Original Article  /  Biliary

Downregulation of AQP2 and AQP2 mRNA expression in kidney medulla of rats with bile duct ligation Yong Wang, Jin-Gang Liu and Ji-Long Han Shenyang, China

BACKGROUND: Obstructive jaundice is a common disease. Acute renal injury, secondary to obstructive jaundice, is one of the main causes of postoperative multiple system failure. This investigation evaluated renal function and renal aquaporin 2 (AQP2) expression changes in obstructive jaundice. METHODS:  Forty male Wistar rats were equally randomized into two groups. Twenty in the obstructive jaundice group were subjected to common bile duct ligation, and then were subdivided into 7- and 14-day obstruction groups, and the other 20 sham-operated rats were also subdivided into 7- and 14-day groups. At the end of each experiment, rats were sacrificed, venous blood was collected from the inferior vena cava, and serum creatinine and urine nitrogen concentrations were measured. At the same time, the medulla of the right kidney was separated and AQP2 expression was assessed. The RT-PCR technique was used to detect AQP2 mRNA expression. RESULTS:  Ligation of the common bile duct caused significant rises in serum bilirubin, creatinine clearance and urine nitrogen. AQP2 expression in the medulla decreased mere significantly (38.35±2.08) in the 7-day ligation group than in the sham-operated group (41.06± 1.04), as did that in the 14-day ligation group, even more than (31.89±1.57). The expression of AQP2 mRNA also decreased more significantly in the 14-day group (0.5429± 0.1107) than in the 7-day group (0.6071±0.1328).

Author Affiliations: Department of Hepatobiliary, Second Affiliated Hospital (Shengjing Hospital), China Medical University, Shenyang 110004, China (Wang Y, Liu JG and Han JL) Corresponding Author:  Yong Wang, MD, PhD, Department of Hepatobiliary, Second Affiliated Hospital (Shengjing Hospital), China Medical University, Shenyang 110004, China (Tel: 86-24-25700868; Email: [email protected]) © 2007, Hepatobiliary Pancreat Dis Int. All rights reserved.

CONCLUSION:  AQP2 expression is inhibited in obstructive jaundice, and so is its gene expression. (Hepatobiliary Pancreat Dis Int 2007; 6: 636-640) KEY WORDS: aquaporin 2;  obstructive jaundice;  k idney medulla  

Introduction

A

cute renal injury, secondary to obstructive jaundice (OJ), frequently precedes acute renal failure or even multiple organ failure. Acute renal injury following common bile duct obstruction remains a major clinical problem. About 6%-8% of patients suffer from acute renal injury, with a mortality of over 68%.[1] Hence is important to understand the pathophysiological changes of acute renal injury and their prevention. Renal injury in OJ has been studied for many years, and at present there are three hypotheses for renal injury: renal ischemia-reperfusion;[2, 3] the destroyed intestinal barrier;[4-6] and bilirubin injury.[7, 8] Clinically, urine volume disorder is more frequently seen than any other symptoms in early renal injury. In general, renal dysfunction can be recognized as a morphological or functional injury. Probably the above three hypotheses can be used to interpret the causes of renal injury, but what is the direct factor for early oliguria? The aquaporins (AQPs) are a family of membrane proteins that function as water channels.[9] Variations in the position and concentration of AQPs have been demonstrated by biochemical and molecular biological techniques.[9] From changes of AQP expression, however, we can obtain information about structural and functional disorders in the kidney.[10] Recently, significant changes of AQP2 expression in experimental renal failure have been reported,[2] but no AQP2 changes in OJ have been observed.

636 • Hepatobiliary Pancreat Dis Int，Vol 6，No 6 • December 15，2007 • www.hbpdint.com

Downregulation of AQP2 and AQP2 mRNA expression in kidney medulla of rats with bile duct ligation

We hypothesize that there is a difference in renal collective duct AQP2 expression after ligation of the common bile duct.

