Published: April 30th, 2021
The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.
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.
The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that transports urine from the kidney to the ureter. The RP transports urine by generating regular rhythmic contractions (peristalsis)1,2,3,4,5, which propels a bolus of urine from the kidney distally to the ureter and ultimately to the bladder, where it is stored until micturition occurs6,7. Loss of this regular activity has dire consequences, including hydronephrosis and kidney failure1,3,8; hence, there is a critical need to study the mechanisms underlying regular, rhythmic RP contractions. Peristaltic contractions originate from the most proximal region of the RP-in the pelvis-kidney junction (PKJ)9,10,11,12,13,14,15 (Figure 1A-C) and propagate distally to push urine from the papilla into the RP (Figure 1B). Electrical pacemaker activity is recorded in the PKJ as spontaneous transient depolarizations10,11,12,13,15,16,17, which are thought to arise from specialized pacemaker cells. These pacemaker cells, previously called atypical smooth muscle cells (ASMCs), are thought to generate or coordinate pacemaker activity and drive the contractions of "typical" smooth muscle cells (SMCs)9,10,11,18,19,20,21,22,23.
ASMCs are most abundant in the proximal RP, at the PKJ (Figure 1A-C), where peristaltic contractions and electrical pacemaker activity originates5,7,8,9,12,13,14,16,17,18,19,20,21,22. A recently published study by this group identified platelet-derived growth factor receptor-alpha (PDGFRα), in combination with smooth muscle myosin heavy chain (smMHC), as a unique biomarker for these interstitial cells (ICs)24, a finding that has been corroborated by other groups25. Based on their morphology and protein expression pattern, these cells were called PDGFRα+ IC type 1 (PIC1)24,26. PIC1s reside in the muscle layer of the PKJ where they display high-frequency, short-duration Ca2+ transients, thought to underlie the generation of pacemaker potentials24. However, other cell types exist in the PKJ, including non-smMHC-expressing PDGFRα+ ICs (PIC2s) in the adventitial layer. Previous reports have suggested that non-smMHC ICs may participate in the regulation of pacemaker activity19. However, further study of non-smMHC ICs is hindered by poor distinction during Ca2+ imaging studies. Typically, heterogeneous cell types within the RP preparations are indiscriminately loaded with Ca2+-sensitive dyes (e.g., Fluo-4). To overcome these challenges and to study a variety of cell types in the RP, genetically encoded Ca2+ indicators (GECIs) can be utilized to selectively express Ca2+-sensitive fluorophores in cell types of interest.
The majority of studies elucidating Ca2+ transient properties in PIC1s/ASMCs were achieved by imaging flat-sheet RP tissue preparations19,21,27. As PIC1s are the only cell type in the PKJ to express smMHC, conditional expression of the GECI, GCaMP, in smMHC+ cells is appropriate to study PIC1s in this configuration. However, as PIC1s and PIC2s both express PDGFRα, conditional expression of GCaMP variants in PDGFRα+ cells prohibit cell distinction in flat-sheet preparations. To circumvent this issue, a vibratome sectioning approach was used to distinguish PIC1s and PIC2s across the PKJ tissue wall24. To reveal these discrete cellular populations, the RP was sectioned coronally, making it possible to identify PIC2s in the adventitia and PIC1s in the muscle wall based on known immunohistochemical labeling and GECI expression patterns. As a result of this novel PKJ imaging approach, PIC1s and PIC2s were found to display distinct Ca2+ signaling properties24. Furthermore, by isolating the most proximal sections of the PKJ region (Figure 2), the pacemaker region of the RP was preserved in a way that had not been accomplished previously. Here, a protocol is described to show how to isolate PKJ preparations from the mouse kidney using vibratome sectioning, how to set up these preparations for Ca2+ imaging experiments, and how to distinguish the different cell types across the PKJ wall.
All mice used and the protocols described in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Institutional Animal Use and Care Committee at the University of Nevada, Reno, NV. Experiments and animal use also conformed to the principles and regulations as described by Grundy28.
