Effect of age on the role of Rho-kinase in short-term partial bladder outlet obstruction

Abstract

Objectives: We examined the expression of Rho-kinase (ROK) isoforms in young and old rabbits' detrusor smooth muscles (SM) during the progression of short-term partial bladder outlet obstruction and correlated them with the time course of obstruction.

Methods: We obtained detrusor samples from bladders after 1, 3, 7, and 14 days of obstruction and also sham-operated control rabbits. We used reverse transcriptase-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting to determine the relative levels of ROK isoform expression at the mRNA and protein levels.

Results: Bladder weight for young rabbits increased between 1 and 7 days' obstruction and came back toward control levels at 14 days' obstruction. In old rabbits, bladder weight increased after obstruction, reaching a maximum at 3 days and remaining at this level throughout the 14 days. In young rabbits, the expression of ROKalpha increased in 1- to 7-day obstructed groups and decreased in the 14-day group, whereas it increased progressively in the old rabbits at both the mRNA and protein levels. There was a significant decrease in the expression of ROKbeta in young obstructed rabbits, which gradually decreased during the course of 1- to 7-day obstruction period and increased after 14 days of obstruction. In old groups, there was a decrease in expression after 1 day of obstruction and values remained at a decreased level throughout the course of the study.

Conclusions: Young rabbit bladders are better able to adapt to bladder outlet obstruction and ROK isoforms respond in a similar way.

Figure 1

RT-PCR analysis of ROK α. A). ROK α mRNA expression in young and old rabbits’ control and obstructed bladders. B) Average expression in different samples of young and old rabbits. Each bar is the mean +/− SEM of 4 obstructed and 4 control rabbits. * = significantly different from control, ¥ = significantly different from young, p < 0.05.

Figure 2

Western blot analyses showing the expression of ROK α at the protein level. A) Protein extracts from detrusor smooth muscles of young and old rabbit’s control and obstructed rabbit bladders probed with antibody against ROK α. B) Panel B depicts the relative expression of ROK α as obtained from scanning the bands. Each bar is the mean +/− SEM of 4 obstructed and 4 control rabbits. * = significantly different from control, ¥ = significantly different from young, p < 0.05.

Figure 3

RT-PCR analysis of ROK β. A) ROK β mRNA expression in young and old rabbits’ control and obstructed bladders. B) Average expression in different samples of young and old rabbits. Each bar is the mean +/− SEM of 4 obstructed and 4 control rabbits. * = significantly different from control, ¥ = significantly different from young, p < 0.05.

Figure 4

Western blot analysis showing the expression of ROK β at the protein level. A) Protein extracts from detrusor smooth muscles of young and old rabbit’s control and obstructed rabbit bladders probed with antibody against of control and obstructed rabbit bladders probed with antibody against ROK β. B) Panel B depicts the relative expression of ROK β as obtained from scanning the bands. Each bar is the mean +/− SEM of 4 obstructed and 4 control rabbits.* = significantly different from control, ¥ = significantly different from young, p < 0.05.