A method for extracting mitochondria-associated membranes and other subcellular components from Sertoli cells

Abstract

The application discloses a method for extracting mitochondria-associated membranes (MAM) and other subcellular components from podocytes, which comprises the following steps: pretreating the podocytes, crushing the podocytes by low-power ultrasonic, separating the crude mitochondrial components by differential centrifugation, and then purifying the high-purity MAM and other subcellular components by density gradient centrifugation and ultracentrifugation. The method replaces the traditional homogenizer grinding with low-power ultrasonic crushing, improves the MAM extraction yield by controlling the ultrasonic parameters, appropriately simplifies the differential centrifugation steps, increases the yield while ensuring the purity, sequentially separates the unbroken cells, the crude mitochondria, the cytoplasm and the endoplasmic reticulum components, and finally obtains the high-purity MAM and other subcellular components by density gradient centrifugation and ultracentrifugation purification. The method effectively improves the yield of the podocyte separation and extraction of the MAM and other subcellular components, reduces the cost, and provides technical support for the research on the podocyte MAM proteomics.

Description

Technical Field

[0001] This invention relates to a method for extracting mitochondrial-associated membranes and other subcellular components, and particularly to a method for extracting mitochondrial-associated membranes and other subcellular components from podocytes. This invention belongs to the field of biotechnology. Background Technology

[0002] The mitochondrial-associated membrane (MAM) is a dynamic contact site between the outer mitochondrial membrane and the endoplasmic reticulum (ER). As a core hub for substance exchange, signal transduction, and energy metabolism regulation between mitochondria and the ER, it plays an irreplaceable role in key physiological processes such as calcium homeostasis, lipid synthesis, and mitochondrial physiology. [1] In the field of kidney disease research, podocyte damage and dysfunction are core components of pathological processes such as proteinuria and glomerulosclerosis, and the disorder of MAM structure and function has been proven to be a potential regulator of podocyte damage. [2–4] Therefore, efficiently obtaining high-purity podocyte MAM components is of significant value for elucidating the pathogenesis of kidney diseases and developing targeted therapies.

[0003] Currently, although methods for extracting MAM cells have been reported in the literature, [5] However, these methods have significant drawbacks when applied to podocytes: Firstly, MAMs, as subcellular organelles, are present in small amounts within cells, thus requiring a large number of cells for extraction. Furthermore, the in vitro differentiation culture of podocyte lines requires a 10-14 day induction period, and the culture process necessitates the use of premium fetal bovine serum and specialized culture medium supplemented with insulin-transferrin-selenium (ITS) factors, resulting in extremely high reagent and time costs. Secondly, existing methods mostly employ glass homogenizers (…). Figure 1 Mechanical grinding is used to disrupt cells, but a large amount of sample adheres to the walls of the homogenizer and the grinding pestle, resulting in high sample loss. This not only causes serious waste of valuable samples but also affects subsequent proteomics and molecular interaction experiments due to insufficient extraction. In addition, the intensity of homogenization is difficult to control precisely, which can easily lead to excessive damage to the mitochondrial and endoplasmic reticulum structures, reducing the integrity and purity of the MAM (mitochondrial matrix endothelial cells), making it difficult to ensure parallel reproducibility between groups, and further affecting the reliability of experimental results.

[0004] Therefore, considering the high cost and preciousness of podocyte culture samples, this invention overcomes the shortcomings of existing homogenization and disruption methods for extracting podocyte MAMs, such as high sample loss, low yield, and insufficient purity. It provides a method for extracting podocyte MAMs based on low-power ultrasonic disruption combined with fractional centrifugation. This method optimizes the cell disruption method and separation process, reducing sample loss while improving the yield and purity of MAMs, providing reliable technical support for MAM research in glomerular diseases.

[0005] References:

[0006] [1]Wang N, Wang C, Zhao H, He Y, Lan B, Sun L, et al. The MAMsStructure and Its Role in Cell Death. Cells 2021;10:657. https: / / doi.org / 10.3390 / cells10030657.

[0007] [2]Li X, Yang Q, Liu S, Song S, Wang C. Mitochondria-associatedendoplasmic reticulum membranes promote mitochondrial fission through AKAP1-Drp1 pathway in podocytes under high glucose conditions. Exp Cell Res 2023;424:113512. https: / / doi.org / 10.1016 / j.yexcr.2023.113512.

