ⅠMolecular Mechanisms Of Vascular Remodeling In Rat Model Of Hyperaldosteronism ⅡTechnique Of Retroperitoneoscopic Radical Nephrectomy Based On Anatomy Of The Perirenal Area

Background:Primary aldosteronism (PA)is a secondary,endocrine-mediated form ofhypertension defined by an autonomous aldosterone overproduction.Recently publishedstudies from various geographic populations reported significantly higher prevalence of PAin hypertensive patients (ranging from 5% to 30%)than the previously published data(ranging from 1% to 2%).Since Conn's original report of the aldosterone-producingadenoma in 1954,many subtypes of primary aldosteronism have been described.The mostcommon subtypes are aldosterone-producing adenoma (APA)and bilateral idiopathichyperaldosteronism (IHA).Because of the deleterious cardiovascular effects of aldosterone,normalization ofcirculating aldosterone level or aldosterone receptor blockade should be an important themanagement plan for all patients with PA.Unilateral laparoscopic adrenalectomy,whichcan result in normalization of hypokalemia in all patients with APA,is supposed to be anexcellent treatment option for patients with unilateral APA.However,the long-term curerate of hypertension after removal of the APA ranges widely in patients,from 33% to 87%.In IHA,the situation is worse as unilateral or bilateral adrenalectomy seldom corrects thehypertension.To date,the mechanisms of persistent hypertension are not completelyelucidated.Remodeling of resistance arteries is a hallmark of arterial hypertension and canbe implicated in the excess cardiovascular damage associated with hypertension.It was alsofound that vascular structural remodeling existed in PA patients' small resistance arteries,where the renin-angiotensin system is shut off.Thus,some scholars hold that vascularremodeling contributes to maintaining high BP values,even when the triggeringmechanisms have vanished in PA.Vascular remodeling is defined as the vascular wall changes from dynamic and trophicstimuli resulting in vascular hypertrophy or rearrangement of vascular wall material.Several types of vascular remodeling have been recently described:Inward remodelingdescribes a decrease in lumen diameter,whereas outward remodeling refers to an increasein lumen diameter.Hypotrophic remodeling describes a decrease in the amount of vascularwall materialno matter whether the lumen diameter is decreased or increased,whereas eutrophic remodeling refers to an absence of change in the amount and properties of wallmaterial regardless of whether the lumen diameter is decreased or increased.In theseprocesses,hypertrophic inward vascular remodeling is the most one and is associated withan increase in wall thickness,an increase in wall-to-lumen ratio,and a decrease in lumendiameter.Chronic sustained hypertension leads to structural changes oft he small and largearteries.These alterations consist of smooth muscle hypertrophy and hyperplasia,increaseddeposition of collagen,and"dilution"or destruction of elastin fibers.In addition,therecould be no growth of vascular wall materials but a"rearrangement"of them,which termed"remodeling."These changes serve to increase wall thickness and the media-to-lumen ratioand to decrease the external and internal diameter of the vessel—all of which contribute toincreased systemic vascular resistance in the small arteries and increased impedance in thelarger arteries.Recent accumulating lines of evidences from clinical and experimental studies havesuggested that direct cardiovascular effect of aldosterone contributes to the development ofcardiovascular injury via MRs in non-epithelial tissue.For instance,aldosterone has beenreported to induce expression of some genes involved in vascular fibrosis,calcification,andinflammation,all of which are considered important in pathology of vascular remodeling.Aldosterone also induce mitogenesis of vascular smooth muscle cells (VSMCs),resultingin vascular structural remodeling under the presence of angiotensinⅡ.However,aldosterone itself without the presence of angiotensinⅡis also considered to causecardiovascular injuries.Vascular structural remodeling in small resistance arteries has beenreported in patients with primary aldosteronism (PA),whose serum aldosterone levels