| Heart failure refers to the syndrome of heart circulation disorder caused by the failure of systolic and/or diastolic functions of the heart and the insufficient outflow of venous blood flow from the heart,resulting in blood pooling in the venous system and insufficient blood perfusion in the arterial system.Heart failure is not an independent disease,but rather the end of the progression of heart disease.Despite many drugs and non-drug treatments available for heart failure,the survival rate for patients with heart failure is still only 50%.With the aging of the population,the number of patients with heart failure has increased dramatically.The treatment of heart failure has become one of the public health problems concerned by the world.Water and sodium retention is the main clinical manifestation of heart failure,especially in patients with advanced decompensated heart failure.Previous studies have shown that the main mechanism is imbalance of fluid and electrolyte balance caused by imbalance of vasoconstriction/sodium retention system(renin-angiotensin-aldosterone system,sympathetic nervous system,arginine vasopressin system)and/or imbalance of vasodilation/sodium excretion system(natriuretic peptide system).Although the application of diuretics,β-receptor blockers,angiotensin converting enzyme inhibitors(ACEI)and angiotensin receptor blockers(ARB)and natriuretic peptide drugs can significantly improve the symptoms and prolong the survival time in patients with heart failure,but for advanced decompensated heart failure with diuretic resistance and/or diluted hyponatremia,still need to explore more convenient and effective treatment measures to solve the water retention of sodium statement and improve the survival rate.In previous clinical practice,our group found that glucocorticoids treatment in patients with heart failure accompanied by water and sodium retention can improve renal function,increase glomerular filtration rate,produce a strong effect of promoting diuresis and improve survival rate.But the pharmacological mechanism is unclear.We through the clinical and animal experiments have confirmed that the glucocorticoids can inhibit the RAAS system,reduce the concentration of the ANG Ⅱand aldosterone,produce powerful diuretic effect.At the same time,we also confirmed through animal experiments that glucocorticoids can up-regulate the expression of NPR-A receptor in the kidney,activate the natriuretic peptide system,thus increasing the blood flow to the kidney,improving renal function and producing strong diuretic effect.It has also been well recognized that patients with HF have abnormal water retention,hypo-osmolality,and hyponatremia secondary to elevations in the levels of the neurohormone arginine vasopressin(AVP).Persistent AVP release regulates downstream aquaporins and sodium channels through renal V2 R activation,leading to water and sodium retention and hyponatremia,which are risk factors for death and hospitalization.and there is a correlation between plasma AVP levels and HF severity/survival of chronic HF patients.Because of the adverse clinical consequences associated with the development of hyponatremia,V2 R antagonists were developed for the treatment of HF patients with hyponatremia.Similarly,does glucocorticoids increase sodium and water excretion by inhibiting V2R? To test the effect of glucocorticoid on the arginine vasopressin system,In this study,acute water loading was given to rats with chronic heart failure to cause a model of advanced heart failure with dilutive hyponatremia,to evaluate the effect of glucocorticoids on left ventricular function in rats,and further explore the molecular mechanism of glucocorticoids promoting water and sodium excretion.Creatinine clearance(Scr),as one of the marker indicators for evaluating glomerular filtration rate(GFR),is susceptible to dietary,physiological,or pathological factors.So it’s not a gold standard for measuring GFR.Cystatin C is expressed stably in most cell and tissues of the human body and is produced at a constant rate.It is completely filtered by glomeruli and reabsorbed and degraded in proximal tubules..GFR is better evaluated than Scr and other endogenous markers because it is generally not susceptible to external factors such as diet,gender,age,and medication.However,previous studies have reported that non-renal factors such as corticosteroids can increase serum cystatin C levels without affecting renal function,and the mechanism is still unclear.Therefore,normal rats were given glucocorticoids,and the gold standard method(inulin clearance)was used to determine the levels of GFR and cystatin C in circulation and tissues to investigate the effect of glucocorticoids on cystatin C and its mechanism.Part One Glucocorticoids improve water and sodium excretion and leftventricular function in