Polypeptide and application thereof in hypertension treatment

Abstract

The invention discloses a fusion polypeptide RAT-1 and application thereof in hypertension treatment. The topological structure of the fusion polypeptide is a kidney targeting head-ACE2 functional segment-MMP responsive connexon-AT1 receptor antagonism segment, and the full-length sequence is as shown in SEQ ID No: 1. The kidney targeting head can be specifically combined with megalin receptors of renal tubular epithelial cells to realize kidney targeting delivery; the ACE2 functional segment can efficiently degrade angiotensin II; the MMP responsive connexon is specifically cut in a focus microenvironment, and an AT1 receptor antagonism segment is released; the AT1 receptor antagonist segment can block the receptor signal channel of angiotensin II. The fusion polypeptide has good plasma stability, shows a powerful and long-acting antihypertensive effect in a spontaneous hypertension rat model, is remarkable in kidney targeting, and can relieve renal fibrosis and protect podocyte functions. The fusion polypeptide disclosed by the invention realizes a synergistic effect of targeted delivery, enzymolysis catalysis, response release and receptor antagonism, and can be used for preparing hypertension treatment medicines and kidney protection medicines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide and its use in the treatment of hypertension. Background Technology

[0002] Hypertension is the most prevalent chronic non-communicable disease globally. A 2023 epidemiological study by the Global Hypertension League showed a standardized prevalence of 23.2% in adults aged 25 and older. Uncontrolled hypertension increases the risk of stroke by 3-5 times and the risk of myocardial infarction by 2-3 times, making it the leading independent risk factor for cardiovascular disease death. While current first-line antihypertensive drugs can achieve stepwise blood pressure control, they have significant clinical limitations: long-term use of thiazide diuretics (such as hydrochlorothiazide) can induce hypokalemia (incidence approximately 8.3%) and impaired glucose tolerance; dihydropyridine calcium channel blockers (such as nifedipine) can cause lower extremity edema (incidence 12.5%) and reflex tachycardia (incidence 7.2%), significantly reducing patient adherence; and angiotensin-converting enzyme inhibitors (such as captopril) can cause dry cough in 15%-20% of patients, with 3%-5% discontinuing treatment due to intolerance.

[0003] Peptide drugs have become a research focus in the field of hypertension treatment due to their advantages such as high targeting, high receptor selectivity, and mild toxicity. However, existing candidate molecules face insurmountable technical bottlenecks: Saralasin, an angiotensin II type 1 receptor antagonist, can effectively antagonize vasoconstriction, but its plasma half-life is only 1.5 hours, requiring continuous intravenous infusion at a rate of 3-5 μg / kg / min to maintain its antihypertensive effect, which greatly limits its clinical application; Atrial natriuretic peptide (ANP), as an endogenous antihypertensive peptide, is easily degraded by neutral endopeptidase (NEP) (t1 / 2≈2.5 min), resulting in a bioavailability of less than 5%, making oral administration completely ineffective; Although bradykinin B2 receptor agonist peptides can exert antihypertensive effects by dilating blood vessels, about 6.8% of users experience allergic reactions such as angioedema, the mechanism of which is directly related to bradykinin-mediated increased vascular permeability. The core of these defects stems from the inherent characteristics of polypeptide molecules: they are easily degraded by serine proteases (such as thrombin) and metalloproteinases (such as matrix metalloproteinases), have a rapid glomerular filtration clearance rate (polypeptides with a molecular weight < 5 kDa can freely pass through the glomerular basement membrane), and have transmembrane transport barriers caused by excessive hydrophilicity.

[0004] Existing technologies have significant shortcomings in addressing these issues: while PEGylation can prolong the half-life (e.g., PEGylated exenatide's half-life is extended to 2 weeks), approximately 20%-30% of receptors produce anti-PEG antibodies, triggering immune complex-related adverse reactions; Fc fusion technology (e.g., aflibercept) introduces a 50kDa Fc fragment, increasing the molecular weight to over 100kDa, resulting in a 40%-50% decrease in tissue penetration; liposome encapsulation technology (e.g., doxorubicin liposomes) typically has a drug loading capacity of less than 10%, and is easily recognized and cleared by the mononuclear-macrophage system (MPS) in the bloodstream, with a stability half-life of less than 6 hours. Therefore, developing novel peptide drugs that combine long-circulating characteristics, targeted delivery capabilities, and high transmembrane transport efficiency is crucial to overcoming the technological bottlenecks in hypertension treatment. Summary of the Invention

[0005] This invention aims to address the problems of existing hypertension treatment drugs, such as poor targeting, short half-life, significant side effects, and insufficient protection against kidney damage caused by hypertension. Existing drugs, such as valsartan and telmisartan, while exerting antihypertensive effects, are widely distributed in the body and lack strong targeting of target organs such as the kidneys, resulting in numerous systemic side effects. Furthermore, their antihypertensive effects are short-lived, requiring frequent dosing. Simultaneously, these drugs offer limited protection against kidney fibrosis and podocyte damage caused by hypertension, failing to meet clinical treatment needs.

[0006] Based on this, the present invention provides a fusion polypeptide RAT-1, designed based on a multifunctional synergistic mechanism of "kidney targeting-enzymatic catalysis-response release-receptor antagonism", and its topology is [kidney targeting head]-[ACE2 functional segment]-[MMP responsive linker]-[AT1 receptor antagonist segment], as follows:

[0007] The kidney-targeting head, with the sequence Cys-Ser-Ser-Ser (CSSS), is designed based on the recognition mechanism of megalin (Lrp2) receptor, which is highly expressed in renal tubular epithelial cells. The megalin receptor can specifically bind to low molecular weight peptides containing the Cys-Ser motif. Introducing this sequence at the N-terminus enhances the selective aggregation of RAT-1 in the proximal convoluted tubules of the kidney and reduces exposure to non-target tissues throughout the body.

[0008] 1. ACE2 functional segment: The sequence is GSYSYKVTLRSPHPLFCG, designed based on the optimized structure of the ACE2 enzyme active site. The key catalytic triplet is retained to ensure hydrolytic activity against angiotensin II (Ang II); rigid residues Phe, Leu, and Pro are introduced to maintain conformational stability, while polar residues Ser and Tyr expose the active site, improving catalytic efficiency; the two ends are connected by flexible Gly to avoid steric hindrance from adjacent domains.

[0009] 2. MMP-responsive linker: The sequence is GPLGLRG, containing the MMP-2 / 9 specific cleavage site GPLG↓LRG. In the lesion microenvironment of hypertensive nephropathy, the expression level of MMP-2 / 9 is significantly increased (3-5 times higher than that of normal tissue), which can specifically cleave this linker to release the downstream AT1 receptor antagonist segment, thereby achieving "lesion-localized activation".

[0010] 3. AT1 receptor antagonist segment: The sequence is DVYARVCY, designed based on the binding conformation of telmisartan to the AT1 receptor. Amino acid sequences complementary to the AT1 receptor pocket are screened through molecular simulation, preserving the pharmacophore characteristics of telmisartan (such as aromatic ring hydrophobic interactions and hydrogen bond binding sites), and competitively antagonizing the binding of Ang II to the AT1 receptor.

[0011] 4. The full-length sequence (SEQ ID NO:1) of the fusion polypeptide RAT-1 is formed by sequentially connecting the above-mentioned domains, and consists of 37 amino acids.

[0012] Based on the fusion peptide RAT-1, the present invention also provides a pharmaceutical composition comprising the fusion peptide RAT-1 and a pharmaceutically acceptable carrier or sustained-release material.

[0013] Preferably, the pharmaceutically acceptable carrier of the present invention includes at least one selected from physiological saline, phosphate buffer, and water for injection. The sustained-release material includes at least one selected from polyethylene glycol, hyaluronic acid, and chitosan. It should be noted that the pharmaceutically acceptable carriers or sustained-release materials described above are conventional applications readily apparent to those skilled in the art in the preparation of compositions. Although not all of them are used in the subsequent embodiments of the present invention, as conventional applications, these carriers and materials should all fall within the scope of protection of the present invention.

