Construction method and application of chronic kidney disease pulmonary arterial hypertension rat model

Abstract

The invention relates to the technical field of rat model construction, in particular to a construction method and application of a chronic nephropathy pulmonary arterial hypertension rat model, and the construction method comprises the following steps: (1) selecting an SPF-level male Sprague Dawley rat which is 8-9 weeks old and has the weight of 280 + / -20 g; (2) performing a 5 / 6 renal resection operation on the rat; (3) one week after the operation, giving 8% high-salt diet to the rats; (4) performing continuous high-salt diet intervention for 14 weeks, and by constructing the CKD-PH disease animal model, simulating pathophysiological characteristics of CKD-PH and researching pathogenesis of CKD-PH, including disorder of an RAAS system, change of serum metabolites and the like; potential treatment drugs aiming at the CKD-PH can be screened and evaluated, and the curative effect of the drugs on the CKD-PH can be evaluated; the biomarker of CKD-PH can be found by analyzing serum metabolites and pathological changes of model animals.

Description

Technical Field

[0001] The present invention relates to the technical field of rat model construction, and particularly to a method for constructing a rat model of chronic kidney disease with pulmonary hypertension and its application. Background Art

[0002] Chronic kidney disease (CKD) is a global health problem, and its association with pulmonary hypertension (PH) has received increasing attention in recent years. Research shows that CKD is an important risk factor for PH, and the incidence of PH in CKD patients increases with the severity of kidney disease. This complication is closely related to poor prognosis.

[0003] At present, there have been a large number of studies on CKD models and PH models, but there is a lack of a stable animal model for chronic kidney disease with pulmonary hypertension (CKD-PH). Most of the existing animal models focus on a single disease state and are difficult to comprehensively simulate the complex pathophysiological characteristics of CKD-PH. This limitation seriously hinders the in-depth study of the pathogenesis of CKD-PH and also restricts the development and evaluation of related treatment strategies.

[0004] In addition, existing models are often difficult to maintain stably for a long time and cannot meet the needs of observing disease progression and long-term treatment effects. At the same time, most models lack multi-dimensional evaluation methods and are difficult to comprehensively reflect the pathological characteristics of CKD-PH, especially the manifestations in aspects such as dysregulation of the renin-angiotensin-aldosterone system (RAAS) and metabolic disorders.

[0005] Therefore, there is an urgent need to establish an animal model that can stably and long-term simulate the pathophysiological characteristics of CKD-PH and develop corresponding multi-dimensional evaluation methods. Such a model should not only reflect the characteristics of CKD and PH, but also reflect the interaction and synergistic effect between the two disease states. Summary of the Invention

[0006] The present invention aims to solve the above technical problems and provides a novel method for constructing a CKD-PH rat model and its application. This method innovatively combines 5 / 6 nephrectomy surgery and high-salt diet intervention to successfully induce a stable CKD-PH state. Through a carefully designed surgical plan and diet intervention, the present invention realizes the synergistic induction of renal function impairment and pulmonary hypertension, effectively simulating the complex pathophysiological characteristics of CKD-PH.

[0007] The present invention discloses a method for constructing a rat model of chronic kidney disease with pulmonary hypertension, comprising the following steps:

[0008] (1) Select SPF-grade male Sprague Dawley rats at 8-9 weeks of age and weighing 280±20 g;

[0009] (2) Perform a 5 / 6 nephrectomy on the rats;

[0010] (3) One week after the operation, feed the rats with an 8% high-salt diet;

[0011] (4) Continuously intervene with a high-salt diet for 14 weeks.

[0012] Specifically, the 5 / 6 nephrectomy includes the following steps:

[0013] (1) Anesthetize the rats with isoflurane, with an induction concentration of 3% and a maintenance concentration of 2.5%;

[0014] (2) Shave the hair and disinfect the surgical area with 75% alcohol;

[0015] (3) Incise the skin, subcutaneous tissue, and muscle to make a 1.5-cm incision;

[0016] (4) Expose the left kidney and remove the upper and lower poles of the left kidney;

[0017] (5) Suture the incision with 4-0 absorbable sutures;

[0018] (6) One week after the operation, completely remove the right kidney.

