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

  • Hikaru Hashitani
  • Michael J. Nguyen
  • Haruka Noda
  • Retsu Mitsui
  • Ryuhei Higashi
  • Keisuke Ohta
  • Kei-Ichiro Nakamura
  • Richard J. LangEmail author
Signaling and cell physiology


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.


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



α-Smooth muscle actin


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


Atypical smooth muscle cell


Basal epithelial cell

BK channel

Large conductance calcium-activated potassium channel


Intracellular concentration of Ca2+




‘T-type’ Ca2+ channel


Ca2+-induced release of Ca2+


4′,6-Diamidino-2-phenylindole dihydrochloride


Dimethyl sulphoxide


Enhanced-yellow fluorescent protein


Focused ion bean scanning electron microscopy


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


Green fluorescent protein


Glibenclamide-sensitive ATP-dependent K+ channels


K channel subunit encoded by KCNQx gene


Hyperpolarization-activated cation nucleotide-gated channel


Interstitial cells


Interstitial cells of Cajal


Inositol triphosphate


An original glass slide product of Matsunami Glass, Osaka, Japan.


Multi-modal Australian ScienceS Imaging and Visualisation Environment


Number of animals


Number of cells


Nerve bundle


Optimal cutting temperature




Phosphate-buffered saline


Platelet-derived growth factor receptor alpha




Physiological salt solution


Renal artery


Renal interstitial cell


Renal vein


Region of interest


Spontaneous transient depolarizations


Typical smooth muscle cell



½ width

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



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.

Compliance with ethical standards


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.

Supplementary material

Movie S2

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

424_2016_1930_MOESM2_ESM.jpeg (12.3 mb)
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)
424_2016_1930_MOESM3_ESM.jpeg (681 kb)
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)


