US20230137870A1

US20230137870A1 - Use of mitochondrial extract to treat and/or prevent kidney injury-related disease

Info

Publication number: US20230137870A1
Application number: US17/912,394
Authority: US
Inventors: Han-Chung CHENG; Chih-Kai Hsu; Hui-Ching TSENG
Original assignee: Taiwan Mitochondrion Application Technology Co Ltd
Current assignee: Taiwan Mitochondrion Application Technology Co Ltd
Priority date: 2020-03-20
Filing date: 2021-03-19
Publication date: 2023-05-04
Also published as: EP4122474A4; US20230016499A1; CN118252850A; CN115335065A; WO2021185377A1; US20230023438A1; CN115297872B; CN115103683A; TWI789724B; CN115135328B; EP4122474A1; CN118236473A; TW202308668A; TW202135837A; TW202135838A; CN115315265B; US20230042445A1; TWI787763B; WO2021185364A1; EP4122444A1

Abstract

The present invention discloses a use of a mitochondrial extract to treat and/or prevent a kidney injury-related disease. Specifically, by administering the mitochondrial extract disclosed in the present invention to a patient having a kidney injury-related disease, the kidney injury-related disease can be effectively alleviated and prevented from deterioration.

Description

TECHNICAL FIELD

The present invention relates to a second use of a mitochondrial extract, and in particular to a use of a mitochondrial extract to treat and/or prevent a kidney injury-related disease.

BACKGROUND

The mitochondrion is an organelle in human cells that provides the ATP required for the normal operation of cells. Moreover, recent studies point out that an increase in the number and the activation of the mitochondria in cells can provide energy required for differentiation of stem cells and help the stem cells successfully differentiate. In other words, the mitochondria feature prominently in human energy metabolism. For example, defects in the mitochondria are likely to cause degenerative or aging-related diseases, such as brain degeneration, muscle weakness, muscle diseases, etc. Many current studies point out that if patients with the Parkinson's or Alzheimer's disease caused by oxidative damage can maintain normal functions of the mitochondria or enhance an antioxidant capacity in the body, neurodegenerative diseases can be stopped from getting worse.
When the kidney tissue is injured for more than 3 months so that the structure or function of the kidney cannot be restored to the original status, it is called a chronic kidney disease. Currently, most clinical treatments are based on drug therapy, supplemented by diet and lifestyle control. However, when the chronic kidney disease gradually worsens with the course of the disease, the patients are faced with kidney fibrosis and gradually lose the kidney functions. In this case, the patients need hemodialysis is or kidney transplantation to sustain life, which is not only a very uncomfortable process for the patients, but also a high burden on national medical cost. In other words, because the pathogenesis and the treatment of the chronic kidney disease neither have been confirmed, there is no effective clinical treatment for the chronic kidney disease. Therefore, in clinical medicine, it is in urgent need to provide a composition or method for effectively treating the chronic kidney disease and renal fibrosis.

SUMMARY

The main objective of the present invention is to provide a second use of a mitochondrial extract, which can effectively alleviate or prevent a kidney injury-related disease, thus achieving the effect of treating the kidney disease or slowing down the progression of the kidney disease.
Therefore, in order to achieve the foregoing objective, the present invention discloses a use of mitochondria to prepare a composition for preventing and/or treating a kidney injury-related disease. Specifically, by administering a certain amount of mitochondrial extracts to a patient having a kidney injury-related disease, the injury in the kidney cells can be alleviated, thus treating the kidney injury-related disease or preventing deterioration.
In the examples of the present invention, the kidney injury-related disease is renal fibrosis, renal inflammation, a chronic kidney disease, an acute kidney disease, a renal tubular injury, renal failure, a prerenal injury, a renal induced injury, a postrenal injury, glomerulitis, pyelonephritis, nephrotic syndrome, uremia, or the like.
In an example of the present invention, the kidney injury-related disease has a mitochondrial injury and at least one of the following symptoms: Proteinuria, edema, oliguria, high urea nitrogen, high creatinine, abnormal uric acid, kidney stone, and an abnormal glomerular filtration rate,
In another example of the present invention, the mitochondria are separated out from stem cells, such as adipose-derived stem cells, CD34+ hematopoietic stem cells, mesenchymal stem cells, bone stem cells, umbilical stem cells, amniotic stem cells, amniotic fluid stem cells, placental stem cells, iPS, or neural stem cells.
In the examples of the present invention, the dose of the mitochondria in the composition ranges from 5 μg to 80 μg, and is preferably 40 μg or more.
The beneficial effects of the present invention are:
When the mitochondrial damage occurs in kidney cells due to fibrosis, oxidative stress or inflammatory environment, administration of the mitochondrial extract or the composition containing the mitochondrial extract provided by the present invention can effectively improve the mitochondrial damage of kidney cells, therefore it can improve or treat renal cell damage or related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a statistical analysis result of a survival rate of renal epithelial cells subjected to treatment with different concentrations of hydrogen peroxide for 24 hours;

