SARA在糖尿病肾病肾小管上皮细胞转分化中的作用与分子机制
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摘要
研究背景
糖尿病肾病(Diabetic nephropathy, DN)是终末期肾脏疾病(End stage renal disease, ESRD)最常见的原因之一。肾小管间质纤维化(Tubulointerstitial fibrosis, TIF)是糖尿病肾病发展到ESRD的最主要病理改变。。肾小管上皮细胞转分化(Epithelial to mesenchymal transition, EMT)是肾间质纤维化的早期且可逆阶段,细胞外基质(Extracellular matrix, ECM)沉积是肾间质纤维化的主要组织学改变。研究表明高糖可导致肾小管上皮细胞EMT及ECM沉积,而转化生长因子-β1(Transforming growth factor, TGF-β1)在这一过程中起关键作用。TGF-β1/Smads信号通路是TGF-β1诱导EMT及ECM沉积的主要途径,受多个因素的调控。
Smad锚着蛋白(Smad anchor for receptor activation, SARA)是TGF-β/Smad信号通路的衔接蛋白(Adaptor protein)。SARA可呈递Smad2和Smad3至活化的TGF-βⅠ型受体(TβⅠR),促进其磷酸化,介导TGF-β1信号由其受体活化到核转位的过程。SARA是Smad2磷酸化及核转位的关键因子,而Smad3的活化不依赖于SARA。研究显示SARA与EMT及纤维化呈负相关,靶向SARA的干预策略能够逆转EMT。基于以上分析,我们认为SARA可能与糖尿病肾病肾小管上皮细胞EMT及ECM沉积有关,为此,本实验开展如下的研究。
目的观察糖尿病肾病患者肾组织中SARA的表达,初步探讨SARA与糖尿病肾病肾间质纤维化的关系。
方法随机选取经临床和肾脏病理确诊为糖尿病肾病及微小病变患者(原发性肾病)各6人。采用HE、Masson、PASM染色观察肾脏普通病理改变并评价肾间质损伤和胶原沉积程度；免疫组化检测肾脏组织SARA表达变化。
结果肾脏普通病理结果显示糖尿病肾病患者肾小球系膜基质中重度增生,结节状硬化,基底膜弥漫性或节段性增厚僵硬,肾小管萎缩并代偿性扩张,肾间质可见多灶性纤维化；对照组患者可见系膜细胞及基质轻度增生,基底膜不厚,轻度的肾小管上皮细胞颗粒样变性及空泡变性，肾间质和肾血管未见明显异常。糖尿病肾病患者肾小管间质损伤指数及胶原沉积面积明显高于对照组。免疫组化显示：对照组患者肾小管上皮细胞有明显的SARA表达,在肾小球系膜细胞也有一定量的表达；而糖尿病肾病患者SARA表达明显下调,尤其在肾小管上皮细胞萎缩及发生肾间质纤维化的区域。
结论糖尿病肾病患者肾组织(特别是肾小管)SARA表达下降；SARA表达下降的同时伴有肾小管间质损伤和胶原沉积,提示SARA可能与糖尿病肾病肾间质纤维化有关。
目的阐明SARA在高葡萄糖诱导的HK-2细胞EMT及ECM沉积中的作用,探讨以SARA为靶点抑制高葡萄糖诱导的HK-2细胞EMT及ECM沉积的策略。
方法用高葡萄糖(30mM D-葡萄糖)刺激HK-2细胞,通过细胞免疫荧光、Western blot及Realtime PCR检测波形蛋白(Vimentin)、紧密连接蛋白(Zona occludens-1, ZO-1)及SARA的表达,通过Western blot及Realtime PCR检测纤维粘连蛋白(Fibronectin, FN)和Ⅰ型胶原(CollagenⅠ, ColⅠ)的表达；转染全长SARA质粒(SARA(WT))及敲除SBD结构域的SARA质粒(SARA(dSBD)),再予高糖刺激,检测HK-2细胞Vimentin、ZO-1、FN、ColⅠ的表达变化。
结果30mM D-葡萄糖刺激后,HK-2细胞ZO-1蛋白及其mRNA表达呈时间依赖性降低,Vimentin、FN和ColⅠ蛋白及其mRNA表达呈时间依赖性升高,而SARA蛋白及其mRNA表达呈时间依赖性下降。与高糖刺激组相比,过表达SARA使HK-2细胞ZO-1表达上调,Vimentin、FN和ColⅠ表达下调；但转染SARA dSBD质粒对高糖诱导的HK-2细胞ZO-1、Vimentin、FN和ColⅠ表达无明显影响。
结论高糖诱导HK-2细胞转分化及ECM沉积；高糖诱导HK-2细胞SARA表达下调；过表达SARA可逆转HK-2细胞转分化,抑制ECM生成,这一效应依赖于SARA的SBD结构域。
目的阐明SARA调控高葡萄糖诱导的肾小管上皮细胞EMT及ECM沉积的分子机制。