[0014] The fusion peptide RAT-1 designed in this invention exhibits significant advantages through the synergistic effect of the "kidney targeting head - ACE2 functional segment - MMP responsive linker - AT1 receptor antagonist segment": the kidney targeting head CSSS can bind with high affinity to the megalin receptor (KD=7.8×10). -10The ACE2 functional segment maintains high catalytic activity (specific activity 140±8 nmol / min / μmol), directly degrading pathogenic Ang II. The MMP-2 / 9 responsive linker specifically cleaves within the lesion microenvironment (cleavage rate >75% after 2 hours), achieving targeted release of the antagonist segment. The AT1 antagonist segment effectively blocks receptor signaling (IC50 = 2.3±0.3 μM). Simultaneously, its plasma half-life reaches 12.3 hours, with a maximum blood pressure reduction of 58.4 mmHg in the SHR model, a duration 3.6 times longer than valsartan, and it significantly reduces renal fibrosis (TGF-β1 and Col IV levels approach normal), demonstrating superior overall performance compared to existing drugs.

[0015] Therefore, the multifunctional synergistic design of RAT-1 in this invention provides a new strategy for the treatment of hypertension, especially suitable for patients with concurrent kidney damage. Its targeted nature can reduce systemic side effects, its long-lasting effect can reduce the frequency of administration, and its renal protective effect can delay disease progression. It is expected to become a novel candidate drug with both antihypertensive and target organ protection capabilities in clinical translation, providing a breakthrough direction for solving the problem of "difficulty in balancing efficacy and safety" in existing treatments. Attached Figure Description

[0016] Figure 1 A schematic diagram of the function of the RAT-1 domain of the fusion peptide. Detailed Implementation

[0017] 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0018] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0019] Example 1: Design and synthesis of the fusion peptide RAT-1

[0020] The fusion peptide designed in this invention (named RAT-1) is based on a multifunctional synergistic mechanism of "renal targeting-enzymatic catalysis-response release-receptor antagonism," and its topology and function ( Figure 1 The sequence is: [Kidney Target Head] - [ACE2 Functional Segment] - [MMP Responsive Linker] - [AT1 Receptor Antagonist Segment], with the specific sequence and design basis as follows:

[0021] 1. Kidney Targeting Head: The sequence is Cys-Ser-Ser-Ser (CSSS), designed based on the recognition mechanism of megalin (Lrp2) receptor, which is highly expressed in renal tubular epithelial cells. Megalin receptor can specifically bind to low molecular weight peptides containing the Cys-Ser motif. Introducing this sequence through the N-terminus can enhance the selective accumulation of RAT-1 in the proximal convoluted tubule of the kidney and reduce exposure to non-target tissues throughout the body.

[0022] 2. ACE2 functional segment: The sequence is GSYSYKVTLRSPHPLFCG, designed based on the optimized structure of the ACE2 enzyme active site.

[0023] The key catalytic triplet is retained to ensure hydrolytic activity against angiotensin II (Ang II);

[0024] Introducing rigid residues Phe, Leu, and Pro to maintain spatial conformational stability, and polar residues Ser and Tyr to expose active sites to improve catalytic efficiency;

[0025] The two ends are connected by flexible Gly to avoid spatial steric hindrance with adjacent structural domains.

[0026] 3. MMP-responsive linker: The sequence is GPLGLRG, containing the MMP-2 / 9 specific cleavage site GPLG↓LRG. In the lesion microenvironment of hypertensive nephropathy, the expression level of MMP-2 / 9 is significantly increased (3-5 times higher than that of normal tissue), which can specifically cleave this linker to release the downstream AT1 receptor antagonist segment, thereby achieving "lesion-localized activation".

[0027] 4. AT1 receptor antagonist segment: The sequence is DVYARVCY, designed based on the binding conformation of telmisartan to the AT1 receptor: through molecular simulation screening, amino acid sequences complementary to the AT1 receptor pocket are selected, retaining the pharmacophore characteristics of telmisartan (such as the hydrophobic interaction of the aromatic ring and hydrogen bond binding sites), which can competitively antagonize the binding of Ang II to the AT1 receptor.