  1. 1.
    Arena E, Nicotina PA, Arena S, Romeo C, Zuccarello B, Romeo G (2007a) Interstitial cells of Cajal network in primary obstructive megaureter. Pediatr Med Chir 29:28–31PubMedGoogle Scholar
  2. 2.
    Arena F, Nicotina PA, Arena S, Romeo C, Zuccarello B, Romeo G (2007b) C-kit positive interstitial cells of Cajal network in primary obstructive megaureter. Minerva Pediatr 59:7–11PubMedGoogle Scholar
  3. 3.
    Biel M, Wahl-Schott C, Michalakis S, Zong X (2009) Hyperpolarization-activated cation channels: from genes to function. Physiol Rev 89:847–885CrossRefPubMedGoogle Scholar
  4. 4.
    Cain JE, Islam E, Haxho F, Blake J, Rosenblum ND (2011) Gli3 repressor controls functional development of the mouse ureter. J Clin Invest 121:1199–1206CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    David SG, Cebrian C, Vaughan ED, Herzlinger D (2005) C-Kit and ureteral peristalsis. J Urol 173:292–295CrossRefPubMedGoogle Scholar
  6. 6.
    Dixon JS, Gosling JA (1970a) Electron microscopic observations on the renal caliceal wall in the rat. Zeitschrift Fur Zellforschung Und Mikroskopische Anatomie 103:328–340CrossRefPubMedGoogle Scholar
  7. 7.
    Dixon JS, Gosling JA (1970b) Fine structural observations on the attachment of the calix to the renal parenchyma in the rat. J Anatomy 106:181–182Google Scholar
  8. 8.
    Dixon JS, Gosling JA (1973) The fine structure of pacemaker cells in the pig renal calices. Anat Rec 175:139–153CrossRefPubMedGoogle Scholar
  9. 9.
    Dixon JS, Gosling JA (1982) The musculature of the human renal calices, pelvis and upper ureter. J Anatomy 135:129–137Google Scholar
  10. 10.
    Englemann TW (1869) Zur Physiologie Des Ureter. Pflugers Archiv Eur J Physiol 2:243–293CrossRefGoogle Scholar
  11. 11.
    Ergang P, Leden P, Bryndova J, Zbankova S, Miksik I, Kment M, Pacha J (2008) Glucocorticoid availability in colonic inflammation of rat. Dig Dis Sci 53:2160–2167CrossRefPubMedGoogle Scholar
  12. 12.
    Gosling JA, Dixon JS (1974) Species variation in the location of upper urinary tract pacemaker cells. Investig Urol 11:418–423Google Scholar
  13. 13.
    Hashitani H, Lang RJ, Mitsui R, Mabuchi Y, Suzuki H (2009) Distinct effects of Cgrp on typical and atypical smooth muscle cells involved in generating spontaneous contractions in the mouse renal pelvis. Br J Pharmacol 158:2030–2045CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hurtado R, Smith CS (2016) Hyperpolarization-activated cation and T-type calcium ion channel expression in porcine and human renal pacemaker tissues. J Anat 228:812–825CrossRefPubMedGoogle Scholar
  15. 15.
    Hurtado R, Bub G, Herzlinger D (2010) The pelvis-kidney junction contains Hcn3, a hyperpolarization-activated cation channel that triggers ureter peristalsis. Kidney Int 77:500–508CrossRefPubMedGoogle Scholar
  16. 16.
    Hurtado R, Bub G, Herzlinger D (2014) A molecular signature of tissues with pacemaker activity in the heart and upper urinary tract involves coexpressed hyperpolarization-activated cation and T-type Ca2+ channels. FASEB J 28:730–739CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Iqbal J, Tonta MA, Mitsui R, Li Q, Kett M, Li J, Parkington HC, Hashitani H, Lang RJ (2012) Potassium and Ano1/Tmem16a chloride channel profiles distinguish atypical and typical smooth muscle cells from interstitial cells in the mouse renal pelvis. Br J Pharmacol 165:2389–2408CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kang H, Park J, Jeong S, Kim J, Moon H, Perez-Reyes E, Lee J (2006) A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels. J Biol Chem 281:4823–4830CrossRefPubMedGoogle Scholar
  19. 19.
    Kang H, Lee H, Jin M, Jeong H, Han S (2009) Decreased interstitial cells of Cajal-like cells, possible cause of congenital refluxing Megaureters: histopathologic differences in refluxing and obstructive Megaureters. Urology 74:318–323CrossRefPubMedGoogle Scholar
  20. 20.
    Klemm M, Exintaris B, Lang R (1999) Identification of the cells underlying pacemaker activity in the guinea-pig upper urinary tract. J Physiol 519:867–884CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lang R (2010) Role of hyperpolarization-activated cation channels in pyeloureteric peristalsis. Kidney Int 77:483–485CrossRefPubMedGoogle Scholar
  22. 22.
    Lang RJ, Klemm MF (2005) Interstitial cell of Cajal-like cells in the upper urinary tract. J Cell Mol Med 9:543–556CrossRefPubMedGoogle Scholar
  23. 23.
