| Objectives: Diabetic nephropathy (DN) is one of the most commonmicrovascular complications of diabetes mellitus (DM), which meanwhile isone of leading causes for chronic renal failure in our country. Therefore,searching for its pathogenesis and effective preventing and controllingmeasures is of great significance. Chinese medicine has shown greatadvantage in preventing and controlling DN. In the opinion of most modernChinese medical scholars in nephropathy, the primary pathogenesis of DN is“blood stasis blocking renal collaterals”, besides “Qi and Yin vacuity” of DM.The principle “activating blood, removing stasis and dredging collaterals” isfollowed in DN treatment, which has become a consensus in the Chinesemedical field of nephropathy. Modern medicine has confirmed that theactivation of renin-angiotensin system (RAS) is the main mechanism involvedin DN. Based on the cognition of over-activation of the classic pathway ofRAS, the angiotensin-converting enzyme-angiotensin Ⅱ-angiotensin type1receptor [ACE-AngⅡ-AT1] pathway, in the development of DN, angiotensinconverting enzyme inhibitor (ACEI) and angiotensin receptor blocker (ARB)have been used as first-line agents in treating and delaying the renal injury ofDM in clinic. However, with the further study on RAS, another functional axis,angiotensin-converting enzyme-related carboxypeptidase2-angiotensin-(1-7)-Mas [ACE2-Ang-(1-7)-Mas] axis, was discovered to be against the function ofACE-AngⅡ-AT1axis. Maintaining and adjusting the balance between thesetwo pathways have become a new concept for diabetic renal injury therapy.Taking blood-stasis-blocking-collaterals syndrome and ACE2-Ang-(1-7)-Masaxis as two important entry points, this research is aimed to explore whether the blood-stasis-blocking-collaterals syndrome of DN is related to thedysfunction of ACE2-Ang-(1-7)-Mas axis, as well as whether the interventionof stasis-removing-and-collaterals-dredging medicine on blood-stasis-blocking-collaterals syndrome also improve the function of ACE2-Ang-(1-7)-Mas axis,by animal experiments and isolated culture of mouse podocytes.Methods:1Verification of stasis-blocking-collaterals syndrome in rats with diabeticnephropathyA totle of32healthy male Sprague-Dawley rats of4weeks old, weighed80-100g were grouped randomly into blank control group (C group, n=8),high-glucose-high-fat control group (H group, n=10), and model group (Mgroup, n=14), after adaptive feeding for1week. Rats in C group were fed withregular rat chow, while others fed with high-glucose-high-fat diet. After4weeks, the rats in M group were injected intraperitoneailly withstreptozotocin (STZ) at the dose of40mg/kg body weight. The rats showingrandom blood glucose≥16.7mmol/L72hours after injection were consideredas successfully duplicated diabetic models. Rats in other two groups wereinjected with citrate buffer solution of equal volume. Random blood glucose(RBG) was measured on the3rd day and at the end of the4th,8th and16thweel after STZ injection. At the end of the16th week, after collection of urineof24hours for urine protein evaluation (UPE), blood samples were collectedby abdominal aortic method under anesthesia, which were used to test bloodviscosity measures, serum lipid profile and platelet parameters. Renal cortexof rats in different groups were examined by light microscope andtransmission electron microscope.2Effect of stasis-removing-and-collaterals-dredging intervention upon stasis-blocking-collaterals syndrome in rats with diabetic nephropathyA total of40experimental rats were divided into C group (n=10) andSTZ-injecting group (n=30). The establishment of models was same as above.Twenty-five diabetic rats were further divided into M group (n=12) andstasis-removing-and-collaterals-dredging group (Z group, n=13). Dosages of medicine in rats were calculated as5.8times as the clinical dosage in human.Rats in Z group were intragastrically fed with suspension of Chinese medicinegranules (1.08g/kg body weight, equal to crude drug4.7g/kg body weight),while rats in other groups with drinking water of equal volume, once daily for16weeks. At the end of the16th week, blood samples were collected byabdominal aortic method to test blood viscosity measures, serum lipid profile,platelet parameters and plasma fibronectin (FN) content. Renal cortex wascryopreserved to test the relative expression