heart failure ratsObjective: Most patients with heart failure are accompanied by renal dysfunction,which further aggravates the symptoms of heart failure.A vicious circle is formed between heart failure and renal dysfunction,thus aggravating the heart and kidney injury and deteriorating the disease condition.Our previous clinical studies have shown that glucocorticoids can improve renal function in patients with chronic decompensated heart failure and produce a strong diuretic effect.However,there are no clear experimental evidences for assessing cardiac function after diuretic action.The purpose of this study was to determine the role of glucocorticoids in improving cardiac and renal function in rats with chronic heart failure with diluted hyponatremia.Methods:1.Left coronary artery ligation was used to cause myocardial infarction model in rats,and chronic heart failure was formed after 12 weeks.The acute water loading model with diluted hyponatremia was established by intraperitoneal injection of 6% body weight deionized water.2.Experimental rats were randomly divided into four groups: sham operation group(CON),heart failure group(CHF),glucocorticoids treatment group(DEX),and glucocorticoids inhibitor group(RU486+DEX).3.The improvement of water and sodium retention by glucocorticoids was evaluated by urine volume,urine sodium,urine osmotic pressure,blood sodium,blood osmotic pressure and other indicators.4.To evaluate the effect of glucocorticoids on renal function in rats with heart failure.5.Left ventricular function in rats with heart failure was evaluated by Millar pressure-volume catheter.Results:1.Physiological parametersIn the acute water loading test,the renal water excretion ability of the heart failure rats was significantly impaired compared with the sham group.Under the combined action of chronic heart failure and acute water loading,CHF rats showed as follows: Less urine,increased blood sodium,decreased blood osmotic pressure,decreased urine sodium,increased urine osmotic pressure.Significantly,within 6 hours after dexamethasone pretreatment,the rats showed a significant increase in urine volume(P < 0.01).At the 6th hour of drug pretreatment,6% body weight water loading was given,followed by dexamethasone-pretreated heart failure rats still exuded additional free water intake(P < 0.01).Due to increased drainage induced by dexamethasone,blood sodium and blood chlorine in rats with heart failure were restored to normal levels,and dilutive hyponatremia in rats with heart failure was successfully reversed.In addition,DEX pretreatment could reverse the decrease in plasma osmotic pressure induced by acute water loading in heart failure rats.DEX results in an increase in urine volume accompanied by an increase in urinary sodium.The beneficial effects of DEX pretreatment can be offset by glucocorticoid receptor(GR)antagonist RU486,suggesting that these effects are GR mediated.2.Effects of glucocorticoids on renal function We studied the effect of glucocorticoid on renal function in rats with heart failure.Compared with the CON group,the creatinine clearance rate of rats with heart failure was significantly reduced(P < 0.01)and the serum creatinine was increased(P < 0.05).Dexamethasone could increase the creatinine clearance rate and decrease the serum creatinine(P < 0.01).This beneficial effect can be blocked by the glucocorticoid receptor(GR)antagonist RU486,indicating that these effects are GR mediated.3.Effect of glucocorticoid on myocardial fibrosis in rats with heart failureWe studied the effect of dexamethasone on cardiac collagen by Western blot and masson staining.We found that dexamethasone inhibit the expression of collagen Ⅰ,but does not affect the expression of collagen Ⅲ.Masson staining also confirmed the inhibitory effect of dexamethasone on cardiac collagen.4.Hemodynamic parametersWe studied the effect of DEX on left ventricular function in heart failure rats through pressure-volume catheter.The end-systolic pressure-volume relationship(ESPVR),maximal slope of systolic pressure increment occurs early during isovolumic contraction(d P/dtmax),preload recruitable stroke work(PRSW),isovolumic relaxation constant(Tau),left ventricular end-diastolic pressure(LVEDP)were collected or calculated.The left ventricular end-diastolic pressure(LVEDP),i.e.the preload of the heart,is the load of the cardiac muscle fibers before the heart contracts,i.e.the blood flow to the ventricles at the end of the diastolic period.LVEDP is the most important marker of heart failure.LVEDP increases when heart failure occurs and goes up when heart failure becomes worse or decompensated.An increase in LVEDP means occurrence of volume overload or fluid retention.The greater the preload,the more pressure is available for the next cardiac contraction.End-systolic pressure volume