wereelevated but serum angiotensinⅡlevel was markedly down-regulated.In addition,aldosterone itself has also been demonstrated to stimulate proliferation of VSMCs.Therefore,aldosterone may directly induce some MR-responsive gen.e associated inregulation of the cell cycle in VSMCs.Nakamura and Suzuki recently reported that murine double minute oncogene 2(MDM2),which was originally cloned from a spontaneously transformed BALB/c 3T3 cellline,was possibly involved in VSMC proliferation through MR by aldosterone in vitro. MDM2 is a nuclear protein that forms a complex with p53 and inhibits p53-mediated geneexpression by concealing its transactivating domain.MDM2 is known to regulate thebiological activity of p53 by preventing p53-mediated apoptosis or reversing p53-inducedG1 block of the cell cycle,thus promoting the entry of cells into S phases throughformation of these complexes.P53 activates the transcription of MDM2 while MDM2conversely inhibits p53-mediated genes,and the two genes are involved in a feed-backregulatory loop.Besides,MDM2 has been considered to be involved in promoting the entryof cells from G2 into M phases by reversing p53-mediated G2 block of the cell cycle.Inaddition,MDM2 is known to interact both physically and functionally with the RB protein,which is also involved in VSMC growth.Finally,MDM2 was also reported to be regulatedby the Ras-driven Raf/MEK/MAP kinase pathway in a p53-independent manner.In theirstudy,Nakamura speculated that MDM2 may be possibly associated withaldosterone-induced vascular structural remodeling of human resistance arteries,whichmay result in persistent hypertension even after resection of aldosterone-producingadrenocortical adenoma.Except hypertrophy or hyperplasia of VSMCs participated in the process of vascularremodeling,increased deposition of extracellular matrix is also important.Thisextracellular matrix is a complex mixture of structural proteins and glycoproteins,includingcollagens,fibronectins,and proteoglycans.These processes are mediated by severalmediators,such as endothelins 1 (ET-1),epidermal growth factor (EGF),platelet-derivedgrowth factor (PDGF)and transforming growth factor-β1 (TGF-β1),et al.TGF-β1 isone of these mediators and it plays an integral part in wound healing and fibrous-tissueformation.In addition,TGF-β1 is known to increase the synthesis of extracellular matrixproteins,inhibit their degradation (such as collagen typesⅠandⅢ)and promote thephenotypic conversion of fibroblasts into myofibroblasts.In this study,we first used a drug delivery system which was implantedsubcutaneously on the back of rat,establishing of a rat model of hyperaldosteronism.Ratswere infused withaldosterone (1μg/h)subcutaneously via a mini-osmotic pump for 4weeks.Systolic BP was monitored by the tail-cuff method.The pathological changes ofaorta were observed and measured under microscope,and vascular changes of mesenteric arteries were evaluated using a pressurized myograph.Then we used reversetranscriptase-polymerase chain reaction (RT-PCR),Western blotting,immunohistochemistry and sirius red staining techniques to assess the expression level ofMDM2,p53,TGF-β1,collagen typesⅠandⅢin VSMCs,respectively.Spironolactone,amineralocorticoid receptor (MR)blocker,has been demonstrated to protect extrarenaltissues from various aldosterone-induced damage.In this study we also examined whetherspironolactone may also inhibit an induction of these aldosterone-induced genes product inVSMCs in vivo.Based on the study above,we aimed to elucidate the molecularmechanisms of vascular remodeling in hyperaldosteronism,and clarify the cause of thepersistent hypertension of APA and improve the prevention and cure measures.PartⅠEstablishment of a rat model of hyperaldosteronismObjective:To introduce a method for establishing a rat model of hyperaldosteronism.Methods:Using a drug delivery system (Osmotic Minipump)which was implantedsubcutaneously on the back of rat,Sprague-Dawley rats were randomly divided into threegroups (n=8 per group):model group,antagonistic group and normal control group.Theyreceived aldosterone (1μg/h),spironolactone (100 mg/kg/d,gastric gavage)plus the equaldoses of aldosterone,vehicle (60% propylene glycol + 10% ethanol + 30% ddH20,V/V)respectively.Systolic blood