Single Nucleotide Polymorphism And Expressive Regulation Of Vitamin D Receptor Gene In Genetic Idiopathic Hypercalciuria Rats

PART ONEEstablishment of Stablly Genetic Hypercalciuric Stone-forming Rats Model and Its Genetic CharacteristicsObjective To establish a colony of genetic hypercalciuria stone-forming(GHS) rats model which can stablely descend and detect its genetic charactertics in order to amassing the experience in establishing genetic rat model.Methods 40 male and 40 female adult Sprague-Dawley rats were placed in individual metabolic cages.Two successsve 24-h urine collections were then obtained to measure the urine calcium excretion.3—4 male and female rats with the highest Ca excretion were chosen for inbreeding to propagate the colony.Results Ratio of GHS rats was obviously increased from generation 4(37.5%) . Urine calcium excretion of more than 90% model rats after generation 7 achieved the level of GHS rats( ( > 1.5mg/24 h). Ca excretion of 12/15 male rats and 9/16 female rats from generation 7 were > 2SD above the mean of the normocalciuric control(NC) rats((1.12+0.36)^*mg/d, (1.15+0.24) ^â–²mg/d) on two successive 24-h urine collections. The significant difference occurred between two groups (F^* ＝27.10, P^*<0.01; F^â–² = 21.16, P^â–²<0.01) .The content of serum calcium,phosphorus and 1,25(OH)₂D₃ were similar(F<2.22, P>0.05).Conclusion Genetic hypercalciuric stone-forming rats model is established successfully.The genetic tendency occurs from generation 4 rats and their various characteristics can be stablely inherited from generation 7 rats. Being similar to human idopathic hypercalciuria, GHS rats are suitable for studying the mechanisms of idiopathic hypercalciuria development and urinary stone forming. PART TWOExpression of mRNA and Protein of Vitamin D Receptor in Genetic Hypercalciuric Stone-Forming(GHS) Rats DuodenumObjective To study the expression of mRNA and protein of Vitamin D receptor(VDR)in genetic hypercalciuric stone-forming (GHS) rats duodenum.Methods Semiquantitative RT-PCR and Western blot methods were used to detect theexpression of mRNA and protein of Vitamin D receptor in 6 genetic hypercalciuricstone-forming rats and 6 normocalciuric control(NC) rats duodenum.Results There was no difference in VDR mRNA expression(P >0.05) but significantdifference in VDR protein expression (P<0.05) among GHS rats and normocalciuriccontrol rats.Conclusion VDR protein expression is higher in GHS rats than in normocalciuriccontrol rats ,which may contribute to the intestinal calcium hyperabsorption.Apossible translational regulation mechanism exists during the process of VDR proteinsynthesis. PART THREEStability of mRNA of Vitamin D Receptor in GeneticHypercalciuric Stone-Forming Rats Duodenum and Effectivenessof Vitamin D on Expression of VDR mRNAObjective To study the stability of mRNA of vitamin D receptor(VDR) in genetichypercalciuric stone-forming(GHS) rats duodenum and effectiveness of Vitamin D onexpression of VDR mRNA .Methods Semiquantitative RT-PCR was used to determine the expression of VDRmRNA in 5 genetic hypercalciuric stone-forming rats and 5 normocalciuric control(NC)rats duodenum respectively before and after administration of 1,25(OH)₂D₃.Transcription termination by actinomycin D was used to detect half-life of VDR mRNAin GHS and control rats.Results There was no difference in VDR mRNA expression(P >0.05) among GHSand normal rats without administration of 1,25(OH)₂D₃ but significantdifference(P<0.05) by administration of 1,25(OH)₂D₃ after 6 h.Conclusion Vitamin D can increase the VDR mRNA expression in GHS ratsduodenum. Half-life of VDR mRNA is prolonged and VDR mRNA stability is increasedin GHS rats. PART FOURConstruction of Rat VDR Protein Expression Plasmid and Analysis of GHS Rats Duodenum VDR cDNAObjective To construct the expression plasmid of rat VDR protein and analyse the genetic hypercalciuria stone-forming(GHS) rats duodenum VDR cDNA sequence. Methods The coding sequence of rat VDR protein gene was amplified by RT-PCR.The PCR product was cloned into the eukaryotic expression vector pcDNA3.1/Zero(+).VDR cDNA sequences of 5 GHS rats and 4 normocalciuric control(NC) rats were analysed. Results A specific band of 2039 bp from PCR amplification was seen in gel electrophoresis.The sequence of the insert in the plasmid was identical to the published coding-region sequence of VDR gene. Differences in basepairs were discovered at three sites when the sequences of VDR cDNA from both normocalciuric and GHS rats were compared with the published rat intestinal VDR cDNA sequence. The first difference encountered at bp 256 is C instead of G; the second at bp 569 is G instead of A; and the third difference is at bp 1658, an A for a G. Alteration at bp1795,a G for A, was also found in GHS rats but not in NC rats.Conclusion The coding sequence of rat VDR protein gene was successfully cloned into the eukaryotic expression vector pcDNA3.1/Zero(+).Coding-region sequences of GHS rats and NC rats intenstial VDR gene are identical,but alteration of bp 1795 in GHS rats has not been found in NC rats.