The Experimental Study Of AQPs In The Kidney Of Sepsis Rats

ObjectiveAquaporins(AQPs) are the main molecular mechanism to maintain the body water balance by water reabsorption and urine concentration.There are eight kinds of AQPs that expressed at the kidney such as AQP1～4,AQP6～8,AQP11,mainly at the proximal convoluted tubules,collecting duct and decending part of Henle loop.AQP 1 has the greatest permeability for water,mediating the water reabsorption from the initial urine.AQP2 is the major type of AQPs expressed at kidney,playing a key role in regulating the water reabsorption.Abnormal expression of AQP1,AQP2 is related to many renal diseases.Sepsis refers to the systemic inflammation caused by infection. Kidney is one of the target organs in sepsis,and the acute kidney injury(AKI) is one of the most common complications of severe sepsis.By now,there are little studies about the relation between the expression of AQPs at septic kidneys and kidney injury.In this experiment,sepsis rats model is used to dynamic observe the expression of AQP 1, AQP2 and the mRNA at the kidney,investigating the relation between the expression of renal AQPs and water metabolism disorder in AKI.MethodsSeptic models were made by cecum ligation perforation(CLP).Sixty-four Wister rats of 6 weeks were randomly divided into two groups:sham group(32) and the septic models(32).Each group was divided into 3-hour,6-hour,12-hour and 24-hour subgroups,with 8 rats in each.The urea,blood and kidney samples were collected.The urine volume,urine osmotic pressure and renal function were observed.The expression of AQPland AQP2 protein in rats' kidneys were detected by immunohistochemistry method,the expressions of AQP1 mRNA,AQP2 mRNA by real-time fluorescent PCR, the pathological changes of the kidneys were observed under light microscopy.Results1 Renal function After CLP,serum creatinine and urea in Wister rats began to increase at 6h,reaching the peak at 24h,the differences among the time points are significant(P＜0.05).There are no significant changes in the sham-group(P＞0.05).2 Changes of urine volume 3h after CLP,the urine volume decreased significantly,after 6h the urine volume gradually increased,at 24h reached the peak. The urine volume in septic models changes significantly(P＜0.05).There are no changes in the sham-group(P＞0.05).3 Urine osmotic pressure 3h after CLP,urine osmotic pressure increased significantly,6h after CLP,urine osmotic pressure decreased,reached the lowest at 24h. The urine osmotic pressure in septic models changes significantly(P＜0.05).There are no changes in the sham-group(P＞0.05).4 Renal pathologic changes 24h after CLP,the kidney changed into dark red and hard.There are spot and patch necrosis under the envelope.Under light microscope, there are no significant changes in kidney structure of surgery group at 3h and 6h.At 12h,the glomerular structure was almost the normal,the proximal convoluted tubule was slightly edema,the lumen structure was unclear,partial nuclear dissolved.At 24h, glomerular loops integrated,cell structure became unclear,tubular epithelial cells fused, nucleus disappeared,inflammatory cells infiltrated.5 Quantitative detection of AQPs gene by real-time PCR(1) Expression of AQP1-mRNA:In septic models,AQPI-mRNA expressed at each time point,and decreased gradually,reaching the lowest at 24h,which were significantly different from that of the sham group at 12h and 24h(P＜0.05).There is no change along with the time in the sham sub -groups(P＞0.05).(2) Expression of AQP2-mRNA:In septic models,AQP2-mRNA expressed at each time point,and decreased gradually,reaching the lowest at 12h,the changes are significant(P＜0.05).In sham group,the expression of AQP2-mRNA increased significantly at 3h(P＜0.05),then decreased significant at 12h and 24h(P＜0.05),there are no significant changes in the sham sub - group(P＞0.05).6Detection of AQPs by Immunohistochemistry method(1)Expression of AQP1 protein:The expression of AQP1 protein appeared in a downward trend,that is,after CLP,the expression of AQP1 protein decreased significantly,reached the lowest at 24h(P＜0.05).Expression of AQP1 protein decreased at 6h,12h,and 24h,which are significantly different from that of sham group(P＜0.05).There was no change of the expression of AQP1 protein in sham groups(P＞0.05).(2) Expression of AQP2 protein:The AQP2 protein expressed at the proximal convoluted tubulesand collecting ducts but not at the glomerular and interstitial.In the septic models,after CLP,the AQP2 protein began to decrease at 12h,reaching the loweat at 24h(P＜0.05),which are significantly different from the sham group(P＜0.05).7 Correlation analysis between AQP1,AQP2,respectively,with the urine,urine osmotic pressure:AQP1,AQP2 were negatively correlated with urine volume in septic models group,respectively(P＜0.05),and were positively correlated with urine osmotic pressure respectively(P＜0.05).Conclusions1 After CLP,expression of AQP1,AQP2 protein decreased along with the time, urine volume increased and urine osmotic pressure decreased.The decreased expression of AQP1,AQP2 protein is one of the main pathological mechanisms that resulting the concentration of urine disorder in septic rats.2 AQP 1 gene and AQP2 gene were involved in the water metabolism mechanism of kidney by regulating the expression of AQP1 and AQP2 protein.The abnormal expression of AQP1 and AQP2 gene is an important cause of water metabolism disorders in septic kidney.