Application of peptide SRD in the preparation of drugs for treating acute kidney injury

Abstract

This invention discloses the application of a peptide SRD in the preparation of drugs for treating acute kidney injury, belonging to the field of biomedical technology. In an in vitro H2O2-induced oxidative stress injury experiment on renal tubular epithelial cells, this invention found that peptide SRD treatment significantly reduced the apoptosis rate of renal tubular cells damaged by oxidative stress, promoted ATP production, and restored mitochondrial respiratory chain function. In an animal model of ischemia-reperfusion-induced acute kidney injury, mice with acute kidney injury injected via the tail vein showed significantly decreased serum creatinine and blood urea nitrogen levels. Pathological observation revealed a significant reduction in the degree of renal tubular injury, a lower histological score, and a significant downregulation of KIM-1, an indicator of the degree of renal tubular injury. This indicates that the antioxidant peptide SRD can improve mitochondrial function in renal tubular cells, effectively reduce renal tubular cell damage, promote the repair of renal tubular epithelial cells in acute kidney injury (AKI), effectively improve renal function, and has a good therapeutic effect on acute kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a polypeptide SRD in the preparation of drugs for treating acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) refers to a sudden decline in kidney function, resulting in the kidneys' inability to properly filter waste products and excess fluid from the blood. Sepsis, severe trauma, ischemia-reperfusion injury, and drug toxicity are the main causes of AKI. Its pathogenesis is complex, involving multiple cell signaling pathways and molecular mechanisms.

[0003] Currently, clinical treatment options for acute kidney injury (AKI) are relatively limited, mainly including symptomatic supportive care such as maintaining electrolyte balance, controlling blood pressure, and correcting acid-base imbalances, as well as renal replacement therapy (e.g., hemodialysis) when necessary. However, these treatments can only relieve symptoms and cannot fundamentally repair damaged kidney tissue, and they also carry certain complications and risks. Although experimental research and clinical exploration of AKI treatment strategies have been continuously expanding and deepening in recent years, and many drugs with potential renal function protection have emerged, there is still a lack of sufficient conclusive evidence to support their widespread application in terms of overall efficacy. Currently, in mitochondrial targeted therapy for AKI, regulating energy metabolism, activating mitophagy, and inhibiting reactive oxygen species (ROS) are the three core strategies. Studies have already confirmed that regulating carnitine palmitoyltransferase I (CPT1) can increase the level of fatty acid oxidation (FAO) regulated by peroxisome proliferator-activated receptor α (PGC1α) to improve renal IRI. Furthermore, key enzymes regulating the tricarboxylic acid cycle (TCA cycle) such as pyruvate dehydrogenase (PDH), citrate synthase (CS), and α-ketoglutarate dehydrogenase (AKGDH) and related complexes in oxidative phosphorylation (such as complex I and complex II) can also improve or restore mitochondrial energy metabolism in IR-AKI, thereby alleviating kidney damage. In targeted autophagy therapy, HIF-1α-BNIP3 and Pink1-parkin eliminate damaged mitochondria and reduce ROS levels by directly inducing or activating autophagy, thereby reducing mitochondrial damage and protecting kidney function. However, currently, no mitochondrial-targeting drugs are approved clinically for the treatment of acute kidney injury. Therefore, new mitochondrial-targeted therapies are urgently needed to improve the clinical outcomes of acute kidney injury. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide the application of a polypeptide SRD in the preparation of a drug for treating acute kidney injury. This polypeptide SRD, as a novel clinical candidate drug for preventing and treating renal ischemia-reperfusion injury, brings good news to kidney transplant patients.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] Application of peptide SRD in the preparation of drugs for treating acute kidney injury.

[0007] The sequence of the peptide SRD is KPPSLSYRCPCRFFESHGGSGGGRGDVY.

[0008] In some specific embodiments, the acute kidney injury is acute kidney injury caused by ischemia-reperfusion.

[0009] Description of the drug:

[0010] Preferably, the drug further includes a pharmaceutically acceptable carrier, such carrier including one of diluents, buffers, suspensions, emulsions, transdermal absorbents, humectants, absorption enhancers, and surfactants;

[0011] The dosage form of the drug of the present invention is a solution;

[0012] The administration routes of the drug of the present invention include intravenous injection, intramuscular injection and subcutaneous injection.

[0013] The drug of this invention can also be used in combination with other drugs for treating acute kidney injury, and the combined use of multiple drugs can greatly improve the success rate of treatment.

