Abstract
The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase.
Highlights
Hyperammonemia is a major factor in the pathogenesis of hepatic encephalopathy (HE) and symptoms including ataxia, stupor, convulsions, and coma have been described in hyperammonemic states
To this aim our approach was threefold: first we evaluated the expression of CPS1, ornithine carbamoyltransferase (OTC), ASS1, argininosuccinate lyase (ASL), ARG1, N-acetylglutamate synthase (NAGS), glutamate-ammonia ligase (GLUL) and GLUD1 in dogs with Congenital portosystemic shunts (CPSS) before and after surgical closure of the shunt
The expression of all selected enzymes involved in the ammonia-metabolizing pathways (CPS1, OTC, ASS1, ASL, ARG1, NAGS, GLUL and GLUD1) was significantly down-regulated in dogs with CPSS compared to healthy liver tissue (Table 3)
Summary
Hyperammonemia is a major factor in the pathogenesis of hepatic encephalopathy (HE) and symptoms including ataxia, stupor, convulsions, and coma have been described in hyperammonemic states. The urea cycle is responsible for the disposal of over 90 percent of surplus nitrogen from dietary or endogenous nitrogen sources [4]. It comprises five key enzymes which catalyse the different steps of incorporation of ammonia into urea (Fig. 1). These are carbamoylphosphate synthetase (CPS1; EC 6.3.4.16), ornithine carbamoyltransferase (OTC; EC 2.1.3.3), argininosuccinate synthetase (ASS1; EC 6.3.4.5), argininosuccinate lyase (ASL; EC 4.3.2.1), and arginase (ARG1; EC 3.5.3.1) (Fig. 1). CPS1 and OTC are located on the inner mitochondrial membrane [5,6]
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