Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Our technique enables us to identify the source and the relative contribution of cytokine producing cells in injured kidneys. Using Brefeldin A to prevent protein secretion allows us to gather more information about the cells producing cytokines in solid organs such as the cell type and the state of these cells.

Our protocol should be easily adaptable to many organ systems. Since Brefeldin A is a cell-permeable small molecule, it should inhibit secretion in most cell types, allowing for detection of cytokines and other secreted proteins.

Tailbone injection is the most technically challenging step because the mouse tailbone is quite small. We recommend practicing this step before performing our protocol on experimental animals.

[Instructor] Begin by putting the cage half on, half off the heating pad for 10 minutes to ensure the mice are warm. After placing the mouse in a restraint device, disinfect the tail using a povidone iodine and alcohol swab three times. Hold the tail horizontally and visualize the lateral tail veins. Then insert a 28 gauge needle, keeping the needle and syringe parallel to the vein towards the direction of the head. Inject 200 microliters of the 1.25 milligram per milliliter BFA solution. The vein should become clear as the blood is replaced with the injection solution. Then remove the needle and press the tail gently until the bleeding stops. Return the mice to the cage and monitor them for additional bleeding or signs of distress. Use a 20 milliliter syringe and perfuse the mouse with 10 to 20 milliliters of PBS through the left ventricle at a two to four milliliter per minute flow rate until the perfusate becomes clear. Remove the kidneys by holding the renal artery and vein close to the papilla and cutting the vessel on the side away from the kidney. Then gently remove the kidney capsule by peeling it off by hand or with a pair of fine sterile forceps. Use a hydrophobic barrier marker pen and outline the tissue sections maintaining at least five millimeters distance from the tissue to the hydrophobic barrier outline. Then add 50 microliters of blocking buffer containing 3% donkey serum and 0.1% triton in 1% bovine serum albumin TBS on top of the section. Incubate room temperature for an hour in a humidified chamber. Dilute the primary antibodies with PBS at the appropriate concentration. To detect cytokines, dilute antibodies directed against TGF beta one, PDGF-D, or CTGF in 50 microliters of PBS. Add Anti-Alpha SMA primary antibody conjugated with Cy3 to the primary antibody solution at 1:200 dilutions to label the myofibroblasts. Remove the blocking buffer and add the primary antibodies to the section, ensuring it does not leak out of the circular hydrophobic outline. Incubate overnight at four degrees Celsius in a humidified chamber. Remove the humidified chamber from the incubator and wash the slides with PBS three times for five minutes. Next, dilute the appropriate secondary antibodies at one to 200 with PBS. Incubate the samples with 50 microliters of secondary antibody solutions for an hour at room temperature in a humidified chamber. Remove the slides from the humidified chamber and wash with PBS for five minutes. Dilute lotus tetragonolobus lectin conjugated with fluorescein in PBS with calcium and magnesium at a concentration of 10 micrograms per milliliter. Then incubate the tissues with 50 microliters of LTL solution for 30 minutes at room temperature in a humidified chamber. Remove slides from the humidified chamber and wash them with PBS with calcium and magnesium for five minutes. Incubate the tissues with 50 microliters of DAPI to stain DNA nuclei for five minutes at room temperature. Wash one time with PBS. Add 20 microliters of antifade mounting reagent to the tissue to mount the cover slips, and then slowly place the cover slip. Wait for 24 hours for the antifade reagent to solidify before imaging. Turn on the inverted microscope with an automated XY stage and select the 20x objective. Place the slide on the microscope stage and locate the tissue section. Then open the image acquisition software and click on live to open the live view window. Find the tissue section ensuring it is in focus. Click on the acquire menu and select scan large image. Set up the area to scan by moving the stage using the joystick to the leftmost part of the tissue section and then click the left arrow. Repeat this for the uppermost, rightmost and bottom tissue segments. Click on the acquisition menu and ensure that the checkbox for multi-channel capture is checked. To capture multi-channel images, click on the lambda tab, click on each channel and set the exposure time to a level where the staining is apparent without saturating any part of the image. Repeat this for each channel to be collected. Then click on Scan. Open the image acquisition software and click on File. Then click on Open and select the image. Right-click on the image window and choose Polygonal ROI. Outline the ROI with a freehand tool. Then outline the LTL positive tubule cells or alpha SMA positive interstitial cells. Click on the measurement tab and then threshold. Set up the upper and lower threshold limits by adjusting the sliders to either side of the positive signal area. Then click on the ROI tab. Finally click on the export icon to save the values. Use spreadsheet software to calculate the percentages of the positive signal area by ROI area.

[Presenter] TGF beta positive vesicles on day three after cisplatin or saline administration are shown in the representative images. TGF beta positive vesicles are observed in PTCs labeled with LTL in cisplatin treated kidneys in the presence of BFA. However, these vesicles were not observed in uninjured or BFA untreated kidneys.

[Instructor] Modification revealed an increase in TGF beta positive area with BFA treatment in cisplatin induced acute kidney injury. Similar to TGF beta, PTGF-D positive or CTGF positive vesicles also accumulated with BFA treatment in cisplatin induced AKI. Quantification demonstrated that PDGF-D positive and CTGF positive areas were also significantly increased with BFA In LTL positive proximal tubule cells BFA injection did not significantly increase plasma BUN levels on day three after cisplatin injection. TGF beta positive vesicles were observed in alpha SMA labeled interstitial cells in cisplatin AKI with BFA treatment. Modification revealed that the TGF beta positive areas in the alpha SMA positive area was significantly increased with BFA treatment in cisplatin AKI. Similar to TGF beta positive vesicles, CTGF positive vesicles also increased with BFA treatment. Quantitation demonstrated that BFA treatment enhanced the ratio of CTGF positive area to alpha SMA positive area In cisplatin induced AKI. Compared to cisplatin induced injury, TGF-beta positive vesicles in PTCs were much smaller in the chronic phase of AA. There was minimal positive staining in LTL positive PTCs. However, the signal intensity of TGF beta in kidney injury molecule one positive PTCs was increased with BFA treatment. The mean intensity level of TGF beta was three times higher with BFA injection than without BFA.

The most critical step is ensuring that the tail vein injection is successful. A failed injection would throw off results. So please look for the signs of success like the vein becoming clear with the injection and please look for the signs of failure like swelling over the tails.

The Brefeldin A injection could be combined with a number of assays including western blot or flow cytometry to evaluate cytokine production on an organ or single cell scale.

Using this technique, we have been able to identify novel cytokines secreted by kidney epithelial cells that were previously thought to come from other cell types. It can be used to settle longstanding debates about the contribution of specific cell types to injury.