A Study Of Traditional Transient Receptor Potential Channel6’s Effect And Mechanism In Regulating The Renal Tubular Epithelial Cell’s Necroptosis In Renal Ischemia-reperfusion Injury

Study BackgroundIschemia-Reperfusion Injury (I/R) is a phenomenon that tissue and organ injuryaggravated after blood flow recovered on the basis of ischemia, which generally happenedin clinical work. In spite of diverse theories, such as oxygen abnormality, calciumabnormality, PH abnormality and so on, its complete mechanism hasn’t been interpretedtotally till now. Due to its physical structure and function, kidney is easily influenced byischemia and anoxia, thus acute kidney injury (AKI) often happened because ofischemia-reperfusion. AKI is a common emergent and intensive clinical symptom referringto multi-disciplines. In renal transplant, nephron-sparing surgery, renal artery stenosis,hydronephrosis, embolism disease, accidental or iatrogenic injuries and so on, AKI causedby I/R is one of inevitable injuries, and the renal tubular epithelial cell injury is the mainreason of renal I/R. Because its injury mechanism hasn’t been interpreted completely, therehaven’t been effective methods of clinically preventing and curing I/R yet. Therefore,discussing renal I/R mechanism has become a significant topic in correlated fields. Strivingto find an effective measure of relieving renal I/R is bound to produce a greater economicand social benefit.The traditional view thought: apoptosis is a cell’s active process of self-regulation, andnecrosis is a relatively irreversible process, which is hard to be stopped or inversed bypharmacological interference. However, at least fairly some part of necrosis was found to belike apoptosis, which was started by specificity factors, and named as “necroptosis”according to a certain access and process. The finding of necroptosis made it possible toregulate cellular necrosis process and have an important influence on life science researchand clinical treatment of diseases. Necroptosis has been confirmed to exist in renalI/R. Andnot Caspase blocker zVAD, but necroptosis’specific inhibitor necrostatin-1was found to improve renal I/R of rats’ model notably. Thereby it is concluded that necroptosis in renalI/R may play the more important role than apoptosis. Necroptosis was regulated bydeacetylase sirtuin-2. Deacetylation of sirtuin-2was the necessary condition of RIP1andRIP3’s direction interaction. Meanwhile enzymatic activity of sirtuin-2depended on the levelof its cofactor NAD+. As we know, the decrease of NAD+activity was related to cellenergy deficiency and oxidative stress, which indicated deacetylation in the process ofnecroptosis was regulated by metabolism, hence it could conclude that necroptosis was alsoregulated by the metabolism factor. The metabolism factor played an significant role inrenal I/R: in the acute phase of renal ischemia, NAD+would exhaust due to anoxia andNAD+level would be compensated quickly after reperfusion. It may explain the reasonwhy there were not extensive cell deaths at first in the process of ischemia, but afterreperfusion there were. Maybe it is because: when reperfusion happened, NAD+level mightobtain supplement again, which made sirtuin-2activity increase, then RIP1and RIP3couldinteract with each other, thus promoting necroptosis to happen. At the same time,necroptosis was always accompanied by the intense inflammatory reaction, which would furtherlead to cell injury by the waterfall effect. It opened a window of chance for us: the cell’s necroptosiswas blocked before reperfusion initiation in order to stop I/R.TRPC6is a member of the traditional transient receptor potential channel (TRPC)family, located on cation channel of cell membrane. After activated, it mainly mediatedcalcium ion inflow. Signal transduction outside and inside the cell was realized by thedownstream signal transduction channel regulated by calcium. Some researches haveproved TRPC6could promote neuron cells to develop and exist. Its expression and NADPoxidase regulated by the internal metabolism factor，which was related to reactive oxygenspecies activity. There are some envidence that TRPC6played a key role in I/R of brainischemia, retina and lungs. TRPC6could be activated by reactive oxygen species and NADPoxidase regulated by the metabolism factor. According to the preliminary experiments, wefound its expression in renal I/R increase and it could be also up-regulated, having the samesubstrate NAD+’s PARP-1level as sirtuin-2. So we would further explore TRPC6’sregulating necroptosis mechanism in renal I/R and provide a new idea and target point forpreventing and curing renal I/R. Materials and Methods1. To monitor differential gene and miRNA change by