Use of sodium phosphate transporter 2a targeting intervention agent in preparation of medicament for preventing and / or treating acute kidney injury

Abstract

The application relates to the field of medical biotechnology, and discloses an application and use of a sodium phosphate transporter 2a targeted intervention agent in preparation of a medicine for preventing and / or treating acute kidney injury. The application relates to the application of the sodium phosphate transporter 2a targeted intervention agent in preparation of the medicine for preventing and / or treating the acute kidney injury. The application discloses a new use of the sodium phosphate transporter 2a targeted intervention agent, and the sodium phosphate transporter 2a targeted intervention agent can be used for preventing and / or treating the acute kidney injury.

Description

Technical Field

[0001] This invention relates to the field of medical biotechnology, specifically to the application and use of a sodium-phosphorus transporter 2a targeting intervention agent in the preparation of a medicament for the prevention and / or treatment of acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a common acute and critical illness among hospitalized patients. The incidence of AKI in Chinese hospitalized patients reaches 18%, and is as high as 30%-50% in critically ill patients. AKI increases the risk of developing or progressing to chronic kidney disease (CKD) by 2.67 times; increases the risk of cardiovascular events by 38%; and increases the risk of all-cause mortality by 1.80 times. The etiology of AKI is diverse, including prerenal, postrenal, and renal causes. Further subdivisions include nephrotoxic drugs, ischemia-reperfusion injury, sepsis, and other causes. Its pathophysiological mechanisms are complex, and there are currently no effective treatments. Therefore, the development of drugs to prevent acute kidney injury is of great significance.

[0003] Sodium phosphate co-transporter 2a (Npt2a) is a phosphate transporter protein expressed at the brush border of the proximal renal tubules. It is responsible for reabsorbing phosphate filtered by the glomeruli back into the bloodstream, and is crucial for maintaining phosphate homeostasis. Npt2a inhibitors lower serum phosphorus levels by inhibiting phosphate reabsorption by proximal renal tubular epithelial cells and promoting urinary phosphate excretion. The Npt2a gene is encoded by SLC34A1, belonging to Solute Carrier Family 34 (SLC34), located on human chromosome 5q35.3. Diseases associated with SLC34A1 gene mutations include renal Fanconi syndrome, hereditary hypophosphatemic rickets with hypercalciuria, and kidney stones. In a rat model of hyperphosphatemia in chronic kidney disease, literature reports that Npt2a inhibitors can promote urinary phosphate excretion and lower serum phosphate levels (Clerin V, Saito H, Filipski KJ, et al. Selective pharmacological inhibition of the sodium-dependent phosphate cotransporter NPT2a promotes phosphate excretion). J Clin Invest(2020;130(12):6510-6522.), suggesting it may become a new drug for treating hyperphosphatemia, a complication of chronic kidney disease. In a rat disease model, long-term administration (once daily) for 8 weeks demonstrated that it promoted urinary phosphorus excretion and reduced serum phosphorus levels in rats. Existing Npt2a inhibitors pf-06869206 and BAY-767 have not yet entered clinical trials, for reasons unknown. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies in the lack of effective drug targets for acute kidney injury.

[0005] To achieve the above objectives, a first aspect of the present invention provides the use of a sodium-phosphorus transporter 2a-targeting intervention agent in the preparation of a medicament for the prevention and / or treatment of acute kidney injury.

[0006] A second aspect of the invention provides the use of sodium-phosphorus transporter 2a as a drug target in the in vitro screening of drugs for the prevention and / or treatment of acute kidney injury.

[0007] Through the above technical solution, the present invention has at least the following advantages compared with the prior art: (1) This invention improves acute kidney injury in mouse models of AKI (cisplatin- and folic acid-induced AKI) of different etiologies by targeting Npt2a and using a sodium-phosphorus transporter 2a-targeting intervention agent for prophylaxis. Specifically, it improves renal function, reduces renal pathological damage, and improves mitochondrial structure and function. Furthermore, the sodium-phosphorus transporter 2a-targeting intervention agent alone has no significant nephrotoxicity. Pretreatment of a cisplatin-stimulated human proximal tubular cell injury model with the sodium-phosphorus transporter 2a-targeting intervention agent can inhibit proximal tubular epithelial cell apoptosis and improve mitochondrial function. Therefore, this invention expands the indications for sodium-phosphorus transporter 2a-targeting intervention agents, not only by lowering blood phosphorus levels but also by enabling the prevention of AKI.

