| [Objective]Aquaporin-2 channel protein (AQP2) is of the most pathophysiological significance among aquaporins by far, which takes a very important role in maintaining the water balance and modulating extracellular fluid volume. AQP2 is predominantly located in the apical plasma membrane and apical vesicles in the collecting duct principal cells. AQP2 is primarily regulated by plasma arginine vasopressin (AVP) via the traditional vasopressin II style receptor (V2R) in vivo through two mechanisms; one is the membrane trafficking mechanism, another is the long-term mechanism. Clinical studies have shown that AQP2 is excreted into the urine, and the excretion increases in response to vasopressin. The urine AQP2 excretion can be regarded as a maker of the affects of the AVP upon the kidney collecting duct principal cells. Therefore, it is necessary to develop an efficient and stable way to detect the urinary AQP2. Based on our previous work, we tried to develop an efficient and stable enzyme-linked immunosorbent assay (ELISA) for measuring urinary AQP2. Using this method, we wanted to observe the rules of excretion of urinary AQP2 under various hydration conditions. As we known, some physiological or pathophysiological conditions are associated with altered abundanceof AQP2 or with abnormality of modulating AQP2 in collecting duct cells. For example, congestive heart failure, hepatic cirrhosis and nephritic syndrome are associated with altered abundance of AQP2. So, it have become an important task to look for some drugs interfering with expression of AQP2. Captopril and Losartan are applied widely to block the renin-angiotensin system (RAS), especially in treating the congestive heart failure. So, we observe their effects upon AQP2 in kidney and urine of the normal rats. In addition, Chinese traditional medicine is a great treasure. Depending on information about Chinese traditional medicine, we selected Astragalus Memberanaxeus (AM), Alisma Orientalis (AO) and Grifola Umbellata (GU) as the targets of our test. [Methods]Rabbit polyclonal antibodies specifically directed against the C-terminal region of AQP2 were raised using a peptide (CELHSPQSLPRGSKA) and this peptide was applied for a standard. The rats were housed individually in metabolic cages and in controlled environment. All rats were allowed free access to tap food and the urine was collected. 1, In order to develop a stable and efficient indirect ELISA to detect the urinary AQP2, urine outputs of Sprague-dawley rats were collected under normal, dehydrated and water loading (80ml/kg) conditions. We used indirect ELISA to detect rat urinary AQP2 excretion under various water metabolic conditions and observed SDS effects upon the results. Meanwhile, the sensibility and the coeffiecient of variation were studied. 2, Captopril and Losartan were applied upon normal rats by intragastric administration. Urine was collected everyday and the urinary AQP2 were measured by indirect ELISA. After treated with drugs for one day, the half rats in every group were anesthetized and the blood was collected. At the time of death, kidneys were dissected on ice and inner medullary regions were removed. After seven days with drugs, the rest half rats were killed in the same way .One of the two innermedullary regions was used to detect AQP2 mRNA and V2R mRNA by RT-PCR. Meanwhile, another was measured for AQP2 protein by western blotting. 3, three kinds of Chinese traditional medicine, AM, AO and GU, were taken as the targets of our test. Normal rats received the three kinds of Chinese traditional medicine by intragastric administration respectively for 7 days, and the dose was equal to 1.2 g-kg^-day"1 Chinese traditional medicine. All urine was measured for urinary AQP2 by ELISA. If the urinary AQP2 concentration was abnormal, two sides of kidney inner medulla would be studied for AQP2 and V2R mRNA and AQP2 protein. [Results]1, It was an important element that urine was treated with SDS previously, because SDS could increase the absorption of urinary AQP2. The rat urine diluted with 0.05% SDS-PBS was optimal to indirect ELISA.2, The indirect ELISA was able to detect as low as 112.5fmol/ml urinary AQP2 with 6.5% and 7% of intra- and inter-assay CVs. After dehydration for 24 hours, urinary AQP2 excretion increased (P<0.01); subsequently rats were given oral water loading, urinary AQP2 excretion drastically decreased (P<0.01). Urinary AQP2 excretion was positively correlated with urine osmolality.3, Captopril and Losartan upon effects of serum Na+, urine volume, urine osmolality and urinay AQP2.The serum Na+ had no significance among the three groups (the normal, captopril and losartan) treated with drugs. With rats treated with drugs, the urine volume of the captopril and losartan groups was higher. The urine osmolality and urinary AQP2 concentration in losartan group had no significance during the cause with this drug. The urine osmolality of captopril group was lower and the osmolality at the beginning, in first day, in seventh day received treatment is 1678+257.8 mosm/kg H2O, 1446.7±215.6 mosm/kg H20,1434±214.9 mosm/kg H2O respectively. But the urinaryAQP2 in captopril group increased from 5.6±1.3 ng/ml at the beginning, 9.2±1.7 ng/ml in the first day with drug to 6.9±2.0 ng/ml in the seventh day with drug.4, Captopril and Losartan on the effects of the expression of AQP2 in kidney inner medulla in normal rats.Compared with the normal, the V2R mRNA of the Captoril and Losartan was no significance in the first and seventh day with drugs respectively. Compared with the normal, the AQP2 mRNA and the AQP2 protein of the Losartan group also were no significance in the first and seventh day with drug respectively. Compared with the normal group, the AQP2 mRNA of the captopril group was reduced by 0.28 and 0.31 in the first and seventh day with drug respectively. Consequently, the AQP2 protein of the captopril was reduced by 0.32 and 0.25 in the first and seventh day with drug respectively. The AQP2 mRNA and protein of the captopril had no significance between the first and the seventh day with drug.5, AM, AO and GU effects upon urine volume, urine osmolality and the urinay AQP2.The urine volume of the AM group was no significance before and after the drugs. The urine volume of the AO group was no significance in statistics, but the urine increased from 10.75±4.2 ml at beginning without drug to 14.25±3.8ml in the seventh day with the drug. The urine volume of the GU increased signicantly from the 9.8±4.2 ml in beginning without drug to 15.8 ± 6.2 ml in the seventh day with the drug.The urinary AQP2 concentration of the AM and AO groups has no change significantly before and after drugs. The urinary AQP2 concentration of the GU group decreased significantly from the 9.47±2.2 ng/ml at beginning without drug to 3.8±2.0 ng/ml in the seventh day with the drug. Consequently, the urinary AQP2 excretion of the GU group decreased signicantly from 560±180 fmol/umol at beginning without drug to 328±32 fmol/umol (P=0.012) in the seventh day with the drug.Comparedwith the normal group, the AQP2 mRNA> V2R mRNA> AQP2 protein of GU had nosignificant change.[Conclusion]1,0.05% SDS is optimal to detect urinary AQP2.2, Indirect ELISA was able to detect as low as 12.5fmol/ml urinary AQP2 with 6.5% and 7% of intra- and inter-assay CVs.3, Captopril and Losartan can increase the urine volume in normal rats, but captopril could also reduce the expression of the kidney inner medullary AQP2 and accelerate the excretion of the urine AQP2 in normal rats.4, The AO and GU could increase the urine volume in normal rats, and the GU also reduced the excretion of the urinary AQP2. But in our test, the GU had no effects upon the expression of AQP2 of kidney inner medulla in normal rats.
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