Response gene to complement 32 regulates the G2/M phase checkpoint during renal tubular epithelial cell repair.

BACKGROUND: The aim of this study was to evaluate the influence of RGC-32 (response gene to complement 32) on cell cycle progression in renal tubular epithelial cell injury. METHODS: NRK-52E cells with overexpressed or silenced RGC-32 were constructed via transient transfection with RGC-32 expression plasmid and RGC-32 siRNA plasmid, and the cell cycle distribution was determined. The expression levels of fibrosis factors, including smooth muscle action (α-SMA), fibronectin (FN) and E-cadherin, were assessed in cells with silenced RGC-32. RESULTS: The cells were injured via TNF-α treatment, and the injury was detectable by the enhanced expression of neutrophil gelatinase-associated lipocalin (NGAL). RGC-32 expression also increased significantly. The number of cells at G2/M phase increased dramatically in RGC-32 silenced cells, indicating that RGC-32 silencing induced G2/M arrest. In addition, after treatment with TNF-α, the NRK-52E cells with silenced RGC-32 showed significantly increased expression of α-SMA and FN, but decreased expression of E-cadherin. CONCLUSIONS: The results of this study suggest that RGC-32 probably has an important impact on the repair process of renal tubular epithelial cells in vitro by regulating the G2/M phase checkpoint, cell fibrosis and cell adhesion. However, the exact mechanism needs to be further elucidated.

The expression of response gene to complement 32 on renal ischemia reperfusion injury in rat.

To investigate the expression of response gene to complement 32 (RGC32) in rat with acute kidney injury (AKI) and to explore the role of RGC32 in renal injury and repair induced by ischemia reperfusion. Rats were randomly divided into two groups, including sham operation group (n = 48) and acute ischemia reperfusion injury (IRI) group (n = 48). Rats were sacrificed following reperfusion 2 h, 6 h, 24 h, 48 h, 72 h, 1 week (w), 2 w, and 4 w. The distribution and expression of RGC32 in renal tissue were observed by means of immunohistochemistry. The mean density of the images detected by Image-Pro Plus 6 was designated as the representative RGC32 expression levels. Meanwhile, RGC32 mRNA expression was measured by qPCR. RGC32 mainly expressed in cytoplasm of proximal tubular epithelial cells. However, RGC32 did not express in renal interstitium and vessels. The expression levels of RGC32 measured by immunohistochemistry at different reperfusion time were 0.0168 ± 0.0029, 0.0156 ± 0.0021, 0.0065 ± 0.0013, 0.0075 ± 0.0013, 0.0096 ± 0.0014, 0.0132 ± 0.0016, 0.0169 ± 0.0014, 0.0179 ± 0.0022, respectively. Compared with the sham group, the level of RGC32 expression in IRI group was significant lower at 24 h, 48 h, 72 h after IRI (p < 0.05). The expression levels of RGC32 mRNA at different reperfusion time measured by qPCR were corroborated the immunohistochemistry finding. The in vitro experiments show the expression of α-SMA and extracellular matrix expression increased signification when the RGC32 was silenced. Our data showed that the RGC32 expression in AKI rat decreased significantly reduces with different reperfusion time and performs a time-dependent manner. RGC32 may play an important role in the pathogenesis of AKI following IRI and repair in rat.

[Rapid screening for MTHFR gene 677C>T polymorphism in Down syndrome using high resolution melting curve and pyrosequencing].