[0019] Specifically, it further includes the following steps:

[0020] At the 8th and 14th weeks of the high-salt diet intervention, conduct the following assessments on the rats:

[0021] (1) Renal function assessment;

[0022] (2) Hemodynamic assessment;

[0023] (3) Cardiac function assessment;

[0024] (4) Pulmonary vascular remodeling assessment;

[0025] (5) Renin-angiotensin-aldosterone system (RAAS) assessment;

[0026] (6) Serum metabolite analysis.

[0027] Specifically, the renal function assessment includes:

[0028] Measure the 24-hour urinary protein, serum creatinine, and serum urea nitrogen levels, and perform renal HE staining and Masson trichrome staining.

[0029] Specifically, the hemodynamic assessment includes:

[0030] Measure the right ventricular systolic pressure (RVSP), mean arterial blood pressure (mABP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) using a BIOPAC MP150 data acquisition system.

[0031] Specifically, the cardiac function assessment includes:

[0032] Determine the ratio of right ventricular mass to left ventricular plus interventricular septum mass (RV / (LV+S)), and measure PAT / PET, heart rate, cardiac output, and stroke volume using echocardiography.

[0033] Specifically, the pulmonary vascular remodeling assessment includes:

[0034] Determine the percentage of pulmonary artery wall thickness and the number of blood vessels per square millimeter, and perform α-SMA and CD31 immunofluorescence staining.

[0035] Specifically, the RAAS assessment includes:

[0036] (1) Measure the concentrations of renin, angiotensin II, aldosterone, and angiotensin (1-7) in plasma using ELISA;

[0037] (2) Perform isometric tension measurement on isolated pulmonary artery rings;

[0038] (3) Perform ACE2 immunofluorescence staining and Western blot analysis.

[0039] 9. The method according to claim 3, wherein the serum metabolite analysis includes:

[0040] (1) Detect metabolites using LC-MS / MS technology;

[0041] (2) Perform differential metabolite analysis using the deSeq2, edgeR, and limma packages of R software;

[0042] (3) Integrate and rank metabolites using robust rank aggregation (RRA);

[0043] (4) Perform KEGG enrichment analysis to determine relevant metabolic pathways.

[0044] An application of a rat model of chronic kidney disease-associated pulmonary hypertension, the rat model being constructed by the method, for the following uses:

[0045] (1) Study the pathogenesis of chronic kidney disease-associated pulmonary hypertension;

[0046] (2) Screen for potential therapeutic drugs for chronic kidney disease-associated pulmonary hypertension;

[0047] (3) Evaluate the effectiveness of treatment strategies for pulmonary hypertension in chronic kidney disease;

[0048] (4) Search for biomarkers of pulmonary hypertension in chronic kidney disease.

[0049] The prominent advantages and unexpected technical effects of the present invention include:

[0050] 1. Simulate the disease characteristics of CKD-PH: After 5 / 6 nephrectomy and high-salt diet, this model showed significant changes in renal function and hemodynamic parameters, accompanied by right ventricular hypertrophy, but no pulmonary vascular remodeling was found, indicating the successful construction of the CKD-PH animal model.

[0051] 2. By constructing this CKD-PH disease animal model, the pathophysiological characteristics of CKD-PH can be simulated, and its pathogenesis can be studied, including the dysregulation of the RAAS system, changes in serum metabolites, etc.; potential therapeutic drugs for CKD-PH can be screened and evaluated, and the efficacy of drugs on CKD-PH can be evaluated; by analyzing the serum metabolites and pathological changes of model animals, biomarkers of CKD-PH can be found; the pathophysiological changes of model rats can be observed for a long time to study the progression process and long-term prognosis of CKD-PH.

[0052] 3. The present invention has developed a comprehensive multi-dimensional evaluation method, including multiple aspects such as renal function, hemodynamics, cardiac function, pulmonary vascular remodeling, RAAS system, and metabolomics analysis. This comprehensive evaluation method enables researchers to deeply understand the pathogenesis of CKD-PH, especially the role of RAAS system dysregulation and metabolic disorders in the disease progression.

