ObjectiveTo investigate the changes and effects of fatty acid metabolism in folic acid （FA）-induced kidney injury and diabetic nephropathy （DN） mouse models. MethodsThe FA-induced kidney injury model was established via intraperitoneal injection of FA. Nine adult C57BL/6J mice were randomly allocated to two groups： saline control group （Saline， n=5） and FA intervention group （n=4）. Mice in the FA group received a single intraperitoneal injection of FA （250 mg/kg）， whereas those in the Saline group were administered an equivalent volume of normal saline. The DN model was constructed using a high-fat diet combined with streptozotocin （STZ） injection. Eleven adult C57BL/6J mice were randomly divided into two groups： normal diet control group （ND， n=6） and DN model group （DN， n=5）. The ND group was fed a normal diet throughout the experiment； the DN group was maintained on a high-fat diet for 8 weeks， followed by a single intraperitoneal injection of STZ （100 mg/kg）， and then continued on the high-fat diet until week 16. （1） Hematoxylin and eosin （HE） and sirius red staining were used to observe pathological changes in the kidneys of the four groups of mice. （2） RNA-Seq was performed on mouse kidney tissues to screen for differentially expressed genes （DEGs）， followed by Gene Ontology （GO）， Kyoto Encyclopedia of Genes and Genomes （KEGG）， and Gene Set Enrichment Analysis （GSEA）. RT-qPCR was used to detect the mRNA levels of fatty acid synthesis and fibrosis-related genes. Lipid droplet accumulation was assessed by Oil Red O staining on frozen kidney sections. （3） Human renal cortical proximal tubular epithelial cells （HK-2） and proximal tubular epithelial cells （PTECs） were treated with 10 ng/mL recombinant human TGF-β1 or high glucose. RT-qPCR was used to measure the mRNA levels of fatty acid synthesis and fibrosis-related genes in HK-2 cells. Lipid droplet accumulation in HK-2 cells and PTECs was detected using Bodipy 493/503 staining. （4） HK-2 cells were treated with 100 µmol/L palmitic acid （PA）. Bodipy 493/503 staining was used to evaluate lipid droplet accumulation. （5） SA-β-gal staining was used to assess senescence in kidney tissues from all four mouse groups. Cellular senescence and the expression of senescence-associated secretory phenotype （SASP） factors in PA-treated HK-2 cells were assessed using SA-β-gal staining， Western blot， and RT-qPCR. The effect of PA-treated PTECs on renal fibroblast activation was assessed by immunofluorescence. Results（1） Both FA and DN groups showed pathological changes in kidney tissue， including renal tubular structural damage and collagen fiber deposition. （2） DEGs in both FA and DN groups were significantly enriched in metabolism-related pathways， especially lipid metabolism pathways. Compared to the Saline control， the expression of fatty acid synthesis genes Srebp1 and Fasn was significantly upregulated in the FA group （Srebp1： t=2.445， P=0.044 4； Fasn： t=2.571， P=0.037 0）. Compared to the ND group， Srebp1， Acc1， and Fasn gene expression was upregulated in the DN group （Srebp1： t=3.354， P=0.010 0； Acc1： t=2.602， P=0.031 5； Fasn： t=2.358， P=0.046 1）. The fibrosis-related gene Col1a1 was also upregulated in both FA and DN groups （FA： t=2.628， P=0.034 0； DN： t=3.602， P=0.007 0）， accompanied by pathological signs of lipid accumulation. （3） In HK-2 cells treated with TGF-β1 or high glucose， the mRNA expression levels of SREBP1 （F=15.41， P=0.004 3； TGFβ： P=0.002 9； HG： P=0.045 2）， ACC1 （F=30.30， P=0.000 7； HG： P=0.001 4）， and FASN （F=18.76， P=0.002 6； TGFβ： P=0.001 7； HG： P=0.031 0） were significantly upregulated. Both treatments also induced lipid accumulation in HK-2 cells and PTECs. （4） There was an increase in senescent cells in the kidneys of both the FA and DN groups. PA treatment induced lipid accumulation， accelerated senescence， and activated fibroblasts in renal tubular epithelial cells. ConclusionsTranscriptional abnormalities of lipid metabolism-related genes were observed in the kidneys of both FA-induced kidney injury and diabetic nephropathy models. Excessive fatty acid synthesis leads to lipid accumulation， which induces senescence in PTECs and ultimately promotes the development and progression of fibrosis.

OBJECTIVETo reveal the role of poly-ADP-ribosylation and DNA methylation in carcinogenic process induced induced by Cr (VI), and to discuss the relations between them.

METHODSThe pre-established Poly (ADP-ribose) glycohydrolase (PARG) deficient cells and 16HBE cells were treated with different concentrations of Cr (VI), and the changes of total genomic DNA methylation level in different groups were detected by methylation immunofluorescent detection, as well as the changes of the activity of methyltransferases. Moreover, RT-PCR and western blotting method were applied to analyze the changes of expression of DNMT1, DNMT3a, DNMT3b and MBD2, upon the protein level.