[0008] [3]Cao Y, Chen Z, Hu J, Feng J, Zhu Z, Fan Y, et al. Mfn2 RegulatesHigh Glucose-Induced MAMs Dysfunction and Apoptosis in Podocytes via PERKPathway. Front Cell Dev Biol 2021;9:769213. https: / / doi.org / 10.3389 / fcell.2021.769213.

[0009] [4]Wei X, Wei 2020;105:154182. https: / / doi.org / 10.1016 / j.metabol.2020.154182.

[0010] [5] Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P. Isolation of mitochondria-associated membranes and mitochondria from animaltissues and cells. Nat Protoc 2009;4:1582–90. https: / / doi.org / 10.1038 / nprot.2009.151. Summary of the Invention

[0011] The purpose of this invention is to provide a method for extracting mitochondrial-associated membranes and other subcellular components from podocytes.

[0012] To achieve the above objectives, the present invention employs the following technical means:

[0013] The present invention discloses a method for extracting mitochondrial-associated membranes and other subcellular components from podocytes. The method includes podocyte pretreatment, podocyte disruption by low-power ultrasound, separation of crude mitochondrial components from the ultrasound-treated cell homogenate by differential centrifugation, resuspension of the crude mitochondrial components, separation of MAM and pure mitochondria by density gradient centrifugation and ultracentrifugation, and finally purification by centrifugation to obtain high-purity MAM and pure mitochondrial components.

[0014] Preferably, the podocyte pretreatment includes the steps of digesting the podocytes with trypsin-EDTA digestion solution, centrifuging, and resuspending the cell pellet in pre-cooled separation reagent 1, wherein the separation reagent 1 contains 225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA and 30 mM Tris–HCl, pH 7.4.

[0015] Preferably, the ultrasonic disruptor used for disrupting podocytes with low power ultrasonication has a power of ≤20W, and the ultrasonication is performed 3-5 times, with each ultrasonication lasting 2 seconds and an interval of 5 seconds. After each ultrasonication, trypan blue staining solution is mixed with the cell suspension, and the proportion of trypan blue-stained positive cells is observed. If the proportion of positive cells is insufficient, ultrasonic disruption is continued. When the proportion of positive cells is 50-60%, ultrasonication is stopped.

[0016] Preferably, the differential centrifugation involves centrifuging the sonicated cell homogenate at 750g for 10 minutes at 4°C, discarding the precipitate and retaining the supernatant; then centrifuging the supernatant at 11000g for 10 minutes at 4°C to obtain the crude mitochondrial extract.

[0017] Preferably, the separation of MAM and pure mitochondria by density gradient centrifugation and ultracentrifugation involves first adding Percoll separation buffer to the ultracentrifuge tube, then slowly adding the resuspended crude mitochondrial component along the tube wall, and then slowly adding mitochondrial resuspension buffer on top to form a three-layer liquid structure; centrifugation results in distinct stratified bands, with the upper milky white band containing the MAM component and the lower milky white band containing the pure mitochondrial component.

[0018] Preferably, the centrifugal purification involves diluting the collected MAM bands with mitochondrial resuspension buffer, centrifuging them at 6300g for 10 min at 4°C in a high-speed centrifuge, collecting the supernatant, centrifuging it at 100000g for 1 h at 4°C with a deceleration level of 3, and the flaky precipitate floating at the bottom of the tube being the MAM component.

[0019] Preferably, the other subcellular components include cytoplasm, pure mitochondria, and endoplasmic reticulum.

[0020] Preferably, the method includes the following steps:

[0021] (1) Pretreatment of podocytes

[0022] 1) Collect podocytes differentiated for 10-14 days using calcium-free methods. 2+ and Mg 2+ After washing the cells with DPBS buffer, trypsin-EDTA digestion solution was added. After digestion, culture medium was added to stop the digestion and the cells were pipetted.

[0023] 2) Collect the cell suspension and centrifuge, discard the supernatant and resuspend all cells in a solution containing Ca. 2+ and Mg 2+ Centrifuge again in DPBS buffer, discard the supernatant, and resuspend all cells in a solution containing Ca. 2+ and Mg 2+ Centrifuge in DPBS buffer;

[0024] 3) Discard the supernatant and resuspend the cell pellet in pre-cooled separation reagent 1, which contains 225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA and 30 mM Tris–HCl, pH 7.4;

[0025] (2) Low-power ultrasound fragmentation of foot cells

[0026] 1) Mix trypan blue staining solution with cell resuspension solution;

[0027] 2) Place the cell suspension on ice to lyse, then adjust the power of the sonicator to ≤20W and sonicate the cell suspension 3-5 times, 2 seconds each time, with a 5-second interval; after each sonication, mix trypan blue staining solution with the cell suspension and observe the proportion of trypan blue positive cells. If the proportion of positive cells is insufficient, continue sonication. Stop sonication when the proportion of positive cells is 50-60%.

