Cyclophosphamide (CYP), a non-specific broad-spectrum anticancer drug, causes serious and uncomfortable side effects in patients including hemorrhagic cystitis (HC).  The cystitis is characterized by bladder inflammation and haemorrhage, pelvic pain, and lower urinary tract symptoms (LUTS) such as increased urinary frequency and urgency.  These symptoms seriously affect the treatment tolerance and prognosis of patients undergoing chemotherapy with CYP [1].  However, before and during the chemotherapy, there are only symptomatic treatments for HC.  Therefore, critical approaches for preventing CYP-induced cystitis are urgently needed.
Hydrogen sulfide (H2S) has recognized as a gasotransmitter and plays key roles in several physiological functions including vasodilation, anti-inflammation, antioxidation, cytoprotection [2].  Therefore, H2S has a possibility to be a promising therapeutic target for CYP-induced cystitis to improve tolerability of chemotherapy with CYP. However, effects of H2S on CYP-induced cystitis have not been investigated yet.
Therefore, in this study, we aimed to evaluate the effects of an H2S donor (NaHS) pretreatment on bladder dysfunction in rats with CYP-induced cystitis and elucidate mechanisms underlying the NaHS-induced effects.

Male Wistar rats (290-320 g) were used.
(1) Study 1: Rats were divided into four groups: Vehicle + Saline (n = 8), Vehicle + CYP (n = 9), NaHS 3 + CYP (n = 10), and NaHS 10 + CYP (n = 8).  The rats were treated with daily intraperitoneal (ip) injections of vehicle (saline, 1 ml/kg) or NaHS (3 or 10 μmol/kg) in saline solution for 7 days (day 1-7).  CYP (150 mg/kg, ip) or saline (5 ml/kg, ip) had been injected two days before the cystometry (day 6).
(2) Study 2: Rats were divided into four groups: CAP(-) + Vehicle + CYP (n = 10), CAP (-) + NaHS 10 + CYP (n = 10), CAP(+) + Vehicle + CYP (n = 16), and CAP(+) + NaHS 10 + CYP (n = 15).  Daily administration of vehicle (saline, 1 ml/kg) or NaHS (10 μmol/kg) and single administration of CYP (150 mg/kg, ip) were performed as the same in Study 1.  To desensitize capsaicin (CAP)-sensitive afferent nerves, CAP solution containing 25 mg/ml CAP in 10% ethanol, 10% Tween-80 and 80% saline was subcutaneously (sc) injected at 125 mg/kg in divided doses on two continuous days (day 3 and 4): 25 and 50 mg/kg at a 12-h interval on the first day (day 3) and 50 mg/kg on the second day (day 4).  Control rats received a corresponding volume of the solvent (10% ethanol, 10% Tween-80 and 80% saline).  On day 8, before anesthesia and cystometry, eye wipe test by an eye drop of CAP solution (10 μl of 100 μg/ml) was performed to confirm CAP-induced desensitization of afferent pathways.
Cystometry was performed one day after the final administration of vehicle or NaHS (day 8).  Under urethane anesthesia (0.8 g/kg, ip), a catheter was inserted into the rat bladder via the bladder dome.  One hour after stabilization, single and continuous cystometry were performed by intavesical instillation of saline at a constant flow rate (4.0 ml/h) via the bladder catheter to measure intravesical pressure.  After cystometry, bladder tissues were collected to perform HE staining.

(1) In continuous cystometry, compared to the control group (Vehicle + Saline), in the Vehicle + CYP group, the intercontraction intervals (ICI) and bladder compliance (Comp) were significantly decreased, and the number of non-voiding contractions (NVCs) was significantly increased (Table 1).  In the NaHS 3 + CYP group, the number of NVCs was significantly decreased compared to the Vehicle + CYP group (Table 1).  In the NaHS 10 + CYP group, ICI and Comp were significantly increased and the number of NVCs was significantly decreased compared to the Vehicle + CYP group (Table 1).  In single cystometry, there was no significant difference in post-voiding residual volume (Rv) among four groups (data not shown).  Based on these data, we performed histological analysis focusing on the following three groups: Vehicle + Saline, Vehicle + CYP, and NaHS 10 + CYP.  In bladder tissues, CYP increased scores of neutrophile infiltration, haemorrhage, and oedema, but these CYP-induced changes were not significantly improved by NaHS pretreatment (Fig. 1).
(2) The number of eye wipes in response to an eye drop of CAP solution was significantly decreased by CAP pretreatment (Fig. 2).  In continuous cystometry, CAP showed a tendency to increase ICI in vehicle- and CYP-treated rats (Table 2).  NaHS-induced improvement of CYP-induced decreases in ICI and Comp and an increase in the number of NVC was not detected in CAP-treated rats (Table 2).  In single cystometry, there was no significant difference in Rv among four groups (data not shown).

(1) CYP injection significantly decreased ICI and Comp and increased the number of NVCs without increasing Rv, suggesting that CYP successfully induced urinary frequency with low bladder distensibility and detrusor overactivity (DO) in rats.  Both doses of NaHS significantly attenuated the CYP-induced increase in NVCs, suggesting that NaHS could suppress the CYP-induced DO.  Moreover, NaHS at a dose of 10 μmol/kg also attenuated the CYP-induced decreases in ICI and Comp, indicating that NaHS partially improved the urinary frequency with low bladder distensibility in CYP-treated rats.  However, in histological analysis, inflammatory scores increased by CYP were not influenced by NaHS in the bladder, indicating that another mechanism rather than anti-inflammation may be involved in NaHS-induced improvement of bladder dysfunction in CYP-treated rats.
(2)  CAP-induced desensitization of bladder afferent pathways is reported to improve DO and hyperalgesia in interstitial cystitis [3].  H2S has a role as a neuromodulator [2], so we investigated mechanisms for NaHS-induced improvement of bladder dysfunction in CYP-treated rats focusing on CAP-sensitive bladder afferent pathways.  In this study, CAP pretreatment successfully desensitized afferent pathways evidenced by CAP-induced decreases in the number of eye wipes in response to an eye drop of CAP solution.  CAP pretreatment by itself showed a tendency to increase ICI without increasing Rv in vehicle- and CYP-treated rats, indicating that hyperexcitability of CAP-sensitive afferent pathways may be involved in the CYP-induced urinary frequency.  In the CAP- and CYP-treated rats, NaHS-induced improvement of decreased ICI and Comp and increased the number of NVCs was not detected.  Therefore, NaHS pretreatment may prevent bladder dysfunction (urinary frequency, low bladder distensibility and DO) in CYP-treated rats by suppressing CAP-sensitive bladder afferent pathways.

Our present data suggest that pretreatment with NaHS partially improved CYP-induced bladder dysfunction at least through suppression of CAP-sensitive bladder afferent pathways.  Therefore, H2S donor pretreatment may be useful for prevention for bladder dysfunction induced by HC, a side effect of CYP chemotherapy.

Monach PA, Arnold LM, Merkel PA. Incidence and prevention of bladder toxicity from cyclophosphamide in the treatment of rheumatic diseases: a data-driven review. Arthritis Rheum. 2010;62:9-21.
Gemici B, Elsheikh W, Feitosa KB, et al. H2S-releasing drugs: anti-inflammatory, cytoprotective and chemopreventative potential. Nitric Oxide. 2015;46:25-31.
Yoshimura N, Oguchi T, Yokoyama H, et al. Bladder afferent hyperexcitability in bladder pain syndrome/interstitial cystitis. Int J Urol 2014;21:18-25.

Continence 2S2 (2022) 100210
DOI: 10.1016/j.cont.2022.100210