[0014] Compared with the prior art, the present invention has at least the following advantages:

[0015] 1) The application of the peptide SRD provided in this invention in the preparation of drugs for treating acute kidney injury. When used at a therapeutically effective dose, this peptide SRD can treat acute kidney injury caused by various etiologies, including ischemia-reperfusion. In vitro and in vivo experiments verified the protective effect of peptide SRD on acute kidney injury (AKI). In an in vitro H2O2-induced oxidative stress injury experiment on renal tubular epithelial cells, peptide SRD treatment significantly reduced the apoptosis rate of renal tubular cells damaged by oxidative stress, promoted ATP production, and restored mitochondrial respiratory chain function. In an animal model of ischemia-reperfusion-induced acute kidney injury, mice with acute kidney injury injected via tail vein showed significantly decreased serum creatinine and blood urea nitrogen levels. Pathological observation revealed a significant reduction in the degree of renal tubular injury, a decrease in histological scores, and a significant downregulation of KIM-1, an indicator of the degree of renal tubular injury. This indicates that the antioxidant peptide SRD can improve mitochondrial function in renal tubular cells, effectively reduce renal tubular cell damage, promote the repair of renal tubular epithelial cells in AKI, effectively improve renal function, and has a good therapeutic effect on acute kidney injury.

[0016] 2) The polypeptide SRD in this invention is a short peptide drug with high biological activity and good safety; it is easy to convert into a drug, its indications have strong expandability, and its application prospects are bright. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0018] Figure 1 The effect of peptide SRD on the viability of human proximal renal tubular epithelial cells (HK-2);

[0019] Figure 2 The effect of peptide SRD on ATP levels in human proximal renal tubular epithelial cells (HK-2) under oxidative stress;

[0020] Figure 3 The effect of peptide SRD on the apoptosis rate of human proximal renal tubular epithelial cells (HK-2) under oxidative stress; Figure (left) is a scatter plot of flow cytometry, and Figure (right) is a quantitative statistical bar chart corresponding to the scatter plot of flow cytometry.

[0021] Figure 4 The effects of peptide SRD on the expression levels of SIRT3, Cleaved-caspase3, and KIM1 proteins in human proximal renal tubular epithelial cells (HK-2) under oxidative stress; among which Figure 4 a is a Western blot image showing the expression levels of SIRT3, Cleaved-caspase3, and KIM1 proteins in human proximal renal tubular epithelial cells (HK-2) under oxidative stress, based on the peptide SRD. Figure 4 b, 4c and 4d are statistical bar charts showing the quantitative effect of peptide SRD on the expression levels of SIRT3, Cleaved-caspase3 and KIM1 proteins in human proximal renal tubular epithelial cells (HK-2) under oxidative stress.

[0022] Figure 5 HE staining and tissue damage scoring results of kidney tissue from IR-AKI mice; among which Figure 5 Image a shows HE staining of kidney tissue from IR-AKI mice; Figure 5 Figure b shows a semi-quantitative scoring of the percentage of damaged renal tubules. Figure 5 c shows the histological changes in the kidneys of IR-AKI mice;

[0023] Figure 6 The effects of peptide SRD on renal function in mice after renal ischemia-reperfusion injury are shown in the figure. The left figure shows the effect of SRD treatment on blood urea nitrogen (BUN) levels in mice; the right figure shows the effect of SRD treatment on serum creatinine (Scr) levels in mice.

[0024] Figure 7 The effect of peptide SRD on ATP levels in renal tissue after renal ischemia-reperfusion injury in mice;

[0025] Figure 8 The effect of peptide SRD on the expression of KIM1, TFAM, and SIRT3 proteins in renal tissue after renal ischemia-reperfusion injury in mice; among which Figure 8 Image a shows the Western blot of KIM1, TFAM, and SIRT3 in kidney tissue of mice after renal ischemia-reperfusion injury treated with peptide SRD. Figure 8 b, 8c and 8d are quantitative statistical bar charts of the expression levels of KIM1, TFAM and SIRT3 proteins in renal tissue of mice after renal ischemia-reperfusion injury treated with peptide SRD.

[0026] Figure 9 The effect of peptide SRD on malondialdehyde (MDA) levels in renal tissue of mice after renal ischemia-reperfusion injury. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the scope of protection of this invention. Any formal equivalent modifications made based on the concept of this invention should be considered within the scope of this invention.

