Polypeptide tcp-6 and its use in the preparation of a medicament for treating acute kidney injury

Abstract

The application belongs to the technical field of medicines, and discloses a polypeptide TCP-6 and application of the polypeptide TCP-6 in preparation of a medicine for treating acute kidney injury. The amino acid sequence of the polypeptide TCP-6 is shown as SEQ ID NO:1. Experiments show that the polypeptide TCP-6 can effectively relieve tubular epithelial cell injury and apoptosis occurring when AKI, and protect kidney function, and no obvious toxic side effects are found, so that the polypeptide TCP-6 can be applied as a medicine for treating acute kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the polypeptide TCP-6 and its application in the preparation of drugs for treating acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome caused by a rapid decline in kidney function due to various etiologies such as ischemia, drugs, or sepsis. It is one of the most common acute and critical illnesses in clinical practice, with a high mortality rate. Due to the lack of effective prevention and treatment methods, AKI often leads to incomplete and maladaptive tissue repair, eventually developing into chronic kidney disease (CKD), imposing a heavy burden on patients and society. Furthermore, AKI can also be secondary to CKD and is closely related to its poor prognosis. Therefore, the prevention and treatment of AKI has become a major global health problem, and the development of drugs for the prevention and treatment of AKI is urgently needed.

[0003] Renal tubular epithelial cells are known to be the primary cells damaged in acute kidney injury (AKI). Under the influence of ischemia, toxins, and other factors, these cells undergo apoptosis, necrosis, and shedding, promoting cast formation and obstructing the tubular lumen. Simultaneously, the damaged tubular epithelium exacerbates the inflammatory response in the kidneys by releasing inflammatory and chemokine-producing factors, further worsening kidney damage. Therefore, protecting renal tubular epithelial cells and promoting their repair is crucial for the treatment of AKI.

[0004] Recent research has found that the extracellular matrix glycoprotein Tenasin C (TNC) is expressed at elevated levels in acute kidney injury. By activating the Wnt / β-catenin signaling pathway, it inhibits renal tubular epithelial cell damage and apoptosis, alleviates renal function impairment, and plays a protective role in the kidneys (Chen et al., Kidney Int. 95:62-74, 2019). Therefore, it has a certain therapeutic effect on acute kidney injury.

[0005] However, tendinin C is a large extracellular matrix protein composed of over 2000 amino acids. Due to its large molecular weight, it is not only difficult to synthesize, leading to high production costs, but also poses risks of allergies and poor absorption and utilization. Therefore, this invention aims to provide a polypeptide drug with a relatively small molecular weight that is effective in treating acute kidney injury. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a polypeptide TCP-6 and its application in the preparation of a drug for treating acute kidney injury. Experiments show that the polypeptide TCP-6 can effectively alleviate renal tubular epithelial cell damage and apoptosis occurring in AKI, protect renal function, and has no obvious toxic side effects; therefore, it can be used as a drug for treating acute kidney injury.

[0007] The present invention provides a polypeptide TCP-6 or a pharmaceutically acceptable salt thereof, the amino acid sequence of said polypeptide TCP-6 being shown in SEQ ID NO: 1.

[0008] ETFTTGLDAPRNLRRVSQTDNSITLEWRNG (SEQ ID NO: 1).

[0009] This invention involves cleaving tendinin C and studying the resulting multiple polypeptide fragments. After screening, it was found that a specific polypeptide TCP-6, composed of 30 amino acids, can effectively alleviate renal tubular epithelial cell damage and apoptosis during AKI, protect renal function, and has no obvious toxic side effects. Therefore, it can be used to prepare drugs for the treatment of acute kidney injury.

[0010] Preferably, the pharmaceutically acceptable salts of the polypeptide TCP-6 include acetate, citrate, hydrochloride, sulfate, phosphate, sulfonate, tartrate, malate, succinate, or fumaric acid.

[0011] The present invention also provides an isolated nucleic acid molecule that encodes the aforementioned polypeptide TCP-6.

[0012] The present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecules.

[0013] The present invention also provides a transformant comprising the above-described recombinant vector.

[0014] The present invention also provides the use of the above-mentioned polypeptide TCP-6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating acute kidney injury.

[0015] Preferably, the acute kidney injury is renal tubular epithelial cell damage and apoptosis caused by ischemia-reperfusion or cisplatin.

[0016] The present invention also provides a medicament for treating acute kidney injury, comprising the above-mentioned polypeptide TCP-6 or a pharmaceutically acceptable salt thereof.

[0017] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0018] More preferably, the pharmaceutically acceptable excipient is at least one of solvents, wetting agents, emulsifiers, thickeners, excipients, suspending agents, disintegrants, fillers, lubricants, or diluents.

