Use of transmembrane glycoprotein nmb ectodomain in treating and ameliorating acute kidney injury

Abstract

This invention discloses the application of the extracellular domain of the transmembrane glycoprotein NMB in the treatment and improvement of acute kidney injury, belonging to the field of medical technology, to study the therapeutic effect and mechanism of the extracellular domain of the transmembrane glycoprotein NMB on acute kidney injury. This invention utilizes the extracellular domain of the transmembrane glycoprotein NMB to treat a disease model of acute kidney injury, finding that the extracellular domain of the transmembrane glycoprotein NMB can effectively alleviate ischemia-reperfusion-induced acute kidney injury, promote kidney tissue repair, and inhibit the progression of acute kidney injury to chronic inflammation and fibrosis.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to the application of the extracellular domain of a transmembrane glycoprotein NMB in the treatment and improvement of acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by rapid deterioration of kidney function. Epidemiological studies show that the incidence of AKI is increasing annually, with an annual growth rate as high as 10%, affecting approximately 20% of hospitalized patients and 50% of intensive care patients. Furthermore, AKI is closely associated with a significantly increased mortality rate. More worryingly, AKI not only exhibits an acute course but also carries the risk of chronic transformation. Existing clinical studies have clearly demonstrated that the transformation from AKI to chronic kidney disease (CKD) (AKI-CKD transition) is directly related to patient prognosis. In a cohort study, AKI patients had an approximately 8-fold increased risk of developing end-stage renal disease (ESRD) compared to a control group without a history of kidney disease. Crucially, research data indicates that patients with ESRD caused by AKI have a significantly higher risk of death within 6 months of onset than those with ESRD caused by other factors. This disease progression pattern not only severely impacts patients' quality of life but also places a heavy burden on the healthcare system.

[0003] Acute kidney injury (AKI) has diverse causes, with ischemia-reperfusion injury being a major one. It typically occurs after a temporary interruption and subsequent restoration of renal blood flow. This condition can be caused by various factors, including major surgeries such as cardiac or renal surgery, traumatic conditions such as massive hemorrhage or severe injury, and shock states such as sepsis or cardiogenic shock. During the ischemic phase, kidney cells cannot obtain sufficient oxygen and nutrients, leading to metabolic disorders, apoptosis, and necrosis. After reperfusion, the restored blood flow brings oxygen and nutrients, but also a hyperotropic environment, which may increase oxidative stress and further damage kidney cells. This damaging microenvironment stimulates intrinsic renal cells and infiltrating immune cells to secrete various damaging and protective factors, leading to either adaptive or adverse repair processes in the kidney.

[0004] Transmembrane glycoprotein NMB (GPNMB), also known as non-metastatic melanoma B glycoprotein or osteoactivin, contains an extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. GPNMB is readily cleaved by metalloproteinases MMP or ADAM10, releasing its extracellular portion (GPNMB-ECD). The GPNMB-ECD contains a polycystic kidney disease (PKD) domain and an integrin recognition (RGD) motif, and can play a crucial role as a signaling molecule in intercellular communication. GPNMB has shown protective effects in some organ injuries and metabolic diseases. For example, GPNMB expression is elevated in cerebral ischemia-reperfusion injury, and intraventricular injection of recombinant GPNMB protein in ischemic mice effectively reduces infarct size. In another study, overexpression of GPNMB-ECD in the liver using a lentiviral vector, which simultaneously increased circulating GPNMB-ECD expression, alleviated organ damage caused by myocardial infarction, improved cardiac function, and reduced scar formation. However, the role of GPNMB-ECD in acute kidney injury has not yet been reported. Summary of the Invention

[0005] Based on the above analysis, the present invention aims to provide an application of the extracellular domain of the transmembrane glycoprotein NMB in the treatment and improvement of acute kidney injury, in order to study the therapeutic effect and mechanism of the extracellular domain of the transmembrane glycoprotein NMB on acute kidney injury.