方法用高葡萄糖(30mM D-葡萄糖)刺激HK-2细胞,Western blot及Realtime PCR检测TGF-β1、Smad2、Smad3表达,Western blot检测p-Smad2、p-Smad3的表达水平；转染全长SARA质粒及SARA(dSBD)质粒,再予高糖刺激,检测Smad2、Smad3、p-Smad2、p-Smad3的表达变化。
结果高糖可诱导TGF-β1、Smad3蛋白及其mRNA表达呈时间依赖性上调；Smad2 mRNA表达呈时间依赖性上调,而其蛋白表达呈时间依赖性下调；高糖可促进Smad2和Smad3磷酸化,但Smad3的活化时间更长。过表达SARA可上调Smad2的蛋白表达,但对其mRNA表达水平无明显影响,过表达SARA对Smad3的蛋白及其mRNA表达无明显影响；转染SARA(dSBD)质粒对Smad2、Smad3的表达无明显影响。过表达SARA可延长Smad2活化时间,而Smad3的活化时间相对缩短,从而改变Smad2和Smad3的相对活化水平。转染SARA(dSBD)对Smad2和Smad3的磷酸化无明显影响。
结论高糖诱导HK-2细胞TGF-β1表达上调,并诱导其通路活化；高糖环境下Smad2和Smad3表达水平改变；SARA通过调节Smad2和Smad3的相对活化水平,调节TGF-β1信号通路的靶效应。
Background Diabetic nephropathy (DN) is one of leading cause of end stage renal disease (ESRD). Tubulointerstitial fibrosis (TIF) is the most common pathological changes in DN. Epithelial to mesenchymal transition (EMT) is the early and reversible stage of TIF and extracellular matrix (ECM) accumulation is the key histological change of TIF. It is established that high glucose can result in EMT and ECM accumulation in renal tubular epithelial cells and Transforming growth factor-β1 (TGF-β1) plays a key role in these process. TGF-β1/Smads is the classical pathway which is involved in TGF-β1 mediated EMT and ECM accumulation. It is reported that TGF-β1/Smad is regulated by many factors such as adaptor protein.
Smad anchor for receptor activation (SARA) is one adaptor protein which can recruit Smad2 and Smad3 to the activated TGF-βreceptorⅠ(TβⅠR) and promote their phosphorylation. SARA plays an essential role in TGF-βinduced Smad2 activation and Smad3 can be phosphorylated in a SARA-independent way. It is showed that SARA is negatively related to EMT and fibrosis and modulation of SARA can reverse the development of EMT.
Therefore, SARA may play a key role in the process of EMT of tubular epithelial cells and ECM accumulation in diabetic nephropathy. To this end, experimental research is carried out as follow.
Objective To investigate the expression of SARA in the kidney and observe the relationship of SARA and TIF in diabetic nephropathy patients.