[0019] Preferably, the dosage form of the drug is tablets, injections, sprays, lyophilized powder for injection, capsules, or coated pills.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] Related experiments showed that the polypeptide TCP-6 proposed in this invention had no obvious toxic side effects in mouse animal experiments. Furthermore, the results of experiments using TCP-6 in two acute kidney injury models—cisplatin (CIS) and ischemia / reperfusion injury (IRI)—showed that, compared with the CIS or IRI model groups, the treatment group, which received TCP-6 via tail vein injection after modeling, exhibited significantly lower levels of renal function indicators such as serum creatinine and blood urea nitrogen. The expression levels of renal tubular epithelial cell injury markers N-GAL and Kim-1, as well as apoptosis-related proteins Fas-L, FADD, p53, and Cleaved Caspase-3, were also significantly reduced. Simultaneously, PAS pathological staining and Caspase-3 immunohistochemical staining results also indicated that renal tubular injury and apoptosis were significantly alleviated after TCP-6 intervention compared to the model group.

[0022] Therefore, TCP-6 significantly inhibits renal tubular epithelial cell damage and apoptosis without obvious toxic side effects, making it suitable for preparing drugs to effectively treat acute kidney injury. Furthermore, as a small-molecule polypeptide, TCP-6 exhibits better drug-like properties and lower production costs compared to the large biomolecule tendinin C, thus possessing broader application prospects. Attached Figure Description

[0023] Figure 1 This study demonstrates the protective effect of different concentrations of TCP-6 against cisplatin-induced apoptosis in human renal tubular epithelial cells in Example 2.

[0024] Figure 2 This is the result of TCP-6 inhibiting cisplatin-induced apoptosis in human renal tubular epithelial cells in Example 2.

[0025] Figure 3 The values ​​represent the serum creatinine levels of each experimental group in Example 3.

[0026] Figure 4 The values ​​represent the blood urea nitrogen levels for each experimental group in Example 3.

[0027] Figure 5 Example 3 describes the detection of the degree to which TCP-6 alleviates renal tubular damage and apoptosis at the protein level.

[0028] Figure 6 The PAS pathological staining and Caspase-3 immunohistochemical staining in Example 3 confirmed the alleviating effect of TCP-6 on renal tubular injury and apoptosis.

[0029] Figure 7 The values ​​represent the serum creatinine levels of each experimental group in Example 4.

[0030] Figure 8 The values ​​represent the serum urea nitrogen levels for each experimental group in Example 4.

[0031] Figure 9 Example 4 describes the extent to which TCP-6 alleviates renal tubular damage and apoptosis at the protein level.

[0032] Figure 10 The PAS pathological staining and Caspase-3 immunohistochemical staining in Example 4 confirmed the alleviating effect of TCP-6 on renal tubular injury and apoptosis. Detailed Implementation

[0033] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments are merely preferred embodiments of this invention and do not constitute a limitation on the scope of protection claimed by this invention. Any modifications, substitutions, or combinations made without departing from the spirit and principle of this invention are included within the scope of protection of this invention.

[0034] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0035] Example 1: Amino acid sequence of polypeptide TCP-6

[0036] This embodiment provides a polypeptide (named TCP-6) with the following amino acid sequence: ETFTTGLDAPRNLRRVSQTDNSITLEWRNG (SEQ ID NO: 1).

[0037] The aforementioned polypeptide TCP-6 has a relatively small number of amino acids (30), so it can be chemically synthesized using conventional solid-phase synthesis methods, or biosynthesized by constructing recombinant expression vectors and recombinant strains.

[0038] Example 2: In vitro experiments showed that peptide TCP-6 inhibited cisplatin-induced damage and apoptosis in human renal tubular epithelial cells.

[0039] 1. Experimental materials:

[0040] Cells: Human renal tubular epithelial cells (HK2).

[0041] Culture medium: DMEM / F12 (1:1) culture medium containing 10% FBS.

[0042] Culture conditions: 37℃ incubator with 5% CO2.

[0043] 2. Experimental treatment and results:

[0044] (I) Cultured human renal tubular epithelial cells were seeded at a rate of approximately 10,000 cells / well in 96-well cell culture plates. After 1 day of culture, the cells were further cultured in serum-free DMEM / F12 (1:1) medium for 12 hours. After changing the medium with serum-free DMEM / F12 (1:1) medium, cisplatin (25 μg / mL) was added to stimulate the cells, and different concentrations (0, 25, 50, 100, 200, 400, 800 ng / mL) of the peptide TCP-6 were added for co-incubation for 12 hours. Cell viability was assessed using the MTT assay after incubation.

[0045] Experimental results are as follows Figure 1 As shown, TCP-6 alleviates cisplatin-induced apoptosis in human renal tubular epithelial cells, and the alleviating effect is concentration-dependent.

