Construction method and application of diuretic-resistant cell model

Abstract

The invention is applicable to the technical field of biomedicine, relates to a construction method and application of a diuretic-resistant cell model, and establishes the diuretic-resistant cell model by using angiotensin II and aldosterone to jointly induce mouse renal manifold M-1 cells. The model is verified by detecting gene and protein expression levels of aquaporin 2, epithelial sodium channel alpha subunit and arginine vasopressin receptor 2. The method is simple to construct and good in repeatability, can truly reflect the pathological characteristics of diuretic resistance, can stably simulate the state of water-sodium retention caused by excessive activation of a renin-angiotensin-aldosterone system, can reproduce the core characteristic of insufficient curative effect of the diuretic, verifies the occurrence effect of the diuretic resistance, and has a good application prospect. And a stable and controllable in-vitro model is provided for researching a molecular mechanism of diuretic resistance and screening prevention and treatment drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and particularly relates to a method for constructing a diuretic resistance cell model and application thereof. BACKGROUND

[0002] Diuretic resistance (DR) is a clinical syndrome that occurs when patients still have insufficient water and sodium excretion and fluid retention after being given a sufficient amount of diuretics. The incidence of DR in patients with advanced heart failure is as high as 20% to 30%, and DR is an independent risk factor for increasing the rehospitalization rate and mortality of patients. One of the core pathological mechanisms of DR is that the use of diuretics will activate the renin-angiotensin-aldosterone system (RAAS) compensatorily, and the overactivation of the RAAS will enhance the reabsorption of water and sodium in the distal tubule and collecting duct of the kidney, thereby offsetting the pharmacological effect of diuretics and forming a vicious cycle.

[0003] At present, the research on diuretic resistance highly depends on clinical observation or in vivo animal experiments. Clinical research has limitations such as large individual differences, many interference factors, and difficulty in deepening the mechanism. Animal experiments have high cost, long cycle, low throughput, and complex ethical approval. At the cellular level, there is currently a lack of an in vitro model that can directly and stably simulate the water and sodium retention caused by the overactivation of the RAAS and reflect the core feature of "insufficient diuretic efficacy". This technical gap seriously hinders the in-depth analysis of the molecular mechanism of diuretic resistance and the efficient screening of targeted therapeutic drugs.

[0004] Therefore, how to provide a method for constructing a diuretic resistance cell model that is simple, has good repeatability, and can truly reflect the pathological characteristics of diuretic resistance is a problem that needs to be solved by those skilled in the art. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for constructing a diuretic resistance cell model to solve the problem that the prior art cannot truly reflect the pathological characteristics of diuretic resistance. In addition, the present application also provides an application of a diuretic resistance cell model in screening drugs for treating diuretic resistance and researching the molecular mechanism thereof.

[0006] To solve the above technical problems, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a method for constructing a diuretic resistance cell model, comprising the following steps:

[0008] Step 1, providing mouse renal collecting duct epithelial cells;

[0009] Step 2, the cells are treated with angiotensin II at a concentration of 0.1 μM and aldosterone at a concentration of 0.1 μM, so as to induce the formation of the model of diuretic-resistant cells.

[0010] Further, on the basis of the combined treatment, the cells are further treated with furosemide at a concentration of 100 μM, so as to verify the diuretic-resistant property of the model.

[0011] Further, the combined treatment time is 48 hours.

[0012] Further, the mouse kidney collecting duct epithelial cells are M-1 cell lines.

[0013] Further, the expression levels of the genes and / or proteins of aquaporin 2, epithelial sodium channel alpha subunit and arginine vasopressin receptor 2 in the model of diuretic-resistant cells are significantly up-regulated compared with untreated normal cells.

[0014] Further, the expression levels of the genes and / or proteins of aquaporin 2, epithelial sodium channel alpha subunit and arginine vasopressin receptor 2 are detected by qPCR and Western blot techniques.

[0015] Further, the model of diuretic-resistant cells is used to study the signal transduction mechanism of the renin-angiotensin-aldosterone system.

[0016] Further, the model of diuretic-resistant cells is cultured in DMEM medium containing 10% fetal bovine serum.

