| Obstructive nephropathy due to congenital or acquired urinary tractobstruction is the first primary cause of acute or chronic renal failure(CRF)and also a major induced cause of urinary tract infection. The mechanism ofchronic urinary tract obstruction is very complicated, including hemodynamicchanges, inflammatory mediators, vascular active substances, oxidative stress,which eventually lead to kidney damage and renal function failure. There is aclosed relationship between renal disease and the system ofRenin-angiotensin-aldosterone (RAAS). At present, the study of angiotensinII(Ang Ⅱ) is more, and the mechanism that AngⅡ through transforminggrowth factor-β (TGF-β) pathway to promote renal interstitial fibrosis (RIF)has more clear. Application of angiotensin converting enzyme inhibitors(ACEI)and/or angiotensin Ⅱ blockers (ARB) caninhibit the damage to structure andfunction of chronic renal disease. But there are still many patients, even ifadministration of plenty of ACEI and ARB are not effective, indicating thereare other factors such as inflammation injury, oxidative stress to participatethis process. Existing studies have shown that inflammatory lesions play animportant role in the progress of chronic disease, therefore anti-inflammatorytherapy has important significance for the prognosis of chronic diseases. Thisinflammatory damage is not caused by infection, but it is endogenous, andclosely related to renal interstitial fibrosis. Moreover, inflammatory mediatorsplay an important role in the process of renal interstitial fibrosis. Studies havepointed out that inflammatory mediators such as monocyte chemotacticprotein1(MCP-1), Interleukin1β(IL-1β), tumor necrosis factor α(TNF-α),induced nitric oxide synthase(iNOS) are important factors of inflammationand fibrosis in chronic renal disease. With the study of RAAS, in addition toAng Ⅱ, aldosterone can also be activated by Serum and glucocorticoid-induced protein kinase1(SGK-1), Nuclear factor κB (NF-κB),oxidative stress and so on, further cause infiltration of renal inflammatorycells and accumulation of extracellular matrix (ECM), eventually leading tothe progress of the RIF. Therefore, blocking activated aldosterone to inhibitinflammatory can delay the progress of chronic renal disease. In our study,renal interstitial fibrosis animal model was established by unilateral ureteralobstruction (UUO), and serum aldosterone, SGK-1, MCP-1, TNF-α, iNOS,IL-1β, NF-κB and α-SMA were detected by Real-time PCR,immunohistochenical method, Western blot. The effect of mineralocorticoidreceptor blocker Eplerenone and Chinese herbs was observed, whether caninhibite the expression of inflammatory mediators by blocking aldosteronesecretion, future inhibite cell phenotype transformation, and providetheoretical and experimental basis.PartⅠ Mechanism and Relationship of Toxin and Stasis on Renal Injury."Toxin" and "Stasis" were briefly introduced with the concept,classification, formation mechanism, the role in the pathogenesis of kidneydisease, and interaction with stasis and toxin, the role of both in kidneydisease, molecular biology and clinical application.Many experimental study showed that renal interstitial fibrosis waspathological change and common pathway of a variety of chronic kidneydiseases, and inflammatory lesions was a original and induced factor in thisprocess. Modern medical studies have shown that infiltration of inflammatorycells and the expression of inflammatory mediators and inducing fibrosiscytokines were attributable to traditional Chinese medicine “endogenoustoxin”, however the accumulation of extracellular matrix in the kidney was aperformance of “stasis”, therefore “toxin” was the cause characterized byinflammation injury,“stasis” was the result characterized by renal interstitialfibrosis, and these two factors were interrelated and they interacted with eachother, caused and aggravated renal interstitial fibrosis, and run through thewhole pathological process of kidney disease. At the same time, we alsosummed up that “toxin and “stasis” can be used as the basic pathogenesis of chronic kidney disease.In clinic, there was a closed relationship between the progress of kidneydisease and infection, and kidney diseases were induced or aggravated byinfection, and nephropathy patients proned to infection. Inflammation injurycaused by infection played an important role in the progress of renalinterstitial fibrosis, and anti-inflammatory therapy may be a therapeutic target."Poison","Yu" pathogenesis theory have provided the theory basis for theclinical treatment of chronic kidney disease, and have important significancefor the progress of chronic kidney disease.PartⅡ Effect of Shenluotong Decoction on pathomorphology in the Ratswith Obstructive NephropathyMethods:48Wistar rats were randomly divided into the shamgroup(n=12), UUO group (n=12), EPL group(n=12), SLT group(n=12). Ratswere free to eat and drink, and temperature was controled in the condition(22±2)℃. After adaptive feeding1WK, except the Sham group’rats, the otherrats were established model of unilateral ureteral obstruction according to themethod of Iwai T. After anesthesia