Stimulation of AQP2 Membrane Insertion in Renal Epithelial Cells In Vitro and In Vivo by the cGMP Phosphodiesterase Inhibitor Sildenafil Citrate (Viagra)
| Title: | Stimulation of AQP2 Membrane Insertion in Renal Epithelial Cells In Vitro and In Vivo by the cGMP Phosphodiesterase Inhibitor Sildenafil Citrate (Viagra) |
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| Authors: | Bouley, Ph.D., Richard; Pastor-Soler, Nuria; Cohen, Ori; McLaughlin, M.D., Margaret Elise; Breton, Sylvie; Brown, Dennis |
| Publisher: | American Journal of Physiology: Renal Physiology |
| Date Published: | June 01, 2005 |
| Reference Number: | 692 |
Vasopressin-stimulated insertion of the aquaporin 2 (AQP2) water channel into the plasma membrane of kidney collecting duct principal cells is a key event in the urinary concentrating mechanism. The paradigm for vasopressin-receptor signaling involves cAMP-mediated protein kinase A activation, which results in the functionally critical phosphorylation of AQP2 on amino acid serine 256. We previously showed that a parallel cGMP-mediated signaling pathway also leads to AQP2 membrane insertion in AQP2-transfected LLC-PK1 (LLC-AQP2) cells and in outer medullary collecting duct principal cells in situ (Bouley R, Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY, Ausiello DA, and Brown D. J Clin Invest 106: 1115-1126, 2000). In the present report, we show by immunofluorescence microscopy, and Western blotting of plasma membrane fractions, that 45-min exposure of LLC-AQP2 cells to the cGMP phosphodiesterase type 5 (PDE5) inhibitors sildenafil citrate (Viagra) or 4-{[3',4'-methylene-dioxybenzyl]amino}-6-methoxyquinazoline elevates intracellular cGMP levels and results in the plasma membrane accumulation of AQP2; i.e., they mimic the vasopressin effect. Importantly, our data also show that acute exposure to PDE5 inhibitors for 60 min induces apical accumulation of AQP2 in kidney medullary collecting duct principal cells both in tissue slices incubated in vitro as well as in vivo after intravenous injection of Viagra into rats. These data suggest that AQP2 membrane insertion can be induced independently of vasopressin-receptor activation by activating a parallel cGMP-mediated signal transduction pathway with cGMP PDE inhibitors. These results provide proof-of-principle that pharmacological activation of vasopressin-independent, cGMP signaling pathways could aid in the treatment of those forms of nephrogenic diabetes insipidus that are due to vasopressin-2 receptor dysfunction.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)
In a majority of the cases of people with congenital NDI, their V2R is mutated and incapable of initiating the molecular pathway that leads to AQP2 insertion.
In their previous work, Brown, et al., found that there exists another molecular pathway by which AQP2 can be stimulated to insert into the cell membrane. When laboratory cell cultures were treated with nitric oxide (NO), L-arginine or atrial natriuretic peptide (ANP), cellular levels of cyclic guanosine monophosphate (cGMP) elevated, resulting in AQP2 insertion.
In their current study, the researchers wanted to know if cGMP elevation – leading to AQP2 insertion – could be stimulated by inhibiting the number of cGMP phosphodiesterases, specifically phosphodiesterase-5 (PDE5) in the cell. PDE5 is a type of enzyme in cells that breaks down cGMP. Because cGMP is continually produced at low levels in cells, this inhibition causes the level of cGMP to increase without the need for hormonal stimulation from outside the cell. To test their hypothesis, the research team treated both laboratory cell cultures and a breed of rats that lacked the hormone, vasopressin, (and thus are incapable of stimulating AQP2 insertion via AVP) with three different cGMP phosphodiesterase inhibitors: sildenafil citrate, MBMQ and IBMX.
Each of these PDE5 inhibitors was added to separate LLC-AQP2 tissue cultures in a laboratory setting. The researchers found significant accumulation of AQP2 in the membranes of cells that express (produce) AQP2. That is, all the PDE inhibitors resulted in a significant increase in the level of AQP2 at or close to the cell membranes. The cells were exposed to the inhibitors for 45 minutes, and the amount of AQP2 accumulated depended on how large a dose of inhibitor they were exposed to.
To understand the mechanics of these results more clearly, the researchers examined the effect of these inhibitors on cellular levels of cAMP and cGMP. They found the inhibitors significantly increased intracellular cGMP levels, as opposed to cAMP levels. Further, the researchers found that when they exposed kidney medullary collecting duct principal cells for 60 minutes, AQP2 accumulated in the apical cell membrane. This is important, for this is the section of the membrane where AQP2 must be to perform its urine concentrating and water balancing function. This same accumulation resulted when the test rats were injected with the PDE5 inhibitor, sildenafil citrate.
Brown, et al.’s study shows that a molecular pathway other than the one initiated by the linkage of AVP and V2R can be stimulated by pharmaceuticals. This work could lead to therapeutic treatment for those forms of NDI that are due to V2R dysfunction. However, there is much more work to do, such as investigating side effects and seeing what combination of pharmaceuticals (i.e., the PDE5 inhibitors) and diet would prove most effective. Finally, most of the urinary concentrating mechanism depends on water reabsorption in that part of the kidney called the cortical collecting duct, and the most obvious effect of the inhibitor injected into the live rats, sildenafil citrate, occurred in the portion of the kidney called the outer medullary collecting ducts.