[0028] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0029] The abbreviations used in this invention have the following meanings:

[0030] ATP, adenosine triphosphate, IRI, ischemia-reperfusion injury

[0031] Scr, serum creatinine, BUN, and blood urinary nitrogen

[0032] PCR, polymerase chain reaction, MDA, malondialdehyde

[0033] In the following examples, the peptide SRD solution used was prepared by the following method:

[0034] Weigh 10.0 mg of peptide SRD powder (the sequence of the peptide SRD is shown in SEQ ID NO.1, specifically KPPSLSYRCPCRFFESHGGSGGGRGDVY, and the peptide SRD was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) and add it to 3.163 mL of HK-2 cell culture medium (stock concentration 1250 µM). Shake well to mix thoroughly, then filter through a 0.22 µm microporous membrane to obtain a sterile peptide SRD solution. Finally, dilute with HK-2 cell culture medium to a final concentration of 100 µM for in vitro experiments.

[0035] 10.0 mg of peptide SRD powder was weighed and added to 2.64 mL of physiological saline (stock concentration 1500 µM), thoroughly shaken and mixed, and then filtered through a 0.22 µm microporous membrane to obtain a sterile peptide SRD solution. Finally, approximately 200 µL (blood drug concentration 100 µM) was injected into each mouse via the tail vein for in vivo experiments.

[0036] Example 1: Effect of different concentrations of peptide SRD on the viability of human proximal renal tubular epithelial cells (HK-2)

[0037] HK-2 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells / well were stimulated with different concentrations of peptide SRD (0, 12.5, 25, 50, 100 µM) for 24 h, and cell viability was detected using a CCK-8 assay kit after 24 h.

[0038] The results are as follows Figure 1As shown in the figure (*P < 0.05, compared with the NC group), the OD value of the 12.5µM peptide SRD group was significantly higher than that of the control group, indicating that the 12.5µM peptide SRD has the effect of increasing the viability of HK-2 cells, while the 25, 50, and 100µM peptide SRD did not affect the viability of HK-2 cells.

[0039] Example 2: Effect of peptide SRD on ATP levels in human proximal renal tubular epithelial cells (HK-2) under oxidative stress

[0040] HK-2 cells were seeded in 6-well plates at a density of 3 × 10⁶ cells / well. 5 The culture medium was 2 mL / well, divided into 4 groups: control group (NC group), H2O2 group, SRD group, and SRD treatment group (H2O2+SRD group). The H2O2 group was stimulated with 110 µM H2O2 for 24 h; the SRD group was treated with 100 µM peptide SRD for 24 h; and the SRD treatment group (H2O2+SRD group) was pretreated with 100 µM peptide SRD for 12 h and then stimulated with 110 µM H2O2 for 24 h. Each group had three replicates.

[0041] After washing each group of cells with PBS, add 200µL of lysis buffer to each well. After lysing for 10 min, pipette the cells into the culture dish until no more cells adhere to the wall. Collect the lysis buffer into an EP tube and centrifuge at 12000r / min for 10 min in a pre-cooled 4℃ high-speed centrifuge. Transfer the supernatant to a new EP tube.

[0042] Establish an ATP concentration gradient standard curve and prepare an appropriate amount of ATP detection working solution according to the kit instructions. Add 100 µL of ATP detection working solution to the detection wells and incubate at room temperature for 5 min. Then add 20 µL of sample or standard to the detection wells, mix quickly, and after at least 2 seconds, measure the RLU value using a chemiluminescence analyzer (Luminometer). Calculate the ATP content of the sample based on the ATP standard.

[0043] The results are as follows Figure 2 As shown (where **P < 0.01, compared with the control group; # (P < 0.05, compared with the H2O2 group): Compared with the control group, peptide SRD had no significant effect on the ATP level of HK-2 cells. Under oxidative stress (H2O2 group), the ATP level of HK-2 cells was significantly reduced. After treatment with peptide SRD (H2O2+SRD group), the ATP level of HK-2 cells significantly recovered. This indicates that peptide SRD can improve the mitochondrial respiratory chain function of HK-2 cells and alleviate oxidative stress damage under oxidative stress.