[0008] (2) The sodium-phosphorus transporter 2a targeted intervention agent provided by the present invention has the advantages of simple administration, high oral bioavailability, good water solubility, and no liver and kidney toxicity. At the same time, prophylactic administration can avoid cisplatin-induced AKI. In the future, for cancer patients, it can reduce cisplatin-related nephrotoxicity, ensure the dosage and efficacy of cisplatin treatment, and the sodium-phosphorus transporter 2a targeted intervention agent has good drug accessibility and moderate price, which is conducive to carrying out subsequent clinical trials. Attached Figure Description

[0009] Figure 1 This is a flowchart of the cisplatin mouse experiment; Figure 2 This is a graph showing the results of cisplatin-treated mouse blood biochemistry tests. Figure 3This is a diagram showing the HE and TUNEL staining results of cisplatin-treated mouse kidney pathology. Figure 4 This is an immunoblot image showing the results of kidney injury and apoptosis proteins in cisplatin-treated mice; Figure 5 This is an immunoblot image of mitochondrial fusion-related proteins in cisplatin-treated mice; Figure 6 This is a flowchart of the folic acid mouse experiment; Figure 7 These are images of the blood biochemistry and pathology results of folic acid-fed mice. Figure 8 This is an immunoblot image showing the results of cisplatin-stimulated HK2 cell damage and apoptosis proteins. Figure 9 This is a graph showing the results of cisplatin stimulation of mitochondrial membrane potential in HK2 cells; Figure 10 This is a diagram showing the results of cisplatin-stimulated mitochondrial oxidative stress in HK2 cells; Figure 11 This is a diagram showing the results of cisplatin-stimulated mitochondrial oxidative respiration in HK2 cells; Figure 12 This is an immunoblot image of the mitochondrial fusion protein in HK2 cells stimulated by cisplatin. Detailed Implementation

[0010] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0011] As previously stated, a first aspect of the present invention provides the use of a sodium-phosphorus transporter 2a-targeting intervention agent in the preparation of a medicament for the prevention and / or treatment of acute kidney injury.

[0012] Preferably, the acute kidney injury includes a clinical syndrome resulting from a rapid decline in kidney function caused by any of the following etiologies: prerenal etiology, postrenal etiology, and renal etiology.

[0013] Preferably, prerenal causes include decreased blood volume, decreased effective arterial blood volume, and changes in intrarenal hemodynamics.

[0014] In this invention, the reduction in blood volume includes fluid loss and bleeding caused by various reasons.

[0015] Preferably, the postrenal etiology includes acute urinary tract obstruction; said acute urinary tract obstruction is an obstruction occurring at any level of the urinary tract from the renal pelvis to the urethra.

[0016] Preferably, the renal etiology includes renal parenchymal injury; said renal parenchymal injury includes damage caused by any one of the following diseases: renal tubular disease, renal interstitial disease, renal vascular disease, and glomerular disease.

[0017] In this invention, the nephrotoxic substances include exogenous toxins and / or endogenous toxins; the exogenous toxins include biological toxins, chemical toxins, antibiotics, and contrast agents; the endogenous toxins include hemoglobin and / or myoglobin.

[0018] Preferably, the clinical syndrome resulting from the rapid decline in renal function meets at least one of the following diagnostic criteria: a. Serum creatinine increases by ≥0.3 mg / dL within 48 hours; b. Serum creatinine increased by ≥1.5 times from baseline within 7 days; c. Serum cystatin C increased ≥1.5 times from baseline within 7 days; d. Average urine output < 0.5 mL / kg / h for 6 hours; e. Elevated clinically validated renal injury biomarkers, including urinary tissue inhibitor of metalloproteinases 2, insulin-like growth factor binding protein 7, and neutrophil gelatinase-associated lipotransferase.

[0019] Preferably, the sodium-phosphorus transporter 2a targeting intervention agent is used in the preparation of drugs for improving renal function, reducing renal pathological damage, inhibiting proximal tubular epithelial cell apoptosis, and improving proximal tubular mitochondrial function.

[0020] Preferably, the sodium-phosphorus transporter 2a targeting intervention includes a reagent that inhibits the transport function of the sodium-phosphorus transporter 2a. Preferably, the sodium-phosphorus transporter 2a targeting intervention is an Npt2a inhibitor or a pharmaceutically acceptable salt thereof; more preferably, the sodium-phosphorus transporter 2a targeting intervention is pf-06869206 or a pharmaceutically acceptable salt thereof.

[0021] Preferably, the effective concentration of the sodium-phosphorus transporter 2a targeting intervention agent is 1-500 mg / kg.

[0022] Preferably, the effective concentration of the sodium-phosphorus transporter 2a targeting intervention agent is 40-500 mg / kg; more preferably, the effective concentration of the sodium-phosphorus transporter 2a targeting intervention agent is 50-200 mg / kg; more preferably, the effective concentration of the sodium-phosphorus transporter 2a targeting intervention agent is 50-100 mg / kg.

[0023] Preferably, the dosage form of the drug is selected from at least one of injections, tablets, capsules, powders, granules, microspheres, and aerosols.

[0024] Preferably, the drug further contains a pharmaceutically acceptable carrier.

[0025] Preferably, the carrier includes a diluent, a disintegrant, an adhesive, a preservative, an isotonic agent, a lubricant, a stabilizer, and a surfactant.

[0026] In one embodiment of the invention, a pharmaceutically acceptable carrier includes a diluent. Non-limiting examples of diluents include sodium hydroxide, water-soluble oils, propylene glycol, glycerin, hydroxyethyl cellulose, and water, as well as mixtures thereof.