of tissue plasminogen activator(t-PA) and plasminogen activator inhibitor-1(PAI-1) mRNA by real-timePCR.3The change of ACE2-Ang-(1-7)-Mas axis of renal cortex in DN rats ofstasis-blood-blocking-collaterals syndrome and effect of stasis-removing-and-collaterals-dredging interventionA total of93experimental rats were divided into C group (n=22) andSTZ-injecting group (n=71). The establishment of models was same as above.Sixty-two diabetic rats were further divided into M group (n=24), irbesartangroup (I group, n=19), Z group (n=19). Interventions were given respectively.At the end of the4th week,6rats were choosed randomly from C and Mgroups respectively to be killed, and other rats were killed at the end of the8thand16th week separately. Renal cortex was cryopreserved to test theexpressions of ACE2, Mas, ACE proteins by immunohistochemical assay andwestern blot, relative expressions of ACE2, Mas, ACE mRNA by real-timePCR, and Ang-(1-7) content by ELISA.4Effect of stasis-removing-and-collaterals-dredging intervention upon renalinjury in diabetic nephropathy of stasis-blood-blocking-collaterals syndromeA total of57experimental rats were divided into C group (n=10) andSTZ-injecting group (n=47). The establishment of models, further division ofSTZ-injecting group and interventions were same as above. Body weight aswell as diet calorie, water intake, urine output and UPE of24hours, weremeasured on the3rd day and at the end of the4th,8th and16th week afterSTZ injection. Meanwhile, urine of24hours was collected to do the urine protein evaluation (UPE). At the sacrifice of rats at the end of the16th week,blood samples were collected to test kidney function; kidney indexes (KI)were calculated; and renal cortex was pathologically examined by lightmicroscope and transmission electron microscope.5Effect of Chinese medicine of stasis-emoving-and-collaterals-dredgingintervention upon high glucose stimulating podocytesThe conditionally immortalized mouse podocytes cultured under growthpermissive condition were divided into normal glucose group (NG group),high glucose group (HG group) and medicated high glucose groups of12different concentrations, administered with corresponding culture mediainterventions for24,48and72hours. Effects of Chinese medicine of differentconcentrations upon cell proliferation under high glucose stimulation wereobserved by MTT test. Based on the comparison of data among differentconcentrations, the optimum concentration was screened for furtherexperiment. Differentiated and mature mouse podocytes cultured undergrowth restrictive condition were divided into NG group, HG group andmedicated high glucose group of the optimum concentration [(H+Z) group],administered with corresponding interventions for24,48and72hours.Collagen-Ⅳ (Col-Ⅳ) content in podocyte supernatant was tested by ELISA,and expressions of ACE and ACE2proteins in podocytes by immuno--cytochemical assay and western blot, relative expressions of ACE and ACE2mRNA by real-time PCR.Results:1Verification of stasis-blocking-collaterals syndrome in rats with diabeticnephropathy1.1General conditionCompared with C and H groups, rats in M group showed polydipsia,polyphagia, polyuria and loss of weight gradually. Rats in C group had ruddypaws and unconspicuous dull-red tail veins, which was similar in H group,while M group had dark-purple paws and conspicuous dark-purple tail veins.1.2Random blood glucose RBG of rats in C and H groups remained constant, with H group higherthan C group (P<0.01). After STZ injection, RBG in M group wassignificantly higher than C and H groups at same time points (P<0.01).1.3UPE of24hoursAt the end of16th week, there was no difference in UPE between C andH groups (P>0.05). Compared with C and H groups, UPE of M group washigher (P<0.01).1.4PathomorphologyRats in C and H groups had normal sized kidneys, with smooth surface,from which glomeruli showed clear and well-distributed structures, with nofusion of foot processes arranged in order. Rats in M group had enlargedkidneys, with unsmooth surface and pale spots on them, from which enlargedswollen glomeruli revealed homogeneously thickened basement membranewith chaotic structure, increased mesangial matrix and segmental fusion ofpodocyte foot processes.1.5Blood viscosity measuresAt the end of16th week, only hematocrit (HCT) of rats in H group waslower than C