Phillips A O, Steadman R, Topley N, Williams J D
Institute of Nephrology, University of Wales College of Medicine, Cardiff Royal Infirmary, United Kingdom.
Am J Pathol. 1995 Aug;147(2):362-74.
Interstitial fibrosis is a marker of progression of renal impairment in diabetic nephropathy. Transforming growth factor (TGF)-beta 1 is one of a group of pro-fibrotic cytokines and growth factors that have been associated with the development of interstitial fibrosis. We have examined the modulating influence of glucose on the production of TGF-beta 1 by cultured human proximal tubular cells. Incubation of growth-arrested human proximal tubular cells (HPTC) (72 hours in serum free medium) in 25 mmol/L D-glucose resulted in increased expression of TGF-beta 1 mRNA (as assessed by reverse transcription polymerase chain reaction). This was apparent after 6 hours and increased up to 120 hours exposure. TGF-beta 1 secretion, however, as measured by specific enzyme-linked immunoassay, was unaffected by exposure to 25 mmol/L D-glucose. Sequential stimulation of HPTC, first with 25 mmol/L D-glucose for 48 hours and then with platelet-derived growth factor (PDGF) isoforms, resulted in a dose-dependent secretion of TGF-beta 1. Pre-exposure to 5 mmol/L D-glucose or 25 mmol/L L-glucose did not prime for TGF-beta 1 release. At 50 ng/ml PDGF this effect was greatest for the AA isoform (AA 31.4 +/- 7.1, AB 20.98 +/- 8.9, BB 7.8 +/- 2.2, P < 0.05 for all versus control, n = 3, mean +/- SEM ng/10(6) cells/24 hours). These effects were blocked by the addition of antibody to the PDGF alpha-receptor. TGF-beta 1 secretion was inhibited in a dose-dependent manner by pretreatment with cyclohexamide, but was not affected by pretreatment with actinomycin D. Stimulation of HPTC with a single dose of PDGF induced TGF-beta 1 mRNA; however, only after application of a second dose of PDGF (after TGF-beta 1 mRNA induction) did TGF-beta 1 protein secretion occur. We also demonstrated that PDGF stimulation of HPTC induced an inherently more stable TGF-beta 1 mRNA transcript. These findings demonstrate that elevated D-glucose concentration alone is insufficient to lead to increased TGF-beta 1 secretion by HPTC despite increased mRNA expression. However, application of a second stimulus such as PDGF, when TGF-beta 1 mRNA expression is increased, leads to increased protein synthesis and secretion of TGF-beta 1. This implies that elevated glucose concentrations might prime proximal tubular cells for TGF-beta 1 synthesis and thus contribute to the development of interstitial fibrosis.
间质纤维化是糖尿病肾病肾功能损害进展的一个标志。转化生长因子（TGF）-β1是一组促纤维化细胞因子和生长因子之一，与间质纤维化的发生有关。我们研究了葡萄糖对培养的人近端肾小管细胞产生TGF-β1的调节作用。将生长停滞的人近端肾小管细胞（HPTC）（在无血清培养基中培养72小时）在25 mmol/L D-葡萄糖中孵育，导致TGF-β1 mRNA表达增加（通过逆转录聚合酶链反应评估）。6小时后即可明显观察到这种增加，并在长达120小时的暴露后持续增加。然而，通过特异性酶联免疫测定法测量，TGF-β1的分泌不受25 mmol/L D-葡萄糖暴露的影响。对HPTC进行顺序刺激，首先用25 mmol/L D-葡萄糖刺激48小时，然后用血小板衍生生长因子（PDGF）异构体刺激，导致TGF-β1呈剂量依赖性分泌。预先暴露于5 mmol/L D-葡萄糖或25 mmol/L L-葡萄糖不会引发TGF-β1释放。在50 ng/ml PDGF时，这种效应在AA异构体中最为明显（AA 31.4±7.1，AB 20.98±8.9，BB 7.8±2.2，与对照组相比，所有P<0.05，n = 3，平均值±SEM ng/10(6)细胞/24小时）。这些效应可通过添加抗PDGFα受体抗体来阻断。用环己酰胺预处理可剂量依赖性地抑制TGF-β1分泌，但放线菌素D预处理对其无影响。用单剂量PDGF刺激HPTC可诱导TGF-β1 mRNA；然而，只有在应用第二剂量的PDGF（在TGF-β1 mRNA诱导后）后才会发生TGF-β1蛋白分泌。我们还证明，PDGF刺激HPTC可诱导本质上更稳定的TGF-β1 mRNA转录本。这些发现表明，尽管mRNA表达增加，但仅升高D-葡萄糖浓度不足以导致HPTC分泌TGF-β1增加。然而，当TGF-β1 mRNA表达增加时，应用第二种刺激物如PDGF会导致TGF-β1的蛋白质合成和分泌增加。这意味着升高的葡萄糖浓度可能使近端肾小管细胞为TGF-β1合成做好准备，从而促进间质纤维化的发展。