[0053] The model of the present invention not only shows significant renal function impairment and pulmonary hypertension characteristics, but also reveals the imbalance of the RAAS system, such as the increase in angiotensin II level and the down-regulation of ACE2 expression. These findings provide a new perspective for understanding the molecular mechanism of CKD-PH. In addition, through metabolomics analysis, the present invention has also identified differential metabolites related to CKD-PH, opening up a way for searching for new biomarkers and therapeutic targets.

[0054] Generally speaking, the CKD-PH rat model and its evaluation method provided by the present invention have the following significant advantages: good stability, can observe the disease progression for a long time; the multi-dimensional evaluation method comprehensively reflects the disease characteristics; reveals the important role of the RAAS system and metabolic disorders in CKD-PH; provides a reliable platform for screening therapeutic drugs and evaluating treatment strategies. These characteristics make the present invention a powerful tool for studying the pathogenesis of CKD-PH, developing new diagnostic and treatment methods, and are expected to promote the research progress in related fields, and ultimately bring benefits to CKD-PH patients. Brief Description of the Drawings

[0055] Figure 1 It is the flow chart for constructing the CKD-PH rat model provided by the embodiments of the present application;

[0056] Figure 2 It is the evaluation results of renal function and kidney sections of the CKD-PH rat model provided by the embodiments of the present application;

[0057] Figure 3 It is the evaluation results of hemodynamics, immunofluorescence, cardiopulmonary pathology, and cardiac function of the CKD-PH rat model provided by the embodiments of the present application;

[0058] Figure 4 It is the evaluation of RAAS dysregulation and the results of isometric tension measurement of pulmonary artery rings in the CKD-PH rat model provided by the embodiments of the present application;

[0059] Figure 5 It is the analysis results of serum metabolites of the CKD-PH rat model provided by the embodiments of the present application;

[0060] Figure 6 It is the relevant analysis results of the identified metabolites of the CKD-PH rat model provided by the embodiments of the present application. Detailed implementation manners

[0061] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0062] Source of materials

[0063] 1. Experimental animals

[0064] Supplier: Guangdong Medical Laboratory Animal Center

[0065] Strain: Sprague Dawley

[0066] 2. Main reagents

[0067] ELISA kits:

[0068] Renin: MEIMIAN Co., Ltd., Beijing, China, Cat.#: MM-034R2;

[0069] Angiotensin II: MLBIO, Cat.#: ml058803;

[0070] Aldosterone: MEIMIAN Co., Ltd., Beijing, China, Cat.#: MM-0555R2;

[0071] Ang(1 - 7): Shanghai Jianglai Industrial Limited By Share Ltd, China, Cat.#: JL21363 - 96T;

[0072] Antibody:

[0073] ACE2: Abcam, Cat.#T55787M;

[0074] CD31: MLBIO, Cat.#ml058803

[0075] α - SMA: Sigma - Aldrich, Cat.#A5228;

[0076] 3. Main Instruments

[0077] BIOPAC MP150 Data Acquisition System: BIOPAC Systems, Inc., Santa Barbara, CA;

[0078] LC - MS / MS System: Thermo Fisher Scientific, USA, Q Exactive HF;

[0079] Western blot Equipment: Millipore;

[0080] Example 1: Construction and Evaluation of CKD - PH Rat Model

[0081] A method for constructing a rat model of pulmonary hypertension in chronic kidney disease of the present invention includes the following steps:

[0082] Step 1. Animal Selection and Grouping:

[0083] Select male Sprague Dawley rats of specific pathogen free (SPF) level, with a body weight of 280 ± 20 g and an age of 8 - 9 weeks. Randomly divide the rats into a sham operation group and a 5 / 6 nephrectomy group.

[0084] Step 2. Surgical Operation:

[0085] Rats in the 5 / 6 nephrectomy group were subjected to 5 / 6 nephrectomy. At 8 weeks of age, the rats were anesthetized with isoflurane (3% induction, 2.5% maintenance), their hair was shaved, and the surgical area was disinfected with 75% alcohol. An incision of 1.5 cm was made through the skin, subcutaneous tissue, and muscle to expose the left kidney. After removing the upper and lower poles of the left kidney, the incision was sutured with 4-0 absorbable sutures. One week after surgery, the right kidney was completely removed and the incision was sutured with 4-0 absorbable sutures. The sham operation group underwent the same incision and suture without nephrectomy.