group (P<0.05). Compared with C group, whole blood viscosity,plasma viscosity and whole blood low shear reductive viscosity of rats in Mgroup were all higher (P<0.01); while HCT and erythrocyte deformabilityindex (EDI) were lower (P<0.05or0.01). Among them, whole blood viscosity,plasma viscosity and EDI were also different from those in H group (P<0.05or0.01).1.6Serum lipid profileAt the end of16th week, total cholesterol (TC), low density lipidcholesterol (LDL) and high density lipid cholesterol (HDL) of rats in H groupwere higher than C group (P<0.05or0.01); while triglyceride (TG) and verylow density lipid cholesterol (VLDL) were not different from C group(P>0.05). Compared with C and H groups, TC, TG, LDL, HDL and VLDL ofrats in M group were all significantly higher (P<0.01).1.7Platelet parameters At the end of16th week, only mean platelet volume (MPV) of rats in Hgroup was higher than C group (P<0.05). Compared with C group, plateletcount (PLT) of rats in M group decreased significantly (P<0.01); MPV andplatelet distributing width (PDW) of M group elevated significantly (P<0.01).And meanwhile, PLT and MPV of rats in M group were also different from Hgroup (P<0.05or0.01). There was no difference in platelet-large cell ratio(P-LCR) among different groups (P>0.05).2Effect of stasis-removing-and-collaterals-dredging intervention upon stasis-blocking-collaterals syndrome in rats with diabetic nephropathy2.1Blood viscosity measuresAt the end of16th week, compared with C group, whole blood viscosity,plasma viscosity and whole blood low shear reductive viscosity of rats in Mgroup were all higher (P<0.01); while HCT and erythrocyte deformabilityindex (EDI) were lower (P<0.01). Compared with M group, whole bloodviscosity with30S-1and200S-1shear rates, plasma viscosity and EDI of Zgroup were improved significantly (P<0.05or0.01).2.2Serum lipid profileAt the end of16th week, compared with C group, TC, TG, LDL, HDLand VLDL of rats in M group were all significantly higher (P<0.01).Compared with M group, all above indexes of Z group were lower (P<0.05or0.01).2.3Platelet parametersAt the end of16th week, compared with C group, platelet count (PLT) ofrats in M group decreased significantly (P<0.01); MPV and plateletdistributing width (PDW) of M group elevated significantly (P<0.05or0.01).Compared with M group, MPV and PDW of Z group were lower (P<0.01).There was no difference in platelet-large cell ratio (P-LCR) among differentgroups (P>0.05).2.4Plasma FN contentAt the end of16th week, plasma FN content of rats in M group washigher significantly than C group (P<0.01). And compared with M group, Z group was lower (P<0.05).2.5Relative expression of t-PA and PAI-1mRNA in renal cortexAt the end of16th week, there was no difference in the relativeexpression of t-PA mRNA among different groups (P>0.05). The relativeexpression of PAI-1mRNA in M group was significantly higher than C group(P<0.05). And its relative expression in Z group was lower than M group(P<0.05).3The change of ACE2-Ang-(1-7)-Mas axis of renal cortex in DN rats ofstasis-blood-blocking-collaterals syndrome and effect of stasis-removing-and-collaterals-dredging intervention3.1Expressions of ACE2, Mas, ACE proteins in renal cortexACE2and ACE expressed mainly in the epithelial cells of glomeruli andtubules of kidney, while Mas approximately expressed in the proximal tubules.There was no significant difference in expressions of ACE2, Mas and ACE inrenal cortex from rats in C group among different time points (P>0.05).Expressions of ACE2and Mas proteins in M group showed downtrend withtime. ACE2expression at the end of16th week was lower than4th week(P<0.05), while Mas expression at the end of16th week was lower than4thand8th week respectively (P<0.05or0.01). But there was no difference inACE expression in M group among different time points (P>0.05).At the end of8th week, there was no significant difference in expressionsof ACE2and Mas among different groups (P>0.05). And there was statisticaldifference in ACE expression only between M and I groups (P<0.05).At the end of16th week, ACE2expression in M group was lower than inC group (P<0.01); and that in I and Z groups were higher than in M group(P<0.05), with Z group higher than I group (P<0.05). Mas expression in Mgroup was lower than in C group (P<0.05); and that in I and Z groups washigher than in M