| [Objective]Study the expression of calcium transporter-related proteins and the changes of urinary calcium excretion in renal tissues and the possible mechanism of RNA interfering with the formation of kidney calcium-sensing receptor(CaSR)in GHS rats.[Method]1.By repeatedly selecting male and female rats with the highest excr etion of urinary calcium,they interbred with each other to produce the next ge neration.Six generations later,the GHS rat model was established.2.Twenty ma le GHS rats and twenty Sprague-Dawley male rats(Normal control rats,NC)w ere randomly divided into 5 groups.Each kidney for group 1 to 4 rats were inj ected with lentivirus expressing CaSR-miRNA via renal venous through surgery.The fifth group received Normal saline intervention as control group.The rats in the 1-4 group were sacrificed at 3 days,7 days,14 days and 21 days after the injection of virus,and the rats in the 5 group were sacrificed at 21 days after the injection,and both kidneys were removed.Before the rats were sacri ficed at the above five time points,the two 24h calcium excretions of the rats in each group were measured by the automatic biochemical analyzer(calcium excretion was measured at each time point in the control group),and the 24-hour calcium excretion was calculated.The mRNA and protein expression level s of calcium transportation-related proteins(CLDN14,TRPV5,NHE3,AQP2,N KCC2)in rat renal epithelial cells were detected by RT-PCR and Western-Blot.[Results]:1.The GHS rats model was successfully constructed.2.Compared wit h control group rats,GHS group rats were treated with CaSR-miRNA in vivo interference for 3-21 days,and 24-hour urinary calcium excretion was reduced.and the difference was statistically significant(P<0.05).The 24-hour urinary c alcium excretion was slightly reduced in the NC group compared with the cont rol group(P>0.05).3.The mRNA expression levels of CLDN14,TRPV5,NH E3,AQP2 and NKCC2 were detected by RT-PCR after in vivo interference.GH S rats groups:compared with the control group,the mRNA expression levels o f CLDN14 and NHE3 were down-regulated within 3-21 days,and the differenc es were statistically significant(P<0.05).The mRNA expression levels of TRP V5,AQP2 and NKCC2 were up-regulated within 7 to 21 days,and the differe nces were statistically significant(P<0.05).NC group rats:compared with the control group,the mRNA expression levels of the above five genes were not s ignificantly changed,and the difference was not statistically significant(P>0.05).4.After in vivo interference with CaSR-miRNA interfering viruses,Western-Blot was used to detect the expression levels of CLDN14,TRPV5,NHE3,A QP2 and NKCC2 proteins.GHS rats groups:compared with the control group,the expression level of CLDN14 protein was down-regulated from 3 to 21 da ys,and the expression level of NHE3 protein was down-regulated from 3 to 14 days,the differences were statistically significant(P<0.05).The expression le vels of TRPV5,AQP2 and NKCC2 proteins were up-regulated within 7 to 21 days,with statistically significant differences(P<0.05).NC group rats:compare d with the control group,there was no significant change in the expression lev els of the above five proteins,and the difference was not statistically significa nt(P>0.05).[Conclusion]:1.By repeatedly selecting male and female rats with the highest excretion of urinary calcium,they interbred with each other to pro duce the next generation.After 6 generations,the genetic hypercalciuric stones forming(GHS)rats model can be established.2.CaSR-miRNA interferes with the virus in vivo to interfere with genetic hypercalciuric stones forming(GHS)rats,the 24-hour urinary calcium excretion decreased after interference,while no such phenomenon was observed in NC rats.3.After in vivo interference b y CaSR-miRNA interfering viruses,mRNA expression levels of CLDN14 and NHE3 genes were down-regulated in GHS group rats,while mRNA expression levels of TRPV5,AQP2 and NKCC2 were up-regulated in GHS rats.The mR NA expression levels of the above genes in NC group rats were not significant ly changed.4.After in vivo interference by casr-mima interfering virus,the expr ession levels of CLDN14 and NHE3 proteins in GHS group were down-regulat ed,while the expression levels of TRPV5,AQP2 and NKCC2 proteins were u p-regulated,while the expression levels of the above proteins in NC group wer e not significantly changed. |
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