Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells.
BACKGROUND
Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling.


METHODS
AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM.


RESULTS
We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+.


CONCLUSIONS
Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation.
Procino
Giuseppe
G
Department of General and Environmental Physiology, University of Bari, Bari, Italy.
Carmosino
Monica
M
Tamma
Grazia
G
Gouraud
Sabine
S
Laera
Antonia
A
Riccardi
Daniela
D
Svelto
Maria
M
Valenti
Giovanna
G
D000199:Actins / Q000378:metabolism; D000818:Animals; D051399:Aquaporin 2; D020346:Aquaporins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005682:Gadolinium / Q000494:pharmacology; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011817:Rabbits; D044169:Receptors, Calcium-Sensing / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D022502:Stress Fibers / Q000378:metabolism
D000199:Actins; D051399:Aquaporin 2; D020346:Aquaporins; D044169:Receptors, Calcium-Sensing; D005576:Colforsin; D005682:Gadolinium; D000242:Cyclic AMP; D011493:Protein Kinase C; D002118:Calcium