Purine nucleoside phosphorylase inhibition ameliorates age-associated lower urinary tract dysfunctions

Abstract

In the aging population, lower urinary tract (LUT) dysfunction is common and often leads to storage and voiding difficulties classified into overlapping symptom syndromes. Despite prevalence and consequences of these syndromes, LUT disorders continue to be undertreated simply because there are few therapeutic options. LUT function and structure were assessed in aged (>25 months) male and female Fischer 344 rats randomized to oral treatment with a purine nucleoside phosphorylase (PNPase inhibitor) 8-aminoguanine (8-AG) or vehicle for 6 weeks. The bladders of aged rats exhibited multiple abnormalities: tactile insensitivity, vascular remodeling, reduced collagen-fiber tortuosity, increased bladder stiffness, abnormal smooth muscle morphology, swelling of mitochondria, and increases in urodamaging purine metabolites. Treatment of aged rats with 8-AG restored all evaluated histological, ultrastructural, and physiological abnormalities toward that of a younger state. 8-AG is an effective treatment that ameliorates key age-related structural and physiologic bladder abnormalities. Because PNPase inhibition blocks metabolism of inosine to hypoxanthine and guanosine to guanine, likely uroprotective effects of 8-AG are mediated by increased bladder levels of uroprotective inosine and guanosine and reductions in urodamaging hypoxanthine and xanthine. These findings demonstrate that 8-AG has translational potential for treating age-associated LUT dysfunctions and resultant syndromes in humans.

Keywords: Aging; Cellular senescence.

Figure 1. 8-Aminoguanine (8-AG) attenuates age-related differences in bladder function.

(A–C) 8-AG decreases voiding frequency (A) (n = young, 13; aged, 21; aged + 8-AG, 30) and increases both the inter-void interval (B) (n = young, 13; aged, 22; aged + 8-AG, 30) and voided volume (C) (n = young, 13; aged, 21; aged + 8-AG, 22). (D and E) In addition, aging decreases abdominal (D) (n = young, 18; aged, 58; aged + 8-AG, 35) and cutaneous (E) (n = young, 13; aged, 21; aged + 8 AG, 23) responses to tactile mechanical stimuli, which is restored to a younger state by 8-AG treatment. Data are presented as mean ± SEM. Ordinary 1-way ANOVA was used to evaluate significance, which was considered at P < 0.05.

Figure 2. 8-Aminoguanine (8-AG) attenuates age-related differences in EBCI.

(A–C) Representative examples of volume-time relationship plots for young (A), aged (B), and aged rats treated with 8-AG (C). 8-AG also attenuates age-related decreases in EBCI (D) (n = young, 13; aged, 21; aged + 8-AG, 23).Data are presented as mean ± SEM. Ordinary 1-way ANOVA was used to evaluate significance, which was considered at P < 0.05.

Figure 3. Ribbon-scanning confocal microscopy coupled with perfusion of the bladder with fluorescent beads shows increased vascular tortuosity with appearance of reduced perfusion in aged bladders.

(A and B) Representative image (from n = young 3; aged 3 and aged + 8-AG 3 rats) (B) showing age-associated differences compared with young bladders (A), which demonstrates mostly straight vessels throughout the tissue. (C) The aged bladder treated with 8-aminoguanine (8-AG) reduces vessel tortuosity, and the tissue no longer appears ischemic, resembling young tissue. n = 9. Panel insets display the reconstruction of the complete bladder with voxels of 0.4 × 0.4 × 10 mm. Yellow boxes display the location of the high-magnification images. Prior to imaging, bladders were cleared using the CUBIC method, and images were acquired by ribbon-scanning confocal microscope. Scale bars: 200 μm (n = young 4; aged 6; aged + 8-AG 4). (D) Doppler flowmeter measurements revealing a significant decrease in bladder blood flow in aged compared with young rats; blood flow defects in the old bladder are reversed to a younger state with 8-AG treatment. Ordinary 1-way ANOVA was used to evaluate significance which was considered at *P < 0.05.

Figure 4. Western immunoblotting revealed significant aging-associated alterations in proteins linked to mitochondrial dynamics and quality control within the bladder mucosa.

(A–D) Mitofusin 2 (MFN2; n = young 14; aged 16; aged + 8-AG 16) (A), a protein involved in mitochondrial fusion; Dynamin-|related protein (B) (DRP-1; n = young 4; aged 5; aged + 8-AG 4), which is involved in mitochondrial fission; Parkin (C) (n = young 4; aged 5; aged + 8-AG 4), which plays a role in mitophagy; and cleaved caspase-3 (D) (n = young 8; aged 9; aged + 8-AG 8), which is activated upon initiation of apoptosis. In all cases, treatment with 8-aminoguanine (8-AG) restored changes similar to a younger state. Representative immunoblotting is inset within each graph. Cleaved caspase-3 (top of inset) is normalized to total caspase-3 (bottom of inset). All samples were run on the same blot, but representative samples were not contiguous. Data are presented as mean ± SEM. Ordinary 1-way ANOVA was used to evaluate significance. *P < 0.05; **P < 0.01.

