This experiment uses selective modulation of the protein kinase C ISO xms to determine their mitochondrial and cellular functions in renal proximal tubular cells under various physiological and pathological conditions. First, isolate the primary renal proximal tubular cells from the renal cortex or RPCs that have oxidative metabolism. Resembling that of renal proximal tubules.
Vivo infect the RPCs with adenoviral vectors that express active and inactive forms of isys of protein. Kinase C then assay the different mitochondrial functions and cell viability to determine the effect of the is zyme on mitochondrial ability to synthesize A TP and prevent cell death. Also perform immuno blot analysis of phosphorylated and protein kinase C epsilon in cellular lysates taken together.
Results show that the activation of protein kinase epsilon negatively regulates mitochondrial ability to generate A TP decreases the cellular levels of a TP and the viability of renal proximal tubular cells. The main advantage of this technique of our existing techniques, such as cell lines, is that primary cultures of renal proximal tubular cells maintain the differentiate functions and oxidated me of renal Proximal. Since protein kinases can be used as targets and potential therapeutic agents, the implications of this technique extend toward therapy, preventing kidney injury or accelerating kidney recovery after injury.
This method can insight the mechanism injury and recovery. It can also be applied to such as the and Per freshly isolated rabbit kidney sequentially with 50 milliliters. Each of sterile D-M-E-M-F 12 medium sterile phosphate buffered saline, pH 7.4 and PBS iron oxide solution to harvest the cortex of each kidney.
First homogenize the tissue using 15 milliliter down homogenizer. Then pass the homogenate through two sterile mesh sieves into a one liter sterile beaker. Rinse the cells remaining on the sieves with deferoxamine containing buffer.
Transfer any tissue left on the bottom. 85 micron sieve to 40 milliliters of deferoxamine containing buffer placed in a conical centrifuge tube. Gently mix the cell suspension with the strong magnet.
Capture the iron filled glomeruli and aspirate the iron attached to the magnet. Adjust the volume of the cell suspension to 40 milliliters and add one milliliter of digestion medium containing collagenase. Incubate at room temperature for 17 minutes.
Gently mixing the suspension. Harvest the cells by centrifugation at 50 times G for two minutes at four degrees Celsius after several washes. Resus suspend the cell pellet in 46 milliliters of medium containing no glucose seed.
The primary RT PCs at a density of one milligram protein per dish culture in two milliliters of glucose free D-M-E-M-F 12 medium on an orbital shaker at 37 degrees Celsius and 5%carbon dioxide. After replenishing the media proceed to infect confluent RPT cells with adenovirus carrying CD NA for either constitutively active protein kinase, C epsilon, or dominant negative protein kinase C epsilon incubate for 24 hours, replenish the media and culture the transfected primary RPCs for another 24 hours. To visualize the mitochondrial morphology of the primary cell monolayer replenish the media, add MIT tracker red five 80 and return the dishes to the incubator for 30 minutes.
Then examine the live monolayers under a fluorescent microscope using a water immersion. Objective two. To assess mitochondrial function, evaluate state three respiration of the RPTC suspension in an oxygen consumption chamber equipped with a Clark type electrode.
First measure state two respiration in the absence of a DP.Then add a DP to a final concentration of 0.4 millimolar. To initiate state three respiration for state four respiration. Add oligo mycin to a final concentration of 0.5 micrograms per milliliter.
Also include a measurement for uncoupled respiration by adding 0.5 micromolar FCCP to cells respiring at state three. Slowly add 20 microliters of 0.5 millimolar JC one solution to each RPTC culture dish and swirl to mix the dye thoroughly. Return the dishes to the incubator for 30 minutes.
Next, place the cultures on ice and perform two ice cold PBS washes. Then harvest the cells. Transfer RPT cells to an einor tube and break up the monolayers into single cell suspension by pipetting.
Analyze the fluorescence by flow cytometry using excitation by a 488 nanometer argon ion laser. Then calculate the mitochondrial membrane potential isolated mitochondria. Facilitate assessment of the integrity of the respiratory chain.
After two washes of the RPTC monolayers with ice cold PBS buffer, harvest the cells and pellet by centrifugation. Wash the pellet once in one milliliter of buffer A then homogenize the cells in a down's homogenizer until most of the cells in the homogenate are broken when inspected under a microscope. Next, remove the cellular debris by low speed centrifugation.