[0027] In one embodiment of the invention, the pharmaceutically acceptable carrier comprises a disintegrant. Non-limiting examples of disintegrants include starch, sodium carboxymethyl starch, hydroxypropyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, alginate, and mixtures thereof.

[0028] In one embodiment of the invention, the pharmaceutically acceptable carrier comprises the adhesive used. Non-limiting examples of the adhesive used include polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, starch paste, gelatin, sodium alginate, and mixtures thereof.

[0029] In one embodiment of the invention, a pharmaceutically acceptable carrier includes a preservative. Non-limiting examples of preservatives include benzyl chloride, benzoic acid, benzyl alcohol, bromonitropropylene glycol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorophenylglycerol ether, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidureus, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thimerosal, and mixtures thereof.

[0030] In one embodiment of the invention, a pharmaceutically acceptable carrier comprises an isotonic agent. Non-limiting examples of isotonic agents include amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), sugar alcohols (such as glycerol, 1,2-propanediol, propylene glycol), 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent includes sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, or water-soluble dextran, including, for example, fructose, glucose, mannose, sorbitol, xylose, maltose, lactose, sucrose, trehalose, dextran, amylopectin, dextrin, cyclodextrin, α- and β-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. Another example of an isotonic agent is a sugar alcohol, wherein the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, hexahexol, xylitol, and arabinitol. Pharmaceuticals comprising each of the isotonic agents listed in this paragraph constitute alternative embodiments of the invention.

[0031] In one embodiment of the invention, a pharmaceutically acceptable carrier comprises a lubricant. Examples of lubricants include magnesium stearate, glyceryl docosanoate, calcium stearate, zinc stearate, stearic acid, silica, talc, polyethylene glycol, mineral oil, carnauba wax, palmitic acid, sodium stearoyl fumarate, sodium lauryl sulfate, glyceryl palmitate, myristic acid, and hydrogenated vegetable oils and fats, as well as other known lubricants, and / or mixtures of two or more thereof. In one embodiment, if present, the lubricant for granulation of the feedstock is magnesium stearate.

[0032] In one embodiment of the invention, a pharmaceutically acceptable carrier includes a stabilizer. Non-limiting examples of stabilizers include hydrophilic polymers, liposomes with hydroxyl or carboxyl groups on their surface, bovine serum albumin nanospheres, human serum albumin nanospheres, and mixtures thereof.

[0033] In one embodiment of the invention, the pharmaceutically acceptable carrier comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term surfactant refers to any molecule or ion consisting of a water-soluble portion (hydrophilic) and a lipophilic portion. For example, surfactants are selected from anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants.

[0034] Preferably, the route of administration of the drug is selected from at least one of oral administration, subcutaneous injection, and intravenous injection.

[0035] Before constructing a mouse AKI model, the inventors of this invention used the aforementioned Npt2a inhibitor to observe the effects of the Npt2a inhibitor on kidney injury by detecting serum creatinine and blood urea nitrogen levels in mice, and by employing techniques such as hematologic staining (HE), Western blotting, and transmission electron microscopy. Simultaneously, the inventors used cell experiments to evaluate the effects of the Npt2a inhibitor on apoptosis, mitochondrial structure, and function of proximal tubular epithelial cells (i.e., proximal renal tubular epithelial cells) by detecting mitochondrial oxidative stress and mitochondrial membrane potential. Therefore, the inventors of this invention discovered that, in addition to its function of lowering serum phosphorus, the Npt2a inhibitor can also improve acute kidney injury, protect mitochondria, and inhibit apoptosis.

[0036] As previously stated, a second aspect of the present invention provides the use of sodium-phosphorus transporter 2a as a drug target in the in vitro screening of drugs for the prevention and / or treatment of acute kidney injury.

[0037] Preferably, the method for screening drugs for the prevention and / or treatment of acute kidney injury includes: screening for candidate drugs that can inhibit the transport function of sodium-phosphorus transporter 2a or downregulate the expression of sodium-phosphorus transporter 2a, thereby screening for drugs that can prevent and / or treat acute kidney injury.

[0038] The present invention will be described in detail below through embodiments.

[0039] Unless otherwise specified, the instruments, reagents, and materials involved in the following examples are all conventional instruments, reagents, and materials that are already available in the prior art and can be obtained through legitimate commercial channels.

[0040] Unless otherwise specified, the experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods already existing in the prior art. The room temperature mentioned in the following examples refers to 25±2℃.

[0041] Npt2a inhibitor: CAS number 2227425-05-8, drug name pf-06869206, molecular formula C15H14ClF3N4O, molecular weight 374.75, white powder, inhibitory efficacy against Npt2a is 380 nM (IC50 is 380 nM).

[0042] Human proximal tubule cells (HK2 cells): purchased from ATCC, catalog number CRL-2190.

[0043] Folic acid powder: purchased from MCE, product number HY-16637.