[0028] (3) Differential centrifugation to separate crude mitochondrial fractions

[0029] 1) Centrifuge the sonicated cell homogenate at 750g for 10min at 4℃. The precipitate consists of cell nuclei and unbroken cells. The supernatant contains cytoplasm and organelles. Discard the precipitate and keep the supernatant.

[0030] 2) Transfer the supernatant to 11000g and centrifuge for 10 min at 4℃. After centrifugation, the precipitate is crude mitochondria, and the supernatant contains cytoplasm and endoplasmic reticulum components.

[0031] 3) Add the pre-cooled separation reagent 2 solution to the supernatant obtained in step 2), mix gently and set aside; the separation reagent 2 contains 225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl, pH 7.4;

[0032] 4) Add pre-cooled mitochondrial resuspension buffer to the crude mitochondrial precipitate obtained in step 2) to obtain crude mitochondrial resuspension.

[0033] (4) Density gradient centrifugation was used to further purify each component.

[0034] 1) Purification treatment of cytoplasmic / endoplasmic reticulum components

[0035] Take an ultracentrifuge tube, add the resuspension obtained in step (3)-3) above, and slowly add the separation reagent 2 solution along the tube wall. Centrifuge at 100000g for 1h at 4℃. After centrifugation, the supernatant is the cytoplasmic component, and the precipitate at the bottom of the tube is resuspended in mitochondrial resuspension buffer, which is the endoplasmic reticulum component.

[0036] 2) Isolation and purification of MAM and pure mitochondria

[0037] a. Take an ultracentrifuge tube and slowly add pre-cooled Percoll separation buffer along the tube wall; slowly add crude mitochondrial resuspension along the tube wall to form clear layers; then slowly add pre-cooled mitochondrial resuspension buffer along the tube wall to form a three-layer liquid structure; centrifuge at 95000g for 30min at 4℃, with the centrifugation deceleration level set to 3.

[0038] b. After centrifugation, distinct stratified bands are formed inside the centrifuge tube: the upper milky white band contains MAM components, and the lower milky white band contains pure mitochondrial components;

[0039] c. Dilute the collected MAM bands with mitochondrial resuspension buffer and centrifuge at 6300g for 10 min at 4°C; collect the supernatant and place it in an ultracentrifuge and centrifuge at 100000g for 1 h at 4°C with a deceleration level of 3; the flaky precipitate floating at the bottom of the tube is the MAM component.

[0040] d. Dilute the collected pure mitochondrial bands with mitochondrial resuspension buffer, mix by inverting, place in a high-speed centrifuge, centrifuge at 6300g for 10min at 4℃, discard the supernatant, and resuspend the precipitate with mitochondrial resuspension buffer to obtain the pure mitochondrial fraction.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] 1. This invention significantly reduces sample loss and increases yield.

[0043] This invention employs low-power ultrasonic disruption instead of traditional homogenizer grinding, avoiding sample adhesion between the homogenizer tube wall and the grinding pestle. Sample loss is reduced from over 20% in traditional methods to below 5%. 15-20 podocyte samples in 10cm culture dishes can stably yield 150-200 μl of purified MAM components, along with appropriate amounts of purified cytoplasm, pure mitochondria, and endoplasmic reticulum components, all suitable for subcellular organelle studies in cell biology experiments. This method effectively improves the yield of podocyte-mediated MAM and other subcellular organelles, reduces costs, and provides technical support for podocyte MAM proteomics research.

[0044] 2. This invention improves the purity and integrity of MAM.

[0045] By precisely controlling the ultrasonic power and the number of disruptions, combined with real-time monitoring of the degree of disruption using trypan blue, we ensured that the cells were fully disrupted and that the mitochondrial and endoplasmic reticulum structures were not excessively damaged. Using a fractional separation strategy combining density gradient centrifugation and two-step ultracentrifugation with Percoll cell separation medium, we achieved efficient separation of MAMs and pure mitochondria, providing a reliable sample guarantee for subsequent molecular mechanism research.