[0006] The objective of this invention is mainly achieved through the following technical solutions:

[0007] This invention provides, in one aspect, the application of the extracellular domain of the transmembrane glycoprotein NMB in the treatment and improvement of acute kidney injury.

[0008] Furthermore, in the aforementioned application, the acute kidney injury is caused by ischemia-reperfusion.

[0009] A second aspect of the present invention also provides the use of the extracellular domain of the transmembrane glycoprotein NMB in the preparation of drugs for the prevention and treatment of acute kidney injury.

[0010] Furthermore, in the aforementioned applications, the drug is an oral preparation or an injection.

[0011] Furthermore, in the aforementioned applications, the drug is used to prevent acute kidney injury from progressing to chronic kidney disease.

[0012] Furthermore, in the aforementioned applications, the drug is used to inhibit the infiltration of F4 / 80 positive macrophages and Ly6G positive neutrophils; and / or,

[0013] The drug is used to downregulate IL-1β and TNF-α expression; and / or,

[0014] The drug is used to promote the proliferation of Ki67-positive cells, reduce TUNEL-positive cells, and upregulate Bcl-2 anti-apoptotic protein; and / or,

[0015] The drug is used to inhibit the deposition of Col1A1, Fibronectin, and α-SMA.

[0016] A third aspect of the present invention also provides the application of the extracellular domain of the transmembrane glycoprotein NMB in the treatment of a disease model of acute kidney injury.

[0017] Furthermore, in the aforementioned application, the disease model is a mouse model.

[0018] Furthermore, in the aforementioned application, the transmembrane glycoprotein NMB extracellular domain solution is administered via intraperitoneal injection.

[0019] Furthermore, in the aforementioned application, the concentration of the transmembrane glycoprotein NMB extracellular domain solution is 2 μg / ml, the dosage is 20 μg / kg, and the administration is performed 5 times.

[0020] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0021] (1) The application of the extracellular domain of transmembrane glycoprotein NMB provided by the present invention in the treatment and improvement of acute kidney injury. The disease model of acute kidney injury was treated with the extracellular domain of transmembrane glycoprotein NMB. It was found that the extracellular domain of transmembrane glycoprotein NMB can effectively reduce acute kidney injury caused by ischemia-reperfusion, promote kidney tissue repair, and inhibit the progression of acute kidney injury to chronic inflammation and fibrosis.

[0022] (2) In this invention, GPNMB-ECD has significant beneficial effects in treating acute kidney injury caused by ischemia-reperfusion: five consecutive intraperitoneal injections at a dose of 20 μg / kg can effectively improve the 7-day survival rate of bilateral ischemia-reperfusion-AKI mice, reducing the mortality rate from 75.0% to approximately 28.6%; in a unilateral ischemia-reperfusion model, it can alleviate kidney tissue damage during the repair period, reduce the gene expression of the kidney injury marker Kim1, reduce apoptotic necrotic cells (TUNEL-positive cells), increase Ki67+ proliferating cells to promote kidney tissue repair, and simultaneously reduce F4 / 80+ macrophages. It inhibits the infiltration of Ly6G+ neutrophils to suppress the inflammatory response; it can also reduce the damage in the transition from AKI to chronic kidney disease, reduce the renal tubular injury score, reduce the expression of DNA damage marker p-H2AX and poor repair marker VCAM1, and inhibit macrophage infiltration and renal fibrosis (reducing Col1A1, Fibronectin, and α-SMA deposition); and it has been determined that 20 μg / kg is the effective therapeutic dose, while high doses (50 μg / kg) have no significant effect, suggesting that there is a reasonable dose window, providing an effective means for the treatment of acute kidney injury caused by ischemia-reperfusion.

[0023] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the written description and the accompanying drawings. Attached Figure Description

[0024] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0025] Figure 1 Kaplan-Meier survival curves for acute kidney injury induced by bilateral renal ischemia-reperfusion (bIRI) injury in mice in Example 1 of this study;

[0026] Figure 2 This is a bar chart showing the relative expression levels of the Kim1 gene in Example 2.

[0027] Figure 3 This is a bar chart showing the relative expression levels of the Gpnmb gene in Example 2.

[0028] Figure 4 This is a TUNEL fluorescence staining image of apoptotic and necrotic cells in Example 2 of this study;

[0029] Figure 5 This is a bar chart showing the quantitative count of TUNEL-positive apoptotic and necrotic cells in Example 2.

[0030] Figure 6This is an immunohistochemical staining image of Ki67-positive cells in Example 2 of this study;

[0031] Figure 7 This is a bar chart showing the quantitative count of Ki67-positive cells in Example 2.

[0032] Figure 8 Immunohistochemical staining image showing the number of F4 / 80 and Ly6G positive cells in Example 2 of this study;

[0033] Figure 9 This is a bar chart showing the quantitative percentage of F4 / 80 and Ly6G positive cell areas in Example 2.

[0034] Figure 10 This is a quantitative bar chart of renal tubular injury scores in Example 3.

[0035] Figure 11 This is an immunoblot image of p-H2AX and VCAM1 proteins in Example 3 of this study;

[0036] Figure 12 This is a bar chart showing the quantitative relative expression levels of p-H2AX and VCAM1 proteins in Example 3 of this study.

[0037] Figure 13 This is an immunohistochemical staining image of F4 / 80 positive cells in Example 3 of this study;

[0038] Figure 14 This is a quantitative bar chart showing the percentage of F4 / 80 positive areas in Example 3 of this study;

[0039] Figure 15 Immunohistochemical staining images of Col1A1, Fibronectin, and α-SMA in Example 3 of this study;

[0040] Figure 16 This is a bar chart showing the quantitative analysis of Col1A1, Fibronectin, and α-SMA immunohistochemical staining in Example 3 of this study;

[0041] Figure 17 This is a quantitative analysis diagram of the relative expression levels of NGAL, VCAM1, cleaved Caspase, and α-SMA proteins in Example 4 of this study. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0043] This invention provides the application of the extracellular domain of the transmembrane glycoprotein NMB in the treatment and improvement of acute kidney injury.

[0044] This invention provides, in one aspect, the application of the extracellular domain of the transmembrane glycoprotein NMB in the treatment and improvement of acute kidney injury.

[0045] Furthermore, in the aforementioned application, the acute kidney injury is caused by ischemia-reperfusion.

[0046] A second aspect of the present invention also provides the use of the extracellular domain of the transmembrane glycoprotein NMB in the preparation of drugs for the prevention and treatment of acute kidney injury.

[0047] Furthermore, in the aforementioned applications, the drug is an oral preparation or an injection.

[0048] Furthermore, in the aforementioned applications, the drug is used to prevent acute kidney injury from progressing to chronic kidney disease.

[0049] Furthermore, in the aforementioned applications, the drug is used to inhibit the infiltration of F4 / 80 positive macrophages and Ly6G positive neutrophils; and / or,

[0050] The drug is used to downregulate IL-1β and TNF-α expression; and / or,

[0051] The drug is used to promote the proliferation of Ki67-positive cells, reduce TUNEL-positive cells, and upregulate Bcl-2 anti-apoptotic protein; and / or,

[0052] The drug is used to inhibit the deposition of Col1A1, Fibronectin, and α-SMA.

[0053] A third aspect of the present invention also provides the application of the extracellular domain of the transmembrane glycoprotein NMB in the treatment of a disease model of acute kidney injury.

[0054] Furthermore, in the aforementioned application, the disease model is a mouse model.

[0055] Furthermore, in the aforementioned application, the transmembrane glycoprotein NMB extracellular domain solution is administered via intraperitoneal injection.

[0056] Furthermore, in the aforementioned application, the concentration of the transmembrane glycoprotein NMB extracellular domain solution is 2 μg / ml, the dosage is 20 μg / kg, and the administration is performed 5 times.

[0057] Example 1: Bilateral renal ischemia-reperfusion (bIRI) experiment in mice.

[0058] 1. Experimental animals

[0059] Eight-week-old male C57BL / 6J mice were purchased and acclimatized in a barrier environment for one week before being randomly divided into three groups: a surgical control + solvent control group (Sham + PBS) of 4 mice, a surgical control + GPNMB-ECD treatment group (Sham + Gp) of 4 mice, a bIRI surgical + solvent control group (bIRI + PBS) of 7 mice, and a bIRI surgical + GPNMB-ECD treatment group (bIRI + Gp) of 7 mice.

[0060] Reagents:

[0061] GPNMB-ECD solution: Dissolve mouse GPNMB-ECD protein lyophilized powder (Sinochem Biotechnology Co., Ltd., model 50475-M08H) in 0.01M PBS to prepare a stock solution with a concentration of 100 μg / ml and store at -20℃. Before the experiment, thaw the GPNMB-ECD solution at room temperature in the dark until it is clear and transparent, and dilute it with 0.01M PBS to a working solution of 2 μg / ml for later use.

[0062] 2. Surgical treatment

[0063] 1) Sham-operated group: The surgical control + solvent control group (Sham + PBS) and the surgical control + GPNMB-ECD (Sham + Gp) underwent sham surgery. The specific treatment process is as follows: Mice were anesthetized by intraperitoneal injection of 0.05% sodium pentobarbital at a dose of 20 mg / kg at room temperature. The skin on the back of the mice was prepared, disinfected with alcohol, and fixed in a prone position on a heating pad. The rectal temperature was measured to keep the body temperature of the mice constant at 37°C. Bilateral longitudinal incisions were made next to the spine, and the skin and muscle layers were cut layer by layer. The kidneys were bluntly picked out with cotton swabs, and the kidney pedicles were clearly exposed. The kidneys were then repositioned and sutured layer by layer. The tissues were disinfected with iodine. The body temperature was maintained after the operation. After the mice naturally woke up, they were put back into the breeding cage.

[0064] 2) bIRI surgery group: bIRI surgery was performed on both the bIRI surgery + solvent control group (bIRI+PBS) and the bIRI surgery + GPNMB-ECD treatment group (bIRI+Gp). The specific procedures were as follows: Mice were anesthetized preoperatively by intraperitoneal injection of 0.05% sodium pentobarbital at a dose of 20 mg / kg at room temperature. The skin on the back of the mice was prepared, disinfected with alcohol, and the mice were fixed in a prone position on a heating pad. Rectal temperature was measured to maintain a constant body temperature of 37°C. Bilateral longitudinal incisions were made beside the spine, and the skin and muscle layers were cut layer by layer. The kidneys were bluntly removed with cotton swabs to clearly expose the renal pedicle. The renal pedicle was clamped with an arterial clamp, and a timer was started (22 minutes). The kidneys were returned to the abdominal cavity, and the skin was temporarily closed. After the timer ended, the arterial clamps were removed, and the kidneys were repositioned after observing normal blood return. The wounds were sutured and disinfected with iodine. Postoperatively, the body temperature was maintained, and the mice were returned to their cages after natural recovery.

[0065] 3. Postoperative care

[0066] Surgery control + solvent control group (Sham+PBS): Mice were given 0.01M PBS solution intraperitoneally 5 times immediately after sham surgery (0h), and at 24h, 48h, 72h and 96h after surgery, with an injection volume of 250μl.

[0067] Surgery control + GPNMB-ECD treatment group (Sham+Gp): Mice were given intraperitoneal injections of GPNMB-ECD solution at a dose of 20 μg / kg immediately after sham surgery (0h) and at 24h, 48h, 72h and 96h after surgery.

[0068] bIRI surgery + solvent control group (bIRI+PBS): Mice were given 0.01M PBS solution intraperitoneally 5 times immediately after bIRI surgery (0h), and at 24h, 48h, 72h and 96h after surgery, with an injection volume of 250μl.

[0069] bIRI surgery + GPNMB-ECD treatment group (bIRI+Gp): Mice were given intraperitoneal injections of GPNMB-ECD solution at a dose of 20 μg / kg immediately after bIRI surgery (0h) and at 24h, 48h, 72h and 96h after surgery.