FIG. 1B shows a statistical analysis result of a survival rate of renal epithelial cells subjected to treatment with different concentrations of hydrogen peroxide and then administered with different doses of mitochondrial precipitates;

FIG. 2A shows a detection and analysis result of a collagen secretion amount from the renal epithelial cells after the cells are subjected to treatment with different concentrations of Advanced glycation end-products (AGEs-B SA) for different lengths of time;

FIG. 2B shows a detection and analysis result of a collagen secretion amount from the renal epithelial cells after the cells are subjected to treatment with different concentrations of AGES-BSA and then administered with different doses of mitochondrial precipitates;

FIG. 2C shows a detection and analysis result of a collagen secretion amount from the renal epithelial cells after the cells are subjected to treatment with different concentrations of hydrogen peroxide and then administered with different doses of mitochondrial precipitates; and

FIG. 3 shows a detection and analysis result of a mitochondrial injury in the renal epithelial cells after the cells are subjected to treatment with hydrogen peroxide or AGEs-BSA and then administered with different doses of mitochondrial precipitates.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides a second use of a mitochondrial extract, which can effectively alleviate a kidney injury-related disease and prevent the progression of the disease by administering a certain amount of mitochondrial extracts or a composition containing the mitochondrial extracts to a patient having the kidney injury-related disease.
Generally speaking, the patient administration dose of the mitochondria disclosed in the present invention ranges from 5 μg to 80 μg, such as 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, 65 μg, 70 μg, or 80 μg, and preferably ranges from 15 μg to 40 μg. Moreover, in order to better treat or alleviate the kidney disease, the mitochondria disclosed in the present invention can be mixed with another component to prepare a composition, where the used component is preferably a material having growth factors, such as a blood product containing growth factors, platelet-rich plasma (PRP), plasma, serum, platelet-rich fibrin (PRF), or the like.
The “mitochondrial extract” mentioned in the present invention refers to mitochondria separated out from cells, and the used separation technique or method should be able to maintain the structural and functional integrity of the mitochondria. For those of ordinary skill in the art to which the present invention pertains, the separation technique or method may be physical or chemical.
The “cells” mentioned in the present invention refer to those having mitochondria, such as adipose-derived stem cells, mesenchymal stem cells, skeletal muscle cells, liver cells, kidney cells, fibroblasts, nerve cells, skin cells, blood cells, and the like.
The “composition” mentioned in the present invention refers to a medical compound, food, functional food, nutritional supplement, etc., and is formed by mixing different components according to different types, to obtain different dosage forms and different administration methods.
The “kidney injury-related disease” mentioned in the present invention refers to a disease caused by damage to kidney cells and has symptoms of a mitochondrial injury, such as renal fibrosis, renal inflammation, a renal disease, an acute kidney disease, a renal tubular injury, renal failure, a prerenal injury, a renal induced injury, a postrenal injury, glomerulitis, pyelonephritis, nephrotic syndrome, uremia, or the like.
In order to prove the efficacy of the mitochondrial extract disclosed in the present invention, detailed description is given below by using several examples with reference to the accompanying drawings.
The mitochondria used in the following examples are taken from human adipose-derived stem cells, but the mitochondria of the present invention are not limited to only coming from the human adipose-derived stem cells. That is, the mitochondria of the present invention can be taken from any human cell.
The doses of the mitochondria used in the following examples are merely exemplary, where the dose of 15 μg of the mitochondria is used as a low-dose representative and the dose of 40 μg is used as a high-dose representative; and the doses are not intended to limit the technical features of the present invention. That is, the doses of 5 μg to 80 μg of the mitochondria disclosed in the present invention can all achieve the effects to be achieved by the present invention.

