Effects Of ACE2 And TGF-β₁/Smad3 Pathway On Renal Fibrosis Induced By High Salt

Objective:To investigate the effects of Angiotensin converting enzyme2（ACE2）on high-salt induced renal fibrosis and its possible mechanisms.Methods:Ⅰ.36 male SD rats were randomly divided into Sham group（n=10）,DOCA group[n=13,DOCA50mg/（kg?w）],DOCA+DIZEgroup[n=13,DOCA50mg/（kg?w）+DIZE 15mg/（kg?d）].Tail artery blood pressure was measured once a week for 8 weeks.24h urinary sodium and potassium were measured.Urine creatinine（Ucr）,urinary microalbumin（mALB）,serum creatinine（Scr）and blood urea（BU）were measured.24h urinary sodium-potassium excretion rate,creatinine clearance rate（Ccr）and the right kidney hypertrophy index were calculated.HE staining and Masson staining were used to observe the histomorphological changes and fibrosis of kidney.The content of Angiotensin（1-7）[Ang（1-7）]was measured by ELISA in renal cortex.The protein expression of collagen I（COL I）,collagen III（COL III）,alpha-smooth muscle actin（α-SMA）,Vimentin,E-cadherin,transforming growth factor-β₁（TGF-β₁）,ρ-Smad3,Angiotensin converting enzyme2（ACE2）,MasR,ACE,AngiotensinⅡ（AngⅡ）,Angiotensin II receptor 1（AT1R）and Angiotensin II receptor 2（AT2R）in renal cortex were determined by Western-blotting.Ⅱ.Ace2 knockout mice were obtained by CRISPR/Cas9 technology and mated with C57 wild type（WT）mice.Gene identification was carried out after reproduction.Male WT mice and Ace2^-/y/y mice were randomly divided into 0.5%normal salt（NS）and 8%high salt（HS）with 10 mice in each group.Tail arterial blood pressure was monitored once 2weeks for 18 weeks.24h urinary sodium,potassium,Ucr,mALB,Scr and BU were measured.24h urinary sodium-potassium excretion rate,Ccr and the kidney hypertrophy index were calculated.HE staining and Masson staining were used to observe the histomorphological changes and fibrosis of kidney.The content of Ang（1-7）was measured by ELISA in renal tissue.The protein expression of COL I,COL III,α-SMA,Vimentin,E-cadherin,TGF-β₁,ρ-Smad3,ACE2,MasR,ACE,AngⅡ,AT1R and AT2R in renal tissue were determined by Western-blotting.Results:Ⅰ.The blood pressure,24h urinary sodium,urinary sodium-to-potassium excretion rate,mALB and the right kidney hypertrophy index of DOCA group were higher than Sham group.Ucr and Ccr were lower than Sham group.DIZE could significantly increase Ccr and reduce the blood pressure,24h urinary sodium,mALB and the right kidney hypertrophy index of DOCA rats.The degree of histomorphology and renal fibrosis in DOCA group were significantly higher than Sham group.DIZE could improve renal damage and fibrosis.Compared with Sham group,the protein expression in DOCA group of COL I,COL III,α-SMA,Vimentin,TGF-β₁,ρ-Smad3,ACE,AngⅡand AT1R were higher,the content of Ang（1-7）and the protein expression of E-cadherin,ACE2,MasR and AT2R were lower in renal cortex.DIZE could significantly reduce the protein expression of COL I,COL III,α-SMA,Vimentin,TGF-β₁,ρ-Smad3,AngⅡand increase the content of Ang（1-7）,the expression of protein E-cadherin,MasR and AT2R in renal cortex.Ⅱ.Ace2^-/y/y male mice could be obtained by mating knockout female mice with WT male mice.There was no difference between blood pressure in each group.Compared with WT+NS group,the 24h urinary sodium and sodium-to-potassium excretion rate were increased and the 24h urinary potassium was decreased in WT+HS group.Compared with Ace2^-/y+NS group,the 24h urinary sodium and sodium-to-potassium excretion rate were increased in Ace2^-/y+HS group.Ace2 gene knockout could significantly change the histomorphology and the fibrosis of kidney,and increased the protein expression of COL I,α-SMA,Vimentin,TGF-β₁,ρ-Smad3,ACE,AngⅡand AT1R,but decreased the protein expression of E-cadherin,MasR and AT2R in kidney.Ace2^-/y+HS group did not show heavier renal damage.There was no statistical differences between the expression of all the protein in kidney above-mentioned compared with WT+HS group expect ACE.Conclusion:Ⅰ.High salt diet can mediate renal fibrosis,down-regulate the protein expression of ACE2,unbalance of intrarenal RAS components,activate the TGF-β₁/Smad3pathway and Epithelial mesenchymal transition（EMT）in salt-sensitive hypertensive rats and normotensive mice.Ⅱ.ACE2 agonist can alleviate renal fibrosis in salt-sensitive hypertensive rats by up-regulating the protein expression of Ang（1-7）,MasR and AT2R in the kidney,increasing degradation of AngⅡ,inhibiting the TGF-β₁/Smad3 pathway and renal EMT.Ace2 gene knockout can induce renal fibrosis by activating the TGF-β₁/Smad3 pathway and renal EMT.Ⅲ.To reveal that down-regulation of Ace2 gene and activation of the TGF-β₁/Smad3pathway involve in the pathophysiological mechanism of renal fibrosis induced by high salt.