Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption

To prevent dehydration, terrestrial animals and humans have developed a sensitive and versatile system to maintain their water homeostasis. In states of hypernatremia or hypovolemia, the antidiuretic hormone vasopressin (AVP) is released from the pituitary and binds its type-2 receptor in renal principal cells. This triggers an intracellular cAMP signaling cascade, which phosphorylates aquaporin-2 (AQP2) and targets the channel to the apical plasma membrane. Driven by an osmotic gradient, pro-urinary water then passes the membrane through AQP2 and leaves the cell on the basolateral side via AQP3 and AQP4 water channels. When water homeostasis is restored, AVP levels decline, and AQP2 is internalized from the plasma membrane, leaving the plasma membrane watertight again. The action of AVP is counterbalanced by several hormones like prostaglandin E2, bradykinin, dopamine, endothelin-1, acetylcholine, epidermal growth factor, and purines. Moreover, AQP2 is strongly involved in the pathophysiology of disorders characterized by renal concentrating defects, as well as conditions associated with severe water retention. This review focuses on our recent increase in understanding of the molecular mechanisms underlying AVP-regulated renal water transport in both health and disease

Regulation of AQP2 localization by Ser256 and S261 phosphorylation and ubiquitination

Vasopressin-induced water reabsorption coincides with phosphorylation of aquaporin-2 (AQP2) at S256 (pS256), dephosphorylation at S261, and its translocation to the apical membrane, whereas treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA) induces AQP2 ubiquitination at K270, its internalization, and lysosomal degradation. In this study we investigated the relationship between S256 and S261 phosphorylation in AQP2 and its ubiquitination and trafficking in MDCK cells. Forskolin stimulation associated with increased pS256 and decreased pS261 AQP2, indicating that MDCK cells are a good model. After forskolin stimulation, TPA-induced ubiquitination of AQP2 preceded phosphorylation of AQP2 at S261, which in the first instance occurred predominantly on ubiquitinated AQP2. Forskolin-induced changes in pS261 were also observed for AQP2-S256A and AQP2-S256D, which constitutively localize in vesicles and the apical membrane, respectively. Although pS261 varies with forskolin as with wild-type AQP2, AQP2-S256A is not increased in its ubiquitination. Our data reveal that pS261 occurred independently of AQP2 localization and suggest that pS261 follows ubiquitination and endocytosis and may stabilize AQP2 ubiquitination and intracellular localization. The absence of increased ubiquitination of AQP2-S256A indicates that its intracellular location is due to the lack of pS256. Furthermore, AQP2-S261A and AQP2-S261D localized to vesicles, which was due to their increased ubiquitination, because changing K270 into Arg in both mutants resulted in their localization in the apical membrane. Although still increased in its ubiquitination, AQP2-S256D-S261D localized in the apical membrane. AQP2-S256D-K270R-Ub, however, localized to intracellular vesicles. Although our localization of AQP2-S261A/D is different from that of others, these data indicate that constitutive S256 phosphorylation counterbalances S261D-induced ubiquitination and internalization or changes its structure to allow distribution to the apical membrane. The vesicular localization of AQP2-S256D-K270R-Ub, however, indicates that the dominant apical sorting of S256D can again be overruled by constitutive ubiquitination. These data indicate that the membrane localization of AQP2 is determined by the balance of the extents of phosphorylation and ubiquitination

Dynamics of Aquaporin 2 Phosphorylation at Serine 256 and Serine 261 upon Co-Expression of Constitutively Active Variants of the Calcium- Sensing Receptor (CaSR) in Renal Cells

Background: We have recently shown that in MCD4 renal cells, cell surface AQP2 expression in cells exposed to CaSR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin. Those findings were in line with data obtained in hypercalciuric subjects displaying at baseline significantly higher AQP2 excretion and no significant increase in AQP2 excretion and urinary osmolarity after acute DDAVP administration compared to normocalciurics (Procino et al Plos One 2012). This indicates that CaSR-AQP2 interplay represents an internal renal defense to mitigate the effects of rising of calcium during antidiuresis on the risk of calcium precipitation. Methods: Human wild-type CaSR (hCaSR-wt) and its constitutively active variants (hCaSR-R990G; hCaSR-N124K) were functionally expressed in renal HEK cells stably expressing hAQP2. The N124K mutation is one of eight naturally occurring activating mutations in subjects with autosomal dominant hypocalcemia, whereas R990G is a gain-of- function of the CASR gene polymorphism. Western blotting analysis of a crude membrane fraction was performed using phospho-specific antibodies. Results: Compared to mock cells, pS256-AQP2 abundance was significantly increased in cells expressing either the wt-CaSR or its activating variants. Of note, we also found a significant increase in pS261-AQP2 in hCaSR-wt expressing cells compared to mock. Interestingly, the expression of pS261-AQP2 was significantly higher in cells expressing the constitutively active CaSR variants with respect to wt-CaSR expressing cells. No change in the pS269 was observed. Conclusions: Since previous data demonstrated that the amount of pS261 significantly decreases in response to short-term vasopressin exposure, it can be speculated that the increase in pS261 observed in cells expressing constitutively active CaSR variants might counteract the vasopressin response

Modulation of cAMP signaling, AQP2 phosphorylation and osmotic water permeability in response to DDAVP or fk under tolvaptan treatment in renal cells.

Background: The vasopressin receptor antagonist tolvaptan has emerged as tool in the management of hyponatremia. However, no direct evidence that the aquaretic effect of tolvaptan is based on impairment of vasopressin stimulated AQP2 phosphorylation and targeting to the plasma membrane has been provided. Methods: MDCK stably expressing hAQP2 or rat kidney slides were exposed to DDAVP or forskolin (FK) stimulation in the presence or in the absence of tolvaptan (10nM). The effect of these treatments on cAMP levels, AQP2 phosphorylation, intracellular calcium concentration and osmotic water permeability was analyzed. Results: In MDCK cells, DDAVP treatment significantly increased cAMP levels paralleled by an increase in p256AQP2. Pretreatment with tolvaptan significantly reduced both effects. Surprisingly, tolvaptan pretreatment strongly reduced the increase in p256AQP2 elicited by FK, a direct activator of adenylyl cyclase. Similar results were obtained in rat kidney slides. In line, tolvaptan prevented the increase in the osmotic water permeability promoted by either DDAVP or FK in MDCK. We therefore analyzed whether tolvaptan had, per se, a cellular effect. Calibration of cellular calcium in MCDK cells revealed that tolvaptan caused a significant increase in intracellular calcium (tolvaptan 64.0±2 nM; ctr 32±1.7 nM). Since p256AQP2 can be de-phosphorylated by PP2A, a calcium depended serine/threonine phosphatase, rat kidney slides were pretreated with tolvaptan and exposed to FK in the presence or absence of caliculyn (5pM) a specific inhibitor of PP2A. Under these conditions tolvaptan failed to prevent FK-induced increase in p256AQP2 suggesting that tolvaptan, activates PP2A. Conclusions: Tolvaptan prevents vasopressin induced increase in p256AQP2, AQP2 trafficking and increase in osmotic water permeability. Moreover tolvaptan increases basal intracellular calcium, which might have relevant consequences in modulating p256AQP2 levels and therefore the clinical response to the drug