group (P<0.01). ACE expression in M group was higher thanin C group (P<0.01); and that in Z group was lower than in M and I groups(P<0.05or0.01).3.2Expressions of ACE2, Mas, ACE mRNA in renal cortex Take rats in C group at the end of4th week as control to quantificate therelative expression. There was no difference in expressions of ACE2, Mas,ACE mRNA in C group among different time points (P>0.05). ACE2mRNAexpression in M group at each time point was lower than that in C group atsame time point (P<0.01), and experienced a course of first rising (P<0.01)then declining (P<0.05) from4th week to16th week. Mas mRNA expressionin M group at4th week was higher than that in C group at same time point(P<0.01), and experienced a declining course from4th week to16th week(P<0.01), which resulted in lower than C group at16th week (P<0.05). ACEmRNA expression in M group at4th week was lower than that in C group atsame time point (P<0.01), and experienced a course of first rising (P<0.01)then declining (P<0.05) from4th week to16th week, which resulted in higherthan C group at16th week (P<0.05).At the end of8th week, there was no significant difference in Mas mRNAexpression among different groups (P>0.05). Compared with C group, ACE2mRNA expression in M group was lower (P<0.01), ACE mRNA expression inM group was higher (P<0.01). Compared with M group, ACE2mRNAexpression in Z group was higher (0.01), and higher than I group as well(P<0.05), while ACE mRNA expressions in both I and Z groups were lowerthan in M group (P<0.05).At the end of16th week, compared with C group, ACE2mRNAexpression in M group was lower (P<0.01), ACE mRNA expression in Mgroup was higher (P<0.01), and Mas mRNA expression in M group showedno difference (P>0.05). Compared with M group, ACE2and Mas mRNAexpressions in Z group were both higher (P<0.05or0.01), with Mas mRNAexpression in Z group higher than I group (P<0.05). There was no statisticaldifference in ACE mRNA expressions among M, I and Z groups (P>0.05).3.3Ang-(1-7) content in renal cortexAt the end of16th week, compared with C group, Ang-(1-7) content inrenal cortex from rats in M group was lower significantly (P<0.01). AndAng-(1-7) content in I and Z groups was higher than in M group (P<0.01). 3.4Linear correlation analysis between blood viscosity and expressions ofACE2, Mas, ACE mRNA and proteins of rats in M groupAt the end of16th week, blood viscosity (including plasma viscosity andwhole blood viscosity under different shear rates) in M group was in negativecorrelation with ACE2mRNA and protein expressions (P<0.05or0.01), andin positive correlation with ACE mRNA and protein expressions (P<0.05or0.01). However, there was no linear correlation between blood viscosity andMas mRNA and protein expressions (P>0.05).4Effect of stasis-removing-and-collaterals-dredging intervention upon renalinjury in diabetic nephropathy of stasis-blood-blocking-collaterals syndrome4.1Dynamic trends of clinical indicators of diabetesAfter4weeks, body weight of rats in M group at each time point waslower than C group at same time point (P<0.01). At the end of16th week,body weights in I and Z groups were higher than in M group (P<0.05or0.01).Compared with C group at same time point, RBG, diet calorie, water intakeand urine output of24hours of rats in M group at each time point were allsignificantly higher (P<0.01). Compared with M group at same time point,RBG, diet calorie and urine output of24hours of rats in I and Z groups ateach time point were not different significantly (P>0.05); while water intakeof24hours of rats in I and Z groups at the end of8th week and that in Z groupat the end of16th week were lower than M group (P<0.05or0.01).4.2UPE of24hoursUPE in M group at each time point was significantly higher than C groupat same time point (P<0.01). At the end of8th week, UPE in I and Z groupswere lower than M group (P<0.05). And at the end of16th week, water intakein Z group was lower than M and I groups (P<0.05or0.01).4.3Kidney functionAt the end of16th week, serum creatinine (Scr), urea nitrogen (BUN) anduric acid (UA) of rats in M group were all higher than C group (P<0.01).Compared with M group, Scr and BUN of I and Z groups were lower (P<0.05or0.01), while UA of Z group was lower than M group as well (P<0.05). 