[0017] Further, the culture environment is a 5% CO2 cell incubator at 37°C.

[0018] In a second aspect, the present application further provides a use of the model of diuretic-resistant cells constructed by the construction method in screening drugs for preventing and / or treating diuretic resistance, characterized in that the use comprises the following steps:

[0019] The candidate drug is applied to the model of diuretic-resistant cells.

[0020] The expression levels or activities of at least one of aquaporin 2, epithelial sodium channel alpha subunit and arginine vasopressin receptor 2 in the model of diuretic-resistant cells are detected.

[0021] The detection results are compared with a model group without the candidate drug, and if the candidate drug can significantly reverse the up-regulation of the expression levels or activities, it is indicated that the candidate drug has the potential for preventing and / or treating diuretic resistance.

[0022] The construction method of the model of diuretic-resistant cells and the use thereof provided by the present application have at least the following beneficial effects compared with the prior art:

[0023] The prior art cannot truly reflect the diuretic resistance pathological characteristics. The present application has simple construction, good repeatability, can truly reflect the diuretic resistance pathological characteristics, can stably simulate the water and sodium retention state caused by excessive activation of the renin-angiotensin-aldosterone system, and can reproduce the core characteristics of the insufficient diuretic effect, verifies the occurrence effect of diuretic resistance, and provides a stable and controllable in vitro model for studying the molecular mechanism of diuretic resistance and screening prevention and treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the scheme of the present application, the drawings required in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0025] Figure 1 The flow chart of the construction method of a diuretic resistance cell model provided for the embodiments of the present application;

[0026] Figure 2 The graph of the influence of different concentrations of aldosterone (Aldo) on the viability of renal collecting duct cells (M-1) in different time in the construction method of a diuretic resistance cell model provided for the embodiments of the present application;

[0027] Figure 3 The graph of the influence of different concentrations of angiotensin II (Ang-II) on the viability of renal collecting duct cells (M-1) in different time in the construction method of a diuretic resistance cell model provided for the embodiments of the present application;

[0028] Figure 4 The gene expression graph of aquaporin 2 (AQP2) of each experimental group in the construction method of a diuretic resistance cell model provided for the embodiments of the present application;

[0029] Figure 5 The gene expression graph of epithelial sodium channel alpha subunit (SCNN1A) of each experimental group in the construction method of a diuretic resistance cell model provided for the embodiments of the present application;

[0030] Figure 6 The gene expression graph of arginine vasopressin receptor 2 (AVPR2) of each experimental group in the construction method of a diuretic resistance cell model provided for the embodiments of the present application;

[0031] Figure 7 The graph of the Western blot detection of the protein expression level of aquaporin 2 (AQP2) of each experimental group in the construction method of a diuretic resistance cell model provided for the embodiments of the present application;

[0032] Figure 8 Figure 2 is a Western blot detection of the protein expression level of aquaporin 2 (AQP2) in each experimental group in the method for constructing a diuretic-resistant cell model according to an embodiment of the present application;

[0033] Figure 9 Figure 3 is a Western blot detection of the protein expression level of epithelial sodium channel alpha subunit (SCNN1A) in each experimental group in the method for constructing a diuretic-resistant cell model according to an embodiment of the present application;

[0034] Figure 10 Figure 4 is a Western blot detection of the protein expression level of epithelial sodium channel alpha subunit (SCNN1A) in each experimental group in the method for constructing a diuretic-resistant cell model according to an embodiment of the present application;

[0035] Figure 11 Figure 5 is a Western blot detection of the protein expression level of arginine vasopressin receptor 2 (AVPR2) in each experimental group in the method for constructing a diuretic-resistant cell model according to an embodiment of the present application;

[0036] Figure 12 Figure 6 is a Western blot detection of the protein expression level of arginine vasopressin receptor 2 (AVPR2) in each experimental group in the method for constructing a diuretic-resistant cell model according to an embodiment of the present application. DETAILED DESCRIPTION

[0037] For the purpose of promoting a fuller understanding of the present application, reference is now made to the following descriptions made in conjunction with the accompanying drawings. In the drawings, the preferred embodiments of the present application are illustrated. However, the present application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. It will fully convey the scope of the application to those skilled in the art.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0039] Example 1

