Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

The overall goal of this procedure is to measure the intracellular calcium concentration changes in response to pharmacological agents. Estimate basal levels of calcium and assess individual channel activity in the podocytes as part of the whole freshly isolated glomerulus. This is accomplished by first clearing blood from rat kidneys by flushing via the abdominal aorta.

Next, the kidneys are harvested and the kidney cortex is isolated and minced with a razor blade. In the third step, the cortical glomeruli are isolated by differential cing. Afterward, the isolated decapitated glomeruli can either be subjected to electrophysiological experiments or loaded with fluorescent dyes and taken for confocal calcium concentration measurements.

Ultimately, these procedures enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents and assess and manipulate calcium conductance at the level of single ion channels. The main advantage of this technique over existing methods is that it allows us to study podocytes as part of the whole isolated glomerulus. Our preparation retains the functional potential of the podocytes.

They remain attached to the capillaries and participate in the homeostasis in the environment that actually can conserves the major parts of the glomeruli filtration apparatus. The implication of this technique extend our pharmacological screening In normal and pathological states, the mechanism of calcium handling by the podocytes serve an essential regulatory role in the development and prevention of renal complications in such diseases as diabetic nephropathy, focal segment omero sclerosis or hypertension. It's a great importance to observe the responsiveness of calcium influx in these cells to drugs, which can be easily screened.

In this preparation, VLA Hunka will demonstrate how to flush the kidney for the following procedures. I'll show glomeruli isolation and V clamp electrophysiology, and Dr.GaN will guide you through confocal calcium imaging. To begin this procedure, place an anesthetized rat on the temperature controlled surgical table Under the microscope, make a midline incision on the abdomen to uncover the vein CVA and the aorta.

Next, insert the ligature around the celiac and superior mesenteric arteries and the abdominal aorta above them. Blunt, dissect the abdominal aorta below the renal arteries. Then place two ligatures around it, but do not ligate.

Clamp the aorta above the ligatures and tie the lower thread. After that, catheterize the aorta with a polyethylene tubing that is attached to a syringe pump filled with PBS below the clamp and fix the catheter with a second ligature. Then remove the clamp and ligate them.

Me enteric and celiac arteries. Infuse the aorta with pre chilled PBS for three minutes at a rate of six milliliters per minute. At the same time, make an incision in the vein of cava near the renal veins to relieve the pressure.

After three minutes, stop the perfusion, excise and encapsulate the kidneys. Place them in PBS solution on ice. Now prepare 30 milliliters of fresh 5%BSA in RPMI 1640 medium.

Using a razor blade and scissors, isolate the cortex of both kidneys, then MIT them until homogenous. Next, push the minced tissue through a 100 mesh stainless steel sieve that has been pre soaked in 5%P-S-A-R-P-M-I solution. Collect the flow through and allow it to pass through a 140 mesh siv.

Subsequently, filter the collected flow through from the 140 mesh sieve using a pre soaked 200 mesh siv. Discard the filtrate and wash the top sieve with 10 to 15 milliliters of the prepared B-S-A-R-P-M-I solution and collect glomeruli from the top of the sieve. Next place the B-S-A-R-P-M-I solution containing G Glomeruli in a 15 milliliter, two bon ice and let the glomeruli sediment at the bottom of the tube for up to 20 minutes.

In this procedure, coat five by five millimeter cover glass chips with molecular weight, 70, 000 to 150, 000 poly lysine and dry them on a heated plate. Next, warm up the experimental solutions to room temperature and fill the patch clamp chamber and pipette Gently mix the glomeruli containing solution. Then apply approximately 50 microliters of it to each polylysine coated cover glass chip.

After that, move the glass chips with glomeruli to the patch clamp chamber. Prefilled with the Beth solution. Perfuse the chamber at a rate of three milliliters per minute for one minute to remove any unattached glomeruli before conducting the conventional patch clamp recording in a cell attached mode.

Now place 500 microliters of the glomeruli fraction in each 0.5 milliliter conical tube and add calcium dyes pure red am and flu oh 4:00 AM.Next, place the tubes on a rotating shaker for up to one hour at room temperature. Then prepare the glass cover slips with polylysine and allow them to dry on the heated plate at 70 degrees Celsius. Once the loading of calcium dies is complete, apply 100 microliters of the glomeruli containing solution to the polylysine coated cover slips and let them stick to the surface for about five minutes.

Then mount the glomeruli attached cover slips onto an imaging chamber and perfuse them with bath solution at a rate of three milliliters per minute to remove the unattached glomeruli and remaining dyes. Afterward, set the confocal laser scanning microscope to a 488 nanometer excitation wavelength. Then set the imaging software to a desired frequency and resolution.

Next, find the glomeruli in brightfield. Subsequently turn on the detection of the fluorescence signal. After that, select a desired focal plane and check the intensity of fluorescence on the flu oh four and firo red channels.

Make sure that the glomerulus is clearly seen in brightfield. Then record the changes in fluorescence intensity for the flu oh four and furor red signals. To import the image sequence, split the channels and use the hyper stack gray scale mode.

In Image J software, open an ROI manager window following the analyze tools, ROI Manager path. Select one or more regions of interest using an oval selection tool and the add T function in the ROI manager. Then select the area in the background and save the selected ROIs.

Next, mark the one row per slice box in the dialogue and click okay in the menu options results. Set measurements. Select mean gray value, and calculate the pixel intensity values for each ROI, which will be displayed in the results window in separate columns.

For each ROI then copy the measured ROI intensity values for each channel into the preferred data analysis software, and subtract the background intensity values from each data point for each time point. Calculate the ratio of intensities of the flu oh four to URA red channels after that plot, the point time changes of calcium transient for each ROI. This image shows the patch clamp pipette attached to the oxide body on the surface of the rat glomerulus.

During an electrophysiological recording, a part of encapsulated glomerulus can be seen in the right lower corner of the image. Here is a representative current trace of the tripy like channels activity from a cell attached patch made on the podocyte to measure the intracellular calcium concentration. Glomeruli are loaded with flu oh 4:00 AM to label intracellular calcium.

This video illustrates the effects of CIN and manganese chloride on calcium influx in the glomeruli. This graph quantifies changes in flu oh four signal intensity recorded from a single podocyte in response to CIN and manganese chloride. As expected, CIN produces the maximum of the fluorescence and manganese chloride quenches the dye and results in the lowest fluorescence intensity.

Application of these drugs allows the researcher to later define exact calcium concentration within the cell. This video demonstrates the effect of 10 micromole a TP on the calcium concentration in the glomeruli. Note that there is an increase in the green flu O four fluorescence and a drop in red fu red fluorescence, which account for an increase intracellular calcium concentration.

This technique offers a unique opportunity to monitor changes in calcium influx at the single ion channel and individual cells level after pharmacological treatments or other manipulations. Thus, this approach represent a very powerful tool considering multiple available genetically modified rodent models. After watching this video, you should have a good understanding of how to perform calcium ation measurements in the podocytes of the, as well as the electrophysiological, which clamp experiments used together all on its own.

These techniques provide an in-depth and mechanistic insight into regulation of calcium handling with podocytes in normal and pathological conditions.