pressure was measured by the tail-cuff method weekly.Allanimals were sacrificed 4 weeks later,then serum Na~+,K~+,aldosterone and PRA weremeasured.Results:The hyperaldosteronism models were successfully established.Two weeks later inaldosterone-infused group,the systolic blood pressure was markedly elevated,serum levelsof K+ and PRA were decreased,and plasma aldosterone level was increased compared withthose in the other two groups,there were statistical significance (P＜0.01),Conclusion:Utilizing the osmotic minipump,the rat model of hyperaldosteronism can besuccessfully established.This model is a simple,effective method with high successful rate and low complication rate.It provides an efficient tool for studying the primaryaldosteronism.PartⅡEffect of aldosterone and mineralocorticoid receptor antagoniston expression of MDM2 and p53 in rat aortic vascular smoothmuscle cells in vivoObjective:To study the effect of aldosterone and mineralocorticoid receptor antagonist onexpression of murine double minute2 (MDM2)and p53 gene in rat aortic vascular smoothmuscle cells in vivo and address a potential role of the interaction of p53 with MDM2 forthe regulation of cellularity.Methods:The rat models of hyperaldosteronism were established as previously describedin partⅠ.Sprague-Dawley rats (n=8 in each group)were randomly divided into threegroups:the normal control group,the aldosterone-infused group,and the antagonistic group.MDM2 and p53 gene expression in rat aortic smooth muscle cells were detected byRT-PCR,Western blotting and immunohistochemical staining.Results:Aldosterone infusion increases MDM2 and p53 gene expression in rat aorta,however the ratio of MDM2/p53 was also increased compared with controls (P＜0.01).Allthe above detrimental effects of aldosterone could be inhibited by spironolactone (P＜0.01).Conclusion:Aldosterone promotes the expression of MDM2 gene in aortic smooth musclecells in vivo,which can be inhibited by spironolactone.However,the increase of p53 geneexpression should be seen as a protective reaction to aldosterone resulting in oxidant stress.The fate of the cells maydepend not only on the presence of p53,but also on the relativeratios of MDM2 and p53 proteins.MDM2 is therefore considered one of themineralocorticoid-responsive genes that regulate cell proliferation of VSMCs,possiblyplaying an important role in aldosterone-induced vascular structural remodeling. PartⅢEffect of aldosterone and mineralocorticoid receptor antagoniston expression of transforming growth factor-beta1,collagentypesⅠandⅢin rat vascular smooth muscle cells in vivoObjective:To investigate the effect of aldosterone and spironolactone on the expressionof transforming growth factor-beta1(TGF-β1),collagen typesⅠandⅢin rat vascularsmooth muscle cells in vivo,which may help to elucidate the molecular mechanismresulting in vascular remodeling in primary aldosteronism.Methods:The rat models of hyperaldosteronism were established as previously describedin partⅠ.Sprague-Dawley rats (n=8 in each group)were randomly assigned to threegroups:the normal control group,the aldosterone-infused group,and the antagonistic group.The pathological changes of aorta were observed and measured under microscope.Mesenteric small arteries were dissected and mounted on a micromyograph,and themedia-to-lumen ratio (M/L)was calculated.TGF-β1 expression in rat vascular smoothmuscle was detected by RT-PCR,Western blotting and immunohistochemical staining.Themessenger RNA level expression of collagen typesⅠandⅢin rat aortic vascular smoothmuscle were detected by RT-PCR.The total collagen and collagen subtypes content in ratvascular wall were detected using polarized light microscopy (Sirius red staining and imageanalysis).Results:(1)With respect to the vascular structure of large aorta,there were no differencesamong three groups;Aldosterone infusion increased media width and media-to-lumen ratioof mesenteric resistance arteries compared with controls (P＜0.01).Spironolactonenormalized media and media-to-lumen ratio;(2)Both mRNA and protein levels ofTGF-β1 of aortic vascular smooth muscle were increased in aldosterone-infused rats(P＜0.01 vs controls);(3)Compared with the other two groups,both collagen typesⅠandⅢin mRNA level were increased in aldosterone-infused rats (P＜0.01);(4)With respect tothe protein level expression of collagen types in aortic media,only collagen typeⅢincreased in aldosterone-infused rats.However,the total collagenand typeⅠandⅢinmesenteric small arteries were significantly greater in aldosterone-infused rats than in the other two groups.