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

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 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.

Functional heterogeneity of PDGFRα (+) cells in spontaneously active urogenital tissues

“…In our previous study, intensely GFP-fluorescent PDGFRα (+) cells were located in both the adventitia and the suburothelial spaces in low magnified micrographs of the proximal renal pelvis, while the coexpression of α-smooth muscle actin in intensely fluorescent PDGFRα (+) cells was not evident. 8 In the present study, lightly GFP fluorescent PDGFRα (+) cells are densely distributed in the renal calyces and developed high-frequency spontaneous Ca 2+ transients. The morphological appearance of these lightly GFP fluorescent PDGFRα (+) cells, the properties of their spontaneous Ca 2+ transients, as well as their robust ATII-induced Ca 2+ response strongly suggested that they are the same population of cells as ASMCs, which are sparsely endowed with the α-smooth muscle actin and thought to be the pacemaker cells that electrically drive typical smooth muscle cells (TSMCs) to generate pyeloureteric peristalsis.…”

“…Distributions of the amplitude (0.43 ± 0.15 ΔF t /F 0 ), half width (624.9 ± 109.4 ms) and frequency (13.8 ± 4.2 minutes −1 , n =30, N = 21) of spontaneous Ca 2+ transients in this lightly fluorescent PDGFRα (+) cells ( Figure 2G) are very similar to that described for ASMCs. 8 Besides the predominant population of muscular PDGFRα (+) cells, intensely fluorescent PDGFRα (+) cells that had a round or oval shape GFP-expressing nuclei occasionally developed, slow, prolonged spontaneous Ca 2+ transients (Figure 2H,I; Supplement Movie S3). In seven PDGFRα (+) cells (n = 4, N = 3), spontaneous Ca 2+ transients had amplitudes of 0.98 ± 0.24 ΔF t /F 0 , the half-width of 2155.7 ± 655 ms) and frequency of 0.68 ± 0.36 minutes −1 .…”

“…Experiments were carried out in the proximal renal pelvis preparations that had been bathed in nifedipine (1-10 μM) to prevent action potential-based Ca 2+ transients in typical smooth muscle cells, with particular focus on the renal calyces where atypical smooth muscle cells (ASMCs) are densely distributed. 8,14 In the musculature of the renal calyx, PDGFRα (+) cells again identified by their nuclear GFP fluorescence ( Figure 2F) exhibited asynchronous spontaneous Ca 2+ transients (Supplement Movie S2). Ca 2+ fluorescence visualized short spindle-shaped cell bodies of PDGFRα (+) cells that were arranged in parallel.…”

“…Properties of spontaneous Ca 2+ transients in this subpopulation of PDGFRα (+) cells were similar to those of intestinal cells that were previously described and distinct from ASMCs. 8 Due to the infrequent encounter with those cells, their pharmacological properties were not investigated.…”

“…Besides the bladder, PDGFRα (+) cells are distributed in other regions of the urinary tract including the renal pelvis, 8 ureteropelvic junction, 9 and urethra 10 where various forms of spontaneous activity are generated in their smooth muscles. PDGFRα mRNA is widely expressed in the male reproductive tissues 11 including the seminal vesicle where PDGFRα (+) cells are distributed in the mucosa.…”