Faroqui, Rashma

In these studies, we examined the effect of estrogen on renal inorganic phosphate (Pi) transport and the roles of its receptor isoforms (ERα and ERβ) and Klotho/FGF23 pathway in this effect in mice. Mice with null mutations (KO) of ERα or ERβ or Klotho and their wild‐type (WT) were placed in metabolic cages and injected daily with 17β‐estradiol or vehicle for 3 days. Food intake, urine volume and Pi excretion were measured daily. The results indicate that estrogen‐treated WT mice exhibited a significant increase in renal Pi wasting despite a reduction in food intake. Molecular studies showed a significant downregulation of NaPi‐IIa and NaPi‐IIc proteins with no change in their mRNA expression levels in the kidney. Estrogen‐induced reduction in food intake, phosphaturia and downregulation of NaPi‐IIa and NaPi‐IIc proteins were observed in ERβ KO but not in ERα KO mice. Further, Klotho KO mice exhibited a significant downregulation of NaPi‐IIa and hypophosphatemia in response to estrogen. In a cell line stably co‐expressing both ERα and ERβ, 24h of estrogen downregulated NaPi‐IIa protein, when cells are transiently transfected with a plasmid containing ORF‐3'UTR but not 5'UTR‐ORF of the mouse NaPi‐IIa transcript. In conclusion, estrogen causes phosphaturia in mice through the downregulation NaPi‐IIa and NaPi‐IIc protein abundance in the kidney. This effect is independent of Klotho/FGF23 pathway and is mediated through the activation of ERα. 3'UTR region of mouse NaPi‐IIa mRNA likely plays an important role in the inhibition of its translation by estrogen.

Estrogen treatment causes renal phosphate (Pi) wasting and hypophosphatemia in rats and humans; however, the signaling mechanisms mediating this effect are still poorly understood. To determine the specific roles of estrogen receptor isoforms (ERα and ERβ) and the Klotho pathway in mediating these effects, we studied the effects of estrogen on renal Pi handling in female mice with null mutations of ERα or ERβ or Klotho and their wild type (WT) using balance studies in metabolic cages. Estrogen treatment of WT and ERβ knockout (KO) mice caused a significant reduction in food intake along with increased renal phosphate wasting. The latter resulted from a significant downregulation of NaPi-IIa and NaPi-IIc protein abundance. The mRNA expression levels of both transporters were unchanged in estrogen-treated mice. These effects on both food intake and renal Pi handling were absent in ERα KO mice. Estrogen treatment of Klotho KO mice or parathyroid hormone (PTH)-depleted thyroparathyroidectomized mice exhibited a significant downregulation of NaPi-IIa with no change in the abundance of NaPi-IIc. Estrogen treatment of a cell line (U20S) stably coexpressing both ERα and ERβ caused a significant downregulation of NaPi-IIa protein when transiently transfected with a plasmid containing full-length or open-reading frame (ORF) 3′-untranslated region (UTR) but not 5′-UTR ORF of mouse NaPi-IIa transcript. In conclusion, estrogen causes phosphaturia and hypophosphatemia in mice. These effects result from downregulation of NaPi-IIa and NaPi-IIc proteins in the proximal tubule through the activation of ERα. The downregulation of NaPi-IIa by estrogen involves 3′-UTR of its mRNA and is independent of Klotho/fibroblast growth factor 23 and PTH signaling pathways.

We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPi-IIa and causes phosphaturia and hypophosphatemia in ovariectomized rats. In the present study, we examined whether E2directly targets NaPi-IIa in the proximal tubule (PT) and studied the respective roles of estrogen receptor isoforms (ERα and ERβ) in the downregulation of NaPi-IIa using both in vivo and an in vitro expression systems. We found that estrogen specifically downregulates NaPi-IIa but not NaPi-IIc or Pit2 in the kidney cortex. Proximal tubules incubated in a “shake” suspension with E2for 24 h exhibited a dose-dependent decrease in NaPi-IIa protein abundance. Results from OVX rats treated with specific agonists for either ERα [4,4′,4″;-(4-propyl-[1 H]-pyrazole-1,3,5-triyl) trisphenol, PPT] or ERβ [4,4′,4″-(4-propyl-[1 H]-pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT + DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa, along with significant phosphaturia and hypophosphatemia. Lastly, heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ERα and ERβ, but not in cells expressing either receptor alone. In conclusion, these studies demonstrate that rat PT cells express both ERα and ERβ and that E2induces phosphaturia by directly and specifically targeting NaPi-IIa in the PT cells. This effect is mediated via a mechanism involving coactivation of both ERα and ERβ, which likely form a functional heterodimer complex in the rat kidney proximal tubule.