An Experimental Study About The Role Of TRPM7 In Regulating Necroptosis In Renal Tubule Ischemic Reperfusion Injury By PARP 1 / NAD+

As a most common reason of acute kidney injury(AKI), renal ischemic reperfusion injury(RIRI) was also one of major factors influencing a transplant kidney’s function recovery of early stage and its long-term survival. So it’s a clinical issue people concerned closely to understand RIRI mechanism further and deeply, as well as to improve or reverse it’s injury. Living and death were two big problems that cells faced. In RIRI, renal tubular epithelial cells were major target cells and infuenced by various injury/repair factors. The interaction of the two factors determined cell’s diversion, living and death. Due to its unique presentation and occurrence mechanism, necroptosis was known as a very important cell death form, which was found in recent years and existed commonly in the issue and cell injury. It had an obvious necrosis feature in pathology, inducing an obvious inflammatory reaction and blocked by the small molecule compound Nec-1(Nec-1) specificity. Necroptosis has been confirmed to exist in RIRI by evidences and regarded to probably play a more important role in RIRI process, compared to apotosis. Therefore, it was meaningful and broadly prospective for AKI treatment to investigate necroptosis’ ocurrence and regulation mechanism in RIRI deeply, as well as to prevent RIRI by blocking cell’s necroptosis.Trallsient receptor potential melastatill related 7(TRPM7), found in recent years, was an important non-selective calcium and magnesium ion channel expressed in the renal tubular epithelial cell. Meanwhile, having kinase activity, it was regarded to probably mediate signal transduction in and out of cells and participate in cell’s multiple physiological/ pathological functions. Some researches have proved that TRPM7 played an important regulating part in neuron ischemic injury, but the mechanism hasn’t been known yet. Moreover, researches on TRPM7’s effect in renal ischemia haven’t been reported yet. Some researches have showed, when deprived of oxygen and energy, ATP production dropped, Na+ /K+-ATP enzyme function exhausted, which could trigger Na+ and Ca2+ to come into cells by TRPM7 channel and activate molecules of TRPM7 downstream to play a regulating role. Now it is found that kinase receptor interacting protein-1(RIP1) has been confirmed to be the key regulating factor of necroptosis, while acetylase sirtuin-2 could make RIP1 deacetylated at the distinctive lysine residue site, and this deacetylation was the necessary condition of direct interaction between RIP1 and RIP3. Sirtuin-2’s enzyme activity depended on metabolic intermediates NAD+ level. NAD+ activity decrease was related to a lack of cellular energy and oxidative stress. Therefore, it could be concluded that necroptosis was regulated by metabolism and the activity of sirtuin-2 could regulate necroptosis process. Meanwhile, present researches found that poly( ADP-ribose) polymerase-1(PARP-1) and sirtuin-2 both belonged to dependent ADP- ribosyltransferase of NAD+, both depended on the cofactor NAD+ level and regulated NAD+ stability. In the condition of NAD+ being, both competitively inhabited each other. In the preliminary experiment, we found both of TRPM7 and PARP-1 expression could increase after reperfusion, and TRPM7 blocker 2-APB could inhibit PARP-1 expression, thus which indicated TRPM7 might influence sirtuin-2 activity by regulating PARP-1/NAD+ level, thereby regulate necroptosis to happen and play a part in RIRI. This study observed the change of TRPM7, PARP-1, RIP1 and RIP3’s expression in SD rats’ renal I/R and analyzed their relationships by function experiments in vitro and vivo. And it investigated the mechanism that TRPM7 regulated SD rats’ renal tubular epithelial cell necroptosis by PARP-1/NAD+ in order to improve renal ischemic reperfusion injury.Methods1、According to document method, SD rats renal ischemia/reperfusion injury models were established by experiments in vivo. Serum creatinine(SCr) and urea nitrogen(BUN) were detected from arterial blood on days 1,2,5 and 7 after kidney ischemia/reperfusion(I/R). Renal injury was observed under a light microscope with HE staining, and TRPM7, PARP-1, RIP1 were tested by immunohistochemistry and Western blotting, as well as their changes and relationships between renal function and pathological injury. Nec-1 intervention group, 2-APB intervention group and Bradykinin intervention group were established