Ubiquitin-conjugating enzyme UBE2Q2 participates in HUWE1-mediated protection on renal tubulointerstitial fibrosis / 生理学报

The ubiquitin-proteasome system plays an important role in protein degradation. The process of ubiquitination requires ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 to complete the coordination. Our previous studies have shown that HUWE1 (HECT, UBA and WWE domain containing 1), as an E3 ubiquitin ligase, can degrade epidermal growth factor receptor (EGFR) to inhibit renal tubulointerstitial fibrosis. However, E2 ubiquitin-conjugating enzymes binding to HUWE1 are still unclear. The aim of the present study was to identify E2 ubiquitin-conjugating enzymes of HUWE1. Real-time PCR was used to identify E2 ubiquitin-conjugating enzyme that may interact with HUWE1. The expression of E2 ubiquitin-conjugating enzyme was detected in kidney of unilateral ureteral obstruction (UUO) mice and HK-2 cells treated with transforming growth factor-β (TGF-β). The results showed that the expressions of E2 ubiquitin-conjugating enzyme UBE2Q2 were significantly down-regulated at both RNA and protein levels in UUO kidneys. The expression of UBE2Q2 was also down-regulated in HK-2 cells stimulated with TGF-β, which was consistent with the change in the expression of HUWE1. These findings indicated that UBE2Q2 expression was synergistic with HUWE1 in the injured kidney. Co-immunoprecipitation (Co-IP) experiments showed that HUWE1 interacted with UBE2Q2 in HK-2 cells. The co-localization of UBE2Q2 and HUWE1 was confirmed by cell immunofluorescence staining. After knocking down UBE2Q2 by siRNA, ubiquitin binding to HUWE1 and EGFR was decreased. In sum, our results demonstrated that UBE2Q2, ubiquitin-conjugating enzyme, works with HUWE1 to mediate ubiquitination and degradation of target protein in kidney.

S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells / 生理学报

The aim of this study was to investigate the role of S100 calcium binding protein A16 (S100A16) in lipid metabolism in hepatocytes and its possible biological mechanism. HepG2 cells (human hepatoma cell line) were cultured with fatty acid to establish fatty acid culture model. The control model was cultured without fatty acid. Each model was divided into three groups and transfected with S100a16 over-expression, shRNA and vector plasmids, respectively. The concentration of triglyceride (TG) in the cells was measured by kit, and the lipid droplets was observed by oil red O staining. Immunoprecipitation and mass spectrometry were used to find the interesting proteins interacting with S100A16, and the interaction was verified by immunoprecipitation. The further mechanism was studied by Western blot and qRT-PCR. The results showed that the intracellular lipid droplet and TG concentrations in the fatty acid culture model were significantly higher than those in the control model. The accumulation of intracellular fat in the S100a16 over-expression group was significantly higher than that in the vector plasmid transfection group. There was an interaction between heat shock protein A5 (HSPA5) and S100A16. Over-expression of S100A16 up-regulated protein expression levels of HSPA5, inositol-requiring enzyme 1α (IRE1α) and pIREα1, which belong to endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway. Meanwhile, over-expression of S100A16 up-regulated the mRNA expression levels of adipose synthesis-related gene Srebp1c, Acc and Fas. In the S100a16 shRNA plasmid transfection group, the above-mentioned protein and mRNA levels were lower than those of vector plasmid transfection group. These results suggest that S100A16 may promote lipid synthesis in HepG2 cells through endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway.

Protective effect of low potassium dextran solution on acute kidney injury following acute lung injury induced by oleic acid in piglets / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Low potassium dextran (LPD) solution can attenuate acute lung injury (ALI). However, LPD solution for treating acute kidney injury secondary to ALI has not been reported. The present study was performed to examine the renoprotective effect of LPD solution in ALI induced by oleic acid (OA) in piglets.</p><p><b>METHODS</b>Twelve animals that suffered an ALI induced by administration of OA into the right atrium were divided into two groups: the placebo group (n = 6) pretreated with normal saline and the LPD group (n = 6), pretreated with LPD solution. LPD solution was injected intravenously at a dose of 12.5 ml/kg via the auricular vein 1 hour before OA injection.</p><p><b>RESULTS</b>All animals survived the experiments with mild histopathological injury to the kidney. There were no significant differences in mean arterial pressure (MAP), creatinin and renal damage scores between the two groups. Compared with the placebo group, the LPD group had better gas exchange parameters at most of the observation points ((347.0 ± 12.6) mmHg vs. (284.3 ± 11.3) mmHg at 6 hours after ALI, P < 0.01). After 6 hours of treatment with OA, the plasma concentrations of NGAL and interleukin (IL)-6 in both groups increased dramatically compared to baseline ((6.0 ± 0.6) and (2.50 ± 0.08) folds in placebo group; and (2.5 ± 0.5) and (1.40 ± 0.05) folds in LPD group), but the change of both parameters in the LPD group was significantly lower (P < 0.01) than in the placebo group. And 6 hours after ALI the kidney tissue concentration of IL-6 in the LPD group ((165.7 ± 22.5) pg×ml(-1)×g(-1) protein) was significantly lower (P < 0.01) than that in placebo group ((67.2 ± 25.3) pg×ml(-1)×g(-1) protein).</p><p><b>CONCLUSION</b>These findings suggest that pretreatment with LPD solution via systemic administration might attenuate acute kidney injury and the cytokine response of IL-6 in the ALI piglet model induced by OA injection.</p>