Preparation method of autologous mitochondria preparation and application thereof

Abstract

The application discloses a preparation method and application of an autologous mitochondrion preparation, relates to the technical field of kidney protection and regeneration, and is characterized in that the mitochondrion is derived from heart tissue of a healthy C57 mouse of 8-10 weeks old, is obtained through a kit combined with a differential centrifugation method, and is characterized in that the obtained mitochondrion is continuous in outer membrane and clear in ridge structure under an electron microscope, and has good structural and functional integrity. In an in-vivo experiment, the mitochondrion is delivered to the renal cortex region of a recipient mouse through a subcapsular injection method 30 minutes before ischemia-reperfusion, and the result shows that the blood creatinine and urea nitrogen levels can be significantly reduced, the necrosis and oxidative stress damage of renal tubular epithelial cells can be reduced, and the renal tissue pathological score can be improved. The application provides a repeatable and standardizable organelle treatment strategy for intervention of kidney transplantation and acute kidney injury related IRI.

Description

Technical Field

[0001] This invention relates to the field of kidney protection and regeneration technology, and more specifically to a method for preparing an autologous mitochondrial preparation and its application. Background Technology

[0002] Renal ischemia-reperfusion injury (IRI) is a crucial pathological basis for acute kidney injury (AKI) and delayed graft function (DGF) after kidney transplantation. It can occur in various clinical settings, including kidney transplantation, partial nephrectomy, hemorrhagic shock, severe infection, and major surgery. During the ischemic phase, interruption of oxygen supply leads to rapid depletion of intracellular ATP, disrupted ion homeostasis, and damage to the cytoskeleton. During the reperfusion phase, the restoration of oxygen supply is accompanied by a surge in reactive oxygen species (ROS), calcium overload, and activation of the inflammatory cascade, further inducing necrosis, apoptosis, or programmed necrosis of tubular epithelial cells (TECs), ultimately resulting in decreased renal function. Although current clinical management of AKI / IRI primarily focuses on supportive care, effective interventions targeting its core pathogenesis are lacking. Therefore, developing new treatment strategies is of great significance.

[0003] Mitochondria are the core organelles for energy metabolism in renal tubular epithelial cells. The proximal tubules of the kidney are highly dependent on oxidative phosphorylation (OXPHOS) for energy, making them particularly sensitive to ischemic injury. Under ischemic conditions: electron transport chain function is suppressed; ATP synthesis is reduced; mitochondrial membrane potential (ΔΨm) decreases; mitochondrial permeability transition pore (mPTP) opens abnormally; and cytochrome c is released, activating the apoptosis pathway. During reperfusion, incomplete restoration of the electron transport chain leads to increased electron leakage, resulting in a significant increase in ROS production, further damaging mitochondrial DNA and membrane lipids, and exacerbating cell damage. Studies have shown that mitochondrial quality control imbalance is closely related to the occurrence of intracellular renal inflammatory response (IRI), including abnormal mitochondrial fusion / division, impaired autophagy, and respiratory chain dysfunction. Therefore, restoring mitochondrial structure and function is considered a key step in intervening in IRI.

[0004] Current intervention strategies for IRI mainly include: antioxidants; anti-inflammatory drugs; ischemic preconditioning or remote preconditioning; and single-molecule target intervention. Although some methods have shown protective effects in animal experiments, their clinical translation is often limited by factors such as administration duration, individual differences, and efficacy stability. Furthermore, IRI involves multi-pathway and multi-stage pathological changes, making it difficult to achieve comprehensive restoration of mitochondrial function by targeting only a single molecule or signaling pathway. Therefore, how to directly supplement or replace damaged mitochondrial function in the early stages of injury has become an important research direction. Mitochondrial transplantation is a recently proposed organelle-level therapeutic strategy. Its basic principle is to isolate structurally and functionally intact mitochondria from healthy tissue and deliver them to damaged tissue to enhance the oxidative phosphorylation capacity of recipient cells and improve tissue function.

[0005] In myocardial ischemia-reperfusion models, studies have confirmed that autologous mitochondrial injection can improve myocardial function and reduce infarct size. Related reviews indicate that mitochondrial transplantation has the potential to reduce tissue necrosis, suppress inflammatory responses, and promote functional recovery in various ischemia-related disease animal models. In the field of kidney injury, studies have attempted to deliver mitochondria via the renal artery route to improve a rat ischemia-reperfusion injury (IRI) model, showing a reduction in renal tubular cell death and improved renal function. However, vascular delivery may be affected by factors such as circulating dilution, reticuloendothelial system clearance, and uneven local perfusion, and carries a potential risk of microembolism. Therefore, exploring more localized, controllable, and reproducible delivery methods is of practical significance. The inventors discovered that subcapsular injection of mitochondria can smoothly and rapidly enter renal tubular epithelial cells without significant kidney damage and has a preventive effect against IRI injury. Furthermore, previous studies by the inventors found that in an autologous kidney transplantation model, perfusion of the donor kidney with mitochondria also has a significant preventive effect against renal ischemia-reperfusion injury. To verify the safety and efficacy of the mitochondrial formulation of this invention, the inventors constructed a transplantation model using C57BL / 6 mice as both donor and recipient. In this model, the major histocompatibility complexes of the donor mice (mitochondrial donors) and recipient mice (mitochondrial recipients) were completely identical, and no immune rejection occurred. This model can effectively simulate the immune microenvironment of autologous mitochondrial transplantation in clinical practice. Therefore, the technical solution verified by this invention can be reasonably expected to be applied to human autologous mitochondrial transplantation.

