【作者】 张昆；

【导师】 傅淑霞； 谭会斌；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:IgA肾病(IgA nephropathy,IgAN)的预后不仅与肾小球硬化、肾功能损伤、高血压、尿蛋白排泄量有关,还与肾间质纤维化(Renal interstitial fibrosis,RIF)关系密切,甚至比肾小球硬化更能反映肾脏受损伤的程度。染色体10上缺失的磷酸酶与张力蛋白同源物基因(Phosphase and tensin homology deleted on chromosome 10 ,PTEN)受上游转化生长因子-β1(TGF-β1)的负性调控,抑制细胞增殖诱导凋亡。已有研究证实PTEN的正常表达能够抑制TGF-β1诱导肺成纤维细胞转分化、增殖以及高糖环境下SD大鼠肾小球系膜细胞肥大和增殖,但其对RIF的影响尚不清楚。本研究旨在探讨IgA肾病肾组织中PTEN表达及其对肾间质纤维化的影响。方法:于河北医科大学第二医院经临床和肾活检病理确诊的原发性IgAN患者47例。所有病人均未用糖皮质激素、细胞毒药物、血管紧张素转换酶抑制剂(ACEI)、血管紧张素ⅡAT1受体阻滞(ARB)治疗,并除外紫癜性肾炎、狼疮性肾炎、乙肝病毒相关性肾炎、肝硬化性肾小球病、银屑病相关性肾炎、甲状腺病相关性肾炎、类风湿性关节炎肾损害、以及肿瘤相关性肾病。合并急性肾小管坏死和急、慢性间质性肾炎的病例也被剔除。10例肾脏肿瘤切除后远端肾组织作对照。肾小管间质的各项病理参数的半定量积分采用Katafuchi等的标准分为4组:Ⅰ组(0分,10例) ;Ⅱ组(1～3分,14例);Ⅲ组(4～6分,13例)和Ⅳ组(7～9分,10例)。。应用免疫组化SP法检测PTEN、TGF-β1、α-平滑肌肌动蛋白(α-SMA)、Ⅲ型胶原(ColⅢ)以及原位杂交法检测PTENmRNA表达。应用北航IMS-2000图像分析系统对肾组织免疫组化以及原位杂交结果进行半定量分析。详细收集患者临床和病理资料。结果:①IgAN患者临床资料:随着IgAN间质病变程度的加重,eGFR和尿渗透压从Ⅰ组到Ⅳ组逐渐降低,各组间差异均有统计学意义(P均<0.05);Ⅳ组UPE高于Ⅰ组和Ⅱ组,组间差异有统计学意义(P<0.05);Ⅲ组和Ⅳ组硬化肾小球数明显高于Ⅰ组和Ⅱ组,而Ⅳ组硬化肾小球数也明显高于Ⅲ组,组间差异有统计学意义(P<0.05);Ⅲ组和Ⅳ组血管积分明显高于Ⅰ组和Ⅱ组,而Ⅳ组血管积分也明显高于Ⅲ组,各组间差异有统计学意义(P<0.05)。②IgANPTEN、PTEN mRNA在肾组织内的表达部位及变化:对照组和IgAN组PTEN表达部位主要在肾小管上皮细胞胞浆,肾小球内无或仅有极少量表达,在肾间质、间质血管及浸润的炎细胞中未见表达;PTEN mRNA表达部位与PTEN相似,此外在肾小管上皮细胞胞核内也有少量表达。对照组和Ⅰ组中,PTEN与PTEN mRNA几乎在每个肾小管上皮细胞胞浆中均有表达,组间差异无统计学意义(P均>0.05);在Ⅱ组、Ⅲ组和Ⅳ组中,随着肾小管间质病变程度的加重,肾间质纤维化、肾小管萎缩和炎细胞浸润逐渐增加,而PTEN的表达量逐渐减少(12.23±1.44,10.04±1.14,8.00±1.98)%,较对照组及Ⅰ组明显减少,各组间差异有统计学意义(P均<0.05);PTEN mRNA的表达量也相应逐渐减少(12.55±1.35,10.38±1.36,8.06±2.21)%,较对照组及Ⅰ组明显减少,各组间差异有统计学意义(P均<0.05)。③IgANTGF-β1、α-SMA、ColⅢ在肾组织内的表达部位及变化:对照组和Ⅰ组中,TGF-β1主要在肾小管上皮细胞管腔面胞浆中表达,肾小球内仅有少量表达,组间差异无统计学意义(P>0.05);在Ⅱ组、Ⅲ组和Ⅳ组中,随着间质病变的加重,TGF-β1除在肾小管上皮细胞的表达外,肾间质、肾小球周围及肾小管周围区域的浸润炎细胞、肾小球系膜细胞均有其表达,且表达量显著增高(9.62±1.82,11.43±1.75,13.20±1.68)%,较对照组和Ⅰ组明显增高,各组间差异有统计学意义(P均<0.05)。对照组和Ⅰ组中,α-SMA表达部位主要在肾间质血管壁平滑肌肌层,肾间质偶有表达,组间差异无统计学意义(P>0.05);在Ⅱ组、Ⅲ组和Ⅳ组中,随着肾小管损伤范围和纤维化程度加重,α-SMA广泛表达于间质区域及一些肾小管细胞胞浆,甚至在病变较重的肾小球系膜区也可见到表达(7.85±0.84,9.40±0.93,10.90±2.00)%,较对照组和Ⅰ组明显增多,各组间差异有统计学意义(P均<0.05)。ColⅢ表达部位主要在肾间质,对照组与Ⅰ组间差异无统计学意义(P均>0.05);Ⅱ组、Ⅲ组和Ⅳ组,随着肾间质纤维化程度加重,ColⅢ表达量逐渐增多,偶可见肾小管上皮细胞少量表达,各组间差异有统计学意义(P均<0.05)。④PTEN、PTEN mRNA与TGF-β1、α-SMA、ColⅢ及其临床资料的相关性分析:肾组织PTEN和PTENmRNA的表达量与eGFR(r =0.791,0.811;P<0.01)、尿渗透压(r =0.739,0.720;P<0.01)呈正相关,与肾组织TGF-β1表达量(r =-0.632,-0.614;P<0.01)、α-SMA表达量(r =-0.669,-0.627;P<0.01)、ColⅢ表达量(r =-0.656,-0.657;P<0.01)、UPE(r =-0.523,-0.606;P<0.01)、肾小球硬化率(r =-0.775,-0.776;P<0.01)以及血管积分(r =-0.850,-0.776;P<0.01)呈负相关。结论:①随着IgAN肾小管间质病变的加重,PTEN和PTEN mRNA表达逐渐减少,甚至缺失,提示PTEN可能是IgAN肾间质纤维化进程中的一种抗纤维化内源性保护分子。②IgAN中TGF-β1可能在基因转录水平下调PTEN表达,诱导肾小管上皮转分化以及细胞外基质的沉积,从而在肾小管间质纤维化过程中起重要作用。更多还原

