Study On The Effect Of Mir-34A On Indoxyl Sulfate Mediating Heart Function Injury In Cardiorenal Syndrome

Cardiorenal syndrome (CRS) is a condition in which a complex inter-relationship between cardiac dysfunction and renal impairment co-exists.A diseased heart has numerous negative effects on kidney function, whist renal insufficiency can significantly impair cardiac function leading to the progression of failure of both organs.CRS includes different kinds of dysfunction of heart and kidney,acute or chronic dysfunction of either organ may induce acute or chronic dysfunction of the other. The primary failing organ can be either heart or kidney.Systemic conditions leading to simultaneous injury and/or dysfunction of the heart and kidney are also proposed to be one of the subtypes of CRS.Since heart diseases and kidney diseases are of higher and higher incidence nowadays,the mobility of CRS has been keeping raising,what makes CRS a important topic needed to be solved.At present,we haven’t made much progress on the understanding and prevention of CRS。Delving into its pathophysiology mechanism has great basic theoretical value and clinical guidance value.Heart-kidney interaction is the key to CRS developing, but its pathophysiology mechanism has not been illuminated.There are common features on pathomechanism among different subtypes of CRS:The primary failing organ causes a series of reaction mechanism such as activation of neuroendocrine system(For example:sympathetic nervous system and RAAS),oxidative stress, inflammatory response, hemodynamics and HP.These mechanisms can be compensatory at first,but contribute to further functional worsening of heart and kidney,or even cause disfunction of the other organ at the beginning.In recent years, clinical and basic research have been confirmed that Angiotensin-Converting Enzyme Inhibitors (ACEI), angiotensin receptor antagonism (ARB), aldosterone receptor antagonist(MR), β-adrenergic receptor blockers and hemodialysis can be prevent and therapy heart and kidney function failure, but can’t prevent CRS progression, this situation remind us there may be other mechanisms which can lead to heart-kidney interaction, and thus promote the development of CRS.The role that uremic toxins play in CRS is being increasingly valued,especially that of IS.There are some researches suggest that IS has these features:(1)It is mainly eliminated through kidney,and can hardly be eliminated through dialysis;(2)Has a dose-dependent effect of promoting rat’s Cardiac fibroblasts fibrosis and cardiomyocyte hypertrophy;(3)Promoting distal tubule hedecrease of Superoxide removal ability of kidney and so on.Thus,it is now considered the feature of IS to cause heart-kidney interactive damage.But there are still some drawbacks to current studied:The specific relationship between IS and HF of CRS patients is not cleared;the mechanism of how IS promote myocardial hypertrophy and myocardial fibroblasts fibrosis has not been elucidated yet.Clarifying those mechanisms will help to understand what role IS plays in CRS,and provide the basis for further screening of therapeutic targets.This research will be summarized as follows:Chapter1Relationship between indole phenol sulfate in Peripheral blood and cardiac function of CRS patientsObjective:Discuss the relationship between IS and cardiac function damage of CRS patients in clinical trials.Methods:We studied90CRF patients(Male53,Female37) who had taken examination or therapy in our hospital during2011,11to2012,12.Selected cases should satisfy the following conditions:(1)Signed informed consent;(2)Glomerular Filtration Rate(GFR)<15ml/min-1.73m2>3个months,with or without evidence of kidney damage;(3)Drug,hemodialysis or any other treatments;(4)Meet the CRS diagnostic standard summarized by Ronco C et al;(5)Excluded:valvular heart disease, rheumatoid disease, hypertrophic cardiomyopathy, DCM, RMD, infective endocarditis, constrictive pericarditis, congenital heart disease, cancer, hyperthyroidism.Take fasting venous blood10ml from every patients,sent7ml of the blood to Zhu Jiang hospital for blood routine examination and blood biochemical examination,and3ml for analyzing indole phenol sulfate content using HPLC with fluorescence detection (HPLC-FLD).Calculate GFR to evaluate renal function using MDRD formula,use cardiac uhrasonography to test the structure and functional index of heart.Put patients into two groups(High-IS group and Low-IS group) in accordance with IS level.Result:After analyzing patients’peripheral blood IS concentration, clinical basic information and cardiac ultrasound we discovered:the proportion of coronary heart disease