Prevention of Radiation-Induced Bladder Injury: A Murine Study Using Captopril

Abstract

Purpose: Pelvic radiation therapy (RT) can cause debilitating bladder toxicities but few clinical interventions exist to prevent injury or alleviate symptoms. From a large genome-wide association study in patients with prostate cancer it was previously reported that SNPs tagging AGT, part of the renin-angiotensin system (RAS), correlated with patient-reported late hematuria, identifying a potential targetable pathway to prevent RT-induced bladder injury. To investigate this association, we performed a preclinical study to determine whether RAS modulation protected the bladder against RT injury.

Methods and materials: C57BL/6 male mice were treated with an oral angiotensin converting enzyme inhibitor (ACEi: 0.3g/L captopril) 5 days before focal bladder X-irradiation with either single dose (SD) 30 Gy or 3 fractions of 8 Gy (8 Gy × 3 in 5 days). RT was delivered using XStrahl SARRP Muriplan CT-image guidance with parallel-opposed lateral beams. ACEi was maintained for 20 weeks post RT. Bladder toxicity was assessed using assays to identify local injury that included urinalysis, functional micturition, bladder-released exosomes, and histopathology, as well as an assessment of systemic changes in inflammatory-mediated circulating immune cells.

Results: SD and fractionated RT increased urinary frequency and reduced the volume of individual voids at >14 weeks, but not at 4 weeks, compared with nonirradiated animals. Urothelial layer width was positively correlated with mean volume of individual voids (P = .0428) and negatively correlated with number of voids (P = .028), relating urothelial thinning to changes in RT-mediated bladder dysfunction. These chronic RT-induced changes in micturition patterns were prevented by captopril treatment. Focal bladder irradiation significantly increased the mean particle count of urine extracellular vesicles and the monocyte and neutrophil chemokines CCL2 and MIP-2, and the proportions of circulating inflammatory-mediated neutrophils and monocytes, which was also prevented by captopril. Exploratory transcriptomic analysis of bladder tissue implicated inflammatory and erythropoietic pathways.

Conclusions: This study demonstrated that systemic modulation of the RAS protected against and alleviated RT-induced late bladder injury but larger confirmatory studies are needed.

Fig. 1.

Animal placement (supine), treatment planning, and focal bladder irradiation using the Small Animal Radiation Research Platform x-ray unit and treatment planning. (A) Visipaque identifying the bladder (beneath white bracket). (B) Placement of bladder-targeted isocenter (red; IsoC_1) and radiation field; the volume of tissue irradiated is shown by the purple square. (C) X-ray beam set-up (transverse, blue column indicated by bracket, bladder identified by white arrow) and dosing. (D) Dose volume histogram shows 100% of the dose targeted to the bladder. Off-target tissue, kidney, and spine (yellow and green circles, respectively, in panel B) show no dose.

Fig. 2.

Assessment of bladder function. (A) Micturition evaluation: pattern of urination showing individual voids and volume of each void measured over a 2-hour period for a single mouse irradiated with 30 Gy and a nonirradiated mouse. (B) Mean volume of individual voids and (C) number of individual voids were determined from averages of values calculated for individual mice (ie, from a single filter paper) exposed to either 30 Gy focal bladder irradiation or to 0 Gy control, and treated with either captopril (C) or vehicle (V), at 4 weeks (n = 6-8) and 15 weeks (n = 4-5) after irradiation. (D) Mean volume of individual voids (large pools >10,000+ pixels and large voids >900 pixels) and (E) number of small individual voids less than 900 pixels were determined from averages of values calculated for individual mice exposed to 8 Gy × 3 or 0 Gy treated with either captopril (C) or vehicle (V), at 4 weeks (n = 5-9) or 15 weeks (n = 5-10) after irradiation. (F) Specific gravity of urine collected at 10 weeks (for 30 Gy exposure) or 15 weeks (for 8 Gy × 3 exposure) or to 0 Gy.

Fig. 3.

Exosome analysis and histopathology of bladder from mice exposed to either 30 Gy or 8 Gy × 3 targeted to bladder or to 0 Gy control and treated with either captopril (Cap), or vehicle (Veh) at 4 hours to1 week after irradiation. (A) Exosome particle size in urine after 30 Gy ± captopril (means ± SEM). *P < .05, **P < .01 (P values were not corrected for multiple comparisons given that the experiments were preliminary in nature because of small sample sizes). (B) Representative image of Gomori Trichrome stained bladder in an untreated mouse, depicting urothelium (U), lamina propria (LP), and detrusor smooth muscle (M). Images of urothelium were used for analysis of urothelial width. Effects of bladder irradiation and captopril treatment on average thickness of urothelial layer in (C) 0 Gy control mice, (D) bladder irradiated mice at 1 week, and (E) at 20 weeks: n = 3 to 6 mice/treatment group. Urothelial layer thickness correlates with micturition parameters of (F) volume of individual voids and (G) number of voids/filter paper in 30 Gy mice. n = 12 mice for correlation studies.

Fig. 4.

Erythropoietin (EPO) concentration in plasma, collected from mice exposed to either 8 Gy × 3 targeted to bladder or to 0 Gy control, as well as mice exposed to hyperbaric oxygen (HBO) as a positive control for EPO production and treated with either captopril (C), or vehicle (V). Blood was collected at 96 hours after the first fraction of irradiation, or at 96 hours after completion of HBO treatment. n = 3 to 5 mice/treatment group. *P < .05.

Fig. 5.

(A) Proportions of circulating neutrophils and monocytes collected in whole blood from mice exposed to either 30 Gy or 8 Gy × 3, targeted to bladder, or to 0 Gy control and treated with either captopril (C), or vehicle (V), at 6, 10, and 15 weeks following irradiation. (B) CCL-2, MIP-1α, and MIP-2 concentration in plasma analyzed by ELISA at 1week after either SD 30 Gy or 8 Gy × 3. n = 3 to 6 mice/treatment group. *P < .05. Abbreviations: CCL-2 = C-C motif chemokine ligand 2, MIP-1α = macrophage inflammatory protein 1α, MIP-2 = macrophage inflammatory protein 2.