[0044] Cisplatin: Purchased from Selleckchem, product number S1166.

[0045] Corn oil: purchased from MCE Company, product number HY-Y1888.

[0046] DMSO: Purchased from MCE, part number HY-Y0329C.

[0047] BCA reagent kit: purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number PC0020.

[0048] ECL colorimetric reagent kit: purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number P0018S.

[0049] Mitosox Red Detection Kit: Purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0061S.

[0050] Physiological saline: purchased from Wuhan Pronosai Life Technology Co., Ltd., product number PB180353.

[0051] Eight-week-old male wild-type mice were purchased from Spefair Company, with mouse number C57BL / 6J.

[0052] RIPA lysis buffer: purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number P00013-B.

[0053] instrument: Transmission electron microscope: purchased from JEOL, model JEM-1400plus2400100.

[0054] Cellular energy metabolism analyzer: purchased from Agilent Technologies, model Seahorse XFe96.

[0055] The following are some examples of experiments: Pretreatment with Npt2a inhibitors when administered to animals: The Npt2a inhibitor was dissolved in DMSO to prepare a stock solution with a concentration of 80 mg / mL, which could be stored long-term at -80°C. Before use, it was diluted with corn oil to a concentration of 8 mg / mL. Mice were administered the Npt2a inhibitor by gavage at a dose of 80 mg / kg. Acute kidney injury was modeled after prophylactic administration.

[0056] Serum creatinine and blood urea nitrogen levels in mice were detected using liquid chromatography.

[0057] Blood calcium and phosphorus levels in mice were detected using ion mass spectrometry.

[0058] HE staining: 1) Dewaxing and hydration: Immerse the sections sequentially in dewaxing solution, anhydrous ethanol, 95% ethanol, 75% ethanol, and 50% ethanol for 3 minutes per bath; 2) Stain with hematoxylin for 5 minutes, rinse with running water for about 10 minutes. Differentiate with 1% hydrochloric acid alcohol for 2 seconds, and ammonia water for 4 seconds. Stain with eosin for 1 minute, and rinse with running water for 5 minutes; 3) Gradient alcohol dehydration: Immerse the sections sequentially in 50% ethanol, 75% ethanol, 95% ethanol, anhydrous ethanol, and dewaxing solution for 3 minutes per bath; 4) Mounting: Remove the sections from the dewaxing solution, air dry in a fume hood, apply a small amount of mounting medium to the sections using a dropper, and mount with a coverslip.

[0059] Western blot method for protein extraction: 1) Protein extraction: Take approximately 20 mg of tissue, add 300 μL of RIPA lysis buffer and 5 grinding beads to each tube, and homogenize. Transfer the homogenate to a clean EP tube and centrifuge at 12,000 rpm for 15 min at 4°C. Transfer the supernatant to a clean EP tube, take a small amount of the original protein solution, dilute it 20 times, and use it for BCA method to determine the protein concentration (the protein concentration determination method should be performed according to the BCA kit instructions). Adjust the protein concentration with RIPA lysis buffer according to the measured protein concentration until the protein concentration in each tube is the same. 1) Add 5X loading buffer, boil in a 95℃ metal bath for 10 min, and store in a -20℃ refrigerator; 2) Electrophoresis: Use 10μl extended pipette tips to sample, with 8μl sample per well (ensuring a total protein content of 30μg); first run the separating gel at a constant voltage of 80V for about 20 min, then adjust the voltage to 120V and run at a constant voltage until the bromophenol blue reaches the bottom of the glass plate (about 50 min), then stop electrophoresis; 3) Transfer: Take a PVDF membrane and immerse it in methanol to activate it for about 1 min. Prepare a transfer sandwich, place the gel on the black side of the filter paper, cover it with the activated PVDF membrane and remove any air bubbles, then cover it with the white side of the filter paper and clamp the sandwich together. According to the positive and negative electrodes, place the transfer clamp into the transfer tank and pour in sufficient transfer buffer. Transfer at a constant current of 400mA for 35 min; 4) Blocking: Open the transfer clamp, determine the front and back sides of the membrane, and cut a small slit in the upper left corner for marking. 5) Primary antibody incubation: Prepare the primary antibody according to the antibody instructions. Discard the blocking solution, wash with TBST for 2 min, add 5 ml of the prepared primary antibody, and incubate overnight at 4°C on a shaker; 6) Secondary antibody incubation: Recover the primary antibody, wash with TBST for 10 min, a total of 3 times, and prepare HRP-labeled secondary antibody of the same species as the primary antibody at a volume ratio of 1:5000 during this period. After washing the membrane, add 5 ml of secondary antibody and incubate at room temperature for 1 h; wash with TBST for 10 min, a total of 3 times; 7) ECL color development: Prepare the required amount of reagents A and B from the ECL color development kit at a volume ratio of 1:1 in the dark. Place the PVDF membrane on the developing instrument tray, evenly drop the developing solution onto the entire PVDF membrane, incubate in the dark for about 10 s, develop and photograph using a Tanon developing instrument, and save the image. Semi-quantitative analysis was performed using ImageJ software based on the grayscale values ​​of the bands.

