| Objective: Renal interstitial inflammation and fibrosis is the common pathological procedure to end-stage renal disease of many kinds of chronic kidney disease.Its pathogenic mechanism is not clarified to this day in which many factors is involved.Cell death plays a key role in inflammation and subsequent fibrosis.Necroptosis is a new type of cell death,which has the main features of cell necrosis such as cell membrane disintegration,organelle swelling,and loss of mitochondrial function,but is also a regulated,caspase-independent programmed cell death.Numerous studies have shown that necroptosis can induce and drive inflammation.Necroptosis plays an important role in mouse models of acute pancreatitis,ischemic injury and neurodegeneration,and clinical Crohn's disease.In addition,necroptosis also plays a pivotal part in various kidney diseases,such as cisplatin-induced renal injury,renal ischemia-reperfusion,kidney transplantation,and chronic renal fibrosis in subtotal nephrectomised rats.In this study,we use the unilateral ureteral obstruction(UUO)model to induce renal interstitial inflammation and fibrosis to investigate the existence of necroptosis.Then we used necrostatins-1(Nec-1),a necroptosis-specific inhibitor,to detect the expression of molecular markers of inflammation and fibrosis in obstructed kidneys.The aim is to explore the renal protective effect and possible mechanism of inhibition of necroptosis,and provide new ideas for the treatment of clinical nephropathy.Methods:1.Animal model:Healthy male C57BL/6J mice were randomly divided into sham operation group(Sham,n=6),unilateral ureteral obstruction operation group(UUO,n=6)and Nec-1 treatment group after ureteral obstruction(UUO+Nec-1,n=6).UUO+Nec-1 group mice were given Nec-1(1mg/kg/day)everyday by intraperitoneal injection for 7 days after surgery.The Sham group performed the same treatment as the model group except ligating ureter.2.Collection of renal tissues and detection of contents:Mice were sacrificed at 7days after operation.Partial renal tissues were fixed in 4% paraformaldehyde for routine pathological staining and immunohistochemical detection.The remaining renal tissue is rapidly frozen in liquid nitrogen and used to extract total protein and total RNA from renal cortex.Immunohistochemistry and Western blot were used to detect the protein expressions of receptor interacting protein 1(RIP1),receptorinteracting protein 3(RIP3),mixed lineage kinase domain-like,(MLKL),tumor necrosis factor-?(TNF-?),Interleukin-1?(IL-1?),monocyte chemotactic protein 1(MCP-1),Transforming growth factor-?1(TGF-?1),?-smooth muscle actin(?-SMA),collagen I(Col 1).Real-time PCR was used to examine the mRNA expression of RIP1,RIP3,MLKL,TNF-?,IL-1?,MCP-1,TGF-?1,?-SMA and Col 1.Results:1.Nec-1 improved UUO-induced renal pathological damageHE staining showed normal structure of the renal tubules in Sham group,and no obvious inflammatory cell proliferation was observed in renal interstitium.UUO group showed obvious mononuclear cell and lymphocyte infiltration,some proximal tubules were swollen or atrophied,some tubular vessels were significantly dilated,and necrotic epithelial cells were seen in the lumen.Compared with UUO group,the interstitial inflammatory infiltration,and the degree of swelling,expanded tubules and necrosis epithelial cells were reduced after Nec-1 treatment.Masson staining showed that a large amount of collagen fibers were deposited in renal interstitium in UUO group,and Nec-1 treatment significantly reduced the degree of fibrosis.2.Nec-1 decreased the expression of necroptosis related indicators induced by UUOImmunohistochemistry results showed that RIP1 was slightly expressed in distal tubule cytosol,while RIP3 and MLKL were weakly expressed in Sham group.RIP1 in UUO group was expressed in cytosol and nucleus of numerous distal tubule epithelial cells.RIP3 was strongly positive in cytosol of proximal tubules,while MLKL was highly expressed in cytosol and nucleus of tubule cells.After Nec-1 intervention,the positive signals of RIP1,RIP3 and MLKL were significantly reduced.Western blot and Real-time PCR showed that the protein and mRNA expression of RIP1,RIP3 and MLKL in UUO renal tissue was significantly higher than that in Sham group.After treatment with Nec-1,the protein and mRNA level of RIP1,RIP3 and MLKL was obviously reduced.3.Nec-1 reduced the expression of inflammatory factors in UUO mice kidneyImmunohistochemistry results showed that the inflammatory factors IL-1? and MCP-1 in Sham group were slightly expressed in tubular cytosol,and were significantly up-regulated in UUO group.In contrast,the positive expressions of IL-1? and MCP-1 were obviously reduced in UUO+Nec-1 group.Western blot and Real-time PCR showed that the protein and mRAN expression of TNF-?,IL-1?,MCP-1 in UUO group was significantly higher than that in Sham group.After Nec-1 treatment,the protein and mRAN level of the inflammatory factors was obviously downregulated.4.Nec-1 treatment attenuated interstitial fibrosisImmunohistochemistry results showed slight expression of Col 1 and TGF-?1 in Sham group,and ?-SMA only expressed in the middle artery smooth muscle cells.In UUO group,the expression of Col 1 was significantly increased in the interstitium.Except of vascular smooth muscle,the positive expression of ?-SMA was also greatly up-regulated in renal interstitial and tubular epithelial cells.TGF-?1 was widely expressed in the cytoplasm of tubules.Western blot and Real-time PCR showed that the protein and mRNA level of Col 1,?-SMA,and TGF-?1 in UUO group was significantly higher than that in Sham group.After treatment with Nec-1,the expression of Col 1,?-SMA,and TGF-?1 was significantly declined.Conclusion:1.Necroptosis plays an important role in pathological changes of unilateral ureteral obstruction of the kidney.2.The renal protective effect of necroptosis-specific inhibitor Nec-1 may be partially achieved by inhibiting inflammation and fibrosis. |
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