Suppression of the PI3K pathway in vivo reduces cystitis-induced bladder hypertrophy and restores bladder capacity examined by magnetic resonance imaging

Abstract

This study utilized magnetic resonance imaging (MRI) to monitor the real-time status of the urinary bladder in normal and diseased states following cyclophosphamide (CYP)-induced cystitis, and also examined the role of the phosphoinositide 3-kinase (PI3K) pathway in the regulation of urinary bladder hypertrophy in vivo. Our results showed that under MRI visualization the urinary bladder wall was significantly thickened at 8 h and 48 h post CYP injection. The intravesical volume of the urinary bladder was also markedly reduced. Treatment of the cystitis animals with a specific PI3K inhibitor LY294002 reduced cystitis-induced bladder wall thickening and enlarged the intravesical volumes. To confirm the MRI results, we performed H&E stain postmortem and examined the levels of type I collagen by real-time PCR and western blot. Inhibition of the PI3K in vivo reduced the levels of type I collagen mRNA and protein in the urinary bladder ultimately attenuating cystitis-induced bladder hypertrophy. The bladder mass calculated according to MRI data was consistent to the bladder weight measured ex vivo under each drug treatment. MRI results also showed that the urinary bladder from animals with cystitis demonstrated high magnetic signal intensity indicating considerable inflammation of the urinary bladder when compared to normal animals. This was confirmed by examination of the pro-inflammatory factors showing that interleukin (IL)-1α, IL-6 and tumor necrosis factor (TNF)α levels in the urinary bladder were increased with cystitis. Our results suggest that MRI can be a useful technique in tracing bladder anatomy and examining bladder hypertrophy in vivo during disease development and the PI3K pathway has a critical role in regulating bladder hypertrophy during cystitis.

Figure 1. MRI visualization demonstrated an increase in the thickness of bladder wall and a decrease in the volume of bladder dome during CYP-induced cystitis.

A spin-echo T2-weighted MR sequence was performed to visualize the urinary bladder in the pelvis on axial sections (A). The same animal was scanned before (B) and after CYP treatment (C-E). At 2 h after cystitis was induced, the anatomy of the urinary bladder appeared similar to control. At 8 h and 48 h post cystitis induction, the thickness of the bladder wall was significantly increased (F) which was accompanied with a decrease in intravesical volume (G). Summary results were from 5 animals before and after CYP injection. *, p<0.05.

Figure 2. PI3K inhibition with LY294002 attenuated cystitis-induced bladder wall thickening and restored bladder capacity.

The urinary bladders from cystitis animals (8 h, A) and cystitis animals treated with PI3K inhibitor LY294002 (CYP 8 h + LY, B) were visualized by MRI. Green circle indicated the outer edge of the urinary bladder (A, B); the pink circle indicated the inner edge of the urinary bladder (A, B). Treatment with LY294002 prevented bladder wall thickening caused by cystitis (C), and also increased the intravesical volume during cystitis (D). n = 4. *, p<0.05.

Figure 3. Postmortem examination by H&E showed PI3K inhibition on bladder wall thickening during cystitis.

Transverse sections (10 µm) of the urinary bladder from control (A), cystitis (B) and cystitis with LY294002 (LY) treatment (C) were stained by H&E (m: muscular layer; u: urothelium; d: bladder dome). The thickness of the bladder wall was increased by cystitis (D). LY294002 treatment reduced bladder wall thickness caused by cystitis (D). Bar  = 200 µm. n = 3. *, p<0.05 vs vehicle control. #, p<0.05 vs CYP cystitis.

Figure 4. PI3K inhibition reduced collagen up-regulation by cystitis.

Trichrome stain (A-C) showed an increased amount of extracellular matrix built-up in the urinary bladder at 8 h of cystitis (B, blue stain). LY294002 (LY) treatment reduced the level of extracellular matrix content in the urinary bladder of cystitis animals (C). Western blot (D) with a specific antibody recognizing type I collagen showed similar results that cystitis increased collagen protein expression which was attenuated by LY294002 treatment (E). Real-time PCR demonstrated that cystitis-induced type I collagen mRNA up-regulation was also inhibited by LY294002 treatment (F). n = 3. Bar n = 200 µm. *, p<0.05 vs vehicle control. #, p<0.05 vs CYP cystitis.

Figure 5. Cystitis-induced up-regulation of pro-inflammatory factors was attenuated by LY294002 treatment.

The levels of tumor necrosis factor alpha (TNFα) (A), interleukin 1α (B) and IL-6 (C) were up-regulated by cystitis examined at 8 h post CYP injection. LY294002 (LY) treatment reversed inflammatory responses in the urinary bladder caused by cystitis. n = 3. *, p<0.05 vs vehicle control. #, p<0.05 vs CYP cystitis.

Figure 6. PI3K inhibition decreased cystitis-induced increment of bladder weight measured in vitro and in vivo.

The bladder mass was calculated by analyzing MRI data and using a formula described in the methodology (A). Postmortem measurement of the bladder weight showed an increase in cystitis animals when compared to control (B); this increment was reduced by LY294002 (LY) treatment (B). n = 4–5. *, p<0.05 vs vehicle control. #, p<0.05 vs CYP cystitis.