[0006] The source of mitochondrial donors directly affects the yield and functional activity of the formulation. Myocardial tissue is a high-energy-consuming tissue with a high mitochondrial density and strong oxidative phosphorylation capacity, making it theoretically more suitable as a mitochondrial donor source. Furthermore, subcapsular renal injection can create a locally closed microenvironment in the renal cortex, which is conducive to the retention and uptake of exogenous mitochondria in the target area, potentially achieving stable protective effects at lower doses. Currently, systematic studies on autologous cardiac mitochondria combined with subcapsular renal injection to improve mouse IRI models lack standardized preparation and quality control protocols. Therefore, it is necessary to establish a mitochondrial transplantation strategy with a clearly defined source, stable preparation, controllable delivery, and a comprehensive efficacy evaluation system.

[0007] Therefore, it is necessary to propose a method for preparing autologous mitochondrial preparations and their applications to solve the above problems. Summary of the Invention

[0008] The purpose of this invention is to solve the problems of poor efficacy, poor targeting, and limited delivery methods in existing intervention methods for renal IRI.

[0009] To achieve the above objectives, the present invention specifically adopts the following technical solution:

[0010] The autologous heart-derived mitochondrial preparation uses mitochondria derived from the heart tissue of healthy C57 mice. Extraction was performed using differential centrifugation and a mitochondrial isolation kit. Electron microscopy confirmed the continuity of the outer membrane and the clear cristae structure, indicating good structural integrity. The concentration is 5 × 10⁻⁶ mitochondria per milliliter. 5 One mitochondria.

[0011] Furthermore, the mitochondria are further labeled with MitoTracker fluorescent markers to trace their distribution in kidney tissue and their uptake by renal tubular epithelial cells.

[0012] Furthermore, the mitochondria were isolated from the heart tissue of healthy male C57 mice aged 8-10 weeks.

[0013] Furthermore, the mitochondria are stored in a pre-cooled mitochondrial storage solution, which can be preserved for a short period at 4°C or for a long period in liquid nitrogen.

[0014] The application of autologous heart-derived mitochondria in the prevention and treatment of renal ischemia-reperfusion injury, the application including the delivery of the above-described mitochondrial preparation to the renal cortex region of recipient mice via subcapsular injection.

[0015] Furthermore, the subcapsular injection dose is 0.2 ml per kidney.

[0016] Furthermore, the injection timing is 30 minutes before the end of the renal ischemia period and the start of reperfusion.

[0017] Furthermore, the application can reduce serum creatinine and blood urea nitrogen levels, and alleviate renal tubular epithelial cell necrosis and oxidative stress damage.

[0018] Furthermore, the application can serve as a therapeutic strategy at the organelle level to improve renal ischemia-reperfusion injury.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. This invention selects heart tissue as the mitochondrial donor. Myocardium is a high-energy-consuming tissue with high mitochondrial density and strong oxidative phosphorylation activity, making it possible to obtain mitochondrial preparations with higher yield and stronger function compared to other tissues.

[0021] 2. This invention pioneers a subcapsular renal injection delivery route. This method can create a localized high-concentration microenvironment in the renal cortex, effectively avoiding the risks of dilution, clearance, and embolism associated with vascular routes, and significantly improving the retention rate of mitochondria in target tissues and cellular uptake efficiency.

[0022] 3. In this invention, a transplantation model is constructed using C57BL / 6 mice as both donor and recipient. The major histocompatibility complexes of the donor and recipient mice are completely identical, and there is no immune rejection reaction. Attached Figure Description

[0023] Figure 1 The aggregation and distribution of exogenous mitochondria injected subcapsularly in mouse kidney tissue.

[0024] Figure 2 Mitochondria extracted from mouse heart tissue can be rapidly introduced into the TEC.

[0025] Figure 3 The results are for serum creatinine and blood urea nitrogen in mice.

[0026] Figure 4 The results of ELISA for serum kidney injury markers in mice.

[0027] Figure 5 The results are WB and PCR of mouse kidney tissue.

[0028] Figure 6 To evaluate the protective effect of subcapsular mitochondrial infusion on the kidneys of IRI mice by verifying renal tubular injury scores, TEC apoptosis, and renal lipid peroxidation levels through kidney tissue sections. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Please see Figures 1 to 6 A method for preparing an autologous mitochondrial preparation and its application. Unless otherwise specified, all reagents used are commercially available analytical grade reagents.

