Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

Abstract

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca²⁺ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca²⁺ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

Figure 1:. Basic kidney anatomy and location of PKJ pacemaker region.

(A) Diagram of the intact kidney showing the orientation of the RP and ureter. The renal artery and renal vein are displayed in red and blue, respectively. (B) The intact kidney can be cut along a sagittal plane to expose the inner aspect of the kidney, including the medulla, papilla (distal medulla where collecting ducts converge), and proximal and distal RP. (C) The medulla and papilla can be excised to completely expose the PKJ and prox RP. (D and E) represent transmitted light images from the PKJ pacemaker region and distal RP, respectively. Sequential sectioning towards the distal end of the pelvis results in the semicircles of muscle in the PKJ region (Di) combining into one, thicker muscular ring (Ei) that encapsulates the entire papilla. Black, dashed rectangles in Di and Ei show approximate areas in coronal kidney sections where transmitted light images were acquired. Orientation of images D and E are 90° anti-clockwise to respective insets (Di and Ei). Scale bars in D and E = 20 μm. Abbreviations: RP = renal pelvis; prox RP = proximal renal pelvis; PKJ = pelvic-kidney junction; PICs = platelet-derived growth factor receptor-alpha-positive interstitial cells; SMC = smooth muscle cell. Please click here to view a larger version of this figure.

Figure 2:. Vibratome sectioning of whole kidneys to generate thin sections.

(A) Kidneys are mounted ureter side down to the base of the vibratome instrument, and a standard blade (attached to the vibratome head) is used to cut sequential sections from the proximal to distal end of the kidney. (B) Diagrammatic representation of a thin section cut from the whole kidney with annotated landmarks. PKJ muscle segments (black dashed rectangle) are often found suspended between parenchymal tissue. (C) Light microscopic image of a proximal kidney section. The appearance of muscle bands suspended between parenchymal tissue indicates the beginning of the proximal PKJ projections (indicated inside white dashed rectangle). (D) Light microscopic image representing the optimal region where multiple (2-3) PKJ segments can be found (areas within white dashed rectangles). Thin PKJ muscle strips are suspended between the kidney parenchyma and align closely with renal arterioles and medulla. (E) Light microscopic image of a distal kidney section. Individual muscle segments have merged to form a single, continuous muscle band (white dashed rectangle) that surrounds the inner papilla (not present in this image). Scale bars C-E = 500 μm. F-H Zoomed (20x) regions from panel D indicate the location of the PKJ (black arrowheads), renal arterioles (white arrowheads), and cut sites for isolating the PKJ (dashed white lines). Scale bars F-H = 100 μm. Abbreviation: PKJ = pelvic-kidney junction. Please click here to view a larger version of this figure.

Figure 3:. Ca²⁺ imaging of vibratome sections.

(A) Representative low-power image (4x) of a vibratome section denoting location of the renal arteriole (asterisk). Scale bar = 200 μm. (B) Zoomed (20x) representative image of the PKJ (labeled) suspended between kidney parenchymal tissue denoting locations of the PKJ muscle (white arrowhead), renal arteriole (asterisk). Scale bar = 50 μm. (C) High-power (40x) image of the PKJ expressing GCaMP in PDGFRα⁺ cells. Scale bar = 20 μm. (D) High-power (20x) image of the PKJ expressing GCaMP in smooth muscle cells. Scale bar = 20 μm. (E) Spatiotemporal map of Ca²⁺ transients sampled from a GCaMP⁺ PDGFRα⁺ cell indicated in panel C. Look up table coded for F/F₀. (F) Spatiotemporal map of Ca²⁺ transients sampled from a GCaMP⁺ PDGFRα⁺ cell indicated in panel D. Look up table coded for F/F₀. (G) Representative data for Ca²⁺ transient frequency (Hz) for GCaMP⁺ PDGFRα⁺ cells and GCaMP⁺ smMHC cells. (H) Representative data for Ca²⁺ transient duration (s) for GCaMP⁺ PDGFRα⁺ cells and GCaMP⁺ smMHC cells. Abbreviations: PKJ = pelvic-kidney junction; PDGFRα⁺ = platelet-derived growth factor receptor-alpha-positive; smMHC = smooth muscle myosin heavy chain. Please click here to view a larger version of this figure.