Methods Experimental animals and reagents This study was approved by the China Medical University Institutional Animal Investigation Committee. AQP2 first antibody: rabbit antiAQP2, catalog No BA0649, Wuhan Boster Biological Technology Co., Ltd., Wuhan, China, and AQP2 second antibody: goat anti-rabbit IgG, product No. A 3687, Sigma-Aldrich Co., USA. Marker ΦX174-Hinc Ⅱ, TakaRa Corp., Dalian, China. Experiments were performed on 40 male Wistar rats weighing 250-300 g. After 7 days of acclimatization, the animals were randomized into two groups matched for body weight. During the entire study, the rats were kept in individual metabolic cages with a natural light/dark cycle, a temperature of (18±2) ℃ and a humidity of (50±2)%. OJ model[11] The rats were anesthetized by intraperitoneal injection of chloral hydrate (300 mg/kg). An epigastric incision 1 cm below the xiphoid and 2 cm long was made. After opening the peritoneum, the common bile duct was ligated with 0 gauge silk. The rats had free access to food and water after recovery. Those rats in the sham-operated (SO) group had a 2-cm incision without the ligation of the common bile duct. The rats in the OJ group were randomly assigned into 7- or 14-day groups (OJ 7 days or 14 days), as were those in the SO group (SO 7 days or 14 days). Serum was collected from the inferior vena cava at the time of sacrifice of rats for measurement of hepatic and renal function. The medulla of the right kidney was separated and immediately frozen in liquid nitrogen at -80 ℃ until measurement of AQP2 abundance and AQP2 mRNA expression. AQP2 abundance Protein preparation The medullary sample was homogenized with a Polytron homogenizer at 3000 rpm in a solution containing 250 nmol sucrose, 1 mmol EDTA, 0.1 mmol phenylmethyl sulfonyl fluoride (fresh), and 20 mmol potassium phosphate buffer (pH 7.6). The protein concentration was determined by the Bradford method with bovine serum albumin as a standard.

Western blotting analysis Protein samples were fractionated electrophoretically with a discontinuous system consisting of 12.5% polyacrylamide resolving gel and 5% stacking gel, followed by transfer to a nitrocellulose membrane at 20 V and 100 mA overnight. AQP2 mRNA expression Total cellular RNA was extracted by the method of Chomczynski and Sacchi.[12] The reverse transcription method was used to synthesize the cDNA probe. In brief, 0.5 μg RNA was homogenized at room temperature. It contained 22 U AMV, 50 μmol oligodt15, 20 U RNase inhibitor, and 1 mmol dNTPs. PCR reagents were phased from TaKaRa Corp., Dalian, China. The reaction volume (25 μl) and contained 3 μl cDNA, 0.1 μl primer, 0.02 mmol dNTP, 10× 2.5 μl buffer, and 1 U Taq polymerase. Approximately 0.1 μl samples were transferred to 2% agarose gel for PCR product analysis of marker ΦX174-Hinc Ⅱ. Following ethidium bromide staining, the amplified products were analyzed using a computer gel imaging system to calculate the densities of PCR products. The F sequence was 5'-GCA TCG GCA TCC TGG TTC-3'; and the R sequence was 5'-GCT GTG GCG TTG TTG TGG-3'. The β-actin F sequence was 5'-CAC CCT GTG CTG CTC ACC GAG GCC-3'; and the R sequence was 5'-CCA CAC AGA TGA CTT GCG CTC AGG-3'. The relative gene expression was defined as the ratio of gene optical density to that of β-actin.

Statistical analysis All data were analyzed with SPSS11.5 software. The results are expressed as mean±SD. Statistical analysis was made with ANOVA or unpaired Student's t test. A P value less than 0.05 was considered statistically significant. All data met Gaussian distribution and homogeneity tests for variance.

Results All rats survived until sacrifice. Ligation of the common bile duct caused significant increase in levels of serum bilirubin, creatinine and urea nitrogen. Fourteen days after the ligation, the increases were more significant, especially in serum direct bilirubin (66.16±10.24 μmol/L, Table 1). AQP2 expression in the medulla decreased more significantly (38.35±2.08) in the 7-day ligation group than in the SO group (41.06± 1.04), as it was seen in the 14-day ligation group,

Hepatobiliary Pancreat Dis Int，Vol 6，No 6 • December 15，2007 • www.hbpdint.com • 637

Hepatobiliary & Pancreatic Diseases International

Table 1. Serum bilirubin, creatinine and blood urine Groups (days) SO 7 SO 14 OJ 7 OJ 14

DBIL (μmol/L) 2.44±1.25 2.86±3.67 45.95±8.39* 66.16±10.24**

BUN (mmol/L) 7.94±0.81 8.54±0.54# 10.17±1.68 11.61±2.98##

Cr (μmol/L) 25.7±6.02 26.4±5.30△ 30.0±7.54 47.8±14.79△△

DBIL: direct bilirubin; BUN: blood urine; Cr: creatinine.*: Compared with SO 7 days group, F=14.550, t=16.215, P=0.000; **: compared with SO 14 days group, F=7.323, t=18.404, P=0.000; # : compared with SO 7 days group, F=3.856, t=1.940, P=0.065; ## : compared with OJ 7 days group, F=1.178, t=1.329, P=0.200; △: compared with SO 7 days group, F=0.082, t=0.276, P=0.778; △△: compared with OJ 7 days group, F=3.753, t=3.390, P=0.003.

Table 2. AQP2 and AQP2 mRNA expression in each group Groups (days) AQP2 density AQP2 mRNA/β-actin SO 7 41.06±1.04 0.8681±0.1356 SO 14 41.45±0.58* 0.8506±0.1224** OJ 7 38.35±2.08 0.6071±0.1328# OJ 14 31.89±1.57 0.5429±0.1107 F value in each group was 94.958, P