by tool drugs to study TRPM7’s effect in signal transduction.2、According to document method, ischemia/reperfusion injury models of SD rats’ renal tubular epithelial cell(NRK-52E)were established by experiments in vitro. Some aspects of the models in vitro were tested and analyzed, such as the ratio of necrosis and apoptosis at different time points of anoxia/reoxygenation, protein expression changes of necroptosis’ key molecule RIP1, PARP-1 and TRPM7. The artificial synthetic inhibition TRPM7’s siRNA fragment was transfected in NRK-25 E by Lipofectamine TM2000 and built TRPM7’s protein pects of necrosis and apoptosis ratio at different time points of anoxia/reoxygenation, protein expression changes of necroptosis’ key molecule RIP1, PARP-1 and TRPM7. And using tool drugs, the cell research from intervention groups Nec-1, 2-APB and Bradykinin analyzed TRPM7’s mechanism of regulating necroptosis by PARP-1/NAD+.Results1、In SD rats’ RIRI models, comparing control group with sham operation group, the levels of SCr and BUN,renal tissues and shape had no significant difference. The levels of SCr and BUN were significantly higher in I/R group than those in the control and sham operation groups. They reached the peak at the time of 24 h after reperfusion, and then gradually declined, falling to be nearly normal about 7d. HE staining results showed acute renal tubular injury appeared obviously both at the time of 24 h, 48 h after reperfusion, and kidney tubules repair started on day 5 and basically completed on day 7. Immunohistochemistry and Western blot results showed that TRPM7 was expressed mainly in the renal tubular epithelial cell. TRPM7’s expression went up remarkably after reperfusion and reached the peak at the time of 24 h, and then began to decline gradually. Changes of PARP-1 and RIP1 expression were remarkably similar. Correlation analysis showed there was a positive relation between TRPM7’s expression change and acute renal tubular injury grading(r=0.71,P<0.05). With the intervention of TRPM7’s non-specific agonist Bradykinin, PARP-1 expression went up and RIP1 expression went down. On the contrary, with the intervention of TRPM7’s non-specific blocker 2-APB, PARP-1 expression went down and RIP1 expression went up.2、In the I/R process of NRK-25 E in vitro, TRPM7 protein expression increased obviously. Necrosis and apoptosis ratio was tested by Flow Cytometer to confirm that necrosis was cells’ main death pattern. With the intervention of necroptosis’ specific inhibitor Nec-1, TRPM7 expression was inhibited apparently. While with TRPM7’s non-specific blocker 2-APB, necrosis rate went up, PARP-1 expression went down and RIP1 expression went up in the I/R model of NRK-25 E in vitro. With Bradykinin intervention, there was a similar effect to necroptosis’ specific inhibitor intervention brought, which presented that necrosis rate went down obviously compared to control groups, PARP-1 expression went up and RIP1 expression went down.3、In NRK-25 E group of TRPM7 low expression, compared to TRPM7 cell group of normal expression, RIP1 expression went up obviously and PARP-1 expression went down obviously in the I/R model in vitro. However, sirtuin-2 expression didn’t change obviously.Conclusion1、In SD rats’ RIRI models, TRPM7 expression went up obviously. Rats’ SCr and BUN levels could be brought down after the intervention of Ca MKII agonist Bradykinin. While SCr and BUN levels went up after TRPM7’s non-specific blocker 2-APB intervention.2、SD rats’ RIRI models in vivo and vitro proved that PARP-1 expression went up and RIP1 expression went down after Bradykinin intervention. On the contrary, PARP-1 expression went down and RIP1 expression went up after 2-APB intervention. Making PARP-1 and sirtuin-2 compete with each other was to regulate necroptosis process ultimately by regulating PARP-1 expression up.3、By establishing NRK-25 E I/R models of TRPM7 low expression, it proved that the necrosis rate, RIP1 expression went up obviously compared to TRPM7 cell group of normal expression and PARP-1 expression went down obviously. Sirtuin-2 expression didn’t change remarkably.To sum up, we thought TRPM7 might activate calcium influx to regulate PARP-1 and regulate sirtuin-2 activity by PARP-1, then take a regulating effect on RIRI by influencing necroptosis process. From a new perspective, this study started with renal tubular epithelial cell necroptosis to investigate possible intervention targets in RIRI mechanism, which may provide a new idea to prevent and treat RIRI.