[0060] Detection of mitochondrial oxidative stress staining: 1) Preparation of MitoSOX Red stock solution: MitoSOX Red was dissolved in DMSO at a concentration of 5 mM; 2) Preparation of working solution: The MitoSOX Red stock solution was diluted to 5 µM; 3) MitoSOX Red staining: The working concentration of MitoSOX Red was added to the culture medium and incubated at 37°C for 20 minutes; 4) Washing: After incubation, the cells were washed with PBS to remove excess MitoSOX Red; 5) Fluorescence detection: The excitation wavelength of MitoSOX Red was 510 nM and the emission wavelength was 580 nM. The red fluorescence was detected using a fluorescence microscope or flow cytometer equipped with appropriate filters.

[0061] Mitochondrial membrane potential detection by staining: Aspirate the cell culture medium from the wells of a six-well plate, wash the cells once with PBS, and add 1 ml of the above cell culture medium. 1) Add 1 ml of JC-1 staining working solution, mix thoroughly, and incubate at 37°C for 20 minutes; 2) During incubation, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5X), and incubate on ice; 3) After incubation at 37°C, aspirate the supernatant and wash the cells twice with JC-1 staining buffer (1X); 4) Add 2 ml of cell culture medium (purchased from Wuhan Pronosei Life Sciences Co., Ltd., catalog number CM-0109); 5) Observe under a laser confocal microscope. The staining kit used was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C2003S.

[0062] Example 1 (1) Preparation of reagents: Cisplatin is dissolved in physiological saline to prepare a cisplatin solution with a concentration of 2 mg / mL.

[0063] (2) Experimental grouping: Male wild-type C57BL / 6J mice at 8 weeks of age were randomly divided into four groups: CON group: 10 μL / g of the control solvent was administered by gavage daily for 6 consecutive days. The control solvent was DMSO and corn oil in a volume ratio of 1:9. Cis group: cisplatin solution was injected intraperitoneally to achieve a cisplatin dosage of 20 mg / kg; mice were sacrificed 72 hours later, tissue samples were collected, and phenotypes were assessed; the modeling method was referenced from Fu Y, Xiang Y, Wei Q, Ilatovskaya D, Dong Z. Rodentmodels of AKI and AKI-CKD transition: an update in 2024. Am J Physiol Renal Physiol. 2024;326(4): F563-F583. Proceed.

[0064] Cis+N group: Npt2a inhibitor 80 mg / kg was administered prophylactically by gavage for 6 consecutive days. On the 4th day of gavage, cisplatin solution was injected intraperitoneally (so that the dose of cisplatin was 20 mg / kg) to establish a cisplatin-induced acute kidney injury model. Group N: Administered 80 mg / kg of Npt2a inhibitor by gavage daily for 6 consecutive days; Blood, urine, and kidney tissue were collected from mice in the CON group, Cis+N group, and N group on day 7 after gavage.

[0065] (3) The acute kidney injury phenotype in mice was evaluated by detecting serum creatinine and serum urea nitrogen levels, and by using techniques such as hematologic staining, transmission electron microscopy, and Western blotting of kidney tissue. (4) Experimental results: Figure 1 The experimental flowchart of cisplatin mice (Cis+N group mice) is shown, which includes daily prophylactic gavage of 80 mg / kg of Npt2a inhibitor for 6 consecutive days, intraperitoneal injection of cisplatin solution on day 4 (so that the dosage of cisplatin is 20 mg / kg) to establish a cisplatin-induced acute kidney injury model, and collection of blood, urine and kidney tissue from mice on day 7.

[0066] Figure 2 Figure A shows the results of serum creatinine detection in cisplatin-treated mice; from Figure 2 As shown in A, compared with the control mice (CON group mice), the serum creatinine of the cisplatin group mice (Cis group mice) was significantly increased, while the use of Npt2a inhibitors could significantly improve the increase in serum creatinine.

[0067] Figure 2 Figure B shows the results of serum urea nitrogen level detection in cisplatin-treated mice; from Figure 2 As shown in B, compared with the control mice (CON group), the cisplatin group mice (Cis group) had significantly higher blood urea nitrogen levels, while the Npt2a inhibitor group could significantly improve the elevated blood urea nitrogen levels.

[0068] Figure 2 C in the figure shows the results of blood calcium level detection in cisplatin-treated mice; from Figure 2 As shown in C, compared with the control group, the cisplatin group of mice had significantly higher blood calcium levels, while the Npt2a inhibitor group showed a significant improvement in elevated blood calcium levels.

[0069] Figure 2 The figure in D shows the results of cisplatin-treated mouse blood phosphorus level detection; from Figure 2 As shown in D, compared with the control group and the cisplatin group, the use of Npt2a inhibitors can significantly reduce blood phosphorus.

