The Mechanism And Clinical Study Of Acute Kidney Injury

BackgroudAcute kidney injury (AKI) has emerged as a common symptom in intensive care unit (ICU). It’s reported that about3.2%of hospitalization patients with AKI in china,and the rate increases to nearly20%in European contruries and American.AKI patients characteristically portends an increase in mortality,the mortality of AKI was as high as50%in ICU patients.However,about20%-30%of AKI patients developed chronic kidney injury and eventually progress into end stage kidney disease (ESRD)and need for renal replacement therapy after "clinical cure". Therefore, early diagnosis,early intervention has greatly improved the prognosis of AKI.For that, the nephrology experts and critical medical resercheres has made a great effort on the mechanism of acute kidney injury. It was confirmed that the epithelial repaired and the prognosis of tubular epithelial cell determined by the death on renal tubular epithelial cell.The death of cell included two kinds:apoptosis and necrosis, the former could be controled and the latter couldn’t. Acute tubular necrosis is the most serious death of renal tubular epithelium. Renal tubular necrosis (ATN) has been considered as unintervated with another necrosis for a long time which seriously blocked the treatment of AKI.Recently,a new form of death which has been confirmed controled and defined as "necroptosis" by cell death named committee in2009.Necroptosis was medicated by receptor interaction protein1(RIP1).The activiation of RIP1included two former:ubquitition and unubquitition. After accepted the signal, ubiquition RIP1activation caspase-8and intrigued apoptosis;When caspase-8channal was blocked,unubiquition RIP1activated NADPH oxidase and generated ROS (reactive oxygen species, ROS), then induce necroptosis, eventually begin autophagy and express the markers of autophagy:LC3-II (Microtubule-associated proteins1light chain3II).Indeed,RIP1represents the molecular target of a new class of cytoprotective agents:the necrostatin-1.The cells and animals model of the cerebral ischemia and reperfusion and myocardial ischemia have confirmed the existence of the necroptosis, and the specifility protective of nee-1.Necroptosis and nec-1provided new theory basis for the restoration of the necrotic cells,and add new intervention targets for the clinical treatment of necrosis. But,whether the exsistence of necroptosis renal tubular necrosis in AKI is still unknown.Meanwhile,the diagnosis and classification of acute kidney injury also arouse the interst of scientists.The three AKI diagnostic and classification criteria:RIFLE (Risk, Injury, Failure, Loss, End stage renal disease)criteria, AKIN(Acute kidney injury network) criteria and the new KDIGO (Kidney Disease:Improving Global Outcomes)criteria were published one by one.RIFLE criteria and AKIN criteria has been widespread applied, more than50,000patients has been identified as AKI by RIFLE criteria,AKIN criteria also been considered as a valiable criteria for AKI in critically ill patients and patients after cardiac surgery. Since the proposed of the AKIN criteria,some studies make a comparison between the RIFLE criteria and AKIN criteria,but the rasults is contradictory. To establish uniform criteria for the definition and classification of AKI, the KDIGO group proposed their own criteria in2011:patients meeting RIFLE criteria or AKIN criteria would be indentified as having AKI.Whether the KDIGO criteria have discernible advantages compared with AKIN criteria and RIFLE criteria with respect to the definition and classification of AKI is not known. Part I The model of necroptosis on tubular epithelail cellular ObjectivesTo establish the model of necroptosis on tubular epithelial cellular(HK-2). Methods1.Human renal proximal tubular epithelail cells (HK-2) culture HK-2cells were grown and maintained in Dulbecco’s modified Eagle’s medium/F12(DMEM/F12)supplemented with penicillin G (100U/ml)and streptomycin (100mg/liter).And the medium contained10%fetal calf serum (FCS) and then incubations were carried out at37℃in a90%relative humidity atmosphere of5%CO2.2. Morphology observation of HK-2cells with different intervenation2.1We photographed the growing HK-2cells cultured in DMEM/F12, observing the variance of HK-2cells morphology in different situation. The groups of growingHK-2were treated as follow:controlgroup; TNF-a+zVAD-frnk+antimy-cinA lh