[0046] 3. This invention is simple to operate and highly repeatable.

[0047] The operation steps of this invention do not require specially customized instruments, and all reagents used are easy to prepare, making it suitable for large-scale laboratory operations. All centrifugation parameters, ultrasonic conditions, and reagent dosages have been standardized, and the repeatability is better than that of traditional grinding methods. Attached Figure Description

[0048] Figure 1 Photograph of a glass homogenizer;

[0049] Figure 2 Flowchart for the isolation and extraction of MAM from podocytes;

[0050] Figure 3 These are photographs of layered bands containing MAM and pure mitochondria, respectively, formed after ultracentrifugation of crude mitochondria.

[0051] Figure 4 This is a photograph of the MAM precipitate obtained after ultracentrifugation purification.

[0052] Figure 5 The results show the purity of MAM and other subcellular components. Detailed Implementation

[0053] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become clearer as a result. However, these examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0054] Example 1: Method for extracting mitochondrial-associated membranes and other subcellular components from podocytes

[0055] 1. Preparation of reagents and instruments

[0056] 1.1 Reagents

[0057] Complete culture medium (RMPI 1640 medium + 10% fetal bovine serum FBS + 1% ITS), PBS buffer, trypsin-EDTA digestion solution, DPBS buffer (containing Ca 2+ and Mg 2+ ), DPBS buffer (without Ca) 2+ and Mg 2+Trypan blue staining solution, separation reagent 1 (containing 225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA and 30 mM Tris-HCl, pH 7.4), separation reagent 2 (containing 225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl, pH 7.4), mitochondrial resuspension buffer (containing 250 mM sucrose, 5 mM Tris-HCl, 0.5 mM EGTA, pH 7.4), and Percoll separation buffer (225 mM mannitol, 25 mM HEPES (pH 7.4), 1 mM EGTA and 30% Percoll (vol / vol)).

[0058] 1.2 Instruments and Equipment

[0059] Clean bench, low-speed centrifuge (including 50ml / 15ml rotor), high-speed centrifuge, ultracentrifuge (including 5ml angle rotor / 13ml horizontal rotor), low-power ultrasonic disruptor (power range 0-100W), constant temperature water bath, inverted microscope, pipettes (2μl-1000μl), sterile 50ml centrifuge tubes, 15ml centrifuge tubes, 1.5ml / 2ml EP tubes, 5ml / 13ml ultracentrifuge tubes.

[0060] 2. Specific operating steps

[0061] 2.1 Pretreatment and Collection of Podocytes

[0062] 1) Collect podocytes differentiated for 10-14 days (20 cells per 10cm culture dish as one sample), remove the culture medium, and rinse with DPBS (Ca-free). 2+ and Mg 2+ After washing the cells twice, add 2 ml of trypsin-EDTA digestion solution to each culture dish and digest the cells in the incubator for about 1 minute. Then add 4 ml of culture medium to stop the digestion and pipette the cells with a Pasteur tube.

[0063] 2) Collect the cell suspension into a 50 ml centrifuge tube, centrifuge the cells at 600 g for 5 minutes at 4°C, discard the supernatant, and resuspend all cells in 15 ml of DPBS (containing Ca2+). 2+ and Mg 2+ Centrifuge the cells again at 600g for 5 minutes at 4°C, discard the supernatant, and resuspend all cells in 5 ml of DPBS (containing Ca2+). 2+ and Mg 2+ In the mixture, the cells were centrifuged at 600g for 5 minutes at 4°C.

[0064] 3) Discard the supernatant and resuspend the cell pellet in 2 ml of pre-cooled separation reagent 1.

[0065] 2.2 Low-power ultrasonic crushing and fragmentation detection

[0066] 1) Mix 30 μl of trypan blue staining solution with 2 μl of cell resuspension solution, and drop the mixture onto a glass slide. At this point, the cells are in an unbroken state and will be used for comparison with cells after they have been broken down.

