| Part1Construction of adenovirus vector-delivered miRNA targeting rat VDR gene and preparation of the adenovirusOBJECTIVE:To design and select the miRNA sequences which can effectively depress the expression of VDR gene, and construct a recombinant adenovirus vector expressing VDR miRNAMATERIALS AND METHODS:Based VDR gene mRNA sequence, four pair of miRNA oligonucletide (SR-63-1-SR-63-4) were designed and synthesized. The oligonucletide were annealed into double strands, then inserted into miRNA expression vector pcDNATM6.2-GW/EmGFPmiR, respectively. The vectors were transformed into competent E.coli DH5αcells. The positive clone was screened with colony polymerase chain reaction and identified by sequencing. VDR cDNA in rat PC-12 cell was cloned into vector pcDNA3.1 to construct the high expression vector containing VDR. The miRNA vector and the high expression vector were co-transfected into 293A cells. Real-time PCR and western blot were used to determine the interference efficacy. The pcDNATM 6.2-GW/EmGFP-VDR-miR vector with the highest RNA interference efficacy was recombined with pAD/CMV/V5-DEST by BP/LR reaction and identified by sequencing. The recombinants were linearized with Pac I and transfected into 293A cells to produce adenovirus. The titer was determined by immune methods.RESULTS:The miRNA oligonucletide sequence was successfully inserted into the plasmid pcDNATM6.2-GW/EmGFPmiR. The third plasmid (SR-63-3) revealed the highest interference efficacy which VDR mRNA knockdown reached 83%. It was confirmed by sequencing that the adenovirus vector was successfully constructed. The mean titer of the positive and negative adenovirus is 1.44×1011ifu/ml and 7.56×1010ifu/ml, respectively。 CONCLUSIONS:The adenovirus vector-delivered miRNA targeting rat VDR was successfully constructed, which could inhabit the mRNA and protein expression of VDR, and the high titer of adenovirus was prepared. Part 2Adenovirus-delivered microRNA targeting VDR reduces intracellular Ca2+ concentration by regulating the express of Ca2+ transport proteins in NRK cellsOBJECTIVE: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. NRK cells (Cellbank, China) were infected with the adenovirus and the cells were collected at 0,48,72,96h after infection. The mRNA and protein levels of VDR and VDR-dependent epithelial Ca2+ transport proteins were detected using real time PCR 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, TRPV5, Calbindin-D28k and PMCAlb in infected NRK cells was significantly lower at 72,96h after infection than that in control cells. No significant difference was found between 2 groups with regard to the mRNA and protein level of TRPV6 and NCX1. Furthermore, VDR knockdown results in a decrease in intracellular Ca2+ concentration in NRK cell lines.CONCLUSIONS:Our study demonstrates that VDR can postively regulate the mRNA and protein expression of TRPV5, Calbindin-D28k and PMCAlb, but not of TRPV6 or NCX1, 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 kidney on hypercalciuria and the mechanisms underlying need to be further addressed. Part 3Effect of silencing VDR gene in kidney on renal epithelial calcium transporter proteins and urinary calcium excretion in genetic hypercalciuric stone-forming ratsOBJECTIVE:Using genetic hypercalciuric stone-forming (GHS) rats, to address the molecular mechanisms that vitamin D receptor (VDR) in kidney may contributes to decreased renal calcium reabsorption in idiopathic hypercalciuria.MATERIALS AND METHODS:Twenty-four eighteen generation male GHS rats with body weight of 200 to 280 g and Twenty-four Sprague-Dawley male rats with body weight-and age-match were used for these studies. GHS and NC rats were divided into six groups with four of each, respectively. Each kidney for group 1 to 4 rats were injected with 2.5×1010 ifu adenovirus expressing VDR-miRNA via renal venous through surgery. Group 5 received empty adenovirus injection with the same amount through the same transduction methods as negative control. Group 6 received vehicle injection with the same volume as sham-operated control. Groups 1 to 4 were killed 3,7,14 and 21 days after injection, respectively. Group 5 and 6 were killed 14 days after injection. Kidney cortical tissue was removed for subsequent analysis including real time PCR and western blot to quantify VDR, CaSR and epithelial Ca2+ transporters mRNA and protein expression levels, respectively. The latter included TRPV5, TRPV6, Calbindin-D9k, Calbindin-D28k, NCX1 and PMCAlb. Two successive 24h urine were collected before being killed, and blood samples were taken. Urine calcium (UCa) and serum Ca2+, phosphorus and PTH levels were measured on an Abbott Aeroset autoanalyzer. Serum 1,25(OH)2D3 was measured by enzyme-linked immunosorbent assay.RESULTS:Basal levels of epithelial Ca2+ transporters were detected in kidney in GHS and NC rats. The mRNA and protein expression levels in kidney tissues of VDR, Calbindin-D28k, and CaSR were significantly higher in GHS rats than in NC rats. In contrast, the expression levels of TRPV5, TRPV6, Calbindin-D9k, and PMCAlb were significantly lower in GHS rats than in NC rats. Each kidney was injected with adenovirus vector via renal venous through surgery. Parallel GFP expression allowed evaluation of infection efficiency in renal tubular epithelia. It showed the high infection efficiency in kidney in vivo throughout days 3-21. A significant VDR mRNA knockdown with markedly lower VDR protein level was observed after 3 days of infection compared with control and negative groups in GHS(P<0.01). The reduction reached the lowest level after 21 days (P<0.01). This effect was not found in NC rats. VDR knockdown in kidney caused significant increase in renal TRPV5 mRNA expression level in GHS rats compared with control and negative groups. The similar negative regulation of VDR on the renal mRNA expression of NCX1 and Calbindin-D9k was observed in GHS rats. The renal mRNA expression levels of Calbindin-D28k, PMCAlb and CaSR were not altered in response to VDR depletion. Western blot analysis consistently demonstrated the similar regulation in the protein abundance as in the mRNA expression levels. The proteins expression of TRPV5, Calbindin-D9k and NCX1 were negatively regulated, while the renal proteins levels of Calbindin-D28k, PMCAlb and CaSR were unaltered. However, this effect was not detected in NC rats. GHS rats excreted significantly more UCa in days 7-21 after VDR knockdown. Although UCa excretion tended to increase in NC rats and in control and negative groups in GHS ratrs after VDR knockdown or injection of vehicle and negative vector, no significant difference was found. Serum Ca2+, phosphorus, PTH and 1,25(OH)2D3 levels did not significantly changed in response to VDR depletion during different time periods in GHS and NC rats.CONCLUSIONS:Our findings suggest reduced renal Ca reabsorption may be associated with down-regulation of TRPV5 by VDR in GHS rats. This appears to be able to explain the mechanisms that elevated VDR levels contribute to the hypercalciuria. However, the details underlying this effect need to be further elucidated.
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