Down-regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy
ABSTRACT Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.
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ABSTRACT: Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media (CM) from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95 and synaptotagmin levels. CM from TSP-1 overexpressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE.This article is protected by copyright. All rights reserved.Journal of Neurochemistry 07/2014; DOI:10.1111/jnc.12810 · 4.24 Impact Factor
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ABSTRACT: Hepatic encephalopathy (HE), a neuropsychiatric abnormality that commonly accompanies cirrhosis of the liver, is often difficult to treat and manage. Changes in chloride homeostasis are involved in the generation of a number of brain disorders. In this study, we considered whether chloride homeostasis is associated with HE. The mRNA levels of the Cl(-) extrusion system (KCC2) and the Cl(-) intrusion system (NKCC1) were detected by real-time RT-PCR in the plasma of 29 cirrhotic patients with HE of grade I-II, 36 cirrhotic patients with HE of grade III-IV, 20 cirrhotic patients without HE and 15 healthy controls. The mRNA levels of KCC2 in cirrhotic patients with mild and severe HE were significantly lower compared to those in cirrhotic patients without HE or in the healthy controls. However, NKCC1 mRNA levels did not differ between the different groups. In addition, for cirrhotic patients with HE, there were significant negative correlations between KCC2 levels and the levels of blood ammonia and hepatic function scores (Child-Pugh and model for end-stage liver disease scores); there was also a significant positive correlation between KCC2 levels and neurological status (Glasgow scores). In conclusion, our study indicates that an imbalance of KCC2 and NKCC1 may be a novel biomarker for detecting HE and for monitoring disease development.Experimental and therapeutic medicine 12/2012; 4(6):1075-1080. DOI:10.3892/etm.2012.721 · 0.94 Impact Factor
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ABSTRACT: Glutamate (Glu) and NMDA decrease Kir4.1 expression in cultured astrocytes.•Glu-induced loss of Kir4.1 is reversed by NMDA receptor antagonists (NMDAra).•Loss of brain Kir4.1 mRNA in hepatic encephalopathy is attenuated by a NMDAra.•Results implicate astrocytic NMDA receptors in Kir4.1 loss in Glu-overexposed brain.Neurochemistry International 10/2014; DOI:10.1016/j.neuint.2014.10.006 · 2.65 Impact Factor