Fan J M, Ng Y Y, Hill P A, Nikolic-Paterson D J, Mu W, Atkins R C, Lan H Y
Department of Nephrology, The First Hospital, Western China University of Medical Sciences, Chengdu.
Kidney Int. 1999 Oct;56(4):1455-67. doi: 10.1046/j.1523-1755.1999.00656.x.
We recently found evidence of tubular epithelial-myofibroblast transdifferentiation (TEMT) during the development of tubulointerstitial fibrosis in the rat remnant kidney. This study investigated the mechanisms that induce TEMT in vitro.
The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of recombinant transforming growth factor-beta1 (TGF-beta1). Transdifferentiation of tubular cells into myofibroblasts was assessed by electron microscopy and by expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin.
NRK52E cells cultured on plastic or collagen-coated plates showed a classic cobblestone morphology. Culture in 1 ng/ml TGF-beta caused only very minor changes in morphology, but culture in 10 or 50 ng/ml TGF-beta1 caused profound changes. This involved hypertrophy, a loss of apical-basal polarity and microvilli, with cells becoming elongated and invasive, the formation of a new front-end back-end polarity, and the appearance of actin microfilaments and dense bodies. These morphological changes were accompanied by phenotypic changes. Double immunohistochemistry staining showed that the addition of TGF-beta1 to confluent cell cultures caused a loss of the epithelial marker E-cadherin and de novo expression of alpha-SMA. An intermediate stage in transdifferentiation could be seen with hypertrophic cells expressing both E-cadherin and alpha-SMA. De novo alpha-SMA expression was confirmed by Northern blotting, Western blotting, and flow cytometry. In particular, cells with a transformed morphology showed strong alpha-SMA immunostaining of characteristic microfilament structures along the cell axis. There was a dose-dependent increase in the percentage of cells expressing alpha-SMA with increasing concentrations of TGF-beta1, which was completely inhibited by the addition of a neutralizing anti-TGF-beta1 antibody. Compared with growth on plastic, cell culture on collagen-coated plates showed a threefold increase in the percentage of cells expressing alpha-SMA in response to TGF-beta1.
TGF-beta1 is a key mediator that regulates, in a dose-dependent fashion, transdifferentiation of tubular epithelial cells into alpha-SMA+ myofibroblasts. This transdifferentiation is markedly enhanced by growth on collagen type I. These findings have identified a novel pathway that may contribute to renal fibrosis associated with overexpression of TGF-beta1 within the diseased kidney.
我们最近在大鼠残余肾的肾小管间质纤维化发展过程中发现了肾小管上皮-肌成纤维细胞转分化（TEMT）的证据。本研究调查了体外诱导TEMT的机制。
将正常大鼠肾小管上皮细胞系（NRK52E）在有无重组转化生长因子-β1（TGF-β1）的情况下，于塑料或I型胶原包被的培养板上培养6天。通过电子显微镜以及α-平滑肌肌动蛋白（α-SMA）和E-钙黏蛋白的表达来评估肾小管细胞向肌成纤维细胞的转分化。
在塑料或胶原包被培养板上培养的NRK52E细胞呈现典型的鹅卵石样形态。在1 ng/ml TGF-β中培养仅引起形态上非常轻微的变化，但在10或50 ng/ml TGF-β1中培养则导致显著变化。这包括细胞肥大、顶端-基部极性和微绒毛丧失，细胞变得细长且具有侵袭性，形成新的前端-后端极性，以及出现肌动蛋白微丝和致密小体。这些形态学变化伴随着表型改变。双重免疫组化染色显示，向汇合的细胞培养物中添加TGF-β1会导致上皮标志物E-钙黏蛋白丧失以及α-SMA的从头表达。在表达E-钙黏蛋白和α-SMA的肥大细胞中可以看到转分化的中间阶段。通过Northern印迹、Western印迹和流式细胞术证实了α-SMA的从头表达。特别是，具有转化形态的细胞沿细胞轴对特征性微丝结构呈现强烈的α-SMA免疫染色。随着TGF-β1浓度增加，表达α-SMA的细胞百分比呈剂量依赖性增加，添加中和性抗TGF-β1抗体可完全抑制这种增加。与在塑料上生长相比，在胶原包被培养板上培养的细胞对TGF-β1反应时，表达α-SMA的细胞百分比增加了三倍。
TGF-β1是一种关键介质，以剂量依赖性方式调节肾小管上皮细胞向α-SMA+肌成纤维细胞的转分化。在I型胶原上生长可显著增强这种转分化。这些发现确定了一条可能导致与患病肾脏中TGF-β1过表达相关的肾纤维化的新途径。
