Dynamic Expression Of Lon Protease 1 In Chronic Kidney Disease

Chronic kidney diseases(CKD)refers to progressive loss of kidney structure and function over a period more than 3 month due to a variety of causes.The damage of the kidney finally leads to the decline of GFR which is called chornic kidney failure(KDIGO guideline).All of the chronic kidney disease eventually develop to a common pathological change-renal fibrosis.Renal tubulointerstitial fibrosis involves an excess accumulation of extracellular matrix(primarily composed of collagen)and usually results in loss of function when normal tissue is replaced with scar tissue.Tubulointerstitial fibrosis having consistently been shown to be the best histological predictor of progression.Although a large body of studies have been performed,there is no effective clinical treatment for renal fibrosis.Thus,exploring the mechanism of fibrosis and discovering an effective therapeutic medicine is an of great importance.Tubulointerstitial fibrosis having consistently been shown to be the best histological predictor of progression.Although a large body of studies have been performed,there is no effective clinical treatment for renal fibrosis.Thus,exploring the mechanism of fibrosis and discovering an effective therapeutic medicine is an of great importance.Lon protease 1(LONP1)is a nuclear DNA-encoded mitochondrial enzyme which is encoded by LONP1 gene in human.It is highly conserved throughout evolution.The interest in Lon has been rapidly increasing in the last years,as it turns out to be deeply involved in a plethora of biological processes,including(but not limited to)the turnover of mitochondrial proteins,the regulation of mitochondria DNA replication,cellular respiration and oxidative phosphorylation,and the maintenance of mitochondrial morphology and dynamics.It has been found of close association with human diseases such as cancer and aging.Given the crucial role of LONP1 in maintaining mitochondria function,we hypothesize that it may play a role in renal tubuleinterstitial fibrosis.There is no study published yet.We will conduct in vitro and in vivo study to abserve the dynamic change of LONP1 in cell injury and Unilateral uretary obstruction.Further,we will confirm the finding in CKD patients by IHC with clinical biopsy sample.Objective:To observe the expression of Lon proteasel in CKD in vitro and in vivo and explore the possible correlation of lonl and kidney pathology features and outcome.Methods:1>Confluent human HK-2 cells(renal tubular epithelial cell line)were induced for cell damage by the treatment of Ang ? at lOnmol for 24 hours.The control cells were treated with same amount of medium.Inflammatory cytokines and fibrosis related molecules were determined by western-blot and real-time PCR,as well as the LONP1 expression.2>129 mice were divided into two groups:sham and UUO.UUO surgery was performed on day 0.Sham group were operated without obstruction.Kidney tissure were collected on day 1,3,7,10,14.Inflammation and fibrosis was evaluated by protein and mRNA quatification and histology examination.3>Kidney biopy samples from 30 pediatric patients with chronic kidney disease were assessed for LONP1 distribution and expression by immunohistochemical staining.Patients were also classified into light,medium and heavy three groups according to the histological fibrosis severity which is determined by Masson staining.Correlation between LONP1 and fibrosis severity was analyzed.Results:(1)In Ang-? induced renal tubular cell damage model,the mRNA expression of LONP1 quantity decreased,(12,24 hours both reduced 50%compare with control group,P<0.001).LONP1 protein expression was significantly reduced(a 45%reduction in 12 hours,24 hours decreased by 40%,P<0.01,but no statistical difference between the two groups).After Ang-?stimulation for 12,24 hours,collagen ? mRNA expression significantly increased compared with control group(12 hours:Ang-? group raised 30%,P<0.05;24 hours raised about 40%,P<0.001).a-SMA was increased at 12 hours and in Ang-? group is about 1.5 times more than that of control group(P<0.05);and at 24 hours it is 1.75 times more than control group(P<0.001).Inflammatory cytokines of IL-1? expression also increased dramatically.At 12 and 24 hours,IL-1? expressed respectively 2 times(P<0.05)and 2.8times(P<0.01)higher than that in control group.Furthermore,using western-blot to test the expression of collagen ? and ?-SMA,we found dosing stimulus increased with time,collagen ? and ?-SMA protein expression were significantly increased compared-with control group:collagen ? of stimulation group at 12 hours was 3.1 times higher compared with the control group(P<0.001),24 hours group was 4.2 times to the control group(P<0.001);a-SMA expression level in 12 hours after stimulation increased about 40%(P<0.05),24 hour increased about 1.6 times(P<0.001)than that of control group.(2)Using HE staining to observe inflammatory cell infiltration.Masson staining to observe the UUO mice kidney histology change,we found UUO module of mice showed with a progressive pathological changes compared with the sham group,first day after surgery only mild renal tubular expanded in mice;then in third day appeared kidney renal tubular expansion and a small amount of collagen fibers deposition;7 days,10 days after surgery appeared part of the renal tubular atrophy and renal interstitial collagen deposition;after 14 days,renal structure damaged obviously,showed tubular atrophy,and a large amount of collagen deposition.As Real-time PCR results showed that the expression of LONP1 has no significant difference between different groups,we used western-blot to detect the LONP1 expression in protein level.Compared with the sham group,LONP1 protein expression on the first day after surgery had no significant change,after 3,7 days of operation,all decreased by about 30%(P<0.05).10,14 days were more apparent,reduced about 61%compared with sham group(P<0.001).By immunohistochemical staining we found that LONP1 mainly expressed in renal tubules.LONP1 expression after 1 day of operation had no significant changes,then decreased along with renal interstitial fibrosis progressed.Real-time PCR results showed that compared with the sham group,the mRNA expression of collagen I,?-SMA,IL-1?,MCP-1 were significantly increased.The expression of E-cadherin mRNA was obviously downward as time goes by.Similarly western-blot results showed that collagen I expression was downregulated at day 3 compared with sham controls and reached a deep reduction at day 14.a-SMA protein production had no obvious change on postoperative day 1,but obviously raised at 3,7,10,14 days(the expression of a-SMA at 14 days after operation increased about 2 times compared with control,P<0.001);the amount of E-cadherin protein expression significantly downregulated as time went by(14 days decreased about 67%,P<0.001);All of above,LONP1 protein expression had negatively correlation with renal tissue fibrosis.(3)corresponding in clinical cases,immunohistochemical staining was applied to observe LONP1 expression in kidney tissues of patients with CKD,we found LONP1 is mainly expressed in the renal tubular,and its expression was significantly decreased in CKD patients compared with the control group.Taking account of pathologic grading,we found that as renal the degree of tubular atrophy,interstitial fibrosis,inflammatory cells infiltration and chronic lesion aggravating,LONP1 expression gradually reduced.Conclusion:In vitro and in animal models,the expression of LONP1 has negative relationship with renal interstitial fibrosis:the more heavier of renal interstitial fibrosis,the less quantity of protein expressed.Likewise,LONP1 expression in kidney decreased significantly in patients with CKD related to the renal tubular atrophy and interstitial fibrosis and inflammatory cells infiltration and chronic lesion.