Inhibition of NMDAR reduces bladder hypertrophy and improves bladder function in cyclophosphamide induced cystitis

Abstract

Purpose: We examined the role of NMDAR in the regulation of bladder hypertrophy and function in a rat model of cyclophosphamide induced cystitis.

Materials and methods: Cystitis was induced by intraperitoneal injection of cyclophosphamide (150 mg/kg body weight). NMDAR phosphorylation (activity) and signal transduction pathways were examined by direct measurement and by specific inhibitors in vivo. Bladder hypertrophy was measured by bladder weight/body weight and type I collagen expression. Bladder function was examined by metabolic recording, conscious cystometry and detrusor muscle strip contractility in response to carbachol.

Results: NMDAR activity measured by the phosphorylation level of the NMDAR1 (NR1) subunit was expressed in the spinal cord but not in the bladder at 48 hours of cystitis. NMDAR inhibition with dizocilpine (MK-801) reduced the cystitis induced increment of bladder weight and type I collagen up-regulation in the bladder. NMDAR regulated type I collagen up-regulation was mediated by the PI3K/Akt pathway. NMDAR inhibition also attenuated cystitis induced urinary frequency measured by metabolic cage and cystometry. Cystitis decreased the responsiveness of detrusor muscle strips to carbachol, which was reversed by MK-801 in vivo. Unlike MK-801 the NMDAR antagonist D-AP5, which could not block central NMDAR activity, had no effect on bladder hypertrophy, type I collagen up-regulation or Akt activation caused by cystitis in the bladder.

Conclusions: Findings suggest that NMDAR activity has a role in cystitis induced bladder hypertrophy and overactivity. NMDAR mediated Akt activation may underlie the mechanism of bladder dysfunction.

Keywords: N-methyl-D-aspartate; collagen; cystitis; hypertrophy; interstitial; overactive; receptors; urinary bladder.

Figure 1

Cystitis induced bladder hypertrophy was blocked by inhibiting NMDAR activity with MK-801 (MK). After CYP and MK-801 treatment bladder was freshly dissected (A). Bladder weight was expressed as ratio of bladder weight to body weight (B to D). Results represent 9 or 10 rats per group. Asterisk indicates p <0.05 vs control. Pound sign indicates p <0.05 vs CYP.

Figure 2

Type I collagen up-regulation induced by cystitis in bladder was blocked by inhibiting NMDAR activity with MK-801 (MK). Western blot (A) shows type I collagen expression in 2 CYP and 2 CYP plus MK-801 rats. Note mean ± SD collagen protein (B). Asterisk indicates p <0.05 vs control. Pound sign indicates p <0.05 vs CYP. Immunostaining for type I collagen (arrows) confirmed Western blot results (C to E). Control and CYP rats received same amount of vehicle as MK-801 amount. Results represent 5 rats per group. Scale bar indicates 50 µm.

Figure 3

In bladder PI3K/Akt pathway was involved in NMDAR mediated collagen production. Akt activity was examined by Akt phosphorylation level (A) normalized to nonphospho-Akt (B). Asterisk indicates p <0.05 vs control. Pound sign indicates p <0.05 vs CYP. Western blots show samples from 2 CYP and 2 CYP plus LY294002 (LY) treated rats (A and C). Relative type I collagen was normalized to β-actin (C and D). PI3K/Akt pathway was involved in NMDAR regulated bladder hypertrophy (E). Control and CYP rats received same amount of vehicle as MK-801 (A and B) and LY294002 amount (C and D). Results represent 4 or 5 rats per group.

Figure 4

Western blot demonstrates that NMDAR NR1 subunit phosphorylation was expressed in spinal cord (SC) but not in bladder during cystitis (A). L6 after CYP injection was used for comparison. Note mean ± SD relative phospho-NR1 level in 3 rats (B). Asterisk indicates p <0.05 vs spinal cord.

Figure 5

Cystitis induced bladder hypertrophy and collagen up-regulation in bladder (A) were not affected by D-AP5. After CYP and D-AP5 treatment bladder weight was measured and expressed as ratio of bladder weight to body weight (B). Asterisk indicates p <0.05 vs control. Western blot of bladder protein extracts was done to analyze type I collagen (C and D) and phospho-Akt (E and F). Control and CYP rats received same amount of vehicle as D-AP5 amount. Results represent 4 rats per group. Western blot shows samples from 2 CYP and 2 CYP plus D-AP5 treated rats (C and E).

Figure 6

Representative results of spontaneous urination metabolic test show that MK-801 inhibited cystitis induced urinary frequency and restored voided volume. Urine was collected by scale connected to computer to record real-time micturition frequency (bars) and voided volume in controls (A), rats with cystitis (B) and rats with cystitis treated with MK- 801 (C). Voiding frequency (D) and voided volume (E) were determined. Control and CYP rats received same amount of vehicle as MK-801 amount. Results represent 3 or 4 rats per group. Asterisk indicates p <0.05 vs control. Pound sign indicates p <0.05 vs CYP.

Figure 7

Urodynamics demonstrated decreased intermicturition interval (IMI) during cystitis, which was reversed by MK-801. Conscious cystometry was performed in controls, and rats with cystitis with and without MK-801 (A to C). s, seconds. Intermicturition interval was recorded and analyzed (D) and bladder compliance was calculated (E). Control and CYP rats received same amount of vehicle as MK-801 amount. Results represent 3 or 4 rats per group. Asterisk indicates p <0.05 vs control. Pound sign indicates p <0.05 vs CYP.

Figure 8

MK-801 in vivo restored contractility impaired by cystitis in detrusor muscle strips from controls, and rats with cystitis with and without MK-801 subjected to 1 µM CCh stimulation (A). Strip tension was expressed as force (B). Asterisk indicates p <0.05 vs control. Pound sign indicates p <0.05 vs CYP. After washing until contractile tone reached baseline 10 µM CCh was applied (A and C). Control and CYP rats received same amount of vehicle as MK-801 amount. Results represent 3 or 4 rats with 2 or 3 strips per rat averaged as 1 point.