Use of mitochondrial extract for treating and / or preventing kidney damage-related diseases

Abstract

To provide a second application of a mitochondrial extract.SOLUTION: The invention relates to an application of a mitochondrial extract for treating and / or preventing kidney injury related diseases. Specifically, the mitochondrial extract provided here is administered to a subject individual afflicted with kidney injury related diseases, thereby effectively improving the kidney injury related diseases and preventing deterioration of the kidney injury related diseases.SELECTED DRAWING: Figure 2B

Description

[Technical Field]

[0001] The present invention relates to secondary uses of mitochondrial extracts, and in particular to the use of mitochondrial extracts for treating and / or preventing diseases associated with kidney damage. [Background technology]

[0002] Mitochondria are organelles present in human cells and supply the ATP necessary for normal cellular activity. Recent studies have shown that increasing the number and activity of mitochondria in cells provides the energy necessary for stem cell differentiation and contributes to successful stem cell differentiation. This indicates that mitochondria play a crucial role in the body's energy metabolism. For example, mitochondrial defects can lead to degenerative or age-related diseases such as Alzheimer's disease, myasthenia gravis, and muscle disorders. Numerous studies have now shown that maintaining normal mitochondrial function and enhancing antioxidant capacity in the body can help prevent the progression of neurodegenerative diseases in patients with Parkinson's disease and Alzheimer's disease, which are caused by oxidative damage.

[0003] Chronic kidney disease (CKD) is a condition in which kidney tissue damage persists for more than three months, rendering the kidney's structure and function irreversible. Currently, clinical treatments primarily involve drug therapy, often supplemented by dietary and lifestyle changes. However, as CKD worsens over time and kidney function is gradually lost due to renal fibrosis, patients may require hemodialysis or kidney transplantation to maintain their lives. This is an extremely painful process for patients and represents a significant burden on national healthcare costs. Therefore, due to the lack of clear understanding of the pathogenesis and treatment methods of CKD, effective clinical treatments for CKD have yet to be developed. Therefore, there is an urgent need in clinical medicine to provide compositions and methods effective for the treatment of CKD and renal fibrosis. Summary of the Invention [Problem to be solved by the invention]

[0004] The main object of the present invention is to provide a secondary use of a mitochondrial extract, which can effectively improve or prevent kidney damage-related diseases, thereby achieving the effects of treating kidney disease and suppressing the progression of kidney disease. [Means for solving the problem]

[0005] To achieve the above-mentioned objectives, the present invention discloses the use of a mitochondrial extract for preparing a composition for preventing and / or treating a kidney damage-related disease. Specifically, when a certain amount of the mitochondrial extract is administered to an individual suffering from a kidney damage-related disease, it can ameliorate kidney cell damage, thereby achieving the effect of treating or preventing the worsening of the kidney damage-related disease.

[0006] In the embodiments of the present invention, the kidney damage-related diseases include renal fibrosis, renal inflammation, chronic kidney disease, acute kidney injury, tubular injury, renal failure, prerenal kidney injury, nephrogenic kidney injury, postrenal kidney injury, glomerulonephritis, pyelonephritis, nephrotic syndrome, and uremia.

[0007] In one embodiment of the present invention, the kidney damage-related disease has, in addition to mitochondrial damage, at least one of the following symptoms: proteinuria, edema, oliguria, elevated urea nitrogen, elevated creatinine, abnormal uric acid levels, stones, and abnormal glomerular filtration rate.

[0008] In another embodiment of the present invention, mitochondria are isolated from stem cells, such as adipose stem cells, CD34+ hematopoietic stem cells, mesenchymal stem cells, bone marrow stem cells, umbilical cord stem cells, amniotic stem cells, amniotic fluid stem cells, placental stem cells, iPS cells, and neural stem cells.