OBJECTIVE: To establish a rapid method for detecting MTHFR gene 677C>T polymorphisms with high-resolution melting curve method (HRM) and pyrosequencing. METHODS: Peripheral blood samples were collected from 155 Down syndrome patients and 182 normal controls from Children's Hospital of Shanghai. The accuracy of three methods including regular HRM, internal control HRM and artificial heterozygosity HRM was compared. Meanwhile, allele frequencies in 10, 30 and 50 mixed samples were measured with pyrosequencing, and the results were compared with that of HRM. RESULTS: Heterozygosity of 677C>T polymorphism could be distinguished by various HRM methods. However, homozygotes CC and TT were only identifiable by internal control HRM and artificial heterozygosity HRM. The accuracy of pyrosequencing for allele frequency has improved with increased sample number. When the number of mixed samples has exceeded 30, the difference between pyrosequencing results and actual values became less than 4%. TT genotype was more frequent in Down syndrome patients than controls (25.2% vs. 14.3%). No significant difference was found in T allele frequency between the two groups (44.9% vs. 40.1%). CONCLUSION: Respectively, internal control HRM and pyrosequencing may be ideal methods for determination of genotypic and allelic frequencies.

Antifungal Drugs and Drug-Induced Liver Injury: A Real-World Study Leveraging the FDA Adverse Event Reporting System Database.

Aims: We aimed to estimate the risk of drug-induced liver injury (DILI) from various antifungal treatments with azoles and echinocandins causing in real-world practice. Methods: We performed disproportionality and Bayesian analyses based on data from the first quarter in 2004 to the third quarter in 2021 in the Food and Drug Administration Adverse Event Reporting System to characterize the signal differences of antifungal drugs-related DILI. We also compared the onset time and mortality differences of different antifungal agents. Results: A total of 2943 antifungal drugs-related DILI were identified. Affected patients tended to be aged >45 years (51.38%), with more males than females (49.03% vs. 38.09%). Antifungal drug-induced liver injury is most commonly reported with voriconazole (32.45%), fluconazole (19.37%), and itraconazole (14.51%). Almost all antifungal drugs were shown to be associated with DILI under disproportionality and Bayesian analyses. The intraclass analysis of correlation between different antifungal agents and DILI showed the following ranking: caspofungin (ROR = 6.12; 95%CI: 5.36-6.98) > anidulafungin (5.15; 3.69-7.18) > itraconazole (5.06; 4.58-5.60) > voriconazole (4.58; 4.29-4.90) > micafungin (4.53; 3.89-5.27) > posaconazole (3.99; 3.47-4.59) > fluconazole (3.19; 2.93-3.47) > ketoconazole (2.28; 1.96-2.64). The onset time of DILI was significantly different among different antifungal drugs (p < 0.0001), and anidulafungin result in the highest mortality rate (50.00%), while ketoconazole has the lowest mortality rate (9.60%). Conclusion: Based on the Food and Drug Administration Adverse Event Reporting System database, antifungal drugs are significantly associated with DILI, and itraconazole and voriconazole had the greatest risk of liver injury. Due to indication bias, more clinical studies are needed to confirm the safety of echinocandins.

Renal proteomic analysis of RGC-32 knockout mice reveals the potential mechanism of RGC-32 in regulating cell cycle.

This study aimed to investigate the exact function of RGC-32 in kidney diseases and explore the potential mechanism of RGC-32 in regulating cell cycle. RGC-32 knockout (RGC-32-/-) mice were generated from C57BL/6 embryonic stem cells. Differentially expressed proteins in the kidney were investigated with the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathway mapping analysis and functional network analysis were also performed. The expressions of Smc3, Smad 2-3, DNA-PK were further confirmed by qPCR. Results showed that 4690 proteins were quantified on the basis of 25165 unique peptides. Comparative proteomic analysis revealed 361 differentially expressed proteins in RGC-32-/- mice (knockout/wild ratio >+/- 1.2 and P<0.05). GO and KEGG pathway mapping analyses showed differentially expressed proteins were involved in spliceosome, fluid shear stress and atherosclerosis protein processing in endoplasmic reticulum, pathways in cancer, viral carcinogenesis, epithelial cell signaling in Helicobacter pylori infection, HTLV-I infection, PI3K-Akt signaling pathway, ubiquitin mediated proteolysis, Parkinson's disease, MAPK signaling pathway, carbon metabolism, Alzheimer's disease, NOD-like receptor signaling pathway, tight junction, Proteoglycans in cancer, phagosome, ribosome, mTOR signaling pathway, and AMPK signaling pathway. Differentially expressed proteins Smc3 (0.821), DNA-PK (0.761), Smad 2-3 (0.631) were involved in cell cycle regulation. mRNA expression of Smad2-3, DNA-PK, and Smc3 was consistent with that from iTRAQ. It is concluded that RGC-32 may affect the expression of many proteins (76 up-regulated and 285 down-regulated) in the kidney, and may regulate the expression of Smc3, DNA-PK and Smad 2-3 to affect the cell cycle.

