The Mechanism Study On Effects Of Hoelen And Qiliqiangxin On The Renal Express Of Aquaporin-2 In Rats With Heart Failre

[Objective]The progressive decrease in left ventricular function is the feature of chronic heart failure（CHF） which results in activation of several neurohormonal compensatory systems,including the sympathetic nervous system,the renin-angiotensin-aldosterone system（RAAS） and arginine vasopressin（AVP）.These compensatory mechanisms serve to maintain perfusion of vital organs but have several deleterious consequences. Renal perfusion and glomerular filtration rate（GFR） decrease and tubular reabsorption of sodium and water increases,results in clinical problems of fluid overload as well as significant morbidities and even mortality,such as acute pulmonary edema.The pathogenic mechanism of fluid overload is not fully understood,and activation of RAAS resulting in salt and water retention has been shown to play a role.On the other hand,this cannot fully explain the pathological changes in CHF subjects as hyponatremia is not uncommonly encountered in these patients.Therefore,water retention is unlikely to be just a passive event following sodium retention.Instead,defective regulation of water reabsorption may play a direct role.AVP is secreted by the posterior pituitary gland in response to reduced cardiopulmonary blood volume,reduced systolic blood pressure,or increased plasma osmolality.Similar to other neurohormones that are activated in CHF,circulating AVP is elevated in patients with CHF.The rate of water reabsorption in the collecting ducts（CDs） is determined by transepithelial water permeability,which is regulated by AVP.AVP regulates water permeability in CDs by increasing the expression and plasma membrane targeting of the membrane-bound water channel aquaporin-2 （AQP-2）.The AQPs are a family of membrane water channels that mediate rapid water transport across cell membranes.AQP-2,the major water channel responsible for water balance,is the predominant vasopressin-regulated water channel of the kidney collecting duct.AQP-2 is localized in the apical plasma membrane and cytoplasmic vesicles in the principal cells of collecting duct,especially in the inner medullary collecting duct.Due to its expression in the final segment of urine production,AQP-2 is a critical water channel in the kidney.Multiple studies have showed that AQP-2 plays the essential roles of water permeability and body water balance in collecting duct,which is tightly regulated by vasopressin.Vasopressin binds to vasopressin binds to vasopressin type 2 receptor（V2-R） located on the principal cells of kidney collecting duct.Water reabsorption in the collecting duct is regulated both by short-term and longterm mechanisms,both of which have been shown to depend critically on AQP-2.Short-term regulation occurs as a result of exocytic insertion of AQP-2 water channels into the apical plasma membrane in response to vasopressin. In addition,long-term regulation of AQP-2 expression has recently been shown to play a major role for regulation of CDs water reabsorption..Although it is clear that an increased baroreceptor-mediated vasopressin release play a critical role for the renal water retention,it is not the only one.Evidence for this comes from a variety of experiments which show that AQP-2 expression can be regulated by vasopressin- independent signaling pathways.Formerly,we demonstrated that in rats with CHF, non-osmotic,baroreceptor-mediated stimulation of AVP resulted in activation of AQP-2 expression in the renal CDs,which accounted for extracellular volume expansion and,eventually,formation of edema..Therefore,down-regulation of renal AQP-2 expression may play a crucial role in the control of CHF symptoms and even the prognosis.Currently diuretic are widely used in clinic therapy,especially in treating the CHF. However,there is little information available on the influence of traditional Chinese medicine.Traditional herbal medicines are widely used in Chinese.Hoelen,a crude herbal drugs,was used as a diuretic and sedative in traditional medicine.Formerly,we demonstrated that the expression of AQP-2 in kidney were decreased obviously in normal rats by the treatment of hoelen.This study was conducted to investigate whether an aqueous extract of hoelen was effective in suppressing the overexpression of renal AQP-2 at the transcriptional and translational levels in a rat with CHF.At the same time,the loop diuretic,frusemide,was used as control.Qiliqiangxin capsule（GLQX） is a traditional Chinese medical formulation.The formula of GLQX has been used to treat CHF,which improved cardiac function, urine volume,and subjective symptoms of patients.We investigated whether GLQX was effective in improving cardiac function and suppressing the overexpression of renal AQP-2 in a rat model of CHF.Chinese traditional medicine is a great treasure for our country.According to the known information about Chinese traditional medicine,we have selected several Chinese traditional medicines for the test in an effort to find one or two medicines that can interfere with the expression of AQP-2. [Methods]Rabbit polyclonal antibodies specifically directed against the C-terminal region of AQP-2 were raised using a peptide（CELHSPQSLPRGSKA） and this peptide was used as a standard.The CHF model was established by ligating the left anterior descending coronary artery to induce myocardial infarction,cardiac geometry and function were evaluated by a doppler echocardiographic system equipped with a 15-MHz linear transducer.The rats were housed individually in metabolic cages under a controlled environment.All rats were given free access to tap food and their urine was collected. 