| Heart failure,the terminal stage of various cardiovascular diseases,is one of the major causes of death in modern society.Water retention is an important pathophysiological mechanism of the development of heart failure with many factors involved in the formation and development of it.Among these factors,the expression of aquaporin2?AQP2?on the apical plasma membrane is a key link in the formation of water retention.AQP2 locates in the intracellular vesicles and apical plasma membrane of renal collecting duct principal cells.Its localization can be regulated by arginine vasopressin?AVP?,calcitonin,nitric oxide?NO?,etc.The redistribution of AQP2 regulated by these factors is so-called short-term regulation of AQP2.Obestatin is a 23-amino acid peptide that is derived from the same 117-residue prepropeptide as ghrelin with GPR39 being its possible receptor.Since its discovery,its research has focused on gastrointestinal function and energy metabolism.In recent years,its research has extended to cardiovascular field and it has been found that it took part in blood pressure modulation,endothelial function and reperfusion injury,but we know little about the relationship and heart failure with scare studies aimed to explore the relationship between obestatin and heart failure.Early studies have shown that in patients with heart failure,especially in patients with cardiorenal syndrome,the concentration of plasma obestatin highly increased.In addition,studies have shown that obestatin act in brain to inhibit thirst,reduce water drinking and then adjust the body water balance,but the role of obestatin playing in the water balance on the periphery is not clear.Since GPR39 was expressed in the kidney and obestatin can repair skeletal muscle cells by endocrine and autocrine / paracrine,we hypothesize that obestatin regulates the localization of AQP2 on the apical plasma membrane of renal collecting duct principal cells by endocrine and autocrine / paracrine and then adjusts the water reabsorption of kidney to regulate water retention of heart failure.We constructed rats model of heart failure to detect the changes of obestatin in plasma and kidney.On this basis,we observed the subcellular distribution of AQP2 and the expression of AQP2 or phosphorylated AQP2 at Ser256?P-AQP2 Ser256?in inner medullary collecting duct 3?IMCD3?to define the effect of obestatin on short-term regulation of AQP2 and discuss its possible mechanism.Part ? Expression of Obestatin in the circulation and kidneys of post-myocardial infarction heart failure ratsObjective:To investigate the expression of obestatin in the circulation and kidneys of the post-myocardial infarction heart failure rats and its biologically possible significance.Methods:6 heart failure rat model were constructed successfully by coronary artery ligation whereas sham group rats?n=5?underwent chest operation without ligation.Took five rats as normal group.The volume of water drunk by rats was measured and urine was collected to measure urine volume before and 8 weeks after the operation.Enzyme-Linked Immunosorbent Assay?ELISA?tested the plasma concentration of obestatin.Immunohistochemistry was used to detect the expression of obestatin in the rats' kidneys and Integral Optical Density?IOD?was measured to analyze the difference among different groups.The level of obestatin expressed in the kidneys of different groups was compared by western blot.Results: The volume of water retention in the heart failure group was significantly higher than that in the normal group and the sham group?22.00±5.44 ml vs 5.80±2.59 ml,P<0.01;22.00±5.44 ml vs 3.00±4.69 ml,P<0.01?;the plasma concentration of obestatin in heart failure group was significantly higher than that in the normal group and the sham group?P<0.05?.Immunohistochemistry proved that obestatin was expressed in renal tubule and collecting tubule.The level of obestatin in the kidneys of the heart failure group was significantly higher than that in the normal group and the sham group?P<0.05?.Conclusion: The expression of obestatin in the circulation and kidneys increased in the heart failure group,which might participate in the development of water retention in heart failure by endocrine and autocrine or paracrine secretion.Part ? The short-term regulatory effect of Obestatin on Aquaporin2Objective:To investigate the short-term regulatory effect of Obestatin on AQP2 in mouse renal medullary collecting duct 3?IMCD3?and to explore its possible mechanism.Methods:IMCD3 cells were cultivated and treated with PBS?different concentration of Obestatin(10-7mmol/L ? 10-6mmol/L)? NA-Obestatin(10-7mmol/L)? dDAVP(10-7mmol/L)and OPC-31260(10-7mmol/L)for 15minutes?30minutes and 60 minutes.The localization of AQP2 was detected by confocal microscopy.The expression of AQP2 and P-AQP2?Ser256?was detected by Western Blot and the proportion of P-AQP2 to AQP2 was measured.Results: 1?Compared with the control group?PBS?,the AQP2 distribution showed no change in the confocal observation after treating with 10-7mmol/L Obestatin or 10-6mmol/L Obestatin for 15 minutes;when treated for 30 minutes and 60 minutes,the AQP2 distribution in the cell membrane decreased.After treating with 10-7mmol/L NA-Obestatin for 15 minutes?30 minutes?60 minutes,the AQP2 distribution showed no change compared with the control group.The distribution of AQP2 in the cell membrane increased after treating with 10-7mmol dDAVP for 30 minutes,and the distribution of AQP2 was not changed at 15 minutes and 60 minutes,compared with the control group.Compared with the control group,the distribution of AQP2 was not changed when treated with 10-7mmol OPC-31260 at the time of 15 minutes;at the time of 30 minutes and 60 minutes,the distribution of AQP2 in the cell membrane was decreased.2?There was no significant change in the expression of AQP2 in both concentrations of Obestatin group(10-7mmol/L?10-6mmol/L)?NA-Obestatin group,d DAVP group and OPC-31260 group at the time of 15 minutes when the expression of P-AQP2?Ser256?and the proportion of P-AQP2 to AQP2 in dDAVP group was significantly higher than that in control group?all P<0.05?while the Obestatin group?NA-Obestatin group and OPC-31260 group showing no significant change in that.After treating for 30 minutes,the expression of AQP2 and P-AQP2?Ser256?in OPC-31260 group was significantly lower than that in control group?all P <0.05?,and the phosphorylation ratio increased?P <0.05?while there was no significant change in the other groups about the expression of AQP2 and P-AQP2?Ser256?and the phosphorylation ratio.After treating for 60 minutes,the expression of AQP2 in the both concentrations of Obestatin group? d DAVP group and OPC-31260 group was significantly lower than that in the control group?all P <0.05?and the NA-Obestatin group had no change;the expression of P-AQP2?Ser256?in both concentrations of Obestatin and OPC-31260 group was significantly lower than that in control group?all P <0.05?while no change showing in dDAVP group and NA-Obestatin group;the phosphorylation ratio of dDAVP group was significantly higher than that of control group?P <0.05?and there was no significant change in the other groups.Conclusion: Obestatin had a short-term regulatory effect on AQP2 in IMCD3 cells and reduced its distribution in the cell membrane probably by increasing the endocytosis of AQP2. |
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