Effect Of Aldosterone On Expression Of Connective Tissue Growth Factor And Proliferation In Human Proximal Tubular Epithelial Cells

ObjectiveIt has been shown in a number of clinical as well asexperimental studies that the prognosis of kidney diseases hadconsanguineous relationship with the inflammation and fibrosisof tubulointerstitial, tubulointerstitial injury plays a key role inthe proceed and development of kidney diseases, it is aconsistent predictor of functional impairment rather thanglomerular damage.Renal tubulointerstitial fibrosis is a common feature ofprogressive renal diseases regardless of the origin. The directcauses of this process are the over-production and blunteddegradation of extracellular matrix (ECM). Various cytokinesare thought to be involved in this process by stimulatingfibroblast proliferation, macrophage infiltration, and ECMaccumulation. Among them, multiple lines of evidence haveshown a close relationship between the expression oftransforming growth factor-β(TGF-β) and the progression oftubulointerstitial fibrosis, suggesting its pathogenic role in renalfibrotic diseases. Studies have provided plausible evidence forTGF-βas a potential therapeutic target against renal fibrosis.However, an important concern is that long-term suppression ofTGF-β, a multifunctional cytokine, might be detrimental.Therefore, to explore novel antifibrotic strategies, it is importantto elucidate the mechanisms and downstream pathways specificto the profibrotic action of TGF-β. Studies have shown thatconnective tissue growth factor (CTGF) acts as a downstreammediator of TGF-β,which expression is increased in a variety ofhuman and experimental diseases characterized by fibrosis,especially in the kidney fibrosis diseases, suggesting its crucialroles in tubulointerstitial fibrosis . In human proximal tubularepithelial cell line cultured with CTGF, result showed CTGFcan promote the proliferation of proximal tubular epithelial cellsand transdifferentiation to myofibroblast(MFB), induce thesynthesis of extracellular matrix(ECM) , hasten the process oftubulointerstitial fibrosis in a dose-dependent manner.Renal tubulointerstitial fibrosis is viewed as the finalcommon pathway to renal failure, regardless of the initiatinginjury. Although the mechanisms of the interstitial are notexactly known, various studies have shown that therenin-angiotensin-aldosterone system (RAAS) plays an importa-nt role in the development and progression of renal diseases.Over the past decades, although more evidence demonstratedconclusively that angiotensin II (Ang II) mediates renal functionand structural damage in various kidney diseases. Recentexperimental and clinical studies also implicate aldosterone(ALD) as an important pathogenesis factor in progressive renaldisease through both hemodynamic effects and direct cellularactions, which has the function of non-dependent Ang IIespecially in stimulating organ fibrosis, but the way is unknown.Hyperaldosteronism has to be found in many animal modelof renal failure and patients of chronic renal failure, ALD playsthe important role of continuous damage in remnant kidney.ALD fosters progressive renal injury, the underlyingmechanisms for the pathological actions are not understood.Recent studies have shown that ALD not only regulated waterand sodium balance, but also promote collagen deposition,fibrosis , such as kidney,heart and ventricles. ALD, whichsynthesized in kidney, can also promote collagen deposition,up-regulate fibrosis cytokine, effect hypertension by the regulateof vessel intensity, effect function of cells by the regulate ofsynthesize and function of Na+-K+-ATPase. ALD has manysimilar affection of CTGF, can stimulate the thicken of tubularmembrane, shrink of tubular epithelial cells, up-regulate TGF-β,induce tubular epithelial cells transdifferentiation to MFB,increase ECM deposition such as collagenⅠ,Ⅳand fibronetion(FN), thus to accelerate tubulointerstitial fibrosis. Variousanimal trials in vitro and in vivo have been demonstrated thataldosterone can stimulate the expression of CTGF in kidney.On the basis of previous studies, what's the effect of ALDon the expression of CTGF in human proximal tubular epithelialcells? Can it enhance the expression of CTGF, thus promote theprogression of tubulointerstitial injury? The purpose of ourstudy is to determine the possible effect of aldosterone onexpression of connective tissue growth factor and proliferationin cultured human proximal tubular epithelial cells in vitro,thereby to explore the role of aldosterone in tubulointerstitialfibrosis and it's mechanism .Methods1 Culture of the human proximal tubular cell (HPTC): HK2was purchased from Cell Culture Center of Peking UnionMedical College. The cells were maintained at 37℃in anatmosphere of 5% CO2 in RPMI 1640, containing 10% FCS,100 U/L penicillin, and 100 mg/L streptomycin. The experimentdivided into two groups: control group and ALD group, toobserve the effect of different concentration(10-11,10-10,10-9,10-8 and 10-7 mol/L) ALD and time (24h,48h and 72h by 10-9mol/L ALD on HK2) on expression of CTGF and proliferationin cultured human proximal tubular epithelial cells.2 The response of CTGF mRNA expression to aldosteronestimulation was observed by reverse transcription polymerasechain reaction (RT-PCR); The proliferate effects of aldosteronewere evaluated by methylene blue assay (MTT).3 The data were shown by　±s, the statistical analysis wasdescribed by SPSS10.0 software. Results1. Successfully cultured the human proximal tubularepithelial cells.2. The results of RT-PCR indicate that ALD can induce the...