| BackgroundPrimary hypertension is the most common non-communicable disease.The prevalence,incidence of primary hypertension and the level of blood pressure are increaed as people get older.With the wide ues of antihypertensive agents,the incidence of cardiovascular and cerebrovascular complications of primary hypertension has witnessed a large decrease,while the incidence of end-stage renal disease(ESRD) due to hypertensive nephropathy has been increased incredibly.According to a clinical cross-sectional survey,119.5 million adults aged 18 years or older had chronic kidney disease in 2012,and the rapid increase in diabetes and hypertension was predicted to drive epidemics of chronic kidney disease.As U.S. renal data system 2013 annual data reported,among the primary diseases of ESRD,hypertensive nephropathy accounted for 24.8% in 2011,which was second to diabetic nephropathy,and the proportion had grown by 8.7% since 2000.The above evidences reveal that the wide use of antihypertensive agents are far from enough to pretect the renal function presently.It is necessary to study the pathogenesis of hypertensive nephropathy further and make ues of appropriate drugs to prevent and cure hypertensive nephropathy.The term "autophagy" was first coined by de Duve,who was a Nobel Prize winner,to describe the presence of single- or double-membrane vesicles that contain parts of the cytoplasm and organelles in various states of disintegration.Autophagy is highly conserved eukaryotic adaptive progress which plays an important role in clearing waste,recycling energy and nutrition,and maintaining intracellular homeostasis by removing aggregated proteins,lipids and damaged organelles.It has been formly demonstrated that inhibiting autophagy can accelarate senescence,while promoting autophagy can extend life span,and during natural aging,the autophagic activity is reduced.If the autophagy of terminally differentiated cells,such as neurons, cardiomyocytes and podocytes and so on,suffers defect will lead to the accumulation of large number of abandoned proteins and cytoplasmic organoids,which will weaken the capacity of coping with the harmful stimulation coming from external environment.It appears entirely that autophagy posseses beneficial effect,but autophagy also has its unfavorable influence.Observations that a large number of autophagosomes accumulate in many dying cells make some scholars consider that it is that autophagy make cells die,which is called programmed cell death type Ⅱ.Maybe it’s because that the intracellur substance is degraded too excessively.Glomerular podocyte is one of integral and important constituents of the glomerular filtration barrier (GFB) and is often exposed to cytotoxic and phlogogenic macromolecules, which has the potential to induce oxidative stress and/or DNA damage. Scaerious and progressive podocyte injury can lead to glomerulosclerosis,even ESRD ultimately,therefore podocyte injury is a key determinant of glomerular diseases including hypertensive nephropathy.Similar to neuron and cardiomyocyte,podocyte,as a terminally differentiated cell,mainly relys on autophagy but not mitosis for clearing damaged DNA and macromolecular substance.High level of basal autophagy has been shown to be present in podocytes in vitro,in vivo,and in human renal biopsies,and plays an important role in maintaining podocyte homeostasis.It suggests now that overactivation of logcal or systemic renin-angiotensin system(RAS) plays a vital role in the development and maintenance of hypertension.Kidney expresses all components of RAS,and intrarenal RAS plays an important role in the development and advance of primary hypertension.Recent study shows that Angiotensin Ⅱ (Ang Ⅱ),which is critial biological active substance contributed to hypertension,can promote glomerular podocyte injury through inhibiting the express of nephrin,which is the key structural protein of podocytic processor can drive oxidative stress.It discovers that Ang Ⅱ can promote autophagy in podocytes.It is still unhknown that what relationship between the Ang Ⅱ induced autophagy and Ang Ⅱ induced podocyte injury.ObjectiveIn this study, we first study the changes of renal cortex RAS and glomerular podocyte autophagy during aging with normotension or hypertension,examine