Methods 12 patients were enrolled into the study, they were diagnosed as DN (n=6) or minimal changes disease (primary kidney disease, control group, n=6) based on clinical manifestations and renal pathology. Renal pathological changes were observed by HE, Masson and PASM staining. Interstitial damage score and collagen deposition score were evaluated. The expression of SARA was tested by immunohistochemistry and the relationship of SARA and tubular interstitial damage in DN was analyzed.
Results HE, Masson and PASM staining showed that there were many lesions in the kidneys of DN patients such as moderate to severe proliferation of mesangial matrix, nodular sclerosis, diffuse or segmental thickening of the basement membrane, tubular atrophy and compensatory expansion, local interstitial fibrosis. Meanwhile, the lesions in the kidneys of minimal change disease patients were less severe. There were mild mesangial cell and matrix proliferation, basement membrane is not thick, mild granular degeneration and vacuolar degeneration in renal tubular epithelial cells and no significant abnormalities in interstitial and renal vascular. Interstitial damage score and collagen deposition score in the kidneys of DN patients were higher than that in control group. Immunohistochemistry showed that the expression of SARA was significantly down-regulated in DN patients compared with that in control group.
Conclusion The expression of SARA is significantly down-regulated in the kidney of DN patients and SARA is negatively related with renal interstitial fibrosis.
Objective To detect the expression of SARA in human renal proximal epithelial cell lines (HK-2) and investigate the role of SARA in the development of high glucose induced EMT and ECM in HK-2.
Methods HK-2 cells were exposed to high glucose (30mM D-glucose). The expressions of Vimentin, ZO-1, FN, ColⅠ, SARA were examined by Western blot and Realtime PCR. Furth more, HK-2 cells were transfected with the plasmids of wild-type SARA (SARA (WT)) and SARA mutant (SARA with SBD deletion, called SARA (dSBD)). Then the expressions of Vimentin, ZO-1, FN, ColⅠwere detected.
Results Stimulation of HK-2 with high glucose resulted in a significant reduce in the expression of ZO-1 and increase in the expressions of Vimentin, FN, ColⅠ. Meanwhile, high glucose reduced the protein and mRNA expression of SARA. Compared with high glucose group, overexpression of SARA in HK-2 unregulated the expression of ZO-1 and downregulated the expression of Vimentin, FN, ColⅠHowever, SARA (dSBD) had no significant effect on the expression ZO-1, Vimentin, FN, ColⅠ
Conclusion High glucose leads to EMT and ECM accumulation and reduces the expression of SARA in HK-2. Overexpression of SARA can reverse the EMT of HK-2 and inhibit the ECM accumulation, while this effect is depend on the SBD domain of SARA.
Objective To investigate the mechanism by which SARA modulates the process of high glucose induced EMT and ECM accumulation in renal tubular epithelial cells.
Methods HK-2 cells was exposed to high glucose (30mM D-glucose) and then the expression of TGF-β1, Smad2, Smad3, p-Smad2, p-Smad3 were detected. Furthermore, HK-2 cells were transfected with the plasmids of wild-type SARA and SARA (dSBD). Then the expressions of Smad2, Smad3, p-Smad2, p-Smad3 were examined.
Results The expression of TGF-β1 and Smad3 increased after stimulation of high glucose in HK-2. However, the Smad2 mRNA expression level increased while its protein expression was downregulated in a time dependent manner. Smad2 and Smad3 were activated by glucose stimulation and Smad3 kept activation for a longer time than Smad2.
Overexpression of SARA increased the protein expression of Smad2 and had no effect on its mRNA expression. Meanwhile, Overexpression of SARA didn't change the protein and mRNA expression of Smad3. SARA (dSBD) had no significant effect on both Smad2 and Smad3 expression.
Overexpression of SARA prolonged the activation time of Smad2 and shortened the activation time of Smad3 and then changed the balance between Smad2 and Smad3. However, SARA (dSBD) had no significant effect on the activation of Smad2 and Smad3.
Conclusion High glucose induces increase of TGF-β1 and Smad3 and decrease of Smad2 protein expression. SARA can modulate the TGF-β1 pathway by regulating the balance between Smad2 and Smad3.