[0070] Figure 3 Image showing HE staining and TUNEL staining results of kidney pathology in cisplatin-treated mice; Figure 3 Figure A shows the HE staining results of cisplatin-treated mouse kidney pathology. Figure 3 As shown in A, compared with the control group, the cisplatin group showed tubular pathological damage, including tubular vacuolation, tubular swelling and rupture. Npt2a inhibitors can improve tubular pathological damage. Figure 3 Figure B shows the TUNEL staining results of cisplatin-treated mouse kidney pathology. Figure 3 As shown in B, compared with the control group, the cisplatin group showed green positive apoptotic cells, mainly distributed in renal tubular cells, and Npt2a inhibitors can significantly improve renal tubular cell apoptosis.

[0071] Figure 4 The image in the middle shows the immunoblotting results of kidney injury and apoptosis proteins in cisplatin-treated mice: Figure 4 Figure A shows the immunoblotting results of the kidney injury protein KIM1 and the apoptosis protein Bcl2 in cisplatin-treated mice. Figure 4 B in the figure shows the quantification of KIM1, a biomarker of kidney injury; from Figure 4 As shown in B, compared with the control group, the cisplatin group mice had increased KIM1 levels, and the Npt2a inhibitor could significantly improve the increase in KIM1. Figure 4 C in the figure shows the quantification of the anti-apoptotic protein Bcl2; from Figure 4 As shown in C, compared with the control group, Bcl2 was downregulated in cisplatin mice, and Npt2a inhibitors could significantly improve the downregulation of Bcl2. Figure 4 The figure shown in D is an immunoblot result of NGAL, a marker protein for acute kidney injury. Figure 4 E in the figure shows the quantification of the renal tubular injury protein NGAL; from Figure 4 As can be seen from E, compared with the control group, the cisplatin group mice had elevated NGAL, and the Npt2a inhibitor could significantly improve the elevation of NGAL.

[0072] Figure 5 Figure A shows the immunoblotting results of mitochondrial fusion-related proteins in cisplatin-treated mice; Figure 5 B in the figure shows the quantification of the mitochondrial fusion protein OPA1; Figure 5 C in the figure shows the quantification of the mitochondrial fusion protein MFN2; from Figure 5 A to Figure 5 As shown in C, the mitochondrial fusion proteins OPA1 and MFN2 in mouse kidneys decreased in the cisplatin group, and the use of Npt2a inhibitors could significantly improve the downregulation of OPA1 and MFN2.

[0073] Example 2 (1) Solution preparation: Folic acid was dissolved in 150mM sodium bicarbonate solution to prepare a folic acid solution with a concentration of 20mg / mL.

[0074] (1) Experimental grouping: Male wild-type C57BL / 6J mice at 8 weeks of age were randomly divided into four groups: CON group: 10 μL / g of control solvent (the control solvent was DMSO and corn oil with a volume ratio of 1:9) was administered by gavage daily for 4 consecutive days. On the 5th day of gavage, blood, urine and kidney tissue were collected from the mice. FA group: Folic acid solution was injected intraperitoneally (to achieve a folic acid dosage of 200 mg / kg); blood, urine, and kidney tissue were collected from mice 24 hours later; the modeling method was referenced in Fu Y, Xiang Y, Wei Q, Ilatovskaya D, Dong Z. Rodent models of AKI and AKI-CKD transition: an update in 2024. Am J Physiol Renal Physiol . 2024;326(4):F 563-F583. Proceeding; FA+N group: The mice were given 80 mg / kg of Npt2a inhibitor by gavage daily for 4 consecutive days. On the 4th day of gavage, folic acid solution was injected intraperitoneally (so that the folic acid dosage was 200 mg / kg) to establish a folic acid-induced acute kidney injury model. On the 5th day of gavage, blood, urine and kidney tissue were collected from the mice. Group N: Mice were administered 80 mg / kg of Npt2a inhibitor by gavage daily for 4 consecutive days. On the 5th day of gavage, blood, urine, and kidney tissue were collected from the mice. (2) The phenotype of acute kidney injury in mice was evaluated by detecting serum creatinine and serum urea nitrogen levels, and by using techniques such as hematologic staining, transmission electron microscopy, and immunoblotting of kidney tissue. (3) Experimental results: Figure 6 This is a flowchart of the folic acid mouse (FA+N group mice) experiment. Figure 6 The study demonstrated the use of Npt2a inhibitors administered prophylactically by gavage at a dose of 80 mg / kg daily for 4 consecutive days. On the 4th day of gavage, folic acid solution was injected intraperitoneally (to bring the folic acid dose to 200 mg / kg) to establish a folic acid-induced acute kidney injury model. On the 5th day of gavage, blood, urine, and kidney tissue were collected from the mice.

[0075] Figure 7 The image shows the blood biochemistry and pathology results of folic acid-fed mice.

[0076] Figure 7 Figure A shows the results of serum creatinine detection in folic acid-fed mice; from Figure 7As shown in A, compared with the control mice (CON group), the serum creatinine level in the folic acid group (FA group) was significantly increased, while the Npt2a inhibitor group could significantly improve the increase in serum creatinine.

