Aromatase Deficiency Causes Altered Expression of Molecules Critical for Calcium Reabsorption in the Kidneys of Female Mice

Abstract

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. Introduction: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. Materials and Methods: Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n ‫ס‬ 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-D 28k , 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-D 28k 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.

Involvement of the transepithelial calcium transport disruption and the formation of epididymal stones in roosters

“…Roosters affected by epididymal lithiasis had increased levels of TRPV6 and CaBP-D28K but not PMCA and NCX1. It is known that modulation of expression of these proteins is under control of different hormones, such as vitamin D3, estrogens, and androgens (Cai et al 1993, Van Abel et al 2002, Kip & Strehler 2004, Hoenderop et al 2005, Oz et al 2007, Hsu et al 2010. On this sense, we showed recently that roosters with lithiasis presented altered levels of vitamin D3, E 2 , and testosterone that paralleled changes in their receptors (VDR, ESR2, and AR respectively) within the epididymal region (Oliveira et al 2011b).…”

“…On this sense, we showed recently that roosters with lithiasis presented altered levels of vitamin D3, E 2 , and testosterone that paralleled changes in their receptors (VDR, ESR2, and AR respectively) within the epididymal region (Oliveira et al 2011b). The vitamin D3 and E 2 responsive systems are directly correlated with TRPV6 and CaBP-D28K expression levels (Hoenderop et al 2001, 2005, Oz et al 2007). However, the hormonal effect on the calcium extrusion step is less clear, although it has been shown that vitamin D3 stimulates PMCA expression in the basolateral membranes of reabsorptive cells in the intestine and kidney (Cai et al 1993, Kip & Strehler 2004.…”

“…Calbindin-D28K is a protein known to buffer intracellular calcium concentrations but it also serves as a calcium source (Schwaller 2009(Schwaller , 2010. This dual role of calbindins within the cell allows the participation of this protein in other biological processes, such as movement of cilia and secretory function, which may be the case for the efferent ductule ciliated cells and epididymal principal cells respectively (Oz et al 2007, Schwaller 2009, 2010.…”

“…The transepithelial calcium reabsorption depends on proteins that mediate the apical calcium entry within the cell, named TRPV5 and TRPV6 (transient receptor potential vanilloid channel), the diffusion of the ion throughout the cytoplasm bound to calcium-binding proteins (CaBP), such as CaBP-D9K or CaBP-D28K, and its extrusion to the extracellular environment in the basolateral membranes of the cells by the Na C /Ca 2C exchanger (NCX) or the plasma membrane calmodulin-dependent calcium ATPase (PMCA; Bindels et al 1991, Van Baal et al 1996, Hoenderop et al 2002, 2005, Bar 2009). This process is orchestrated by several hormones that regulate the expression and activity of the aforementioned proteins, including vitamin D3, androgens, and estrogens (Cai et al 1993, Hoenderop et al 2001, 2005, Van Abel et al 2002, Dick et al 2003, Meyer et al 2007, Oz et al 2007, Suzuki et al 2008, Hsu et al 2010, Yang et al 2011. Interestingly, the efferent ductules of roosters express high levels of vitamin D3 receptor (VDR), androgen receptor (AR), and estrogen receptors (ESR1 and ESR2 -also known as ERa and ERb respectively; Kwon et al 1997, Dornas et al 2007, 2008, Oliveira et al 2011a.…”

Roosters affected by epididymal lithiasis present local alteration in vitamin D3, testosterone and estradiol levels as well as estrogen receptor 2 (β) expression

“…High levels of vitamin D3 and E 2 are associated with increased calcium reabsorption, as they promote the expression of the transient receptor potential vanilloid channels (TRPV5), calbindin-D28k (CaBP-D28K), and plasma membrane calcium ATPase (PMCA), including in birds (Cai et al 1993, Hoenderop et al 2001, Van Abel et al 2002, Dick et al 2003, Kip & Strehler 2004, Oz et al 2007). These proteins participate on the entry of calcium within the cell, its diffusion throughout the cell cytoplasm to the basolateral membranes and calcium extrusion to the extracellular environment respectively (Hoenderop et al 2005).…”