Adenovirus‐delivered microRNA targeting the vitamin D receptor reduces intracellular Ca²⁺ concentrations by regulating the expression of Ca²⁺‐transport proteins in renal epithelial cells

Abstract

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. MATERIALS AND 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.

Roux-en-Y gastric bypass surgery reduces bone mineral density and induces metabolic acidosis in rats

“…An unexpected finding of our study was that renal calcium excretion was increased in RYGB rats; this was already seen in the initial phase of the study with intestinal calcium malabsorption and massive bone loss, but also later in the study. A decrease in intestinal calcium availability normally leads to 1,25(OH) 2 D-mediated upregulation of renal CALB1 and TRPV5 expression (53) to minimize urinary calcium loss. Despite higher 1,25(OH) 2 D levels, urinary calcium concentration and absolute calcium excretion were increased in RYGB rats throughout the study, and there was no change in renal CALB1 and TRPV5 expression when examined at study end.…”

Leukotriene B4 Enhances Innate Immune Defense against the Puerperal Sepsis Agent Streptococcus pyogenes

“…Fluorescence was measured with excitation at 494 nm and emission at 516 nm. The assay was performed using a Fluo4-NM Calcium Kit (Invitrogen) according to the manufacturer’s instructions and adapted according to previous works (47, 48). …”

Distal Convoluted Tubule

“…In addition, expression of calbindin-d28K and NCX1 are reduced (PMCA1b expression is unchanged) (192); Ca 2+ supplementation reversed these effects, and also increase PMCA1b expression. The final demonstration of the central importance of vitamin D in regulating expression of the renal transcellular pathway proteins comes from ablation of VDR in mice, which display reduced TRPV5 expression (507), or knockdown of VDR by microRNA in a rat cell line, which reduces expression of TRPV5, Calbindin D28K, NCX1 and PMCA1b (571). The observation that dietary Ca 2+ supplementation reverses the effects of both VDR and 1α (OH)ase knockout on transcellular pathway proteins suggests that dietary Ca 2+ regulates their expression independently of 1,25(OH) 2 D 3 .…”