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Objective:To investigate the expression of urinary exosomal miR-150-5p in different age groups and its relationship with kidney aging.Methods:Seventy-six subjects were recruited at the geriatrics department of Beijing Friendship Hospital from January 2014 to December 2016, with 15 in the young group, 35 in the middle-aged group, and 26 in the elderly group.We detected the expression of urinary exosomal miR-150-5p by real-time PCR from urine samples of different age groups and used Logistic regression analysis to evaluate the correlation of different levels of miR-150-5p with age, gender, history of hypertension, history of coronary heart disease, and hyperlipidemia.Additionally after overexpression and knockdown of miR-150-5p in human renal tubular cells of the HK2 cell line, we analyzed the expression levels of epithelial-to-mesenchymal transition(EMT)-related proteins by Western blotting.Results:Logistic regression analysis showed that age was closely related to estimated urinary exosomal miR-150-5p levels( P<0.05), and estimated urinary exosomal miR-150-5p levels in the elderly group(1.33±0.41)were significantly higher than those in the young group(0.88±0.40)( P<0.05). In addition, estimated urinary exosomal miR-150-5p levels became inversely proportional to the glomerular filtration rate as age increased. In vitro experiments showed that the expression levels of ZO-1 and E-cadherin in epithelial cells were slightly decreased after knockdown of miR-150-5p in renal tubular epithelial cells of the HK-2 cell line. Conclusions:The expression of miR-150-5p in urinary exosomes may be slightly increased in the elderly.It could inhibit the occurrence of renal EMT, and its expression is correlated with renal function.Mir-150-5p may also affect the expression of adhesion molecule-related proteins in HK-2 cells.MiR-150-5p in urinary exosomes is expected to become one of the genetic markers associated with renal aging.
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OBJECTIVE: To explore the role of N~6-methyladenosine(m~6A) catalytic enzymes(methyltransferases and demethylases) in cadmium-induced oxidative damage in human renal epithelial cells(HK-2 cells), and to analyze the correlation between nuclear factor-erythroid 2-related factor 2(NRF2) and m~6A catalytic enzymes. METHODS: i) HK-2 cells in logarithmic growth phase were randomly divided into control group and 6 cadmium sulfate treatment groups, then treated with 0, 2, 4, 8, 16, 32 and 64 μmol/L cadmium sulfate solution for 24 hours. The cell survival rates were detected by CCK-8 assay, and the appropriate doses of cadmium sulfate were selected for subsequent experiments. ii) HK-2 cells in logarithmic growth phase were randomly divided into control group and low-, medium-, and high-dose groups, and treated with 0, 4, 8, and 16 μmol/L cadmium sulfate solution respectively for 24 hours. Subsequently, the levels of reactive oxygen species(ROS) were detected by fluorescence probe. The mRNA expression of NRF2, the m~6A methyltransferases such as methyltransferase like proteins(METTL) 3, METTL14, METTL16 and the m~6A demethylases such as fat mass and obesity associated protein(FTO), AlkB family of nonheme Fe(Ⅱ)/α-ketoglutarate(α-KG)-dependent dioxygenases 5(ALKBH5) were determined by real-time polymerase chain reaction. RESULTS: i) The survival rate of HK-2 cells was more than 60.00% and lower than that of the control group(P<0.05) after the cells were stimulated with 16 μmol/L of cadmium sulfate. Therefore, 4, 8 and 16 μmol/L of cadmium sulfate were selected as the stimulation concentrations in the follow-up experiments. ii) The relative expression of NRF2, METTL3, METTL14 and METTL16 in HK-2 cells in low-dose group increased(all P<0.05), while the levels of ROS and the relative mRNA expression of NRF2, METTL3, METTL14, METTL16 and FTO in HK-2 cells in medium and high-dose groups increased(all P<0.05) when compared with the control group. There was no significant difference in the expression of ALKBH5 mRNA among these 4 groups(P>0.05). In the correlation analysis, NRF2 mRNA expression was positively correlated with the mRNA expression of METTL3 and METTL16 [Pearson correlation coefficient(r) = 0.61 and 0.66, respectively, all P<0.05]. There was no correlation between NRF2 mRNA expression and METTL14, FTO and ALKBH5(r=0.53, 0.48, and 0.01 respectively, all P>0.05). CONCLUSION: Cadmium sulfate may increase intracellular ROS level, up-regulate NRF2 expression and activate NRF2 signaling pathway as well as enhance the expression of METTL3 and METTL16 in HK-2 cells, thus increasing intracellular oxidative damage and decreasing the cell survival rate.
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Objective: To investigate the effect of type I interferon receptor 1 (IFNAR1) on Pseudorabies virus (PRV) replication. Methods: A stable cell line in which porcine kidney epithelial cells (PK 15) knocked out the IFNAR1 gene was constructed using a lentiviral-mediated CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR) gene editing technique. The function of IFNAR1 was verified by cell viability assay, fluorescence observation, flow cytometry detection, titer determination, and Real-time PCR. Results: With the prolongation of PRV infection, knocking out the IFNAR1 gene can significantly promote transcription of PRV-TK mRNA, translation of PRV-gE protein, and virulence of progeny virus. Conclusion: IFNAR1 plays an important role in inhibiting the proliferation of PRV.
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Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human hereditary disorder characteristic of development of bilateral multiple fluid-filled kidney cysts. Accumulated evidence has suggested that primary cilium of renal epithelial cell plays a key role in cystogenesis. In this article we will give an overview on the basic information about polycystic kidney disease (PKD) and summarize the recent progresses in studies of regulation of polycystin-1 and -2 trafficking to cilia. We will also discuss the possible role of trafficking defects of polycystins on the pathogenesis of ADPKD.
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This study was undertaken to examine the effect of ethanol on Na+-dependent phosphate (Na+-Pi) uptake in opossum kidney (OK) cells, an established renal proximal tubular cell line. Ethanol inhibited Na+-dependent component of phosphate uptake in a dose-dependent manner with I50 of 8.4%, but it did not affect Na+-independent component. Similarly, ethanol inhibited Na+-dependent uptakes of glucose and amino acids (AIB, glycine, alanine, and leucine). Microsomal Na+-K+-ATPase activity was not significantly altered when cells were treated with 8% ethanol. Kinetic analysis showed that ethanol increased Km without a change in Vmax of Na+-Pi uptake. Inhibitory effect of n-alcohols on Na+-Pi uptake was dependent on the length of the hydrocarbon chain, and it resulted from the binding of one molecule of alcohol, as indicated by the Hill coefficient (n) of 0.8-1.04. Catalase significantly prevented the inhibition, but superoxide dismutase and hydroxyl radical scavengers did not alter the ethanol effect. A potent antioxidant DPPD and iron chelators did not prevent the inhibition. Pyrazole, an inhibitor of alcohol dehydrogenase, did not attenuate ethanol-induced inhibition of Na+-Pi uptake, but it prevented ethanol-induced cell death. These results suggest that ethanol may inhibit Na+-Pi uptake through a direct action on the carrier protein, although the transport system is affected by alterations in the lipid environment of the membrane.