This protocol yields intact pacemaker regions of the renal pelvis, the smooth muscle organ within the kidney that pumps urine into the ureter and into the bladder. Unlike isolating cells from the renal pelvis, this approach provides intact in situ environments of renal pelvis pacemaker regions and a more physiological preparation for studying renal pelvis pacemaking. An individual that has never performed this technique may struggle to cut uniform sections.

A first-time user must ensure the kidney is level on the vibratome stage and doesn't speed up the cutting process. Using internal tissue forceps and internal dissection scissors, pinch the intestines and lift them away from the abdominal wall. Simultaneously cut the underside of the intestines free from the body at the proximal duodenum and distal colon to gain access to the retroperitoneal space containing the kidneys.

Gently pinch and lift the distal end of the ureter with tissue forceps. Using the dissection scissors, cut underneath the pinched ureter towards the kidney until it has become liberated from the surrounding connective tissue. Once the kidneys are exposed, extract them individually.

Fill a silicon elastomer-coated dish with ice cold KRB solution. Transfer the kidney to the dissection dish and ensure that it is completely submerged. Use fine spring scissors and internal forceps to remove adipose tissue from the base of the kidney to expose the distal renal pelvis, or RP, and proximal ureter.

Remove the proximal ureter and a portion of the distal RP from the base using fine spring scissors and forceps. Pierce the outer renal capsule with fine tip forceps, angling the tips away from the kidney body. Using forceps with each hand, pinch the loose ends of the capsule and peel them apart until the remaining renal capsule membrane is removed entirely.

Insert a razor blade into the blade holder of the vibratome instrument and adjust the blade clearance angle to approximately 18 degrees. Adjust the blade parameters as mentioned in the text manuscript, ensuring that the kidney section thickness does not exceed 150 micrometers, which would negatively impact calcium ion imaging experiments. Use blunt-ended forceps to gently grasp and remove the prepared kidney from ice cold KRB solution.

Immediately place the kidney on absorbent paper for approximately two to four seconds to remove excess external moisture. Gently roll the kidney across the absorbent paper to ensure that all sides of the parenchyma have dried so that there is optimal adhesion of the kidney to the vibratome stage. Immediately apply a thin layer of cyanoacrylate glue to the base of the vibratome specimen plate and use blunt-ended forceps to place the kidney ureter side down on the area covered in glue.

Gently apply downward pressure to the top of the kidney with the flat edge of the forceps for approximately 10 to 20 seconds to dry the glue. Firmly secure the specimen plate to the bottom of the buffer tray and adjust the level of KRB solution so that the top of the kidney is fully immersed. For automatic vibratome sectioning, select the start and end positions of the vibratome blade cutting cycle 0.5 to 1 centimeter clear of the kidney to ensure that the entire kidney plane is getting sectioned.

Start the automatic cutting process, making sure that the blade makes contact with the kidney. Using forceps, collect sections that are liberated from the kidney and immediately transfer them to individual wells. Continue the sectioning protocol and use a light microscope to identify PKJ regions in the kidney slices until the PKJ regions become more apparent.

Fill a silicon elastomer-coated imaging dish with ice cold KRB solution. Transfer an individual kidney slice to the dish. Insert Minutien pins around the periphery of a kidney slice to secure the section to the base of the imaging dish.

Place the imaging dish on the stage of an upright spinning disc confocal microscope and immediately start perfusing with KRB solution. Use a low magnification water immersion objective lens to locate the kidney slice. Center the imaging field on areas of the slice where the PKJ is present using landmarks.

Once the PKJ is located, use a higher magnification water immersion objective lens to magnify the area of interest. Distinguish different cells of interest in different types of calcium ion transient durations in the PKJ wall. Once cells of interest have been identified, adjust the laser intensity to yield a good signal-to-noise ratio and record images at a temporal sampling frequency between 16 and 32 Hertz.

Light microscopic images of whole kidney sections show annotated landmarks of a single PKJ region. Multiple PKJ regions present close to the inner medulla and the distal kidney. The locations of the PKJ renal arterials and PKJ boundaries are shown here.

A PKJ section from a mouse expressing GCaMP in PDGFR alpha positive cells is shown here. The thin PKJ wall suspended between parenchymal tissue can be distinguished using landmarks such as the renal arteriole. The expression of GCaMP6f in this specific transgenic tissue is spread across the entire width of the PKJ across both the muscle and adventitial layers.

In the PDGFR alpha GCaMP6f positive kidney slices, a network of cells that typically extends over the width of the PKJ wall is fluorescent and displays oscillating calcium ion transients of various durations and frequencies. In SMC GCaMP3 positive kidney slices, a layer of GCaMP3 positive cells are present in the muscle layer. A spatiotemporal map shows that PDGFR alpha positive cells located in the PKJ adventitia elicit long duration low-frequency calcium ion transients, whereas SMCs fired shorter duration calcium ion transients more frequently.

Similarly, an array of fluorescent calcium ion signals within and surrounding collecting ducts and oscillating calcium ion transients in the SMCs were also observed. When attempting this procedure, it is important the kidney is as level as possible during vibratome sectioning to ensure uniform kidney slices are liberated from the block. Following this procedure, other methods such as single-cell isolation, immunohistochemistry and molecular studies can be performed to increase our understanding of pacemaker mechanisms in the renal pelvis.

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