Involvement of JAK-STAT signaling/function after cyclophosphamide-induced bladder inflammation in female rats

Abstract

Cytokines are upregulated in a variety of inflammatory conditions and cytokine/receptor interactions can activate JAK-STAT signaling. Previous studies demonstrated upregulation of numerous cytokines in the urinary bladder following cyclophosphamide (CYP)-induced cystitis. The role of JAK-STAT signaling in urinary bladder inflammation and referred somatic sensitivity has not been addressed. The contribution of JAK-STAT signaling pathways in CYP-induced bladder hyperreflexia and referred somatic hypersensitivity was determined in CYP-treated rats using a JAK2 inhibitor, AG490. Acute (4 h; 150 mg/kg ip), intermediate (48 h; 150 mg/kg ip), or chronic (75 mg/kg ip, once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Phosphorylation status of STAT-3 was increased in urinary bladder after CYP-induced cystitis (4 h, 48 h, chronic). Blockade of JAK2 with AG490 (5-15 mg/kg ip or intravesical) significantly (P < or = 0.05) reduced bladder hyperreflexia and hind paw sensitivity in CYP-treated rats. These studies demonstrate a potential role for JAK-STAT signaling pathways in bladder hyperreflexia and referred pain induced by CYP-induced bladder inflammation.

Fig. 1.

Upregulation of phosphorylated (p) STAT3 expression in whole urinary bladder with cyclophosphamide (CYP)-induced cystitis (4 h, 48 h, and chronic) using Western blotting techniques. A: representative example of a Western blot of whole urinary bladder (20 μg) for pSTAT3 expression in control rats and those treated with CYP for varying duration. Total STAT3 staining was also determined. B: histogram of relative pSTAT3 band density in all groups examined normalized to total STAT in the same samples presented as a percentage of control STAT3 activation (p-STAT3 expression). pSTAT3 expression in urinary bladder is significantly increased at all time points with CYP treatment. pSTAT3 expression is significantly greater at 48-h CYP treatment compared with 4-h or chronic CYP treatment. *P ≤ 0.01. Data are a summary of n = 5 for each group.

Fig. 2.

Bladder function recordings in CYP-treated [4 h (A) and 48 h (B)] rats treated with vehicle or the JAK2 inhibitor, AG490 (5 mg/kg; intravesical instillation). Intravesical administration of AG490 reduced voiding frequency after CYP-induced cystitis. A: continuous cystometrogram recordings in a 4-h CYP-treated + vehicle (top trace) and same rat treated with AG490 (bottom trace). Arrows point to some nonvoiding bladder contractions (NVCs). In CYP-treated rats further treated with intravesical instillation of AG490, voiding frequency was reduced. B: continuous cystometrogram recordings in a 48-h CYP-treated + vehicle (top trace) and same rat treated with AG490 (bottom trace). Arrows point to some NVCs induced by CYP treatment (see Table 1). NVCs were not significantly affected by AG490 treatment (see Table 1). With intravesical instillation of AG490 in CYP-treated rats, voiding frequency was reduced. The x-axis represents time (s) and the y-axis represents intravesical pressure (cmH₂O).

Fig. 3.

Intraperitoneal (ip) treatment with AG490, a JAK2 inhibitor, reduced referred somatic sensitivity after CYP treatment (4 h). As expected, CYP treatment + vehicle reduced paw withdraw threshold (allodynia). Pretreatment with AG490 (5 or 15 mg/kg ip) significantly reduced paw withdrawal responses induced by CYP treatment (4 h). Both concentrations of AG490 produced similar changes in paw withdraw threshold. *P ≤ 0.05 compared with control. **P ≤ 0.01 compared with CYP + vehicle. Data are a summary of n = 6 for each group.