2000 Global Researcher Conference Proceeding
March 10 - 12, 2000
|Conference:||2000 Global Researcher Conference|
|Title:||Vasopressin-V2-receptor dependent and independent regulation of collecting duct aquaporin-2 expression and trafficking|
|Authors:||Nielsen, Soren; Promeneur, Dominique; Christensen, Birgitte Monster; Kwon, Tae-Hwan; Knepper, Mark; Frokiaer, Jorgen|
|Institutions:||University of Aarhus, National Institutes of Health, The Water and Salt Research Center, University of Aarhus|
Aquaporin-2 (AQP2) is the predominant vasopressin regulated water channel of the kidney collecting duct and is critically involved in acute and chronic regulation of body water balance. This study was undertaken to test if there are differences in vasopressin-V2-receptor mediated regulation of AQP2 expression versus vasopressin-V2-receptor mediated regulation of AQP2 trafficking in order to provide a rationale for treatment of diabetes insipidus.
For this purpose vasopressin-deficient Brattleboro rats were subjected to treatment with vasopressin-V2-receptor antagonists, lithium or thirsting. AQP2 levels were high in BB rats corresponding to 52 ± 8% of levels in normal Wistar rats. Treatment of BB rats with an AVP-V2-receptor antagonist (SR 121463A, 0.8 mg/day) for 48 hours resulted in a significant increase in urine output to 170 ± 9% of control levels. Moreover the treatment was associated with a marked reduction in total kidney AQP2 protein levels (42 ± 10% vs 100 ± 8%), inner medullary AQP2 protein levels (53 ± 8% vs 100 ± 20%) and AQP2 mRNA levels (36 ± 7% vs 100 ± 21%). The levels of AQP2 phosphorylated in the PKA phosphorylation consensus site (Ser-256 of AQP2) was relatively high in untreated Brattleboro rats compared to normal Wistar rats demonstrating that phosphorylation in the PKA site is taking place in vasopressin-deficient BB rats. However phosphorylated AQP2 (and non-phosphorylated AQP2) was exclusively found in intracellular vesicles demonstrating that there was no targeting to the plasma membrane. Treatment of BB rats with SR 121463A dramatically reduced phosphorylated AQP2 levels to 3 ± 1% of control levels (100 ± 17%). Lithium-treatment for 1 month, known to reduce adenylyl cyclase activity, also resulted in a very extensive down regulation of total AQP2 protein levels in BB rats to 15 ± 6% of levels in control BB rats (100 ±10%) and was associated with an increase in urine output to 220% of control levels. The marked down regulation of AQP2 levels in response to AVP-V2-receptor antagonist treatment or lithium treatment strongly indicates that the high AQP2 expression in BB rats depends in part on activation of AVP-V2-receptors and that the signalling cascade(s) involve adenylyl cyclase and hence cAMP. BB rats subjected to complete water restriction produced only a small but significant increase in AQP2 mRNA levels (235 ± 33%) and total AQP2 protein levels (156 ± 22%). Semiquantitative immunoelectron microscopy confirmed the increase in AQP2 abundance but revealed no significant change in the AQP2-labeling density of the apical plasma membrane of collecting duct principal cells in untreated and thirsted BB rats. In conclusion, the expression and phosphorylation of AQP2 in BB rats is in part dependent on AVP-V2-receptor signalling, and the AVP-V2-mediated regulation of AQP2 trafficking and AQP2 expression is effectively decoupled in BB rats indicating that there are differences in the AVP-V2-receptor mediated regulation of AQP2 trafficking and expression.
The results indicate that AQP2 expression levels may be surprisingly high in some forms of nephrogenic or central diabetes insipidus, and raises the possibility to design drugs that induce AQP2 plasma membrane targeting to reduce the severe polyuria seen in these patients.
When the antidiuretic hormone, arginine vasopressin (AVP), binds with the vasopressin-2 receptor (V2R), a molecular sequence occurs which directs aquaporin-2 (AQP2) to the apical membrane of the principal cells of the kidney collecting duct. AQP2s must first be expressed within the cell, then directed to the cell membrane. Both of these steps are mediated by the AVP/V2R bond. Nielsen, et al., sought to determine if both of these steps occur together or if the expression process and the targeting to the cell membrane occur separately.
To do this, they subjected one group of Brattleboro (BB) rats (which are genetically deficient in AVP) to treatment with a V2R antagonist (a compound that binds with V2R, thus preventing AVP from doing so). They subjected another group of rats to lithium treatment, and another to a water fast. Treatment with the AVP antagonist resulted in a significant increase in urine value and a marked reduction in aquaporin-2 (AQP2) levels. AQP2 undergoes the phosphorylating process in BB rats, but in the antagonist-treated BB rats, the phosphorylated AQP2 was found only inside the cell. This demonstrates that the AQP2s were not targeted to the cell membrane. The BB rats treated with lithium expressed very low levels of AQP2 and experienced high urine volume output. These experiments with the two groups of rats demonstrated that the AVP/V2R bond is necessary for high numbers of AQP2 in the rats. BB rats that were put on a water fast showed a small but significant increase in AQP2 numbers, but showed no more AQP2s in the cell membrane than the control group.
Taken together, these findings indicate that there are differences between the AVP/V2R mediated expression of AQP2 and the AVP/V2R mediated directing of AQP2 to the cell membrane. Thus, it is possible that some NDI patients may have a significant amount of AQP2 in their kidney collecting duct principal cells even though they might show few in their cells’ apical membranes. This finding supports the idea of designing drugs that specialize in delivery of AQP2s to cell membranes.