Effect And Mechanism Of EZH2 Inhibitor 3-DZNeP In Renal Tubular Cell Injury Induced By Oxalate

Purpose:The incidence and recurrence rate of kidney stones are high,and the pathogenesis of kidney stones remains unclear so far.Therefore,renal stone is a long-term problem for urologists in clinical practice.The concentration of oxalic acid in urine is closely related to the formation of kidney stones.Therefore,the hyperoxaluria model was established for research.Previously we successfully constructed a kidney stone model on mice,and gene chip technology was used to compare the expression levels of different genes between the control group and the model group.The expression of EZH2,an epigenetic molecule that has been evaluated recently in various diseases,was significantly increased.By reviewing the relevant literature,we found that in a variety of kidney injury models,using 3-DZNe P to target the expression of EZH2 can significantly reduce kidney injury and improve renal function.Therefore,we aimed to identify the potiential role of 3-DZNe P in acute kidney injury induced by oxalate.Methods:The degree of damage of renal tubular epithelial cells stimulated by high oxalate and the role 3-DZNe P plays in it was evaluated by immunohistochemistry,werstern blot,CCK-8detection,lactate dehydrogenase cytotoxicity detection,reactive oxygen species detection and other methods.In terms of mechanism exploration,the classic cell signal pathways closely correlated with cell proliferation and 3-DZNe P was identified.Furthermore,pathway inhibitors and small interfering RNA(si RNA)or short hairpin RNA(sh RNA)were used to knock down the expression of related genes to explore the regulatory effects of upstream transcription factors on downstream target gene transcription.Results:(1)EZH2 inhibitor 3-DZNe P can alleviate kidney injury and reduce stone formation induced by hyperoxaluria rat modelFirst of all,we found that the renal tubular damage in the model group was significantly increased,renal function was significantly impaired,kidney crystals were significantly formed when compared with the control group;the increased protein expression of EZH2 and H3K27me3 can be observed,and the expression was higher in the 4th week hyperoxaluria model than in the 2nd week.PCR and immunohistochemistry results also showed that EZH2 was highly expressed in the model group,and mainly expressed in the nucleus of renal tubular epithelial cells.Secondly,we used the EZH2 inhibitor 3-DZNe P to inhibit the high expression of EZH2 and found that 3-DZNe P significantly reduced renal tubular damage in the hyperoxaluria model group,reduced the formation of renal crystals,improved renal function,and apoptosis index cleaved-caspase3 and inflammation indicators IL-6 and MCP-1 were significantly down-regulated,which indicated that the apoptosis and inflammation levels of renal tubular epithelial cells were also significantly decreased.(2)EZH2 inhibitor 3-DZNe P can alleviate oxalate-induced renal tubular epithelial cell damageFirst of all,CCK-8,LDH release experiment and reactive oxygen species(ROS)detection experiment found that different concentrations of oxalate and different time periods stimulated renal tubular epithelial cells(NRK-52E)can reduce cell viability and increase intracellular ROS.Secondly,we found that the expression of intracellular apoptosis index cleaved-caspase3 and inflammation index IL-6 and MCP-1 increased,which indicated that oxalate would promote NRK-52 E cell apoptosis and increase inflammation.The TUNEL experiment also confirmed that high oxalate stimulation can induce apoptosis.However,the stimulation of oxalic acid did not lead to the increased expression of EZH2 in NRK-52 E cells,but the modified H3K27me3 increased induced by oxalate,which indicated that there is no change for the expression level of EZH2 in NRK-52 E stimulated by oxalic acid,but its activity increases.Finally,we pretreated NRK-52 E cells with 3-DZNe P and then stimulated them with oxalic acid.Compared with the oxalic acid-stimulated group,the expression of EZH2 and H3K27me3 was significantly down-regulated in the treatment group,followed by the significantly decreased expression level of cleaved-caspase3,IL-6 and MCP-1.TUNEL experiment also found that the apoptosis of 3-DZNe P group was significantly reduced.The results showed that the cell viability and intracellular oxidative stress level were also significantly amelioated by 3-DZNe P.Subsequently,we knocked down EZH2 using Sh RNA and found that the cells in the Sh-EZH2 group had significantly lower expressions of cleaved-caspase3,IL-6 and MCP-1 and lower levels of oxidative stress than the Sh-NC group under high oxalic acid stimulation.(3)EZH2 inhibitor 3-DNZe P reduces oxalate-induced kidney injury by inhibiting the JNK/Fox O3 a pathwayFirst,we found that stimulated by oxalic acid,the three sub-pathways of the MAPK pathway,ERK,JNK,and P38 were activated.The expression of Fox O3 a is also up-regulated.And then 3-DZNe P can inhibit the up regulated expression of p-ERK,p-JNK,p-P38 and Fox O3 a.The expression of Fox O3 a is also significantly down-regulated after knocking down EZH2.Then we added CC90003,SP600125,and SB203580 to block the ERK,JNK and P38 pathways respectively after 3-DZNe P pretreatment and oxalic acid stimulation and found that only SP600125 can significantly reduce the expression of Fox O3 a.Furthermore we knocked down Fox O3 a with si RNA and found that it could reduce cell apoptosis.Secondly,in vivo experiments also showed that the expression of p-ERK,p-JNK,p-P38 and Fox O3 a in the model group was significantly increased,and 3-DZNe P could reverse this phenomenon.Conclusion:3-DZNe P can significantly improve cell apoptosis and ameliorate oxidative stress,thereby reducing kidney crystallization.3-DZNe P may play a role by down-regulating EZH2 and then inhibiting the phosphorylation of JNK and then down-regulating the expression of Fox O3 a.JNK/Fox O3 a may be a signaling pathway that mediates 3-DZNe P to reduce kidney injury induced by high oxalate.Therefore,the use of 3-DZNe P to target EZH2 is expected to become a potential therapeutic strategy for the treatment and prevention of kidney stone diseases in the future.