P53 inhibitor pifithrin-α prevents the renal tubular epithelial cells against injury.

The injury and repair of renal tubular epithelial cells play an important role in the pathological process of acute kidney injury (AKI). This study aimed to clarify the role of cell cycle change in renal tubular epithelial cell injury and repair in vivo and in vitro. Sprague-Dawley rats received bilateral renal pedicle clamping for 45 min (ischemia) followed by reperfusion. Pifithrin-α, a p53 inhibitor, was administered at 24 h before renal ischemia and 3 and 14 days after reperfusion. Results showed the tubular epithelial cells in M phase increased significantly at 2 h to 72 h after ischemia/reperfusion (I/R), while pifithrin-α decreased them. Renal I/R caused renal tubular epithelial damage in rats, which was improved by pifithrin-α. The α-SMA mRNA expression was up-regulated significantly after I/R, while it was down-regulated by pifithrin-α.NRK-52E cells were cultured in vitro, cell damage was induced by addition of TNF-α, and then cells were treated with pifithrin-α. Cells treated with TNF-α alone in G2/M phase increased significantly, but they were reduced in the presence of pifithrin-α. In NRK-52E cells treated with pifithrin-α for 6 h, NGAL mRNA expression was significantly lower than in cells without pifithrin-α treatment. After NRK-52E cells were treated with pifithrin-α for 24 h, α-SMA and FN mRNA expression was significantly lower than in cells without the treatment. In summary, pifithrin-α can facilitate the progression of renal tubular epithelial cells through G2/M phase, protecting them against injury.

Immune response of dendritic cells capturing antigens from apoptotic U937 cells induced by artesunate / 中国实验血液学杂志

The objective of study was to investigate whether U937 cells-loaded dendritic cells (DCs) could induce anti-leukemic immune activity. The apoptosis of U937 cells was induced by artesunate (ART). DCs derived from peripheral blood mononuclear cells of health donors were loaded with apoptotic U937 cells, and induced to maturation in the presence of TNF-alpha. Matured DCs were cocultured with autologous T-lymphocytes, and combined with IL-2 in order to induce the leukemia-specific CTL. The phenotypes of DCs and T lymphocytes were tested by flow cytometry. The ability of DC capturing antigens was measured by Dextran-FITC endocytosis. The IL-12p70 level was assayed by ELISA kit. The proliferation of CTL and CTL activity were measured by MTT assay. The results showed that the apoptotic rate of the U937 cells was 51.2% when U937 cells were induced by 1 microg/ml ART for 48 hours in vitro. DCs had the most powerful ability of endocytosis in its immature phase. Apoptotic U937 cells could not induce the features of DC maturation, and apoptotic U937 cell-pulsed immature DCs could be matured with TNF-alpha. The IL-12p70 level secreded by apoptotic U937 cell-loaded mature DCs (mDC-(Apo)U937) was higher than that of non-loaded mDC. The proliferation of autologous T lymphocytes co-cultured with mDC-(Apo)U937 was significantly remarkable and the content of CD8(+) CTL was significantly higher in comparison with any other groups. CTL induced by mDC-(Apo)U937 had stronger killing effect on U937 cells than NB4 (p < 0.01). It is concluded that the mDC-(Apo)U937 can effectively generate T cell-mediated dendritic antileukemic responses in vitro.