4.4KIAt the end of16th week, KI of rats in M group was higher than C group(P<0.01). And KI in I and Z groups were lower than M group (P<0.05).4.5Observation of renal cortical structures by light microscopeRenal cortex of rats in C group showed normal structures. Renal cortex ofM group revealed obvious hypertrophy of glomerular, slightly thickenedbasement membrane, expansion of mesangial matrix, expanded capillaries,narrow renal capsule and disarranged renal tubular epithelial cells. Comparedwith M group, renal capsules in I and Z groups were wider; and themesangial proliferations were both alleviated.4.6Observation of ultrastructures of glomeruli by transmission electronmicroscopeGlomeruli from rats in C group showed normal ultrastructures. And thosefrom M group revealed homogeneously thickened basement membrane withnot clear structure, increased mesangial matrix and segmental fusion ofpodocyte foot processes. Compared with M group, glomeruli from rats in Iand Z groups showed clearer ultrastructures with segmentally thickenedbasement membrane and partially segmental fusion of podocyte footprocesses.5Effect of Chinese medicine of stasis-emoving-and-collaterals-dredgingintervention upon high glucose stimulating podocytes5.1MTT testThe optical density (OD) of HG group at each time point wassignificantly lower than NG group at same time point (P<0.05or0.01).Chinese medicine of several concentrations could reverse the suppressingproliferation effect of high glucose at each time point. Among differentconcentrations, the OD of100μg/ml medicated high glucose group was thehighest, and higher than HG group (P<0.01). The optimum concentration was100μg/ml.5.2Col-Ⅳ content in podocyte supernatantThere was no significant difference in Col-Ⅳ content of podocyte supernatant among different groups podocyte at24hours (P>0.05). Col-Ⅳcontents of podocyte supernatant of HG group at48and72hours were bothhigher than NG group at same time points (P<0.01). And Col-Ⅳ content ofpodocyte supernatant of (HG+Z) group at48hours was significantly lowerthan HG group at same time point (P<0.05).5.3Expressions of ACE and ACE2proteins in podocytesThe ACE and ACE2proteins expressed specificly in the cytoplasm ofpodocytes. Compared with NG group at48hours, the ACE protein expressionof HG group was higher (P<0.05), while the ACE2protein expression of HGgroup was lower (P<0.01). Compared with HG group at48hours, the ACEprotein expression of (HG+Z) group was higher (P<0.05), while the ACE2protein expression of (HG+Z) group was lower (P<0.05). However, there wasno difference in expressions of ACE and ACE2proteins among differentgroups at24and72hours (P>0.05).5.4Relative expressions of ACE and ACE2mRNA in podocytesCompared with NG group at same time point respectively, ACE mRNAexpression of HG group at each time point was higher significantly (P<0.05),and ACE2mRNA expression of HG group was lower significantly only at48and72hours time points (P<0.05or0.01). Compared with HG group at sametime point respectively, just at48hours time point, ACE mRNA expression of(HG+Z) group was lower (P<0.05), and ACE2mRNA expression of (HG+Z)group was higher (P<0.01).Conclusions:1DN rats had dark purple paws and tail veins; and their blood in highviscosity, hypercoagulable state. These were in accordance with the features ofblood-stasis-blocking-collaterals syndrome in Chinese medicine, whichaffirmed the existence of blood-stasis-blocking-collaterals syndrome.2The stasis-removing-and-collaterals-dredging therapy played a benefitrole in regulating blood-stasis-blocking-renal-collaterals syndrome in DN.3The function of ACE2-Ang-(1-7)-Mas axis was down-regulated in DMrats. The stasis-removing-and-collaterals-dredging intervention for16weeks could up-regulate its function, which effect was superior to thepositive control drug irbesartan.4The stasis-removing-and-collaterals-dredging therapy alleviated therenal injury in diabetic nephropathy of stasis-blood-blocking-collateralssyndrome effectively, which effect was slightly superior to the positivecontrol drug irbesartan.5High glucose stimulation had effects of suppressing proliferation,promoting Col-Ⅳ secretion, up-regulating ACE expression anddown-regulating ACE2expression upon mouse podocytes. Chinese medicineof stasis-removing-and-collaterals-dredging intervention for48h could reversethe negative effects of high glucose stimulating podocytes. |
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