[0044] Example 3: Effect of peptide SRD on apoptosis rate of human proximal renal tubular epithelial cells (HK-2) under oxidative stress

[0045] HK-2 cells were seeded in 6-well plates at a density of 3 × 10⁶ cells / well. 5 Cells per well, culture medium 2 mL / well, divided into 4 groups, grouped in the same manner as in Example 2. Each group had three replicates. Cell apoptosis was detected using the Annexin V-APC / PI apoptosis detection kit, specifically as follows: Exfoliated cells were collected from the culture medium, digested with trypsin without EDTA, centrifuged at 1000 rpm for 5 min to collect cells, washed twice with pre-chilled PBS, and centrifuged at 1000 rpm for 5 min. Cells were resuspended in 400 µL of 1x Annexin V binding buffer per tube, resulting in a concentration of approximately 1×10⁻⁶. 6 Cells / mL. Add 5 µL of Annexin V-APC staining solution to the cell suspension, mix gently, and incubate at 4°C in the dark for 5 min. Add 6 µL of PI staining solution, mix gently, and incubate at 4°C in the dark for 5 min. Immediately analyze using flow cytometry or fluorescence microscopy.

[0046] The results are as follows Figure 3 As shown (where ****P < 0.0001, compared with the control group; #### P < 0.0001 (compared with the H2O2 group): Compared with the control group, peptide SRD had no significant effect on the apoptosis rate of HK-2 cells. Under oxidative stress (H2O2 group), the apoptosis rate of HK-2 cells increased significantly (approximately 8.48-fold). After treatment with peptide SRD (H2O2+SRD group), the apoptosis rate of HK-2 cells decreased significantly (approximately 2.92-fold). This indicates that peptide SRD can significantly inhibit HK-2 cell apoptosis and alleviate oxidative stress damage under oxidative stress.

[0047] Example 4: Effects of peptide SRD on the expression levels of SIRT3, Cleaved-caspase3, and KIM1 proteins in human proximal renal tubular epithelial cells (HK-2) under oxidative stress

[0048] HK-2 cells were seeded in 6-well plates at a density of 3 × 10⁶ cells / well. 5Cells per well, cultured in 2 mL / well, divided into 4 groups, as described in Example 2. Each group had three replicates. Cells were harvested, and total protein was extracted from each group. Protein concentration was measured using a BCA kit. 30 µg of total protein from each group was loaded onto a PVDF membrane and separated by SDS-PAGE. The membrane was then wet-transferred to a PVDF membrane and blocked with 5% skim milk-PBS for 1 h. The following antibodies were added: SIRT3, Cleaved-caspase3, KIM1 (1:1000 dilution), and internal control β-actin (1:5000 dilution). The membrane was incubated overnight at 4°C on a shaker. The next day, the membrane was washed, and secondary antibody (horseradish enzyme-conjugated goat anti-rabbit, 1:5000 dilution) was added and incubated at room temperature for 1 h. Horseradish peroxidase-labeled enhanced chemiluminescence (ECL reagent) was used for color development. The PVDF membrane was immersed in the color development solution, and images were acquired using a gel imaging system. ImageJ software was used to analyze the gray values ​​of the target protein and the internal control β-actin bands, and the ratio of the two was used to represent the relative expression level of the target protein.

[0049] The results are as follows Figure 4 As shown (where *P<0.05, ****P<0.0001 compared with the control group); ## (P < 0.01, compared with the H2O2 group): Compared with the control group, peptide SRD had no significant effect on the expression levels of the renal protective molecule SIRT3, the apoptosis-executing protein Cleaved-caspase3, and the renal injury molecule KIM1 in HK-2 cells. Under oxidative stress (H2O2 group), the expression level of SIRT3 in HK-2 cells was significantly decreased, while the expression levels of Cleaved-caspase3 and KIM1 were significantly increased. After treatment with peptide SRD (H2O2+SRD group), the expression level of SIRT3 in HK-2 cells significantly recovered, while the expression levels of Cleaved-caspase3 and KIM1 were significantly decreased. This indicates that peptide SRD can protect HK-2 cells under oxidative stress, resist oxidative stress damage, and inhibit HK-2 cell apoptosis.