【Abstract】 Objective: The prognosis of IgA Nephropathy (IgAN) has correlation with glomerular sclerosis、hypertension、severity of proteinuria、renal function impairment, also closely with renal interstitial fibrosis(RIF). Phosphase and tensin homology deleted on chromosome 10(PTEN), downregulated by TGF-β, can inhibit cell proliferation and induce apoptosis. Substantial evidence suggests the roles of PTEN in the development of idiopathic pulmonary fibrosis, rheumatoid arthritis and Diabetic Nephropathy. But the relationship between PTEN and RIF remains unknown. Our studies examined the potential correlation between the expression of PTEN and RIF in IgA nephropathy.Methods: Forty-seven patients diagnosed as primary IgA nephropathy by renal pathology at the second affiliated hospital of Hebei Medical University were involved in this study. All patients were not treated with glucocorticosteroid、cytotoxic drug、ACEI and ARB. Patients with Henoch-Schonlein Nephritis、Lupus Nephritis、HBV-GN associated glomerulonephritis、hepatic cirrhosis glomerulopathy、psoriasis associated glomerulonephritis、thyropathy、renal damage induced by rheumatoid arthritis and cancer associated nephropathy were excluded. Patients complicating acute renal tubular necrosis、acute or chronic interstitial nephritis also need to be excluded. 10 specimens from normal renal tissue of renal carcinoma as control group. All tissue were diagnosed as normal which evaluated by light microscopy and immunofluorescence. Tubulointerstitial lesion(TIL) was classified by using Katafuchi scale, including no TIL(Group I), mild TIL(Group II), moderate TIL(Group III) and severe TIL(Group IV). The expression of PTEN、TGF-β1、α-SMA and ColⅢin renal tissue were detected by immunohistochemistry, PTENmRNA were detected by in situ hybridization. The semi-quantitative analysis of renal tissue immunohistochemistry and in situ hybridization results were measured applicating Beihang IMS-2000 image analysis system. Collecting detailed clinical and pathological data.Results:①Clinical data: With the progress of RIF in IgA nephropathy, eGFR and urine osmotic pressure were gradually decreased from Group I to Group IV (P<0.05); UPE in Group IV was higher than in Group I and Group II (P<0.05); the rate of sclerosis glomeruli in Group III and Group IV was obviously higher than in Group I and Group II, and the rate of sclerosis glomeruli in Group IV was also higher than in Group III (P<0.05); the scores of vascular lesion in Group III and Group IV was obviously higher than in Group I and Group II, and the scores of vascular lesion in Group IV was also higher than in Group III (P<0.05).②The expression of PTEN and PTENmRNA in renal tissues of IgAN: Renal tissues from renal biopsy in control group and IgAN groups show abundant expression of PTEN in endochylema of renal tubular epithelial cell, negligible expression in glomeruli, and no expression in renal interstitium, blood vessel and inflammatory cells; The expression of PTENmRNA was similar with PTEN, and little expression in the nucleus of renal tubular epithelial cells. In control group and Group I, there are expression of PTEN and PTENmRNA in the cytoplasm of almost every tubular epithelial cells(P>0.05); With the progress of RIF in IgA nephropathy, the expression of PTEN and PTEN mRNA decreased gradually (12.23±1.4,12.55±1.35;10.04±1.14,10.38±1.36;8.00±1.98,8.06±2.21)%, there was significant differences in three groups(P<0.05).