The Effects Of The Diuretic And Chinese Traditional Medicine On The Urine Excretion Of Aquaporin-2 Channel Protein In The Normal Rats

[Objective]Aquaporin family, firstly found in early 1990s, has been a hot research topic inrecent years. Its discovery was a milestone for people in the understanding of themechanism of water transportation in cells. Among the aquaporins, aquaporin-2channel protein (AQP2) is one of those known by far that have the mostpathophysiological significance, which plays a very important role in maintaining thewater balance and modulating extracellular fluid volume. AQP2 is predominantlylocated in the apical plasma membrane and apical vesicles inside the collecting ductprincipal cells, and it is primarily regulated in vivo by plasma arginine vasopressin(AVP) via the traditional vasopressin type 2 receptor (V₂R) through two mechanisms:one is the membrane trafficking mechanism and the other is the long-term mechanism.Clinical studies have shown that part of AQP2 can be excreted into the urine, and theexcretion increases with the increasing vasopressin. Thus, the urine AQP2 excretioncan be viewed as an index of the effects of AVP on the kidney collecting ductprincipal cells. It is well known that some physiological or pathophysiologicaldiseases are associated with the altered abundance of AQP2 or with the abnormality of modulating AQP2 in the collecting duct cells. For example, congestive heartfailure, hepatic cirrhosis and nephritic syndrome are associated with alteredabundance of AQP2. Thus, it is an important task to search for certain drugs that caninterfere with the expression of AQP2. Currently diuretic are widely used in clinictherapy, especially in treating the congestive heart failure. Although there are somereports on the influence of diuretic on the urine AQP2, little information is availableon its influence on the mutation of kidney AQP2 gene. Here we conduct a study onthe influence of diuretic on the AQP2 in both urine and kidney under normalconditions. On the other hand, Chinese traditional medicine is a great treasure for ourcountry. According to the known information about Chinese traditional medicine, wehave selected several Chinese traditional medicines for the test in an effort to find oneor two medicines that can interfere with the expression of AQP2.[Methods]Rabbit polyclonal antibodies specifically directed against the C-terminal region ofAQP2 were raised using a peptide (CELHSPQSLPRGSKA) and this peptide wasused as a standard. The rats were housed individually in metabolic cages under acontrolled environment. All rats were given free access to tap food and their urinewas collected.Experiment 1: Furosemide, antisterone and dihydrocholorothiazide were appliedto normal rats by intragastric administration. Urine was collected every day and theurinary AQP2 were measured by an indirect ELISA. After being treated with drugsfor seven days, the rats in each group were anesthetized and the blood was collected.At the time of death, kidneys were dissected on ice and the inner medullary regionswere collected. One of the two inner medullary regions was used to detect AQP2mRNA and V₂R mRNA by RT-PCR, and the other was used to measure AQP2 protein by western blotting.Experiment 2: Eight Chinese traditional medicines were selected for our test.Normal rats received Chinese traditional medicine by intragastric administration for 7days, and the dose was equal to 1.2 g·kg^-1·day^-1 Chinese traditional medicine. Allurine was measured for urinary AQP2 by ELISA.Experiment 3: Normal rats received Grifola Umbellata (GU) and Indian Bread (IB)by intragastric administration for 15 days. At the time of death, kidneys weredissected on ice and the inner medullary regions were collected. One of the two innermedullary regions was used to detect AQP2 mRNA and V₂R mRNA by RT-PCR, andthe other was used to measure AQP2 protein by western blotting.[Results]1. Effects of furose, dihydrocholorothiazide and antisterone on serum Na⁺, urinevolume, urine osmolality and urinary AQP2No significant difference in serum Na⁺ was observed between the three groups(the furose group, the dihydrocholorothiazide group and the antisterone group) andthe control group after 7 days of treatment. All the three groups showed a significantincrease in urine volume, which was of statistical significance (P＜0.001); and theurine volume from the furose group reached its maximum after treating for 4 days.After the treatment, the urine osmolality of the furose group was lower than thecontrol group while that of the dihydrocholorothiazide group was higher, both ofwhich were of statistical significance (P＜0.001). The urine osmolality of theantisterone group, however, showed no difference of statistical significance from thecontrol group.2. Effects of furose, dihydrocholorothiazide and antisterone on the urinary AQP2 concentration and excretion in normal ratsThere was no significant difference in urinary AQP2 concentration among thenormal rats before the treatment. After treating with the three diuretics, the urinaryAQP2 concentration increased significantly (P＜0.001), but there was no statisticallysignificant difference between the three groups. All three diuretics resulted in thesignificant increase in the rate of urine AQP2 excretion (P＜0.001), among which theeffects of furose were more significant than the other two.3. Effects of furose, dihydrocholorothiazide and antisterone on the expression ofAQP2 in kidney inner medulla in normal ratsAfter 7 days of treatment, the AQP2/β-actin mRNA quality and the V₂-R/β-actinmRNA quality of the furose group were significantly higher than the control group (P＜0.001); those of the dihydrocholorothiazide group were significantly lower (P＜0.001); while those of the antisterone group showed no difference of statisticallysignificance.4. Effects of a 7-day treatment with several Chinese traditional medicines on urinevolume and urinary AQP2Among the eight Chinese traditional medicines tested, Ginseng, Tangshen,Chinese Angelica, Plantain Seed and Pepperweed Seed had no effects on altering theurine volume and the urinary AQP2 concentration. However, the Grifola Umbellata(GU) and Indian Bread (IB) groups showed a significant increase in urine volume (P＜0.001) and a statitically significant decrease in urinary AQP2 concentration.5. Effects of a 15-day treatment with GU and IB on serum Na+, urine volume andurine osmolalityNo significant difference in serum Na⁺ was observed between the GU and IBgroups and the control group after 15 days of treatment. However, the GU group and the IB group showed an increase of statistical significance in urine volume (P＜0.001)and a decrease of statistical significance in urine osmolality (P＜0.001).6. Effects of a 15-day treatment with GU and IB on urinary AQP2 in normal ratsThe urinary AQP2 concentration of the GU and IB groups showed a sharpdecrease during the first 5 days of the treatment, which was followed by a continuousincrease until no significant difference was observed on the 15th day of treatmentcompared to the urinary AQP2 concentration prior to treatment. On the other hand,the rates of urine AQP2 excretion of both groups showed a decrease of statisticalsignificance after 5 days of treatment, after which these rates tended to increase andbegan to show a difference of statistical significance after 9 days of treatment. Incomparison with IB, GU had a more significant influence on increasing the urineAQP2 excretion.7. Effects of GU and IB on the expressions of AQP2 gene, V2-R gene and AQP2protein in kidney inner medulla of normal ratsAfter 15 days of treatment, the AQP2/β-actin mRNA quality and AQP2 protein ofthe GU and IB groups were significantly different from those of the control group (F=121.642, P=0.001). The AQP2 mRNA of both groups was significantly lower thanthe control group (P＜0.001), while the change of the GU group was more significant(P＜0.011). On the other hand, both groups showed significantly lower qualities ofV₂-R/β-actin mRNA than the control group, but there was no significant differencebetween the two groups (P＞0.05).[Conclusions]1. All three widely used diuretics, furose, dihydrocholorothiazide and antisterone, canincrease the volume of urine in rats and the excretion of AQP2 protein. Among them, furose decreases the urine osmolality and increases the expression of kidney innermedullary AQP2 and V₂-R genes as well as the expression of kidney AQP2 protein;oppositely, dihydrocholorothiazide increases the urine osmolality and reduces theexpression of the kidney inner medullary AQP2 and V₂-R mRNA; while antisteroneshows little influence on the expression of kidney AQP2 gene.2. Among the eight Chinese traditional medicines tested in the present study, both GUand IB can increase the urine volume and the urine osmolality and reduce the urineAQP2 excretion in the early stage, although later the urine excretion of AQP2 revertsto increase with the progression of treatment. They also help to suppress theexpression of the kidney inner medullary AQP2 and V₂-R genes as well as theexpression of AQP2 protein during the 15 days of treatment. In comparison with IB,GU shows a more significant effect on the expression of AQP2 mRNA and AQP2protein but a similar influence on the expression of V₂-R mRNA.