[0050] Example 5: Study on the role of peptide SRD in the treatment of acute kidney injury due to ischemia-reperfusion

[0051] This embodiment evaluates the efficacy of peptide SRD in treating acute kidney injury due to ischemia-reperfusion injury. The specific testing method is as follows:

[0052] Six- to eight-week-old male C57BL / 6J mice, weighing approximately 20g, were selected. Mice were randomly divided into four groups of five mice each: sham operation group (Sham group), peptide treatment group (Sham+SRD group), ischemia-reperfusion acute kidney injury group (IRI group), and peptide SRD treatment group (IRI+SRD group). In the sham operation group (Sham group), the abdominal cavity was incised after anesthesia to expose the renal pedicle without clamping; the abdominal cavity was closed after 45 minutes. In the peptide SRD treatment group (Sham+SRD group), peptide SRD (stock concentration 1500µM) was prepared with physiological saline and injected via the tail vein at 12h and 24h after the sham operation (blood concentration 100µM, approximately 60mg / kg). In the ischemia-reperfusion acute kidney injury group (IRI group), mice were anesthetized, fixed in a supine position, and the midline of the abdomen was incised. Both renal pedicles were freed and clamped, and the mice were placed in an incubator; the clamps were released after 45 minutes. When both kidneys changed from dark red to bright red, it indicated successful renal reperfusion. The muscle layers were then sutured (continuous sutures, without gaps), followed by interrupted sutures on the skin. Immediately after the procedure, each mouse was injected intraperitoneally with 0.5 mL of warm, sterile saline. The peptide SRD treatment group (IRI+SRD group) received peptide SRD (1500 µM stock concentration) prepared with saline and administered via tail vein injection at 12 h and 24 h post-surgery (100 µM blood concentration). Two days later, blood was collected from the heart to measure serum creatinine and blood urea nitrogen levels. Kidney tissue was harvested, paraffin-embedded, and sectioned (for subsequent HE staining). Proteins were extracted from the kidney tissue to measure KIM1, TFAM, and SIRT3 protein levels. Results were as follows:

[0053] 1) Histological changes in the kidneys after treatment with peptide SRD for ischemia-reperfusion acute kidney injury

[0054] Processing of paraffin-embedded tissue specimens: During the experiment, after removing the kidneys from the experimental mice, they were cut in half along their long axis. One half of the kidney tissue was placed in a 4% paraformaldehyde solution and fixed at 4°C for 24 hours. The next day, the fixed kidney tissue was dehydrated using the following steps: the tissue was sequentially immersed in ethanol solutions of different concentrations, namely, 50% ethanol for 90 min, 70% ethanol for 90 min, 85% ethanol for 90 min, 95% ethanol for 90 min, 100% ethanol for 60 min, and then immersed in another bottle of 100% ethanol for 60 min. Subsequently, the tissue was cleared by immersing it sequentially in a mixture of ethanol and xylene (volume ratio 1:1) for 60 min, xylene for 60 min, and another bottle of xylene for 60 min. Finally, the tissue was immersed in a suspension of xylene and paraffin (volume ratio 1:1) at 62°C for 90 min, and then sequentially immersed in two bottles of paraffin solution at 62°C for 120 min each, completing the pre-embedding treatment of the tissue.

[0055] The processed tissue was placed in a paraffin-containing mold, and its position was adjusted to ensure proper placement. It was then placed on the cooling platform of a paraffin embedding machine for cooling. After cooling, the tissue was cut into 4µm thick slices, which were then placed in a slide bleaching machine to fully unfold. The unfolded slices were then gently lifted and placed on glass slides pre-coated with poly-L-lysine. The slides were numbered using a marker for subsequent identification and handling. The slides were then placed in a 70°C oven for drying for 1 hour to ensure close adhesion between the tissue slices and the slides.

[0056] After completing the above steps, remove the slides from the oven for use in subsequent experiments; perform HE staining on paraffin sections of mouse kidney tissue: place the slides in a 60℃ oven for 20 minutes. Immediately after removing the slides from the oven, immerse them in xylene I for 15 minutes → xylene II for 15 minutes → anhydrous ethanol for 5 minutes → 95% ethanol for 5 minutes → 75% ethanol for 5 minutes → distilled water for 5 minutes;

[0057] Staining: Hematoxylin staining for 7 min → rinse with tap water for 5 min → rinse with distilled water → differentiate with hydrochloric acid ethanol for 3 s → rinse with tap water for 5 min → rinse with distilled water → eosin staining for 5 min → rinse with tap water for 5 min (depending on the intensity of eosin staining, decide whether to place in anhydrous ethanol) → dry the slide with a hairdryer → xylene I for 3 min → xylene II for 5 min. Mount the slide with neutral resin and cover with a coverslip. Observe the histological changes in the kidney tissue on the second day after AKI treatment with peptide SRD under a microscope (HE×400 magnification, scale bar 50µm).