24-h urine was collected throughout the study,urine volume and urinary AQP-2 were determined daily.At the end of 4 weeks,rats were sacrificed and trunk blood was collected in heparinized tubes for the determination of plasma sodium,osmolality, creatinine,and hormones.Rapidly,the kidneys were removed and dissected into cortex and medulla.The renal medulla was snap frozen in liquid nitrogen and stored at—80℃for subsequent protein and RNA extraction.Half of the kidneys were make frozen section for immunofluorescence.The rats were randomized into CHF group（CHF）,high dosage hoelen treated group（HH）,medium dosage hoelen treated group（HM）,low dosage hoelen treated group（HL）,high dosage Qiliqiangxin treated group（QH）,medium dosage Qiliqiangxin treated group（QM）,low dosage Qiliqiangxin treated group（QL）,furosemide treated group（F） and control group（CTL）.These groups received different treatments for 1 or 4 weeks via intragastric administration.Experiment 1:High dosage hoelen(2.4 g·kg^-1·d^-1),medium dosage hoelen(1.2 g·kg^-1·d^-1),low dosage hoelen(0.6 g·kg^-1·d^-1) and furosemide(20 mg·kg^-1·d^-1) were applied to CHF rats by intragastric administration.Urine was collected every day and the urinary AQP-2 were measured by an indirect ELISA.After being treated with drugs for 1 or 4 weeks,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 regions were collected.One of the two inner medullary regions was used to detect AQP-2 mRNA and V2R mRNA by Real-time PCR,and the other was used to measure AQP-2 protein by western blotting.Experiment 2:High dosage qiliqiangxin(1.0 g·kg^-1·d^-1),medium dosage qiliqiangxin(0.5g·kg^-1·d^-1),low dosage qiliqiangxin(0.25 g·kg^-1·d^-1),were applied to CHF rats by intragastric administration.Urine was collected every day and the urinary AQP-2 were measured by an indirect ELISA.After being treated with drugs for 1 or 4 weeks,the rats in each group were anesthetized,Cardiac geometry and function were evaluated by a doppler echocardiographic system,and the blood was collected.At the time of death,kidneys were dissected on ice and the inner medullary regions were collected.One of the two inner medullary regions was used to detect AQP-2 mRNA and V2R mRNA by Real-time PCR,and the other was used to measure AQP-2 protein by western blotting.[Results]1.Changes of CHF rats on the expression of renal AQP-2 and cardiac functionThe urine volume was significantly decreased in CHF-1 rats compared with CTL-1 groups（P＜0.01）.Accordingly,the urinary osmolality was significantly increased.Moreover,the expression of renal AQP-2 mRNA and AQP-2 protein were obviously increased in CHF-1 groups（P＜0.01）.The value of EF and FS were significantly decreased in CHF-1 rats compared with those in CTL-1 rats （P＜0.01）.Plasma AVP and BNP levels of CHF-1 rats were higher than the CTL-1 rats.Similarly,the urine volume was significantly decreased in CHF-4 rats,and the expression of renal AQP-2 mRNA and AQP-2 protein were significantly increased, both of which were of statistical significance（P＜0.001）.2.Effects of hoelen on the expression of renal AQP-2 and cardiac function in CHF ratsAs expected,the continuous administration of hoelen at a dose of 2.4g·kg^-1·d^-1, 1.2 g·kg^-1·d^-1 and 0.6 g·kg^-1·d^-1 for 1 week caused a significant increase in urine volume.The expression of renal AQP-2 mRNA and AQP-2 protein in HH-1,HM-1 and HL-1 groups were considerablely depressed than that in CHF-1 group.In particular, the aquaretic effect of hoelen was a dose-dependent manner,suggesting that this effect is based on a pharmacological property.Interestingly,the plasma AVP levels and V2-RmRNA were unchanged.The expression of renal AQP-2 mRNA,AQP-2 protein and V2-R mRNA were significant decreased in HH-4,HM-4 and HL-4 groups.The plasma BNP was also significant decreased.3.Effects of furosemide on the expression of renal AQP-2 and cardiac function in CHF ratsBoth F-1and F-4 groups showed a significant increase in urine volume,which was of statistical significance（P＜0.001） and a statitically significant decrease in urinary osmolality.On the 9 days treatment with furosemide,the maximum effect of diuretic was seen.Although urine volume was reduce after 9 days,it still higher than CHF groups.However,the value of EF and FS were not changed in both F-1 and F-4 groups（P＞0.05）.There was no significant difference in plasma AVP levels or the expression of renal AQP-2 between the F-1 group and CHF-1 group.However,the expression of renal AQP-2 in F-4 group were significantly increased,and the plasma AVP was also increased. 4.Effects of qiliqiangxin on the expression of renal AQP-2 and cardiac function in CHF ratsThe continuous administration of qiliqiangxin for 1 week caused a significant increase in urine volume.However,the plasma AVP levels of the QH-1,QM-1 or QL-1 group showed no difference in statistical significance from the CHF-1 group.The index of cardiac function were not changed in QH-1,QM-1 or QL-1 group（P＞0.05）. Similarly,daily urine output was significantly increased and urinary concentration was marked decreased during 4 weeks treatment with Qiliqiangxin.The plasma BNP was significantly decreased in QH-4,QM-4 or QL-4 group,while the change of the QH-4 group was more significant（P＜0.01）.On the other hand,the AQP-2 mRNA and AQP-2 protein of the QH-4,QM-4 or QL-4 group were significantly decreased from those of CHF-4 group.[Conclusions]1.Our findings suggest that one week hoelen treatment exhibits an aquaretic effect in rats with CHF.This mechanism of action may be associated with inhibition of AQP-2 in the renal medulla through vasopressin-independent signaling pathways.2.Four weeks hoelen treatment decreased the expression of renal AQP-2 mRNA and AQP-2 protein.Accordingly,the urine volume was significantly increased,and the urinary osmolality was significantly decreased.The expression of renal AQP-2 protein and the cardiac function may be causal relation mutually.3.After one week treatment of furosemide,daily urine output was significantly increased compared with CHF-1 rats,while furosemide shows little influence on the expression of kidney AQP-2.4.Four weeks treatment with furosemide increased the vasopressin level,which stimulated water reabsorption via the renal APQ2.These changes are most likely compensatory phenomena,which prevent an excess loss of sodium and water.5.In one week treatment with qiliqiangxin,the urine volume was significantly increased,however,there are no evidence show that qiliqiangxin repressed the expression of renal AQP-2 directly.6.Four weeks qiliqiangxin treatment improved the cardiac function of CHF rats, which partly contributes to attenuating the expression of AQP-2 in kidney.