The Role And Mechanism Of Klotho In Inhibiting The Inflammatory State Of CKD

Backgroud The morbidity,hospitalization and mortality of chronic kidney disease(CKD)increased year by year,which has become the focus of global attention.With the gradual progress of CKD,the disease can involve other organs besides kidney,and then eventually developed for end stage renal disease(ESRD).Therefore,it is particularly important to find new therapeutic targets to delay the progress of CKD.Inflammation plays an important role in the development and progression of CKD.A number of clinical studies have shown that compared with healthy person,serum levels of inflammatory factors such as C-reactive protein(CRP),interleukin-6(IL-6),tumor necrosis factor(TNF-?)in CKD patients was significantly increased.Vaziri et al confirmed that the colonic epithelial barrier of CKD patients and CKD animal models are damage,their intestinal flora are disorders,and thus lead to systemic inflammation and uremia symptoms deteriorates.Studies have confirmed that CKD patients with elevated serum levels of inflammatory factors can inhibit vasodilatation(inhibition of nitric oxide synthesis),stimulate vasoconstriction,and reduce renal blood flow.Inflammation can also induce the release of cytokines and increase the production of adhesion molecules,which promote T cell conglutinate and migrate into the renal interstitial,and further promote the accumulation of a large number of fibrosis factors leading to renal fibrosis.The Recent studies have confirmed that inflammation is closely related to CKD and ESRD's important complications-cardiovascular disease.inflammatory factors can lead to myocardial injury,left ventricular hypertrophy and atherosclerosis and other lesions.Thus,inflammatory factors are confirmed that involved in CKD and its complications,development.Inhibition of inflammation can effectively prevent the progress of CKD.Klotho plays a key role in renal anti-inflammatory and repair kidney damage.Klotho mainly expressed in renal tubular epithelial cells,the mouse with Klotho silencing appear multiple organ dysfunction and shorten the phenomenon of life.In the Klotho-deficient mouse model,its characterization is very similar to that of uremic patients and is accompanied by complications such as vascular endothelium damage,mineral metabolic disorders,and even cardiovascular disease.Intraperitoneal injection of Klotho protein,uremic symptoms are alleviated,suggesting that CKD is relevant to the shortage of renal Klotho secretion.Secretory Klotho protein can act as a hormone-like substance into the blood circulation to play a cytoprotective effect.We and other scholars have found that e GFR and serum Klotho protein levels were positively correlated in CKD patients.Previous studies have shown that in CKD patients,with the progression of the disease,renal tubular epithelial cells Klotho m RNA gradually reduced.In the model of AKI mice induced by ischemic perfusion injury,as the renal function deteriorated,Klotho levels were declining,meanwhile serum levels were elevated.In the diabetic mouse model,decreased expression of Klotho can lead to increased serum inflammation.With decreased renal function,The decrease of Klotho is accompanied by increased expression of RIG-I and IL-6.In the study of senescence of inflammatory cells,the activation of RIG-I / NF-?B signaling pathway is closely related to the increase of inflammatory factors.Thus,activatio n of the RIG-I / NF-?B signaling pathway causes a significant increase in the release of inflammatory factors that may be a key step in CKD progression when Klotho levels fall.These results suggest that Klotho is closely related to the inflammatory response of cells and organs to the body,and maintaining the normal level of serum Klotho is important for suppressing inflammation and inflammation.The elevated levels of inflammation of CKD patients will increase the renal function damage,and further lead to metabolic waste can not be effectively ruled out.After dialysis treatment,most of the small and medium molecular weight water-soluble waste can be effectively removed.However,i Indoxyl sulfate(IS)and other uremic protein tuberculosis toxins because of the combination with the plasma protein can not be effectively removed.Previous studies have shown that IS accumulation in the body increases the ROS production in renal tubular epithelial cells,vascular endothelial cells,monocytes / macrophages,which will result in oxidative stress damage to renal tubules and vascular function,and aggravated CKD and its complications Condition.IS increased serum levels accompanied by TNF-?,IL-6 and other inflammatory factors increased,which will increase the renal microinflammatory state,which led to reduced Klotho secretion,making further deterioration of renal function.Macrophages are not only secreting more inflammatory factors after stimulation with IS,but also increase the infiltration of the renal int erstitial,which will accelerate the process of renal fibrosis.IS can increase the inflammatory response,increase the level of oxidative stress,stimulate renal interstitial