[0070] 4. Postoperative observation

[0071] Mice were monitored for body weight and their general condition was recorded daily. 1 ml of warm saline was injected intraperitoneally to replenish fluids. Mice mortality was observed and recorded daily at 8:00 AM and 8:00 PM.

[0072] 5. Experimental Results

[0073] like Figure 1As shown, the 7-day survival rate of mice in both the surgery control + solvent control group (Sham + PBS) and the surgery control + GPNMB-ECD treatment group (Sham + Gp) was 100%. After bIRI surgery, the 7-day mortality rate of untreated mice (bIRI + PBS group) was 75.0%, while treatment with GPNMB-ECD (bIRI + Gp group) reduced the mortality rate of bIRI-AKI mice to approximately 28.6% (P < 0.05), indicating that GPNMB-ECD can improve the survival rate of bIRI-AKI mice.

[0074] Example 2: Unilateral Renal Ischemia-Reperfusion (uIRI) Experiment in Mice

[0075] 1. Experimental animals

[0076] Twelve-week-old male C57BL / 6J mice were purchased and acclimatized in a barrier environment for one week before being randomly divided into three groups: a surgical control group (Sham) of 4 mice, a uIRI surgery + solvent control group (uIRI + PBS) of 6 mice, and a uIRI surgery + GPNMB-ECD treatment group (uIRI + Gpnmb) of 6 mice.

[0077] Reagents:

[0078] GPNMB-ECD solution: Dissolve mouse GPNMB-ECD protein lyophilized powder (Sinochem Biotechnology Co., Ltd., catalog number 50475-M08H) in 0.01M PBS to prepare a stock solution with a concentration of 100 μg / ml and store at -20℃. Before the experiment, thaw the GPNMB-ECD solution at room temperature in the dark until it is clear and transparent, and dilute it with 0.01M PBS to a working solution of 2 μg / ml for later use.

[0079] 2. Surgical treatment

[0080] 1) Sham surgery group; The surgical control group (Sham) underwent sham surgery. The specific procedure was as follows: Mice were anesthetized by intraperitoneal injection of 0.05% sodium pentobarbital at a dose of 20 mg / kg at room temperature. The skin on the back of the mice was prepared and disinfected with alcohol. The mice were fixed in a prone position on a heating pad, and the rectal temperature was measured to keep the body temperature of the mice constant at 37°C. A longitudinal incision was made on the left side next to the spine. The skin and muscle layers were cut layer by layer. The kidney was bluntly picked out with a cotton swab. After the kidney pedicle was clearly exposed, the kidney was repositioned and then sutured layer by layer. The tissue was disinfected with iodine. The body temperature was maintained after the operation. After the mice woke up naturally, they were put back into the breeding cage.

[0081] 2) uIRI surgery group: Six mice underwent uIRI surgery + solvent control group (uIRI+PBS) and uIRI surgery + GPNMB-ECD treatment group (uIRI+Gpnmb). The specific treatment process was as follows: Mice were anesthetized preoperatively by intraperitoneal injection of 0.05% sodium pentobarbital at a dose of 20 mg / kg at room temperature. The skin on the back of the mice was prepared, disinfected with alcohol, and fixed in a prone position on a heating pad. Rectal temperature was measured to maintain a constant body temperature of 37℃. A longitudinal incision was made on the left side of the spine, and the skin and muscle layers were cut layer by layer. The kidney was bluntly removed with a cotton swab, clearly displaying the renal pedicle. The renal pedicle was clamped with an arterial clamp, and a timer was started (45 minutes). The kidney was returned to the abdominal cavity, and the skin was temporarily closed. After the timer ended, the arterial clamp was removed, and after observing normal renal blood return, the kidney was repositioned, the wound was sutured, and disinfected with iodine. Postoperatively, the body temperature was maintained, and the mice were returned to their cages after natural recovery.