Example 1: Culture of Renal Epithelial Cell Strains

The culture of renal epithelial cell strains was performed in a cell culture medium that contains MEM-α Earl's salt and 5% fetal calf serum in a 37° C. environment (containing 5% carbon dioxide). When the cells were cultured to reach the completeness of 80%, the cell culture medium was removed and the phosphate buffer solution was used for rinsing the cells. Afterwards, the phosphate buffer solution was removed and 0.25% trypsin or 2.21 mM EDTA was added in to react for 20 min, then MEM-α containing 5% fetal calf serum was added in to neutralize the trypsin, and cells in suspension were collected and centrifuged. Then, the cells were counted, and diluted by using MEM-α containing 5% fetal calf serum to a final concentration of 5×10⁴cells per ml, for use in the subsequent subculture or analysis.

Example 2: Extraction of Mitochondria

The human adipose-derived stem cells were cultured to obtain 1.5×10⁸cells, and the Duchenne phosphate buffer solution (DPBS) was used to flush the cells and then was removed. Trypsin was added in to react for 3 min, and then a stem cell culture liquid (Keratinocyte SFM (1X) liquid, bovine pituitary extract, or 10 wt % fetal calf serum) was added in to terminate the reaction. Afterwards, the cells were collected and centrifuged (600 g for 10 min), and the supernatant was removed. Then, 80 ml IBC-1 buffer solution (the buffer solution is compounded of 225 mM mannitol, 75 mM sucrose, 0.1 mM EDTA, and 30 mM Tris-HCl with pH of 7.4) was added to the cells, and centrifugation was conducted after homogenization, to obtain a precipitate which was the mitochondria (referred to as a mitochondrial precipitate in the following description). 1.5 ml IBC-1 buffer solution and a proteolytic enzyme inhibitor were added to the mitochondrial precipitate, and then the mitochondrial precipitate was placed aside in a 4° C. environment, for use in the following examples.

Example 3: Injury Test for Renal Epithelial Cells (1)

The renal epithelial cells cultured in Example 1 were subcultured in a 96-well plate, where the concentration per well was 5×10⁴cells/200 μL. After 8-hour culturing, the supernatant was removed and the phosphate buffer solution was used for rinsing. Afterwards, 2004, cell culture liquid not containing 5% fetal calf serum was added to each well for culturing for 8 hours, and after culturing, different concentrations of hydrogen peroxide (0.3 mM, 0.5 mM, 1 mM, 3 mM, 5 mM, and 10 mM) were separately added for treatment. After 24-hour culturing with the different concentrations of hydrogen peroxide, the supernatant was removed from each well and a cell culture liquid (100 μL/well) containing 10% alamar blue was added. After culturing for 3 to 4 hours, fluorescent signal measurement (Excitation/Emission: 560/590 nm) was performed, to obtain a result shown in FIG. 1A.
It can be known from the result of FIG. 1A that, after the renal epithelial cells are subjected to treatment with different concentrations of hydrogen peroxide for 24 hours, these cells are likely to get injured, where when the concentration of the hydrogen peroxide is 1 mM or more, there is a significant increase in death of the renal epithelial cells due to the injury. Specifically, after the renal epithelial cells are treated for 24 hours by using hydrogen peroxide with a concentration of 1 mM, the survival rate of the renal epithelial cells is 81.4%; after the renal epithelial cells are treated for 24 hours by using hydrogen peroxide with a concentration of 5 mM, the survival rate of the renal epithelial cells is 31.3%; and after the renal epithelial cells are treated for 24 hours by using hydrogen peroxide with a concentration of 10 mM, the survival rate of the renal epithelial cells is 24.2%. Such a result shows that treating the renal epithelial cells with the hydrogen peroxide can indeed construct an injury and death pattern of the renal epithelial cells; and moreover, as the concentration of the added hydrogen peroxide increases, the injury in the renal epithelial cells worsens and the number of dying renal epithelial cells also rises.

