The role of nitric oxide in bladder urothelial injury after bladder outlet obstruction.

Abstract

To investigate the role of nitric oxide (NO) in the pathogenesis of injury to the bladder mucosa after bladder outlet obstruction (BOO). The response of bladder mucosa to BOO consists of thickening with fibrous connective tissue; NO is a multipurpose messenger important in blood vessel relaxation, neuronal communication and inflammatory activities of macrophages, and is synthesized by NO synthase (NOS), with three distinct isoforms; inducible (iNOS), endothelial (eNOS) and neuronal (nNOS). Fifteen male guinea pigs had a silk ligature placed around the bladder neck to induce BOO; controls included five sham-operated animals. The animals were killed at 2, 4, 6, 8 and 12 weeks after BOO. NADPH-diaphorase staining was used as a nonspecific method to determine NOS isoform distribution. Single- and double-label immunofluorescence histochemistry for iNOS, eNOS, nNOS and macrophage colony stimulating factor (M-CSF) were used, with laser scanning confocal microscopy to assess the results. The relative amount of iNOS mRNA was evaluated using reverse-transcription-polymerase chain reaction (RT-PCR), and a standard method to determine the degree of apoptosis. Bladder mucosa had a markedly increased intensity of NADPH-diaphorase staining 2 weeks after BOO. iNOS immunoreactivity was markedly greater in the bladder mucosa 2 weeks after BOO than in controls. The immunolocalization of iNOS and M-CSF was identical in bladder mucosa after BOO. RT-PCR showed stronger iNOS mRNA expression in the obstructed bladder mucosa 2 weeks after BOO than in controls. eNOS and nNOS expression was detected only 8 weeks after BOO, with markedly more immunoreactivity 12 weeks after initiating BOO. Increased eNOS and nNOS immunoreactivity strongly correlated with the induction of apoptosis in the bladder mucosa. There was overexpression of iNOS by activated macrophages in the early stages of BOO. This overexpression did not induce apoptosis in the iNOS-expressing cells of bladder mucosa in the early stages. After long-term BOO bladder mucosa showed strong eNOS and nNOS immunoreactivity, correlating with apoptosis. Thus we suggest that eNOS and nNOS, by triggering cell death, may be important in eliminating hyperplastic urothelial cells, reflecting the plasticity of the bladder response to obstructive stimuli.