Hanmin Wang

Hypoxia-induced down-regulation of microRNA-34a promotes EMT by targeting the Notch signaling pathway in tubular epithelial cells.

Background Hypoxia-induced renal tubular cell epithelial–mesenchymal transition (EMT) is an important event leading to renal fibrosis. MicroRNAs (miRNAs) are small non-coding RNA molecules that bind to their mRNA targets, thereby leading to translational repression. The role of miRNA in hypoxia-induced EMT is largely unknown. Methodology/Principal Findings miRNA profiling was performed for the identification of differentially expressed miRNAs in HK-2 cells under normal and low oxygen, and the results were then verified by quantitative real time RT-PCR (qRT-PCR). The function of miRNAs in hypoxia-induced renal tubular cell EMT was assessed by the transfection of specific miRNA inhibitors and mimics. Luciferase reporter gene assays and western blot analysis were performed to validate the target genes of miR-34a. siRNA against Jagged1 was designed to investigate the role of the miR-34a-Notch pathway in hypoxia induced renal tubular cell EMT. miRNA-34a was identified as being downregulated in hypoxic renal tubular epithelial cells. Inhibition of miR-34a expression in HK-2 cells, which highly express endogenous miR-34a, promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker Z0-1, E-cadherin and increased expression of the mesenchymal markers α-SMA and vimentin. Conversely, miR-34a mimics effectively prevented hypoxia-induced EMT. Transfection of miRNA-34a in HK-2 cells under hypoxia abolished hypoxia-induced expression of Notch1 and Jagged1 as well as Notch downstream signals, such as snail. Western blot analysis and luciferase reporter gene assays showed direct evidence for miR-34a targeting Notch1 and Jagged1. siRNAs against Jagged1 or Notch1 effectively prevented miR-34a inhibitor-induced tubular epithelial cell EMT. Conclusions/Significance Our study provides evidence that the hypoxia-induced decrease of miR-34a expression could promote EMT in renal tubular epithelial cells by directly targeting Notch1 and Jagged1, and subsequently, Notch downstream signaling.

Hypoxia-induced Bmi1 promotes renal tubular epithelial cell–mesenchymal transition and renal fibrosis via PI3K/Akt signal

In vitro and in vivo evidence shows that activation of HIF-1a/Twist-Bmi1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis via modulation of PI3K/Akt/Snail signaling by facilitating EMT.

CIAPIN1 inhibits the growth and proliferation of clear cell renal cell carcinoma

Our previous studies indicated a direct correlation with loss of CIAPIN1 and carcinogenesis of tumor in human gastric cancer. Here we presented that the expression of CIAPIN1 was absent or significantly decreased in 102 cases of clear cell renal cell carcinoma (CCRCC) tissues (P<0.05). Up-regulating CIAPIN1 by adenoviral vectors exhibited significant inhibition of CCRCC-derived cell growth in vitro and in vivo with G1 cell cycle arrest. Simultaneously, CIAPIN1-induced growth suppression was found partially to regulate various proteins, including inhibition of cyclinD1, cyclinE, cdk2, cdk4, p-Rb and VEGF, but up-regulation of p27Kip1 and Rb.

ABCG2 protects kidney side population cells from hypoxia/reoxygenation injury through activation of the MEK/ERK pathway.