[0028] 5. RAT-1 full-length sequence (SEQ ID NO:1): formed by sequentially linking the above domains, with a total length of 37 amino acids, the sequence is: CSSSGSYSYKVTLRSPHPLFCGGPLGLRGDVYARVCY (sequence characteristics: target head 4aa + ACE2 functional segment 18aa + linker 7aa + antagonist segment 8aa).

[0029] 6. The final RAT-1 full-length sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. After purification, the peptide was detected by UPLC and the purity was 98.7%. The molecular weight was detected by MALDI-TOF MS and the measured molecular weight was approximately 4287.1 Da, which was consistent with the theoretical value, confirming that the synthesis was correct.

[0030] Example 2: Functional Verification Experiment of RAT-1

[0031] 1. Validation of renal targeting (SPR method)

[0032] (1) Megalin protein (50 μg / mL) was immobilized on a CM5 chip by amine coupling at a density of 12000 RU, and BSA was immobilized in the blank channel as a control;

[0033] (2) RAT-1 and the mutant RAT-1ΔT (sequence: GSYSYKVTLRSPHPLFCGGPLGLRGDVYARVCY) with the missing target head were diluted to 0.1-10 μM with running buffer (PBS + 0.05% Tween-20) and injected sequentially into the chip channels (flow rate 30 μL / min, binding for 120 seconds, dissociation for 300 seconds).

[0034] (3) Use BIAevaluation 4.1 software to fit the binding curve and calculate the equilibrium dissociation constant (KD).

[0035] (4) Results: The KD of RAT-1 and megalin was 7.8 × 10⁻⁶. -10 The KD of M,RAT-1ΔT is 3.2×10 -7 M indicates that the target head CSSS can significantly enhance the specific binding to megalin (affinity increased by 400 times).

[0036] 2. MMP-2 / 9 responsive cleavage verification (1) RAT-1 (100 μM) and MMP-2 / 9 (10 nM) were incubated in reaction buffer (50 mM Tris-HCl + 10 mM CaCl2 + 0.05% Brij-35, pH 7.5) at 37°C, and samples were taken after 0-4 hours;

[0037] (2) Negative control: RAT-1 reacted with inactivated MMP-2 / 9 (heated at 80°C for 30 minutes) or MMP-1;

[0038] (3) HPLC was used to detect the cleavage products (N-terminal fragment: CSSSGSYSYKVTLRSPHPLFCGPLG; C-terminal fragment: LRGDVYARVCY) and calculate the cleavage rate.

[0039] (4) Results: RAT-1 was cleaved at a rate of 76.5%-82.6% after 2 hours and over 87% after 4 hours under the action of MMP-2 / 9; the cleavage rate was <5% when reacting with MMP-1 or inactivating enzyme, proving that the linker has a specific response to MMP-2 / 9.

[0040] 3. Validation of ACE2 functional segment activity (Ang II hydrolysis experiment)

[0041] (1) Incubate RAT-1 (0.1-2 μM) and Ang II (10 μM) in ACE2 reaction buffer (50 mM HEPES + 150 mM NaCl, pH 7.5) at 37°C for 30 minutes;

[0042] (2) Detect the concentrations of AngⅡ (m / z 1046.5→909.4) and its hydrolysis product Ang-(1-7) (m / z 899.5→775.4), and calculate the degradation rate and specific activity.

[0043] (3) Results: The specific activity of Ang II degradation of RAT-1 was 140±8 nmol / min / μmol (142±10 nmol / min / μmol for recombinant ACE2). The ΔACE2 degradation rate of the mutant RAT-1 lacking the ACE2 functional segment was < 3%, indicating that the ACE2 functional segment retained catalytic activity.

[0044] 4. Verification of AT1 receptor antagonistic activity (calcium flow assay)

[0045] (1) CHO-AT1 cells loaded with Fluo-4 AM (5 μM) were pre-incubated with RAT-1 (0.1-100 μM) for 10 minutes and then Ang II (100 nM) was added.

[0046] (2) Detect the fluorescence intensity at 525 nm and calculate IC50.