③The expression of TGF-β1、α-SMA and ColⅢin renal tissues of IgAN: In control group and Group I, TGF-β1 expressed mainly in cavosurface of renal tubular epithelial cell, but little in glomeruli, there was no difference between the groups(P>0.05); With the progress of RIF in IgA nephropathy, TGF-β1 in addition to the expression of renal tubular epithelial cells, also have a certain expression in renal interstitium, inflammatory cells surrounding renal glomeruli and tubules regions and mesangial cells, and the expression of TGF-β1 was significantly increased(9.62±1.82,11.43±1.75, 13.20±1.68)%; there were significant differences among three groups (P<0.05).In control and Group I,α-SMA expressed mainly in renal vascular smooth muscle wall, occasionally in renal interstitium, there was no difference between the groups(P>0.05); With the progress of RIF in IgA nephropathy,α-SMA widely expressed in interstitium and endochylema of renal tubular epithelial cell,even in glomerular mesangial region(7.85±0.84, 9.40±0.93, 10.90±2.00)%, there was significant differences among the three groups, and compared with control group and Group I (P<0.05). In control group and Group I, ColⅢexpressed mainly in renal interstitium, there was no difference between the groups(P>0.05); With the progress of RIF in IgA nephropathy, the expression of ColⅢincreased gradually, occasionally little in renal tubular epithelial cell, there was significant differences among the three groups (P<0.05).④The correlation between pathological and clinical data: The expression of PTEN and PTENmRNA in renal tissues has positive correlations with eGFR(r =0.791,0.811;P<0.01), with urine osmotic pressure(r =0.739,0.720; P<0.01); negative correlations with the expression of TGF-β1(r =-0.632, -0.614; P<0.01), with the expression ofα-SMA(r =-0.669, -0.627; P<0.01), with the expression of ColⅢ(r =-0.656, -0.657; P<0.01), with urinary protein excretion for 24 hours(r =-0.523, -0.606; P<0.01), with the rate of sclerosis glomeruli(r =-0.775, -0.776; P<0.01), with the scores of vascular lesion(r =-0.850,-0.776;P<0.01).Conclusion:①With the increase of TIL in IgAN, the expression of PTEN was gradually decreased, even deletion; These suggested that PTEN may be an anti-fibrosis endogenous protection element in RIF of IgA nephropathy.②Moreover, TGF-βsignaling induces epithelial to mesenchymal transition and extracellular matrix accumulation possibly through a mechanism dependent on the downregulation of PTENmRNA.更多还原