Breast cancer resistance protein 1 (BCRP1/ABCG2) is used to identify the side population (SP) within a population of cells, which is enriched for stem and progenitor cells in different tissues. Here, we investigated the role of extracellular signal-regulated kinase (ERK) 1/2 in the signaling mechanisms underlying ischemic/hypoxic conditions in kidney SP cells. Kidney SP cells were isolated using Hoechst 33342 dye-mediated fluorescein-activated cell sorting and then incubated under hypoxia/reoxygenation (H/R) with or without verapamil, a selective BCRP1/ABCG2 inhibitor. ABCG2 expression, ERK activity, cell viability, metabolic activity, and membrane damage were tested after H/R treatment. To evaluate the role of ERK 1/2 on the expression and function of ABCG2, the expression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which preferentially activates ERK, was upregulated by transfection with the recombinant sense expression vector pcDNA3.1-MEK and downregulated by pretreatment with U0126, a specific MEK inhibitor. We found that hypoxia activated ERK activity in the kidney SP cells but not in non-SP cells both in vitro and in vivo. Overexpression of MEK mimicked hypoxia-induced ABCG2 expression. Contrarily, U0126 inhibited hypoxia- and MEK-upregulated ABCG2 expression. Furthermore, H/R induced significant increases in nuclear, metabolic, and membrane damage in both SP cells and non-SP cells; however, this H/R-induced cytotoxicity was much more severe in non-SP cells than in SP cells. Notably, the viability of kidney SP cells was enhanced by MEK overexpression and inhibited by U0126. Verapamil treatment reversed MEK-induced viability of kidney SP cells. When administered systemically into animals with renal ischemia/reperfusion injury, the SP cells significantly improved renal function, accelerated mitogenic response, and reduced cell apoptosis. However, this improved therapeutic potential of SP cells was significantly reduced by pretreatment with verapamil. Collectively, these findings provide evidence for a crucial role for the MEK/ERK-ABCG2 pathway in protecting kidney SP cells from ischemic/hypoxic injury.

Treatment of acute hypernatremia in severely burned patients using continuous veno–venous hemofiltration with gradient sodium replacement fluid: a report of nine cases

Dear Editor, In severely burned patients, various healthcare-related factors, such as mechanical ventilation, solute dieresis, and secondary sepsis, may cause hypernatremia that can result in a mortality rate ranging from 33.5 to 66 % among burn victims [1, 2]. The treatment of hypernatremia usually includes restriction of sodium intake, supplementation with salt-free water, and diuretic administration, but the efficacy of these measures is dubious in many cases and often associated with a high rate of complications and mortality [3]. In the study reported here, we successfully treated hypernatremia in nine severely burned patients using continuous veno– venous hemofiltration (CVVH). The study cohort comprised nine patients with acute hypernatremia who were being treated at the Burn Intensive Care Unit. Inclusion criteria were: hypernatremia was not corrected after conventional treatments for 24 h or disturbance of consciousness or acute kidney injury. All nine patients suffered full-thickness skin and deep partial-thickness burns and had an average total burn surface area of 73 % (range 40–90 %). Seven patients suffered concurrent acute kidney injury and had an average serum creatinine level of 292.3 ± 206.0 lmol/L. Eight patients received mechanical ventilation, and two patients needed vasoactive drugs. The mean serum sodium level of the nine patients was 168.2 ± 5.3 mmol/L after hospital admission (at 6.0 ± 3.1 days), and the patients displayed symptoms related to hypernatremia, such as nausea, vomiting, convulsions, drowsiness, and coma. All patients received 36.25 h (average) of CVVH treatment using the Prismaflex HF 1000 Set system (Gambro, Lund, Sweden), which has an effective membrane area of 0.9 m and 90 % predilution route; replacement fluid infusion was set at 2 L/h and blood flow was set at 200 mL/min. The replacement fluid was supplemented with 3 % sodium chloride to adjust the sodium concentration, using the formula [y (mmol/L) = 480 9 (x ? 0.9)/(4 ? x) ? 34.5], where y is the sodium concentration in the replacement fluid and x is the additional 3 % NaCl (in liters). The original sodium level in the replacement fluid was set to be lower than the serum sodium level by 8 mmol/L and to subsequently undergo a reduction rate of 2.47 ± 0.24 mmol/ L every 4 h. After the treatment, all patients showed a significant serum sodium reduction (168.2 ± 5.3 vs. 142.0 ± 1.84 mmol/L, respectively; p \ 0.01), and the serum sodium reduction rate was 0.67 ± 0.13 mmol/L/h with no complications (Fig.1). Likewise, the serum urea and creatinine concentrations decreased from 26.5 ± 8.9 to 18.3 ± 12.2 mmol/L and from 292.3 ± 206.0 to 141.4 ± 35.7 lmol/L (p \ 0.05), respectively. The APACHE II (Acute Physiology and Chronic Health Evaluation II) score (18.7 ± 8.3 vs. 10.8 ± 6.0, respectively; p = 0.018) and Glasgow Coma Score (12.6 ± 2.2 vs. 14.9 ± 0.3, respectively; p \ 0.01) were also significantly improved. With the exception of one patient who died of systematic Pseudomonas aeruginosa infection, all patients recovered and were eventually discharged. To date, few studies have evaluated the use of hemodialysis in treating hypernatremia [4, 5]. Our