[0040] Culture medium and drug mother liquor preparation: 500 mL DMEM medium, 50 mL fetal bovine serum (FBS), 5 mL penicillin-streptomycin double-antibiotic solution were mixed to prepare the culture medium; 5 mg of Ang-II was dissolved in 4.8 mL of 1% PBS (PBS:ddH2O=1:999) to obtain a 1 mmol / L Ang-II mother liquor, which was filtered with a 0.22 μM filter to remove bacteria and stored in a -20°C refrigerator; 1 mg of Aldo was dissolved in 300 ul of 0.01% ethanol (absolute ethanol:ddH2O=1:9999) to obtain a 10 mmol / L Aldo mother liquor, which was filtered with a 0.22 μM filter to remove bacteria and stored in a 4°C refrigerator; 10 mg of furosemide was dissolved in 1 mL of 0.1M NaOH to obtain a 30 mmol / L furosemide, which was filtered with a 0.22 μM filter to remove bacteria and stored in a -20°C refrigerator. The working concentration of furosemide was 0.1 mmol / L.

[0041] Cell culture: After the medium of mouse renal collecting duct primary cells (M-1) was changed and subcultured, the cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double-antibiotic solution, and the culture environment was a 5% CO2 cell incubator at 37°C. Before the drug treatment, the M-1 cells were incubated in a serum-free medium for 24 h.

[0042] CCK8 detection of cell viability to screen suitable drug concentration: M-1 cells in the logarithmic growth phase were inoculated in a 96-well plate at a density of 5×10³ cells per well in a volume of 100 μL / well. After the cells were completely adhered, drug treatment was performed. The experiment was divided into a blank group (containing only the culture medium and the CCK-8 reagent without cells), a control group and a drug administration group. In the drug administration group, the cells were incubated with fresh complete medium containing different concentrations of aldosterone (Aldo, 10, 100, 1000 nM) or angiotensin II (Ang-II, 10, 100, 1000 nM); in the control group, the same volume of complete medium without drugs was added. Each concentration had 6 replicate wells, which were incubated in a 37°C incubator for 24 h and 48 h (the same grouping conditions were repeated for the 48 h group). At the predetermined time point, 10 μL of CCK-8 reagent was directly added to each well, which was further incubated at 37°C in the dark for 2 h. Then, the absorbance value of the cells at a wavelength of 450 nm was detected using an enzyme label instrument, the cell viability was calculated, and the proliferation curve was drawn. The OD value of the blank well was used for calibration. The cell viability=OD (experiment)-OD (blank) / OD (control)- OD (blank) x 100%.

[0043] In this embodiment, as Figures 2-3As shown, with the increase of Aldo and Ang-II concentration, the cell viability of M-1 cells showed a downward trend, and when the concentration of both was > 10 nM, the cell viability decreased significantly. When the concentration of Aldo was ≥ 100 nM, the cell viability gradually increased with the extension of incubation time and tended to be stable; while the concentration of Aldo and Ang-II was 100 nM, and the incubation time was 48 h, the cell viability decreased significantly, but the survival rate was maintained above 75%, indicating that at this concentration and time, the drug can fully exert the pathological effect, while avoiding large-scale apoptosis or necrosis. Based on this, 100 nM (0.1 μM) of Aldo and Ang-II were used to act on M-1 cells for 48 h in the subsequent experiments.

[0044] Cell grouping: M-1 cells were grown to about 80%, and then trypsinized and plated in 96-well plates. After the cells in the 6-well plates grew to 70-80% confluence, the cells were synchronized by culturing in serum-free DMEM medium for 24 h. According to the previous CCK8 results, the cells were treated with Ctrl group (complete medium), Ang-II group (Ang-II, 0.1 μM), Ald group (Aldo, 0.1 μM), 2A group (Ang-II 0.1 μM + Aldo 0.1 μM), Angf group (Ang-II 0.1 μM + furosemide 100 μM), Aldf group (Aldo 0.1 μM + furosemide 100 μM), and 2Af group (Ang-II 0.1 μM + Aldo 0.1 μM + furosemide 100 μM) for 48 h. This grouping was used for subsequent qPCR and Western blot experiments.