[0046] (II) The cultured human renal tubular epithelial cells were inoculated at 1.5 × 10⁻⁶. 6 Cells were seeded in 6-well cell culture plates and cultured for 1 day, then cultured in serum-free DMEM / F12 (1:1) medium for 12 hours. After changing the medium with serum-free DMEM / F12 (1:1) medium, cisplatin (25 μg / mL) was added to stimulate the cells, and the peptide TCP-6 (100 ng / mL) was added and incubated for 12 hours. After incubation, cell proteins were collected for Western blotting. The group treated with cisplatin stimulation with recombinant tendinin C protein (100 ng / mL) served as a positive control.

[0047] Experimental results are as follows Figure 2 As shown, after cisplatin stimulation of human renal tubular epithelial cells, the protein levels of renal tubular injury markers N-gal, apoptosis-related proteins Cleaved Caspase-7 and FADD were significantly increased. However, after TCP-6 intervention, the levels of each protein were significantly decreased, indicating that TCP-6 can inhibit cisplatin-induced apoptosis of human renal tubular epithelial cells.

[0048] Example 3: Inhibitory effect of peptide TCP-6 on acute kidney injury in a mouse cisplatin model

[0049] 1. Laboratory animals:

[0050] C57 mice, male, 8 weeks old, weighing 20-22g, SPF grade.

[0051] First, the animals were weighed and numbered by earrings. Twenty-four healthy mice weighing 20-22g were selected and randomly divided into four groups of six each: sham surgery group, cisplatin model group, high-dose drug intervention group, and low-dose drug intervention group.

[0052] 2. Experimental Groups:

[0053] (I) Sham-operated group: Mice were injected intraperitoneally with physiological saline at a dose of 20 μL / g. The numbers were recorded and verified, and the mice were placed in the corresponding cages.

[0054] (II) Cisplatin model group: mice were injected intraperitoneally with physiological saline containing cisplatin (1 mg / mL) at a dose of 20 μL / g. The number of the injections was recorded and verified, and the mice were placed in the corresponding cages.

[0055] (III) Low-dose drug intervention group: In addition to the cisplatin model group, TCP-6 saline solution was injected intraperitoneally. The injection dose of TCP-6 was 150 μg / kg body weight.

[0056] (IV) High-dose drug intervention group: In addition to the cisplatin model group, TCP-6 saline solution was injected intraperitoneally. The injection dose of TCP-6 was 300 μg / kg body weight.

[0057] 3. Experimental procedure:

[0058] TCP-6 water-soluble powder was diluted with sterile saline to prepare injection solutions of 15 μg / mL (low dose) and 30 μg / mL (high dose). All groups were housed in cages. The sham-operated group was observed only. The cisplatin model group received only a tail vein injection of sterile saline containing cisplatin. At 24 and 48 hours post-surgery, mice in the low-dose and high-dose intervention groups were injected with the corresponding concentrations and doses of TCP-6 saline solution, respectively. 72 hours after cisplatin injection, all mice were sacrificed, blood samples were collected, and the left kidney was harvested. The tissues were fixed in 10% neutral buffered formaldehyde and frozen in liquid nitrogen, respectively. After dehydration, embedding, sectioning, and slide preparation, the formaldehyde-fixed tissues were subjected to PAS pathological staining and Caspase-3 immunohistochemical staining, respectively. Proteins were extracted from the frozen tissue homogenate, and the expression levels of renal tubular injury marker N-gal and apoptosis-related proteins Fas-L, p53, and Cleaved Caspase-3 were detected by Western blotting.

[0059] like Figure 3-4 As shown, the serum creatinine and blood urea nitrogen levels in the cisplatin model group were significantly higher than those in the sham surgery group. After TCP-6 intervention, the serum creatinine and blood urea nitrogen levels in both the high-dose and low-dose drug intervention groups were significantly lower.

[0060] like Figure 5 As shown, compared with the cisplatin model group, the protein levels of tubule damage markers N-gal, apoptosis-related proteins Fas-L, p53, and cleaved caspase-3 were significantly reduced after high- and low-dose TCP-6 intervention. Figure 6 Immunohistochemical staining also confirmed the alleviating effect of TCP-6 on renal tubular damage and apoptosis.

[0061] Example 4: Inhibitory effect of peptide TCP-6 on acute kidney injury in a mouse ischemia / reperfusion model

[0062] 1. Laboratory animals:

[0063] C57 mice, male, 8 weeks old, weighing 20-22g, SPF grade.

[0064] First, the animals were weighed and numbered by earrings. Eighteen healthy mice weighing 20-22g were selected and randomly divided into three groups of six each: sham surgery group, ischemia / reperfusion model group, and drug intervention group.

