The Study On The Role Of Connective Tissue Growth Factor In Diabetic Nephropathy And The Effect Of Pentoxifylline On Its Expression

Object:Transforming growth factorβ(TGF-β) is generally accepted to be the main pro-fibrotic factor in the diabetic nephropathy (DN), so it plays a vital role in the pathogenesis of DN. However, the powerful anti-inflammatory, and anti-proliferative effects of TGF-βlimit it to be the therapeutic target of DN. Connective tissue growth factor (CTGF) is the main downstream effector of profibrotic TGF-βaction, but it does not mediate the advantageous anti-inflammatory and anti-proliferative effects. CTGF becomes the perfect intervention point of DN, so it appears especially important to study the TGF-β-CTGF pathway, to discuss the role of CTGF in the pathogenesis of DN and to find an effective medicine which can inhibit the expression of CTGF while has no effect on the expression of TGF-β. In this research we use cultured mesangial cells to study the mechanism by which high glucose induces CTGF expression in vitro and use experimental diabetic rat model to research the important role of CTGF in extracellular matrix (ECM) acccumulation in the early stage of DN and in diabetic glomerulosclerosis and interstitial fibrogenesis in the later period of DN in vivo. Furthermore we use pentoxifylline (PTX) as the intervention medicine to study its inhibitory effect on CTGF expression and to explore the significance of CTGF as therapeutic target of DN.Method:1. In vitro, we use cultured rat mesangial cells to study the influence of different concentration of high glucose (15mM,30mM) on the mRNA and protein expression of TGF-P and CTGF, the protein expression of P-Smad2/3 and the Smad7 and the FN mRNA expression in the different exposure time (24h,48h,72h) Furthermore we add TGF-βneutral antibody in the cultured medium to observe the change of CTGF and FN expression.2. We use streptozotocin (STZ) to induce experimental diabetic rat model and then use the method of RT-PCR, immunohistochemistry and western-blot to examine the dynamic changes of CTGF expression in the kidney cortex at different phase of the onset of diabetes (2w,4w,8w, 12w), and to study the relationship between CTGF expression and renal hypertrophy, the extracellular matrix (ECM) accumulation of early stage of DN, and in diabetic glomerulosclerosis and interstitial fibrogenesis in the later period of DN. Simultaneously we use ELISA method to examine the change of urine CTGF content.3. We add different concentration of PTX (0.1mM,0.3mM, 1mM) in the mesangial cell cultured medium of high glucose to observe its influence on TGF-β,CTGF mRNA and the protein expression. Moreover we study the influence of PTX intervention on the expression of CTGF in diabetic rat renal cortex and its significance in DN therapy.Result:1. High glucose can increase TGF-β,CTGF mRNA and protein expression in mesangial cells(P<0.05), and in time and dose dependence, otherwise at the same time P-Smad2/3 expression increases and Smad7 expression decreases(P<0.05). The blockage of TGF-βcan decrease glucose induced CTGF mRNA and protein expression by 86.4% and 91.8%(P<0.05).2. Diabetic rat renal cortex mRNA and CTGF expression begin increase at the second week of the onset of diabetes and gradually increase alongwith the course of diabetes development, they reach the peak at 12w (P<0.05). Its expression appears in renal glomerulus in the early phase of DN and also appears in renal tubular interstitial tissue in the later period of DN. The relevant analysis demonstrates positive correlation between the expression of CTGF and TGF-β, the renal index, the renal glomerular volume, the mesangial matrix index, the fibrosis degree of renal tubule tubular interstitial fibrogenesis (P<0.05). The diabetic rat urine CTGF content gradually elevates(P<0.05)along with the course of diabetes, it has positive correlation with 24h urineμMA,BUN and Cr(P<0.05).3. 0.3mM PTX may suppress high glucose induced CTGF mRNA expression in the mesangial cell by 31.5% and suppress the protein expression by 18.6%. When the PTX dosage increases, the suppressive effect becomes more remarkable, 1mM PTX suppresses CTGF mRNA and the protein expression by 80.8% and 68.9%, respectively(P<0.05), but it has no obvious influence on the TGF-βexpression(P>0.05). In vivo, the CTGF mRNA and protein expression in the renal cortex were significantly lower in the PTX treated group compared with DM group in the same time course(P<0.05), moreover this kind of inhibition started from the second week continuously to the 12th week. In 2w,4w,8w,12w it may supress the CTGF protein expression by 98.8%,87.8%,72.4% and 79.1%, respectively. However, PTX cannot inhibit TGF-βexpression in 2w,4w,8w, whereas in 12w in the PTX group, the TGF-βexpression was lower t compared with DM group(P<0.05).Conclusion:1. High glucose upregulated CTGF mRNA expression and its protein expression mainly through TGF-β-Smads pathway.2. CTGF plays a vital role in the DN developing process, its expression increases through the whole process of DN. In the early time, it mediated the renal hypertrophy and the glomerular ECM accumulation, and in the later period it mediated diabetic glomerulosclerosis and interstitial fibrogenesis.3. The examination of urine CTGF content can become one of the DN diagnostic index.4. PTX can suppress CTGF expression effectively, but it has no direct inhibition to the TGF-βexpression, so from this we can investigate the mechanism of PTX to treat DN from a new aspect.