糖尿病肾病(Diabetic nephropathy, DN)是终末期肾脏疾病(End stage renal disease, ESRD)最常见的原因之一。肾小管间质纤维化(Tubulointerstitial fibrosis, TIF)是糖尿病肾病发展到ESRD的最主要病理改变。。肾小管上皮细胞转分化(Epithelial to mesenchymal transition, EMT)是肾间质纤维化的早期且可逆阶段,细胞外基质(Extracellular matrix, ECM)沉积是肾间质纤维化的主要组织学改变。研究表明高糖可导致肾小管上皮细胞EMT及ECM沉积,而转化生长因子-β1(Transforming growth factor, TGF-β1)在这一过程中起关键作用。TGF-β1/Smads信号通路是TGF-β1诱导EMT及ECM沉积的主要途径,受多个因素的调控。
Smad锚着蛋白(Smad anchor for receptor activation, SARA)是TGF-β/Smad信号通路的衔接蛋白(Adaptor protein)。SARA可呈递Smad2和Smad3至活化的TGF-βⅠ型受体(TβⅠR),促进其磷酸化,介导TGF-β1信号由其受体活化到核转位的过程。SARA是Smad2磷酸化及核转位的关键因子,而Smad3的活化不依赖于SARA。研究显示SARA与EMT及纤维化呈负相关,靶向SARA的干预策略能够逆转EMT。基于以上分析,我们认为SARA可能与糖尿病肾病肾小管上皮细胞EMT及ECM沉积有关,为此,本实验开展如下的研究。
目的观察糖尿病肾病患者肾组织中SARA的表达,初步探讨SARA与糖尿病肾病肾间质纤维化的关系。
方法随机选取经临床和肾脏病理确诊为糖尿病肾病及微小病变患者(原发性肾病)各6人。采用HE、Masson、PASM染色观察肾脏普通病理改变并评价肾间质损伤和胶原沉积程度；免疫组化检测肾脏组织SARA表达变化。
结果肾脏普通病理结果显示糖尿病肾病患者肾小球系膜基质中重度增生,结节状硬化,基底膜弥漫性或节段性增厚僵硬,肾小管萎缩并代偿性扩张,肾间质可见多灶性纤维化；对照组患者可见系膜细胞及基质轻度增生,基底膜不厚,轻度的肾小管上皮细胞颗粒样变性及空泡变性，肾间质和肾血管未见明显异常。糖尿病肾病患者肾小管间质损伤指数及胶原沉积面积明显高于对照组。免疫组化显示：对照组患者肾小管上皮细胞有明显的SARA表达,在肾小球系膜细胞也有一定量的表达；而糖尿病肾病患者SARA表达明显下调,尤其在肾小管上皮细胞萎缩及发生肾间质纤维化的区域。
结论糖尿病肾病患者肾组织(特别是肾小管)SARA表达下降；SARA表达下降的同时伴有肾小管间质损伤和胶原沉积,提示SARA可能与糖尿病肾病肾间质纤维化有关。
目的阐明SARA在高葡萄糖诱导的HK-2细胞EMT及ECM沉积中的作用,探讨以SARA为靶点抑制高葡萄糖诱导的HK-2细胞EMT及ECM沉积的策略。
方法用高葡萄糖(30mM D-葡萄糖)刺激HK-2细胞,通过细胞免疫荧光、Western blot及Realtime PCR检测波形蛋白(Vimentin)、紧密连接蛋白(Zona occludens-1, ZO-1)及SARA的表达,通过Western blot及Realtime PCR检测纤维粘连蛋白(Fibronectin, FN)和Ⅰ型胶原(CollagenⅠ, ColⅠ)的表达；转染全长SARA质粒(SARA(WT))及敲除SBD结构域的SARA质粒(SARA(dSBD)),再予高糖刺激,检测HK-2细胞Vimentin、ZO-1、FN、ColⅠ的表达变化。
结果30mM D-葡萄糖刺激后,HK-2细胞ZO-1蛋白及其mRNA表达呈时间依赖性降低,Vimentin、FN和ColⅠ蛋白及其mRNA表达呈时间依赖性升高,而SARA蛋白及其mRNA表达呈时间依赖性下降。与高糖刺激组相比,过表达SARA使HK-2细胞ZO-1表达上调,Vimentin、FN和ColⅠ表达下调；但转染SARA dSBD质粒对高糖诱导的HK-2细胞ZO-1、Vimentin、FN和ColⅠ表达无明显影响。
结论高糖诱导HK-2细胞转分化及ECM沉积；高糖诱导HK-2细胞SARA表达下调；过表达SARA可逆转HK-2细胞转分化,抑制ECM生成,这一效应依赖于SARA的SBD结构域。
目的阐明SARA调控高葡萄糖诱导的肾小管上皮细胞EMT及ECM沉积的分子机制。
方法用高葡萄糖(30mM D-葡萄糖)刺激HK-2细胞,Western blot及Realtime PCR检测TGF-β1、Smad2、Smad3表达,Western blot检测p-Smad2、p-Smad3的表达水平；转染全长SARA质粒及SARA(dSBD)质粒,再予高糖刺激,检测Smad2、Smad3、p-Smad2、p-Smad3的表达变化。