[0058] The results are as follows Figure 5 As shown (where ****P < 0.0001 compared with the sham surgery group); ####(P < 0.0001, compared with the IRI group): SRD treatment had no significant effect on mouse renal tissue pathology. Following renal ischemia-reperfusion injury, mice showed significant shedding of renal tubular epithelial cells, with detached cell fragments and casts visible within the lumen. Necrosis of renal tubular epithelial cells was observed, along with significant tubular dilation and a small amount of inflammatory cell infiltration in the tubulointerstitium. In contrast, peptide SRD treatment significantly reduced renal tissue pathological damage, decreasing renal tubular epithelial cell shedding and necrosis, and significantly reducing inflammatory cell infiltration. The histological changes in the kidneys closely resembled those of normal renal tissue. A semi-quantitative scoring was performed based on the percentage of damaged tubules: 0 points indicated no damage; 1 point indicated damage < 25%; 2 points indicated damage between 25% and 50%; 3 points indicated damage between 50% and 75%; and 4 points indicated damage > 75%. The results showed that kidney damage worsened in the IRI group 2 days after AKI, and the damage score was significantly higher than that in the sham surgery group. However, the renal tubular damage score was significantly reduced after SRD treatment, suggesting that SRD treatment can effectively reduce the degree of kidney damage in AKI.

[0059] 2) Changes in renal function after treatment with peptide SRD for ischemia-reperfusion acute kidney injury

[0060] Renal function tests: Blood samples were collected 2 days after the operation. The samples were centrifuged at 4500 r / min for 2 min, and the serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured using a serum creatinine and blood urea nitrogen assay kit.

[0061] The results are as follows Figure 6 As shown (where *P<0.05, ****P<0.0001 compared with the sham surgery group); # P < 0.05 #### (P < 0.0001 compared with the IRI group): SRD treatment had no significant effect on renal function in mice, but serum creatinine and blood urea nitrogen levels were significantly elevated after renal ischemia-reperfusion injury. However, after peptide SRD treatment, serum creatinine and blood urea nitrogen levels in mice almost returned to normal. The results suggest that peptide SRD treatment can restore renal function in mice, thereby alleviating acute kidney injury caused by ischemia-reperfusion injury.

[0062] 3) Changes in renal tissue ATP levels after treatment with peptide SRD for ischemia-reperfusion acute kidney injury

[0063] Two days after AKI, kidney tissue was harvested and ATP levels were measured according to the instructions of the enhanced ATP assay kit. The specific procedure was as follows: 200 µL of lysis buffer was added to every 20 mg of fresh kidney tissue, and the tissue was homogenized using a glass homogenizer. The sample was boiled for 2 min. After lysis, the sample was centrifuged at 12000 rpm for 5 min at 4°C, and the supernatant was collected for subsequent measurements. A standard curve was determined according to the kit instructions. An appropriate amount of ATP assay working solution was prepared, requiring 100 µL of ATP assay working solution per sample or standard. The reagent was thawed on ice. An appropriate amount of ATP assay reagent was taken and diluted with ATP assay reagent diluent at a ratio of 1:4. 100 µL of ATP assay working solution was added to the test wells and incubated at room temperature for 5 min. 20 µL of standard was added to the test wells and quickly mixed with a pipette. After at least 2 seconds, the RLU value was measured using a chemiluminescence analyzer. The concentration of ATP in the sample was calculated based on the standard curve; the protein concentration was measured using the BCA method, and the ratio was used to reflect the ATP content per unit protein.

[0064] The results are as follows Figure 7 As shown (where ****P < 0.0001, compared with the sham surgery group); ### (P < 0.001, compared with the IRI group): SRD treatment did not significantly change the ATP level in mouse kidney tissue. However, ATP levels in mouse kidney tissue were significantly downregulated after acute kidney injury due to renal ischemia-reperfusion injury. Treatment with peptide SRD significantly restored ATP levels in mouse kidney tissue. These results suggest that peptide SRD treatment can significantly improve mitochondrial respiratory chain function in renal tubular cells and enhance cellular oxidative phosphorylation, thereby alleviating acute kidney injury due to ischemia-reperfusion injury in mice.

[0065] 4) Changes in renal injury markers KIM, SIRT3, and TFAM proteins after treatment with peptide SRD for ischemia-reperfusion acute kidney injury.