Example 4: Injury Test for Renal Epithelial Cells (2)

The renal epithelial cells cultured in Example 1 were subcultured in a 96-well plate, where the concentration per well was 1×10⁴cells/200 μL. After 8-hour culturing, the supernatant was removed and the phosphate buffer solution was used for rinsing. Afterwards, 2000 μL cell culture liquid not containing 5% fetal calf serum was added to each well for culturing for 8 hours. After culturing, hydrogen peroxide with the concentrations of 1 mM and 3 mM was separately added in for treatment for 4 hours, and then different doses (15 μg and 40 μg) of mitochondrial precipitates (prepared in Example 2) were administered to the cells treated with the different concentrations of hydrogen peroxide, to perform culturing for 24 hours. After culturing completion, the supernatant was removed and a cell culture liquid (100 μL/well) containing 10% alamar blue was added. After culturing for 3 to 4 hours in a 37° C. environment, fluorescent signal measurement (Excitation/Emission: 560/590 nm) was performed after culturing completion, to obtain a result shown in FIG. 1B.
It can be known from the result of FIG. 1B that, the survival rate of the renal epithelial cells decreases because the cells are injured after treatment with the hydrogen peroxide. When the renal epithelial cells are injured due to induction by the hydrogen peroxide and then a certain amount of mitochondrial precipitates are added, the survival rate of the renal epithelial cells can be significantly improved. Moreover, as the administration dose increases, the survival rate of the renal epithelial cells also rises.
It can be known from the result that, the mitochondrial extract disclosed in the present invention can indeed protect the renal epithelial cells against injuries caused by oxidation or inflammation, and can repair the injured renal epithelial cells, thus efficiently avoiding the renal epithelial cells from death. In other words, the mitochondrial extract disclosed in the present invention or a composition containing the mitochondrial extract can indeed alleviate and/or prevent a kidney injury or kidney disease due to oxidative stress.

Example 5: Fibrosis Test for Renal Epithelial Cells (1)

The renal epithelial cells cultured in Example 1 were cultured in a 6-well plate by using a culture liquid containing 5% fetal calf serum, where the concentration per well was 1×10⁵cells/2 ml. After 24-hour culturing, the supernatant was removed and the phosphate buffer solution was used for rinsing. Afterwards, 1 ml cell culture liquid not containing 5% fetal calf serum was added to each well for culturing for 8 hours. Then, different concentrations (100 μg/ml and 400 μg/ml) of Advanced Glycation End product-BSA (AGES-BSA) were administered to perform culturing for 4 hours. After culturing completion, the cell culture medium containing AGEs-B SA was removed and the phosphate buffer solution was used for rinsing. Then, 1 ml cell culture liquid not containing 5% fetal calf serum was added to each well for culturing for 24 and 48 hours separately, and the supernatant was separately collected after culturing completion. A collagen secretion assay was performed by using the Sircol™ Soluble Collagen Assay Kit, to obtain a result shown in FIG. 2A.
It can be known from the result of FIG. 2A that, whether the renal epithelial cells are treated with a high (400 μg/ml) or low (100 μg/ml) concentration of AGEs-B SA, the secretory expression amount of collagen from the renal epithelial cells can be improved, and rises as the treatment time increases. It indicates that AGEs-BSA can indeed induce lesions to the renal epithelial cells and cause fibrosis, which is the early stage of the chronic kidney disease. Moreover, if the secretory expression amount of collagen continuously increases, the chronic kidney disease is caused.

Example 6: Fibrosis Test for Renal Epithelial Cells (2)

The process of this example was substantially identical with that in Example 5, but had the following differences. In this example, after the cell culture medium containing AGEs-B SA was removed, a cell culture liquid not containing 5% fetal calf serum and different doses (15 μg and 40 μg) of mitochondrial precipitates (prepared in Example 2) were added to each well for separately culturing for 24 hours. After culturing completion, the supernatant was separately collected and a collagen secretion assay was performed by using the soluble collagen assay kit, to obtain a result shown in FIG. 2B.
It can be known from the result of FIG. 2B that, the administration of the mitochondrial precipitates can reduce the amount of collagen secretion induced by AGEs-BSA in the renal epithelial cells, which indicates that the mitochondria or a composition containing the mitochondria disclosed in the present invention can efficiently alleviate and/or prevent kidney fibrosis or the related kidney diseases.