fibrosis and inhibit Klotho and other anti-inflammatory factor synthesis to accelerate CKD and complications.In summary,we hypothesize that the decrease in Klotho expression in the kidney and the reduction of Klotho in circulation may be closely related to the inflammatory state of CKD patients in the development of CKD,and therefore participate in the development and progression of CKD and its complications.The correlation between Klotho and inflammatory factors in serum of CKD patients and whether Klotho can directly inhibit uremic protein binding toxin-induced release of monocyte inflammatory factors is still lack of systematic study.To this end,we will use enzyme linked immunosorbent assay(ELISA)to detect serum levels of Klotho and inflammatory factors in patients with CKD,clear the relationship between Klotho levels and inflammatory factors in CKD patients;and to investigate the role and mechanism of Klotho in inhibiting the release of monocyte inflammatory factors induced by IS in vitro,and to find a new target for the occurrence and development of CKD and its complications.Methods Vivo study 1.Clinical research: Collect serum samples from 286 CKD2-5 stage patients with non-dialysis,The levels of Klotho,IL-6,TNF-? and CRP were detected by enzyme-linked immunosorbent assay(ELISA),determinate IS Level by High Performance Liquid Chromatography(HPLC).The levels of inflammatory factors(CRP,IL-6,TNF-?),Klotho and IS were analyzed by one-way ANOVA.The correlation between inflammatory factors and e GFR,Klotho,IS was analyzed by grade correlation(Spearman).Partial correlation analysis(ALB and blood phosphorusas control variable)is used to reveal the correlation between inflammatory factors and Klotho.2.Human samples research: Choose 5 healthy volunteers and 5 CKD5 non-dialysis patients and collecte their peripheral venous blood from morning fasting.Serum samples were collected after centrifugation and centrifuged.Mononuclear cells(The mononuclear-macrophage system in the peripheral blood is mainly mononuclear cells,is macrophages in the tissue.)by western blot in two groups of mononuclear cells.All trial participants had signed informed consent,and the study was approved by the Ethics Committee of the Second Affiliated Hospital of the Third Military Medical University.This study was conducted in accordance with the Helsinki Declaration and the ethical review of the international guidelines for epidemiological research.Vitro study 1.IS-induced monocyte inflammatory factor release and the mechanism(three groups of experiments):(1)THP-1 cells cultured in vitro were divided into control group,2mg / L IS group,10 mg / L IS group and 50 mg / L IS group.Cells were cultured for 24 h to extract the total cell protein.The expression of RIG-I,p NF-?B and TNF-? was detected by Western Blot,and the levels of TNF-? in supernatant were measured by ELISA.(2)THP-1 cells were divided into the following groups: Control group,IS group(containing 50 mg / L IS),PDTC group(containing 10?M PDTC),IS +10?M PDTC group(10?M PDTC pretreatment 1h,then add 50 mg / L IS continued to be incubated for 24 hours).Western blot was used to detect the expression of RIG-I,p NF-?B and TNF-?.(3)THP-1 cells were divided into 6 groups: Control group,IS group,Con si RNA group,RIG si RNA group,IS + Con si RNA group,IS + RIG si RNA group.The latter four groups were treated with si RNA for transfection followed by continuous concentration of IS for 24 h.The expression of RIG-I,p NF-?B and TNF-? in the cells was measured.2.Klotho inhibits IS-induced monocyte inflammatory factor release: THP-1 cells were subjected to experiments in the following groups: Control group,IS group(50mg / L IS),Klotho group(including 400 pmol / L Klotho),IS + Klotho group(containing 400 pmol / L Klotho medium for 1 h,incubated with 50 mg / L IS for 24 h).The expression of RIG-I and p NF-?B protein was detected by Western Blot.The changes of TNF-? in culture supernatant were detected by ELISA.Result 1.Analysis of the correlation between serum Klotho protein level and inflammatory factors in CKD patients In this study,286 patients with primary CKD,5 healthy volunteers and 5 uremic patients were recruited.According to the 2007 US Kidney Disease Foundation(KDOQI)renal function staging criteria,CKD will be divided into five stage.Among them,CKD 1 was 3 cases and CKD 2 was 33 cases(CKD 1 ~ 2 patients were close to normal range,36 cases could be used as control group),51 cases of CKD 3,55 cases of CKD 4,144 cases of CKD 5.CKD staging was not related to gender,age,BMI and ALB(P> 0.05).The levels of serum CRP,IL-6,TNF-? and IS were significantly higher in patients with CKD than those in CKD stage 1-2,and the levels of Klotho protein were significantly decreased.The situation gradually deteriorated with the severity of CKD.Statistical analysis showed:(1)Single factor analysis of variance in multiple comparison Tukey tests reveal that serum CRP,IL-6,TNF-? and indole sulfate levels gradually increased(P <0.01)with decreased e GFR in CKD patients,meanwhile Klotho protein content decreased gradually(P <0.01).