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Hypoxia can cause Epithelial-mesenchymal transition (EMT) in renal tubular cells, and in turn, renal fibrosis. We tested the expression of TRIM46, a member of tripartite motif-containing (TRIM) family proteins, and mesenchymal markers under hypoxia. Our results showed that hypoxia significantly enhanced expression of TRIM46 in HK2 human renal proximal tubular epithelial cells. Our data further showed that hypoxia led to upregulated expression of mesenchymal markers including α-smooth muscle actin, vimentin, and Snail, and downregulated expression of epithelial marker E-cadherin, coupled with an increased abundance of nuclear β-catenin. However, such effects were reversed when TRIM46 expression was knocked down. TRIM46 overexpression had similar effects as hypoxia exposure, and such effects were reversed when cells were treated with XAV-939, a selective inhibitor for β-catenin. Furthermore, we found that TRIM46 promoted ubiquitination and proteasomal degradation of Axin1 protein, a robust negative regulator of Wnt/β-catenin signaling activity. Finally, increased TRIM46 coupled with decreased Axin1 was observed in a rat renal fibrosis model. These data suggest a novel mechanism contributing to EMT that mediates hypoxia-induced renal fibrosis. Our results suggest that selectively inhibiting this pathway that activates fibrosis in human kidney may lead to development of a novel therapeutic approach for managing this disease.

IntroductionRenal fibrosis is one of the common pathologies of chronic kidney disease. This study aimed to investigate the function of ubiquitin specific peptidase 49 (USP49) in renal fibrosis and to explore the underlying mechanismMaterial and methodsAfter analyzing the correlation between UPS49 and Smad2/3 pathways, we explored the effect of transforming growth factor-β1 (TGF-β1) on the expression of USP49. Then, the USP49 knockdown and ectopic expression human kidney-2 (HK-2) cell lines were constructed to investigate the role of USP49 in fibrosis, by determining the expression of epithelial-to-mesenchymal transition (EMT) markers (E-cadherin, α-SMA, and vimentin), phosphorylated Smad2/3 (p-Smad2/3), and protein phosphatase magnesium-dependent1A (PPMIA). Coimmunoprecipitation and ubiquitination analyses were used to determine the direct interaction between USP49 and PPM1A. The PPM1Aoverexpressed HK-2 cells were further introduced to evaluate the effects of USP49 on fibrosis. The unilateral ureteral obstruction (UUO) rats were introduced to confirm the UPS49 function in renal fibrosis in vivo.ResultsUSP49 was negatively correlated with Smad2/3 pathway, and TGF-β1 inhibited the USP49 expression. In HK-2 cells, USP49 overexpression suppressed the activity of α-SMA and p-Smad-2/3 and activated E-cadherin, vimentin, and PPMIA, whereas USP49 knockdown displayed the reverse effects. USP49 could form a complex with PPM1A. USP49 positively regulated PPM1A expression through deubiquitination. Moreover, the fibrotic effects of USP49 knockdown were significantly attenuated with ectopic expression of PPM1A. The anti-fibrotic effect was confirmed with low expressed USP49 and PPM1A in vivo.ConclusionsUSP49 might exert anti-fibrotic effects via regulating PPM1A/Smad2/3, and USP49 might be an effective target for the treatment of renal fibrosis.

Renal fibrosis is considered as the final pathway of all types of kidney diseases, which can lead to the progressive loss of kidney functions and eventually renal failure. The mechanisms behind are diversified, in which the mammalian target of rapamycin (mTOR) pathway is one of the most important regulatory pathways that accounts for the disease. Several processes that are regulated by the mTOR pathway, such as autophagy, epithelial-mesenchymal transition (EMT), and endoplasmic reticulum (ER) stress, are tightly associated with renal fibrosis. In this study, we have reported that the expression of tripartite motif-containing (TRIM) protein 6, a member of TRIM family protein, was highly expressed in renal fibrosis patients and positively correlated with the severity of renal fibrosis. In our established in vitro and in vivo renal fibrosis models, its expression was upregulated by the Angiotensin II-induced nuclear translocation of nuclear factor-κB (NF-κB) p50 and p65. In HK2 cells, the expression of TRIM6 promoted the ubiquitination of tuberous sclerosis proteins (TSC) 1 and 2, two negative regulators of the mTORC1 pathway. Moreover, the knockdown of TRIM6 was found efficient for alleviating renal fibrosis and inhibiting the downstream processes of EMT and ER in both HK2 cells and 5/6-nephrectomized rats. Clinically, the level of TRIM6, TSC1/2, and NF-κB p50 was found closely related to renal fibrosis. As a result, we have presented the first study on the role of TRIM6 in the mTORC1 pathway in renal fibrosis models and our findings suggested that TRIM6 may be a potential target for the treatment of renal fibrosis.

A new species was formed when protein P 23 (one segment of ciliate Euplotes octocarinatus centrin) was added to a solution of Eu 3+ . The interaction between P 23 and Eu 3+ was investigated by cyclic voltammetry, pulse voltammetry and electrochemical impedance spectroscopy in 10 mM N -2-hydroxyethylpiperazine- N -2-ethanesulfonic acid (HEPES) buffer (pH 7.4) using a pyrolytic graphite electrode. The formal potential ( E o ′) of Eu 3+ shifted from −0.61 to −0.84 V (versus saturated calomel electrode) after P 23 was added to the Eu 3+ solution. The diffusion coefficient ( D ), the charge-transfer coefficient ( α ) and the electron transfer standard rate constant ( k s ) were obtained in the absence and the presence of P 23 . The affinity constant of Eu 3+ and P 23 was determined to be (1.89 ± 0.51) × 10 4 M −1 . The electrochemical investigation of europium bound to the protein provided useful data for the studies of calcium-binding proteins.

A highly hydrophilic, nontoxic, and conductive effect of colloidal gold nanoparticles (GNP) and multi-walled carbon nanotubes (MWCNT) on pyrolytic graphite electrode has been demonstrated. The direct electron transfer of catalase (CAT) was achieved based on the immobilization of MWCNT/CAT-GNP on a pyrolytic graphite electrode by a Nafion film. The immobilized catalase displayed a pair of well-defined and nearly reversible redox peaks in 0.1 M phosphate buffer solution (PBS) (pH 6.98). The dependence of E°′on solution pH indicated that the direct electron transfer reaction of catalase was a single-electron-transfer coupled with single-proton-transfer reaction process. The immobilized catalase maintained its biological activity, showing a surface controlled electrode process with an apparent heterogeneous electron transfer rate constant (k s) of 1.387±0.1 s−1 and charge-transfer coefficient (α) of 0.49, and displayed electrocatalytic activity in the electrocatalytic reduction of hydrogen peroxide. ...