[0082] 3. Postoperative care

[0083] Surgery + solvent control group (Sham): Mice were injected intraperitoneally with PBS solution 250 μl five times immediately after sham surgery (0 h) and at 24 h, 48 h, 72 h and 96 h after surgery.

[0084] uIRI surgery + solvent control group (uIRI+PBS): Mice were injected intraperitoneally with PBS solution 250μl at five times immediately after uIRI surgery (0h) and at 24h, 48h, 72h and 96h after surgery.

[0085] uIRI surgery + GPNMB-ECD treatment group (uIRI+Gpnmb): Mice were injected intraperitoneally with GPNMB-ECD solution at a dose of 20 μg / kg immediately after uIRI surgery (0h) and at 24h, 48h, 72h and 96h after surgery.

[0086] 4. Postoperative monitoring

[0087] Mouse weight was monitored and general condition was recorded daily. Kidney tissue was taken from mice on day 7 after sham surgery and uIRI surgery for testing.

[0088] 5. Experimental Results:

[0089] like Figure 2 As shown, compared with the surgery + solvent control group (Sham), the gene expression level of the kidney injury marker Kim1 was significantly increased on day 7 after uIRI surgery, and compared with the uIRI surgery + solvent control group (uIRI + PBS), the gene expression level of Kim1 in the uIRI surgery + GPNMB-ECD treatment group was significantly decreased (P<0.05).

[0090] like Figure 3As shown, the expression of the Gpnmb gene was detected by qPCR. Compared with the surgery + solvent control group (Sham), the expression of Gpnmb increased on day 7 after uIRI surgery. Moreover, compared with the uIRI surgery + solvent control group (uIRI + PBS), the exogenous supplementation of GPNMB-ECD in the uIRI surgery + GPNMB-ECD treatment group (uIRI + Gpnmb) significantly increased the expression of the Gpnmb gene in the kidney tissue (P < 0.001).

[0091] like Figure 4 and Figure 5 As shown, apoptotic and necrotic cells were labeled using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Compared with the surgery + solvent control group (Sham), the number of apoptotic and necrotic cells in the kidney tissue of mice was significantly increased on day 7 after uIRI surgery. Moreover, compared with the uIRI surgery + solvent control group (uIRI + PBS), the number of apoptotic and necrotic cells in the uIRI surgery + GPNMB-ECD treatment group (uIRI + Gpnmb) was significantly reduced after treatment (P < 0.05).

[0092] like Figure 6 and Figure 7 As shown, compared with the surgery + solvent control group (Sham), on day 7 after uIRI surgery, the kidney tissue initiated a spontaneous repair program, and the number of Ki67+ proliferating cells in the tissue increased. Moreover, compared with the uIRI surgery + solvent control group (uIRI + PBS), the number of Ki67+ proliferating cells in the uIRI surgery + GPNMB-ECD treatment group (uIRI + Gpnmb) mice was significantly increased after treatment (P<0.05), indicating that the tissue damage repair capacity was further enhanced.

[0093] like Figure 8 and Figure 9 As shown, compared with the surgery + solvent control group (Sham), the infiltration of F4 / 80+ macrophages and Ly6G+ neutrophils into the kidney tissue was significantly increased on day 7 after uIRI surgery, indicating enhanced intra-tissue inflammatory response. Moreover, compared with the uIRI surgery + solvent control group (uIRI+PBS), the number of F4 / 80+ and Ly6G+ cells was significantly reduced after GPNMB-ECD treatment in the uIRI surgery + Gpnmb group (uIRI+Gpnmb) (P<0.05), indicating a decrease in the number of inflammatory cells and a reduction in inflammatory response.

[0094] Example 3: Examination of kidney tissue from mouse unilateral ischemia-reperfusion (uIRI) experiments.

[0095] 1. Experimental animals: Same as in Example 2.

[0096] Reagents: Same as in Example 2.

[0097] 2. Surgical treatment: Same as in Example 2.

[0098] 3. Postoperative management: Same as in Example 2

[0099] 4. Postoperative monitoring: Mouse weight was monitored and general condition was recorded daily. Kidney tissue was collected from mice on day 14 after sham surgery and uIRI surgery for testing.

