BOO deregulates proliferation and apoptosis in the developing bladder

Cell proliferation in the fe ta l bladder

I found that 30 days o f BOO resulted in enhanced growth o f the fetal bladder as assessed by whole bladder weight, protein and DNA measurements. This is similar to the growth observed in the obstructed fetal bladder by other investigators in the fetal sheep (Karim et al, 1993; Peters et al, 1992b). Assessing PCNA protein expression, a surrogate marker of cell proliferation, I found that experimental fetal BOO was associated with a significantly

increased percentage o f proliferating detrusor SMC, versus sham controls, with no

significant difference in the lamina propria or urothelial cells between the two groups. In addition, the average area o f detrusor SMC was increased after 30 days o f BOO.

Furthermore, in obstructed bladders only, a prominent population o f proliferating cells

was noted between muscle bundles; these cells probably represent fibroblast-like cells which may be implicated in the increased collagen in this area, as seen by Masson’s

trichrome and elastin van Geison staining in obstructed bladders. If these alterations in

proliferation and hypertrophy have occurred for a significant period during BOO, my results are consistent with the conclusion that both proliferation and hypertrophy o f

o f the new, larger SMC may be an adaptation in the fetal bladder that attempts to overcome the urinary flow impairment in my experimental model.

Apoptosis in the fe ta l bladder detrusor

The response to BOO, in terms o f cell turnover, however, appears more complex when programmed cell death is also considered. Within detrusor muscle bundles, I found that BOO was associated with a significant increase in the percentage o f apoptotic SMC; if the assumption that the clearance o f cells undergoing programmed cell death is equal in

the sham and obstructed groups is correct, the extent o f apoptosis is approximately three

times higher after BOO. This upregulation o f apoptosis may occur for a number o f reasons. First, it may represent a deletion o f ‘excess’ cells in a rapidly proliferating population. Such a phenomenon has been observed as a strategy to control cell numbers in metanephric kidney development (Coles et al, 1993; Welham et al, 2002) and during embryogenesis and epidermal development in the fruit fly (Li et al, 1999; Namba et al, 1997). Such a function may allow (Welham et al, 2002) some detrusor SMC to be deleted as their neighbouring SMC hypertrophy and fill the limited space. Conversely, the

apoptosis I documented may represent a pathological loss o f potentially functional muscle cells which might help to overcome BOO. Overall, it appears the balance o f growth in the fetal bladder is mediated by cell proliferation and hypertrophy with this process modified by programmed cell death. Further studies are necessary to determine what factors are involved in controlling the fine balance o f proliferation and apoptosis; investigations o f the proto-oncogene, c-myc (Rohn et al, 1998), show this protein can stimulate cell proliferation in the presence o f appropriate survival factors and trigger

apoptosis in their absence. Such dual capacity may have an important impact on cell turnover in the obstructed fetal bladder and warrant further investigation.

Apoptosis in the fe ta l bladder lamina propria

In the lamina propria layer, I documented a significant, approximately, three-fold

increase in apoptosis in obstructed bladders, whereas, unlike the detrusor layer, the

percentage o f proliferating cells was not different firom sham controls. Furthermore, the

number o f cells per unit area significantly decreased in this layer. Collectively, these observations suggest that enhanced death may have led to a decrease in cell density in the lamina propria.

In the early stages o f murine bladder development, the organ is relatively simple, with a prominent mesenchymal layer enveloping a simple epithelial layer (Baskin et al, 2001;

Smeulders et al, 2001). Through a poorly-understood process o f mutual inductive events (Baskin et al, 2001), murine bladder mesenchyme differentiates into the detrusor muscle layer and the lamina propria, while the epithelium acquires a complex, multi-layered phenotype. While it is not established whether ovine lamina propria or adjacent

epithelium have similar inductive roles in the experimental period o f observation in my thesis, it is interesting to note that attenuation of the lamina propria layer was

accompanied by ‘simplification’ of the urothelium, which had only one or two layers of

be made o f the possibility that the simplification o f the urothelium may also result directly from the mechanical effects o f increased urine volume within the obstructed bladder. Furthermore, as the bladder matures, neurovascular bundles traverse the lamina propria (Levy and Wight, 1990); while I did not formally localise apoptotic nuclei to particular cell types in the lamina propria, my impression was that most dying cells were not in vessel walls and they were therefore likely to be interstitial cells o f the lamina propria.