Protective Effects Of Klotho Protein In Renal Ischemia/Reperfusion Injury

Background: Renal ischemia-reperfusion injury(IRI)is one of the most common causes of acute kidney injury(AKI),and renal tubular necrosis is also the most typical pathological change of AKI.Inflammation is thought to play an important role in renal ischemia-reperfusion injury.It has been reported that the accumulation of monocytes / macrophages and the high expression of cytokines and chemokines in kidney may be associated with I/R-induced AKI.P38 MAPK is the most important mitogen-activated protein kinase(MAPK)family member,which involved in the inflammation.It is activated in ischemia-reperfusion injury,and regulates the downstream transcription factors to translocate into the nucleus and regulates the transcription of the target gene,contribute to cytokines or chemokines production.NF-?B is one of the key transcription factors that directly regulates the transcription of cytokines and chemokines and plays an important role in triggering inflammatory cascade.NF-?B was significantly activated in I/R-injuried kidneys.Inhibition of NF-?B activity could improve the renal function and histological damage,and decrease the expression of cytokines in I/R-injuried kidneys.Klotho was recognized as an anti-inflammatory and anti-senescence protein,and can be used to inhibit the secretion of cytokines.The exact mechanism of anti-inflammatory effects was less investigated.We hypothesized that ischemia and hypoxia in tubular epithelial cells results in the activation of MAPK signal,which actives NF-?B,leading to the upregulation of m RNA of cytokines or chemokines,finally recruits inflammatory cells.Incubation of soluble Klotho protein may improve renal I/R injury and inflammation by inhibiting p38 MAPK and downstream NF-?B pathway.Objective: To investigate the roles of P38 MAPK and NF-?B in the I/R-induced inflammation and how Klotho protein improve the inflammatory responses in I/R-injuried kidneys.Methods: In vivo experiments:we used 7-8 weeks BALB/c male mice to develop the renal IRI model by clipping bilateral renal pedicles(I/R group,n=5).In Klotho group,5 mice were intravenous injected with soluble Klotho protein before the development of I/R injury.five mice were performed sham operation(sham group).Renal function(Scr and BUN)was evaluated by biochemical method.Renal tubule injuries were observed by hematoxylin-eosin(HE)staining.The expression of KIM-1,macrophage marker F4/80,MCP-1,p-P38 MAPK,p-ERK1/2,p-JNK,p-P65 were detected by using IHC.The expression of KIM-1,MCP-1,P38,p-P38,ERK1/2,p-ERK1/2,JNK,p-JNK,P65,p-P65 and Klotho protein were estimated by using western blot.In vitro experiment: HK-2 cells were cultured in different hypoxia and reoxygenation group according to the different designated time of incubation.MCP-1 m RNA and Klotho m RNA were tested by real-time PCR.P38 MAPK,p-P38 MAPK,P65,p-P65,MCP-1 and Klotho protein were detected by using western blot.The activation of p-P65 and p-P38 were observed by immune fluorescence staining.Results: In vivo,the Scr of the sham group and the I/R group were 9.40±3.13?mol/L and 82.00±22.12?mol/L respectively,P<0.01.The BUN of the sham group and the I/R group were 10.20±1.13mmol/L and 52.90±6.24 mmol/L respectively,P<0.01.Klotho injection could rescue kidney function significantly,the Scr and BUN were 23.02±15.44?mol/L and 14.72±9.15 mmol/L respectively,compared with I / R group,P<0.01.IRI can reduce kidney Klotho expression to 0.16±0.09,the expression of Klotho in sham group was 0.76 ± 0.22.HE staining showed that renal tissue was damaged severely with renal tubular epithelial cells necrosis,which was alleviated significantly with addition of Klotho.IHC staining showed that KIM-1,F4/80 and MCP-1 production were higher than those in sham group.Western blotting analysis showed that the expression of KIM-1 in I/R group was 1.75 times higher than that in sham group(P<0.01).IHC showed that the overexpression of p-P38 MAPK in I/R group was mainly concentrated in renal tubular epithelial cells,but p-ERK was mainly activated in renal interstitial cells.The expression of p-P65 in the nucli of renal tubular epithelial cells was also increased.Western blotting showed that the expression of phosphorylated P38 MAPK and phosphorylated ERK in I/R group increased by 153.39±22.20% and 149.26±16.87%,respectively,compared with the control group(P <0.01 and 0.02,respectively).Phosphorylated P65 increased by 145.83 ± 18.6% compared with sham group,P = 0.02.The expression of p-P38 MAPK,p-ERK,p-P65,MCP-1 and F4/80 in the Klotho group were lower than those in the I/R group.In vitro,the expression of MCP-1 m RNA and protein,p-P38 and p-P65 increased gradually after hypoxia 24 hours and reoxygenation incubation in different time,and reached to the highest in 12 hours(H24R12).IF staining showed that p-P65 was concentrated in nucli of H24R12 incubated HK-2 cells.P38 MAPK inhibitor SB203580 dose-dependently inhibited p-P38 MAP,MCP-1 m RNA and protein expression in H/R-incubated HK-2 cells.P65 inhibitor BAY11-7082 dosedependently inhibited MCP-1 m RNA and protein levels in H/R incubated HK-2 cells.SB203580 also dose-dependently reduced H/R-induced p-P65 protein expression.Klotho can inhibit the expression of MCP-1 gene and protein,p-P38 MAPK and p-P65 expression in H/R incubated HK-2 cells in a concentration-dependent manner.Immunofluorescence staining also demonstrated that Klotho protein incubation(800 pmol/L)reduced the expression of p-P38 MAPK and p-P65 in H/R-cultured HK-2 cells.Conclusions: 1.In the mouse I/R kidney injury model,the expression of MCP-1 and macrophages infiltration were increased.Activated P38 MAPK was expressed in renal tubular epithelial cells,however phosphorylated ERK is enriched in interstitial cells.The phosphorylation of NF-?B subunit P65 increased in the I/R mouse kidney,and the process was regulated by P38 MAPK activation.IRI causes Klotho gene and protein downregulation,and soluble Klotho protein treatment alleviates I/R kidney injury,including reduction of histological damage and inflammation.It might be associatied with blocking the P38 MAPK / NF-?B signal.2.H/R incubated HK-2 cells increased the secretion of MCP-1.Inhibition of P38 MAPK and NF-?B activation in vitro can inhibit H/R-induced MCP-1 expression. Preincubated with Klotho protein blocked H/R-induced overexpression of MCP-1 by inhibiting the activation of P38 MAPK and P65 in HK-2 cells.