This method can help answer key questions about renal metabolism, such as fatty acid oxidation, which is closely related to the development of renal fibrosis and various other nephropathies. The main advantage of this technique is using a high-throughput platform to screen for compounds that regulate mitochondrial bioenergetics in renal tubular epithelial cells. The implications of this technique extend toward therapy of renal metabolic disease because it allows for high-throughout screening of compounds that regulate mitochondrial metabolism in renal epithelial cells.
To prepare the collagen-coated 60-millimeter Petri dishes, add two milliliters of a pre-filtered 20-millimolar acetic acid followed by 35 microliters of collagen I solution to each dish. Then, incubate the plate at room temperature for an hour, air-dry it, and expose it to UV.Next, rinse off the acetic acid residues using PBS. Perform three rinses.
Then, store the dish in a 37 degree Celsius carbon dioxide-free cell culture incubator. After preparing the mouse for surgery, remove the fur from the chest and abdomen using a depilatory cream. Then, disinfect the skin with iodine, and wipe away the iodine residue.
Next, make an incision to open the abdominal cavity, and expose the heart and kidneys. Now, set up a perfusion pump at 32 milliliters per minute, and remove any bubbles in the tubing before starting the perfusion. Use a 27-gauge needle at the end of the line.
To proceed, insert a needle into the left ventricle through the heart apex. As soon as the buffer fills the heart, poke a hole in the right atrium to create an exit for the perfusion buffer. Next, switch to the digestion buffer, and reduce the pump speed slightly to 30 milliliters per minute for the digestion.
After 20 milliliters of digestion buffer has been perfused, remove both kidneys for the tubular cell isolation. To process the harvested kidneys, first, remove the renal capsules and medulla. Then, mince both kidneys into tiny pieces, and incubate them in 10 milliliters of a digestion buffer in a 37 degree Celsius oven with gentle rotation for five minutes.
After the digestion, remove any undigested tissues by passing the buffer through a 70-micron filter. After collecting the digested tissue, add 10 milliliters of culture media to stop the digestion. Next, centrifuge the filtered tissue suspension at 50 g for five minutes to pellet the tubular cells.
Then, transfer the supernatant to a new tube, add five milliliters of culture media, and repeat the centrifugation to collect any remaining tubular cells. Now, resuspend the first pellet in 20 milliliters of culture media, and centrifuge it at 50 g for five minutes to further purify the collected cells. Dispose of the supernatant.
Then, resuspend both pellets in 10 milliliters of culture medium. Next, count the living cells using trypan blue staining. Then, seed up to 10 million cells onto a 60-millimeter collagen-coated dish, and let the tubular cells attach overnight.
To start, seed P1 tubular cells at 40, 000 cells into wells of a freshly prepared 96-well microplate coated with collagen. Use 100 microliters of culture media per well. Optimization of cell density and compound concentration in the extracellular flux assay is key to a successful experiment.
We recommend a pilot titration experiment to identify optimal cell density and compound concentration. Next, hydrate the sensor cartridge. First, lift the sensor cartridge, and fill each well of the plate with 200 microliters of calibration solution.
Then, reposition the cartridge to submerge the sensors, and incubate the assembly overnight at 37 degrees Celsius. The next day, aspirate off the old media, and replace it with 175 microliters of glucose or fatty acid assay media. Then, continue the incubation for an hour without carbon dioxide.
During the cell incubation, load port A of the cartridge with 25 microliters of oligomycin, load port B with FCCP, load port C with rotenone/antimycin A, and load port D and all other ports with water. Now, store the cartridge in the carbon dioxide-free incubator until the setup is ready for calibration. Proceed by turning on the extracellular flux analyzer and the controller.
Next, open the Analyzer Software, choose a standard assay, and press the Assay Wizard option. Then, under the Compounds tab, assign the compounds to the appropriate ports. Under the Background Correction tab, select the wells that contain no cells.
Next, use the Groups and Labels tab to label the experimental groups. Then, under the Protocol tab, set the appropriate mix and measure cycles using the available commands. These settings are indicated in the text protocol, and once they are input, save the template for future use.
Now, press End Wizard to proceed. Now, press Start to begin the calibration. The analyzer then ejects the plate holder and prompts for the cartridge plate to be inserted.
This calibration process takes 20 to 25 minutes to complete. Once the system is calibrated, press the prompt command to exchange the calibration plate for the cell plate, and continue the run. When the run is completed, save the OCR data, and remove the plate from the analyzer.
Now, add Hoechst to each of the assay wells, and incubate the plate for five minutes at 37 degrees Celsius. Then, count the cells using 355-nanometer excitation and a 460-nanometer emission, and normalize the OCR data to the cell count. In this protocol, a heterogeneous population of kidney cells, incompletely digested tubules, and other tissue debris were cultured on collagen-coated plates.
The day after isolation, the culture media was centrifuged to remove debris, and the tubular cells were re-plated. Subsequently, the cells attached more easily, and by the fifth day in culture, they were at 80 to 90%confluency. The renal TECs were then sub-cultured and grew to confluency within one week.
Similar growth occurred over the next two passages. The monolayer of cells took a dome formation, lifting off the plate and staying attached by tight junctions. The mitochondrial respiration of isolated primary TECs was measured using described extracellular flux analysis of the OCR at different plating densities as described.
Cells plated at greater densities had proportional increases in OCR. 40, 000 cells per well covered the surface of the plate better than any other plating densities, thus making it the optimal concentration to test interactions between cells and compounds. For example, media containing fatty acid substrate was applied to TECs along with 2DG, an inhibitor of glycolysis, to directly evaluate fatty acid oxidation in TECs at passages one and two.
While attempting this procedure, it's important to always keep record of the exact volumes and concentrations in each well. Alterations in the final concentrations of the components can cause the whole experiment to fail. After its development, this technique will pave the way for researchers in the nephrology field to explore the role of tubular fatty acid metabolism in the pathogenesis of various nephropathies.
