Inhibition of Endogenous Hydrogen Sulfide Production Decreases Hypoxia Induced Erythropoietin Release By Hep3B Cells.

Abstract

Introduction and Objective: Anemia of end stage renal disease (ESRD) affects over 90% of all hemodialysis patients and is a tremendous concern both for patients and health care providers. Natural erythropoietin (EPO) production is stimulated by local hypoxic conditions within the kidney tubular cells. Unfortunately over time, patients require exogenous EPO stimulating agents to treat their anemia, however they can become resistant to these agents thus necessitating increasing doses with resultant greater side effects. Novel therapeutics to overcome anemia of ESRD are necessary. Recent evidence suggests that endogenously produced hydrogen sulfide (H2S) may act as an oxygen sensor. Given the known involvement of other small molecules such as nitric oxide (NO) in natural EPO production and the observation of diminished urinary H2S levels in ESRD patients on hemodialysis, we postulated that H2S may be the primary mediator of EPO production during hypoxic conditions. Materials/Methods: Hep3B cells are a well described model for EPO production. These cells were incubated under hypoxic conditions (1% O2) for 24 hours at 37°C. During hypoxia cells were treated with varying concentrations of the H2S donor, GYY 4137, the substrate for H2S biosynthesis, L-cysteine, and various inhibitors of H2S production, including DL-propargylglycine (PAG) and hydroxylamine (HA). RNA was collected following the end of hypoxia and EPO expression was measured by real time RT-PCR. Results: In comparison to normoxic conditions, cells subjected to hypoxic conditions showed a significant increase in erythropoietin expression. Upon inhibiting endogenous H2S production using either HA and PAG, we found a significant decreases in EPO expression (p=0.013). Subsequent administration of GYY 4137 (500 uM) rescued the effect of these inhibitors, inducing a significant increase in EPO mRNA expression (p= 0.0012) compared to normoxic controls. Conclusions: These data are the first to show a novel influence of endogenous H2S on EPO production under hypoxic conditions. The use of novel oral H2S donors may represent a more efficacious and cost-effective alternative to expensive standard therapies in treatment of anemia of ESRD.