Example 7: Fibrosis Test for Renal Epithelial Cells (3)

The process of this example was substantially identical with that in Example 6, but had the following difference. The stimulant that induces fibrosis in the renal epithelial cells was changed from AGEs-BSA to hydrogen peroxide, to obtain a result shown in FIG. 2C.
It can be known from the result of FIG. 2C that, the treatment of the renal epithelial cells with the hydrogen peroxide can induce an increase in the collagen secretion amount, that is, the treatment of the renal epithelial cells with the hydrogen peroxide can indeed construct a pattern of renal cell fibrosis. After mitochondrial precipitates are administered to the renal epithelial cells subjected to treatment with the hydrogen peroxide, the collagen secretion amount in the renal epithelial cells can be obviously reduced, which indicates that the mitochondria or a composition containing the mitochondria disclosed in the present invention can efficiently alleviate and/or prevent kidney fibrosis or the related kidney diseases.

Example 8: Functional Injury Test of Mitochondria in the Renal Epithelial Cells

The renal epithelial cells cultured in Example 1 were subcultured in a 96-well plate by using a cell culture liquid containing 5% fetal calf serum, where the concentration per well was 5×10⁴cells/200 μL. After 24-hour culturing, the supernatant was removed and the phosphate buffer solution was used for rinsing; and then 1 ml cell culture liquid not containing 5% fetal calf serum was added to each well to perform culturing for 8 hours. After culturing, 3 mM hydrogen peroxide and 100 μg/ml AGEs-BSA were separately administered to perform culturing for 4 hours; and then the cell culture liquid containing hydrogen peroxide or AGEs-BSA was removed, and the phosphate buffer solution was used for rinsing. Afterwards, 1 ml cell culture liquid not containing 5% fetal calf serum and different doses (15 μg and 40 μg) of mitochondrial precipitates (prepared in Example 2) were added to each well, to perform culturing for 24 hours separately. After culturing completion, the phosphate buffer solution was used for rinsing, and a buffer solution containing 10 μM JC-1 staining reagent was then added in to react for 10 min at 37° C. After rinsing, fluorescent signal measurement (Excitation/Emission: 488/530 nm) was performed, to obtain a result shown in FIG. 3 .
It can be known from the result of FIG. 3 that the expression level of the JC-1 monomer in the renal epithelial cells subjected to treatment with only the hydrogen peroxide or AGEs-BSA increases, which indicates that mitochondria in the renal epithelial cells are injured due to an inflammatory environment caused by the hydrogen peroxide or AGEs-BSA. When the renal epithelial cells are treated first by using the hydrogen peroxide or AGEs-BSA and then by using different doses of mitochondrial precipitates, the expression level of the JC-1 monomer therein obviously decreases. Moreover, as the dose of the mitochondrial precipitates increases, the expression level of the JC-1 monomer in the renal epithelial cells decreases.