[0009] In the present embodiment, the amount of mitochondria in the composition is 5-80 μg, preferably 40 μg. [Effects of the Invention]

[0010] The beneficial effects of the present invention are as follows: When mitochondrial damage occurs in renal cells due to environmental factors such as fibrosis, oxidative stress, or inflammation, the mitochondrial extract provided by the present invention or a composition containing the same can be administered to effectively ameliorate the mitochondrial damage in renal cells, thereby achieving the effect of improving or treating renal cell damage and related diseases. [Brief explanation of the drawings]

[0011] [Figure 1A] This shows the results of a statistical analysis of the survival rate of renal epithelial cells 24 hours after treatment with different concentrations of hydrogen peroxide. [Figure 1B] This is the result of a statistical analysis of the survival rate after treating renal epithelial cells with different concentrations of hydrogen peroxide and then administering different doses of mitochondrial precipitates. [Figure 2A] This shows the results of detecting and analyzing the amount of collagen secreted by renal epithelial cells after treating them with different concentrations of advanced glycation end products (AGEs-BSA) for different periods of time. [Figure 2B] Renal epithelial cells were treated with different concentrations of AGEs-BSA and administered different doses of mitochondrial precipitates, and then the amount of collagen secreted by the renal epithelial cells was detected and analyzed. [Figure 2C] The figures show the results of detecting and analyzing the amount of collagen secreted by renal epithelial cells after treating them with different concentrations of hydrogen peroxide and administering different doses of mitochondrial precipitates. [Figure 3] The results show that renal epithelial cells were treated with hydrogen peroxide or AGEs-BSA, and then various doses of mitochondrial precipitates were administered, followed by detection and analysis of the state of mitochondrial damage in the renal epithelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a secondary use of the mitochondrial extract, namely, that by administering a certain amount of the mitochondrial extract or a composition containing the same to an individual suffering from a nephropathy-related disease, the nephropathy-related disease can be effectively improved and the worsening of the nephropathy-related disease can be prevented.

[0013] Typically, the dose of mitochondria provided by the present invention administered to an individual is 5 to 80 μg, for example, 5, 10, 15, 20, 25, 30, 40, 50, 60, 65, 70, or 80 μg, with a preferred dose being 15 to 40 μg. Furthermore, to achieve superior therapeutic or ameliorative effects for kidney disease, the mitochondria provided by the present invention may be combined with other components to prepare a composition, and the combined components preferably contain growth factors, such as blood products containing growth factors, platelet-rich plasma (PRP), plasma, serum, or platelet-rich fibrin.

[0014] In the present invention, the term "mitochondrial extract" refers to mitochondria isolated from cells, and the isolation technique or method used must be capable of maintaining the structural and functional integrity of mitochondria. To those skilled in the art of the present invention, the isolation technique or method may be physical or chemical.

[0015] In the present invention, the term "cell" refers to cells having mitochondria, such as adipose stem cells, mesenchymal stem cells, skeletal muscle cells, liver cells, kidney cells, fibroblasts, nerve cells, skin cells, and blood cells.

[0016] In the present invention, the "composition" may be a pharmaceutical composition, food, functional food, nutritional supplement, etc., and may be a combination of various ingredients according to type, and may have various dosage forms and various administration methods.

[0017] In the present invention, "nephropathy-related diseases" refer to diseases caused by damage to renal cells, and have symptoms of mitochondrial damage, such as renal fibrosis, renal inflammation, kidney disease, acute kidney injury, tubular injury, renal failure, prerenal kidney injury, nephrogenic kidney injury, postrenal kidney injury, glomerulonephritis, pyelonephritis, nephrotic syndrome, and uremia.

[0018] In order to demonstrate the effects of the mitochondrial extract provided by the present invention, several examples will be described in detail below with accompanying drawings.

[0019] Although the mitochondria used in the following examples are derived from human adipose-derived stem cells, the mitochondria of the present invention are not limited to those derived from human adipose stem cells. In other words, the mitochondria of the present invention can be derived from any cell in the human body.

[0020] The doses of mitochondria used in the following examples are merely examples, and although a dose of 15 μg of mitochondria represents a low dose and a dose of 40 μg represents a high dose, they in no way limit the technical features of the present invention. That is, the mitochondria provided by the present invention can achieve the effects that the present invention aims to achieve at any dose between 5 and 80 μg. [Example]

[0021] Culture of renal epithelial cell lines

[0022] Renal epithelial cell lines were cultured in a cell culture medium containing MEM-α Earl's salt and 5% fetal bovine serum at 37°C (containing 5% carbon dioxide). When the cell culture reached 80% confluence, the cell culture medium was removed, the cells were washed with phosphate buffer, and after removing the phosphate buffer, 0.25% trypsin / 2.21 mM EDTA was added and incubated for 20 minutes. MEM-α containing 5% fetal bovine serum was then added to neutralize the trypsin. The suspended cells were collected and centrifuged, after which the cells were counted. Then, the cells were resuspended in MEM-α containing 5% fetal bovine serum until the final concentration reached 5x10. 4The cells were diluted to 100 cells / mL for subsequent subculture or analysis. [Example]