relationship(ESPVR)describes the maximal pressure that can be developed by the ventricle at different LV filling volumes.ESPVR is considered to be the most reliable index of ventricular contractility.In the experiment,we changed the preload by blocking the inferior vena cava of rats,and plotted the pressure at the end point of ventricular contraction and the corresponding volume value under different loads,so as to get a straight line with a certain slope.ESPVR flattens and shifts to the right during heart failure,representing a decrease in myocardial contractility.Because ESPVR is independent of changes in preload,afterload,and heart rate,it is a better indicator of systolic function than other hemodynamic parameters such as ejection fraction,cardiac output,d P/dtmax,and stroke volume.The stroke work,i.e.cardiac contractility,not only varies with afterload but also with preload.Therefore,the linear relationship between stroke work and end-diastolic volume is used as the index to measure cardiac contractility,which is called preload recruitable stroke work(PRSW).It is insensitive to preload by definition,but it is also remarkably insensitive to changes in afterload at the expense of lower inotropic sensitivity.During heart failure,myocardial contractility is reduced,and the slope of the PRSW becomes shallow.The maximal slope of systolic pressure increment(d P/dtmax)occurs in the early stage of isovolumetric contraction and is one of the parameters associated with cardiac contractility.However,d P/dtmax is affected by preload,afterload,heart rate,and cardiac hypertrophy,makes it a poor contractility index.Isovolumic relaxation constant(Τau)represents the exponential decay of the ventricular pressure during isovolumic relaxation.Several studies have shown that Tau is a preload-independent measure of isovolumic relaxation.With an increased tau(i.e.,slowing of relaxation),a higher mean left atrial pressure is required to achieve normal filling volumes.In heart failure,there is an increase of LVEDP and right atrial pressure.Isovolumic relaxation is impaired and Τau is prolonged.Result shows a typical changes in left ventricle pressure-volume loop throughout the cardiac cycle in four groups.The data showed that the rats with CHF had much lower ESPVR(red line in the loops)than those normal controls.DEX restored ESPVR in the rats with CHF.There was no differences on d P/dtmax and PRSW between the four groups.The data also indicated that the rats with CHF had an increased Tau(i.e.,slowing of relaxation)compared with those normal controls,and DEX could remarkably decrease Tau.It is of note that acute water load resulted in a dramatic increase of LVEDP in the rats with CHF,and LVEDP was decreased by DEX.These favorable effects induced by DEX were abolished by RU486.Part Two Glucocorticoid reverses water excretion in rats with heart fail-ure through arginine vasopressin pathwayObjective: Decompensated heart failure with hyponatremia,cardiac output decreases,the relative lack of effective circulating blood volume not only activate the sympathetic nervous system,the RAAS system,has also led to AVP release increased significantly,AVP V2 receptor(AVP V2R)expression increased,so as to activate downstream adenylate cyclase,promote the manifold water channel protein expression increased,the body ‘s ability to drain water decreases,eventually lead to water retention dilution and hyponatremia.The purpose of our study was to investigate whether glucocorticoids can increase the excretion of free water by inhibiting the V2R-AQPS pathway in animals.Methods: Quantitative Western Blot was used to detect the expression of V2 receptor in renal medulla,and immunohistochemistry was used to determine the expression site of V2 receptor.The expressions of aquaporin 2(AQP2)and aquaporin 3(AQP3)in renal internal medullary were detected by quantitative Western Blot,and the expression sites of aquaporins were determined by immunohistochemistry.Results:1.Effects of glucocorticoid on plasma AVP in rats with heart failurePrevious studies have found elevated plasma AVP levels in CHF patients with hyponatremia.In order to further study the effect of glucocorticoid on the plasma AVP level,we measured the plasma AVP level of rats in each group by ELISA.We did not find significant statistical difference in plasma AVP level between CHF group and normal rats(P > 0.05),which may be related to the significant decrease in plasma osmotic pressure and the temporary downregulation of AVP level in heart failure rats after water loading.Consistent with our hypothesis,dexamethasone treatment significantly reduced plasma AVP levels in heart failure rats(P < 0.05).This suggests that the diuretic effect of dexamethasone is achieved by regulating the arginine vasopressin pathway.However,the plasma AVP level of DEX+RU486 group was abnormally