Conditionally Immortalized Human Proximal-Tubular Epithelial Cells Isolated from the Urine of a Healthy Subject Express Functional Calcium- Sensing Receptor (CaSR)

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Abstract

Background: The calcium-sensing receptor (CaSR) is a G protein coupled receptor, which plays an essential role in regulating Ca2+ homeostasis. Here we show that conditionally immortalized proximal tubular epithelial cell line (ciPTEC) obtained by immortalizing and subcloning cells exfoliated in the urine of a healthy subject expresses functional endogenous CaSR. Methods: Primary cells isolated from human urine sediment were infected with SV40T and hTERT vectors. Subconfluent cell layers were transferred to 33°C and selected by antibiotics for 15 days. Cells were subcloned and expanded to 70% confluence at 33°C. After maturation at 37°C for 10 days, the cloned cells were used. Results: The obtained ciPTEC cells expressed ZO-1 protein and aquaporin 1 thus confirming their epithelial and PT origin respectively. The expression of the endogenous CaSR in ciPTEC was confirmed by Western blotting revealing the immunodetection of both forms at 130 and ~200 kDa, corresponding to the monomeric and mature receptor. Of note, functional studies with Fura2-AM indicated that the physiological agonist, calcium (Ca2+), and the calcimimetic NPS-R568, induced a significant increase in cytosolic calcium, proving a high sensitivity of the endogenous receptor to low concentrations of its agonists. Cytosolic calcium levels were 46.2±2.22% (vs ATP 100%) after stimulation with 2.5μM Ca2+ and to the 37±1.76% (vs ATP 100%) after stimulation with 2.5μM NPS-R568. Calcium depletion from the ER using CPA (cyclopiazonic acid) abolished the increase in cytosolic calcium elicited by NPS-R568 confirming the origin of calcium exit from intracellular stores. Conclusions: We conclude that human proximal tubular ciPTEC cells express functional CaSR and respond to its activation with a release of calcium from the ER. These cell lines represent a valuable tool for research into the disorder associated with gain or loss of function of the CaSR by producing cell lines from patients. 

Autore Pugliese

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  • VALENTI G.;TAMMA G.;SVELTO M.;RANIERI M.

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Anno di pubblicazione

2013

ISSN

1046-6673

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