Raloxifene Intervention On 1-α-hydroxylas Of Cadmium-induced Renal Injury

Objective: In this research, extract and culture SD rat renal tubular epithelial cells,also,establishment the cell model that exposure to CdCl2 and use RAL to intervene, observe the survival rate of cells exposure to CdCl2 and under the intervene of RAL after 24 h, also, observe the effect of RAL on the 1-α-hydroxylase of cells induced by CdCl2, for the new treatment of osteoporosis caused by cadmium.Methods:Selecte 10 newborn SD rats, use Percoll density gradient to purified and centrifugation renal tubular epithelial cells of SD rat, use morphological to identificate the extracted cells. Use low glucose medium which include 15% FBS to culture the primary culture of the cells and 10% FBS to culture the subculture. All the experiment cells were used in third generations.Use different dose of CdCl2 to intervene cells and evaluate the survival after 24 hour by MTT.Use same dose of CdCl2 and different dose of RAL to intervene cells and evaluate the survival after 24 h by MTT. The test group, group A:(the blank group), use 10 ml DMEM to cultured cells; group B(CdCl2 group): use 10 ml CdCl2(20μmol/L) to intervene cells; group C(RAL group): use 10 ml CdCl2(20μmol/L)and RAL(0.1μmol/L) to intervene cells.collect cells after 24 hour. RT-PCR:1. Use Trizol method to abstract total RNA of each group;2. RT:Use Tiangenn kit to RT total RNA;3. Amplified fragment:RTPCR;4. test the mRNA expression levels of 1-α-hydroxylase in each group. Use SPSS 18.0 statistical software to analysis research data, use the x ±s to describe measurement data,use t test to comparie between the two group,use one-way ANOVA to comparie among groups, the difference was statistically significant if P<0.05.Results: the extracted primary cells showed a uniform spherical shape before adherent, most cells attached completely after culture 2 day and 4~5 day completely adherent, the cultured cells is large and linked closely between cells,Integrated into the film or multilayer growth. Nuclear was oval,large and common have multiple nucleolus, multilateral cobblestone paving. consistent with other literature; In group A, the cells attached closely and grow well; In group B,the cells attached not closely and a great quantity of cells lose its normal morphological, a great quantity of cells death; compared with group B, the cells of group C attached more closely and fewer cells change its normal morphological. MTT test show: The cell survival rate of each CdCl2 groups were reduced significantly compared to the control group after 24h(P﹤0.01),Moreover,it was inversely proportional to CdCl2 dose, 20μmol/L CdCl2 can reduce the survival rate from 100.00% to 51.82% after 24h; the cell survival rate of each RAL groups were raise significantly compare to the CdCl2 group(P﹤0.01), 0.1μmol/L RAL can raise the survival rate from 100.00% to 168.85% after 24 h. RT-PCR test: the mRNA expression level of 1-α-hydroxylase in RAL group(group B) was reduced significantly compared to group A(P﹤0.01),and was raise significantly compared to the CdCl2 group(group B)(P﹤0.05).Conclusions: CdCl2 is toxic to renal tubular epithelial cells and it is a dose-dependent relationship. Raloxifene can protect the renal tubular epithelial cells that exposed to CdCl2, and the effect is different with different dose. Raloxifene have effect on the 1-α-hydroxylase.the changing of 1-α-hydroxylase is caused by the damage of tubular epithelial cells,which is induced by CdCl2. Probably,it can regulate calcium and phosphorus metabolism by this way and then intervene osteoporosis that caused by CdCl2,which can establish new foundation for the treatment of osteoporosis caused by CdCl2 in clinical.