Aromatase deficiency causes altered expression of molecules critical for calcium reabsorption in the kidneys of female mice *.
Kidney stones increase after menopause, suggesting a role for estrogen deficiency. ArKO mice have hypercalciuria and lower levels of calcium transport proteins, whereas levels of the klotho protein are elevated. Thus, estrogen deficiency is sufficient to cause altered renal calcium handling.


BACKGROUND
The incidence of renal stones increases in women after menopause, implicating a possible role for estrogen deficiency. We used the aromatase deficient (ArKO) mouse, a model of estrogen deficiency, to test the hypothesis that estrogen deficiency would increase urinary calcium excretion and alter the expression of molecular regulators of renal calcium reabsorption.


METHODS
Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n = 5-12/group) were used to measure urinary calcium in the fed and fasting states, relative expression level of some genes involved in calcium reabsorption in the distal convoluted tubule by real-time PCR, and protein expression by Western blotting or immunohistochemistry. Plasma membrane calcium ATPase (PMCA) activity was measured in kidney membrane preparations. ANOVA was used to test for differences between groups followed by posthoc analysis with Dunnett's test.


RESULTS
Compared with WT, urinary Ca:Cr ratios were elevated in ArKO mice, renal mRNA levels of transient receptor potential cation channel vallinoid subfamily member 5 (TRPV5), TRPV6, calbindin-D28k, the Na+/Ca+ exchanger (NCX1), and the PMCA1b were significantly decreased, and klotho mRNA and protein levels were elevated. Estradiol treatment of ArKO mice normalized urinary calcium excretion, renal mRNA levels of TRPV5, calbindin-D(28k), PMCA1b, and klotho, as well as protein levels of calbindin-D28k and Klotho. ArKO mice treated with estradiol had significantly greater PMCA activity than either untreated ArKO mice or WT mice.


CONCLUSIONS
Estrogen deficiency caused by aromatase inactivation is sufficient for renal calcium loss. Changes in estradiol levels are associated with coordinated changes in expression of many proteins involved in distal tubule calcium reabsorption. Estradiol seems to act at the genomic level by increasing or decreasing (klotho) protein expression and nongenomically by increasing PMCA activity. PMCA, not NCX1, is likely responsible for extruding calcium in response to in vivo estradiol hormonal challenge. These data provide potential mechanisms for regulation of renal calcium handling in response to changes in serum estrogen levels.
Oz
Orhan K
OK
Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9153, USA. Orhan.Oz@UTSouthwestern.edu
Hajibeigi
Asghar
A
Howard
Kevin
K
Cummins
Carolyn L
CL
van Abel
Monique
M
Bindels
Rene Jm
RJ
Word
R Ann
RA
Kuro-o
Makoto
M
Pak
Charles Y C
CY
Zerwekh
Joseph E
JE
D000042:Absorption; D000818:Animals; D001141:Aromatase / Q000172:deficiency*; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D053565:Hypercalciuria / Q000235:genetics / Q000378:metabolism; D007669:Kidney Calculi / Q000235:genetics / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D000090265:Klotho Proteins; D008593:Menopause / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000096:biosynthesis / Q000235:genetics; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000235:genetics; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis / Q000235:genetics; D050916:TRPV Cation Channels / Q000096:biosynthesis / Q000235:genetics
C576499:Calb1 protein, mouse; D064092:Calbindin 1; D064026:Calbindins; D015220:Calcium Channels; C499849:NCX1 protein, mouse; D064030:S100 Calcium Binding Protein G; D019831:Sodium-Calcium Exchanger; D050916:TRPV Cation Channels; C492240:Trpv5 protein, mouse; C446923:Trpv6 protein, mouse; D004958:Estradiol; D001141:Aromatase; D005966:Glucuronidase; D000090265:Klotho Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases; C506954:Atp2b1 protein, mouse; D002118:Calcium