gene detection chip and miRNAmonitoring chip, and make network analysis of miRNA target gene, analysis of proteinnetwork module, and screen differential gene and predict its possible action by NCBI GEOdatabase.2. To detect TRPC6’s expression in HK-2cells byimmunofluorescence staining.3. According to literature method, the hypoxia-reoxygenation model was set up bymineral oil and PBS added with Calcium and Magnesium, and the cell’s I/R process wassimulated in vitro.4. To build, screen and amplify silent HK-2cells of TRPC6by the RNAI technique.5. To utilize the flow cytometry to detect HK-2cell’s necrosis/apoptosis ratio in theprocess of hypoxia-reoxygenation Injury by AnnexinV-FITC/PI detection kit.6. To detect HK-2cell’s TRPC6, RIP1, PARP-1, sirtuin-2expression by western-blot.7. To establish an animal model of rats’renal I/R by means of cutting left kidney andclipping right renal artery with artery clamps for40min.8. To detect a rat’s serum creatinine level by Olympus AU2700AutomaticBiochemical Analyzer.9. To observe the morphological change of an ischemia-reperfusion rat’s renal injuryby renal histopathological HE staining.10. To make renal tissues immunohistochemical staining by a highly sensitive two-stepimmunohistochemical detection kit to observe expression changes of RIP1、RIP3、TRPC6、CaMKII、PARP-1in renal tissues.11. To detect renal tissues TRPC6, PARP-1, RIP1, sirtuin-2protein expression byWestern-blot.12. To utilize medicines, such as OAG, SKF96365and KN-93to study TRPC6channel and CaMKII’s action in signal transduction.Major Results1. Differential expression genes and miRNA, including TRPC6were screened andbioinformatics analysis was made.2. HK-2cell expressed TRPC6. TRPC6expression increased obviously in the processof cell hypoxia-reoxygenation.3. In the model of HK-2cell in vitro hypoxia-reoxygenation, cell necrosis was dominant in the cell’s death, exceeding cell apoptosis ratio.4. The cell necrosis rate of TRPC6’s silent HK-2cell in the hypoxia-reoxygenationinjury model went up obviously.5. TRPC6’s agonist OAG interference could make HK-2cell’s necrosis rate descendobviously in the hypoxia-reoxygenation injury model and have the similar effect likenecroptosis specific inhibitor nec-1. But TRPC6blocking agent SKF96365and CaMKIIinhibitor KN-93interference made HK-2cell’s necrosis rate go up obviously in thehypoxia-reoxygenation injury model.6. In the condition of hypoxia-reoxygenation, PARP-1expression of TRPC6’s silentHK-2cell was obviously down-regulated. Sirtuin-2expression was influenced by TRPC6silence. RIP1expression was up-regulated obviously after TRPC6silence.7. In the condition of hypoxia-reoxygenation, HK-2’s PARP-1expression of OAGinterference group went up and PARP-1expression of SKA96365and KN-93group wasdown-regulated. On the contrary, RIP1expression of OAG group was down-regulated andPARP-1expression of SKA96365and KN-93group went up.8. In the rat’s model of renal ischemia reperfusion, TRPC6was expressed mainly onthe renal tubular epithelial cell membrane, a little in glomerulus, too. After reperfusion-24h,48h, TRPC6expression of the renal tubular epithelial cell increased obviously and it beganto decrease on the5thday. The change trend of PARP-1, RIP1and sirtuin-2’s proteinexpression was similar to TRPC6’s.9. In the rat’s model of renal ischemia reperfusion, the serum creatinine level of ratswith OAG interference went down obviously,RIP1went up. However, the serum creatininelevel of rats with TRPC6blocking agent SKF96365interference was obviously went up,RIP1went down,compared with the ischemia reperfusion group.Conclusion1. By means of gene detection chip, network analysis of miRNA target gene andanalysis of protein network module,we found TRPC6was the differential gene of I/R andpredicted it might play an important role in regulating I/R.2. In the hypoxia-reoxygenation injury model of culturing HK-2cell in vitro, TRPC6expression obviously increased, CaMKII regulated PARP-1expression up after calciuminflow. Cell’s necroptosis was regulated by competitive action between PARP-1and sirtuin-2.Adopting RNAI technique, the necrosis rate of silent HK-2cell of TRPC6went upobviously, PARP-1expression went down, sirtuin-2expression changed not obviously, andRIP1expression also went up evidently in the hypoxia-reoxygenation injury model.3. In the rat’s model of renal ischemia reperfusion, TRPC6expression went upobviously. TRPC6’s agonist OAG improved rats’ serum creatinine level, TRPC6blockingagent SKF96365and CaMKII inhabitor KN-93accelerated serum creatinine rising.4. To sum up, TRPC6mediated calcium signal transduction in I/R by CaMKII,andregulated necroptosis of the renal tubular epithelial cell by means of PARP-1’s interactionwith sirtuin-2.