[0023] Mitochondrial extraction

[0024] Human adipose stem cells were cultured at a cell count of 1.5x10 8 The cells were cultured until they were homogenized. After rinsing with Dulbecco's phosphate buffer solution (DPBS), the DPBS was removed. Trypsin was added and incubated for 3 minutes. Stem cell culture medium (Keratinocyte SFM (1X) liquid, bovine pituitary extract, 10 wt% fetal bovine serum) was added to terminate the incubation. The cells were then collected and centrifuged at 600 g for 10 minutes. The supernatant was removed, and 80 mL of IBC-1 buffer (225 mM mannitol, 75 mM sucrose, 0.1 mM EDTA, 30 mM Tris-HCl pH 7.4) was added to the cells. The cells were homogenized and centrifuged. The resulting precipitate was called mitochondria (hereafter referred to as the mitochondrial precipitate). 1.5 mL of IBC-1 buffer and protease inhibitors were added to the mitochondrial precipitate, which was then stored at 4°C and used in the following examples. [Example]

[0025] Renal epithelial cell injury test (1)

[0026] The renal epithelial cells cultured in Example 1 were subcultured in a 96-well plate at a concentration of 5x10 per well. 4After 8 hours of incubation, the supernatant was removed and the wells were washed with phosphate buffer. 200 μL of cell culture medium without 5% fetal bovine serum was added to each well and incubated for 8 hours. After incubation, the wells were treated with various concentrations of hydrogen peroxide (0.3, 0.5, 1, 3, 5, and 10 mM). After 24 hours of incubation with various concentrations of hydrogen peroxide, the supernatant was removed from each well and 100 μL of cell culture medium containing 10% Alamar Blue was added (100 μL / well). After 3–4 hours of incubation, fluorescence fingerprinting (Excitation / Emission: 560 / 590 nm) was performed. The results are shown in Figure 1A.

[0027] The results in Figure 1A show that renal epithelial cells began to develop damage after 24 hours of incubation with various concentrations of hydrogen peroxide. At hydrogen peroxide concentrations of 1 mM or higher, the incidence of cell death significantly increased. Specifically, the viability of renal epithelial cells after 24 hours of treatment with 1 mM hydrogen peroxide was 81.4%, after 24 hours of treatment with 5 mM hydrogen peroxide was 31.3%, and after 24 hours of treatment with 10 mM hydrogen peroxide was 24.2%. These results demonstrate that treatment of renal epithelial cells with hydrogen peroxide can indeed establish a model of renal epithelial cell injury and death. Furthermore, increasing the concentration of hydrogen peroxide increases the severity of cell injury and cell death. [Example]

[0028] Renal epithelial cell injury test (2)

[0029] The renal epithelial cells cultured in Example 1 were subcultured in a 96-well plate at a concentration of 1 x 10 per well. 4After culturing for 8 hours, the supernatant was removed, washed with phosphate buffer, and 200 μL of cell culture medium without 5% fetal bovine serum was added to each well. The cells were then cultured for 8 hours, treated with 1 mM and 3 mM hydrogen peroxide for 4 hours. The cells were then cultured for 24 hours with different doses (15 μg and 40 μg) of mitochondrial precipitate (prepared in Example 2). After culturing, the supernatant was removed, and 100 μL of cell culture medium containing 10% Alamar Blue was added (100 μL / well). The cells were cultured for 3-4 hours at 37°C. Fluorescence fingerprint measurements (Excitation / Emission: 560 / 590 nm) were performed at the end of the culture period. The results are shown in Figure 1B.

[0030] The results in Figure 1B show that renal epithelial cells treated with hydrogen peroxide are damaged and have reduced cell viability. However, administration of a certain amount of mitochondrial precipitate to renal epithelial cells damaged by hydrogen peroxide can significantly improve the viability of the renal epithelial cells, and that the viability of the renal epithelial cells increases with increasing dose.

