University of Tokyo School of Medicine, Japan.
Am J Physiol Renal Physiol. 2010 Sep;299(3):F568-76. doi: 10.1152/ajprenal.00659.2009. Epub 2010 Jun 9.
Uremic toxins can deteriorate renal function, but little is known about its mechanism. Because tubular injury is central to progression of chronic kidney disease (CKD), we investigated the effects of a representative uremic toxin indoxyl sulfate (IS) on tubular cells. IS induced endoplasmic reticulum (ER) stress in cultured human proximal tubular cells, demonstrated by the increase in C/EBP homologous protein (CHOP) in the immunoblots. Moreover, administration of an oral adsorbent AST-120 reduced serum IS concentration and decreased tubular expression of CHOP in immunohistochemistry in 5/6-nephretomized, CKD model, rats. Furthermore, we disclosed that IS inhibited proliferation of tubular cells in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2'-deoxyuridine assay, whereas the results of trypan blue exclusion and lactate dehydrogenase assay showed that IS did not promote cell death. This inhibition was mitigated by small interfering (si) RNA against CHOP. Furthermore, IS increased the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21). Surprisingly, this was mediated by the inflammatory cytokine interleukin (IL)-6, the expression of which was decreased by siRNA against activating transcription factor 4, another ER stress marker; however, the induction of IL-6 and p21 by IS was not suppressed by siRNA targeted to CHOP, suggesting that they were downstream of ER stress, but independent of CHOP. Moreover, we found that their upregulation was dependent on ERK, using the ERK pathway inhibitor U-0126. Collectively, we demonstrated that IS induced ER stress in tubular cells and inhibited cell proliferation via two pathways downstream of ER stress, namely CHOP and ERK-IL-6-p21. These are possible targets for suppressing progression of CKD.
尿毒症毒素可损害肾功能，但对其作用机制知之甚少。由于肾小管损伤是慢性肾脏病（CKD）进展的核心，我们研究了代表性尿毒症毒素硫酸吲哚酚（IS）对肾小管细胞的影响。IS 在培养的人近端肾小管细胞中诱导内质网（ER）应激，免疫印迹中 C/EBP 同源蛋白（CHOP）的增加证明了这一点。此外，在 5/6 肾切除、CKD 模型大鼠中，口服吸附剂 AST-120 降低了血清 IS 浓度，并降低了免疫组化中肾小管 CHOP 的表达。此外，我们发现 IS 在 3-(4,5-二甲基噻唑-2-基）-5-(3-羧甲氧基苯基）-2-(4-磺苯基）-2H-四唑和 5-溴-2'-脱氧尿苷测定中抑制肾小管细胞增殖，而台盼蓝排斥和乳酸脱氢酶测定的结果表明 IS 并未促进细胞死亡。用 CHOP 的 siRNA 可减轻这种抑制作用。此外，IS 增加了细胞周期蛋白依赖性激酶抑制剂 p21(WAF1/CIP1)（p21）。令人惊讶的是，这是由炎症细胞因子白细胞介素（IL）-6 介导的，其表达通过针对激活转录因子 4 的 siRNA 降低，激活转录因子 4 是另一种 ER 应激标志物；然而，IS 诱导的 IL-6 和 p21 不受针对 CHOP 的 siRNA 的抑制，表明它们是 ER 应激的下游产物，但独立于 CHOP。此外，我们发现它们的上调依赖于 ERK，使用 ERK 通路抑制剂 U-0126。总之，我们证明了 IS 在肾小管细胞中诱导 ER 应激，并通过 ER 应激的两条下游途径（即 CHOP 和 ERK-IL-6-p21）抑制细胞增殖。这些可能是抑制 CKD 进展的靶点。