[0077] Figure 7 Figure B shows the results of mouse serum urea nitrogen level detection; from Figure 7 As shown in B, compared with the control mice, the folic acid group mice had significantly higher blood urea nitrogen levels, while the Npt2a inhibitor group showed a significant improvement in the elevated blood urea nitrogen levels.

[0078] Figure 7 C in the figure shows the results of the detection of blood calcium levels in mice; from Figure 7 As shown in C, compared with the control mice, the folic acid group mice had significantly higher blood calcium levels, while the Npt2a inhibitor group showed a significant improvement in elevated blood calcium levels.

[0079] Figure 7 The figure D shows the results of mouse blood phosphorus level detection; from Figure 7 As indicated by D, there was no significant difference in blood phosphorus levels among the groups.

[0080] Figure 7 E in the image shows the results of HE staining for kidney pathology; from Figure 7 As can be seen from E in the data, compared with the control group, the folic acid group showed tubular pathological damage, including tubular vacuolation, tubular swelling and rupture. Npt2a inhibitors can significantly improve tubular pathological damage.

[0081] Figure 7 F in the figure shows TUNEL apoptosis staining of mouse kidney sections; from Figure 7 As shown in F, compared with the control group, the folic acid group showed green positive apoptotic cells, mainly distributed in renal tubular cells. Npt2a inhibitors can significantly improve renal tubular cell apoptosis.

[0082] Example 3 Since Npt2a is mainly expressed in the proximal tubular epithelial cells of the kidney, and the proximal tubule is the main site of involvement in AKI, this embodiment examines the effect of Npt2a inhibitors on a cisplatin-induced human proximal tubular cell injury model.

[0083] (1) Prepare the solution: Preparation of cisplatin solution: Dissolve cisplatin in DMEM medium to prepare a 4mM cisplatin solution; Preparation of Npt2a inhibitor solution: Dissolve Npt2a inhibitor in DMSO to obtain a 200 mM Npt2a inhibitor solution.

[0084] (2) Experimental method: Human proximal tubule cells (HK2 cells) were resuscitated in DMEM medium and then stored at a density of 2 × 10⁻⁶ cells / cells. 5 Inoculate 10 cells / well into 16-well plates and incubate at 37°C in a 5% CO2 incubator. Divide the plates into the following 4 groups: Con (control group): No treatment was given; Cis (cisplatin-stimulated group): When the cell density reached 70%, cisplatin solution (final concentration of cisplatin was 10 μM) was added to induce renal tubular injury. Cis+N (cisplatin stimulation + Npt2a inhibitor group): When the cell density reached 70%, Npt2a inhibitor solution (final Npt2a inhibitor concentration of 20 μM) was added for pretreatment for 1 h, and then cisplatin solution (final cisplatin concentration of 10 μM) was added to induce renal tubular injury. N (Npt2a inhibitor alone group): When the cell density reached 70%, Npt2a inhibitor was added (final concentration of Npt2a inhibitor was 20 μM). Each group was incubated at 37 degrees Celsius in a 5% CO2 incubator for 24 hours. Cells were then collected and subjected to Western blotting, transmission electron microscopy, and techniques such as cell energy metabolism analyzer, detection of mitochondrial oxidative stress and mitochondrial membrane potential to assess apoptosis, mitochondrial structure and function.

[0085] Figure 8 Figure A shows the immunoblotting results of cisplatin-stimulated HK2 cell damage and apoptosis proteins; Figure 8 B in the figure shows the quantitative results of the cell damage protein KIM1; Figure 8 C in the figure shows the quantitative results of the pro-apoptotic protein BAX; Figure 8 D in the figure represents the quantitative results of the apoptosis protein Bcl2; Figure 8 E in the figure represents the quantitative results of the cell damage protein NGAL; from Figure 8 It was found that, compared with the control group, the cisplatin-stimulated group showed upregulation of the damaging proteins NGAL and KIM1, upregulation of the pro-apoptotic protein BAX, and downregulation of the anti-apoptotic protein Bcl2, which could be significantly improved by the use of Npt2a inhibitors.

[0086] Figure 9 The diagram shows the results of cisplatin-stimulated mitochondrial membrane potential in HK2 cells; from Figure 9 It can be seen that, compared with the control group, the cisplatin-stimulated group had mitochondrial membrane potential disruption (reduction of multimeric JC-1 and increase of monomeric JC-1), and the use of Npt2a inhibitors could significantly improve mitochondrial membrane potential disruption.

[0087] Figure 10 The diagram shows the results of cisplatin-stimulated mitochondrial oxidative stress in HK2 cells; from Figure 10It can be seen that, compared with the control group, the cisplatin-stimulated group had increased mitochondrial oxidative stress, and the use of Npt2a inhibitors could significantly improve the increase in mitochondrial oxidative stress.