[0086] Step 3, Dietary intervention:

[0087] One week after surgery, the rats in the 5 / 6 nephrectomy group were given a high-salt diet (8%), and the sham operation group was given a normal-salt diet (0.4%).

[0088] Step 4, Model evaluation:

[0089] At 8 weeks and 14 weeks after surgery, a series of evaluations were performed on the rats: After anesthesia with isoflurane, the hemodynamic parameters of the rats were measured using a BIOPAC MP150 data acquisition system, including right ventricular systolic pressure (RVSP), systolic blood pressure (SBP), and diastolic blood pressure (DBP); the ratio of right ventricular mass to the mass of the left ventricle plus interventricular septum (RV / (LV+S)) was calculated to evaluate the degree of right ventricular hypertrophy; tissues such as the lung, kidney, and heart were taken for HE staining and Masson trichrome staining for multi-organ pathological changes examination; the ratio of pulmonary vascular wall thickness to total vascular diameter (WT%) and the number of blood vessels per square millimeter (mm-2) were measured to evaluate pulmonary vascular remodeling; the expression analysis of angiotensin-converting enzyme 2 (ACE2) in the pulmonary vascular endothelium, the activity evaluation of key vascular components in the renin-angiotensin-aldosterone system (RAAS), and the analysis of changes in serum metabolites were performed, etc.

[0090] Quality control measures:

[0091] Experimental repeatability: All experiments were repeated 3 times to ensure the repeatability of the experimental results.

[0092] Statistical analysis: n refers to the number of animals, and the data are expressed as the standard deviation (SD) ± mean; Student's t-test was used for comparison between two groups; one-way analysis of variance (ANOVA) was used for multi-group comparison, followed by Tukey's post hoc test; for data that significantly deviated from the normal distribution, non-parametric tests (such as the Mann–Whitney U test) were applied, and ROC curve analysis was used to evaluate the diagnostic accuracy of candidate metabolites; the R 4.1.1 DeSeq2 software package was used for statistical analysis of metabolomics data; statistical significance was set at *P<0.05, **P<0.01.

[0093] Control of influencing factors: Strictly control the experimental environment (such as temperature, humidity), animal feeding conditions (such as diet, lighting), and surgical operation procedures to ensure the reliability of experimental results.

[0094] Twenty-one SPF-grade male Sprague Dawley rats, weighing 280 ± 20 g and aged 8 - 9 weeks, were selected from the Guangdong Provincial Laboratory Animal Center for Medical Research. They were randomly and evenly divided into a sham operation group and a 5 / 6 nephrectomy group (at 8 weeks and 14 weeks).

[0095] Perform different surgical operations and dietary interventions on the three groups according to the above methods, and the operation procedures are as Figure 1 shown.

[0096] After a high-salt diet for 8 weeks and 14 weeks postoperatively, renal function was evaluated in rats of different groups, and the results are as Figure 2 shown: ① The 24-hour urinary protein level was measured by colorimetry: The 24-hour urinary protein level in the 5 / 6 nephrectomy group was the highest at 8 weeks and decreased slightly at 14 weeks; ② The serum creatinine level was measured by enzyme-linked immunosorbent assay (ELISA): The serum creatinine level in the 5 / 6 nephrectomy group increased continuously compared with the sham operation group; ③ The serum urea nitrogen level was measured by colorimetry: The serum urea nitrogen level in the 5 / 6 nephrectomy group increased continuously compared with the sham operation group; ④ Changes in kidney weight: The kidney weight in the 5 / 6 nephrectomy group decreased compared with the sham operation group, with the lowest kidney weight at 8 weeks and a significant recovery at 14 weeks; ⑤ HE staining and Masson trichrome staining were used to observe the changes in kidney structure and collagen deposition in kidney sections of rats in each group: The glomeruli that were swollen and sclerosed in the 5 / 6 nephrectomy group at 14 weeks gradually returned to normal size compared with those at 8 weeks, but there were still progressive dilatation and persistent renal interstitial fibrosis, and progressive fibrosis existed in the kidneys of the 5 / 6 nephrectomy surgery group. The results suggest that 5 / 6 nephrectomy and a high-salt diet successfully induced the progression of chronic kidney disease and there was a compensatory mechanism.

