Altered central TRPV4 expression and lipid raft association related to inappropriate vasopressin secretion in cirrhotic rats

Abstract

Inappropriate vasopressin (AVP) release causes dilutional hyponatremia in many pathophysiological states such as cirrhosis. The central molecular mechanisms that mediate inappropriate AVP release are unknown. We tested the hypothesis that changes in the expression or trafficking of TRPV4 in the central nervous system may contribute to inappropriate AVP release in the bile duct ligation (BDL) model of cirrhosis in the rat. Four weeks after surgery, BDL rats demonstrated significantly increased plasma vasopressin and plasma renin activity (PRA), hypervolemia, and decreased plasma osmolality. These effects were blocked by providing BDL rats with 2% saline to drink for 15 days. TRPV4 protein expression was significantly increased in brain punches from BDL rats containing the supraoptic nucleus (SON) of the hypothalamus (100% +/- 11 to 157% +/- 4.8), and this effect was blocked in BDL rats given saline. Immunohistochemistry demonstrated a significant increase in TRPV4-positive cells and the percentage of AVP neurons that also were TRPV4-positive in the SON of BDL rats. In the hypothalamus of BDL rats, TRPV4 lipid raft association increased compared with sham (from 100% +/- 2.1 to 326.1% +/- 16). This effect was significantly attenuated in BDL rats given 2% saline to drink (174% +/- 11). In the brain stem, TRPV4 lipid raft association was reduced by BDL and inversely related to plasma AVP and PRA. We speculate that changes in TRPV4 expression and compartmentalization within lipid rafts could contribute to a feed-forward mechanism related to AVP release in cirrhosis.

Fig. 1.

Linear regression analysis of plasma renin activity (PRA) and vasopressin (AVP) from sham-ligated rats (Sham), bile duct ligation rats (BDL), and BDL rats given 2% saline to drink (BDL+saline).

Fig. 2.

Average daily fluid intake from Sham and BDL rats given water or 2% saline to drink for 2 wk. Values are means ± SE, *Statistically significant, P < 0.05. Parentheses indicate number of animals.

Fig. 3.

A: digital images of representative Western blot analysis for TRPV4 from brain punches taken from SON of sham-ligated rats, BDL rats, and BDL rats given 2% saline to drink (BDL+sal). Summary of densitometric analysis of TRPV4 immunoreactivity normalized with GAPDH from sham, BDL, and BDL+ saline. Values are expressed as means ± SE. ***Statistically significant, P < 0.001. Parentheses indicate number of animals. B: linear regression analysis of AVP and transient receptor potential vanilloid 4 (TRPV4) immunoreactivity in the SON of sham, BDL, and BDL+ saline rats. C: linear regression analysis of PRA and TRPV4 immunoreactivity in the SON of sham, BDL, and BDL+ saline rats.

Fig. 4.

Digital images of representative Western blot analysis for TRPV4 from brain punches taken from PVN (A), OVLT (B), and SFO (C) from sham, BDL rats, and BDL rats given 2% saline to drink (BDL+sal). Graph represents summary data of densitometric analysis of TRPV4 immunoreactivity in sham, BDL, and BDL+ saline from these regions. Values are expressed as means ± SE. Numbers in parentheses represent number of animals.

Fig. 5.

Digital images of representative Western blot analysis for TRPV4 from brain punches taken from NTS (A) and RVLM (B) from sham, BDL rats. Graph illustates densitometric analysis of TRPV4 immunoreactivity in sham and BDL. Values are expressed as means ± SE. Numbers in parentheses represent number of animals.

Fig. 6.

Digital images of representative examples of immunohistochemistry for TRPV4 (green: A and B) and AVP (red: C and D) staining in the SON of sham (A and C) and BDL rats (B and D). Merged images (E and F) illustrate colocalization of TRPV4 and AVP in the SON of sham and BDL rats. Scale bar in each image is 100 μm.

Fig. 7.

Quantitative analysis of changes in AVP and TRPV4 staining in the SON from sham-ligated and BDL rats. *Significantly different from sham, P < 0.01. **Significantly different from sham, P < 0.001. A: average number per section of AVP-positive neurons. B: average number per section of TRPV4-positive neurons. C: percentage of AVP-positive neurons also positive for TRPV4. D: percentage of TRPV4-positive neurons also positive for AVP. Numbers in parentheses represent number of animals.

Fig. 8.

Digital images of representative examples of immunohistochemistry for TRPV4 (green: A and B) and AVP (red: C and D) staining in the PVN of sham (A and C) and BDL rats (B and D). Merged images (E) and (F) illustrate colocalization of TRPV4 and AVP in the SON of sham and BDL rats. Scale bar in each image is 100 μm.

Fig. 9.

Quantitative analysis of changes in AVP and TRPV4 staining in the PVN from sham-ligated and BDL rats. A: average number per section of parvocellular AVP-positive neurons. B: average number per section of magnocellular AVP-positive neurons. C: average number per section of parvocellular TRPV4-positive neurons. D: average number per section of parvocellular TRPV4-positive neurons. *Significantly different from sham, P < 0.01. **Significantly different from sham, P < 0.001. Numbers within parentheses represent number of animals.

Fig. 10.

TRPV4 and flotillin distributions over sucrose density gradients from total hypothalamus. Total hypothalamus of sham animals was homogenized, fractionated, and the crude plasma membrane fractions were homogenized by the Na₂CO₃ method, and the fractions were resolved by SDS-PAGE and Western blot analysis. The fraction number 4 corresponds to the lipid raft fraction.

Fig. 11.

A: TRPV4 and flotillin distributions over sucrose density gradients in the hypothalamus of BDL rats and BDL rats given 2% saline to drink (BDL+sal). The fraction number 4 corresponds to the lipid raft fraction. TRPV4 immunoreactivity was quantified by densitometry using the Scion program, and the respective values were normalized by the flotillin densitometry in four independent experiments. Values are expressed as means ± SE, ***Statistically significant different from sham, P < 0.001. **Statistically significant different from sham, P < 0.01. ^aStatistically significant difference from BDL, P < 0.001. B: linear regression analysis of AVP and TRPV4 immunoreactivity in lipid raft (fraction #4) from total hypothalamus in all three groups. C: linear regression analysis of PRA and TRPV4 immunoreactivity in lipid raft (fraction number 4) of total hypothalamus from sham, BDL, and BDL+ saline.

Fig. 12.

A: TRPV4 and flotillin distributions in sucrose density gradients in the brain stem of sham, BDL rats, and BDL rats given 2% saline to drink (BDL + sal). The fraction number 4 corresponds to the lipid raft fraction. Values are expressed as means ± SE. ***Statistically significant different from sham, P < 0.001. ^aStatistically significant different from BDL, P < 0.001. B: linear regression analysis of vasopressin (AVP) and TRPV4 immunoreactivity in lipid raft (fraction number 4) of total brain stem from sham, BDL, and BDL+ saline. C: linear regression analysis of PRA and TRPV4 immunoreactivity in lipid raft (fraction number 4) of total brain stem from sham, BDL, and BDL+ saline.