[0067] 2) Lyse the cell suspension on ice for 15 minutes. Then, adjust the ultrasonic homogenizer power to the lowest setting (≤20W) and sonicate the cell suspension in the EP tubes. Sonicate each sample 3-5 times (2 seconds each time, with a 5-second interval to avoid heat generation). After the third sonication, mix 30 μl of trypan blue staining solution with 2 μl of cell suspension, add it to a glass slide, and observe it under an inverted microscope. Compare the mixture with the unbroken state and observe the proportion of trypan blue-positive (blue) cells. If the proportion of positive cells is insufficient, continue sonication until the proportion of positive cells reaches 50-60%. Stop sonication. Reserve 10-20 μl of cell homogenate (H) and store it at -20℃ for subsequent MAM purity testing.

[0068] 2.3 Differential centrifugation separation of crude extract components

[0069] 1) Collect the cell homogenate after sonication into a 1.5ml EP tube, centrifuge at 750g for 10min at 4℃, the precipitate consists of cell nuclei and unbroken cells, the supernatant contains cytoplasm and organelles, discard the precipitate and keep the supernatant;

[0070] 2) Transfer the supernatant to a new 1.5ml EP tube and centrifuge at 11000g for 10min at 4℃. The precipitate after centrifugation is crude mitochondrial fraction (Mc). The supernatant contains cytoplasm and endoplasmic reticulum components.

[0071] 3) Collect the supernatant obtained in step 2) into a 5ml centrifuge tube, add 2ml of pre-cooled separation reagent 2 solution, and mix gently for later use;

[0072] 4) Add about 150 μl of pre-cooled mitochondrial resuspension buffer to the crude mitochondrial precipitate obtained in step 2). Gently resuspend the mitochondria using a 200 μl pipette tip with the tip cut open, avoiding damage to the mitochondria and disrupting their interaction with the endoplasmic reticulum. Keep 10-20 μl of the crude mitochondrial resuspension for purity testing. Then add mitochondrial resuspension buffer to the remaining liquid to a final volume of 2 ml and mix well for later use.

[0073] 2.4 Density gradient centrifugation was used to further purify the components.

[0074] 2.4.1 Purification of Cytoplasmic / Endoplasmic Reticulum Components

[0075] Take a 5ml ultracentrifuge tube, add the resuspension obtained in step 3) of 2.3 above, and then slowly add 1ml of separation reagent 2 solution along the tube wall, so that the liquid is 0.5cm away from the tube opening. Place the centrifuge tube on the angle rotor of an ultracentrifuge and centrifuge at 100000g for 1h at 4℃. After centrifugation, the supernatant is the cytoplasmic component (cytosol, Cyto). The precipitate at the bottom of the tube is resuspended in 200ul of mitochondrial resuspension buffer, which is the endoplasmic reticulum component (ER).

[0076] 2.4.2 Isolation and purification of MAM and pure mitochondria

[0077] 1) Take a 13ml ultracentrifuge tube and slowly add 8ml of pre-cooled Percoll separation buffer along the tube wall; slowly add 2ml of crude mitochondrial resuspension along the tube wall to form clear layers; then slowly add 2ml of pre-cooled mitochondrial resuspension buffer along the tube wall to form a three-layer liquid structure; after balancing, place it on the horizontal rotor of an ultracentrifuge and centrifuge at 95000g for 30min at 4℃, setting the centrifugation deceleration level to 3.

[0078] 2) After centrifugation, distinct stratified zones form inside the centrifuge tube (see...). Figure 3 The upper milky white band contains MAM components, and the lower milky white band contains pure mitochondrial fraction (Mp) components. Use a long-needle syringe to slowly rotate and aspirate about 1 ml of the upper MAM band; use another syringe to aspirate about 600 μl of the pure mitochondrial band from the bottom of the tube.

[0079] 3) Dilute the collected MAM bands 5-fold with mitochondrial resuspension buffer, mix thoroughly by inverting, and centrifuge at 6300g for 10 min at 4°C. Collect the supernatant, transfer it to a 5 ml ultracentrifuge tube, balance it, and centrifuge at 100000g for 1 h at 4°C with a deceleration level of 3. After centrifugation, the MAM fraction consists of flaky precipitate floating at the bottom of the tube (see...). Figure 4 Carefully aspirate the solution using a 200 μl pipette tip and mix it with mitochondrial resuspension buffer to obtain the MAM component.

[0080] 4) Dilute the collected pure mitochondrial bands 5 times with mitochondrial resuspension buffer, mix by inverting, place in a high-speed centrifuge, centrifuge at 6300g for 10 min at 4℃, discard the supernatant, and resuspend the precipitate with mitochondrial resuspension buffer to obtain the pure mitochondrial fraction.