[0097] The BIOPAC MP150 data acquisition system was used to measure hemodynamic parameters and evaluate cardiac function in rats of different groups, and the results are as Figure 3Shown as follows: ① RVSP and RV / (LV+S) ratio (Fulton Index): The levels of RVSP and RV / (LV+S) in the 5 / 6 nephrectomy group were significantly increased at 14 weeks; ② mABP, SBP, DBP: The systemic blood pressure in the 5 / 6 nephrectomy group was significantly increased; ③ Percentage of pulmonary artery wall thickness (PA Wall Thickness(%)) and number of vessels per square millimeter (Number of Vessel(mm-2)): No significant differences were found among the three groups; ④ Immunofluorescence staining of α-SMA (red) and CD31 (green): There were no significant differences in the expression of α-SMA and CD31 in the pulmonary artery among groups; ⑤ Echocardiography measurement of PAT / PET, HR (BMP), CO, SV: The PAT / PET ratio in the 5 / 6 nephrectomy group was decreased compared with the sham operation group, while the values of CO, HR, and SV were increased; ⑥ HE staining of heart sections to measure the cross-sectional area of cardiomyocytes: The cross-sectional area of cardiomyocytes in the 5 / 6 nephrectomy group was significantly increased compared with the sham operation group. The results suggest that 5 / 6 nephrectomy combined with high-salt diet can successfully induce pulmonary hypertension and heart failure, but no smooth muscle thickening or distal pulmonary vascular remodeling was found.

[0098] The RAAS imbalance was evaluated and the isometric tension of pulmonary artery rings was measured in rats of different groups, and the results were as Figure 4 shown as follows: ① The concentrations of renin, angiotensin II (Ang II), aldosterone (ALD), and Ang(1-7) in plasma were measured by ELISA kit: Compared with the sham operation group, the level of Ang II in the 5 / 6 nephrectomy group was significantly increased, the level of Ang(1-7) was significantly decreased, and the levels of renin and aldosterone were both significantly increased, but the increase at 8 weeks was higher than that at 14 weeks; ② Isometric tension measurement of isolated pulmonary artery rings: The plasma of the 5 / 6 nephrectomy group at 14 weeks could significantly induce the contraction of pulmonary artery rings, and the plasma-induced contraction of pulmonary artery rings could be significantly inhibited by using losartan (Ang II receptor inhibitor); ③ Immunofluorescence staining of ACE2: ACE2 was expressed in endothelial cells, and ACE2 in the 5 / 6 nephrectomy group was significantly decreased, and the expression of CD31 was not significantly different; ④ WB results of ACE2 in different groups: Compared with the sham operation group, the expression of ACE2 in the 5 / 6 nephrectomy group was down-regulated. The results suggest that the renin-angiotensin-aldosterone system is imbalanced in this model, and it affects pulmonary vascular contraction through changes in plasma metabolites.

[0099] The serum metabolites of rats in different groups were analyzed by LC-MS / MS technology, and the results were as Figure 5 、 6Shown as follows: ① Differentially expressed metabolites between the sham operation group and the 5 / 6 nephrectomy + high-salt diet-induced group were analyzed using the deSeq2, edgeR, and limma packages through R software. Red represents upregulated differentially expressed metabolites, blue represents downregulated differentially expressed metabolites, and gray represents metabolites excluded by the threshold criteria of |log2 fold change (FC)| > 1 and p-value < 0.05: 1460, 1070, and 972 upregulated differentially expressed metabolites were identified respectively; ② Robust rank aggregation (RRA) was used to integrate, rank, and identify all metabolites: finally, 400 upregulated differentially expressed metabolites were obtained; ③ Venn diagram analysis: 377 upregulated differentially expressed metabolites were identified; ④ KEGG enrichment analysis: 17 upregulated differentially expressed metabolites were determined; ⑤ There was a correlation among the 17 metabolites, and there were significant differences in the expression of the top five upregulated metabolites, and all showed obvious AUC-ROC values. ⑥ Time series analysis: All metabolites could be divided into four groups, and the upregulated differentially expressed metabolite diacylglycerol (DAG) was in the first group (i.e., gradually increasing over time). ⑦ KEGG pathway analysis revealed the involvement of 83 signaling pathways related to 5 major upregulated metabolites, including the renin-angiotensin-aldosterone system.

