The Effect Of High Concentration Calcium On Cell Injury And OPN Expression In Rat Renal Epithelial Cells

Objective: To determine the effects of high concentration calcium on cell injury andOPN expression in rat renal epithelial cells(NRK cells),and discuss the effects ofhypercalciuria in calcium-containing kidney stone formation and mechanisms underlyingsuch effects.Methods: NRK cells were cultured3,6and24hours in calcium concentration1mg/ml、2mg/ml and3mg/ml DMEM complete medium respectively(group1,2,3)andcontrol group were treated with DMEM complete medium.Proliferation activity of NRKcells were examined by Cell Counting Kit-8(CCK-8) test;OPN concentration in culturesupernatant was detected by enzyme-linked immunosorbent assay(ELISA);Apoptosis rateof NRK cells were examined by Flow cytometry;OPN gene expression and protein levelswere detected by Western Blot and real time RT-PCR respectively.Results:(1) OD values in control group and group1,2,3were3.147±0.060、2.844±0.309、2.321±0.089、1.897±0.611(after3h);3.472±0.097、2.878±0.127、2.545±0.06、2.222±0.047(after6h);3.851±0.096、1.831±0.076、0.669±0.093、0.569±0.004(after24h),which were measured via CCK-8test.The differences had statistical significance ingroup1,2,3compared with the control group at different time points(P<0.05),except thedifference3h in group1(P>0.05).At different time points,the differences also had statisticalsignificance within group1,2,3(P<0.05),except the difference between3h and6h withingroup1(P>0.05).(2) Apoptosis rate in control group and group1,2,3were2.36%、3.07%、4.53%、5.40%(after3h)，2.36%、4.75%、7.28%、7.83%(after6h)，2.36%、41.76%、56.84%、62.96%(after24h).(3) OPN concentration in culture supernatant(μg/ml) were10.461±1.573、14.820±1.213、17.150±0.841、19.893±0.611(after3h)，10.461±1.573、23.791±1.518、26.307±0.876、28.607±0.733(after6h)，10.461±1.573、26.882±1.100、28.948±1.818、24.377±0.641(after24h).The differences had statistical significance in group1,2,3at different time points compared with the control group(P<0.05). At the sametime point,the differences also had statistical significance within group1,2,3(P<0.05),except the difference between6h and24h within group2(P>0.05).(4) OPNexpression levels of group1,2,3after3,6h were higher than that of the control group,andthe differences had statistical significance(P<0.05).At the same time point, the differenceswithin group1,2,3had statistical significance(P<0.05),except the difference betweengroup2and group3after3h(P>0.05).(5) OPN mRNA relative expressing quantity ofgroup1,2,3after3(6h)were2.803(9.989)times,5.195(12.999)times,8.380(27.929)timesthat of the control group respectively, which were increased apparently compared with thecontrol group.Conclusions: High concentration calcium can inhibit NRK cells proliferationactivity,induce apoptosis,up-regulate expression of OPN mRNA and increase the secretionof OPN in NRK cells. It is presumed that hypercalciuria play a role in kidney stoneformation via inducing cell injury,up-regulating expression of OPN gene and promotingadhesion of crystals to renal tubular epithelial cells.