[0045] In this embodiment, as Figures 4-6 As shown, compared with the Ctrl group, the relative expression level of AQP2 mRNA in the 2A group was significantly increased; compared with the 2A group, the relative expression levels of AQP2 mRNA in the Ald group, Angf group and 2Af group were significantly decreased (P < 0.05).

[0046] Compared with the Ctrl group, the relative expression level of SCNN1A mRNA in the Ald group and 2A group was significantly increased, and the increase level of the two groups was almost the same, and the increase level of the Ald group was slightly higher; compared with the Ald group and 2A group, the relative expression level of SCNN1A mRNA in the Ang-II group and Angf group was significantly decreased (P < 0.05).

[0047] The relative expression level of AVPR2 mRNA was significantly increased in Ald group and 2A group compared with Ctrl group, and the increase level was almost the same in the two groups, and the increase level of 2A group was slightly higher; compared with Ald group and 2A group, the relative expression level of SCNN1A mRNA in Ang-II group, Angf group and Aldf group was significantly decreased (P < 0.05).

[0048] Among them, Ctrl group, Ang-II group (0.1 μM Ang-II), Ald group (0.1 μM Aldo), 2A group (0.1 μM Ang-II+0.1 μM Aldo), Angf group (0.1 μM Ang-II+100 μM furosemide), Aldf group (0.1 μM Aldo+100 μM furosemide), 2Af group (0.1 μM Ang-II+0.1 μM Aldo+100 μM furosemide), *P < 0.05.

[0049] Example 2

[0050] qPCR measurement of AQP2, SCNN1A, AVPR2 gene expression: The primers of AQP2, SCNN1A and AVPR2 genes were designed by using NCBI, PrimerBank and other websites, and the sequences are shown in Table 1. After the cells in the 6-well plate were grouped and treated for 48 h, the culture medium was discarded, and the cells were washed twice with PBS solution. The total RNA of each group was extracted by using total RNA extraction kit, and the cDNA was reverse transcribed at 37℃ for 15 min and 85℃ for 5 s (3 repeats for each group). The amplification system was 20 μL system: 2 × Taq Pro Universal SYBRqPCR Master Mix 10 μL, upstream and downstream primers 0.4 μL each, cDNA 2 μL, ddH2O 7.2 μL. PCR amplification was performed under the condition of 5℃ for 5 s, 60℃ for 30 s, and 40 cycles, and the Ct value of each sample was read, and the 2 -ΔΔCt .

[0051] Table 1

[0052]

[0053] Western blot detection of AQP2, SCNN1A, AVPR2 protein levels: After the end of cell grouping treatment, total protein was extracted from each group, and 200 μL lysis buffer was used to lyse cells in each well. After centrifugation at 4°C, the supernatant was extracted. According to the BCA protein detection kit and the enzyme label instrument, the protein concentration of each group was determined and corrected to the same concentration, and then boiled in 100°C boiling water for 10 min to denature the protein. Denatured protein was subjected to protein electrophoresis in a 10% SDS-PAGE gel according to 15 μL protein loading per well, and transferred to a polyvinylidene fluoride (PVDF) membrane at 200 mA constant current. After washing the transferred membrane with TBST for 3 times, it was blocked at room temperature for 2 h, then incubated with diluted primary antibody at 4°C overnight, the next day it was transferred to secondary antibody diluent at room temperature for 1 h, washed with TBST for 3 times, and finally soaked in ECL working solution for chemiluminescence detection.

[0054] In this embodiment, the AQP2 protein expression levels in each group of M-1 cells are as shown in Figures 7-8 Compared with the Ctrl group, the AQP2 protein expression levels of the Ang-II group, the 2A group, the Aldf group and the 2Af group [were (1.46±0.1), (1.71±0.06), (1.37±0.22), (1.38±0.24) respectively] were significantly increased; the AQP2 protein expression level of the 2A group was higher than that of the Ang-II group, the Aldf group and the 2Af group. (P < 0.05)

[0055] Among them, Ctrl group, Ang-II group (0.1 μM Ang-II), Ald group (0.1 μM Aldo), 2A group (0.1 μM Ang-II+0.1 μM Aldo), Angf group (0.1 μM Ang-II+100 μM furosemide), Aldf group (0.1 μM Aldo+100 μM furosemide), 2Af group (0.1 μM Ang-II+0.1 μM Aldo+100 μM furosemide), *P < 0.05.