[0100] Van Gieson staining of blood vessels:

[0101] Van Gieson staining was performed on formalin-fixed, paraffin-embedded blood vessel sections. The sections were dewaxed and hydrated, and nuclear staining was performed with Mayer hematoxylin. Subsequently, the sections were stained with Van Gieson solution (acid fuchsin and picric acid), and collagen fibers were stained red / pink. After dehydration and clearing, the sections were mounted and examined under an optical microscope. The collagen content in the blood vessel wall was quantified by measuring the percentage of the red-stained area relative to the total blood vessel area using iViewer 6.0 section analysis software (Unic Technologies Inc., Beijing, China).

[0102] Isometric tension measurement of isolated pulmonary arteries:

[0103] PA was isolated under a stereomicroscope to obtain an ex vivo PA ring with a length of 2 mm. The ex vivo PA ring was placed in Krebs-Henseleit solution (containing 118.0 mM NaCl, 25.0 mM NaHCO3, 4.7 M KCl, 1.2 mM aH2PO4, 1.8 mM CaCl2, 1.2 mM MgSO4, and 11.1 mM glucose) containing 95% O2 and 5% CO2 to equilibrate and reach the baseline tension state. To evaluate the viability of the PA ring, 60 mM KCl was used to induce its contraction. When the PA ring reached its maximum contraction amplitude, 10 μM Ach (acetylcholine) was used to evaluate its endothelial integrity; then phenylephrine (1 μM) was used to induce optimal contraction in calcium solution. After the contraction induced by phenylephrine, the PA ring was waited to recover to its baseline tension. To evaluate the effect of plasma on contraction, the plasma of the sham nephrectomy group and the 5 / 6 nephrectomy group was added to Krebs-Henseleit solution respectively, and angiotensin II (Ang II) was used as a positive control to observe the change of vascular tension.

[0104] Western blot:

[0105] RIPA lysis buffer (50 mmol / L Tris pH 7.4, 150 mmol / L NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS), phenylmethylsulfonyl fluoride (PMSF; 100 mmol / L GBCBIO), protease inhibitor mixture (E2, Roche), and phosphatase inhibitor mixture (E3, Sigma) were added at a ratio of 100:2:1:1 (RIPA:PMSF:E2:E3) to prepare a solution for lysing tissues. After three rounds of homogenization, tissue extracts were obtained by centrifugation at 4 °C. The protein concentration was quantified using the Bradford assay. Equal amounts of soluble proteins were mixed with SDS sample buffer, boiled, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% PBST milk powder (PBS containing 0.1% Tween-20) for 1 hour and incubated overnight at 4 °C with a primary antibody diluted in PBST containing 1% BSA (Sigma). After washing three times with PBST, the membrane was incubated with an HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibody. After washing five times with PBST, protein bands were observed using the ECL system (Millipore).

[0106] Example 2: Application of the rat model of chronic kidney disease-induced pulmonary arterial hypertension

[0107] This example demonstrates various applications of the CKD-PH rat model constructed and evaluated based on Example 1:

[0108] (1) Study the pathogenesis of CKD-PH: Using this model, the roles of the dysregulation of the RAAS system, abnormal pulmonary vascular function, and metabolic disorders in the pathogenesis of CKD-PH can be deeply studied. For example, by analyzing the changes in ACE2 expression and the elevation of angiotensin II levels, the key role of the imbalance of the RAAS system in the pathogenesis of CKD-PH can be revealed.

[0109] (2) Screen potential therapeutic drugs: This model can be used to evaluate the therapeutic effects of various drugs on CKD-PH. For example, the effects of RAAS inhibitors, vasodilators, or metabolic regulators on the blood pressure, pulmonary vascular remodeling, and cardiac function of model rats can be tested.

[0110] (3) Evaluate the effectiveness of treatment strategies: This model can be used to evaluate the long-term effects of different treatment regimens. For example, the differences in improving the symptoms of CKD-PH between single-drug treatment and combination drug therapy strategies can be compared.