[0047] (3) Results: The IC50 of RAT-1 against Ang II-induced calcium flow was 2.3 ± 0.3 μM, indicating that the AT1 antagonistic segment can specifically block AT1 receptor activation (the positive control telmisartan IC50 was 0.12 ± 0.02 μM).

[0048] 5. In vivo hypotensive experiment in spontaneously hypertensive rats (SHR)

[0049] (1) Materials: SPF grade SHR (12 weeks old, SBP≥160 mmHg), WKY rats, and non-invasive tail artery blood pressure monitor (Softron BP-98A).

[0050] (2) Grouping: SHRs were randomly divided into a model control group, a low-dose RAT-1 group (0.5 mg / kg), a high-dose RAT-1 group (1 mg / kg), a RAT-1ΔT group (1 mg / kg), and a telmisartan group (10 mg / kg), with 8 animals in each group. The drugs were administered via tail vein injection for 7 days.

[0051] (3) Measure SBP and kidney / plasma drug concentration ratio before and after drug administration.

[0052] (4) Results: Seven days after administration, the SBP of the high-dose RAT-1 group decreased by 58.4 mmHg (32.7%), which was significantly better than that of the RAT-1ΔT group (28.4 mmHg, 15.9%), and better than that of the clinical drug telmisartan (36.1 mmHg, 20.1%). The kidney / plasma concentration ratio of the high-dose RAT-1 group (8.62±0.71) was significantly higher than that of the RAT-1ΔT group (2.10±0.32) and the telmisartan group (0.30±0.04), indicating that kidney-targeted delivery can increase local drug concentration and directly enhance the antihypertensive effect.

[0053] Example 3: Plasma stability test of RAT-1

[0054] 1. Experimental objective: To verify the stability of RAT-1 in human plasma and to evaluate its in vivo metabolic characteristics.

[0055] 2. Materials

[0056] Samples: RAT-1, control peptide "YV" (sequence: Tyr-Val, used as a control peptide for easy degradation).

[0057] Human plasma: Anticoagulated blood from healthy volunteers, centrifuged at 3000×g for 10 minutes, supernatant collected, and stored at -80℃;

[0058] 3. Experimental Procedure

[0059] (1) Plasma incubation system: RAT-1 was diluted with physiological saline to 1 mg / mL and added to human plasma preheated at 37°C at a ratio of 1:9 (v / v) (final concentration 0.1 mg / mL). After gentle mixing, it was incubated in a water bath at 37°C.

[0060] (2) Sampling: Take 500 μL of incubation solution at 0, 2, 4, 8, 12 and 24 hours respectively, immediately add 1 mL of pre-cooled methanol (containing internal standard Leu-Enkephalin) to terminate the reaction, vortex for 30 seconds, centrifuge at 4℃ and 12000×g for 15 minutes, and take the supernatant and filter it through a 0.22 μm filter membrane;

[0061] (3) HPLC-MS detection

[0062] Column: ZORBAX Eclipse Plus C18 (2.1×150mm, 3.5μm);

[0063] Mobile phase: A (0.1% formic acid in water), B (0.1% formic acid in acetonitrile), gradient 0-15 min 10%-90% B, flow rate 0.3 mL / min;

[0064] Mass spectrometry conditions: Electrospray ionization (ESI+) source, scan range m / z 300-1500, RAT-1 characteristic ion m / z = 4567.3 ([M+2H]). 2+ The characteristic ion m / z of the control peptide "YV" is 297.1 ([M+H]). + );

[0065] (4) Data calculation: The remaining rate of intact peptides at different time points was calculated with the peak area at 0 hours as 100%. The first-order kinetic curve was fitted using GraphPad Prism 8 to calculate the half-life (t1 / 2).

[0066] 4. The experimental results are shown in Table 1.

[0067] RAT-1 exhibits good stability in human plasma, retaining 47.8% of its intact peptide after 24 hours, with a fitted half-life of t1 / 2 = 12.3 hours. In contrast, the control peptide "YV" is almost completely degraded after 24 hours (remaining at 0.8%), with a half-life of only 1.7 hours. These results indicate that the sequence design of RAT-1 (including rigid residues and target head protection) significantly enhances its resistance to plasma protease degradation.

