Constitutive expression of inducible nitric oxide synthase in healthy rat urothelium?

BACKGROUND: Contrasting findings have been reported regarding a possible constitutive expression of inducible nitric oxide synthase (iNOS) in a normal mammalian bladder. The current study was designed to further investigate such putative iNOS expression. MATERIALS AND METHODS: The experiments were conducted with paraffin-embedded archival material from the urinary bladder of 6 normal, male Sprague-Dawley rats. In addition, two normal female mice (C57BL/6) were sacrificed and the urinary bladders were harvested. The occurrence of iNOS mRNA was examined by the RNAScope in situ hybridization method. Protein expression of iNOS and 3-nitrotyrosine (the latter used as an indicator of oxidative stress) was investigated by immunohistochemistry. RESULTS: No expression of iNOS mRNA was observed in the bladder tissue. iNOS protein and 3-nitrotyrosine were strongly expressed in the urothelium. iNOS was also expressed perinuclearly in the detrusor. CONCLUSIONS: Although the RNAScope methodology could not demonstrate mRNA for iNOS in the normal urinary bladder, the results by immunohistochemistry strongly suggest the occurrence of iNOS in particular, in the urothelium. Positive reactivity for 3-nitrotyrosine may indicate ongoing oxidative stress of the urothelium. The finding of perinuclear iNOS immunoreactivity could suggest an intracrine signaling function by iNOS to the nucleus.

The extracellular matrix proteoglycan versican is strongly expressed in the urothelium of healthy rats.

Objective: We have previously demonstrated protein expression of the extracellular matrix degrading protein ADAMTS5 in the nuclei of urothelial cells in healthy rats. The proteoglycan versican constitutes one of the main substrates for this protease. In this follow up study we investigated a potential co-localization of versican and ADAMTS5 in the urinary bladder wall.Material and Methods: The study was conducted with archive material (paraffin embedded bladder tissue from our previous study, i.e., 8 male Sprague-Dawley rats). Protein expression of versican was investigated by immunohistochemistry. Furthermore, the occurrence of versican mRNA was examined by in-situ hybridization.Results: Positive immunoreactivity for versican was evident in the urothelium but also, weakly, in the detrusor. This expression was localized only in the cytoplasm, leaving the nuclei devoid of reactivity. Interestingly, versican mRNA was only sparsely observed in the urothelial cells.Conclusions: We found by immunohistochemistry that the substrate for ADAMTS5, versican, was localized in the cytosol of urothelial cells. This demonstrates a difference regarding the expression of ADAMTS5, which was emphasized in the nuclei. This could imply an additional, non-enzymatic, function of ADAMTS5 in the urothelium.

The extracellular matrix-degrading protein ADAMTS5 is expressed in the nuclei of urothelial cells in healthy rats.

OBJECTIVE: The aim of this study was to investigate whether protein expression of the extracellular matrix-degrading protease ADAMTS5 can be demonstrated in the urinary bladder of healthy rats, and, if so, to determine the localization of this enzyme. MATERIALS AND METHODS: The experiments were conducted with eight inbred male Sprague-Dawley rats. Immunohistochemistry was used to investigate the expression of ADAMTS5 in the urinary bladder. Negative controls were established by either excluding the primary antibody or applying the antibody after it had been preabsorbed with its immunogenic peptide. Confocal microscopy was used to visualize the distribution of ADAMTS5 in the urinary bladder tissue. RESULTS: Immunoreactivity for ADAMTS5 was demonstrated in the urothelium and in the detrusor. This expression was localized not only in the cytoplasm, but also in the nuclei. Confocal microscopy corroborated these findings. CONCLUSION: Expression of ADAMTS5 was demonstrated in the cytoplasm as well as in the nuclei of the urothelium and detrusor cells, suggesting that it may play a role at the transcriptional level.

Bladder pain syndrome/interstitial cystitis ESSIC type 3C: high expression of inducible nitric oxide synthase in inflammatory cells.

OBJECTIVE: Bladder pain syndrome/interstitial cystitis (BPS/IC) includes a heterogeneous collection of underlying pathological conditions. Compared to the classic IC with a Hunner lesion, now denominated ESSIC type 3C, the non-Hunner type of BPS/IC appears different in a number of respects. In a previous study, measuring luminal nitric oxide (NO) in the bladder of patients with BPS/IC, it was reported that all patients with ESSIC type 3C had high levels of NO. The aim of the present study was to investigate the source of inducible nitric oxide synthase (iNOS) and thereby the cellular origin of NO production via iNOS. MATERIAL AND METHODS: Immunohistochemistry, with two different anti-iNOS antibodies, was used to study 10 patients with BPS/IC ESSIC type 3C who expressed high levels of intraluminal NO. These results were compared with four patients with non-Hunner BPS/IC. To substantiate further the involvement of iNOS in this condition, the protein expression of nitrotyrosine, a marker for iNOS activation, was also assessed. RESULTS: On routine histopathology, the tissues of type 3C patients exhibited inflammatory infiltrates of varying intensity. Strong immunoreactivity for both iNOS and nitrotyrosine was noted within the urothelium but also within the inflammatory infiltrates in the lamina propria of these subjects. CONCLUSIONS: The findings of a clearly detectable protein expression of iNOS in both the urothelium and the inflammatory infiltrates in bladder biopsies from patients with BPS/IC ESSIC type 3C suggest that the production of NO, in this entity, may occur in different tissue compartments.