[0111] (4) Search for biomarkers of CKD-PH: Through metabolomic analysis of model rats, new biomarkers related to the progression of CKD-PH can be discovered. For example, differential metabolites such as diacylglycerol (DAG) found in the experiment may become potential diagnostic or prognostic markers.

[0112] Through these applications, the CKD-PH rat model provided by the present invention provides a powerful tool for deeply studying the pathophysiological mechanism of this disease and developing new diagnostic and treatment strategies.

[0113] The above are only examples of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. A method for constructing a rat model of chronic kidney disease and pulmonary hypertension, characterized in that , including the following steps: (1) Select SPF male Sprague Dawley rats aged 8-9 weeks and weighing 280±20g; (2) performing a 5 / 6 nephrectomy on the rats; (3) One week after surgery, the rats were given an 8% high-salt diet; (4) Continue high-salt diet intervention for 14 weeks.

2. The method according to claim 1, characterized in that , the 5 / 6 nephrectomy surgery comprises the following steps: (1) anesthetizing the rat with isoflurane, wherein the induction concentration is 3% and the maintenance concentration is 2.5%; (2) Shave and disinfect the surgical area with 75% alcohol; (3) Cut the skin, subcutaneous tissue, and muscle, making a 1.5 cm incision; (4) Expose the left kidney and remove the upper and lower poles of the left kidney; (5) Use 4-0 absorbable sutures to close the incision; (6) One week after surgery, the right kidney was completely removed.

3. The method according to claim 1, characterized in that , further comprising the following steps: At the 8th and 14th weeks of the high-salt diet intervention, the rats were evaluated as follows: (1) Assessment of renal function; (2) Hemodynamic assessment; (3) Cardiac function assessment; (4) Assessment of pulmonary vascular remodeling; (5) renin-angiotensin-aldosterone system (RAAS) assessment; (6) Analysis of serum metabolites.

4. The method according to claim 3, characterized in that , the renal function assessment includes: The levels of 24-hour urine protein, serum creatinine, and serum urea nitrogen were measured, and kidney HE staining and Masson's trichrome staining were performed.

5. The method according to claim 3, characterized in that , the hemodynamic assessment includes: Right ventricular systolic pressure (RVSP), mean arterial pressure (mABP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured using a BIOPAC MP150 data acquisition system.

6. The method according to claim 3, characterized in that , the cardiac function assessment includes: The ratio of right ventricular mass to left ventricular plus septal mass (RV / (LV+S)) was determined, and PAT / PET, heart rate, cardiac output, and stroke volume were measured by echocardiography.

7. The method according to claim 3, characterized in that , the pulmonary vascular remodeling assessment includes: The percentage of pulmonary artery wall thickness and the number of blood vessels per square millimeter were measured, and α-SMA and CD31 immunofluorescence staining was performed.

8. The method according to claim 3, characterized in that , the RAAS assessment includes: (1) ELISA was used to measure the plasma concentrations of renin, angiotensin II, aldosterone, and angiotensin (1-7); (2) Perform isometric tension measurements on isolated pulmonary artery rings; (3) Perform ACE2 immunofluorescence staining and Western blot analysis.

9. The method according to claim 3, characterized in that , the serum metabolite analysis includes: (1) Metabolite detection using LC-MS / MS technology; (2) differential metabolite analysis was performed using the deSeq2, edgeR, and limma packages of R software; (3) Use robust rank aggregation (RRA) to integrate and rank metabolites; (4) KEGG enrichment analysis was performed to determine the relevant metabolic pathways.

10. Application of a rat model of chronic kidney disease and pulmonary hypertension, characterized in that , the rat model is constructed by the method according to any one of claims 1 to 9 and is used for the following purposes: (1) To study the pathogenesis of pulmonary hypertension in chronic kidney disease; (2) Screening for potential therapeutic drugs for pulmonary hypertension in chronic kidney disease; (3) evaluate the effectiveness of treatment strategies for pulmonary hypertension in chronic kidney disease; (4) To identify biomarkers of pulmonary hypertension in chronic kidney disease.

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