[0068] Table 1 Summary of Experimental Results

[0069]

[0070] 5. Summary

[0071] RAT-1 has a long half-life in human plasma, providing a metabolic basis for its long-term effect in vivo, which is superior to easily degraded control peptides.

[0072] Example 4: Experiment on the blood pressure reduction effect of RAT-1 in the SHR model

[0073] 1. Experimental objective: To verify the in vivo antihypertensive effect of RAT-1 in spontaneously hypertensive rats (SHR) and compare it with the clinical drug valsartan.

[0074] 2. Materials and Animals

[0075] Animals: 24 SPF-grade male SHR (16 weeks old, weight 300±20g, systolic blood pressure SBP≥190mmHg) were randomly divided into 3 groups (n=8).

[0076] Reagents: RAT-1, valsartan, physiological saline;

[0077] Instruments: PA-C40 implantable arterial catheter, physiological signal recorder.

[0078] 3. Experimental Procedure

[0079] (1) Catheter implantation: After SHR anesthesia (1% sodium pentobarbital, 50 mg / kg ip), PA-C40 catheter was implanted into the abdominal aorta under aseptic conditions. The patient recovered for 72 hours after the operation, during which baseline blood pressure was monitored (recorded once per hour and the average value was taken).

[0080] (2) Group administration

[0081] RAT-1 group: 1 mg / kg RAT-1 (diluted with normal saline, volume 1 mL / kg) was injected via tail vein.

[0082] Valsartan group: 10 mg / kg valsartan (0.5% carboxymethyl cellulose sodium suspension) was administered by gavage.

[0083] Saline group: 1 mL / kg of normal saline was injected via the tail vein;

[0084] (3) Blood pressure monitoring: SBP was recorded continuously for 96 hours after drug administration, and the average value was taken every 6 hours. The maximum blood pressure reduction (ΔSBPmax) and the duration of blood pressure reduction were calculated (T>50% blood pressure reduction: the duration of the maximum blood pressure reduction ≥50%).

[0085] (4) Tissue distribution detection: 24 hours after administration, two rats in each group were sacrificed, and kidney tissue and plasma were collected. The drug concentration was detected by ELISA (coated with anti-RAT-1 antibody, prepared by the inventors) and the kidney / plasma concentration ratio was calculated.

[0086] 4. The experimental results are shown in Tables 2 and 3.

[0087] Blood pressure reduction: Six hours after RAT-1 administration, SBP dropped to 156±5 mmHg, with a maximum reduction of 56.3 mmHg, which was significantly greater than that of valsartan (36.2 mmHg, p<0.01).

[0088] Duration: The blood pressure-lowering effect of RAT-1 can be maintained for more than 72 hours (72 hours SBP is still 17.2% lower than baseline), and the blood pressure-lowering time at T>50% is 52.4 hours, which is 3.6 times that of valsartan (14.7 hours);

[0089] Tissue distribution: The kidney / plasma concentration ratio of RAT-1 was 8.7, which was significantly higher than that of valsartan (0.3), indicating that it had stronger renal targeting.

[0090] Table 2. Changes in systolic blood pressure (mmHg, x±s, n=8)

[0091]

[0092] *Note: *p<0.01 vs saline group; #p<0.05 vs valsartan group (ANOVA-Tukey method)

[0093] Table 3 Comparison of Key Indicators

[0094]

[0095] 5. Summary

[0096] RAT-1 demonstrated potent and long-lasting antihypertensive effects in the SHR model, and its renal targeting was significantly better than that of valsartan, laying the foundation for reducing systemic side effects.

[0097] Example 5: Experiment on the renal protective mechanism of RAT-1

[0098] 1. Experimental objective: To investigate the protective effect and mechanism of RAT-1 against Ang II-induced kidney injury.

[0099] 2. Materials and Models

[0100] Model: 24 male SD rats (12 weeks old) were used to establish a renal fibrosis model by continuous perfusion of Ang II (1000 ng / kg / min) via subcutaneous implantation of an osmotic pump (verified after 2 weeks: urinary protein >30 mg / 24 h, TGF-β1 increased).