the possible mechanism of renal injury during aging with hypertension further.Then we respectively intervene rats with angiotensin converting enzyme inhibitors (Benazepril) and angiotensin Ⅱ receptor blockers(Losartan),and observe the effects of benazepril and losartan on glomerular podocyte autophagy in aged spontaneously hypertensive rats(SHRs),and investigate the new mechanism of renal protective effect of ACEI/ARB further.Methods(1) Normotensive Wistar-Kyoto-rats(WKYs) were allocated to three groups by age:3-month-old group,13-month-old group,22-month-old group.Sex,age matched spontaneously hypertensive rats(SHRs) were served as controls.Each group had 6 rats.Blood pressure was monitored.Level of urinary albumin,urinary creatinine,serum creatinine(SCR),blood urea nitrogen(BUN), serum and renal cortex renin activity and Ang II were detected.The kidney ultrastructural changes and podocyte autophagosomes were observed under light and transmission electron microscopy.Expression of ATiR,AT2R,nephrin,Beclin 1,LC3B-Ⅱ,Atg5 and p62 from glomeruluses were analyzed by western blot.(2) Male 18-mont-old WKYs were distributed as NORM group(2ml physiological saline per day);SHRs were randomized into 4 groups randomly:CTRL group(2ml physiological saline per day),ACEI group(10mg/kg benazepril per day),ARB group(30mg/kg losartan per day) and Combined group(10mg/kg benazepril and 30mg/kg losartan per day).Each group had 6 rats,and the experiment was conducted for 4 months.Blood pressure were monitored regularly.At the end of experiment,we detected the level of urinary protein,urinary creatinine,SCR,BUN,serum and renal cortex renin activity and Ang Ⅱ.The kidney ultrastructural changes were observed under light and transmission electron microscopy;The expression of nephrin, Beclin 1,LC3B-Ⅱ,Atg5 and p62 in glomeruluses were analyzed by western blot.Results(1) Blood pressure of WKYs or SHRs had no significant change with age(P>0.05),but blood pressure of SHRs was significantly higher than that of age-matched WKYs(P<0.05).(2) During aging with normotension or hypertension,urinary albumin/creatinine ratio were increased significantly(P<0.05),and urinary albumin/creatinine ratio of SHRs was significantly higher than that of age-matched WKYs(P<0.05).(3) There was no significant difference in SCR and BUN during aging with normotension or hypertension(P>0.05).However when comparison executed bteween tow groups,SCR and BUN of 3-month-old SHR group and 13-month-old SHR group were higher significantly than that of age-matched WKY groups(P<0.05).(4) Level of serum and renal cortex renin activity and Ang Ⅱ had no significant change during aging with normotension(P>0.05).An age-dependent decrease of renal cortex Ang Ⅱ appeared in hypertensive rats(P<0.05),but level of serum and renal cortex renin activity and serum Ang Ⅱ had no significant difference(P>0.05).(5) The age-related structural changes,including glomerulosclerosis,tubular atrophy and interstitial fibrosis were observed both in normtensive and hypertensive rats with age,but these pathological changes were more serious in hypertensive rats.What’s more,autophagosomes accumulated relatively during aging with normotension.(6) The expression of AT1R,AT2R, Beclin 1 from glomeruluses had no significant difference(P>0.05),while that of LC3B-Ⅱ,Atg5 and p62 were increased significantly during aging with normotension(P<0.05);The expression of AT1R was increased significantly,while that of nephrin,Beclin 1,LC3B-Ⅱ,Atg5 and p62 were decreased during aging with hpertension(P<0.05).(7) After drug intervention,the blood pressure and urinary albumin/creatinine ratio of four SHR groups were still significantly higher than that of NORM group,but the blood pressure and urinary albumin/creatinine ratio of ARB group and Combined group were significantly lower than that of CTRL group(P<0.05).(8) After drug intervention,there was no significant difference in the level of SCR and BUN among these five groups(P>0.05);(9) After drug intervention,the level of cortex AngⅡ of four SHR groups was significantly lower than that of NORM group respectively,while level of cortex Ang II of ARB group was significantly higher than that of CTRL group(P<0.05);(10) After drug intervention,the renal ultrastructural observation displayed that glomeruluses shrinked,the epithelial cell foot processes fused even effaced,and the renal tubule atrophied focally in four SHR groups,but these pathological changes were most serious in CTRL group and alleviated relatively in Combined group.The autophagosomes of NORM group and Combined group were significantly more than that of CTRL group(P<0.05).