with10%chloral hydrate, the rats weresliced through the left middle abdominal skin, separated the left ureter, ligatedrespectively on ureteral1/3and middle1/3ligation with silk, cut off the ureter,then sutured in skin by layer to layer. The rats in Sham group were onlyseparated the left ureter, but not ligated and cutted off the ureter. At3daysafter operation, renal pathology of modeling rats showed a significant increasewith infiltration of inflammatory cells, as well as at5days after operation,pathological changes showed renal collagen deposition, therefore this modelwas successfully copied. The success rate of this surgery was92%, and fiverats were died after UUO operation. Rats were supplemented with alternaterats. After operation, the rats were treated with100mg/kg Eplerenone in EPLgroup and26g/kg in SLT group; others in sham and UUO group administeredwith the same volume of saline once a day. At the10day after surgery, allrats were sacrificed and specimens of blood were got to detect index and leftkidney was cut to be measured the index of kidney weight/body weight(KW/BW). Parts of left kidney were prepared respectively for HE andMASSON staining.The data were counted by SPSS13.0software. Before comparison of eachgroup, tests of normality and homogeneity of variance were given. If the datacan achieve such criteria, the results were analyzed with One way ANOVA.The data were expressed as mean±SD. All results are considered significantat P<0.05and P<0.01.Results:1Index of kidney weight, body weight and kidney weight/body weightAfter surgery,5rats were died and rats were supplemented with alternaterats. The general situation of Sham group’s rats were in good condition in thewhole experimental process: daily diet and water were stable, the growth ofweight was gradual, hair was shiny, activity was agile, and response toexternal stimuli was sensitive. At1day after surgery, all rats showed thatwater intake, food intake and weight were down-regulated. At6days aftersurgery, the general situation of UUO group’s rats were not optimal: activitywas decreased significantly, and hair was loose and dull. The weight of UUOmodeling animals were not increased but decreased. At10days after surgery,the weight of modeling animals were increased slowly, and there weresignificant differences compared with Sham group(P<0.01, P<0.05);compared with UUO group, obstructed kidney weight and kidney weight/body weight were also decreased significantly(P<0.01, P<0.05).After UUOoperation, food intake, water intake and urine volume in UUOmodeling ratswere decreased, but there was no significant difference between each group.2The Result of PathomorphologyHE staining showed that basic structure of kidney was normal, tubularepithelial cells arranged regularly, interstitial edema was almost nought and ithad few inflammation cells in Sham group. There were a large number ofinflammation cells infiltration, and renal tubular epithelial cells weredenatured, shed and turgid in UUO group. There were some inflammation cellinfiltration and renal tubular epithelial cells were turgid in treatment groups, but it was lighter than UUO group. Masson staining showed that a littlecollagen deposition in glomerular/tubular basement membrane and renalinterstitium in Sham group. The structure were disordered and tubularinterstitial collagen were significantly increased in UUO group. There weresome degree of collagen deposition in treatment groups, but it wassignificantly reduced compared with the UUO group.3The count of renal interstitial inflammatory cellsCompared with the Sham group, the number of inflammatory cells wasincreased significantly in the UUO group (P<0.01). After treatment, thenumber of inflammatory cells was significantly lower in both EPL group andSLT group (P<0.01, P<0.05). Compared with Medulla area, the number ofinflammatory cells was significantly heighest in cortical area; Compared withcortical area, the number of inflammatory cells was significantly decreased inboundary area (P<0.01, P<0.05).Part Ⅲ Effect of Shenluotong Decoction on Inflammatory Mediators inthe Rats with Obstructive NephropathyMethods: The methods of animal grouping, model establishment,medication and statistics were the same to PartⅡ,10days. The expression ofMCP-1in kidneys were detected by ELISA, and serum content of TNF-α,iNOS and IL-1β were detected by radioimmunoassay. The mRNA and proteinexpression of MCP-1and TNF-α were detected by Real time-PCR,immunohistochemistry and Western Blot respectively.Results:1Serum content of inflammatory mediatorsCompared of the rats in Sham group, serum content of MCP-1, TNF-α,iNOS and IL-1β were increased significantly (P<0.01, P<0.05). Compared ofthe rats in UUO group, serum content of MCP-1, TNF-α, iNOS and IL-1βwere decreased significantly in both EPL group and SLT group (P<0.01,P<0.05).2The results of Real time-PCRCompared with the rats in Sham group, the mRNA expression of MCP-1 and TNF-α were enhanced obviously in UUO group(P<0.01). Compared withthe rats in UUO group, the high expression of MCP-1mRNA and TNF-αmRNA was down-regulated respectively in both EPL and SLT group(P<0.01).3The results of