RESULTSAfter treated by Cr(VI) for 24 h, the healthy 16HBE cells showed a significant lower level of genomic DNA methylation; however, there was no significant changes (P > 0.05) found in PARG deficient cells by immunofluorescence assay. When the dose of Cr (VI) reached 5.0 µmol/L, the activity of methyltransferases in 16HBE cells and PARG deficient cells (49.33 ± 2.65, 80.05 ± 2.05) decreased by 20% and 50% comparing with contrast group (99.27 ± 1.10, 99.30 ± 0.60) . After treated by Cr (VI) for 24 h, the expression of mRNA and protein level among DNMT1, DNMT3a, DNMT3b and MBD2 decreased significantly in healthy 16HBE cells; and the expression of DNMT1 and DNMT3a decreased in PARG deficiency cells. The relevant expression levels of mRNA of DNMT1 were separately (0.99 ± 0.09), (0.79 ± 0.10), (0.59 ± 0.13) and (0.39 ± 0.02) (F = 247.17, P < 0.01), the expression levels of protein were separately (1.00 ± 0.03), (0.69 ± 0.15), (0.65 ± 0.10) and (0.55 ± 0.13) (F = 214.12, P < 0.01), the expression levels of DNMT3a mRNA were separately (1.00 ± 0.04) , (0.93 ± 0.11) , (0.79 ± 0.07) , (0.59 ± 0.05) (F = 498.16, P < 0.01) , and the expression levels of protein were separately (1.00 ± 0.14) , (0.97 ± 0.11) , (0.79 ± 0.17) , (0.57 ± 0.15) (F = 390.11, P < 0.01) when the dose of Cr (VI) at 0, 0.3, 1.2 and 5.0 µmol/L. However, there were no significant changes of expression found in DNMT3b and MBD2.

CONCLUSIONPoly-ADP-ribosylation could regulate the activity of DNMT3b and MBD2, protect cells against the DNA methylation alteration induced by Cr(VI) and maintain the global genomic DNA methylation level.

Cell Line ; Chromium ; toxicity ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; drug effects ; DNA-Binding Proteins ; metabolism ; Epithelial Cells ; metabolism ; Genome ; Humans ; Poly Adenosine Diphosphate Ribose ; metabolism ; RNA, Messenger ; genetics

OBJECTIVETo investigate the effect of poly-ADP-ribosylation in hexavalent chromium Cr(VI) induced cell damage.

METHODSThe study object, poly (ADP-ribose) glycohydrolase (PARG) deficient human bronchial epithelial cells (16HBE cells), was constructed previously by our research group. Normal 16HBE cells and PARG-deficient cells were treated with different doses of Cr (VI) for 24 h to compare the differences to Cr (VI) toxicity, meanwhile set up the solvent control group. On this basis, 5.0 µmol/L of Cr (VI) was selected as the exposure dose, after the exposure treatment, total proteins of both cells were extracted for two dimension fluorescence difference gel electrophoresis (2D-DIGE) separation, statistically significant differential protein spots were screened and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS/MS), and further validated by Western blot.

RESULTSAfter Cr (VI) treatment, the survival rate of PARG-deficient cells was higher than normal 16HBE cells. When the doses reached up to 5.0 µmol/L, the survival rate of 16HBE cells and PARG-deficient cells were respectively (59.67 ± 6.43)% and (82.00 ± 6.25)%, the difference between which was significant (t = -4.32, P < 0.05). 18 protein spots were selected and successfully identified after 2D-DIGE comparison of differential proteins between normal 16HBE cells and PARG-deficient cells before and after exposure. The function of those proteins was involved in the maintenance of cell shape, energy metabolism, DNA damage repair and regulation of gene expression. The differential expression of cofilin-1 was successfully validated by Western blot. The expression level of cofilin-1 in the 16HBE cells increased after Cr (VI) exposure with the relative expression quantity of 1.41 ± 0.04 in treated group and 1.00 ± 0.01 in control group, the difference of which was statistically significant (t = -18.00, P < 0.05), while the expression level in PARG-deficient cells had no statistically significant difference (t = -8.61, P > 0.05).

CONCLUSIONMost of the identified differential proteins are closely related to tumorigenesis, suggesting that poly-ADP-ribosylation reaction may resist the cytotoxicity of Cr(VI) by inhibiting Cr (VI) induced tumorigenesis, which provides important reference data to clarify the mechanisms of poly-ADP-ribosylation in Cr (VI) induced cell damage.

Bronchi ; Cell Transformation, Neoplastic ; genetics ; Chromium ; Cofilin 1 ; DNA Repair ; Epithelial Cells ; Glycoside Hydrolases ; deficiency ; physiology ; Humans ; Tandem Mass Spectrometry