patients、creatinine, GFR, BNP, LVPW, E/e’of high-IS group(IS>35ng/dL)is higher than low-IS group.Further Linear regression analysis reported that IS concentration may be related gender, NYHA classification, hyperlipidemia, coronary heart disease, cerebral infarction, diabetes, antiplatelet drugs, diuretics, statins, GFR, BNP, LVDd, IVST, LVEF, LVMI, DCT, E/e.To further clarify the effect of IS,we have also used linear stepwise regression analysis,and the outcome showed diuretics, GFR, BNP, LVEF, LVMI and E/e can significantly affect IS concentration;while diuretics, BNP, LVMI and E/e maybe positively related to IS,GBFR and LVEF maybe negatively related on the other hand.Conclusion:Accumulation of IS in CRF patient is significantly associated with heart and kidney function.To clear wether IS plays a bridging role in CRS-causing cardiac damage,further research will be done using animal model.Chapter2:Relationship between indole phenol sulfate and cardiac function of CRS rat modelObjective:Confirm the relationship between IS and cardiac function damage through animal experiment.Methods:We chose male SD rats,divided them into control group (Sham+Sham), infarction group(MI+Sham), the STNx group (Sham+STNx) and infarction+STNx group(MI+STNx).Monitor rats’blood pressure using non-invasive blood pressure monitoring system,detecting rats cardiac structure and function using echocardiography,calculate rat’s GFR,use HPLC-FLU to calculate rats’blood serum IS level,detect rats’ infarct size,interstitial fibrosis of the infarction area,cardiomyocyte hypertrophy and other related indexes,inspect miR-34a expression in rats’infarction issue by the method of quantitative PCR.Result:1. Comparison of modeled rats’ survival rate and infarct size:Survival rate of control group (Sham+Sham), infarction group(MI+Sham), the STNx group (Sham+STNx) and infarction+STNx group(MI+STNx) were100%,59.7%,91.7%and100%respectively. There was no significant statistically difference of infarction size between infarction group and infarction+STNx group.2. Comparison of BP of all groups of rats:After14weeks past,compared to other two groups,STNx group and MI+STNx group had a significantly increase in BP, while the systolic blood pressure of MI+STNx group was obvious lower than that of STNx group.3. Comparison of heart weight:Heart weight and left ventricle weight of MI+STNx group were significantly higher than MI+Sham group,and that of Sham+STNx group were higher than MI+Sham group(P<0.01).4. Ultrasonic cardiogram:LVEF of MI+Sham group and MI+STNx group rats were obviously lower than other two groups,and LVEF of MI+STNx group was even lower than MI+Sham group(P<0.01).LVMI and E/e’of Sham+STNx was significantly higher than other groups(P<0.01).5. Hemodynamic changeCompared with MI+Sham group,MI+STNx rats had a significant increase in systolic pressure diastolic pressure and LVEDP()<0.05),and a significant reduce in CO(P<0.05).Compared with Sham+Sham group,the dP/dt max and dP/dt min of MI+Sham group and MI+STNx group were significantly lower(P<0.05);but MI+Sham group and MI+STNx group had no statistical difference (P>0.05) in dP/dt max and dP/dt min. Compared with Sham+Sham group,the systolic function related indicators such as PRSW and ESPVR of MI+Sham group and MI+STNx group significantly reduced (P<0.05).6.Myocardial interstitial fibrosisSirius scarlet dye showed that compared with Sham+Sham group,myocardial interstitial fibrosis of left ventricular non-infarct zone were significantly higher in other there groups(P<0.01);compared with MI+Sham group, myocardial interstitial fibrosis of left ventricular non-infarct zone ofMI+STNx group was significantly increased (P<0.01).7.Cross sectional area(CSA) of ardiac muscle cellsAccording to the results of HE staining, compared with MI+Sham group,CSA of ardiac muscle cells was significant higher in MI+STNx group(P<0.05);compared with Sham+Sham group, CSA of ardiac muscle cells was significant higher in MI+Sham group and Sham+STNx group(P<0.05).8.Renal function and plasma IS levelCompared with Sham+Sham group and MI+Sham group,in Sham+STNx group and MI+STNx group,GFR was significant lower,CrCl,blood creatinine level,IS were significant higher.