    Lang R, Exintaris B, Teele M, Harvey J, Klemm M (1998) Electrical basis of peristalsis in the mammalian upper urinary tract (review). Clin Exp Pharmacol Physiol 25:310–321CrossRefPubMedGoogle Scholar
  24. 24.
    Lang RJ, Takano H, Davidson ME, Suzuki H, Klemm MF (2001) Characterization of the spontaneous electrical and contractile activity of smooth muscle cells in the rat upper urinary tract. J Urol 166:329–334CrossRefPubMedGoogle Scholar
  25. 25.
    Lang R, Davidson M, Exintaris B (2002) Pyeloureteral motility and ureteral peristalsis: essential role of sensory nerves and endogenous prostaglandins. Exp Physiol 87:129–146CrossRefPubMedGoogle Scholar
  26. 26.
    Lang R, Hashitani H, Tonta M, Parkington H, Suzuki H (2007a) Spontaneous electrical and Ca2+ signals in typical and atypical smooth muscle cells and interstitial cell of Cajal-like cells of mouse renal pelvis. J Physiol 583:1049–1068CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lang RJ, Hashitani H, Tonta MA, Suzuki H, Parkington HC (2007b) Role of Ca2+ entry and Ca2+ stores in atypical smooth muscle cell autorhythmicity in the mouse renal pelvis. Br J Pharmacol 152:1248–1259CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lang R, Hashitani H, Tonta M, Bourke J, Parkington H, Suzuki H (2010) Spontaneous electrical and Ca2+ signals in the mouse renal pelvis that drive pyeloureteric peristalsis. Clin Exp Pharmacol Physiol 37:509–515CrossRefPubMedGoogle Scholar
  29. 29.
    Kj P, Gw H, Lee H, Spencer N, Ward S, Smith T, Sanders K (2006) Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ. Am J Physiol-Cell Physiol 290:C1411–C1427Google Scholar
  30. 30.
    Metzger R, Schuster T, Till H, Stehr M, Franke FE, Dietz HG (2004) Cajal-like cells in the human upper urinary tract. J Urol 172:769–772CrossRefPubMedGoogle Scholar
  31. 31.
    Metzger R, Schuster T, Till H, Franke FE, Dietz HG (2005) Cajal-like cells in the upper urinary tract: comparative study in various species. Pediatr Surg Int 21:169–174CrossRefPubMedGoogle Scholar
  32. 32.
    Miyakawa T, Mizushima A, Hirose K, Yamazawa T, Bezprozvanny I, Kurosaki T, Iino M (2001) Ca2+-sensor region of Ip3 receptor controls intracellular Ca2+ signaling. EMBO J 20:1674–1680CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Nguyen MJ, Angkawaijawa S, Hashitani H, Lang RJ (2013) Nicotinic receptor activation on primary sensory afferents modulates autorhythmicity in the mouse renal pelvis. Brit J Pharmacol 170:1221–1232CrossRefGoogle Scholar
  34. 34.
    Nguyen MJ, Higashi R, Ohta K, Nakamura KI, Hashitani H, Lang RJ (2015) Autonomic and sensory nerve modulation of peristalsis in the upper urinary tract. Auton Neurosci. doi: 10.1016/J.Autneu.2015.07.425 PubMedGoogle Scholar
  35. 35.
    Nguyen MJ, Hashitani H, Lang RJ (2016) Angiotensin receptor-1a knockout leads to hydronephrosis not associated with a loss of pyeloureteric peristalsis in the mouse renal pelvis. Clin Exp Pharmacol Physiol 43:535–542CrossRefPubMedGoogle Scholar
  36. 36.
    Ohta K, Sadayama S, Togo A, Higashi R, Tanoue R, Nakamura K (2012) Beam deceleration for block-face scanning electron microscopy of embedded biological tissue. Micron 43:612–620CrossRefPubMedGoogle Scholar
  37. 37.
    Santicioli P, Maggi CA (1998) Myogenic and neurogenic factors in the control of pyeloureteral motility and ureteral peristalsis. Pharmacol Rev 50:683–722PubMedGoogle Scholar
  38. 38.
    Solari V, Piotrowska AP, Puri P (2003) Altered expression of interstitial cells of Cajal in congenital ureteropelvic junction obstruction. J Urol 170:2420–2422CrossRefPubMedGoogle Scholar
  39. 39.
    Thakur P, Nehru B (2015) Inhibition of neuroinflammation and mitochondrial dysfunctions by carbenoxolone in the rotenone model of Parkinson’s disease. Mol Neurobiol 51:209–219CrossRefPubMedGoogle Scholar
  40. 40.
    Tsuchida S, Suzuki T (1992) Pacemaker activity of the pelvicalyceal border recorded by an intracellular glass microelectrode. Urol Int 48:121–124CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Hikaru Hashitani
    • 1
  • Michael J. Nguyen
    • 2
  • Haruka Noda
    • 1
  • Retsu Mitsui
    • 1
  • Ryuhei Higashi
    • 3
  • Keisuke Ohta
    • 3
  • Kei-Ichiro Nakamura
    • 3
  • Richard J. Lang
    • 2
    Email author
  1. 1.Department of Cell PhysiologyNagoya City University Graduate School of Medical SciencesNagoyaJapan
  2. 2.Department of Pharmacology, School of Biomedical SciencesMonash UniversityClaytonAustralia
  3. 3.Department of AnatomyKurume University School of MedicineKurumeJapan

Personalised recommendations