increased(P < 0.01).2.Glucocorticoid down-regulated V2 R expression in heart failure ratsRenal reuptake of water is mainly regulated by V2 R expression in the lateral basolateral membrane of the collecting duct.In the study of heart failure rats,it was found that the increase of serum AVP concentration led to the up-regulation of downstream AQP2,which was the main cause of water retention in heart failure.In order to study the effect of glucocorticoid on renal V2 R in heart failure rats,we detected the expression of V2 R in renal internal medulla by Western blot.The results showed that the expression of V2 R in CHF rats was higher than that in the control group(P < 0.05).However,DEX treatment reduced the V2 R of CHF collection tubes(P < 0.05).The inhibitory effect of DEX on V2 R can be blocked by GR antagonist RU486(P < 0.05)(Fig 2A).This suggests that the inhibitory effect is mediated by glucocorticoid receptors.Immunohistochemical results suggested that V2 R was located on the basolateral membrane side of the collecting tube and further verified WB results(Fig 2B).3.Glucocorticoid down regulated the expression of AQP2 in renal medullary tissue of rats with heart failureActivation of V2 R regulates AQP2 shuttle to apicle surface,resulting in increased permeability of the kidneys to free water.In order to determine the effect of glucocorticoids on AQP2,the expression and site of AQP2 protein were detected by western blot and immunohistochemistry.AQP2 is expressed in the apical membrane of the collecting tube of the inner medullum of the kidney,and the western blot has a protein band at 25 KD and a glycolated protein band at 35-45 kd.We found that compared with the sham operation group,AQP2 was significantly increased in the renal medullating duct of the heart failure rats(P < 0.05),while DEX pretreatment significantly reduced the expression of AQP2(P < 0.05).Glycosylated AQP2 expresses the same trend(Fig 3A).We identified the histological localization of AQP2 by immunohistochemistry and further confirmed the results of WB.(Fig 3 B)4.Glucocorticoid down regulated the expression of AQP3 in the renal medulla of rats with heart failureActivation of V2 R regulates AQP3 to shuttle to the basolateral membrane and reabsorb water from the host cells into the blood.To determine the effect of glucocorticoids on AQP3,their expression was detected by western blot and immunohistochemistry.AQP3 was expressed in the basement membrane of the collecting duct of the inner medullum of the kidney.There was a protein band at 32 KD and a glycolated protein band at 40-55 kd of the western blot.We found that the expression of AQP3 in the inner medullated collection tube in heart failure rats was significantly increased compared with that in the sham group(P < 0.05),and the expression of AQP3 in the glycinated group was significantly decreased by DEX(P < 0.01)(Fig 4A).We confirmed the histological localization of AQP3 by immunohistochemistry and further confirmed the results of WB.(Fig 4 B)Part Three Glucocorticoid reverses sodium excretion in heart failurerats through arginine vasopressin pathwayObjective: V2 R also activates sodium channels during heart failure,increasing the expression of sodium channels in the renal intramedullary and leading to sodium retention in the body.This part of the experiment determined the regulatory effect of glucocorticoid on salt metabolism in heart failure rats through the study of sodium channel and serum and glucocorticoid-induced kinase 1(SGK1)and epithelial sodium channel(ENa C),sodium potassium dichlorocotransporter(NKCC2)and sodium potassium atpase(NKA).Methods: The expression of SGK1 in renal medulla was detected by quantitative Western Blot.The expressions of ENa C,NKCC2 and NKA in the collecting tube of the renal medulla were detected by quantitative Western Blot.Immunohistochemistry was used to determine the expression sites of sodium channel related proteins.Results:1.Glucocorticoid down-regulated alpha-enac expression in renal medulla of heart failure ratsThe abundance of ENa C expression in kidney of rats with heart failure and diluted hyponatremia was increased(P < 0.05).Pretreatment with dexamethasone could decrease the expression of ENa C in the kidney of heart failure rats(P < 0.05).GR inhibitor RU486 reversed this effect of dexamethasone(P < 0.05).It is suggested that the inhibition of ENa C by dexamethasone is mediated by glucocorticoid receptor.2.Glucocorticoid down-regulated NKCC2 expression in renal medullary tissue of heart failure ratsThe expression of NKCC2 in renal internal medulla of rats with heart failure and diluted hyponatremia was significantly higher than that of the sham group(P < 0.05).Dexamethasone pretreatment significantly reduced the expression abundance of NKCC2 in heart failure rats(P < 0.05).However,rats given RU486 did not