Altered iron homeostasis in ammonia-exposed cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy

Introduction:

Recent data suggest that cognitive impairment in cirrhotic patients may not fully resolve after an acute episode of hepatic encephalopathy (HE). It was proposed that this permanent deterioration of cognitive functions may result from heme oxygenase 1 (HO1) mediated astrocyte senescence. As HO1 liberates ferrous iron from heme, we analysed effects of ammonia on iron homeostasis in cultured rat astrocytes and in post mortem human brain tissue.

Results:

Exposure of cultured rat astrocytes to NH₄Cl (5mM, 72h) strongly modulated the fluorescence intensities of two Fe² chelating fluorescence reporter dyes in a way indicative for an elevation of intracellular unbound ferrous iron: The fluorescence intensity of RhoNox1 increased, and Rhodamine B-[(2,2'-bipyridine-4-yl)-aminocarbonyl]benzylester (RDA) fluorescence decreased. RDA fluorescence strongly colocalized with MitoTracker fluorescence and RhoNox1 fluorescence colocalized with LysoTracker. NH₄Cl upregulated mRNA levels of the iron exporter ferroportin and reduced mRNA levels of the accessory factors ceruloplasmin and hephaestin in a glutamine synthesis dependent way. Likewise, siRNA mediated downregulation of GS prevented upregulation of FPN mRNA levels by NH₄Cl in astrocytes. Exposure of cultured astrocytes to ferrous iron (25µM, 18h) changed mRNA expression levels of iron metabolism related genes similar to NH₄Cl. At the protein level, NH₄Cl downregulated the expression of both, ferroportin as well as ceruloplasmin.Chelation of unbound iron by the iron chelators 2,2'-bipyridine and pyridoxal isonicotinoyl hydrazone abolished NH₄Cl-induced alterations of RhoNox1 and RDA fluorescence, transcription of iron related genes, as well as proliferation inhibition, respectively. Expression levels of genes related to iron metabolism such as natural resistance-associated macrophage protein, ceruloplasmin or hepcidin were also changed in post mortem brain samples of patients with liver cirrhosis and HE but not in those without HE.

Discussion:

The present study suggests a role of HO1 and iron for ammonia-induced astrocyte senescence. Expression levels of genes related to iron metabolism were also changed in cerebral cortex of patients with liver cirrhosis and HE. These data indicate that iron metabolism is altered in brain in HE.