further, we used transmission electron microscopy to observe the ultrastructure of the renal cortex in each group. Compared with the Sham group, the IRI group showed more vacuolar degeneration in the renal tubular epithelial cells, and the nuclear chromatin was condensed into high-electron-density clumps, accumulating along the inner side of the nuclear membrane, which is an early marker of apoptosis. Meanwhile, compared with the IRI group, the IIA-12 treatment group showed a small number of cytoplasmic vacuoles, normal nuclei, and intact microvilli. Figure 3 (E).

[0166] Example 4

[0167] Compound IIA-12 improves IRI-induced inflammatory response in mouse kidney tissue.

[0168] To investigate the effect of IIA-12 on renal tubular inflammation induced by IRI, we used IHC and WB to detect the expression of inflammatory factors in renal tissue. IHC and semi-quantitative analysis showed that the expression levels of TNF-α and IL-6 proteins in IRI renal tissue were significantly increased compared with the Sham group, while the expression levels of TNF-α and IL-6 proteins in the IRI+IIA-12 group showed a dose-dependent decrease. Figure 4 Further Western blot analysis and semi-quantitative analysis of IL-1β, TNF-α, and IL-6 in the kidney tissue of each group showed that, compared with the Sham group, the expression levels of inflammatory factors in the kidney tissue of mice in the IRI group were significantly increased, while in the IRI+IIA-12 group, the expression of inflammatory factors showed a dose-dependent decrease. Figure 4 The results of DG staining (WB) and IHC staining were consistent.

[0169] Example 5

[0170] Furthermore, based on our in vivo mouse experiments, we used the human renal tubular epithelial cell line (HK-2) to evaluate the effect of IIA-12 on H / R-induced cell damage. Prior to this, we used the CCK-8 assay to verify the effect of IIA-12 on cell viability to explore the appropriate dosage of IIA-12 in cell experiments, ultimately using a dose of 5 μM for subsequent cell experiments. Figure 5 (A). First, we used Western blotting to determine the effects of IIA-12 on the expression of renal tubular injury markers and apoptosis-related proteins. Semi-quantitative analysis of the results showed that the expression levels of KIM-1 and BAX in the H / R group were significantly higher than those in the Control group, while the expression level of BCL-2 was significantly lower than that in the Control group. After IIA-12 treatment, compared with the H / R group, the expression levels of KIM-1 and BAX proteins were significantly decreased, while the expression level of BCL-2 was significantly increased. There was no significant difference in the expression levels of KIM-1, BCL-2, and BAX proteins between the IIA-12-treated group and the Control group. Figure 5 (WB). Secondly, we used flow cytometry to detect the apoptosis levels induced by H / R before and after IIA-12 treatment. Annexin V+7AAD- represented the early apoptotic cell population, and Annexin V+7AAD+ represented the late apoptotic cell population. The results showed that after H / R modeling, the number of both early and late apoptotic cells increased. Compared with the H / R group, the proportion of apoptotic cells in the H / R+IIA-12 group was significantly decreased, consistent with the changes in apoptotic protein levels detected by Western blotting. There was no significant difference in apoptosis levels between the IIA-12 treatment group and the Control group. Figure 5 Medium EF)

[0171] Example 6

[0172] Compound IIA-12 reduces H / R-induced inflammatory responses in HK-2 cells.

[0173] In in vitro experiments, we further used Western blotting and RT-qPCR to detect the expression levels of AHR, FOXO3, and inflammatory cytokine proteins before and after IIA-12 treatment. First, in the H / R group, the expression level of AHR was significantly higher than that in the Control group, while the expression level of FOXO3 was significantly lower than that in the Control group. Compared with the H / R group, AHR expression in HK-2 cells was significantly decreased and FOXO3 level was significantly increased after IIA-12 treatment. Figure 6 (AC). Secondly, Western blot analysis and semi-quantitative analysis showed that the expression levels of IL-1β and TNF-α proteins in the H / R group were significantly higher than those in the Control group, while the expression levels of IL-1β and TNF-α proteins were significantly decreased in the H / R+IIA-12 group. Figure 6 Finally, RT-qPCR was used to analyze the transcriptional expression of IL-6 gene in each group of cells. The results showed that compared with the Control group, the expression of IL-6 gene in the H / R group was significantly increased, while the expression of IL-6 gene was significantly decreased after IIA-12 treatment. Figure 6 (F). The results of the above cell experiments are consistent with the results in mice.

[0174] In summary, the results show that in a mouse model of ischemia-reperfusion kidney injury, IIA-12 firstly significantly reduced creatinine and blood urea nitrogen levels and improved renal function; secondly, it significantly reduced renal tubular apoptosis and renal tissue inflammation, and promoted tubular repair after injury; finally, the effect of IIA-12 was dose-dependent. In an in vitro H / R-induced HK-2 cell injury model, IIA-12 significantly reduced cell apoptosis, decreased the release of inflammatory factors from HK-2 cells, and improved cell damage, consistent with the results of the in vivo model.

Claims

1. The application of IIA compounds in the preparation of drugs for treating kidney injury-related diseases, wherein the structural formula of the IIA compound is shown in Formula 1: ， Formula 1; in, X is an amino group or a C1-C6 group, a straight-chain or branched alkyl group, an alkoxy group, a hydroxy group, an amino group, an amide group, an ester group, a hydroxyalkyl group, or an alkoxyalkyl group; R is a substituted or unsubstituted five- or six-membered aromatic heterocycle, including: phenyl, tolyl, naphthyl, quinolinyl, indolyl, furanyl, thiophene, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrroleyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl. The kidney injury-related diseases include acute kidney injury and inflammatory kidney injury. The acute kidney injury includes sepsis-induced acute kidney injury, ischemia-reperfusion kidney injury, drug-induced kidney injury, contrast agent-induced nephropathy, and folic acid-induced acute kidney injury.

2. The use of the IIA compound as described in claim 1 in the preparation of a therapeutic drug for kidney injury-related diseases, characterized in that, The IIA is IIA-12, and the structural formula of IIA-12 is shown in Formula 2: ， Equation 2.

3. A pharmaceutical preparation for treating kidney injury, characterized in that, It comprises IIA-12 as described in claim 2, with pharmaceutically acceptable excipients.

4. The pharmaceutical preparation for treating kidney injury as described in claim 3, characterized in that, The drug formulation can be administered orally or by injection.

5. The pharmaceutical preparation for treating kidney injury as described in claim 3, characterized in that, The dosage form of the pharmaceutical preparation is any one of tablets, capsules, granules, injections, or lyophilized powder for injection.

6. The use of the IIA compound as described in claim 1 or 2 in the preparation of a medicament for treating kidney injury-related diseases, characterized in that, The kidney injury-related disease is ischemia-reperfusion kidney injury, and the IIA compound is IIA-12.

7. The application as described in claim 6, characterized in that, The IIA-12 is the active pharmaceutical ingredient, and its effective mass percentage in the preparation of the drug is 0.05% to 95%.

8. The application as described in claim 6, characterized in that, Depending on the mass of the organism, the concentration of IIA-12 used is 5 to 100 mg / kg.

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