Neurotrophin/receptor expression in urinary bladder of mice with overexpression of NGF in urothelium

Abstract

Urothelium-specific overexpression of nerve growth factor (NGF) in the urinary bladder of transgenic mice stimulates neuronal sprouting in the urinary bladder, produces increased voiding frequency, and results in increased referred somatic hypersensitivity. Additional NGF-mediated pleiotropic changes might contribute to the increased voiding frequency and pelvic hypersensitivity observed in these transgenic mice, such as modulation of other growth factor/receptor systems. Chronic overexpression of NGF in the urothelium was achieved through the use of a highly urothelium-specific uroplakin II promoter. In the present study, we examined NGF, brain-derived neurotrophic factor (BDNF), and associated receptor [p75(NTR), tyrosine kinase (Trk)A, TrkB] transcript and protein expression in urothelium and detrusor smooth muscle of NGF-overexpressing (OE) and littermate wild-type mice, using real-time quantitative reverse transcription-polymerase chain reaction, ELISAs, and semiquantitation of immunohistochemistry. We focused on these growth factor/receptors given the established roles of NGF/TrkA, NGF/p75(NTR), and BDNF/TrkB systems in bladder function. Increased voiding frequency in NGF-OE mice was confirmed by examining urination patterns. BDNF, TrkA, and TrkB protein expression was significantly (P ≤ 0.01) reduced and p75(NTR) protein expression was significantly (P ≤ 0.01) increased in urinary bladder of NGF-OE mice. The NGF-OE-induced changes in neurotrophic factor/receptor expression in urinary bladder may represent compensatory changes to reduce voiding frequency in the NGF-OE mouse.

Fig. 1.

Modulation of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) transcript expression in littermate wild-type (WT) and NGF-overexpressing (NGF-OE) mice in urothelium and detrusor smooth muscle. WT urothelium samples were set equal to 100% and normalized to the relative expression of the housekeeping gene 18S. WT detrusor samples were expressed relative to WT urothelium samples and normalized to the relative expression of the housekeeping gene 18S. A: NGF mRNA expression in urothelium (U). B: NGF mRNA expression in detrusor. C: BDNF mRNA expression in urothelium. D: BDNF mRNA expression in detrusor. Values are means ± SE for sample size (n = 5–7). **P ≤ 0.0001, *P ≤ 0.01 vs. WT.

Fig. 2.

A–F: BDNF immunoreactivity (IR) in urothelium (U; A, B, D, E) and detrusor smooth muscle (sm) (A, C, D, F) of littermate WT (A–C) and NGF-OE (D–F) mice. Comparable BDNF IR was present in urothelial cells of WT and NGF-OE urothelium (A, B, D, E). BDNF-IR expression was reduced in detrusor sm of NGF-OE mice (D, F) compared with detrusor sm of WT mice (A, C). L, lumen. Calibration bar: 50 μm (B, C, E, F), 125 μm (A, D). G: summary histogram of BDNF expression in U and detrusor sm in WT and NGF-OE mice. Values are means ± SE (n = 5–7). *P ≤ 0.01.

Fig. 3.

BDNF, tyrosine kinase (Trk)B, and TrkA content in the urinary bladders of WT and NGF-OE transgenic mice. BDNF (A), TrkB (B), and TrkA (C) content in whole urinary bladder was determined in WT and NGF-OE. Urinary bladder NGF, TrkB, and TrkA content was significantly (*P ≤ 0.01) decreased in NGF-OE transgenic vs. WT mouse bladders. Values are means ± SE (n = 5–7).

Fig. 4.

Modulation of p75^NTR, TrkA, and TrkB receptor transcript expression in littermate WT and NGF-OE mice in urothelium and detrusor smooth muscle. WT urothelium samples were set equal to 100% and normalized to the relative expression of the housekeeping gene 18S. WT detrusor samples were expressed relative to WT urothelium samples and normalized to the relative expression of the housekeeping gene 18S. A: p75^NTR mRNA expression in urothelium (U). B: p75^NTR mRNA expression in detrusor. C: TrkA mRNA expression in U. D: TrkA mRNA expression in detrusor. E: TrkB mRNA expression in U. F: TrkB mRNA expression in detrusor. Values are means ± SE for sample size (n = 5–7). #P ≤ 0.05, *P ≤ 0.01 vs. WT.

Fig. 5.

p75^NTR IR in urothelium (U) (A–F) and detrusor smooth muscle (sm) (A, B, D, E) of littermate WT (A–C) and NGF-OE (D–F) mice. Comparable p75^NTR IR was present in urothelial cells of WT and NGF-OE urothelium (A–F). p75^NTR IR was expressed in detrusor sm of WT mice (A, B) and increased in detrusor sm of NGF-OE mice (D, E). L, lumen. Calibration bar: 50 μm (B, C, E, F), 125 μm (A, D). G: summary histogram of p75^NTR expression in the U and detrusor sm in WT and NGF-OE mice. Values are means ± SE for n = 5–7. *P ≤ 0.01.

Fig. 6.

TrkA IR in urothelium (U) (A–F) and detrusor smooth muscle (sm) (C, F) of littermate WT (A–C) and NGF-OE (D–F) mice. TrkA IR was significantly (P ≤ 0.01) reduced in urothelial cells of NGF-OE mice (A–F). TrkA IR was also significantly (P ≤ 0.01) reduced in detrusor sm of NGF-OE mice (C, F). G: higher-power image of TrkA IR in urothelial cells in littermate WT mice. Trk staining was visualized with Cy3-conjugated secondary antibody (red areas). YOYO-1 identified nuclear profiles of urothelial cells and was visualized with Cy2-conjugated secondary antibody (green areas). L, lumen. Calibration bar: 50 μm (B, E), 125 μm (A, C, D, F), 30 μm (G). H: summary histogram of TrkA expression in U and detrusor sm in WT and NGF-OE mice. Values are means ± SE for n = 5–7. *P ≤ 0.01.

Fig. 7.

TrkB IR in urothelium (U) (A–D) and detrusor smooth muscle (sm) (B–D) of littermate WT (A, B) and NGF-OE (C, D) mice. TrkB IR was significantly (P ≤ 0.01) reduced in urothelial cells of NGF-OE mice (A, C). TrkB IR was also significantly (P ≤ 0.01) reduced in detrusor sm of NGF-OE mice (B, D). E: higher-power image of TrkB IR in urothelial cells in littermate WT mice. Trk staining was visualized with Cy3-conjugated secondary antibody (red areas). YOYO-1 identified nuclear profiles of urothelial cells and was visualized with Cy2-conjugated secondary antibody (green areas). L, lumen. Calibration bar: 50 μm (A–D), 30 μm (E). F: summary histogram of TrkB expression in the U and detrusor sm in WT and NGF-OE mice. Values are means ± SE for n = 5–7. *P ≤ 0.01.