1. Generate PDGFRα-GCaMP6f and SMCGCaMP3 mice
2. Prepare and inject transgenic mice with tamoxifen to induce conditional expression of GCaMP
3. Prepare solutions
4. Prepare silicon elastomer-coated dissection and microscope imaging dishes
5. Kidney dissection
6. Prepare the kidney for vibratome sectioning
7. Prepare and calibrate the vibratome instrument
8. Vibratome sectioning the kidney
9. Kidney slice Ca2+ image acquisition
10. Ca2+ imaging analysis
In situ Ca2+ imaging of the PKJ can reveal important cellular activity of RP pacemaker cells. By using mice that express genetically encoded Ca2+ indicators (such as GCaMP), driven by cell-specific promotors, information about RP pacemaking can be obtained with accuracy and detail that is not possible from Ca2+ imaging experiments from flat-sheet RP preparations. The beginning of the PKJ is distinguished by the sudden appearance of semi-circles of muscle suspended between kidney parenchymal tissue (Figure 2C; proximal PKJ enclosed in dashed box). During subsequent rounds of sectioning, the inner medulla becomes distinguishable from the surrounding cortical tissue. Under a light microscope, the inner medulla appears striated in regions, lighter in color compared to cortical tissue and discontinuous on its long axis with the rest of the kidney (Figure 2B,D). At this point, more PKJ regions will start to appear. Examples of this are shown in Figure 2D (dashed rectangles, H and G) where 3 semi-circles of muscle are suspended by parenchymal tissue. These muscle bands will be closely apposed to the inner papilla and will typically neighbor a renal arteriole (Figure 2D, dashed rectangles; Figure 2F-H, black arrowheads). As more distal sections are derived, these bands of muscle will integrate to form a more complete, unified structure, indicating the end of the PKJ region (Figure 2E).
Figure 3A,B shows a PKJ section at low power (4-10x) from a mouse expressing GCaMP in PDGFRα+ cells (GCaMP6f expressed by inducible Cre-recombinase driven by Pdgfra). Using landmarks such as the renal arteriole (Figure 3A; asterisk), experimenters should be able to readily distinguish the thin PKJ wall suspended between parenchymal tissue (Figure 3B; asterisks). 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 (Figure 3C).
In PDGFRα+ GCaMP6f+ kidney slices, a network of cells that typically extends over the width of the PKJ wall will be fluorescent (Figure 3C) and display oscillating Ca2+ transients of various durations and frequencies. PDGFRα+ cells in the PKJ wall display two different types of Ca2+ transient durations. In the adventitial layer (orientated closer to cortex), PDGFRα+ cells present as a network of cells and their processes are defined. Adventitial PDGFRα+ cells exhibit low-frequency (4 ± 2.7 Hz) and long-duration (1 ± 0.67 s) Ca2+ transients. The second layer of PDGFRα+ cells, present in the muscle layer (orientated closer to the medulla), exhibit similar Ca2+ transient frequencies and durations as SMC GCaMP3+ cells (described below) as they are the same cell type.
In SMC GCaMP3+ kidney slices, a layer of GCaMP3+ cells is present in the muscle layer (Figure 3D). There will be no fluorescent signal in the adventitial layer (Figure 3D; asterisk). GCaMP3+ SMCs in the muscular layer typically exhibit high-frequency (10 ± 4 Hz) and short-duration (632 ± 74 s) Ca2+ transients. PDGFRα+ cells located in the PKJ adventitia elicit long-duration, low-frequency Ca2+ transients (Figure 3E, Video 1). However, Ca2+ imaging experiments from tissue expressing GCaMP3 driven by the Myh11 promotor is restricted to the muscular aspect of the PKJ (Figure 3D). Compared to PDGFRα+ cells in the adventitia, SMCs fired shorter duration Ca2+ transients more frequently (Figure 3F, Video 2).
In addition to understanding signaling properties in PKJ PDGFRα+ ICs, the application of this technique to study other cell types in the vibratome-sectioned kidney has been demonstrated in this paper. Upon close examination of the renal medulla (in mice expressing GCaMP6f in PDGFRα+ cells), an array of fluorescent Ca2+ signals within and surrounding collecting ducts (Video 3) was observed. Medullary PDGFRα+ cells fired spontaneous Ca2+ transients of variable frequency and duration. These Ca2+ imaging studies of kidney vibratome sections could also be expanded to studying renal arterioles (~50-80 mm diameter) that often neighbor PKJ muscle segments (Figure 2F,G; white arrows). Ca2+ imaging of renal arterioles (from tissue expressing GCaMP in smooth muscle cells) demonstrates oscillating Ca2+ transients in SMCs (Video 4).
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.
Video 1: Spontaneous Ca2+ transients in GCaMP+ PDGFRα+ cells in PKJ vibratome sections. Abbreviations: PKJ = pelvic-kidney junction; PDGFRα+ = platelet-derived growth factor receptor-alpha-positive. Please click here to view this video. (Right-click to download.)
Video 2: Spontaneous Ca2+ transients in GCaMP+ smooth muscle cells in pelvic-kidney junction vibratome sections. Please click here to view this video. (Right-click to download.)
Video 3: Spontaneous Ca2+ transients in GCaMP+ PDGFRα+ cells in renal medullary vibratome sections. Abbreviation: PDGFRα+ = platelet-derived growth factor receptor-alpha-positive. Please click here to view this video. (Right-click to download.)