[0031] 1. Mitochondrial extraction and quality control: Healthy male C57 mice aged 8–10 weeks were selected as mitochondrial donors. After anesthesia, the mice underwent rapid thoracotomy to remove heart tissue, which was immediately placed in pre-cooled PBS buffer to remove residual blood. Large blood vessels and adipose tissue were removed, and the myocardial tissue was retained for later use. The entire procedure was performed at 4°C or on ice to maximize mitochondrial activity. The myocardial tissue was homogenized and then minced to approximately 1 mm. 3 Size; add pre-chilled mitochondrial separation buffer; homogenize using a mechanical homogenizer at 4°C (60Hz, 30s) until the tissue is in a homogeneous suspension. Centrifuge the tissue differentially, first at 1000g, 4°C, 5min to remove cell nuclei and undisturbed tissue. Collect the supernatant, then centrifuge at 3500g, 4°C, 10min to obtain mitochondrial precipitate. Discard the supernatant; gently resuspend in pre-chilled mitochondrial storage solution; keep on ice for later use.

[0032] 2. Establishment of a renal ischemia-reperfusion injury model and subcapsular injection of mitochondria into the kidney.

[0033] Healthy C57 mice of the same strain were selected as recipients. After anesthesia, a midline abdominal incision was made to expose both kidneys and their pedicles. The renal pedicles were clamped with non-invasive vascular clamps for 20 minutes to establish an ischemia model. The clamps were then released to restore blood flow and complete reperfusion, thus establishing a renal IRI model. Thirty minutes before reperfusion ended, mitochondrial transplantation was performed. A Hamilton's needle was inserted parallel to the renal capsule under a microscope, and the subcapsular space was gently dissected to form a 5×10⁻⁶ mitochondrial graft. 5 0.2 ml of a mitochondrial solution at a concentration of / ml was injected into the renal capsule, ensuring the capsule remained filled without rupture or fluid leakage. The following experimental groups were established: the Sham group (kidney exposed only, without clamping), the IRI group, and the IRI + mitochondrial group. Simultaneously, the IRI group and the IRI + normal saline (NS) group were established to verify that the subcapsular injection method had no significant impact on the results.

[0034] 3. Verification of mitochondrial entry into renal tubular epithelial cells. Donor mitochondria were labeled using MitoTracker series fluorescent dyes, membrane-bound fluorescent dyes, or mitochondrial-specific fluorescent proteins, followed by subcapsular injection into the kidney. After administration, kidney tissue was harvested and sections prepared. Combined with nuclear staining and immunofluorescence staining with renal tubular markers, the localization of exogenous mitochondria in the renal cortex and renal tubular epithelial cells was observed using a fluorescence microscope. Results are as follows: Figure 1 , 2 As shown.

[0035] 4. Evaluation of the efficacy of mitochondrial transplantation in improving renal intraepithelial neoplasia (IRI)

[0036] Blood samples were collected 24 h after reperfusion to detect serum creatinine and blood urea nitrogen (BUN), and serum samples were collected to detect KIM-1 and NGAL injury markers. Simultaneously, protein and RNA were extracted from kidney tissue, and Western blot and PCR analyses were performed to analyze the protein expression and RNA content of KIM-1 and NGAL. The results showed that the levels of serum creatinine (Scr), blood urea nitrogen (BUN), and kidney injury markers were significantly reduced in the mitochondrial transplantation group. Figures 3 to 5 As shown in the figure. Kidney tissue was harvested 24 hours after ischemia-reperfusion for HE staining. Semi-quantitative damage scoring was performed based on indicators such as renal tubular dilation, brush border detachment, cast formation, and necrosis. Results showed that renal tubular damage was significantly reduced in the mitochondrial transplantation group, as shown in the figure. Figure 6 As shown.

[0037] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A method for preparing an autologous mitochondrial preparation, characterized in that: The mitochondria were derived from the heart tissue of healthy C57 mice and extracted using differential centrifugation combined with a mitochondrial isolation kit; their concentration was 5 × 10⁻⁶ per milliliter. 5 One mitochondria.

2. The method for preparing an autologous mitochondrial preparation according to claim 1, characterized in that: The mitochondria are further labeled with MitoTracker fluorescent markers to trace their distribution in kidney tissue and their uptake by renal tubular epithelial cells.

3. The method for preparing an autologous mitochondrial preparation according to claim 2, characterized in that: The mitochondria were isolated from the heart tissue of healthy male C57 mice aged 8-10 weeks.

4. The method for preparing an autologous mitochondrial preparation according to claim 3, characterized in that: The mitochondria are stored in a pre-cooled mitochondrial storage solution, which can be preserved for a short period at 4°C or for a long period in liquid nitrogen.

5. The application of a mitochondrial preparation made from an autologous mitochondrial agent in the prevention and treatment of renal ischemia-reperfusion injury, characterized in that: The application includes delivering the mitochondrial formulation of any one of claims 1 to 4 to the renal cortex region of recipient mice via subcapsular injection.

6. The application according to claim 5, characterized in that: The dosage for subcapsular injection into the kidneys is 0.2 ml per kidney.

7. The application according to claim 6, characterized in that: The injection was administered 30 minutes before the end of the renal ischemia period and the start of reperfusion.

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