[0101] Grouping: The model rats were randomly divided into 3 groups (n=8): RAT-1 treatment group, valsartan control group, and PBS control group; 8 normal rats were set up as blank control.

[0102] 3. Experimental Procedure (1) Dosing Regimen:

[0103] RAT-1 group: 1 mg / kg RAT-1 was injected via tail vein once every 3 days for 4 weeks;

[0104] Valsartan group: Valsartan 30 mg / kg / day by gavage for 4 weeks;

[0105] PBS control group: equal volume of PBS was injected via tail vein once every 3 days; (2) detection indicators

[0106] Electron microscopy: Renal cortical tissue was taken, fixed with glutaraldehyde, and then podocyte morphology was observed under transmission electron microscopy. Cell counts were performed per mm. 2 Number of podocytes lost in the glomerulus;

[0107] Western Blot: Detection of protein expression of TGF-β1 (fibrosis marker) and Col IV (collagen IV, a marker of basement membrane thickening) in renal tissue (primary antibodies: anti-TGF-β1; anti-Col IV);

[0108] Masson staining: After staining kidney tissue sections, Image-Pro Plus calculates the percentage of fibrosis area.

[0109] 4. The experimental results are shown in Table 4.

[0110] Four weeks after Ang II perfusion, rats in the PBS control group showed significant kidney damage: TGF-β1 and Col IV levels were significantly elevated, and podocytes were lost in large numbers (38.7 cells / mm²). 2 );

[0111] RAT-1 treatment group: TGF-β1 decreased to 8.2 ng / mg, Col IV percentage was 15.8%, and podocyte loss was 12.9 cells / mm². 2 All were significantly lower than those in the valsartan group (p<0.05), and close to the level of the blank control group;

[0112] Mechanism suggests that RAT-1 protects podocyte structure by targeting the kidneys to degrade Ang II and blocking AT1 receptors, thus doubly inhibiting the TGF-β1-mediated fibrosis pathway.

[0113] Table 4. Indicators of renal fibrosis (x±s, n=8)

[0114]

[0115] *Note: *p<0.05 vs PBS group; **p<0.01 vs PBS group; #p<0.05 vs valsartan group

[0116] 5. Summary

[0117] RAT-1 can significantly alleviate Ang II-induced renal fibrosis and protect podocyte function. Its renal protective effect is superior to that of valsartan, which is directly related to the enhanced local drug concentration through targeted delivery.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fusion polypeptide, wherein the fusion polypeptide is RAT-1, characterized in that, The topological structure of RAT-1 is as follows: kidney targeting head - ACE2 functional segment - MMP responsive linker - AT1 receptor antagonist segment, wherein the amino acid sequence of RAT-1 is shown in SEQ ID NO:

1.

2. The fusion polypeptide according to claim 2, characterized in that, The amino acid sequence of the kidney targeting head is CSSS.

3. The fusion polypeptide according to claim 2, characterized in that, The amino acid sequence of the ACE2 functional segment is GSYSYKVTLRSPHPLFCG.

4. The fusion polypeptide according to claim 2, characterized in that, The amino acid sequence of the MMP-responsive linker is GPLGLRG.

5. The fusion polypeptide according to claim 2, characterized in that, The amino acid sequence of the AT1 receptor antagonist segment is DVYARVCY.

6. A pharmaceutical composition, characterized in that, The composition comprises the fusion peptide RAT-1 of claim 1 and a pharmaceutically acceptable carrier or sustained-release material.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutically acceptable carriers include at least one of physiological saline, phosphate buffer, and water for injection.

8. The pharmaceutical composition according to claim 6, characterized in that, The sustained-release material includes at least one of polyethylene glycol, hyaluronic acid, and chitosan.

9. Use of the fusion polypeptide RAT-1 as described in claim 1 in the preparation of a drug for treating hypertension.

10. Use of the fusion polypeptide RAT-1 as described in claim 1 in the preparation of a nephroprotective drug.

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