Expression levels of genes involved in bladder function depended on the degree of tissue remodeling
To investigate gene expression profiles of bladder remodeling due to BOO, we generated an experimental BOO model in rats. The proportion of bladder smooth muscle increased markedly in all rats exposed to experimental partial outlet obstruction compared with those in control rats. Variations in the degree of muscular hypertrophy were observed among rats, and rats were categorized into two groups according to the degree of muscular hypertrophy: the strong muscular hypertrophy (SH) group and the mild hypertrophy (MH) group (Fig. 1A). We then analyzed total gene expression patterns in the groups by RNA-seq (control group, n = 3; BOO group, n = 6; Table S1). We first confirmed the mRNA expression levels of the hypoxia markers Vegf and Hif1α. Vegf levels were significantly higher in all BOO rats than in control rats and were positively correlated with the thickness of the detrusor muscle (r2 = 0.9023, Fig. 1B). In contrast, Hif1α levels increased about 4-fold only in SH group rats (Fig. 1C). HIF1α protein is known to be transiently upregulated in hypoxia, whereas HIF1α mRNA is frequently downregulated 13–15. The reduction of HIF1α mRNA expression in hypoxia is an important component of the cellular adaptation to hypoxia. Therefore, SH group rats exhibiting high levels of Hif1α mRNA were presumed to be in the termination of hypertrophy stage and to have lost protection against hypoxia. In contrast, MH group rats exhibiting low levels of Hif1α mRNA were thought to be in the initiation of hypertrophy stage.
Next, we compared the expression patterns of some genes involved in bladder functions from RNA sequencing results between the three groups. Aquapolin 3 (Aqp3) and matrix metallopeptidase 8 (Mmp8) were markedly upregulated in the SH group compared with those in the control group, but only slightly increased in the MH group (Fig. 1D). In contrast, cholinergic receptor muscarinic 3 (Chrm3) and Chrm2 were downregulated in the SH group compared with those in the control group and slightly decreased in the MH group (Fig. 1D). Moreover, transient receptor potential cation channel subfamily v member 4 (Trpv4) and nitric oxide synthase 3 (Nos3) were increased only in the MH group (Fig. 1D). These results suggested that the expression levels of some genes related to bladder function were altered during bladder remodeling following BOO. Furthermore, the expression patterns were different for every gene.
Total gene expression profiles in the MH and SH groups
To compare total gene expression patterns between the MH and SH groups, we further analyzed the results of RNA sequencing. In total, 4,213 genes exhibited a more than 2-fold increase in expression in the SH group compared with those in the control group, whereas 2,803 genes were increased in the MH group (Fig. 2A). Among these upregulated genes, 1,813 genes were upregulated in both the SH and MH groups (Fig. 2A). In contrast, 507 genes exhibited less than half the expression in both the SH and MH groups compared with the control group (Fig. 2A). The 1,813 upregulated genes in both the MH and SH groups were categorized using GO analysis (Fig. 2B). The results showed that many of the genes were categorized into the molecular functions category, including cytokines and cytokine receptors (Fig. 2B), consistent with the results of previous studies using BOO model rats 4,16.
Next, we focused on genes showing alterations in expression levels only in the MH group. We showed that 471 genes exhibited a more than 2-fold increase in expression in the MH group compared with those in the control and SH groups, whereas 214 genes in the MH group were expressed at levels less than half those in the other two groups (Fig. 2C). Some genes, including Dbp, stabilin 1 (Stab1), ATP binding cassette subfamily C member 5 (Abcc5), and neuronatin (Nnat) were markedly increased in the MH group compared with those in the control and SH groups (Fig. 2D). Some other genes, including apoptosis inducing factor mitochondria associated 1 (Aifm1) and vascular cell adhesion molecule 1 (Vcam1) were significantly downregulated in the MH group (Fig. 2D). Genes showing altered mRNA expression levels may play roles in bladder remodeling at the initiation of the hypertrophy stage.
Dbp was highly expressed in the MH group
The above results suggested that DBP was upregulated during the early phase of hypertrophy. DBP is a transcriptional factor belonging to the circadian clock family and plays roles in the control of circadian rhythm via regulation of target gene expression 17. Thus, we hypothesized that DBP may regulate the expression of genes involved in bladder functions at the initiation of bladder remodeling due to BOO. First, to confirm the expression levels of Dbp in rat BOO models, we quantified Dbp mRNA levels by RT-qPCR. Dbp mRNA levels were higher in the MH group than in the control and SH groups (Fig. 3A). We then assessed DBP protein expression by immunohistochemistry using anti-DBP antibodies. DBP protein was detected in the nuclei of bladder smooth muscle cells in the MH group, but not in the control and SH groups (Fig. 3B).
DBP regulated AQP3 and TRPV4 expression under hypoxic conditions in human urinary bladder cells
Next, we assessed whether DBP was upregulated under hypoxic conditions in human bladder cells. Hypoxia was induced in J82 urinary bladder cancer cells by treatment with cobalt chloride (COCl2) solution. Protein levels of the hypoxia marker HIF1α and mRNA levels of VEGF were increased by COCl2 in a concentration-dependent manner (Fig. 4A and 4B). Under hypoxic conditions, protein and mRNA levels of DBP were higher than those under normal conditions (Fig. 4A and 4B). mRNA levels of the period circadian regulator 1 (Per1) gene, which is known to be transcriptionally activated by DBP, were increased under hypoxic conditions and decreased by knockdown of DBP (Fig. 4B). These results suggested that DBP was upregulated in response to hypoxia and could control gene expression via transcriptional regulation in human bladder cells. To investigate whether DBP contributed to bladder functions under hypoxic conditions, we quantified mRNA levels of genes related to bladder function using DBP-knockdown cells (Fig. 4B). AQP3 is an intrinsic membrane protein belonging to the aquaporin family and functions as a water channel in many cell types involved in fluid transport 18. TRPV4 is a cation channel of the transient receptor potential superfamily and is implicated in sensing the filling state of the bladder 19. NOS3 is an endothelial nitric oxide synthase that is involved in the induction of apoptosis in the bladder mucosa 20. Rat homologs of these genes were markedly upregulated in the MH group (Fig. 1D). In human bladder cells, mRNA levels of Aqp3, Trpv4, and Nos3 were increased by treatment with 500 µΜ COCl2 (Fig. 4B). The increases in Aqp3 and Trpv4 under hypoxic conditions were blocked by Dbp knockdown; however, the expression levels of Nos3 were not affected (Fig. 4B). These results suggested that DBP upregulated Aqp3 and Trpv4 genes under hypoxic conditions in the human urinary bladder.