Expression and function of CXCL12/CXCR4 in rat urinary bladder with cyclophosphamide-induced cystitis

Abstract

Chemokines, otherwise known as chemotactic cytokines, are proinflammatory mediators of the immune response and have been implicated in altered sensory processing, hyperalgesia, and central sensitization following tissue injury or inflammation. To address the role of CXCL12/CXCR4 signaling in normal micturition and inflammation-induced bladder hyperreflexia, bladder inflammation in adult female Wistar rats (175-250 g) was induced by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every 3rd day for 10 days) time points. CXCL12, and its receptor, CXCR4, were examined in the whole urinary bladder of control and CYP-treated rats using enzyme-linked immunosorbent assays (ELISAs), quantitative PCR (qRT-PCR), and immunostaining techniques. ELISAs, qRT-PCR, and immunostaining experiments revealed a significant (P < or = 0.01) increase in CXCL12 and CXCR4 expression in the whole urinary bladder, and particularly in the urothelium, with CYP treatment. The functional role of CXCL12/CXCR4 signaling in micturition was evaluated using conscious cystometry with continuous instillation of saline and CXCR4 receptor antagonist (AMD-3100; 5 microM) administration in control and CYP (48 h)-treated rats. Receptor blockade of CXCR4 using AMD-3100 increased bladder capacity in control (no CYP) rats and reduced CYP-induced bladder hyperexcitability as demonstrated by significant (P < or = 0.01) increases in intercontraction interval, bladder capacity, and void volume. These results suggest a role for CXCL12/CXCR4 signaling in both normal micturition and with bladder hyperreflexia following bladder inflammation.

Fig. 1.

Time-dependent changes in CXCL12 protein expression in whole urinary bladder following cyclophosphamide (CYP) treatment as determined with enzyme-linked immunosorbent assays (ELISAs). CXCL12 expression increased significantly (*P ≤ 0.01) at 48 h and chronic time points compared with control urinary bladders; n = 4 for control and each experimental condition.

Fig. 2.

Time-dependent and regional changes in CXCL12 and CXCR4 mRNA expression in urinary bladder as detected by quantitative PCR (qRT-PCR). Following 48 h and chronic CYP treatment, CXCL12 mRNA expression was significantly increased in both the urothelium (A; **P ≤ 0.01) and the detrusor (B; **P ≤ 0.01). CXCR4 mRNA expression was increased significantly after 48 h CYP treatment in both the urothelium (C; *, P ≤ 0.05) and the detrusor (D; *P ≤ 0.05); n = 5–7 for each group.

Fig. 3.

Time-dependent and regional changes in CXCR7 mRNA expression in urinary bladder as detected by qRT-PCR. CXCR7 mRNA expression increased significantly in both the urothelium (A; *P ≤ 0.05) and the detrusor (B; *P ≤ 0.05) following chronic CYP treatment but not 4 or 48 h CYP treatment (n = 5–7 for each group).

Fig. 4.

Regulation of CXCL12 (A–D) and CXCR4 (E–H) expression in cryostat sections of urinary bladder after CYP treatment. The urothelium was outlined in red, and images were thresholded (A, B, E, and F). All pixels above threshold are depicted in yellow. Corresponding fluorescence images of CXCL12 expression in control or after 48 h CYP treatment are shown in C and D, respectively. Fluorescence images of CXCR4 expression in urinary bladder of control or after 48 h CYP treatment are shown in G and H, respectively. L, lumen; U, urothelium; Sub U, suburothelium. Calibration bar represents 50 μm.

Fig. 5.

Summary histogram of CXCL12 (A)- and CXCR4-immunoreactivity (IR) (B) expression in the urothelium of the urinary bladder of control rats and those treated with CYP (48 h). Values are means ± SE (n = 3–8). **P ≤ 0.01.

Fig. 6.

Representative cystometrogram recordings of effects of CXCR4 receptor blockade in control (no inflammation) rats using continuous intravesical infusion of saline. Pre-AMD-3100 (A) and post-AMD-3100 (B) drug treatments in control rats (no CYP treatment) with continuous intravesical instillation of saline. CXCR4 receptor blockade with intravesical infusion of AMD-3100 (5 μM) increased both bladder capacity (measured as the amount of saline infused in the bladder at the time when micturition commenced) and void volume compared with pretreatment conditions (A). Bladder function recordings in A and B are recorded from the same rat.

Fig. 7.

Summary histograms of the effects of CXCR4 receptor blockade with AMD-3100 intravesical infusion in control rats (no CYP treatment). A: infusion of AMD-3100 (5 μM) significantly (**P ≤ 0.01) increased intercontraction interval. B: bladder capacity was also significantly (**P ≤ 0.01) increased along with a significantly (*P ≤ 0.05) increased void volume (C). Values are means ± SE (n = 4–6).

Fig. 8.

Representative cystometrogram recordings of effects of CXCR4 receptor blockade in CYP-treated (48 h) rats using continuous intravesical infusion of saline. Pre-AMD-3100 (A) and post-AMD-3100 (B) drug treatments in 48 h CYP-treated rats with continuous intravesical instillation of saline. CXCR4 receptor blockade with intravesical infusion of AMD-3100 (5 μM) increased both bladder capacity (measured as the amount of saline infused in the bladder at the time when micturition commenced) and void volume compared with pretreatment conditions (A). Bladder function recordings in A and B are recorded from the same rat.

Fig. 9.

Summary histograms of effects of CXCR4 receptor blockade with AMD-3100 intravesical infusion in 48 h CYP-treated rats. A: infusion of AMD-3100 (5 μM) significantly (**P ≤ 0.01) increased intercontraction interval. B: bladder capacity was also significantly (**P ≤ 0.01) increased along with a significantly (**P ≤ 0.01) increased void volume (C). Values are means ± SE (n = 6).