[0088] Figure 11 Figure A shows the OCR results of cisplatin-stimulated mitochondrial oxidative respiration in HK2 cells; from Figure 11 As shown in A, compared with the control group, the mitochondrial oxidative respiration OCR intensity decreased in the cisplatin-stimulated group, and the use of Npt2a inhibitors could significantly improve it.

[0089] Figure 11 Figure B shows the results of cisplatin-stimulated mitochondrial basal respiration values ​​in HK2 cells; from Figure 11 As shown in B, compared with the control group, the mitochondrial basal respiratory value of the cisplatin-stimulated group decreased, and the use of Npt2a inhibitors could significantly improve it.

[0090] Figure 11 Figure C shows the results of cisplatin-stimulated maximum mitochondrial respiration in HK2 cells; from Figure 11 As shown in C, compared with the control group, the mitochondrial maximum respiratory value decreased in the cisplatin-stimulated group, and the use of Npt2a inhibitors could significantly improve the decrease in mitochondrial maximum respiratory value.

[0091] Figure 11 Figure D shows the results of cisplatin-stimulated ATP synthesis in HK2 cells; from Figure 11 As shown in D, compared with the control group, ATP synthesis decreased in the cisplatin-stimulated group, and the use of Npt2a inhibitors could significantly improve the decrease in ATP synthesis.

[0092] Figure 11 E in the figure shows the result of cisplatin stimulation of residual mitochondrial respiration values ​​in HK2 cells; from Figure 11 As can be seen from E, compared with the control group, the mitochondrial residual respiration value decreased in the cisplatin-stimulated group, and the use of Npt2a inhibitors can significantly improve the decrease in residual respiration value.

[0093] Figure 12 A in the figure shows the immunoblotting results of cisplatin-stimulated mitochondrial fusion protein in HK2 cells; Figure 12 B in the figure shows the quantitative results of the mitochondrial fusion protein OPA1; Figure 12 B in the figure shows the quantitative results of the mitochondrial fusion protein MFN2; from Figure 12 It was found that, compared with the control group, the mitochondrial fusion proteins OPA1 and MFN2 were downregulated in the cisplatin-stimulated group, and the use of Npt2a inhibitors could significantly improve the downregulation of OPA1 and MFN2.

[0094] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Application of sodium-phosphorus transporter 2a-targeting intervention in the preparation of drugs for the prevention and / or treatment of acute kidney injury.

2. The application according to claim 1, characterized in that, The acute kidney injury includes a clinical syndrome resulting from a rapid decline in renal function caused by any of the following etiologies: prerenal etiology, postrenal etiology, and renal etiology. The prerenal causes include decreased blood volume, decreased effective arterial blood volume, and changes in intrarenal hemodynamics; The postrenal etiology includes acute urinary tract obstruction; the acute urinary tract obstruction is an obstruction occurring at any level of the urinary tract from the renal pelvis to the urethra. The renal etiology includes renal parenchymal injury; the renal parenchymal injury includes damage caused by any one of the following diseases: renal tubular disease, renal interstitial disease, renal vascular disease, and glomerular disease; The clinical syndrome resulting from the rapid decline in renal function meets at least one of the following diagnostic criteria: a. Serum creatinine increases by ≥0.3 mg / dL within 48 hours; b. Serum creatinine increased by ≥1.5 times from baseline within 7 days; c. Serum cystatin C increased ≥1.5 times from baseline within 7 days; d. Average urine output < 0.5 mL / kg / h for 6 hours; e. Elevated clinically validated renal injury biomarkers, including urinary tissue inhibitor of metalloproteinases 2, insulin-like growth factor binding protein 7, and neutrophil gelatinase-associated lipotransferase.

3. The application according to claim 1, characterized in that, The sodium-phosphorus transporter 2a targeting intervention agent is used in the preparation of drugs for improving renal function, reducing renal pathological damage, inhibiting proximal tubular epithelial cell apoptosis, and improving proximal tubular mitochondrial function.

4. The application according to any one of claims 1-3, characterized in that, The sodium-phosphorus transporter 2a targeting intervention includes a reagent that inhibits the transport function of the sodium-phosphorus transporter 2a.

5. The application according to any one of claims 1-3, characterized in that, The effective concentration of the sodium-phosphorus transporter 2a targeting intervention agent is 1-500 mg / kg.

6. The application according to claim 5, characterized in that, The effective concentration of the sodium-phosphorus transporter 2a targeting intervention agent is 40-500 mg / kg.

7. The application according to any one of claims 1-3, characterized in that, The dosage form of the drug is selected from at least one of the following: injection, tablet, capsule, powder, granule, microsphere, and aerosol.

8. The application according to any one of claims 1-3, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

9. The application according to claim 8, characterized in that, The carrier includes diluents, disintegrants, binders, preservatives, isotonic agents, lubricants, stabilizers, and surfactants.

10. Use of sodium-phosphorus transporter 2a as a drug target in in vitro screening of drugs for the prevention and / or treatment of acute kidney injury.

Images