Uremic Toxin Indoxyl Sulfate Impairs Hydrogen Sulfide Formation in Renal Tubular Cells

Abstract

Hydrogen sulfide (H2S) is the third gasotransmitter that recognized as a cytoprotectant. Recent study demonstrated that exogenous supplement of H2S ameliorates functional insufficiency in chronic kidney disease (CKD). However, how H2S system is impaired by CKD has not been elucidated. The uremic toxin indoxyl sulfate (IS) is known to accumulate in CKD patients and harm the renal tubular cells. This study therefore treated proximal tubular cells LLC-PK1 with IS to see how IS affects H2S formation. Our results showed that H2S release from LLC-PK1 cells was markedly attenuated by IS when compared to that in control cells. The H2S donors NaHS and GYY-4137 significantly attenuated IS-induced tubular damage by enhancing cell survival in dose-dependent manners. Inhibition of remnant H2S production in the IS-treated cells by AOAA exacerbated tubulotoxicity. These results clearly indicated IS impairs H2S formation. We then examined protein and mRNA expression of H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). IS downregulated both CBS and 3-MST protein and mRNA expressions after 24-72 h of treatment. CSE protein and mRNA levels were lowered at 24 h and 24-48 h, respectively, by IS. The low expression of CBS and 3-MST at both protein and mRNA levels could be reversed by AhR inhibition. As transcription factor specificity protein 1 (Sp1) regulates gene expression of H2S-producing enzymes, we further showed that IS significantly decreased the DNA binding activity of Sp1, this however did not affect Sp1 protein expression. AhR inhibition reversed low Sp1 activity caused by IS. Together these results, we conclude that IS activates AhR which then attenuates Sp1 function on regulation of H2S-producing enzyme expression. Attenuation of H2S formation contributed to uremic toxin-mediated renal tubular cell damage.