结果高糖可诱导TGF-β1、Smad3蛋白及其mRNA表达呈时间依赖性上调；Smad2 mRNA表达呈时间依赖性上调,而其蛋白表达呈时间依赖性下调；高糖可促进Smad2和Smad3磷酸化,但Smad3的活化时间更长。过表达SARA可上调Smad2的蛋白表达,但对其mRNA表达水平无明显影响,过表达SARA对Smad3的蛋白及其mRNA表达无明显影响；转染SARA(dSBD)质粒对Smad2、Smad3的表达无明显影响。过表达SARA可延长Smad2活化时间,而Smad3的活化时间相对缩短,从而改变Smad2和Smad3的相对活化水平。转染SARA(dSBD)对Smad2和Smad3的磷酸化无明显影响。
结论高糖诱导HK-2细胞TGF-β1表达上调,并诱导其通路活化；高糖环境下Smad2和Smad3表达水平改变；SARA通过调节Smad2和Smad3的相对活化水平,调节TGF-β1信号通路的靶效应。
Background Diabetic nephropathy (DN) is one of leading cause of end stage renal disease (ESRD). Tubulointerstitial fibrosis (TIF) is the most common pathological changes in DN. Epithelial to mesenchymal transition (EMT) is the early and reversible stage of TIF and extracellular matrix (ECM) accumulation is the key histological change of TIF. It is established that high glucose can result in EMT and ECM accumulation in renal tubular epithelial cells and Transforming growth factor-β1 (TGF-β1) plays a key role in these process. TGF-β1/Smads is the classical pathway which is involved in TGF-β1 mediated EMT and ECM accumulation. It is reported that TGF-β1/Smad is regulated by many factors such as adaptor protein.
Smad anchor for receptor activation (SARA) is one adaptor protein which can recruit Smad2 and Smad3 to the activated TGF-βreceptorⅠ(TβⅠR) and promote their phosphorylation. SARA plays an essential role in TGF-βinduced Smad2 activation and Smad3 can be phosphorylated in a SARA-independent way. It is showed that SARA is negatively related to EMT and fibrosis and modulation of SARA can reverse the development of EMT.
Therefore, SARA may play a key role in the process of EMT of tubular epithelial cells and ECM accumulation in diabetic nephropathy. To this end, experimental research is carried out as follow.
Objective To investigate the expression of SARA in the kidney and observe the relationship of SARA and TIF in diabetic nephropathy patients.
Methods 12 patients were enrolled into the study, they were diagnosed as DN (n=6) or minimal changes disease (primary kidney disease, control group, n=6) based on clinical manifestations and renal pathology. Renal pathological changes were observed by HE, Masson and PASM staining. Interstitial damage score and collagen deposition score were evaluated. The expression of SARA was tested by immunohistochemistry and the relationship of SARA and tubular interstitial damage in DN was analyzed.
