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

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 Ca 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 Ca3.1 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 Cachannel blocker, Ni (10-200 μM), reduced the frequency while the L-type Ca channel blocker (1 μM nifedipine) reduced the amplitude of propagating Ca waves and contractions in the TSMC layer. Upon complete suppression of Ca entry through TSMC Ca channels, ASMCs displayed high-frequency (6 min) Ca transients, and ICs distributed into two populations of cells firing at 1 and 3 min, respectively. IC Ca transients periodically (every 3-5 min) summed into bursts which doubled the frequency of ASMC Ca 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(+), Ca3.1(+) ICs can accelerate ASMC Ca signalling.