Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

A novel porcine ex vivo retina culture model for oxidative stress induced by H2O2.

Oxidative stress is a key player in many ophthalmic diseases. However, the role of oxidative stress in most degenerative processes is not yet known. Therefore, accurate and practical models are required to efficiently screen for therapeutics. Porcine eyes are closely related to the human eye, and can be obtained from the abattoir as a by-product of the food industry. Therefore, they offer excellent opportunities for the development of culture models with which to pre-screen potential therapies, while reducing the use of laboratory animals. To induce oxidative stress, organotypic cultures of porcine retina were treated with different doses of hydrogen peroxide (H2O2; 100, 300 and 500µM) for three hours. On days 3 and 8, the retinas were conserved for histological and Western blotting analyses and for evaluation of gene expression, which determined the number of retinal ganglion cells (RGCs), the activation state of glial cells, and the expression levels of several oxidative stress markers. H2O2 treatment led to a reduction in the number of RGCs and to an increase in apoptotic RGCs. In addition, a dose-dependent increase of microglia and an elevation of CD11b expression was observed. On day 3, a reduction of IL-1ß, and an increase of iNOS, as well as of HSP70 mRNA were found. On day 8, an increase in TNF-α and IL-1ß mRNA expression was detected. In conclusion, this ex vivo model offers an opportunity to study the molecular mechanisms underlying certain eye disorders and to test new therapeutic approaches to diminish the effects of oxidative stress.

The novel induction of retinal ganglion cell apoptosis in porcine organ culture by NMDA - an opportunity for the replacement of animals in experiments.

Some of the advantages of retina organ culture models include their efficient and easy handling and the ability to standardise relevant parameters. Additionally, when porcine eyes are obtained from the food industry, no animals are killed solely for research purposes. To induce retinal degeneration, a commonly used toxic substance, N-methyl-D-aspartate (NMDA), was applied to the cultures. To this end, organotypic cultures of porcine retinas were cultured and treated with different doses of NMDA (0 [control], 50, 100 and 200µM) on day 2 for 48 hours. On day 7, the retinas were cryo-conserved for histological, Western blot and quantitative rt-PCR (qrt-PCR) analyses. NMDA treatment was found to significantly increase retinal ganglion cell (RGC) apoptosis in all the treated groups, without a profound RGC loss. In addition, the intrinsic apoptotic pathway was activated in the 50µM and 100µM NMDA groups, whereas induced nitric oxide synthase (iNOS) expression was increased in the 200µM group. A slight microglial response was detectable, especially in the 100µM group. NMDA treatment induced apoptosis, oxidative stress and a slight microglia activation. All these effects mimic a chronic slow progressive disease that especially affects RGCs, such as glaucoma. A particular advantage of this model is that mediators that can interact in the very early stages of the onset of RGC death, can be easily detected and potential therapies can be tested.