Adenovirus-delivered microRNA targeting the vitamin D receptor reduces intracellular Ca²⁺ concentrations by regulating the expression of Ca²⁺-transport proteins in renal epithelial cells.
What’s known on the subject? and What does the study add? Experimental data have shown that VDR overexpression in the duodenum and kidney cortex is a biological characteristic of genetic hypercalciuric stone-forming rats (GHS rat), and a link between idiopathic calcium stone formation and the microstatellite marker D12S339 (near the VDR locus) has been proven in humans. Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca²⁺ concentration in NRK cell lines. The effect of the elevated VDR level in the kidney on hypercalciuria and the underlying mechanisms need to be further addressed.


OBJECTIVE
• To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects.


METHODS
• The adenovirus vector-delivered microRNA targeting rat VDR was constructed. We infected the normal rat kidney epithelial cell line NRK (Cellbank, China) with the adenovirus and then collected the cells at 0, 48, 72, 96, 120 h after infection. The mRNA and protein levels of VDR and VDR-dependent epithelial Ca2+ transport proteins were detected using real-time polymerase chain reaction and Western blot assays, respectively. • Fluorescent Ca²⁺ indicator Fluo-4 NW (Fluo-4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500-IX71, Japan) were used to detect the cytosolic free Ca²⁺ concentration at different time points after infection.


RESULTS
• The mRNA and protein level of VDR, transient receptor potential vanilloid receptor subtype 5 (TRPV5), calbindin-D28k and plasma membrane Ca²⁺-ATPase (PMCA1b) in infected NRK cells was significantly lower at 72 and 96 h after infection than that in control cells. • There was no significant difference between the two groups in the mRNA and protein level of TRPV6 and the Na⁺/Ca²⁺-exchanger (NCX1). • Furthermore, VDR knockdown results in a decrease in intracellular Ca²⁺ concentration ([Ca²⁺]i) in NRK cell lines.


CONCLUSIONS
• Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in [Ca²⁺]i in NRK cell lines. • The effect of the elevated VDR level in the kidney on hypercalciuria and the mechanisms underlying need to be further addressed.
Xi
Qilin
Q
Department of Urology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, HuBei, China.
Wang
Shaogang
S
Ye
Zhangqun
Z
Liu
Jihong
J
Yu
Xiao
X
Zhu
Zhaowei
Z
Su
Shiqiang
S
Bai
Jian
J
Li
Cong
C
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods*; D006935:Hypercapnia / Q000235:genetics / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics*; D018613:Microscopy, Confocal; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000096:biosynthesis / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019459:Urothelium / Q000378:metabolism* / Q000473:pathology
D015220:Calcium Channels; D035683:MicroRNAs; D012367:RNA, Viral; D018167:Receptors, Calcitriol; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium