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Renal fibrosis is the common pathological process of various chronic kidney diseases (CKD). Recent studies indicate that mitochondrial fragmentation is closely associated with renal fibrosis in CKD. However, the molecular mechanisms leading to mitochondrial fragmentation remain to be elucidated. The present study investigated the role of regulators of calcineurin 1 (RCAN1) in mitochondrial fission and renal interstitial fibrosis using conditional knockout mice in which RCAN1 was genetically deleted in tubular epithelial cells (TECs). TEC-specific deletion of RCAN1 attenuated tubulointerstitial fibrosis and epithelial to mesenchymal transition (EMT)-like phenotype change after unilateral ureteral obstruction (UUO) and ischemia reperfusion injury (IRI) through suppressing TGF-β1/Smad3 signaling pathway. TEC-specific deletion of RCAN1 also reduced the tubular apoptosis after UUO by inhibiting cytochrome c/caspase-9 pathway. Ultrastructure analysis revealed a marked decrease in mitochondrial fragmentation in TECs of RCAN1-deficient mice in experimental CKD models. The expression of mitochondrial profission proteins dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff) was also downregulated in obstructed kidney of TEC-specific RCAN1-deficient mice. Furthermore, TEC-specific deletion of RCAN1 attenuated the dysfunctional tubular autophagy by regulating PINK1/Parkin-induced mitophagy in CKD. RCAN1 knockdown and knockout similarly improved the mitochondrial quality control in HK-2 cells and primary cultured mouse tubular cells stimulated by TGF-β1. Put together, our data indicated that RCAN1 plays an important role in the progression of tubulointerstitial fibrosis through regulating the mitochondrial quality. Therefore, targeting RCAN1 may provide a potential therapeutic approach in CKD.

High glucose (HG)-induced mitochondrial dynamic changes and oxidative damage are closely related to the development and progression of diabetic kidney disease (DKD). Recent studies suggest that regulators of calcineurin 1 (RCAN1) is involved in the regulation of mitochondrial function in different cell types, so we investigate the role of RCAN1 in mitochondrial dynamics under HG ambience in rat glomerular mesangial cells (MCs). MCs subjected to HG exhibited an isoform-specific up-regulation of RCAN1.4 at both mRNA and protein levels. RCAN1.4 overexpression induced translocation of Dynamin related protein 1 (Drp1) to mitochondria, mitochondrial fragmentation and depolarization, accompanied by increased matrix production under normal glucose and HG ambience. In contrast, decreasing the expression of RCAN1.4 by siRNA inhibited HG-induced mitochondrial fragmentation and matrix protein up-regulation. Moreover, both mitochondrial fission inhibitor Mdivi-1 and Drp1 shRNA prevented RCAN1.4-induced fibronectin up-regulation, suggesting that RCAN1.4-induced matrix production is dependent on its modulation of mitochondrial fission. Although HG-induced RCAN1.4 up-regulation was achieved by activating calcineurin, RCAN1.4-mediated mitochondrial fragmentation and matrix production is independent of calcineurin activity. These results provide the first evidence for the HG-induced RCAN1.4 up-regulation involving increased mitochondrial fragmentation, leading to matrix protein up-regulation.

Parathyroid hormone-related protein (PTHrP) is known to be up-regulated in both glomeruli and tubules in patients with diabetic kidney disease (DKD), but its role remains unclear. Previous studies show that PTHrP-induced hypertrophic response in mesangial cells (MCs) and epithelial-mesenchymal transition (EMT) in tubuloepithelial cells can be mediated by TGF-β1. In the present study, although long-term PHTrP (1–34) treatment increased the mRNA and protein level of TGF-β1 in primary rat MCs, fibronectin up-regulation occurred earlier, suggesting that fibronectin induction is independent of TGF-β1/Smad signaling. We thus evaluated the involvement of epidermal growth factor receptor (EGFR) signaling and found that nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species mediates PTHrP (1–34)-induced Src kinase activation. Src phosphorylates EGFR at tyrosine 845 and then transactive EGFR. Subsequent PI3K activation mediates Akt and ERK1/2 activation. Akt and ERK1/2 discretely lead to excessive protein synthesis of fibronectin. Our study thus demonstrates the new role of PTHrP in fibronectin up-regulation for the first time in glomerular MCs. These data also provided new insights to guide development of therapy for glomerular sclerosis.

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. TGF-β1 is a major cytokine mediating the production of various extracellular matrix (ECM) proteins. The aim of this study is to elucidate the effect of parathyroid hormone (PTH) on TGF-β1 and high glucose-induced upregulation of ECM proteins in primary mesangial cells from Sprague-Dawley rat. The results showed that PTH pretreatment prevented TGF-β1 and high glucose-induced Smad2/3 phosphorylation and consequent upregulation of fibronectin and type IV collagen within 4 h. The inhibitory effect of PTH is due to PTH1R activation, because knocking down PTH 1 receptor (PTH1R) by RNA interference reversed the inhibitory effect of PTH on TGF-β1 and high glucose-induced Smad2/3 phosphorylation and ECM upregulation. Furthermore, it is found that PTH1R associated with TGF-β type II receptor (TβR II) and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH alone. The internalization of TβR II might reduce the amount of membrane TβR II, attenuate the sensitivity of mesangial cells to TGF-β1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-β1 and high glucose. Further studies are needed to know whether the endocytic receptors are to be degraded or recycled, and evaluate the role of PTH in TGF-β1 signaling more comprehensively.

This study aimed to investigate the effects of ethanol on the expression of caveolin‑1 (CAV‑1) in HepG2 hepatocarcinoma cells. Ethanol‑treated HepG2 cells were investigated using the in vitro model to determine whether ethanol can influence the expression of CAV‑1. Cell viability was measured using the colorimetric 3‑(4, 5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Expression of CAV‑1 was detected using western blot analysis. Quantitative PCR (qPCR) was used to determine CAV‑1 mRNA levels. The distribution of CAV‑1 in HepG2 cells was analyzed using immunofluorescence. The MTT assay results revealed that cell viability was not altered at ethanol concentrations of1.0%, while ethanol concentrations1.0% caused cell shedding, but not cell fragmentation. Western blot analysis showed significant differences in the levels of CAV‑1 expression between the control group and the 1.0% ethanol‑treated group at 6, 12 and 24 h (all P0.05). qPCR showed significant differences in the expression levels of caveolin‑1 mRNA between the control group and the 1.0% ethanol‑treated group at 6 h, 12 h and 24 h (all P0.05). Immunofluorescence demonstrated that CAV‑1 was distributed discontinuously at the boundaries of HepG2 cells. The results indicate that ethanol may increase the expression of CAV‑1 in HepG2 cells.

A great deal of evidence highlighted the pathophysiologic importance of TGF-β1/Smad3 pathway in masangial extracellular matrix (ECM) accumulation, but some alternative signaling pathways are also involved. TGF-β was shown recently to induce rapid and transient epidermal-like growth factor receptor (EGFR) transactivation and subsequent fibronectin expression via heparin-binding epidermal-like growth factors (HB-EGF) release and binding in mesangial cells, which is independent of Smad2 activation. However, whether TGF-β could induce persistent EGFR transactivation remains to be identified. The present study demonstrates that in addition to transient EGFR transactivation, TGF-β1 can also induce continuous EGFR transactivation by a non-ligand-dependent pathway in rat mesangial cells. This sustained EGFR transactivation is mainly due to Src kinase-mediated persistent EGFR tyrosine phosphorylation at Y845 rather than Y1173. TGF-β1-induced early Smad3 phosphorylation is independent of transient EGFR transactivation and ERK1/2 activation initiated by HB-EGF release, whereas Src-mediated chronic EGFR transactivation and ERK1/2 activation participate in Smad3 activation in a relatively modest and delayed manner. Therefore, the present study further clarifies the mechanisms of EGFR transactivation in the TGF-β-initiated ECM upregulation and raises the possibility that targeting EGFR may provide a viable alternative strategy for inhibiting TGF-β in chronic kidney disease.

Pathogenic Vibrio spp., Aeromonas spp. and Plesiomonas shigelloides are associated with human gastroenteritis and wound infections, as well as fish diseases. The comprehensive and accurate identification of these pathogens is crucial for the current public health. The present study describes the development of a multiplex assay for the simultaneous identification of ten bacterial pathogens in a single reaction by using a multiplex ligation reaction based on probe melting curve analysis (MLMA). The specificity for target genes was 100%, as assessed with a panel of 67 bacterial pathogens, which indicated no cross-reactions. The detection limit of this assay ranged from 0.8 × 107 CFU/mL to 1.5 × 108 CFU/mL at the pure bacterial culture level and from 0.1 ng to 1.0 ng at the DNA level. The MLMA assay was used to detect ten species of pathogens in 269 clinical and seafood samples, and for further validation, the results were compared with the conventional culture method. The results indicated greater than 90% sensitivity and 100% specificity for each bacterial pathogen tested, and the kappa correlation for all the pathogens ranged from 0.95 to 1.00. Overall, this assay is well suited for public health laboratories for its high throughput, accuracy, and low cost.

The present study aimed to investigate the effects of alcohol on intestinal epithelial barrier permeability and expression of the tight junction-associated proteins, zonula occludens-1 (ZO-1) and claudin-1. An alcohol-treated Caco-2 intestinal epithelial cell monolayer in vitro model was used to investigate whether alcohol is able to induce intestinal barrier dysfunction and decrease expression of ZO-1 and claudin-1. MTT assay results revealed unaltered cell viability at alcohol concentrations of

Flow cytometry, BrdU incorporation, and western blotting were used to investigate whether caveolin-1 (Cav-1) is involved in cell cycle progression of renal glomerular mesangial cells. Wide type mesangial cells re-entered the cell cycle after 22 h incubation with 20% fetal bovine serum (FBS) and Cav-1 knockout cells remained in G0/G1 phase after 48 h, which indicated that Cav-1 knockout mesangial cells were G0/G1 arrest. The protein level of cyclin D1 significantly increased after incubation with 20% FBS for 6 h in wide type mesangial cells, and 12 h in Cav-1 knockout cells. It suggested that cyclin D1 upregulation was delayed in knockout mesangial cells. Cav-1 re-expression in Cav-1 knockout mesangial cells increased the ratio of S phase from 4.8% to 15.26%, and decreased the ratio of G0/G1 phase from 90.96% to 77.84%, which implied that Cav-1 re-expression can reverse cell cycle arrest. It concludes that Cav-1 may promote the proliferation of mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We have recently shown that epidermal growth factor receptor (EGFR) transactivation mediates high glucose (HG)-induced collagen I upregulation through PI3K-PKCbeta1-Akt signaling in mesangial cells (MC). Phospholipase Cgamma1 (PLCgamma1) interacts with activated growth factor receptors and activates classic PKC isoforms. We thus studied its role in HG-induced collagen I upregulation in MC. Primary rat MC were treated with HG (30 mM) or mannitol as osmotic control. Protein kinase activation was assessed by Western blotting and collagen I upregulation by Northern blotting. Diabetes was induced in rats by streptozotocin. HG treatment for 1 h led to PLCgamma1 membrane translocation and Y783 phosphorylation, both indicative of its activation. Mannitol was without effect. PLCgamma1 Y783 phosphorylation was also seen in cortex and glomeruli of diabetic rats. HG induced a physical association between EGFR and PLCgamma1 as identified by coimmunoprecipitation. PLCgamma1 activation required EGFR kinase activity since it was prevented by the EGFR inhibitor AG1478 or overexpression of kinase-inactive EGFR (K721A). Phosphoinositide-3-OH kinase inhibition also prevented PLCgamma1 activation. HG-induced Akt S473 phosphorylation, effected by PKCbeta1, was inhibited by the PLCgamma inhibitor U73122. PLCgamma1 inhibition or downregulation by small interference RNA also prevented HG-induced collagen I upregulation. Our results indicate that EGFR-PLCgamma1 signaling mediates HG-induced PKCbeta1-Akt activation and subsequent collagen I upregulation in MC. Inhibition of EGFR or PLCgamma1 may provide attractive therapeutic targets for the treatment of diabetic nephropathy.