[0056] In this embodiment, the SCNN1A protein expression levels in each group of M-1 cells are as shown in Figures 9-10 Compared with the Ctrl group, the SCNN1A protein expression level of the 2a group (1.58±0.05) was significantly increased. The SCNN1A protein expression levels of the other groups did not change significantly. (P < 0.05)

[0057] The AVPR2 protein expression levels in each group of M-1 cells are as shown in the table. Compared with the Ctrl group, the AVPR2 protein expression levels of the 2a group and the 2Af group [1.49 ± 0.11 and 1.26 ± 0.19, respectively] were significantly increased; the AVPR2 protein expression level of the 2A group was higher than that of the 2Af group. The AVPR2 protein expression levels of the other groups did not change significantly (P < 0.05).

[0058] In this embodiment, the AVPR2 protein expression levels in each group of M-1 cells are as shown in the table. Compared with the Ctrl group, the AVPR2 protein expression levels of the 2a group and the 2Af group [1.49 ± 0.11 and 1.26 ± 0.19, respectively] were significantly increased; the AVPR2 protein expression level of the 2A group was higher than that of the 2Af group. The AVPR2 protein expression levels of the other groups did not change significantly (P < 0.05). Figures 11-12

[0059] The AVPR2 protein expression levels in each group of M-1 cells are as shown in the table. Compared with the Ctrl group, the AVPR2 protein expression levels of the 2a group and the 2Af group [1.49 ± 0.11 and 1.26 ± 0.19, respectively] were significantly increased; the AVPR2 protein expression level of the 2A group was higher than that of the 2Af group. The AVPR2 protein expression levels of the other groups did not change significantly (P < 0.05).

[0060] As can be seen from the above, the combined treatment of Ang-II and Aldo can synergistically up-regulate the expression of SCNN1A, AVPR2 and AQP2 at the gene and protein levels (P < 0.05), and the effect is more significant than that of single treatment, which reflects the integrated action of RAAS signals at the molecular level.

[0061] Example 3

[0062] ​To verify whether the model can reproduce the core feature of "insufficient diuretic effect" of clinical DR, the present embodiment carries out a diuretic intervention experiment: on the basis of the three groups of "Ang-II alone, Aldo alone, Ang-II+Aldo combined treatment", three groups of diuretic intervention groups of "Ang-II+ furosemide, Aldo + furosemide, Ang-II+Aldo + furosemide" are added, and the expression changes of core molecules (AQP2, SCNN1A, AVPR2) before and after adding drugs are compared to analyze the intervention effect of diuretics. From the results, there are significant differences in the response of different treatment groups to diuretics: compared with the Ang-II alone group, the AQP2 protein expression level in the Ang-II + furosemide group is significantly decreased; while compared with the Aldo alone group, the expression of AQP2, SCNN1A and other molecules in the Aldo + furosemide group is not significantly inhibited. It shows that furosemide can partially antagonize the water reabsorption pathway promoted by Ang-II, while the sodium reabsorption pathway dominated by Aldo is not sensitive to furosemide, because furosemide mainly affects the cAMP-PKA-AQP2 pathway by inhibiting the sodium transport in the thick ascending limb of the loop of Henle to antagonize water reabsorption. When Ang-II and Aldo are combined with furosemide, the effect of diuretics shows the characteristics of "partially effective but not complete": the gene and protein expression levels of AQP2, SCNN1A and AVPR2 all decrease significantly, but the down-regulation amplitude cannot completely reverse the up-regulation trend of Ang-II and Aldo, and the pathological state of increased sodium and water reabsorption in the collecting duct still persists. This experimental result is highly consistent with the pathological performance of DR patients in the clinic: when RAAS is overactivated to cause DR (such as in the late stage of heart failure), even if furosemide is used, the compensatory effect of RAAS still causes "the diuretic natriuretic and diuretic effects are offset, and water and sodium retention are difficult to alleviate". It can be seen that the model constructed in the present embodiment not only can simulate the molecular characteristics of overactivation of RAAS, but also can reproduce the core phenotype of "insufficient diuretic effect" of DR, which fully proves that it can be used as a reliable tool for in vitro research on the mechanism of DR and screening of targeted intervention drugs.