[0065] 2. Experimental Groups:

[0066] (I) Sham-operated group: Mice were anesthetized at room temperature with 1% sodium pentobarbital at a dose of 10 μL / g. After anesthesia, mice were fixed on a surgical board, and the abdomen was disinfected. The abdominal cavity was opened layer by layer along the midline of the abdomen, from the lower end of the sternum to 0.5 cm above the pubic symphysis. After finding the bilateral renal pedicles, the layers were sutured. After local disinfection, the mice were verified, marked, and placed in the appropriate cages.

[0067] (II) Ischemia / Reperfusion Model Group: Anesthesia, fixation, disinfection, and laparotomy were performed as in the sham surgery group. After laparotomy, the bilateral renal pedicles were located and slightly freed. Both renal pedicles were clamped using atraumatic miniature arterial clamps (the right side was clamped first, followed by the left). Successful clamping was indicated by the kidneys changing from bright red to dark purple. The mice were placed on a 37°C metal heating plate. After 30 minutes, the arterial clamps were released, and the kidneys changed from dark purple to pink, indicating successful reperfusion. The mice were sutured layer by layer. After local disinfection, the markings were verified and recorded, and the mice were placed in the appropriate cages. Physiological saline solution was then injected intravenously.

[0068] (III) Drug intervention group: Same as the ischemia / reperfusion model group, but with intravenous injection of TCP-6 saline solution.

[0069] 3. Experimental procedure:

[0070] The water-soluble TCP-6 powder was diluted with sterile saline to prepare an injection solution of 15 μg / ml. Each group was housed in cages. The sham-operated group was observed only. The ischemia / reperfusion model group received only the same dose of sterile saline injected via the tail vein as the drug intervention group. The drug intervention group received a 10 μL / g dose of the prepared TCP-6 saline solution at 24 and 36 hours post-operation. Mice in each group were sacrificed 48 hours post-operation, blood samples were collected, and the left kidney was harvested. The kidneys were fixed in 10% neutral buffered formaldehyde and frozen in liquid nitrogen, respectively. After dehydration, embedding, sectioning, and slide preparation, the formaldehyde-fixed tissues were subjected to PAS pathological staining and Caspase-3 immunohistochemical staining, respectively. Proteins were extracted from the frozen tissue homogenate, and the expression levels of renal tubular injury markers N-gal and Kim-1, and apoptosis-related proteins FADD, p53, and Cleaved Caspase-3 were detected by Western blotting.

[0071] 4. Experimental Results:

[0072] like Figure 7-8 As shown, the serum creatinine and blood urea nitrogen levels in the ischemia / reperfusion model group were significantly higher than those in the sham surgery group, but after TCP-6 intervention, the serum creatinine and blood urea nitrogen levels decreased significantly.

[0073] like Figure 9 As shown, compared with the ischemia / reperfusion model group, the protein levels of renal tubular injury markers N-gal, Kim-1, apoptosis-related proteins FADD, p53, and Cleaved Caspase-3 were significantly reduced after TCP-6 intervention.

[0074] like Figure 10 As shown, the results of PAS pathological staining and Caspase-3 immunohistochemical staining indicate that, compared with the ischemia / reperfusion model group, the damage and apoptosis of renal tubules were significantly alleviated after TCP-6 intervention.

[0075] The above examples demonstrate that the peptide TCP-6 can significantly alleviate the elevation of serum renal function indicators induced by cisplatin and ischemia / reperfusion injury, and significantly reduce renal tubular damage and apoptosis levels. Therefore, TCP-6 could be a novel drug for effectively inhibiting acute kidney injury.

[0076] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A polypeptide TCP-6 or a pharmaceutically acceptable salt thereof, characterized in that, The amino acid sequence of the polypeptide TCP-6 is shown in SEQ ID NO:

1.

2. The polypeptide TCP-6 or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Pharmaceutically acceptable salts of the polypeptide TCP-6 include acetate, citrate, hydrochloride, sulfate, phosphate, sulfonate, tartrate, malate, or succinate.

3. An isolated nucleic acid molecule, comprising, The nucleic acid molecule encodes the polypeptide TCP-6 of claim 1.

4. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 3.

5. A transformant characterized in that, The transformant comprises the recombinant vector of claim 4.

6. The use of the polypeptide TCP-6 of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating acute kidney injury.

7. Use according to claim 6, characterized in that, The acute kidney injury referred to here is renal tubular epithelial cell damage and apoptosis caused by ischemia-reperfusion or cisplatin.

8. A medicament for treating acute kidney injury, characterized by, Includes the polypeptide TCP-6 of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

9. The medicament according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The medicament according to claim 9, characterized in that, The pharmaceutically acceptable excipient is at least one of the following: solvent, wetting agent, emulsifier, thickener, suspending agent, disintegrant, filler, lubricant, or diluent.

11. The medicament according to claim 8, characterized in that, The dosage form of the drug is tablet, injection, spray, lyophilized powder for injection, capsule or coated pill.

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