[0031] These results demonstrate that the mitochondrial extract provided by the present invention can indeed protect renal epithelial cells, prevent the occurrence of renal epithelial cell damage caused by oxidative or inflammatory responses, repair damaged renal epithelial cells, and effectively prevent the death of renal epithelial cells. In other words, the mitochondrial extract provided by the present invention or a composition containing the same can certainly have the effect of improving and / or preventing kidney damage or kidney disease caused by oxidative stress. [Example]

[0032] Renal epithelial cell fibrosis test (1)

[0033] The renal epithelial cells cultured in Example 1 were cultured in a 6-well plate in a culture medium containing 5% fetal bovine serum at a concentration of 1 x 10 per well. 5After 24 hours of incubation, the supernatant was removed and washed with phosphate buffer. 1 ml of cell culture medium without 5% fetal bovine serum was added to each well and incubated for 8 hours. Different concentrations of advanced glycation end products (AGEs-BSA) (100 μg / ml and 400 μg / ml) were added and incubated for 4 hours. After incubation, the AGEs-BSA-containing cell culture medium was removed and washed with phosphate buffer. 1 ml of cell culture medium without 5% fetal bovine serum was added to each well and incubated for 24 and 48 hours, respectively. After incubation, the supernatant was collected and collagen secretion was measured using a soluble collagen assay kit (Sircol® Soluble Collagen Assay Kit). The results are shown in Figure 2A.

[0034] The results in Figure 2A show that the expression level of collagen secretion in renal epithelial cells increased in both renal epithelial cells treated with high concentrations (400 μg / ml) and low concentrations (100 μg / ml) of AGEs-BSA, and that the expression level of collagen secretion increased as the treatment time increased, indicating that AGEs-BSA indeed induces lesions in renal epithelial cells and produces a state of fibrosis. This is the early stage of chronic kidney disease, and if the expression level of collagen secretion continues to increase, chronic kidney disease will develop. [Example]

[0035] Renal epithelial cell fibrosis test (2)

[0036] The steps of this example were generally the same as those of Example 5, except that after removing the cell culture medium containing AGEs-BSA, cell culture medium without 5% fetal bovine serum and different doses (15 μg and 40 μg) of mitochondrial precipitate (prepared in Example 2) were added to each well, and then cultured for 24 hours. After the culture was completed, the supernatants were collected and collagen secretion was measured using a water-soluble collagen measurement kit. The results are shown in Figure 2B.

[0037] The results in Figure 2B reveal that administration of mitochondrial precipitates can reduce the amount of collagen secretion in renal epithelial cells induced by AGEs-BSA, demonstrating that the mitochondria provided by the present invention or a composition containing the same can effectively improve and / or prevent renal fibrosis and associated kidney diseases. [Example]

[0038] Renal epithelial cell fibrosis test (3)

[0039] The steps of this example were generally the same as those of Example 6, except that the stimulant that induces fibrosis in renal epithelial cells was changed from AGEs-BSA to hydrogen peroxide. The measurement results are shown in Figure 2C.

[0040] The results in Figure 2C demonstrate that treating renal epithelial cells with hydrogen peroxide induces an increase in collagen secretion. This means that treating renal epithelial cells with hydrogen peroxide can indeed create a renal cell fibrosis model. Adding mitochondrial precipitates to renal epithelial cells that had been treated with hydrogen peroxide significantly reduced the amount of collagen secreted from the renal epithelial cells, demonstrating that the mitochondria or compositions containing them provided by the present invention can effectively ameliorate and / or prevent renal fibrosis and related kidney diseases. [Example]

[0041] Testing for impairment of mitochondrial function in renal epithelial cells

[0042] The renal epithelial cells cultured in Example 1 were subcultured in a 96-well plate in a cell culture medium containing 5% fetal bovine serum at a concentration of 5x10 per well. 4The cells were cultured for 24 hours at 200 μL / well. The supernatant was removed, washed with phosphate buffer, and 1 mL of cell culture medium without 5% fetal bovine serum was added to each well. The cells were then cultured for 8 hours. After incubation, 3 mM hydrogen peroxide and 100 μg / mL AGEs-BSA were added to each well for 4 hours. The cell culture medium containing hydrogen peroxide or AGEs-BSA was then removed, washed with phosphate buffer, and 1 mL of cell culture medium without 5% fetal bovine serum and different doses (15 μg and 40 μg) of mitochondrial precipitate (prepared in Example 2) were added to each well and cultured for 24 hours. After incubation, the cells were washed with phosphate buffer, and a buffer containing 10 μM JC-1 staining reagent was added. The cells were incubated at 37°C for 10 minutes. After washing, fluorescence fingerprint measurements (Excitation / Emission: 488 / 530 nm) were performed. The results are shown in Figure 3.