(2)Spearman analysis showed that CRP,TNF-? and IL-6 were negatively correlated with e GFR and Klotho(P <0.01),and positively correlated with IS(P <0.01).Serum Klotho and e GFR are positively correlated(P <0.001).Serum IS,P and uric acid were negatively correlated with e GFR(P <0.001).(3)Partial correlation analysis(control variables are ALB and blood phosphorus)shows that Klotho and TNF-? still had a significant correlation(P <0.001),while the correlation between IL-6,CRP and Klotho decreased(P <0.001).In order to further validate the above results,this study used CD14 + magnetic bead sorting method to extract 5 healthy individuals and 5 CKD(stage 5)patients serum mononuclear cells were detected.Western blot analysis revealed that the expression levels of RIG-I,p NF-?B and TNF-? in mononuclear cells of CKD patients were significantly increased compared with healthy controls.At the same time,ELISA test results also showed that patients with CRP,IL-6,TNF-? levels were significantly higher than healthy controls.These results indicate that mononuclear cells in uremic patients are in an activated state and are associated with RIG-I / NF-?B signaling pathway.The above results suggest that IS is likely to activate this signal path during the progress of CKD.2.IS induced monocyte inflammatory factor expression and its molecular mechanism In order to detect whether IS can cause monocyte inflammatory response,In this study,using 0,2 mg / L,10 mg / L,50 mg / L IS solution incubated with activated THP-1 macrophages for 24 hours,the culture supernatant was then detected by ELISA,The changes of TNF-? m RNA in cells were detected by q PCR method..The results showed that IS could enhance the production of THP-1 macrophage inflammatory factor TNF-?,and the level of TNF-? protein in culture supernatant was also increased,the level of TNF-? m RNA in the cells was significantly higher,showing a dose-dependent effect.Meanwhile,Western Bolt test also showed that IS could enhance the levels of p NF-?B and RIG-I protein,and increased with the increase of IS concentration,showing a dose-dependent effect.This suggests that IS may activate the RIG-I / NF-?B signaling pathway of macrophages.In order to determine the role of the signaling pathway,firstly use NF-?B specific inhibitor PDTC and IS itreat macrophages,and then detect the expression of TNF-?.The results showed that compared with the IS group,PDTC + IS could significantly reduce the expression of p NF-?B and the expression and release of TNF-? in the macrophages In order to verify the effect of IS on RIG-I,specific RIG-I si RNA was used to inhibit its expression.RIG-I si RNA and consi RNA were transfected into THP-1 macrophages,and then treated with IS,and finally the expression levels of TNF-?,p NF-?B and RIG-I were measured.The experiments showed that the expression of RIG-I in IS + RIG-I si RNA group was also significantly lower than that in IS + consi RNA group.At the meantime,its p NF-?B expression level also decreased significantly.Furthermore,the levels of TNF-? secreted by macrophages into the culture supernatant were also reduced.These results suggest that RIG-I si RNA can inhibit the activation of NF-?B signaling pathway and decrease the expression of macrophage inflammatory factors by inhibiting the expression of RIG-I gene.3.Klotho inhibits IS-induced monocyte inflammatory status In order to detect whether exogenous Klotho can affect the release of monocyte inflammatory factors,we used Klotho to pre-incubate macrophages for 1 hour,then add IS-treated macrophages for 24 hours,and finally use Western blot to detect the expression of RIG-I and p NF-?B.The results showed that the Klotho treatment alone did not inhibit the activation of the RIG-I / NF-?B signaling pathway compared with the control group.However,compared with the IS treatment group,Klotho significantly inhibited the increas e in IS-induced RIG-I and p NF-?B levels.This suggests that Klotho can inhibit the activation of the RIG-I / p NF-?B signaling pathway caused by IS.Furthermore,the increase in TNF-? levels in the culture supernatant caused by IS was also significantly reversed by Klotho.Experimental results suggests that exogenous Klotho can inhibit IS-induced monocyte inflammatory responses.conclusion 1.With renal function and serum levels of Klotho gradually decreased in CKD patients,the mononuclear cell inflammatory response increased.This is closely related to the activation of RIG-I / NF-?B signaling pathway.2.In CKD patients increased IS can be through the RIG-I / NF-?B signaling pathway to activate macrophages,and promote the expression and release of inflammatory factors.3.Exogenous Klotho protein attenuates IS-induced mononuclear cell inflammation by inhibiting RIG-I / NF-?B signaling pathway In this study,activation of RIG-I / NF-?B signaling pathway was associated with exacerbation of monocyte inflammatory status.Exogenous Klotho reduced the activation of RIG-I / NF-?b,which significantly reduced IS-induced mononuclear Cell inflammation levels.Exogenous Klotho can effective inhibiti inflammatory state of CKD,which can provide a new treatment ideas on the of theclinical treatment.