(P<0.01)9.The expression of type I collagen,type Ⅲ collagen and miR-43aImmunohistochemical staining analysis showed in myocardial non-infarct zone,the expression of type I collagen and type III collagen significantly increased in STNx+MI group and Sham+STNx group than the other two groups(P<0.05);quantitative PCR detection show that in myocardial non-infarct zone,the expression of miR-34a significantly increased in STNx+MI group and Sham+STNx group than other two groups(P<0.05).The expression of type III collagen and miR-34a was higher in STNx+MI group and Sham+STNx group(P<0.05).10.Correlation analysis of miR-34a,IS and cardiac hypertrophy,myocardial fibrosisCorrelation analysis showed the expression of miR-34a in rats’ myocardial non-infarct zone was positive related to peripheral blood creatinine (r=0.881, P<0.001), LVEDP (r=0.746, P<0.746), myocardial fibrosis size(r=0.897, P<0.001), CSA of ardiac muscle cells(r=0.834, P<0.001), Type I collagen(r=0.927, P<0.001), type III collagen(r=0.911, P<0.911), peripheral blood IS content (r=0.903, P<0.001),and was negative related to dP/dtmin (r=0.417, P=0.417), PRSW (r=0.658, P<0.001), GFR (r=0.938, P<0.001), creatinine clearance (r=0.944, P<0.001),which suggest miR-34a may be involved in the process of IS mediate heart function failure in CRS.Conclusion:Researches of this chapter built CRS model through myocardial infarction+STNx rats,confirmed that IS is closely related to myocyte hypertrophy,myocardial fibrosis of the fibroblast, heart and renal damage.This model preliminary confirmed MI+STNx can lead to deterioration of heart and renal function and myocardial remodeling intensifing in rats.miR-34a may be involved in the process of IS mediate heart function failure in CRS.Chapter3:miR-34a’s effect in indole phenol sulfate promoting cardiomyocyte hypertrophy and myocardial fibroblasts fibrosisObjective:Clear the relationship between miR-34a and IS promoting cardiomyocyte hypertrophy and cardiac fibroblasts fibrosis.Methods:Firstly indicated the cultured newborn mice cardio-myocytes(NCMS) and newborn mice cardio-fibroblasts(NCFs).Respectively build over expression vector and suppress expression vector, according to experimental design, control group,IS group,asnti-miR-34a+IS group,miR-34a group and miR-34a+IS group were divided. Did no intervention measures to the control group, IS group was given IS stimulating,asnti-miR-34a+IS group was given IS stimulating on the basis of anti-miR-34a transfected NCMs、NCFs,miR-34a group wa given just miR-34atransfected NCMs and NCFs, miR-34a+IS group was given IS stimulating on the basis of miR-34a group.Final concentration of IS stimulating was20μM.Collected NCMs、NCFs48hours later, extracted RNA for Real time PCR detection,extacted protein for western blotting detection of protein expression level,and NCMs hypertrophy and NCFs fibrosis were determined by3H labeled leucine.Results:1.Expression of miR-34a of each groupsBetween NCMs groups,expression of miR-34a had a significant statistical difference(F=199.976, P<0.001),between NCFs groups,expression of miR-34a had a significant statistical difference(F=482.752, P<0.001).Compared with control group, expression of miR-43a in IS groups ofNCMs groups and NCFs groups have increased by about2.15times and1.91times(P<0.001), in miR-34a groups of NCMs groups and NCFs groups have increased by about2.63times and2.44times(P<0.01),in anti-miR-34a groups of NCMs groups and NCFs groups have increased by about0.61times and0.73times(P<0.001),in IS+anti-miR-43a groups of NCMs groups and NCFs groups have increased by about0.78times and0.74times(P<0.001).2.Effect of IS and miR-34a on uptake ratio of NCMs、NCFs3H labeled leucineThe difference of NCMs and NCFs3H labeled leucine uptake rate have statistically significant (P<0.001) among five groups. In which, the3H labeled leucine uptake rate was highest in miR-34a group, but lowest in anti-miR-34a group (P<0.05). These results suggest that miR-34a can promote NCMs and NCFs uptaking3H labeled leucine.3.Effect of IS and miR-43a on Notch-1protein expression of NCMs and NCFs groupsWestern blotting test showed,compared with control group,Notch-1protein expression of IS group and miR-34a group reduced,Notch-protein expression of anti-miR-34a group and IS+miR-34a group incuced,which suggest miR-34a may inhibit the expression of Notch-1by inducing expression of miR-34a.Conclusion:IS can promote myocardial hypertrophy and myocardial fibroblasts fibrosis possibly through miR-34a,this effect may partially work by acting on Notch- We can draw the following conclusion through the above parts of experiment above:1.IS accumulation in CRS patients is closely related to heart and renal funtion;2.This study verified IS is closely related to myocardial hypertrophy,myocardial fibroblasts fibrosis,heart and kidney damage by establishing rats myocardial infarction+STNx model.miR-34a may be involved in the process of IS mediate heart function failure in CRS.3.IS can promote myocardial hypertrophy and myocardial fibroblasts fibrosis possibly through miR-34a,this effect may partially work by acting on Notch-1.