[0100] 5. Experimental results: Exogenous GPNMB-ECD can alleviate renal tissue damage during the transition from uIRI-AKI to chronic kidney disease, inhibit chronic inflammatory response, and suppress renal fibrosis.

[0101] Renal tubular injury was scored on renal tissue samples taken from mice in both sham surgery and uIRI groups 14 days after surgery. Figure 10 As shown: Compared with the surgery + solvent control group (Sham), the injury score of the uIRI surgery + solvent control group (uIRI+PBS) was significantly increased (10.0±0.1 vs 1.2±0.2 points, P<0.0001); compared with the uIRI surgery + solvent control group (uIRI+PBS), the injury score of the uIRI surgery + GPNMB-ECD treatment group (uIRI+Gpnmb) mice was significantly reduced after treatment with Gpnmb-ECD protein (7.2±0.8 vs 10.0±0.1 points, P<0.05).

[0102] Further analysis using Western blot revealed the expression levels of DNA damage marker p-H2AX and renal tubular repair impairment marker VCAM1. Figure 11 and Figure 12 As shown in the figure. GAPDH-normalized grayscale analysis revealed that, compared to the surgery + solvent control group (Sham), the expression of p-H2AX and VCAM1 was significantly increased in the uIRI surgery + solvent control group (uIRI + PBS); compared to the uIRI surgery + solvent control group (uIRI + PBS), the expression of p-H2AX and VCAM1 was significantly decreased in the uIRI surgery + GPNMB-ECD treatment group (uIRI + Gpnmb) after treatment with Gpnmb-ECD protein (P < 0.01). These results indicate that exogenous Gpnmb-ECD protein can significantly improve chronic tubular injury induced by renal ischemia-reperfusion injury.

[0103] The degree of macrophage infiltration in the kidney tissue of uIRI mice was assessed by F4 / 80 immunohistochemical staining, such as Figure 13 Representative images obtained from immunohistochemical staining showed that the surgery + solvent control group (Sham) had only a small number of scattered F4 / 80 positive cells; the uIRI surgery + solvent control group (uIRI + PBS) showed a large number of dark F4 / 80 positive macrophages infiltrating, mainly distributed in the renal tubulointerstitial region; while the macrophage infiltration was significantly reduced in the uIRI surgery + GPNMB-ECD treatment group (uIRI + Gpnmb). Figure 14 Quantitative analysis showed that the F4 / 80 positive area in the kidney tissue of mice in the uIRI surgery + GPNMB-ECD treatment group (uIRI+Gpnmb) was significantly lower than that in the uIRI surgery + solvent control group (uIRI+PBS) (P<0.001). These results indicate that exogenous Gpnmb-ECD protein can inhibit macrophage infiltration in the chronic phase of AKI, suggesting that it may improve renal prognosis by regulating the chronic inflammatory response.

[0104] like Figure 15 Immunohistochemical staining of kidney tissue showed that 14 days after uIRI surgery, significant deposition of collagen type I α1 chains (ColA1) and fibronectin (FN), as well as increased α-SMA expression, were observed in the kidney tissue of mice in the uIRI surgery + solvent control group (uIRI + PBS), indicating significant renal fibrosis. Figure 16 As shown, compared with the uIRI surgery + solvent control group (uIRI + PBS), the positive areas of ColA1, FN, and α-SMA in the uIRI surgery + GPNMB-ECD treatment group (uIRI + Gpnmb) were significantly reduced after Gpnmb-ECD protein intervention (P < 0.001). These results indicate that exogenous Gpnmb-ECD protein effectively alleviates chronic renal fibrosis caused by renal ischemia-reperfusion injury by inhibiting extracellular matrix deposition and myofibroblast activation.

[0105] Example 4: Dosage control experiment of unilateral renal ischemia-reperfusion (uIRI) in mice 1. Experimental animals: Same as in Example 2.