Video 4: Low-amplitude Ca2+ transient activity in the vibratome sections of the renal arteriole. Please click here to view this video. (Right-click to download.)
The RP consists of a heterogeneous population of cells with differential cell densities observed in various RP regions. PIC1s (previously referred to as ASMCs) are most abundant in the PKJ, where pacemaker activity originates24. The protocol, described here, allows investigators to isolate the pacemaker region from the rest of the mouse kidney. By cutting sections of PKJ using a vibratome, the pacemaker regions of the RP (identified as muscle bands attached to parenchyma) are kept intact, thus affording the use of in situ imaging to accurately study RP pacemaker cells when combined with cell-specific fluorescence reporters.
While this approach can provide new insights into RP pacemaking, there are some considerations that experimenters should be familiar with to improve imaging outcomes and sectioning efficiency. For an untrained user, this method of PKJ isolation and imaging is easier to learn than typical sharp dissection of flat-sheet RP preparations. Sharp dissection of RP from whole kidneys requires weeks of consistent practice to successfully isolate viable tissues for physiology experiments. As this vibratome sectioning protocol requires little sharp dissection knowledge, it is accessible to users who do not have experience dissecting other smooth muscle structures.
However, there are some critical points to note for this protocol. Successfully adhering kidneys to the vibratome specimen plate requires dexterity and patience. If the kidney is orientated incorrectly and leans to one side, oblique rather than straight sections will be cut. Due to the delicate nature of the PKJ, the oblique angle can often destroy the muscle bands of the pacemaker region. Furthermore, imaging of oblique sections results in poor imaging acquisition as cell networks are not typically in the same focal plane. The procedure is also time-consuming, with sectioning of a single kidney often taking up to an hour to complete, during which time the setup requires monitoring.
While the movement of the vibratome can be sped up, if the speed is increased too much (>20% than what is recommended in the protocol), the blade will shred rather than cleanly cut the kidney, resulting in loss of delicate PKJ structures. Similarly, a cutting speed that is too low can cause the section to become jagged. Optimization of cutting speed and blade amplitude is essential. Care must also be taken in handling vibratome sections. Due to their delicate nature, PKJ muscles are easily disrupted during handling and can tear. A well-trained user will be able to harvest approximately 1-2 PKJ regions per 4 kidney slices that are suitable for Ca2+ imaging experiments. Typically, PKJ sections that do not meet Ca2+ imaging criteria have: 1) poor GCaMP expression, 2) a contorted PKJ wall, or 3) a broken PKJ wall. For data analysis, approximately 3-4 cells per field of view (FOV) could be sampled.
Whilst there are many cells in the FOV of PDGFRα-GCaMP6f and SMC-GCaMP3 PKJ sections, small tissue movements often exclude cells from analysis. This can usually be resolved by applying a stabilization protocol to images. Under conditions where preparations do not move, at least 3-5 cells can be sampled from PDGFRα-GCaMP6f sections and 5-6 cells from SMC-GCaMP3 sections. Typically, the time taken from animal sacrifice (optimum age for mice is 8-16 weeks) to performing Ca2+ imaging experiments is 2-3 h, which is adequate to ensure tissue integrity, if tissues are incubated in ice-cold solutions throughout the procedure when required. In summary, a vibratome cutting protocol has been described here to generate intact preparations of RP PKJ regions from the mouse kidney. This technique allows the preservation of RP pacemaker regions for in situ Ca2+ imaging studies to investigate RP pacemaker mechanisms.
The authors have nothing to disclose.
This project was funded by R01 DK124509 from NIDDK.
|24-well culture plate
|To store kidney slices in
|35 mm x 10 mm Petri dishes
|Kidney slice calcium imaging dish
|48-well culture plate
|To store kidney slices in
|60 mm x 15 mm Petri dish
|Kidney sharp dissection dish
|To dry the kidney before applying glue
|The Jackson Laboratory
|The Jackson Laboratory
|The Jackson Laboratory
|The Jackson Laboratory
|For adhering the kidney to the specimen plate
|Extra-Fine Bonn Scissors
|Fine Science Tools
|Used for internal dissecting scissors
|Fine Science Tools
|Used for external dissecting scissors
|Fine Science Tools
|Used for fine dissection of kidney
|Gillette Silver Blue double-edge blades
|For insertion into blade holder of vibratome
|For calcium imaging analysis
|Pins were cut in half to reduce their length
|Student Adson Forceps
|Fine Science Tools
|For gently holding and moving the kidney
|Student Dumont Forceps
|Fine Science Tools
|Used for internal dissecting forceps
|Vannas spring scissors
|Fine Science Tools
|For sharp dissection and cleanup of isolated kidney
|Optional component for calibrating blade movement during cutting
|VT1200 S Vibrating Blade Microtome
|Configuration 1 is used in our protocol
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