遗传高钙尿结石大鼠肾组织钙敏感受体mRNA异常表达及其意义

Abnormal expression of calcium sensing receptor mRNA in kidney of rats with genetic hypercalciuric stone-forming and implication

目的观察肾钙敏感受体(CaSR)在特发性高钙尿症(IH)发病中的作用。方法雄性遗传性高钙尿结石(GHS)大鼠和正常野生型SD大鼠各6只,相同的饮食和饲养条件下,检测各组大鼠血Ca^(2+)、P5^+、1,25(OH)_2D_3和连续2个24 h尿Ca^(2+)的水平,使用半定量逆转录-聚合酶链反应(RT-PCR)检测肾组织钙敏感受体mRNA表达,PCR产物纯化后进行DNA直接测序。结果两组大鼠血Ca^(2+)、P5^+、1,25-(OH)_2D_3水平相似,均在正常范围内,而GHS组平均24 h尿Ca^(2+)排泄量为正常对照(NC)组的3.36倍[分别为(2.66±0.64)、(0.79±0.06)mg/d],两组比较差异有统计学意义(t=5.82,P<0.01);GHS组CaSR mRNA平均相对吸光度(A)值为1.354±0.108,NC组为0.602±0.218,差异有统计学意义(t=6.19,P<0.01);CaSR基因第7外显子区域cDNA测序结果与Genebank(Accession:NM-016996)公布的碱基序列比较,GHS组有4只大鼠存在第3072位碱基G/A替换,使第1024位谷氨酸(Glu)变为赖氨酸(Lys),其余2只和NC组均与Genebank公布的碱基序列一致。结论肾组织CaSR mRNA高表达及其第7外显子Glu1024Lys替换可能是特发性高钙尿症发病的分子机制之一。

Objective To study the effect of calcium sensing receptor （CaSR） on idiopathic hypercaleiuria formation. Methods The enrolled included 6 male rats with genetic hyperealeiurie stoneforming （GHS） and 6 male normal wild-type SD rats subject to the same diet in the same feeding condition. The level of serum calcium, phosphorus, 1,25 （OH）2D3 and two successive 24-h urinary calcium were examined respectively. Semi-quantitative RT-PCR method was used to detect the expression of CaSR mRNA in renal tissues. Purified PCR products were directly DNA sequenced. Results The levels of serum calcium,phosphorus and 1,25 （OH）2D3 were similar in the GHS group and normal control （NC） group. However,the average content of 24-h urine calcium excretion in the GHS group was 2, 36 times of that in the NC group [ （ 2.66 ± 0.64 ） and （ 0.79 ± 0.06） mg/d respectively） ] （ t = 5.82, P 〈 0.01 ）. The average relative absorbanee （A） value of CaSR mRNA in the GHS group was significantly higher than that in the NC group [ （ 1. 354 ±0. 108） and （0.602 ±0. 218） respectively] （ t =6.19 ,P 〈0.01 ）. The comparison of the CaSR seventh exon sequence with that published in the Genebank（Aeeession：NM-016996） databases revealed that the substitution of A for G at basepairs 3072 which resulted in Lysine instead of Glutamine at the 1024th amino acid site was found in 4 GHS rats. The polymorphisms site was not found in the other two GHS rats and total NC group rats. Conclusion The high expression of CaSR mRNA and the substitution of Lysine for Glutamine at the 1024th amino acid site of the CaSR seventh exon in the renal tissues may be one of the molecular mechanisms of idiopathic hyperealeiuria formation.