Figure 5. Concurrent imaging and mechanical testing of bladder collagen fibers in the detrusor layer of young versus aged bladder.

(A) The collagen fibers in young bladder are highly tortuous (occurred in 3 of 3 rats) enabling large stretch with very little change in load (stress). (D) This soft phase of the stress-stretch curve is followed by a transition to a stiff phase at higher stretch (asterisk indicates onset of stiff phase or critical stretch). (B) There is a substantial decrease in tortuosity in the detrusor layer of aged bladders (occurred in 3 of 3 rats) compared with young bladders, leading to a premature recruitment of collagen fibers. (E) The early recruitment leads to shortening of the soft phase and early shift to the stiff phase in aged versus young bladders (best fit graph with individual data points plotted; n = 3 each for young, aged, aged + 8-AG). (C–F) This trend was reversed after 8-aminoguanine (8-AG) treatment, resulting in partial recovery in tortuosity (C) (occurred in 3 of 3 rats), rightward shift of stress-stretch curve toward the young bladder curve (E), and recovery in critical stretch for onset of stiff phase (F). Data are presented as mean ± SEM. Scale bars: 100 mm. Ordinary 1-way ANOVA was used to evaluate significance. *P < 0.05.

Figure 6. Concurrent multiphoton imaging and biomechanical testing of bladder collagen fibers in lamina propria of young versus aged rat bladder.

(A) The collagen fibers in the lamina propria of young bladder are recruited (straightened) under physiological loading levels (occurred in 3 of 3 rats). (B) In contrast, the aged bladder did not expand sufficiently to recruit collagen fibers, even at physiological loads (occurred in 3 of 3 rats). (C) Collagen fibers in the bladders of aged animals treated with 8-AG were able to be recruited (straightened) at physiological loading levels (occurred in 3 of 3 rats). Aged bladders also exhibit increased wall thickness as compared with younger rat bladders. (D) Bladder wall thickness in old rats was restored toward a younger state with 8-AG treatment (n = young, 4; aged, 4; aged + 8-AG, 4). Data are presented as mean ± SEM. Scale bars: 100 mm. Ordinary 1-way ANOVA was used to evaluate significance. *P < 0.05; **P < 0.01.

Figure 7. Representative transmission electron microscopy images of bladder smooth muscle in young rats, aged rats, and aged rats (n = 3 each) treated with 8-aminoguanine (8-AG).

(A and B) These images reveal abnormal detrusor smooth muscle (SM) morphology in aged rats. Panel B includes separation and degeneration of cells as compared with young rats (A) (3 of 3 rats). However, the abnormal morphology in aged rats is restored to a younger state by 8-AG treatment (C; 3 of 3 rats). (D–F) Higher-magnification transmission electron microscopy images revealing substantial swelling and disruption of smooth muscle mitochondria (asterisk denote mitochondria) in aged bladders (E; 3 of 3 rats) compared with young bladders (D; 3 of 3 rats); these anomalies were restored to a younger state by 8-AG treatment (F; 3 of 3 rats). Scale bars: 1 µm (magnification, 15,000×) (A–C) and 600 nm (magnification, 30,000×) (D–F).

Figure 8. Aged bladder detrusor exhibited alterations in various biomarkers.

(A–C) Senescent biomarker p16 (A) (n = young, 8; aged, 8; aged + 8-AG, 8), catalase activity (B) (n = young, 11; aged, 10; aged + 8-AG, 8), and cleaved caspase-3 (C) (cleavage product shown in lower panel [17-19kDa], compared with uncleaved caspase-3 in upper panel; 35kDa; n = young, 8; aged, 9; aged + 8-AG, 7). (D) Aged bladder detrusor smooth muscle exhibit alterations in α-smooth muscle actin (n = young, 5; aged, 5; aged + 8-AG, 4). (E) Significant differences were deterred in PARP activation, as indicated by cleavage product at 89 kDa (lower band, yellow asterisks), compared with uncleaved PARP (upper band, 116 kDa) (n = young, 8; aged, 9; aged + 8-AG, 7). 8-AG treated in aged rats restored these biomarkers to those of a younger state. Representative immunoblotting is inset within each graph. All samples were run on the same blot, but representative samples were not contiguous. Data are presented as mean ± SEM. Ordinary 1-way ANOVA was used to evaluate significance. *P < 0.05.

Figure 9. Purine metabolome measurements in young, aged, and aged rats treated with 8-aminoguanine.

(A) In aged rats, endogenous urinary 8-aminoguanine (8-AG) is below assay detection limits (n = young, 3; aged, 4; aged + 8-AG, 3). (B) However, aged rats have higher urinary hypoxanthine levels (n = young, 4; aged, 4; aged + 8-AG, 6); both of these abnormalities are restored to younger levels with 8-AG treatment. (C) In addition, guanosine levels (n = young, 8; aged, 8; aged + 8-AG, 10) are altered with age and recovered with 8-AG treatment. Ordinary 1-way ANOVA was used to evaluate significance. *P < 0.05.