group; nec-1+control; nec-1+TNF-a+zVAD-fmk+antimycinAlh group.2.2Morphorlogy of HK-2cells determined by microscope in different treatment:The group of HK-2with different stimulation as follows:control group; TNF-a+zVAD-fmk+antimycinA1h group.3. The viability of the cells were measurementedThe viability of the cells was measured by the (CCK-8) test, as described previously.HK-2cells were seeded in96-well culture plates containing sera-free meium at an initial density of50,000cells per well. After a12hour incubation,nec-l(20umol/l) at37℃,andTNF-a(10ng/ml) and/or zVAD-fink (50umol/L) and/or antimycinA(10umol/l)were added to some of the wells and every group has four well.The groups of growing HK-2were treated as follow:(l):Control group:The HK-2cell were cultured without any treatments;(2):TNF-a group:The HK-2cells were culured with TNF-a (10ng/ml) for6hours;(3):TNF-a+zVAD-fink group:The HK-2cells were culured with TNF-a (10ng/ml) and zVAD-fmk (50umol/l) for6hours; (4):TNF-a+zVAD-fmk+antimycinA for30minutes group:The HK-2cells were cultured with TNF-a (lOng/ml) and zVAD-frnk (50umol/1) for6hours then removed clear liquid and added antimycinA (10umol/l) for30minutes.(5):TNF-a+zVAD-frnk+antimycinA forl hour group:The HK-2cells were cultured with TNF-a (10ng/ml)and zVAD-fmk(50umol/l) for6hours then removed clear liquid and added antimycinA (10umol/l) for1hour.(6):TNF-a+zVAD-fink+antimycinA for2hours group:The HK-2cells were cultured with TNF-a (lOng/ml) andzVAD-fink (50umol/l) for6hours then removed clear liquid and added antimycinA(10umol/l) for2hours.(7)-(12):as described as (1)-(6),only before that treated with nec-l(20umol/l) for8h. Next,fresh sera-free medium containing10%CCK-8was administered to the cells, which were incubated for2hours at37℃. The colorimetric reaction was measured by absorption at450run. Because the concentration of the colored product is proportional to the number of viable cells, cell viability was calculated as the percentage of untreated cells that are considered100%viable.4.The evaluation of the expression of LC3-ⅡTo measure the expression of LC3-IIby western-blot:ten thousand HK-2cells per well were cultured for12hr in6-well culture plates containing sera-free medium. All drugs was then added to some of the wells and the cells were incubated (as described above). The cells were then been carried out as the instruction.5.Statistical AnalysisAll measurement data were expressed as mean±standard deviation. Statistical analyses were conducted with SPSS13.0for Windows, comparing continuous variables of groups used One-way ANOVA,LSD method was used as multiple comparison for homoscedasticity, and Dunnett’s T3method was used as multiple comparison for heterogeneity of variance. Significance was difined as P<0.05.Results1.The variance of the morphology of HK-2cellular:The normal cellular stepping on the culture bottleformly ovoidly and irregularly and with a ovoid nuclear in the centre of cellular.But,for the group treated with TNF-a+zVAD-fmk+antimycinA processing one hour,the cells and organelles inflation,fragmentation.However,in the group of pretretmented with nee-1,the inflation and fragmentation of cellular is improved. By electron microscopy,visiable processing fractured cell membrane, rounded mitochondria,disappearing ridge, appearing many autophagy bubble with bilateral membrane.2. Nec-1specifily improved the viability of HK-2cellularThe viability of HK-2renal tubular epithelial cell among different group,the difference was statistically significant (F=410.780, P=0.000). In the group of stimulated with TMF-a+zVAD-fmk+antmycinA processing1hour,pretreatment with nec-1would increased the viability by38.59%compare the group untreated with nec-1(/*<0.05). But in other groups, no similar variance was observed.3. The expression of LC3-II by western-blot.Significant difference of the expression of LC3-Ⅱ in the groups were observed (F=115.748,P<0.0001).The significant increasing of the expression in LC3-II were measured in the group (3) to group (6)(P<0.05), but only in the group of intervated withTNF-a+zVAD-fmk+antimycinA for one hour,pretreated nec-1could signifi-cantly decrease the expression of LC3-Ⅱ (P<0.05).ConclusionsOur study suggested that the exsitance of necroptosis on renal epithelial cell in vitro which company with the decresing viability of cellular,the flation of cell and organism,and the increased expression of LC3-II.Nec-1,the specificity