(5)Spironolactone down-regulates the expression of TGF-β1 andcollagen typeⅠ(P＜0.01),and could not downregulate the expression of collagen typeⅢcompletely (P＜0.05).The differences were of statistical significance.Conclusion:Aldosterone and its receptor antagonist can regulate the expression ofTGF-β1,and collagen typesⅠandⅢin vascular smooth muscle cells,which mayinfluence the process of vascular remodeling in primary aldosteronism. Background:To carry out radical nephrectomy successfully,the most fundamentalprinciple is en-bloc dissection of the tumorous kidney outside the renal fascia.Astandardized procedure for retroperitoneoscopic radical nephrectomy (RRN)is lacking,soaccurate and thorough anatomical consideration of the renal area is crucial.Theretroperitoneum lies between the posterior parietal peritoneum anteriorly and thetransversalis fascia posteriorly.Since Gerota described the fascial layer around the kidneyin 1895,the structure of the retroperitoneal space has been studied for many years byanatomists and radiologists.Currently,the generally accepted viewpoints were proposed byMeyers and other scholars in a series of articles.According to their opinions,there at leastpresented three fasciae structures around the kidney:the anterior renal fascia (Gerotafascia),the posterior renal fascia (Zuckerkandl fascia),and the lateroconal fascia.Theretroperitoneum was divided into three distinct compartments by perirenal fasciae:(1)theanterior pararenal space between the parietal peritoneum and the anterior renal fascia.Superiorly it extends to the dome of the diaphragm,inferiorly it communicates with thepelvis;(2)the perirenal space between the anterior and posterior renal fasciae;(3)theposterior pararenal space between the posterior renal fascia and the transversalis fasciacontains only fat.It is open towards the pelvic cavity inferiorly but is limited medially byfusion of the posterior renal fascia with the fascia of the quadratus lumborum and psoasmuscles.Accurate anatomical recognization of the perirenal space and the fasciaeenveloping the kidney is the key to perform radical nephrectomy,no matter whether viaopen or laparoscopic approach.Objective:To study the endoscopic anatomical structures in retroperitoneal space,and tointroduce our experiences in retroperitoneoscopic radical nephrectomy accordingly.Methods:RRN was carried out at our institution in 100 patients from March 2006 toDecember 2008.Following the principle"outside the renal fascia"of radical nephrectomy,the entire surgical procedure was described as being along"two spaces"and"two poles".The ventral aspect of the kidney was dissected in the anterior pararenal space between theparietal peritoneum and the anterior renal fascia.The dorsal aspect was dissected in the anterior psoas space between the posterior renal fascia and the lumbar muscles.Cephalicdissection was upward to the subdiaphragmatic area,whereas caudal dissection was downto the iliac fossa.Important anatomical structures such as the parietal peritoneum and itsreflexion,anterior renal fascia,lateroconal fascia,posterior renal fascia,and psoas muscleswere identified during the procedure.Results:All procedures were successful except two cases which necessitated conversion toopen surgery.The commonest intraoperative complication was peritoneal effractions (11cases).No major intraoperative complications occurred.Median operative time was 65minutes (range,55-185 minutes).Median estimated blood loss was 60 mL (range,25-600mL).Surgical margins for all 100 specimens were negative for tumor.Local recurrences orport-site metastasis were not observed during a mean follow-up period of 13 months (range,1-31 months).Conclusion:Based on the accurate anatomical visualization of the perirenal area and thefascial structures,our RRN technique enabled us to enter the correct anatomical planesposteriorly and anteriorly,and carry out the RRN smoothly while simultaneously adheringto oncological principles.