(11) After drug intervention, the expression of nephrin,Beclin 1,LC3B-Ⅱ,Atg5 and p62 in CTRL group were suppressed significantly compared with NORM group(P<0.05);While compared with CTRL group,the expression of nephrin, Beclin 1,LC3B-Ⅱ and Atg5 in ACEI group and the expression of nephrin, Beclin 1.LC3B-Ⅱ,Atg5 and p62 in ARB group and Combined group were induced significantly(P<0.05).Conclusions(1) In this study,age-related structural changes,including glomerulosclerosis, tubular atrophy and interstitial fibrosis were presenced in both normtensive WKYs and hypertensive SHRs with age,but these pathological changes were more serious in SHRs.As for function,urinary albumin/creatinine ratio were increased apparently during aging with normotension or hypertension,and urinary albumin/creatinine ratio of SHRs was higher than that of age-matched WKYs.It shows that aging promotes kidney structural changes and functional recession,and hypertension accelerates these recession.(2) The expression of LC3B-Ⅱ,Atg5,p62 from glomeruluses were increased during aging with normotension.On the contrary,the expression of Beclin l,LC3B-II,Atg5,p62 from glomeruluses were decreased during aging with hypertension.What’s more,autophagosomes accumulated relatively during aging with normotension under electron microscopy.It reveals that glomerular podocyte autophagic activity is suppressed relatively during aging with normotension,but induced relatively during aging with hypertension.(3) The expression of nephrin from glomeruluses were decreased during aging with hypertension,and electron microscopy shown the foot processes were fused and effacement apparently in eldly SHRs,which reveals that glomerular podocytes are damaged when aging with hypertension. What’ more,the level of renal cortex Ang Ⅱ and the expression of AT2R were decreased,and the expression of AT1R was increased,leading to a hypothesis that the podocyte autophagy is induced by Ang Ⅱ through the upregulated AT1R,and once the autophagy activity become so high that many kinds of structual macromolecule including nephrin is digested excessively,the podocytes will be damaged.It may be one of possible mechanisms of renal damage during aging with hypertension.(4) Compared with CTRL group,the urinary albumin/creatinine ratio of ARB group and Combined group were significantly lower,and the expression of nephrin from glomeruluses was higher after ACEI/ARB intervention.In addition,it was shown from light and transmission electron microscopy that the pathological changes of kidney in Combined group was more alleviative than that of three other SHR groups. All of above indicates that ACEI/ARB posseses renal protective effect.(5) After ACEI/ARB intervention,the number of autophagosomes in glomerular podocytes of ACEI group,ARB grpup and Combined group grew,especially the autophagosomes of Combined group was much more than that of CTRL group,indicating that the accumulation of autophagosomes in glomerular podocytes present in SHRs after ACEI/ARB intervention,and the autophagy activity was supressed.(6) Combined with these results of this study,we could make the conclusions that the autphagy activity of glomerular podocytes of eldly SHRs is so high that the structual macromolecule including nephrin is degraded too much,bring about the podocytes being damaged,even being died,which can force the hypertensive nephropathy to initiate and progress.After ACEI/ARB intervention,the autphagy activity of glomerular podocytes is downregulated,leading to the result that the synthetic rate is farther than the degradative rate of structual macromolecule,and pathological changes of kidney is alleviated and renal function is improved. So far,we can hypothesize that the renal protective effect of ACEI/ARB may be related to mediating glomerular podocyte autophagy,but it needs more specific experiments to verify. |
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