Experimental Investigates Of Vasoactivating Agents Change The Renal Aquaporins Expression After Common Bile Duct Recanalization

ObjectiveAcute renal injury caused by obstructive jaundice is frequently the main factor predisposing to acute renal failure, and the mortality is more than 68%. Renal tubule necrosis is the main reason. We investigated the renal structure and functional injury in experiment. At the same time aquaporin 1 expression in proximal tubule and aquaporin 2 and 3 expression in collective tubule were measured. Compared with renal structure and function, we might find the reason of renal injury in obstructive jaundice. Also we would investigate the renal protective effect of vasoactivative agents such as dobutamine and dopamine. The aquaporin 1,2 and 3 expression and message RNA expression would be detected in rats injected with vasoactivative agents.Methods110 male Wistar rats, weighting from 250 to 300g, were studied. After 7 days acclimatization, animals were randomized into six groups matched for body weight. One hundred rats were anesthetized with an intraperitoneal injection of 300mg/kg Chloral Hydrate. Epigastric incision (2 cm long) was made 1 cm below xiphoid. After mobilizing the common bile duct, a 0 silk suture was passed behind the common bile duct, then ligated the common bile duct with a 2 mm diameter Silastic catheter in order to producing a biliary blockage. Another 10 rats only underwent sham operation without any abdominal interruption. Seven days later, all rats received second surgery. The catheter was taken out to allowthe common bile duct re - open in the experimental group. The rats in control group underwent another simply open procedure. After second surgery, the rats in experimental group were randomly assigned into five groups;obstructive jaundice, 5ug/kg/min dobutamine, 10 ug/kg/min dobutamine, 5ug/kg/min dopamine and lOug/kg/min dopamine group, which would be administered drugs via a femeral vein cannula with different rate;0.9% Normal Saline, 5ug/kg/min dobutamine, lOug/kg/min dobutamine, 5ug/kg/min dopamine or lOug/kg/min dopamine respectively. After 2 hours injection, half of the rats in each experimental group were sacrificed and the left would be sacrificed 24 hours later. Then serum of each rat was collected to test liver function and kidney function. Right kidney were kept in the 80 C refrigerator ( Hitachi) , which would be used to measure aquaporin 1 expression by western blotting technique and aquaporin 1 mRNA expression by RT - PCR technique. The kidney was homogenized with polytron homogenizer at 3000 rpm. The protein concentration was determined by the method of Braford with bovine serum albumin as a standard. Protein samples were electrophoretically fractionated with a discontinuous system consisting of a 12. 5% poly aery lamide resolving gel and 5% stacking gel, followed by transfer to a nitrocellulose membrane at 20V and 100mA overnight. Total cellular RNA was extracted from kidney by the method of Chomczynski and Sacchi. Reverse transcription was used to synthesize cDNA probe. In brief 0.5 ug RNA was homogenized at room temperature in 20 jxl. The PCR reagents are from TaKaRa corporation, Dalian, China. Reaction volume is 25(xl,and cDNA 3jxl, primer 0. 1 ,xl, dNTP 0.02mmol/L, 10 x buffer 2. 5jjl1, Taq polymerase 1U. And 0. 1 |xl samples were transferred to a 2% agarose gel for PCR product analysis. X174 - Hinc II (TakaRa corporation) was used as the relative molecular mass standard. Following ethidium bromide staining, the amplificate products were analyzed using computer gel imaging analysis system. The densities of PCR products were calculated. The gene relative express quantity is equal to the ratio of gene optical density for calculation and that of J3 actin.Reults1. All rats finished the experiments, and the skin color of ear and tail became yellow three days later in experimental groups. After sacriface, the serum and kidney were kept for measuring. By using Hitachi 7600 Automated Blood Analyzer to analyse the blood samples, we found that common bile duct ligation resulted in significant rise in the plasma levels of bilirubin, creatine and urea nitrogen compared with normal levels. However after second surgery, the plasma bilirubin levels decreased significantly. At the same time the renal function in experimental groups had nearly no change compared with the sham operation group. Using histochemistry technique we found obvious changes in renal structure.2. The aquaporin 1 expression in obstructive jaundice group decreased significantly and the aquaporin 1 mRNA expression elevated at the same time. While the aquaporin 1 expression in dobutamine 5 ug group was nearly the same with the sham operation group at the time of bile duct recanalizaion. Twenty four hours later the expression decreased significantly. The aquaporin 1 expression in dopamine 5ug group and lOug group increased significantly at the time of bile duct recancalization. Just like those in dobutamine group, the expression decreased significantly 24 hours later. While the aquaporin lmRNA expression in each group was just in reverse. The aquaporin 2 expression in obstructive jaundice group was decreased. But the expression elevated in 24 hours even higher than that in sham operation group. The aquaporin 2 expression changes in dopamine groups was just like this. Aquaporin 2 expression in experimental group suffered from dobutamine 5 ug/kg/min was nearly the same with that in normal group. While that was better than the group suffered from dobutamine 10 ug/kg/ min. The relative expression of aquporin 2 mRNA in each group just got the reverse result from the western blotting results. Aquaporin 3 expression in obstructive jaundice group decreased at the time of recanalization, but there was no significant change between obstructive jaundice group and sham operation group 24 hours later. Renal aquaporin 3 expression and aquaporin 3 expression in experi-mental group suffered from dobutamine 5 ug/kg/min were nearly the same with that in sham operation group. While that was better than the group suffered from dobutamine 10 ug/kg/min. At the time point of recanalization the aquaporin 3 expression in dobutamine lOug group was lower than that in 5ug group. The relative expression of aquporin 3 mRNA in each group just got the reverse result from the western blotting results.Conclusion1. Renal proximal tubule and collective tubule structure were injuried in obstructive jaundice.2. At the time of recanalizaion the serum direct bilirubin decreased significantly with the renal function nearly no change.3. The aquaporin 1, 2 and 3 in proximal or collective tubules fluctuated significantly. Vasoactive agents such as dopamine and dobutamine could do effects on modulating aquaporin expression and water reabsorption ability.