(P)RR Promotes Renal Fibrosis By Inhibiting NPRA Through Twist1/2 And HDAC1/2

BackgroundThe cardiovascular disease and renal disease can have cause-and-affected each other.In cardiovascular disease,especially hypertension,elevated blood pressure damages the kidneys and gradually exacerbates cardiovascular and kidney dysfunction.In renal diseases,when renal fibrosis and inflammation occur,renin secretion changes,tubular fluid reabsorption disorders and renal hemodynamic disorders will aggravate the defects of cardiovascular and renal homeostasis.However,the specific mechanism by which renal pathological changes aggravate cardiovascular disease remains unclear.The(pro)renin receptor[(P)RR],(P)RR is a new member of renin-angiotensin system(RAS)discovered in 2003.(P)RR is expressed in many organs,especially the collecting duct of the renal cortex.(P)RR combined with renin can improve the efficiency of angiotensinogen I(Ang I),which in turn generates angiotensinogen II(Ang II).(P)RR is involved in water-salt balance,acid-base balance,and autophagy by amplifying the effects of Ang II,which plays a role in hypertension,diabetic retinopathy,and renal fibrosis.Atrial natriuretic peptide(ANP)is mainly produced in atrial and ventricular myocytes in response to cardiac wall stretching and various stimuli such as endothelin and alpha-adrenergic factors.In the kidney,ANP binds to Natriuretic peptide receptor-A(NPRA)to produce the second messenger cyclic guanosine monophosphate(c GMP),which promotes sodium production and counter the effects of RAS system activation.In recent years,antagonism between Ang II and NPRA has been found,but the relationship between(P)RR independent of Ang II and NPRA remains unclear.Meanwhile,the role of NPRA in renal fibrosis induced by(P)RR independent of Ang II remains to be investigated.ObjectiveThe purpose of this study was to investigate the effect of(P)RR on renal fibrosis independent of Ang II,to provide a new direction and theoretical basis for clinical understanding of the relationship between cardiovascular and renal diseases.Method(1)Animal models of Apolipoprotein E-knockout(Apoe-/-)male mice were constructed by subcutaneously injecting prorenin or an equivalent dose of normal saline with an implanted capsule under osmotic pressure micropump.They were also fed a high-fat diet for 8 weeks and given losartan intragastric administration(except for Ang II).(2)The systolic blood pressure of mice was measured and recorded by noninvasive sphygmomanometer,and the weight of mice was measured and recorded by electronic scale.(3)Enzyme-linked immunoassay assay were used to detect the levels of c GMP,plasminogen activator inhibitor-1(PAI-1)and connective tissue growth factor(CTGF)in mouse kidney.(4)H&E staining and Masson staining were used to observe renal inflammation and fibrosis.(5)Western Blot(WB)and Quantitative Real-Time PCR(q PCR)were used to detect the expression levels of(P)RR and Histone deacetylases1(HDAC1),Histone deacetylases2(HDAC2),Twist1,Twist2,CTGF,PAI-1,and NPRA in kidney.Results1.In comparison with the control group,the renal m RNA and protein expressions of(P)RR in the prorenin group was significantly increased after subcutaneous injection of prorenin(P < 0.05 for each).There were no significant differences in body weight and systolic blood pressure between the two groups(P > 0.05 for each).2.In comparison with the control group,the renal levels of cGMP,PAI-1 and CTGF in the prorenin group were significantly improved(P < 0.05 for each).Compared with the control group,mice in the prorenin group showed renal interstitial inflammatory cell infiltration,renal tubule structure disorder and renal fibrosis,and the quantitative level of fibrosis was statistically significant(P < 0.05).3.Compared to the control group,the renal m RNA and protein expressions of Twist1/2 and HDACH1/2 in the prorenin group were significantly improved,but the renal m RNA and protein expressions of NPRA were significantly decreased(P < 0.05 for each).4.In vitro cultured mesangial cells,in comparison to blank group,the protein expressions of PAI-1 and CTGF were significantly increased in Prorenin group(P < 0.05 for each).Compared to the prorenin group,(P)RR knockdown significantly decreased the protein expression of PAI-1 and CTGF(P < 0.05 for each).In comparison to the prorenin group,(P)RR overexpression significantly increased the protein expression of PAI-1 and CTGF(P < 0.05 for each).5.In vitro cultured mesangial cells,the(P)RR was activated by different concentrations of prorenin.In comparison to the control group,m RNA and protein expressions of Twist1/2 and HDACH1/2 in renal mesangial cells raised with the increase of prorenin concentration,while m RNA and protein expressions of NPRA reduced with the increase of prorenin concentration(P < 0.05 for each).6.In vitro cultured mesangial cells,compared with blank group,NPRA protein expression was significantly reduced in the prorenin group,while Twist1/2 and HDAC1/2 protein expression were significantly increased(P < 0.05 for each).Compared with the prorenin group,(P)RR knockdown significantly increased NPRA protein expression,while significantly decreased Twist1/2 and HDAC1/2 protein expression(P < 0.05 for each).Compared with the prorenin group,(P)RR overexpression significantly decreased NPRA protein expression,while significantly increased Twist1/2 and HDAC1/2 protein expression(P < 0.05 for each).Compared with the prorenin group,TSA(HDAC1/2 inhibitor)significantly improved NPRA protein expression and significantly reduced Twist1/2 and HDAC1/2 protein expression(P < 0.05 for each).Compared with the prorenin group,Harmine(Twist1/2 inhibitor)significantly increased NPRA protein expression,while significantly decreased Twist1/2 and HDAC1/2 protein expression(P < 0.05 for each).7.In vitro cultured mesangial cells,compared with blank group,the protein expressions of PAI-1 and CTGF were significantly improved in the prorenin group(P < 0.05 for each).Compared to the prorenin group,the protein expressions of PAI-1 and CTGF in ANP group were significantly reduced(P < 0.05 for each).Compared to ANP group,overexpression of NPRA significantly decreased the protein expression of PAI-1 and CTGF(P < 0.05 for each).Compared with ANP group,(P)RR knockdown significantly reduced the protein expression of PAI-1 and CTGF(P < 0.05 for each).Compared with ANP group,TSA(HDAC1/2 inhibitor)significantly reduced the protein expression of PAI-1 and CTGF(P < 0.05 for each).Compared with ANP group,Harmine(Twist1/2 inhibitor)significantly reduced the protein expression of PAI-1 and CTGF(P < 0.05 for each).In comparison to ANP group,NPRA knockdown significantly increased the protein expression of PAI-1 and CTGF(P < 0.05 for each).Conclusion1.Renal(P)RR activation increases the expression of fibrosis markers CTGF and PAI-1,renal interstitial inflammatory cell infiltration,renal tubule structure disorder,and renal fibrosis,leading to the Ang II independent renal fibrosis.2.(P)RR activation in the kidney inhibits NPRA expression by up-regulating Twist1/2 and HDAC1/2 expression.3.Renal(P)RR promotes Ang II independent renal fibrosis by inhibiting NPRA through Twist1/2 and HDAC1/2.