reverse this effect of dexamethasone(P > 0.05).This indicates that the inhibition of NKCC2 by dexamethasone may not have passed the classical GS pathway.3.glucocorticoid had no effect on the expression of NKA in renal intramedullary tissues of rats with heart failureThe results showed that the expression of NKA in the CHF group was not statistically different from that in the sham operation group(P > 0.05).Similarly,dexamethasone pretreatment had no effect on NKA expression in heart failure rats(P > 0.05).4.Glucocorticoid down-regulated SGK1 expression in renal medullary tissue of heart failure ratsWe found that the expression of SGK1 in the kidney of heart failure rats was significantly higher than that of the sham group(P < 0.05).Pretreatment with dexamethasone could down-regulate the expression of SGK1 in the kidney of rats with heart failure(P < 0.05).However,no GS inhibitor RU486 was found to reverse the expression of SGK1 in heart failure animals(P > 0.05).Part Four Corticosteroids significantly increase cystatin C levels in thecirculation by promoting cystatin C production in ratsObjective: Cystatin C is a new endogenous GFR marker,but there are also reports of diabetes,thyroid disease,acute kidney injury,cancer,heart dysfunction and steroid drugs that have a significant effect on the level of Cystatin C.Therefore,we designed this study to determine the effect of glucocorticoids on circulation and renal function of Cystatin C,and to ensure that glucocorticoids can increase the production of cystatin C in tissues.Methods:1.SD rats were randomly divided into 3 groups: normal control group(CON),dexamethasone group(DEX),and glucocorticoid inhibitor group(DEX+RU486).2.Measurement of inulin clearance.3.Enzyme-linked immunosorbent(ELISA)assay for cystatin C in kidney,brain,intestine,liver and lung.Results:1.Effects of DEX on plasma cystatin C levels and inulin clearanceAfter 2-day treatment,the rats receiving DEX had much higher cystatin C levels in the circulation compared with those treated with vehicle.The effect of DEX on cystatin C levels was totally abolished by glucocorticoid receptor(GR)antagonist RU486.However,there was no different between three groups in renal inulin clearance.2.Effect of DEX on the production of cystatin C in tissuesTo test the effect of DEX on production of cystatin C,we collected five organ tissue homogenates,such as kidney,brain,intestine,liver and lung.The cystatin C levels were consistently higher in DEX-treated rats compared those treated with vehicle.Consistent with the findings in plasma cystatin C levels,the effect of DEX on cystatin C in the above tissues was also abolished by RU 486.Conclusions:1.In rats with heart failure,water and sodium excretion in the kidney is reduced and cardiac function is severely impaired,resulting in water and sodium retention and dilutive hyponatremia.Dexamethasone treatment can significantly increase the water and sodium excretion capacity,restore normal plasma osmotic pressure,and correct dilutive hyponatremia in heart failure rats.Dexamethasone can also increase the systolic force of the left ventricle in heart failure rats,restore normal left ventricular end-diastolic pressure,and improve the left heart function in heart failure rats.The effects of dexamethasone on sodium excretion,diuresis and cardiac function were eliminated by glucocorticoid receptor inhibitor RU486,suggesting that these effects were mediated by glucocorticoid receptors.2.The plasma AVP level and renal V2 R expression of heart failure rats were significantly higher than those of normal rats.Dexamethasone pretreatment can reduce plasma AVP and down-regulate renal V2 R protein expression abundance.The down-regulation effect of dexamethasone on plasma AVP and renal V2 R can be eliminated by RU486.It indicates that glucocorticoid can down-regulate the arginine vasopressin system.3.The increased expression of AQP2 and AQP3 in renal medullary collecting ducts in heart failure rats resulted in impaired water excretion in vivo,and dexamethasone increased water excretion by down-regulating the expression of AQP2 and AQP3 in kidney.At the same time,heart failure also causes increased expression of ENa C and NKCC2 in the kidney,which causes sodium excretion disorder.Dexamethasone increases sodium excretion by down-regulating the expression of ENa C and NKCC2 in the kidney.These results indicate that glucocorticoid regulates the protein expression of the downstream sodium hydrate channel through the arginine vasopressin pathway and increases sodium hydrate excretion in rats with heart failure associated with diluted hyponatremia.4.Corticosteroids could promote the production of cystatin C in the tissues,resulting in an elevation in serum cystatin C without negatively affecting kidney function. |
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