Modeling Hypoxia/Reoxygenation Injury in Proximal Tubular Epithelial Cells

Our research focuses on modeling renal ischemia-reperfusion injury using kidney proximal tubular cells to study damaged mechanisms and explore therapeutic strategies. Reproducing the complex ischemic environment of the kidney in vitro remains difficult, especially cold ischemia and immune interactions. To begin, remove the culture medium from the IM-PTECs to refresh the cells.

Add two milliliters of fresh L3 medium without interferon gamma into the wells of a six-well plate. Place the cells in a hypoxia incubator set at 1%oxygen and 5%carbon dioxide at 37 degrees Celsius for 48 hours. After incubation, remove the IM-PTEC plates from the hypoxia incubator and replace the medium with fresh L3 medium without interferon Gamma.

Place the cells into a standard cell culture incubator under normal oxygen conditions and 5%carbon dioxide at 37 degrees Celsius for 24 hours. To perform RT-qPCR, prepare the primer sets by mixing them to a final concentration of 10 micromolar. Prepare the reaction mix by combining the required reagents along with primers and nuclease-free water per well.

Mix gently to ensure homogeneity. Using a pipette, dispense eight microliters of the RT-qPCR mix into each well of a 384-well PCR plate, and add two microliters of the complementary DNA sample to each well, allowing a small droplet to remain on the edge of the well. After all samples have been added, tap the plate gently until all droplets fall completely into the wells, and seal the plate firmly with its membrane.

Spin the 384-well PCR plate to remove air bubbles and ensure even distribution. Now, place the sealed plate into the RT-qPCR system. Set the program, and start the measurement.

Turn on the oxygen consumption rate and extracellular acidification rate analyzer to let it warm up for at least five hours before starting the experiment. Hydrate a sensor cartridge overnight at 37 degrees Celsius in a non-carbon dioxide incubator. On the next day, prepare the assay medium for 196-well cell culture microplate by adding Dulbecco's Modified Eagle Medium with appropriate supplements and mixing gently until homogeneous.

Next, prepare the inhibitor solutions by mixing the required components. Then remove the hydrated sensor cartridge from the incubator. Using a multichannel pipette and a loading guide, load 25 microliters of each inhibitor into its corresponding port.

Now aspirate the growth medium from the cell culture microplate and add 175 microliters of assay medium tempered at 37 degrees Celsius into each well using a multi-channel pipette. Place the cell culture microplate in a 37 degrees Celsius, non-carbon dioxide incubator, and incubate for 45 to 60 minutes before starting the assay. After incubation, insert the calibration plate containing the loaded sensor cartridge into the analyzer for calibration.

Once calibration is complete, place the cell culture microplate into the analyzer for measurement. After the measurement run, remove and save the cell culture microplate for subsequent quantification of total protein for data normalization. 48 hours of hypoxia followed by 24 hours of reoxygenation produced the greatest increase in caspase-3 activity while maintaining cell viability comparable to that of normoxic controls.

After hypoxia and reoxygenation, there was a significant increase in the expression of the kidney injury markers Kim-1 and Ngal, as well as in the expression of markers of renal partial epithelial to mesenchymal transition and fibrosis, Acta2 and Col1a1. Mitochondrial respiration analysis showed that after the ischemic challenge, all respiration parameters, including basal respiration, ATP production, proton leak, maximal respiration, and spare capacity, were reduced, indicating hindered mitochondrial metabolism. There is a lack of standardized cell-based models to study kidney ischemia-reperfusion injury under controlled metabolic conditions.

Our protocol is simple, cost-effective, and reproducible, only requiring standard laboratory equipment and an hypoxia incubator. Identifying novel therapeutic targets and validating protecting compounds using both in vitro and in vivo kidney models.