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Interstitial cell modulation of pyeloureteric peristalsis in the mouse renal pelvis examined using FIBSEM tomography and calcium indicators

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Abstract

Typical and atypical smooth muscle cells (TSMCs and ASMCs, respectively) and interstitial cells (ICs) within the pacemaker region of the mouse renal pelvis were examined using focused ion beam scanning electron (FIB SEM) tomography, immunohistochemistry and Ca2+ imaging. Individual cells within 500–900 electron micrograph stacks were volume rendered and associations with their neighbours established. ‘Ribbon-shaped’, Ano1 Cl channel immuno-reactive ICs were present in the adventitia and the sub-urothelial space adjacent to the TSMC layer. ICs in the proximal renal pelvis were immuno-reactive to antibodies for CaV3.1 and hyperpolarization-activated cation nucleotide-gated isoform 3 (HCN3) channel sub-units, while basal-epithelial cells (BECs) were intensely immuno-reactive to Kv7.5 channel antibodies. Adventitial to the TSMC layer, ASMCs formed close appositions with TSMCs and ICs. The T-type Ca2+channel blocker, Ni2+ (10–200 μM), reduced the frequency while the L-type Ca2+ channel blocker (1 μM nifedipine) reduced the amplitude of propagating Ca2+ waves and contractions in the TSMC layer. Upon complete suppression of Ca2+ entry through TSMC Ca2+ channels, ASMCs displayed high-frequency (6 min−1) Ca2+ transients, and ICs distributed into two populations of cells firing at 1 and 3 min−1, respectively. IC Ca2+ transients periodically (every 3–5 min−1) summed into bursts which doubled the frequency of ASMC Ca2+ transient firing. Synchronized IC bursting and the acceleration of ASMC firing were inhibited upon blockade of HCN channels with ZD7288 or cell-to-cell coupling with carbenoxolone. While ASMCs appear to be the primary pacemaker driving pyeloureteric peristalsis, it was concluded that sub-urothelial HCN3(+), CaV3.1(+) ICs can accelerate ASMC Ca2+ signalling.

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Abbreviations

α-SMA:

α-Smooth muscle actin

Ano1:

Antoctamin-1 Ca2+-activated Cl channel encoded by the ANO1 gene

ASMC:

Atypical smooth muscle cell

BEC:

Basal epithelial cell

BK channel:

Large conductance calcium-activated potassium channel

[Ca2+]i :

Intracellular concentration of Ca2+

Cap:

Capillary

CaV3.x:

‘T-type’ Ca2+ channel

CIRC:

Ca2+-induced release of Ca2+

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

DMSO:

Dimethyl sulphoxide

eYFP:

Enhanced-yellow fluorescent protein

FIB SEM:

Focused ion bean scanning electron microscopy

F t /F 0 :

Ratio (F t /F 0) of the fluorescence at time t and baseline fluorescence at t = 0

GFP:

Green fluorescent protein

KATP :

Glibenclamide-sensitive ATP-dependent K+ channels

KV7.x:

K channel subunit encoded by KCNQx gene

HCN:

Hyperpolarization-activated cation nucleotide-gated channel

ICs:

Interstitial cells

ICC:

Interstitial cells of Cajal

IP3 :

Inositol triphosphate

MAS:

An original glass slide product of Matsunami Glass, Osaka, Japan. http://www.matsunami-glass.co.jp/english/life/clinical_g/data18.html

MASSIVE:

Multi-modal Australian ScienceS Imaging and Visualisation Environment

N:

Number of animals

n:

Number of cells

NB:

Nerve bundle

OCT:

Optimal cutting temperature

P:

Papilla

PBS:

Phosphate-buffered saline

PDGFRα:

Platelet-derived growth factor receptor alpha

PG:

Prostaglandin

PSS:

Physiological salt solution

RA:

Renal artery

RIC:

Renal interstitial cell

RV:

Renal vein

ROI:

Region of interest

STDs:

Spontaneous transient depolarizations

TSMC:

Typical smooth muscle cell

U:

Urothelium

½ width:

Half-amplitude duration measured as the time between 50% peak amplitudes on the rising and falling phases

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Acknowledgements

The work was supported by Grant-in-Aid for Scientific Research (No. 26670705) from the Japan Society for Promotion of the Science (JSPS) to H.H. The authors acknowledge the use of the imaging facilities within the Multi-modal Australian ScienceS Imaging and Visualization Environment (MASSIVE) at the Monash University node of the National Imaging Facility.

Author information

Correspondence to Richard J. Lang.

Ethics declarations

Funding

The work was supported by Grant-in-Aid for Scientific Research (No. 26670705) from the Japan Society for Promotion of the Science (JSPS) to H.H.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institutions at which the studies were conducted.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Electronic supplementary material

Video of the 3 dimensional reconstruction of the proximal renal pelvis, highlighting individual cells of interest. (MP4 8368 kb)

Movie S2

Video of the 3 dimensional reconstruction of the proximal renal pelvis, highlighting individual cells of interest. (MP4 8368 kb)

Fig. S1

Three regions of the renal pelvis block (ai-iii) were milled and photographed, the resulting micrograph stacks of ortho-slices (bi-iii) were aligned and processed using 3D visualization software. Structures and cells of a similar morphology were colour coded; extraneous structures such as blood vessels and capsules were made relatively transparent (bi-iii) or removed for (ci-iii) for clarity. The number of ASMCs (pale green cells di-iii) and ‘ribbon-shaped’ fibroblast-like interstitial cells (ICs pale and dark blue cells di-iii) decreased and increased, respectively, with distance from the papilla base. Axes: X green, Y red and Z blue. Calibration bars 50 μm (b), 1e15 nm (b) and 0.2e14 nm (d). (JPEG 12.3 MB)

Fig. S2

Sequential standard electron micrographs (slice numbers indicated) illustrating that macrophages (ai-iii arrows) and nerve bundles (bi-iii arrows), which travelled considerable distances through the FIBSEM stacks, were volume rendered (aiv yellow cell, biv purple structure, respectively) to reveal their location and associations with neighbouring cells such as BECs (ci-ii). (JPEG 680 kb)

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Hashitani, H., Nguyen, M.J., Noda, H. et al. Interstitial cell modulation of pyeloureteric peristalsis in the mouse renal pelvis examined using FIBSEM tomography and calcium indicators. Pflugers Arch - Eur J Physiol 469, 797–813 (2017). https://doi.org/10.1007/s00424-016-1930-6

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Keywords

  • Pyeloureteric peristalsis
  • Upper urinary tract
  • Focused ion bean scanning electron microscopy
  • Calcium
  • Smooth muscle cells