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DNA demethylation catalysed by the ten-eleven translocation (TET) protein is an important step during extensive global epigenetic reprogramming in mammals. However, whether TET proteins play a key role in DNA demethylation during the development of bovine pre-implanted embryos is still unclear. In this study, we utilized dimethyloxallyl glycine (DMOG), a small-molecule inhibitor of the TET protein, to impede the enzymatic activity of TET and explore subsequent effects on bovine parthenogenetic embryo development. We first detected the expression of the TET family, consisting of TET1, TET2 and TET3, in bovine MII stage oocytes and found that TET3 is more highly expressed than TET1 and TET2. Treatment with 1 mM DMOG increased 5mC levels (30.4% vs 79.8% at the 8-cell stage for satellite I, 25.3% vs 40.6% at the 8-cell stage for α-satellite, 20.5% vs 73.5% at the blastocyst stage for satellite I and 16.6% vs 30.0% at the blastocyst stage for α-satellite) at every bovine parthenogenetic embryo developmental stage. At the same time, DNA methylation level of satellite DNA and pluripotency gene promoters increased significantly. Real-time PCR analysis results indicated that the transcription levels of NANOG and OCT-4 decreased in the DMOG-treated group. Furthermore, TET inhibition negatively affected blastocyst formation, resulting in a decline in the blastocyst rate (17.1 ± 1.3% vs 24.1 ± 0.6%); however, the percentage of apoptotic cells was significantly increased according to the results of a TUNEL assay. Additionally, expression levels of the apoptosis-related gene BAX were up-regulated, while the expression of BCL-2 was down-regulated. In conclusion, these results support that TET plays important roles in bovine parthenogenetic embryo development by influencing DNA methylation reprogramming, gene expression and apoptosis.

Liver cirrhosis is recognized as being the consequence of immune-mediated hepatocyte damage and repair processes. However, the regulation of these immune responses underlying liver cirrhosis has not been elucidated. In this study, we used GEO datasets and bioinformatics methods to established coding and non-coding gene regulatory networks including transcription factor-/lncRNA-microRNA-mRNA, and competing endogenous RNA interaction networks. Our results identified 2224 mRNAs, 70 lncRNAs and 46 microRNAs were differentially expressed in liver cirrhosis. The transcription factor -/lncRNA- microRNA-mRNA network we uncovered that results in immune-mediated liver cirrhosis is comprised of 5 core microRNAs (e.g., miR-203; miR-219-5p), 3 transcription factors (i.e., FOXP3, ETS1 and FOS) and 7 lncRNAs (e.g., ENTS00000671336, ENST00000575137). The competing endogenous RNA interaction network we identified includes a complex immune response regulatory subnetwork that controls the entire liver cirrhosis network. Additionally, we found 10 overlapping GO terms shared by both liver cirrhosis and hepatocellular carcinoma including "immune response" as well. Interestingly, the overlapping differentially expressed genes in liver cirrhosis and hepatocellular carcinoma were enriched in immune response-related functional terms. In summary, a complex gene regulatory network underlying immune response processes may play an important role in the development and progression of liver cirrhosis, and its development into hepatocellular carcinoma.

Resolvin D1 (RvD1) is a lipid-derived mediator generated during the resolution inflammation. While the immunoresolvent effects of Resolvins have been extensively studied in leukocytes, actions of Resolvins on intrinsic kidney cells have received little attention. The podocyte plays a central role in glomerular function, and podocyte damage can lead to proteinuria and glomerulosclerosis. This study examined whether RvD1 has renoprotective effects upon podocytes. We investigated a mouse model of adriamycin (ADR) nephropathy featuring rapid induction of podocyte damage and proteinuria followed by glomerulosclerosis. We identified a progressive loss of synaptopodin expression over a 28 day time-course of ADR nephropathy which was associated with increased acetylation of 14-3-3β and reduced synaptopodin phosphorylation. Groups of mice were given once daily RvD1 treatment (4 ng/g body weight/day) starting either 30 min (early treatment) or 14 days (late treatment) after ADR injection and continued until mice were killed on day 28. Early, but not late, RvD1 treatment attenuated ADR-induced proteinuria, glomerulosclerosis and tubulointerstitial fibrosis, modified macrophages from an M1 to M2 phenotype. Early RvD1 treatment prevented the down-regulation of synaptopodin expression and changes in 14-3-3β acetylation and synaptopodin phosphorylation. In a podocyte cell line, RvD1 was shown to prevent rapid TNF-α-induced down-regulation of synaptopodin expression. In transfection studies, TNF-α-induced a decrease in synaptopodin phosphorylation and an increase in acetylation of 14-3-3β, resulting in disassociation between 14-3-3β and synaptopodin. RvD1 prevented TNF-α induced post-translational modification of synaptopodin and 14-3-3β proteins, and maintained the synaptopodin/14-3-3β interaction. Furthermore, replacement of lysine K51, or K117+K122 in 14-3-3β with glutamine, to mimic lysine acetylation, significantly reduced the interaction between 14-3-3β and synaptopodin. In conclusion, our studies provide the first evidence that RvD1 can protect against podocyte damage by preventing down-regulation of synaptopodin through inhibition of 14-3-3β/synaptopodin dissociation. RvD1 treatment may have potential application in the treatment of chronic kidney disease.

The role of podocytes in the development and progression of glomerular disease has been extensively investigated in the past decade. However, the importance of glomerular endothelial cells in the pathogenesis of proteinuria and glomerulosclerosis has been largely ignored. Recent studies have demonstrated that endothelial nitric oxide synthatase (eNOS) deficiency exacerbates renal injury in anti-GBM and remnant kidney models and accelerates diabetic kidney damage. Increasing evidence also demonstrates the importance of the glomerular endothelium in preventing proteinuria. We hypothesize that endothelial dysfunction can initiate and promote the development and progression of glomerulopathy. Administration of adriamycin (ADR) to C57BL/6 mice, normally an ADR resistant strain, with an eNOS deficiency induced overt proteinuria, severe glomerulosclerosis, interstitial fibrosis and inflammation. We also examined glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice, an ADR susceptible strain, by immunostaining, TUNEL and Western blotting. Interestingly, down-regulation of eNOS and the appearance of apoptotic glomerular endothelial cells occurred as early as 24 hours after ADR injection, whilst synaptopodin, a functional podocyte marker, was reduced 7 days after ADR injection and coincided with a significant increase in the number of apoptotic podocytes. Furthermore, conditioned media from mouse microvascular endothelial cells over-expressing GFP-eNOS protected podocytes from TNF-α-induced loss of synaptopodin. In conclusion, our study demonstrated that endothelial dysfunction and damage precedes podocyte injury in ADR-induced nephropathy. Glomerular endothelial cells may protect podocytes from inflammatory insult. Understanding the role of glomerular endothelial dysfunction in the development of kidney disease will facilitate in the design of novel strategies to treat kidney disease.