Effects Of Simvastatin On EPCR And TM In Human Renal Proximal Tubular Epithelial Cells (HKC)

Objective: Besides renal function, patients with chronic nephrosis often face the threat of hemorrhage or blood coagulation which may lead to subsequent hemorrhage or thrombosis. Therefore, it's of great significance to look into the pathogenesis of the two conditions for the sake of prevention and better treatment. It also will affect the prognosis of chronic nephrosis.Anti-coagulative system plays the key role in preventing the body from hemorrhage or blood coagulation. EPCR is one of the important members of this system. EPCR (Endothelial ProteinC Receptor), a kind of purified protein, is a new member of family of ProteinC, it serves as an indicator of VEC's (vascular endothelial cells) function. It was observed in lab test that combining EPRC with ProteinC could highly activated the latter, the activation rate reached up to 4-20 times compared with no combination. And as a result, the highly activated ProteinC inhibited the thrombosis by means of inactivating the clotting factors in the endothelial cells. In addition, it was recorded that the combining of EPCR and ProteinC strengthened human body's anti-inflammatory function: after the injection of activated ProteinC, the mortality rate of severe sepsis among patients went down clearly.Some inflammatory factors such as TNF-αand IL-1βcan down-regulate mRNA of EPCR, and the expression of EPCR will be suppressed by such down-regulation. We will observe the expression of EPCR in HKC by means of FCM(flow cytometry) and RT-PCR, discover the effects of TNF-αon the expression of EPCR in HKC and find the effects of simvastatin on the expression of EPCR and TM. After this, we will approach the action of EPCR and TM in the kidney tubules interstitial fibrosis. The following are about the research and its results. PartⅠEffects of TNF-αon the expression of EPCR in HKC.Method: First the cell cultures of HKC was done respectively, then measure the amount of expression of EPCR in them separately by means of FCM(flow cytometry). After that, add TNF-α(0ng/ml,2ng/ml,10ng/ml, and 20ng/ml each time)into them and measure the expressions of EPCR again after 3,6, 12and 24hours successively. At last make comparisons among them to identify the effects of TNF-αon the expression of EPCR.Results:Ⅰ. The High expression of EPCR in HKC was seen. It became 97.7%.Ⅱ. 2ng/ml TNF-αwas able to down-regulate EPCR mRNA(p<0.01)clearly. The stronger the density 0f TNF-αwas, the more obvious the suppression effect was.Ⅲ. Adding TNF-α3 hours later, the down-regulation effect was seen clearly. The suppression effects was more obvious when the time later.Conclusion:I. The expression of EPCR in HKC is high.II. the originally high expression of EPCR in HKC was down-regulated by adding TNF-α, and the longer the administration time and the stronger the density 0f TNF-αwas, the more obvious the suppression effect was.III. The down--regulation of the expression of EPCR in HKC is possibly one of the mechanisms through which inflammatory factors such as TNF-αlead to kidney lesion. EPCR can be one of the monitoring indexes that reflect the level of the pathological of kidney tubules.PartⅡEffects of simvastatin on the TNF-α's down-regulation of EPCR and TM.Method: First prepare the HKC culture. When the cells grew to 80% confluence, they were divided into three groups. The first group is the positive control group. TNF-α (20ng/ml) was added into it as the cell culture medium. Wait for 24hours and observe the the expression of EPCR and TM. The second group is the blank controller group in which no medicine was added. The third group was the experimental group. This group was divided ito three teams. Simvastatin(0.1μmol/L) was added into the first team as the culture medium. Wait for 3,6 and 12 hours each, then substitute TNF-α(20ng/ml) as the new culture medium for Simvastatin(0.1μmol/L) and wait for another 24 hours. The methods of the second and the third teams were the same as the first, but the density of simvastatin was 1μmol/L in second team and 10μmol/L in the third team. In addition, The whole experiment setting was duplicated 5 times and each time the experimental procedures were repeated 3 times. Finally, measure the expression of EPCR mRNA in every group to ascertain the effects of Simvastatin on TNF-α's down-regulation of EPCR and TM.Results:I. In the positive control group(20ng/ml TNF-α, 24hours), the expression of EPCR and TM in HKC was clearly suppressed(p<0.01).II. In all groups , the suppression effect of TNF-αon the expression of EPCR and TM in HKC was diminished by Simvastatin . The action of Simvastatin was affected by the dosage and administration time of Simvastatin. The more the dosage was, the more clearly the action was. The longer the time was, the more clearly the action was.Conclusion:Ⅰ.The down--regulation of the expression of EPCR in HKC is one of the mechanisms, through which inflammatory factors such as TNF-αlead to kidney lesion.Ⅱ.Simvastatin was capable of diminishing the toxic effect of inflammatory factors and regenerating the expression of anti-inflammatory factors. It was able to protect kidney. This is probably one of the mechanisms through which simvastatin inhibit renal interstitial fibrosis.