Beyond neurons: Involvement of urothelial and glial cells in bladder function

Abstract

Aim: The urothelium, or epithelial lining of the lower urinary tract (LUT), is likely to play an important role in bladder function by actively communicating with bladder nerves, smooth muscle, and cells of the immune and inflammatory systems. Recent evidence supports the importance of non-neuronal cells that may extend to both the peripheral and central processes of the neurons that transmit normal and nociceptive signals from the urinary bladder. Using cats diagnosed with a naturally occurring syndrome termed feline interstitial cystitis (FIC), we investigated whether changes in physiologic parameters occur within 3 cell types associated with sensory transduction in the urinary bladder: 1) the urothelium, 2) identified bladder dorsal root ganglion (DRG) neurons and 3) grey matter astrocytes in the lumbosacral (S1) spinal cord. As estrogen fluctuations may modulate the severity of many chronic pelvic pain syndromes, we also examined whether 17beta-estradiol (E2) alters cell signaling in rat urothelial cells.

Results: We have identified an increase in nerve growth factor (NGF) and substance P (SP) in urothelium from FIC cats over that seen in urothelium from unaffected (control) bladders. The elevated NGF expression by FIC urothelium is a possible cause for the increased cell body size of DRG neurons from cats with FIC, reported in this study. At the level of the spinal cord, astrocytic GFAP immuno-intensity was significantly elevated and there was evidence for co-expression of the primitive intermediate filament, nestin (both indicative of a reactive state) in regions of the FIC S1 cord (superficial and deep dorsal horn, central canal and laminae V-VIl) that receive input from pelvic afferents. Finally, we find that E2 triggers an estrus-modifiable activation of p38 MAPK in rat urothelial cells. There were cyclic variations with E2-mediated elevation of p38 MAPK at both diestrus and estrus, and inhibition of p38 MAPK in proestrous urothelial cells.

Conclusion: Though urothelial cells are often viewed as bystanders in the processing of visceral sensation, these and other findings support the view that these cells function as primary transducers of some physical and chemical stimuli. In addition, the pronounced activation of spinal cord astrocytes in an animal model for bladder pain syndrome (BPS) may play an important role in the pain syndrome and open up new potential approaches for drug intervention.

Fig. 1

Cross-section of urinary bladder wall. H&E stained cross-section of the cat urinary bladder wall.

Fig. 2

E2 triggers an estrus-modifiable activation of p38 MAP kinase in rat urothelial cells. Urothelial cells isolated from rats in diestrus, proestrus, and estrus phases were treated with 10 nM of β-estradiol (or vehicle, saline) for 60 min. Levels of phospho-p38 in cellular whole cell lysates were determined after Western blotting. *P < 0.05; Mann–Whitney rank sum test; n = 2–4.

Fig. 3

Diameter distribution of identified DRG neurons in FIC and normal cats. Mean number (±SEM) of identified (fast-blue dye labeled) DRG neurons in a minimum of 10 sections of the S1 DRG from FIC (n = 4) and normal (n = 4) animals.

Fig. 4

Increased GFAP immuno-intensity from FIC as compared to unaffected cat spinal cord. GFAP immuno-intensity levels in the S1 spinal cord superficial and deep dorsal horn; lamina × (surrounding the central canal) and the sacral parasympathetic nucleus (SPN region) from cats with FIC are higher compared with similar regions from unaffected cats (unpaired t-test; n = 4 each, FIC and unaffected).

Fig. 5

GFAP immuno-intensity in FIC and normal cat spinal cord. The immunointensity of glial fibrillary acidic protein (GFAP), an intermediate filament protein found in astrocytes, is significantly elevated in lower lumbosacral (S1) FIC spinal cord. a: Representative image of astrocytes (green—GFAP astrocytic marker; blue-4′,6-diamidino-2-phenylindole (DAPI)—used to stain cell nuclei) in S1 spinal cord dorsal horn (DH) region from a healthy cat as compared with similar S1 DH spinal cord region from an FIC cat (in b). c: FIC astrocytes (GFAP, green) co-express the intermediate filament, nestin (red), indicating a reactive state (blue—DAPI, nuclear marker). Scale bars: (a,b) 100 μm; (c), 10 μm.