Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

The overall goal of this procedure is to register single channel activity and to measure intracellular calcium concentration changes in cystic monolayers freshly isolated from rodent models of polycystic kidney disease. This is accomplished by first flushing the PCK rec kidneys via abdominal aorta in order to remove blood from the renal tissues. The second step is to isolate the epithelial monolayer of the cysts.

Next, the isolated monolayer can be used for conventional patch clamp recordings of ion channels in the apical membranes of epithelial cells. The isolated tissue can also be used for loading with calcium dyes. For quantifying intracellular calcium concentration changes in the epithelial cells.

These parameters measured in the native environment of cysts can be compared to healthy tubules in order to study the process of cyst agenesis. The main advantage of this technique is that it allows studying chi channel activity and calcium flux in the renal cysts. This analysis provides physiologically irrelevant data obtained from freshly isolated tissues.

With this approach, we can monitor single channel activity in the freshly isolated epithelia. It is unique method because it can be used to precisely characterize iron channels behavior using different modifications of the patch technique. We are able to test channel's response to various drugs in cystic and in healthy tubs.

The freshly isolated cyst monolayer can be used for intracellular calcium imaging. This technique allows pharmacological screening and functional studies of intracellular calcium homeostasis in cystic cells, which is, which are important means to discover new treatments for the prevention of polycystic kidney disease. Blood lefting care will show the surgical approach used to clear kidneys from blood.

The isolation procedures of the kidney and cysts will be shown first. Begin this procedure by performing a laparotomy and flush the kidneys with PBS through the abdominal aorta of an anesthetized rat. To do so, connect a polyethylene tubing catheter to a syringe pump filled with PBS.

Cut the skin and the abdominal wall along the abdominal midline and shift the abdominal organs with cotton swabs to get access to the descending aorta with branching mesenteric and celiac arteries. Next place the first ligature around the mesenteric and celiac arteries and the second ligature around the aorta. Under the diaphragm.

Gently separate the aorta ADOS by blunt, dissecting the connective tissues around it within forceps. Then place the third ligature about three millimeters below the left renal artery and the fourth ligature above the iliac arteries bifurcation. Tie the fourth ligature and attach a vessel clamp around the aorta between the left renal artery and the third ligature.

Make an incision between the clamp and the fourth ligature to catheterize the aorta. Then use the third ligature to firmly fix the catheter. Now, release the clamp.

Make sure that the blood pulse is visible in the catheter to ensure proper installation. Then start profusion and tie the first and second ligatures. After that, quickly cut the left renal vein to relieve pressure.

Continue flushing for one to two minutes until the organs are completely blanched.Blanc. Subsequently, cut the renal blood vessels, urethra and surrounding connective and adipose tissues. To excise the kidneys, make a short tear in the kidney capsule and peel the kidneys to encapsulate them.

Then place the kidneys in ice cold PBS. Next, prepare five by five millimeter and 25 by 25 millimeter. Cover glass chips coated with poly L lysine.

Cut the kidneys with a razor blade along the frontal plane into one to two millimeter thick slices. Then place one of the slices under a stereo microscope. Locate the cysts, which appear as round shaped cavities using fine tipped forceps.

Dissect the internal epithelial layer of a cyst as thin as possible to obtain a monolayer area. After that, attach it to a glass chip covered with polylysine and expose the cysts apical side up To provide access for the pipettes to perform electrophysiology, fill the patch clamp chamber with bath solution. Then transfer the cover glass chip with isolated tissues to the chamber.

Select a cell in the epithelial monolayer and approach a typical membrane with a patch pipette. After that, conduct a conventional patch clamp experiment in cell attached mode. In this procedure, place the isolated cysts and split open tubules In a three and a half centimeter dish containing PSS, add five micromolar flu, eight HD, and 0.05%onic acid to help disperse the acet to meth lesters.

Then protect the sample from light and incubate on a slow shaker at room temperature for 30 to 40 minutes. After incubation, wash out the incubation media with clean PSS. Then place the tissue under a confocal microscope to further perform fluorescence imaging.

Turn on the confocal acquisition software. Subsequently turn on the argon laser to 488 nanometers of excitation and adjust the emission to 520 over 25 nanometers. Then locate the tissue in bright field afterward.

Monitor flu oh eight H fluorescence in the tissue sample with a frequency of 0.25 hertz or higher. Apply a TP or other pharmacological modulators to induce an increase in intracellular calcium concentration. Shown here are the representative current traces for the epithelial sodium channel activity measured in cell attached patches of freshly isolated cysts from 16 week old PCK rats after the treatment of benzoyl and drinking water for 12 weeks.

Dashed lines indicate the respective current state with the C and op n denoting the closed and open states. This graph summarizes the observed epithelial sodium channel activity in which benzoyl a selective inhibitor of the epithelial sodium channel, significantly decreased the epithelial sodium channel activity. This video shows the cystic mono layer loaded with flu oh eight H before and after the application of 10 micromolar a TP.The increase in green fluorescent signal intensity can be seen when intracellular calcium concentration goes up in response to a TP.This graph summarizes the effect of a TP on calcium levels in the cell.

Monolayer of PCK rats application of a TP causes a rapid increase in the intracellular calcium level, which returns to basal level within two or three minutes. This technique offers a unique opportunity to monitor changes in calcium flux and iron channel activity in the cystic kidneys. Besides characterizing activity of the cellular sodium channel and calcium influx in PCK rates, this approach may be used for analyzing other iron channels in multiple AR and A-D-P-K-D models.