Abstract
Background and Aims
Hyperparathyroidism (SHPT) which caused by disorders of PTH metabolism is one of the most common complications in CKD patients. In the progression of SHPT, expression of CaSR in the parathyroid gland decreases, which impairs the regulation of PTH secretion. It is technically challenging to precisely manipulate PTH secretion in hyperplastic human parathyroid tissue. As an emerging technology, optogenetics allows the reversible control of neuronal activity and cellular precesses using lights. In this study, we establish an optogenetic approach that bypasses CaSR to regulate the level of PTH secretion in human parathyroid cells. It is hoped to provide a new idea for the prevention and treatment of SHPT.
Method
First, we isolated and identified human parathyroid gland by SHPT patients. The primary cell culture of chief cells were performed and identification. After that, the cells were transfected with the lentivirus carrying ChETA-eYFP or eYFP gene. Lights stimulation of the cultured human parathyroid cells was performed after ChETA expression. The PTH level was measured by ELISA before and after stimulation. Meanwhile, Fura-2/AM was loaded to normal or ChETA expressing chief cells for calcium imaging by microscope.
Results
The parathyroid glands of three SHPT patients were obtained after total parathyroidectomy (Fig.1A). Hematoxylin & Eosin (HE) staining was performed. The tissue showed typical histological characteristics of SHPT: asymmetric enlargement of the chief cells and the nodular distribution (Fig.1B).
Parathyroid cells were cultured by primary culture method. By the 7th day of cultivation, the chief cells were grown in uniform size and aggregation state (Fig. 2A). The cells were stained with immunofluorescence, results showed that most of the isolated cells expressed parathyroid hormone (PTH), calcium-sensing receptors (CaSRs) and Vitamin D receptors (VDR) (Fig. 2B).
We used lentivirus carrying the CMV-ChETA-eYFP construct to transfect cultured human parathyroid cells. Comparing with eYFP, most of the chief cells were labeled and expressed green fluorescence successfully after 48 hours (Fig.3).
We constructed a blue light-emitting diode to stimulated the transfected human parathyroid chief cells for 0.5h (20 Hz with stimulus intervals of 50 ms). Culture medium was collected before and after stimulation for 1h and 6h, and PTH was determined by ELISA. The results showed that there was no significant difference in PTH level between ChETA group and eYFP group before stimulation, however, PTH decreased significantly after 1 hour of stimulation in ChETA group, and gradually returned to the control group after 6 hours (Fig.4).
After that, we used a calcium fluorescence assay to reveal light-induced changes of intracellular Ca2+. In ChETA group, blue light stimulation led to a significant increase in fluorescent signals, but a significant decrease when turned off, and the signal strength increased again when it was turned on in a rhythmic manner. But the fluorescent signal of eYFP group remained stable throughout the whole process (Fig.5).
Conclusion
We used optogenetics to bypass CaSR rhythmic suppression of PTH secretion in human parathyroid cells. It is hoped that this study will provide new strategies for the treatment and prevention of SHPT
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