Indoxyl sulfate inhibits proliferation of human proximal tubular cells via endoplasmic reticulum stress

Kawakami T, Inagi R, Wada T, Tanaka T, Fujita T, Nangaku M. Indoxyl sulfate inhibits proliferation of human proximal tubular cells via endoplasmic reticulum stress. Am J Physiol Renal Physiol 299: F568-F576, 2010. First published June 9, 2010; doi:10.1152/ajprenal.00659.2009.-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.