[0066] Two days after AKI, kidney tissue was harvested for protein extraction, and protein concentration was detected using a BCA kit. 15 µg of total protein from each group was loaded onto a PVDF membrane and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membrane was then wet-transferred to a PVDF membrane and blocked with 5% skim milk-PBS for 1 h. The following antibodies were added: SIRT3, TFAM, KIM1 (1:1000 dilution), and internal control β-actin (1:5000 dilution), and incubated overnight at 4°C on a shaker. The next day, the membrane was washed, and a secondary antibody (horseradish peroxidase-labeled goat anti-rabbit, 1:5000 dilution) was added and incubated at room temperature for 1 h. Horseradish peroxidase-labeled enhanced chemiluminescence (ECL reagent) was used for color development. The PVDF membrane was immersed in the developing solution, and images were acquired using a gel imaging system. ImageJ software was used to analyze the grayscale values ​​of the target protein and the internal control β-actin bands, and the ratio of the two bands represented the relative expression level of the target protein.

[0067] The results are as follows Figure 8 As shown (where *P<0.05, ***P<0.001 compared with the sham surgery group); # P < 0.05 ## (P < 0.01, compared with the IRI group): SRD treatment did not significantly change the expression levels of KIM1, TFAM, and SIRT3 proteins in mouse kidney tissue. After renal ischemia-reperfusion acute kidney injury in mice, KIM1 protein expression was significantly upregulated, while TFAM and SIRT3 protein expression were significantly downregulated. After peptide SRD treatment, KIM1 protein expression was significantly downregulated, while TFAM and SIRT3 protein expression were significantly upregulated. These results suggest that peptide SRD treatment can significantly alleviate ischemia-reperfusion acute kidney injury in mice, improve mitochondrial biosynthesis in renal tubular cells, thereby improving the quality control system of renal tubular cells and reducing kidney injury.

[0068] 5) Changes in MDA (malondialdehyde) levels in renal tissue after treatment with peptide SRD for ischemia-reperfusion acute kidney injury.

[0069] Two days after AKI, kidney tissue was collected to detect the MDA level in the kidney tissue according to the instructions of the MDA detection kit. The specific procedure was as follows: 20 mg of kidney tissue was taken from each sample and added to about 200 µL of PBS. Then, the tissue was homogenized using a glass homogenizer. After homogenization, the tissue was centrifuged at 12,000 r / min for 10 min and the supernatant was collected for subsequent measurement.

[0070] Protein concentration was determined using a BCA protein concentration assay kit. TBA stock solution and MDA detection working solution were prepared according to the instructions, and a standard curve was constructed. 0.1 ml of PBS was added to a centrifuge tube as a blank control, 0.1 ml of the above-mentioned standards at different concentrations were added to construct the standard curve, and 0.1 ml of sample was added for measurement. Then, 0.2 ml of MDA detection working solution was added; after mixing, the mixture was heated at 100°C for 15 min; after cooling to room temperature in a water bath, it was centrifuged at 1000 rpm for 10 min at room temperature; 200 µL of the supernatant was added to a 96-well plate, and the absorbance was measured at 532 nm using a microplate reader. MDA content calculation: After calculating the MDA content in the sample solution, the initial MDA content in the sample was expressed as the protein content per unit weight, i.e., MDA content / protein concentration.

[0071] The results are as follows Figure 9 As shown (where ***P < 0.001 compared with the sham surgery group); ## (P < 0.01, compared with the IRI group): SRD treatment did not significantly change the MDA level in mouse renal tissue. MDA levels in mouse renal tissue were significantly increased after renal ischemia-reperfusion acute kidney injury. Treatment with peptide SRD significantly downregulated MDA levels in mouse renal tissue. These results suggest that peptide SRD treatment can significantly reduce oxidative stress damage to renal tubular cells and decrease lipid peroxidation levels, thereby alleviating ischemia-reperfusion acute kidney injury in mice.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. The application of polypeptide SRD in the preparation of drugs for treating acute kidney injury, characterized in that, The sequence of the polypeptide SRD is KPPSLSYRCPCRFFESHGGSGGGRGDVY, wherein the acute kidney injury is acute kidney injury caused by ischemia-reperfusion.

2. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

3. The application according to claim 2, characterized in that, The drug is a solution.

4. The application according to claim 3, characterized in that, The drug is administered via intravenous injection.

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