[0106] Reagents:

[0107] GPNMB-ECD solution: Dissolve mouse GPNMB-ECD protein lyophilized powder (Sinochem Biotechnology Co., Ltd., catalog number 50475-M08H) in 0.01M PBS to prepare a stock solution with a concentration of 100 μg / ml and store at -20℃. Before the experiment, thaw the GPNMB-ECD solution at room temperature in the dark until it is clear and transparent, and dilute it with 0.01M PBS to a working solution of 5 μg / ml for later use.

[0108] 2. Surgical treatment: Same as in Example 2.

[0109] 3. Postoperative care:

[0110] Surgery + solvent control group (Sham): Mice were given intraperitoneal injections of PBS solution at 250 μl each, immediately after sham surgery (0 h) and at 24 h, 48 h, 72 h and 96 h after surgery.

[0111] uIRI surgery + solvent control group (uIRI+PBS): Mice were given intraperitoneal injections of PBS solution at 250 μl each, immediately after uIRI surgery (0 h) and at 24 h, 48 h, 72 h and 96 h after surgery.

[0112] uIRI surgery + GPNMB-ECD treatment group (uIRI+Gpnmb): Mice were given intraperitoneal injections of GPNMB-ECD solution at a dose of 50 μg / kg immediately after uIRI surgery (0h) and at 24h, 48h, 72h and 96h after surgery.

[0113] 4. Postoperative monitoring:

[0114] Mouse weight was monitored and general condition was recorded daily. Kidney tissue was collected from mice in surgical control and on day 7 after uIRI surgery for testing.

[0115] 5. Experimental Results:

[0116] Total protein was extracted from mouse kidney tissue 7 days post-surgery using protein lysis buffer. Semi-quantitative Western blot analysis was performed on the expressed kidney injury markers NGAL and VCAM1, apoptosis marker cleaved-caspase 3, and fibrosis marker α-SMA. Specific results are shown below. Figure 17 As shown, compared with the surgery + solvent control group (Sham), the expression of NGAL, VCAM1, cleaved-caspase3, and α-SMA was significantly increased in the uIRI surgery + solvent control group (uIRI+PBS), while the expression levels of these damage markers did not change significantly in the uIRI surgery + GPNMB-ECD treatment group (uIRI+Gpnmb). This example verifies that high-dose GPNMB-ECD (50 μg / kg) administration failed to alleviate renal tissue damage in the transition phase of uIRI-AKI to chronic kidney disease.

[0117] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of the extracellular domain of transmembrane glycoprotein NMB in the treatment and improvement of acute kidney injury.

2. The application according to claim 1, characterized in that, The acute kidney injury was caused by ischemia-reperfusion.

3. Use of the extracellular domain of the transmembrane glycoprotein NMB in the preparation of drugs for the prevention and treatment of acute kidney injury.

4. The application according to claim 3, characterized in that, The drug is an oral preparation or an injectable preparation.

5. The application according to claim 4, characterized in that, The drug is used to prevent acute kidney injury from progressing to chronic kidney disease.

6. The application according to claim 4, characterized in that, The drug is used to inhibit the infiltration of F4 / 80 positive macrophages and Ly6G positive neutrophils; and / or, The drug is used to downregulate IL-1β and TNF-α expression; and / or, The drug is used to promote the proliferation of Ki67-positive cells, reduce TUNEL-positive cells, and upregulate Bcl-2 anti-apoptotic protein; and / or, The drug is used to inhibit the deposition of Col1A1, Fibronectin, and α-SMA.

7. Application of the extracellular domain of a transmembrane glycoprotein NMB in a disease model for treating acute kidney injury.

8. The application according to claim 7, characterized in that, The disease model is a mouse model.

9. The application according to claim 8, characterized in that, The transmembrane glycoprotein NMB extracellular domain solution was administered via intraperitoneal injection.

10. The application according to claim 8, characterized in that, The extracellular domain solution concentration of the transmembrane glycoprotein NMB was 2 μg / ml, the dosage was 20 μg / kg, and the administration was performed 5 times.

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