Pellet the crude mitochondria from the supernatant at 15, 000 times G for 10 minutes at four degrees Celsius and perform three washes of the pelleted mitochondria in buffer c resuspend the mitochondrial pellet in 50 to 100 microliters of the assay buffer and freeze in liquid nitrogen to assay NADH ubiquinone oxid reductase activity. Add 500 microliters of the assay buffer with additions and mitochondria also include a blank sample that has all the additions but no mitochondria. Equilibrate the plate at 30 degrees Celsius with mixing for five minutes.
Next, place the plate in the spec and add 10 microliters of 3.25 millimolar ubiquinone to start the reaction in each. Well record the absorbance at 340 nanometers for three minutes in 22nd intervals. In a similar assay, measure the activity of cytochrome C oxidase using reduced cytochrome C as substrate for the A TP synthase reaction.
Prepare mixes for assaying total APAs activity and oligo mycin insensitive APAs activity of each treatment group. Incubate the samples in reaction mix at 31 degrees Celsius for 10 minutes. Start the reaction by adding a TP substrate and incubate at 31 degrees Celsius for five minutes.
Transfer 200 microliters of the incubation mixture to a tube containing 50 microliters of three molar TCA, precipitate the proteins on ice for 10 minutes. Then pellet by centrifugation. Next load a 96 well plate with 50 microliters of each phosphate standard and 50 microliters of each sample supernatant in duplicate.
Then add 250 microliters of Sumner reagent to each well incubate at 30 degrees Celsius for 15 minutes. Then record the absorbance at 595 nanometers. Wash the RPTC monolayers twice with ice cold PBS buffer.
Harvest the cells in one milliliter PBS and transfer the cell suspension to an einor tube. Pellet the RPTC samples by centrifugation and determine a TP content using the luciferase method of an A TP bioluminescence assay kit. For analysis of cell viability in 35 millimeter dishes, use five RPTC monolayers for three control treatment groups of cells positive for on osis cells positive for apoptosis and a no stain control to two dishes.
Add two microliters of 50 millimolar cisplatin to the second set. Add two microliters of 500 millimolar turt butyl hydroperoxide. After staining the cells using propidium iodide as detailed in the accompanying text, wash the monolayers with the binding buffer.
Next, stain the cells using ANNEXIN five conjugated with ZI as detailed in the text protocol. Then gently harvest the cells and suspend them in 500 microliters of binding buffer. Using a rubber policeman.
Use a pipette to disperse the cells into a single cell suspension and transfer the cell suspension to flow cytometry tubes. Disperse the cells into a single cell suspension. Proceed immediately to quantify the fluorescence for PI and FE by flow cytometry adenoviral delivery of CD NA coating the CONSTITUENTLY active and the inactive mutants of protein kinase C epsilon results in significantly increased protein levels of protein kinase C epsilon in RPTC and in mitochondria.
As expected, the constitutively active form of the enzyme is phosphorylated in RP tcs and the dominant negative enzyme is not phosphorylated. The presence of active protein kinase C epsilon decreases mitochondrial respiration in RPTC regardless of the substrate used to energize mitochondria. But the inactive protein kinase C epsilon has no effect on mitochondrial respiration.
Interestingly, activation of protein kinase C epsilon in RP TCS reduces the activity of complexes one in four. This decrease in RPTC respiration is associated with increases in the mitochondrial membrane potential. The dominant negative mutant has no such effect.
These changes are also accompanied by decreases in the activity of a TP synthase in mitochondria isolated from RPTC over expressing the active protein kinase C epsilon. As a result, the A TP content of RPTC over expressing the active protein kinase C epsilon is decreased sustained activation of protein kinase. C epsilon has profound effects on mitochondrial morphology as evidenced by mitochondrial fragmentation.
Protein kinase C activation, but not inhibition. Also results in changes in RPTC morphology causing cell shrinkage and elongation of surviving cells. These mitochondrial effects of over expressing the active protein kinase C epsilon in RPCs also correlated with cell death by both ons and apoptosis.
After watching this video, you should have a good understanding of how to over express proteins in primary cultures using adenoviral technique and how to assess mitochondrial capacity for a TP synthesis. Once mastered, this technique can be done in four hours if it's performed properly. And deficiently, Don't forget that working with adenovirus access thet, so take precautions such as appropriate.