[0043] The results in Figure 3 show that renal epithelial cells treated only with hydrogen peroxide or AGEs-BSA showed increased expression of JC-1 monomer, indicating that mitochondria in renal epithelial cells were damaged by the inflammatory environment created by hydrogen peroxide or AGEs-BSA. In contrast, renal epithelial cells treated first with hydrogen peroxide or AGEs-BSA and then with different doses of mitochondrial precipitate showed a significant decrease in JC-1 monomer expression. Furthermore, the expression of JC-1 monomer in renal epithelial cells decreased as the dose of mitochondrial precipitate increased. [Example]

[0044] Animal testing

[0045] Ten-week-old C57BL / 6 mice were housed under constant temperature and humidity with a 12:12-hour light / dark cycle. A mouse model of renal injury was performed using an ischemia-reperfusion (I / R) model. The procedure was as follows: First, 150 mg / kg of phenobarbital was injected intraperitoneally into the abdomen of the mouse to induce coma. Then, surgery was performed on the left kidney. The left kidney was then excised and the renal artery to the kidney was blocked with a vascular clamp. After 30 minutes of occlusion, the vascular clamp was removed and blood flow was restored. This completed the I / R renal injury model.

[0046] Mice undergoing I / R kidney injury model treatment were injected with different doses of mitochondria (15 μg and 40 μg) into the kidney via the renal artery, creating high-dose and low-dose mitochondria groups. The control group (I / R group) was injected with phosphate buffer. After treatment, the kidneys of each group were returned to the body and the wounds were sutured. Blood samples were collected on day 1 (D1) and day 2 (D2) for serum creatinine (creatinine) and blood urea nitrogen (BUN). The blood was then centrifuged and separated to collect serum, which was then analyzed for urea nitrogen and creatinine content. Serum creatinine was measured using a mouse creatinine assay kit (Crystal Chem, model number 80350), and serum urea nitrogen was measured using a urea assay kit (Abcam, model number ab83362). Mice from each group were sacrificed on day 7 (D7) after transplantation. The left kidney of each mouse was perfused and formalin-fixed, then paraffin-embedded and sectioned into tissue sections for H&E staining. The staining results were examined histologically for morphological changes due to ischemic injury. Renal injury was assessed using Jablonski's semiquantitative standard, with 0 representing normal tissue, 1 representing less than 5% tubular damage, 2 representing 5% to 25% tubular damage, 3 representing 25% to 75% tubular damage, and 4 representing >75% tubular damage.

[0047] The results are shown in Table 1 below. Compared with the control group, the I / R group showed a significant increase in serum urea nitrogen and creatinine levels. This indicates that the I / R group indeed caused renal injury. Furthermore, the renal injury score results showed that the I / R group had a score of 3-4, indicating severe renal tubular damage. Meanwhile, compared with the I / R group, the mice in the mitochondrial group had significantly lower serum urea nitrogen and creatinine levels, indicating that mitochondria can effectively ameliorate renal injury. The renal injury score results also indicated that administering mitochondria can restore damaged kidney cells and ameliorate renal tubular damage. Furthermore, the ameliorative effect of renal injury increased with increasing mitochondrial dosage.

[0048] Table 1: Analysis results of serum and kidney tissue sections after different treatments in each group of mice. JPEG0007736347000001.jpg86118

[0049] The above results indicate that when mitochondrial damage occurs in renal cells due to environmental factors such as fibrosis, oxidative stress, or inflammation, administering the mitochondrial extract provided by the present invention or a composition containing the same can effectively improve the mitochondrial damage in renal cells, thereby achieving the effect of improving or treating renal cell damage and related diseases.

Claims

[Claim 1] A composition for preventing and / or treating a kidney damage-related disease, comprising mitochondria, A composition, wherein the amount of the mitochondria in the composition is 15 μg to 40 μg / 200 μL.

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