Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells
Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells
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DOI：
10.1111/j.1523-1755.2004.00958.x
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发表时间：
2004-11-01
影响因子：
19.6
通讯作者：
Stahl, RAK
中科院分区：
文献类型：
--
作者：
Wolf, G;Schroeder, R;Stahl, RAK
背景资料。临床和实验观察表明，蛋白尿不仅是慢性肾病的标志，而且可能与进展为终末期肾功能衰竭有关。滤过的蛋白质被肾小管上皮细胞摄取，使这个系统不堪重负，可能导致肾小管合成各种促炎症和促纤维化的细胞因子，包括转化生长因子-β(TGF-β)。转化生长因子-β首先通过与特定受体结合发挥作用。我们在体外系统中利用定义明确的小鼠近端肾小管上皮细胞系(MCT细胞)研究了无脂肪酸牛血清白蛋白是否调节了转化生长因子-β的特异性受体的表达。将MCT(和LLC-PK1)细胞置于含不同浓度白蛋白的无血清培养液中。用实时聚合酶链式反应(PCR)检测肾素和血管紧张素原的转录本，用酶联免疫吸附试验(ELISA)检测分泌的血管紧张素Ⅱ(Ang II)。部分细胞还用AT(1)受体拮抗剂氯沙坦处理。用Northern分析检测转化生长因子-β受体I和II型的mRNA水平，用蛋白质印迹法检测蛋白丰度。为了测试上调的转化生长因子-β受体的功能结果，将MCT细胞与白蛋白预先孵育，然后用低剂量的转化生长因子-β处理，这种转化生长因子-β本身通常不会诱导IV型胶原的表达。Western blotts检测到磷酸化Smad2的下游信号事件。用[I-125]转化生长因子-β1进行Scatchard分析，以估计亲和力和特异性结合部位的数量。将不同长度的与CAT报告基因相连的转化生长因子-βII型启动子瞬时导入MCT细胞，检测其转录活性。MCT细胞与0.5-10 mg/mL白蛋白孵育后，II型转化生长因子-β受体的mRNA和蛋白表达增加，而不影响I型受体。白蛋白孵育12小时后，II型转化生长因子-β受体蛋白表达增加，48小时后仍可检测到。白蛋白介导的II型转化生长因子-β受体基因的增加在1莫莫/L氯沙坦存在下被减弱，这表明局部肾素-血管紧张素系统参与了这一过程。经白蛋白处理的MCT细胞明显增加血管紧张素原和肾素转录本的表达，并向培养上清液中分泌更多的Ang II。转录活性分析表明，含有激活蛋白(AP-1)结合位点的启动子片段是白蛋白诱导的转化生长因子-βII型受体转录所必需的。结合分析表明，白蛋白处理显著增加了总结合部位以及对转化生长因子-β的亲和力。这一效应具有功能后果，因为白蛋白处理的MCT细胞与下游Smad2的转化生长因子-β介导的磷酸化反应更强，并且与对照细胞相比也增加了IV型胶原的表达。我们的发现表明，白蛋白上调培养的近端肾小管上皮细胞上的配体结合的转化生长因子-β受体。白蛋白诱导的局部Ang II产生的激活似乎是这种效应的原因。这可能放大了转化生长因子-β对肾小管上皮细胞的基质刺激作用，并可能成为蛋白尿对肾小管上皮细胞的病理生理作用最终导致肾小管间质纤维化的新机制。
Background. Clinical and experimental observations suggest that proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. Filtered proteins are taken up by tubular cells, and overwhelming this system may lead to tubular synthesis of various proinflammatory and profibrogenic cytokines, including transforming growth factor-beta (TGF-beta). TGF-beta acts by first binding to specific receptors. We studied in an in vitro system using a well-defined mouse proximal tubular cell line (MCT cells) whether fatty acid-free bovine albumin modulates expression of specific receptors for TGF-beta.Methods. MCT (and LLC-PK1) cells were challenged in serum-free medium with different concentrations of albumin. Activation of a local renin-angiotensin system was tested by real-time polymerase chain reaction (PCR) for renin and angiotensinogen transcripts and determination of secreted angiotensin II (Ang II) by enzyme-linked immunosorbent assay (ELISA). Some cells were also treated with the AT(1) receptor antagonist losartan. TGF-beta receptor types I and II mRNA levels were determined by Northern analysis whereas protein abundance was measured by Western blots. To test for a functional consequence of up-regulated TGF-beta receptors, MCT cells were preincubated with albumin and subsequently treated with low-dose TGF-beta that normally does not induce collagen type IV expression by itself. Downstream signaling events were detected by Western blots for phosphorylated Smad2. Scatchard assays with [I-125] TGF-beta1 were performed to estimate affinity and number of specific binding sites. Different length TGF-beta type II promoter constructs linked to CAT reporter were transiently transected into MCT cells to determine transcriptional activity.Results. Incubation of MCT cells with 0.5 to 10 mg/mL albumin leads to an increase in type II TGF-beta receptor mRNA and protein expression without influencing type I receptors. An increase in type II TGF-beta receptor protein expression was detected after 12 hours of albumin incubation and was still detectable after 48 hours. The albumin-mediated increase in type II TGF-beta receptor mRNA was attenuated in the presence of 1 mumol/L losartan, suggesting involvement of a local renin-angiotensin system. MCT cells treated with albumin significantly increased expression of angiotensinogen and renin transcripts and also secreted more Ang II into the culture supernatant. Analysis of transcriptional activity showed that promoter segments containing activating protein (AP-1) binding sites are necessary for albumin-induced transcription of the TGF-beta type II receptor. Binding assays revealed that albumin treatment significantly increased the overall binding sites as well as the affinity for TGF-beta. This effect had functional consequences because MCT cells pretreated with albumin reacted with a stronger TGF-beta-mediated phosphorylation of down-stream Smad2 and also increased collagen IV expression compared with control cells.Conclusion. Our findings indicate that albumin up-regulates ligand-binding TGF-beta receptors on cultured proximal tubular cells. Albumin-induced activation of local Ang II production appears to be responsible for this effect. This may amplify the matrix-stimulatory actions of TGF-beta on tubular cells and could be a novel mechanism for how proteinuria exhibits pathophysiologic effects on tubular cells ultimately leading to tubulointerstitial fibrosis.