Results HE, Masson and PASM staining showed that there were many lesions in the kidneys of DN patients such as moderate to severe proliferation of mesangial matrix, nodular sclerosis, diffuse or segmental thickening of the basement membrane, tubular atrophy and compensatory expansion, local interstitial fibrosis. Meanwhile, the lesions in the kidneys of minimal change disease patients were less severe. There were mild mesangial cell and matrix proliferation, basement membrane is not thick, mild granular degeneration and vacuolar degeneration in renal tubular epithelial cells and no significant abnormalities in interstitial and renal vascular. Interstitial damage score and collagen deposition score in the kidneys of DN patients were higher than that in control group. Immunohistochemistry showed that the expression of SARA was significantly down-regulated in DN patients compared with that in control group.
Conclusion The expression of SARA is significantly down-regulated in the kidney of DN patients and SARA is negatively related with renal interstitial fibrosis.
Objective To detect the expression of SARA in human renal proximal epithelial cell lines (HK-2) and investigate the role of SARA in the development of high glucose induced EMT and ECM in HK-2.
Methods HK-2 cells were exposed to high glucose (30mM D-glucose). The expressions of Vimentin, ZO-1, FN, ColⅠ, SARA were examined by Western blot and Realtime PCR. Furth more, HK-2 cells were transfected with the plasmids of wild-type SARA (SARA (WT)) and SARA mutant (SARA with SBD deletion, called SARA (dSBD)). Then the expressions of Vimentin, ZO-1, FN, ColⅠwere detected.
Results Stimulation of HK-2 with high glucose resulted in a significant reduce in the expression of ZO-1 and increase in the expressions of Vimentin, FN, ColⅠ. Meanwhile, high glucose reduced the protein and mRNA expression of SARA. Compared with high glucose group, overexpression of SARA in HK-2 unregulated the expression of ZO-1 and downregulated the expression of Vimentin, FN, ColⅠHowever, SARA (dSBD) had no significant effect on the expression ZO-1, Vimentin, FN, ColⅠ
Conclusion High glucose leads to EMT and ECM accumulation and reduces the expression of SARA in HK-2. Overexpression of SARA can reverse the EMT of HK-2 and inhibit the ECM accumulation, while this effect is depend on the SBD domain of SARA.
Objective To investigate the mechanism by which SARA modulates the process of high glucose induced EMT and ECM accumulation in renal tubular epithelial cells.
Methods HK-2 cells was exposed to high glucose (30mM D-glucose) and then the expression of TGF-β1, Smad2, Smad3, p-Smad2, p-Smad3 were detected. Furthermore, HK-2 cells were transfected with the plasmids of wild-type SARA and SARA (dSBD). Then the expressions of Smad2, Smad3, p-Smad2, p-Smad3 were examined.
Results The expression of TGF-β1 and Smad3 increased after stimulation of high glucose in HK-2. However, the Smad2 mRNA expression level increased while its protein expression was downregulated in a time dependent manner. Smad2 and Smad3 were activated by glucose stimulation and Smad3 kept activation for a longer time than Smad2.
Overexpression of SARA increased the protein expression of Smad2 and had no effect on its mRNA expression. Meanwhile, Overexpression of SARA didn't change the protein and mRNA expression of Smad3. SARA (dSBD) had no significant effect on both Smad2 and Smad3 expression.
Overexpression of SARA prolonged the activation time of Smad2 and shortened the activation time of Smad3 and then changed the balance between Smad2 and Smad3. However, SARA (dSBD) had no significant effect on the activation of Smad2 and Smad3.
Conclusion High glucose induces increase of TGF-β1 and Smad3 and decrease of Smad2 protein expression. SARA can modulate the TGF-β1 pathway by regulating the balance between Smad2 and Smad3.
引文
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