inhibitor of necroptosis could improve the viability of HK-2cellular,resume cell from necrosis and decrese autophrology. Part II:The comparison of the diagnosing criteria of acute kidney injuryObjectivesTo compare the value of RIFLE criteria,AKIN criteria and KDIGO criteria in diagnosing and classifing the acute kidney injury in critically ill patients.MethodsPatients admitted to the Department of Intensive Medicine of Guangdong General Hospital between October2009and July2010were evaluated retrospectively. Exclusion criteria were patients:with chronic renal failure requiring dialysis or requiring dialysis1month before admission to the ICU; who had received RRT or developed AKI before admission to the ICU; who did not have measurement of Scr in the first48h after admission to the ICU.The following information was recorded:age, sex, disease history, Scr, Acute Physiology and Chronic Health Evaluation (APACHE) II score, length of hospital stay, and hospital mortality. AKI was based on changes in serum creatinine in1week. The baseline Scr level was the lowest value in the first48h after ICU admission or the first test value. RIFLE, AKIN and KDIGO criteria were applied to define and classify AKI The mortality (in-hospital and at1-year) were the observed endpoints. Analyses were undertaken using SPSS verl3.0(SPSS, Chicago, IL, USA) and Medcalc ver9.2.10(MEDCAL,Beligum).Continuous variables were expressed as the mean±standard deviation or median (interquartile range). Categorical data were presented as the percentage of the number of cases. Comparisons among the KDIGO, AKIN, and RIFLE criteria with respect to in-hospital mortality and1-year mortality were done using Fridman M test,if there are significcant difference exsit,and the paired chi-square test for continuous variables and categorical variables, respectively. Multiple comparison were corrected with Bonferroni method.Multivariate logistic regression analysis was employed to evaluate the association between AKI identified by the three criteria with regard to hospital mortality and1-year mortality. This discrimination was assessed by measuring the AUROC. A two-tailed P value<0.05was considered significant. ResultsA total of524(356males) critically ill patients were assessed. The mean age was56.54±16.79years. A total of156(29.8%) patients received ventilation. The median length of hospital stay was13days, and28(5.3%) patients had RRT;②95patients were diagnosesed AKI by RIFLE criteria,and135patients was AKI according to the AKIN criteria,but the number of AKI increasing to140identified by KDIGO criteria. The incidence of AKI as defined by KDIGO and AKIN criteria was higher than that defined by RIFLE criteria by8.6%and7.7%, respectively, and this difference wasn’t statistically significant (18.1%vs25.8%vs26.7%, X2=0.133,P=0.936). A total of64(12.2%) patients were dead in hospital,and34(34.1%) AKI patients dead according to the RIFLE criteria;46(37.8%) patients were dead identified by AKIN criteria and47(33.6%) patients dead diagnosed by KDIGO criteria.X2was applied to compare the difference of the three criteria in predictive the mortality,and no signifiacnt difference was observd (34.1%vs33.6%vs37.8%,X2=4.000, P=0.135).Logical regression suggested that AKI was associated with mortality as identified by any of the three criteria (P<0.001),the odds ratio (OR) is9.194,95%CI:(5.018,16.845) vs12.006,95%CI:(6.277,22.964) vs11.006,95%CI:(5.78,20.956),respectively.The area under the receiver-operator characteristic curve (AUROC) for in-hospital mortality was0.778,95%CI:(0.710,0.844)(P<0.001) for KDIGO criteria,0.780,95%CI:(0.713,0.846) CP<0.001) for AKIN criteria, and0.732,95%CI (0.656,0.809)(P O.001) for RIFLE criteria, respectively.④422patients were followed up in one year,and90(21.3%)patients dead,included27AKI patients identified by RIFLE criteria,37AKI patients according to AKIN criteria and38AKI patients diagnosed by KDIGO criteria,but the difference among the three criteria was not significant:X2=3.000,P=0.223.The AUROC showed that the three criteria had similar and significant abilities to predict the1-year mortality:0.648for KDIGO criteria,0.644for AKIN criteria, and0.611for RIFLE criteria. ConclusionsKDIGO criteria were not better than AKIN and RIFLE criteria for diagnosing and predicting the prognosis of AKI in critically ill patients. More prospectively study is necessary for the identification of value of KDIGO criteria.