[0081] 3. Sample preservation and purity verification

[0082] The obtained cell homogenates (H), cytoplasm (cyto), crude mitochondria (Mc), pure mitochondria (Mp), MAM, and endoplasmic reticulum (ER) were analyzed for protein concentration and then stored at -80°C for long-term preservation. Based on previous research [Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. NatProtoc 2009;4:1582–90. https: / / doi.org / 10.1038 / nprot.2009.151.A. Lewis, S.-Y.Tsai, T.-P. Su, Detection of Isolated Mitochondria-Associated ER Membranes Using the Sigma-1 Receptor, in: MG Waugh (Ed.), Lipid Signal. Protoc., Springer New York, New Jersey], the results were analyzed. York, NY, 2016: pp. 133–140. https: / / doi.org / 10.1007 / 978-1-4939-3170-5_11.], Comprehensive detection of markers for various subcellular organelles to verify the purity of MAM:

[0083] 1) β-tubulin, as a marker of cytoplasm, is mainly enriched in the cytoplasm, but lacks it in MAM, mitochondria (Mc and Mp), and endoplasmic reticulum;

[0084] 2) COX4 is a protein located in the inner mitochondrial membrane, while MAM is formed by the contact between the outer mitochondrial membrane and the endoplasmic reticulum. Therefore, COX4 is enriched in mitochondria (Mc and Mp) but lacks in the cytoplasm, MAM and endoplasmic reticulum.

[0085] 3) Grp75 and VDAC1 are proteins located in the outer mitochondrial membrane, and are therefore enriched in mitochondria (Mc and Mp), present in the MAM, but lacking in the cytoplasm and endoplasmic reticulum.

[0086] 4) Careticulin is a marker of the endoplasmic reticulum, and is therefore enriched in the endoplasmic reticulum, present in the MAM, and lacking in the cytoplasm and pure mitochondria.

[0087] 5) Sigma1R, as a marker of MAM, is enriched in MAM but lacks it in pure mitochondria.

[0088] The Western Blot results of the above markers are shown below. Figure 5 The results show that the purity of MAM and other subcellular components obtained from podocytes is high, which can be used for further research on MAM and other subcellular components.

Claims

1. A method for extracting mitochondrial-associated membrane (MAM) and other subcellular components from podocytes, characterized in that, The method includes the following steps: pretreatment of podocytes, disruption of podocytes by low-power ultrasound, separation of crude mitochondrial components from the cell homogenate after ultrasound by differential centrifugation, resuspension of the crude mitochondrial components, separation of MAM and pure mitochondria by density gradient centrifugation and ultracentrifugation, and finally purification by centrifugation to obtain high-purity MAM and pure mitochondrial components.

2. The method as described in claim 1, characterized in that, The podocyte pretreatment includes digesting the podocytes with trypsin-EDTA digestion solution, centrifuging, and resuspending the cell pellet in pre-cooled separation reagent 1, wherein separation reagent 1 contains 225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA and 30 mM Tris–HCl, pH 7.

4.

3. The method as described in claim 1, characterized in that, The ultrasonic disruptor used for disrupting podocytes with low power ultrasound has a power of ≤20W. Ultrasound is performed 3-5 times, with each ultrasound lasting 2 seconds and an interval of 5 seconds. After each ultrasound, trypan blue staining solution is mixed with the cell suspension, and the proportion of trypan blue-stained positive cells is observed. If the proportion of positive cells is insufficient, ultrasound disruption is continued. When the proportion of positive cells is 50-60%, ultrasound is stopped.

4. The method as described in claim 1, characterized in that, The differential centrifugation involves centrifuging the sonicated cell homogenate at 750g for 10 minutes at 4°C, discarding the precipitate and retaining the supernatant; then centrifuging the supernatant at 11000g for 10 minutes at 4°C to obtain the crude mitochondrial extract.

5. The method as described in claim 1, characterized in that, The separation of MAM and pure mitochondria by density gradient centrifugation and ultracentrifugation involves first adding Percoll separation buffer to the ultracentrifuge tube, then slowly adding the resuspended crude mitochondrial components along the tube wall, and then slowly adding mitochondrial resuspension buffer on top, forming a three-layer liquid structure. After ultracentrifugation, distinct stratified bands are formed, with the upper milky white band containing MAM components and the lower milky white band containing pure mitochondrial components.