ImmunohistochemistryThe expression of MCP-1and TNF-α were weaker in Sham group, aswell as both were strengthed, and mainly located in renal tubular epithelialcells cytoplasm in UUO group. Compared with the rats in UUO group, theexpression of MCP-1and TNF-α were decreased in range and terror in twotreatment groups.4The results of Western BlotCompared with the rats in Sham group, the protein expression of MCP-1and TNF-α were enhanced obviously in UUO group. Compared of the rats inUUO group, the protein expression of MCP-1and TNF-α were decreasedsignificantly in both EPL group and SLT group (P<0.01).Part Ⅳ Effect of Shenluotong Decoction on Serum aldosterone andSGK-1in the Rats with Obstructive NephropathyMethods: The methods of animal grouping, model establishment,medication and statistics were the same to PartⅡ,10days. Serum aldosteronewas detected by radioimmunoassay, and the mRNA and protein expression ofSGK-1were detected by Real-time PCR and Western blot respectively.Results:1The results of RadioimmunoassayCompared of the rats in Sham group, the secretion of serum aldosteronewas increased significantly in UUO group(P<0.01). Compared of the rats inUUO group, the content of serum aldosterone was expressed with a downwardtrend, but this reduction wasn’t statistically significant in EPL group(P>0.05).Serum aldosterone was reduced in SLT group significantly (P<0.01).2The results of Real-time PCRCompared of the rats in Sham group, the mRNA expression of SGK-1were enhanced obviously in UUO group(P<0.01). Compared with the rats inUUO group, the mRNA expression of SGK-1were down-regulated significantly (P<0.01).3The results of Western BlotCompared of the rats in Sham group, the protein expression of SGK-1were enhanced obviously in UUO group(P<0.01). Compared with the rats inUUO group, the protein expression of SGK-1were down-regulatedsignificantly (P<0.01).Part Ⅴ Effect of Shenluotong Decoction on α-SMA and NF-κB in theRats with Obstructive NephropathyMethods: The methods of animal grouping, model establishment,medication and statistics were the same to PartⅡ,10days. The proteinexpression of NF-кB and α-SMA were detected by immunohistochemistry andWestern Blot.Results:1The results of ImmunohistochemistryIn Sham group, there were weak expression of NF-κ B mainly in renaltubular epithelial cells; Compared of the rats in Sham group, the expression ofNF-κ B significantly increased and obviously in the proximal tubule in UUOgroup.In ELP and SLT groups, the expression of NF-κ B were significantlydecreased in range and strength.In Sham group, there were a few expressions of α-SMA only in vascularsmooth muscle cells. The expression ofα-SMA were remarkably increased inUUO group than that in corresponding Sham group with the expanding scopeof tubulointerstitium in cortical and corticomedullary area. Compared with therats in UUO group, the expression of α-SMA were reduced obviously in bothEPL group and SLT group.2The results of Western BlotCompared of the rats in Sham group, the protein expression of α-SMAand NF-кB were enhanced obviously in UUO group(P<0.01). Compared withthe rats in UUO group, the protein expression of α-SMA and NF-кB weredown-regulated significantly (P<0.01). Conclusions:1“toxin” and “stasis” were the basic pathogenesis of chronic kidneydisease.“toxin” was the cause characterized by inflammation injury,“stasis”was the result characterized by renal interstitial fibrosis, and these two factorswere interrelated and they were interacted with each other, caused andaggravated renal interstitial fibrosis, and run through the whole pathologicalprocess of kidney disease.2The animal models of renal interstitial fibrosis were established byunilateral ureteral obstruction. Inflammatory cells infiltration and Collagentissues in the kidney were increased remarkably in UUO modeling rats.Chinese herbs and eplerenone can prevent kidney damage induced by UUO,reduce inflammatory cells infiltration and delay the progress of interstitialfibrosis.3Inflammatory is an important original factor of obstructive nephropathy,and inflammatory mediators play an important role in this process.Inflammatory mediators, such as MCP-1, TNF-α, IL-1β and iNOS, areimportant factors in mediating inflammation and interstitial fibrosis of chronickidney disease. Chinese herbs and eplerenone can down-regulate theexpression of MCP-1, TNF-α, IL-1β and iNOS, future inhibit infiltration ofinflammatory cells and inflammatory injury, and delay the progress ofinterstitial fibrosis.4In addition to the Ang II, aldosterone may also induce renalinflammatory and infiltration of inflammatory cells through activation ofSGK-1. Chinese herbs and eplerenone can reduce the content of serumaldosterone, down-regulate mRNA and protein expression of SGK-1, futureinhibit inflammatory and interstitial fibrosis of Obstructive Nephropathy.5Chinese herbs and eplerenone can down-regulate the expression ofinflammatory mediators by NF-κB pathway, inhibit signal transduction,thereby inhibit cell phenotype transformation, reduce renal inflammatory, anddelay the progress of renal interstitial fibrosis. |
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