Glomerular matrix accumulation is a hallmark of diabetic renal disease. Serine/threonine kinase PKC-β1 mediates glucose-induced Akt S473 phosphorylation, RhoA activation, and transforming growth factor (TGF)-β1 upregulation and finally leads to matrix upregulation in mesangial cells (MCs). It has been reported that glucose-induced PKC-β1 activation is dependent on caveolin-1 and the presence of intact caveolae in MCs; however, whether activated PKC-β1 regulates caveolin-1 expression and phosphorylation are unknown. Here, we showed that, although the caveolin-1 protein level had no significant change, the PKC-β-specific inhibitor LY-333531 blocked caveolin-1 Y14 phosphorylation in high glucose (HG)-treated MCs and in the renal cortex of diabetic rats. The Src-specific inhibitor SU-6656 prevented the HG-induced association between PKC-β1 and caveolin-1 and PKC-β1 membrane translocation, whereas PKC-β1 small interfering RNA failed to block Src activation, indicating that Src kinase is upstream of PKC-β1 activation. Although LY-333531 blocked PKC-β1 membrane translocation, it had no effect on the PKC-β1/caveolin-1 association, suggesting that PKC-β1 activation requires the interaction of caveolin-1 and PKC-β1. PKC-β1-mediated Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation in response to HG were prevented by SU-6656 and nonphosphorylatable mutant caveolin-1 Y14A. In conclusion, Src activation by HG mediates the PKC-β1/caveolin-1 association and PKC-β1 activation, which assists in caveolin-1 Y14 phosphorylation by Src kinase. The downstream effects, including Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation, require caveolin-1 Y14 phosphorylation. Caveolin-1 is thus an important mediator of the profibrogenic process in diabetic renal disease.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent data have linked the serine/threonine kinase protein kinase B (Akt) to matrix modulation. Here, we studied its role in high glucose-induced collagen elaboration by mesangial cells.Primary rat mesangial cells were treated with high glucose levels (30 mmol/l) or mannitol as osmotic control. Western blots, northern blots, ELISA and immunohistochemistry were used for assessment. Diabetes was induced in rats by streptozotocin.Phosphorylated Akt at S473 (pAktS473), corresponding to Akt activation, was seen in diabetic glomeruli. In mesangial cells, high glucose levels induced pAktS473 by 20 min. This was sustained to 72 h, while mannitol had no effect. Akt activation by kinase assay and phosphorylation on threonine 308 was also observed. Phosphoinositide 3-kinase (PI3K) inhibitors LY294002 (20 micromol/l) and wortmannin (100 nmol/l) prevented pAktS473. Collagen IA1 transcript and collagen I protein upregulation by high glucose levels were inhibited by PI3K blockade, as was collagen I secretion into the medium (ELISA). Dominant-negative Akt overexpression also inhibited high glucose-induced collagen IA1 transcript and collagen I protein production. Since signalling through the epidermal growth factor receptor (EGFR) can activate PI3K-Akt, we studied its activation by high glucose levels. EGFR was correspondingly activated by 10 min; mannitol had no effect. EGFR activation was also seen in glomeruli from diabetic rats and co-localised with collagen IA1 in diabetic glomeruli. Specific EGFR inhibition (AG1478, 5 micromol/l or dominant-negative EGFR) blocked high glucose-induced pAktS473, phosphorylation on threonine 308 and activation of the EGFR downstream target p44 extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase. Finally, EGFR inhibition also blocked high glucose-induced collagen I upregulation at transcriptional and protein levels.We conclude that EGFR-PI3K-Akt signalling mediates high glucose-induced collagen I upregulation in mesangial cells and that this pathway is activated in diabetic glomeruli. Targeting its components may provide a new therapeutic approach to diabetic kidney disease.

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein-Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.