6. The method as described in claim 1, characterized in that, The centrifugal purification process involves diluting the collected MAM bands with mitochondrial resuspension buffer, centrifuging them at 6300g for 10 min at 4°C, collecting the supernatant, centrifuging it at 100000g for 1 h at 4°C with a deceleration level of 3, and then centrifuging it at the bottom of the tube as a sheet-like precipitate.

7. The method as described in claim 1, characterized in that, The other subcellular components include cytoplasm, pure mitochondria, and endoplasmic reticulum.

8. The method as described in claim 1, characterized in that, The method includes the following steps: (1) Pretreatment of podocytes 1) Collect podocytes differentiated for 10-14 days using calcium-free methods. 2+ and Mg 2+ After washing the cells with DPBS buffer, trypsin-EDTA digestion solution was added. After digestion, culture medium was added to stop the digestion and the cells were pipetted. 2) Collect the cell suspension and centrifuge, discard the supernatant and resuspend all cells in a solution containing Ca. 2+ and Mg 2+ Centrifuge again in DPBS buffer, discard the supernatant, and resuspend all cells in a solution containing Ca. 2+ and Mg 2+ Centrifuge in DPBS buffer; 3) Discard the supernatant and resuspend the cell pellet in pre-cooled separation reagent 1, which contains 225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA and 30 mM Tris–HCl, pH 7.4; (2) Low-power ultrasound fragmentation of foot cells 1) Mix trypan blue staining solution with cell resuspension solution; 2) Place the cell suspension on ice to lyse, then adjust the power of the sonicator to ≤20W and sonicate the cell suspension 3-5 times, 2 seconds each time, with a 5-second interval; after each sonication, mix trypan blue staining solution with the cell suspension and observe the proportion of trypan blue positive cells. If the proportion of positive cells is insufficient, continue sonication. Stop sonication when the proportion of positive cells is 50-60%. (3) Differential centrifugation to separate crude mitochondrial fractions 1) Centrifuge the sonicated cell homogenate at 750g for 10min at 4℃. The precipitate consists of cell nuclei and unbroken cells. The supernatant contains cytoplasm and organelles. Discard the precipitate and keep the supernatant. 2) Transfer the supernatant to 11000g and centrifuge for 10 min at 4℃. After centrifugation, the precipitate is crude mitochondria, and the supernatant contains cytoplasm and endoplasmic reticulum components. 3) Add the pre-cooled separation reagent 2 solution to the supernatant obtained in step 2), mix gently and set aside; the separation reagent 2 contains 225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl, pH 7.4; 4) Add pre-cooled mitochondrial resuspension buffer to the crude mitochondrial precipitate obtained in step 2) to obtain a crude mitochondrial component resuspension. (4) Density gradient centrifugation was used to further purify each component. 1) Purification treatment of cytoplasmic / endoplasmic reticulum components Take an ultracentrifuge tube, add the resuspension obtained in step (3)-3) above, and slowly add the separation reagent 2 solution along the tube wall. Centrifuge at 100000g for 1h at 4℃. After centrifugation, the supernatant is the cytoplasmic component, and the precipitate at the bottom of the tube is resuspended in mitochondrial resuspension buffer, which is the endoplasmic reticulum component. 2) Isolation and purification of MAM and pure mitochondria a. Take an ultracentrifuge tube and slowly add pre-cooled Percoll separation buffer along the tube wall; then slowly add crude mitochondrial component resuspension along the tube wall to form clear stratification; then slowly add pre-cooled mitochondrial resuspension buffer along the tube wall to form a three-layer liquid structure. Centrifuge at 95000g for 30 min at 4℃, with the centrifugation deceleration level set to 3. b. After centrifugation, distinct stratified bands are formed inside the centrifuge tube: the upper milky white band contains MAM components, and the lower milky white band contains pure mitochondrial components; c. Dilute the collected MAM bands with mitochondrial resuspension buffer and centrifuge at 6300g for 10 min at 4°C; collect the supernatant and place it in an ultracentrifuge and centrifuge at 100000g for 1 h at 4°C with a deceleration level of 3; the flaky precipitate floating at the bottom of the tube is the MAM component. d. Dilute the collected pure mitochondrial bands with mitochondrial resuspension buffer, mix by inverting, place in a high-speed centrifuge, centrifuge at 6300g for 10min at 4℃, discard the supernatant, and resuspend the precipitate with mitochondrial resuspension buffer to obtain the pure mitochondrial fraction.

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