[0063] Compared with the prior art, the method for constructing a diuretic-resistant cell model and the application thereof described in the above embodiments can truly reflect the pathological characteristics of diuretic resistance. The present application has the advantages of simple construction, good repeatability, and the ability to truly reflect the pathological characteristics of diuretic resistance. A diuretic-resistant cell model is successfully constructed by stimulating M-1 cells with 100 nM Ang-II and Aldo for 48 h. The model is verified at the gene and protein levels, successfully simulates the pathological characteristics of sodium and water retention in the collecting duct caused by overactivation of RAAS, and presents the core phenotype of DR that is "partially reversed but cannot be completely corrected" under the intervention of diuretics, and has stability and repeatability. The construction of the model not only provides a reliable in vitro experimental platform for in-depth analysis of the molecular mechanisms of RAAS regulating the function of the collecting duct and the occurrence of DR, but also lays a solid foundation for subsequent screening of drugs targeting the RAAS signaling pathway or core effect molecules DR.

[0064] Obviously, the above-described embodiments are only preferred embodiments of the present application, but not all the embodiments. The preferred embodiments of the present application are shown in the drawings, but do not limit the patent scope of the present application. The present application can be realized in many different forms, and conversely, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or equivalently replace some technical features thereof. Any equivalent structure made by using the content of the present application specification and drawings, directly or indirectly applied in other related technical fields, is also within the patent protection scope of the present application.

Claims

1. A method for constructing a diuretic-resistant cell model, characterized by, The method comprises the following steps: Step 1, providing mouse renal collecting duct epithelial cells; Step 2, jointly treating the cells with angiotensin II at a concentration of 0.1 μM and aldosterone at a concentration of 0.1 μM, so as to induce the formation of the diuretic-resistant cell model.

2. The method of claim 1, wherein the cell model is resistant to a diuretic. On the basis of the joint treatment, further treatment is performed using furosemide at a concentration of 100 μM, so as to verify the diuretic-resistant property of the model.

3. The method for constructing a diuretic-resistant cell model according to claim 1, characterized in that, The joint treatment time is 48 hours.

4. The method for constructing a diuretic-resistant cell model according to claim 1, characterized in that, The mouse renal collecting duct epithelial cells are M-1 cell lines.

5. The method of claim 2, wherein the cell model is resistant to a diuretic. The gene and / or protein expression levels of aquaporin 2, epithelial sodium channel α subunit and arginine vasopressin receptor 2 in the diuretic-resistant cell model are significantly up-regulated compared with untreated normal cells.

6. The method of claim 5, wherein the cell model is resistant to a diuretic. The gene and / or protein expression levels of aquaporin 2, epithelial sodium channel α subunit and arginine vasopressin receptor 2 are detected by qPCR and Western blot techniques.

7. The method of claim 1, wherein the cell model is resistant to a diuretic. The diuretic-resistant cell model is used for studying the signal pathway mechanism of the renin-angiotensin-aldosterone system.

8. The method of claim 1, wherein the cell model is resistant to a diuretic. The diuretic-resistant cell model is cultured in DMEM medium containing 10% fetal bovine serum.

9. The method of claim 8, wherein the cell model is resistant to a diuretic. The culture environment is a 5% CO2 cell incubator at 37°C.

10. Use of a diuretic-resistant cell model constructed according to the construction method of any one of claims 1 to 9 for screening drugs for the prevention and / or treatment of diuretic resistance, characterized in that, The application comprises the following steps: The candidate drug is applied to the diuretic-resistant cell model; The expression level or activity of at least one of aquaporin 2, epithelial sodium channel α subunit and arginine vasopressin receptor 2 in the diuretic-resistant cell model is detected; The detection result is compared with the model group without the candidate drug, and if the candidate drug can significantly reverse the up-regulation of the expression level or activity, it indicates that the candidate drug has the potential for preventing and / or treating diuretic resistance.

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