Bladder wall fibrosis is associated with a variety of lower urinary tract disorders including various forms of cystitis, outlet obstruction and spinal cord injury (SCI). Fibrosis can decrease bladder compliance and capacity adversely impacting voiding and storage functions. However, many studies report contrary findings where rodents with SCI exhibit increased bladder compliance and capacity [1]. We propose that this discrepancy is due to the use of females in rodent based SCI studies and their reduced tendency to develop fibrosis. It has been demonstrated that males are predisposed to developing organ fibrosis, which appears to involve sex differences in the regulation of angiotensin II type 1 versus angiotensin II type 2 receptors (AT1Rs, AT2Rs) [2]. To our knowledge, differences in SCI-induced fibrosis have not been evaluated between male and female mice. Accordingly, our aim was to measure differences in bladder wall fibrosis in male and female mice with SCI and characterize the involvement of angiotensin receptor signalling.

Male/female 8 to 12 weeks old C57Bl/6 mice were anesthetized using 2% isoflurane and their spinal cords transected between the T8-T9 segments, while sham control animals underwent a laminectomy without transection. At this time, mice were also implanted subcutaneously with micro-osmotic pumps (Alzet, model 1004) that released 1 mg/kg/day PD123319 (AT2R antagonist), 10 mg/kg/day losartan (AT1R antagonist) or vehicle (saline) for 28 days at 0.11 µl/hr. Following surgeries, animals were treated with analgesics and prophylactic antibiotics and their bladders manually expressed twice a day for up to seven days. Four to six weeks following injury, animals were sacrificed and their bladders isolated and divided into two strips that were used for length-tension recordings or saved in 10% buffered formaldehyde for histology (modified Verhoeff Van Gieson). Slides were imaged using brightfield montage microscopy (Olympus Fluoview microscope) and collagen content quantified in at least 6 comparable sections from each bladder strip using NDP and HCImage software (Hamamatsu Photonics). Experiments were carried out on n ≥ 4 mice and the unpaired Student’s t-test used to determine differences between transected versus sham controls and mice with and without drug treatment.

Histological evaluation of bladder sections from SCI mice showed significantly increased total collagen content in males versus females and sham controls (Fig. 1A). Male SCI mice treated with losartan showed diminished bladder collagen content compared to vehicle controls. Conversely, female SCI mice treated with PD123319 showed increased collaged content compared to vehicle treated mice (Fig 1B). The hypothetical mechanism of sex dependent differences in AT1/2R mediated bladder fibrosis and graphical representation of bladder collagen content in the test groups are summarized in Fig 1C and 1D, respectively. Functional assessments using isolated bladder strips showed that length-tension profiles were left-shifted in males (Fig. 2A) following SCI exhibiting greater passive tension indicative of increased stiffness (Fig. 2B). AT2R inhibitor treatment of females resulted in decreased compliance compared to untreated females. Alternatively, AT1R inhibitor treatment of males with SCI resulted in markedly increased compliance.

These data suggest that female mice may be protected against the development of bladder fibrosis due to altered expression and/or estrogen inhibition of AT1Rs, while males are prone to bladder fibrosis due to altered expression and/or testosterone inhibition of AT2Rs [3].

While renin-angiotensin system has well-known effects regulating systemic blood pressure, it also has a significant role in modulating tissue inflammation and fibrosis. It also is thought to have marked sex-linked differences; estrogen inhibits AT1Rs which have profibrotic actions, while testosterone inhibits AT2Rs which promote antifibrotic activity. Our data support this theory and suggests that AT1R blocker, losartan, may have therapeutic relevance in decreasing fibrosis in adult male or post-menopausal female patients with SCI.

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