Example 9: Experiment on Animals

10-week-old C57BL/6 mice were used and raised at constant temperature and humidity, with a 12-hour light/dark cycle. An ischemia-reperfusion (referred to as an “I/R kidney injury pattern” in the following description) pattern was used as the kidney injury pattern of the mice, and the steps were as follows: First, intraperitoneal injection was conducted and 150 mg/Kg phenobarbital was injected into the abdomen of each mouse. When the mouse was unconscious, surgery was performed on the left kidney of the mouse. The left kidney was moved to the outside from the incision, and then the blood vessel for the renal artery to flow into the kidney was blocked with a vascular clamp. After blocking for 30 min, the vascular clamp was removed to allow the blood flow to pass, thus completing the I/R kidney injury pattern.
For the mice subjected to treatment of the UR kidney injury pattern, different doses (15 μg and 40 μg) of mitochondria were delivered to the kidney by means of injection through the renal artery vessel, to form a high mitochondrial dose group and a low mitochondrial dose group. For the control group (I/R group), the phosphate buffer solution was injected. After completion of treatment for the mice in each group, the kidneys were separately moved back into the bodies and the wounds were sutured; and on the first day (D1) and the second day (D2) after surgery for each group of mice, the blood samples were collected, and the serum creatinine (Cr) and the blood urea nitrogen (BUN) were measured. Afterwards, the drawn blood was centrifuged and the serum was collected after separation, to analyze the contents of urea nitrogen and creatinine in the serum, where the detection of serum creatinine was made by using a mouse creatinine analysis kit (Brand: Crystal Chem; Model: 80350) and the detection of urea nitrogen was made by using a urea analysis kit (Brand: abcam; Model: ab83362). When the mice in each group were sacrificed on the 7^thday (D7) after transplantation, the left kidney of each mouse was perfused and fixed with formalin, then was paraffin-embedded and histologically sectioned, and finally was subjected to H&E staining. According to morphological changes caused by an ischemic injury based on histological studies for the staining result, the degree of kidney injuries was scored as follows by using the Jablonski's semi-quantitative criteria: 0 for normal tissue; 1 for the renal tubular injury area of less than 5%; 2 for the renal tubular injury area of more than 5% to less than 25%; 3 for the renal tubular injury area of greater than 25% to less than 75%; and 4 for the renal tubular injury area of greater than 75%.
The foregoing results are shown in the following table 1. It can be known from the results of table 1 that, compared to the control group, the expression amounts of the urea nitrogen and the creatinine in the serum of the FR group obviously increase, which indicates that the FR kidney injury pattern can indeed cause a kidney injury. Moreover, it can be known from the scoring result of the kidney injury that, the score of the FR group is 3-4, indicating a severe renal tubular injury. However, compared to the FR group, the contents of urea nitrogen and creatinine in the serum of the mice in the group administered with the mitochondria obviously decrease, which indicate that the administration of the mitochondria can effectively alleviate the kidney injury. It can be further known from the scoring result of the kidney injury that, the administration of the mitochondria can recover the injured kidney cells and alleviate the renal tubular injury; and as the administration dose of the mitochondria increases, a better effect of alleviating the kidney injury can be achieved.

TABLE 1

Analysis results of serum and kidney tissue sections
after different treatment for the mice in each group

		Low	High
		mitochondrial	mitochondrial
		dose group	dose group
Control group	I/R group	(15 μg)	(40 μg)

D1: Urea	19 ± 1.8	61 ± 2.6	25 ± 2.3	24 ± 2.4
nitrogen in the
serum (mg/dL)
D2: Urea	21 ± 1.6	119 ± 2.2	63 ± 1.9	41 ± 2.1
nitrogen in the
serum (mg/dL)
D1: Creatinine	0.23 ± 0.024	0.41 ± 0.021	0.31 ± 0.023	0.29 ± 0.025
in the serum
(mg/dL)
D2: Creatinine	0.22 ± 0.03	0.59 ± 0.022	0.42 ± 0.026	0.34 ± 0.031
in the serum
(mg/dL)
D7: Scores of	0	3.7 ± 0.32	2.9 ± 0.29	1.8 ± 0.35
kidney injury

The foregoing results show that, when the mitochondria in the kidney cells are injured due to fibrosis, oxidative stress or inflammation, the administration of the mitochondrial extract disclosed in the present invention or a composition containing the mitochondrial extract can effectively alleviate the mitochondrial injury in the kidney cells, thus alleviating or treating the kidney cell injury or its related diseases.

Claims

What is claimed is:

1. A use of mitochondria to prepare a composition for preventing and/or treating a kidney injury-related disease.

2. The use of claim 1, wherein the kidney injury-related disease is kidney fibrosis.

3. The use of claim 1, wherein the kidney injury-related disease is kidney inflammation.

4. The use of claim 1, wherein the kidney injury-related disease has a symptom of a mitochondrial injury.

5. The use of claim 1, wherein the kidney injury-related disease is a chronic kidney disease.

6. The use of claim 1, wherein the kidney injury-related disease is a renal tubular injury.

7. The use of claim 6, wherein the symptom of the renal tubular injury is that the injury area is greater than 25%.

8. The use of claim 1, wherein the dose of the mitochondria in the composition is 5 μg or more.

9. The use of claim 1, wherein the dose of the mitochondria in the composition is 40 μg or more.