Twist overexpression promoted epithelial-to-mesenchymal transition of human peritoneal mesothelial cells under high glucose

BACKGROUND Long-term peritoneal dialysis (PD) results in functional and structural alterations of the peritoneal membrane. Previous studies have suggested that high glucose (HG) could induce transdifferentiation of peritoneal mesothelial cells into myofibroblasts, but the molecular mechanisms of HG-induced epithelial-to-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) are unclear. This study was undertaken to elucidate the effects and mechanisms of Twist on HG-induced EMT of HPMCs. METHODS HPMCs were exposed to 5.6 mM glucose [normal glucose (NG)], 50 mM glucose (HG) or 50 mM glucose with Si-Twist or pcDNA3.1-Twist. Western blot and immuocytochemistry were performed to determine Twist, E-cadherin and α-smooth muscle actin (α-SMA) protein expression. MMP2 and MMP9 were detected by zymography. Rats were daily instilled with PD fluid and lipopolysaccharide (LPS) or sodium chloride during 6 weeks. Histological analyses were carried out in parietal peritoneum. Twist was detected by western blotting. RESULTS Twist and α-SMA protein and immuocytochemistry were significantly increased in HG-conditioned media compared to NG media. E-cadherin protein was lower in pcDNA3.1-Twist-transfected HPMCs compared to pcDNA3.1 cells. Twist protein was upregulated 12 h after HG stimulation. MMP9 was increased in pcDNA3.1-Twist-transfected HPMCs compared to pcDNA3.1 cells. Exposure of rat peritoneum to PD fluid and LPS resulted in an increase of extracellular matrix deposition. Twist and α-SMA were stained in the PD fluid group and compared to the control group. Twist protein was significantly increased in the PD group. CONCLUSIONS In conclusion, HG-induced Twist expression might contribute to EMT of HPMCs. Twist may control EMT of HPMCs by regulating MMP9.

Serum Response Factor Accelerates the High Glucose-Induced Epithelial-to-Mesenchymal Transition (EMT) via Snail Signaling in Human Peritoneal Mesothelial Cells

Background Epithelial-to-Mesenchymal Transition (EMT) induced by glucose in human peritoneal mesothelial cells (HPMCs) is a major cause of peritoneal membrane (PM) fibrosis and dysfunction. Methods To investigate serum response factor (SRF) impacts on EMT-derived fibrosis in PM, we isolated HPMCs from the effluents of patients with end-stage renal disease (ESRD) to analyze alterations during peritoneal dialysis (PD) and observe the response of PM to SRF in a rat model. Results Our results demonstrated the activation and translocation of SRF into the nuclei of HPMCs under extensive periods of PD. Accordingly, HPMCs lost their epithelial morphology with a decrease in E-cadherin expression and an increase in α-smooth muscle actin (α-SMA) expression, implying a transition in phenotype. PD with 4.25% glucose solution significantly induced SRF up-regulation and increased peritoneal thickness. In immortal HPMCs, high glucose (HG, 60 mmol/L) stimulated SRF overexpression in transformed fibroblastic HPMCs. SRF-siRNA preserved HPMC morphology, while transfection of SRF plasmid into HPMCs caused the opposite effects. Evidence from electrophoretic mobility shift, chromatin immunoprecipitation and reporter assays further supported that SRF transcriptionally regulated Snail, a potent inducer of EMT, by directly binding to its promoter. Conclusions Our data suggested that activation of SRF/Snail pathway might contribute to the progressive PM fibrosis during PD.

The T-box transcription factor Brachyury promotes renal interstitial fibrosis by repressing E-cadherin expression.

BackgroundEpithelial-to-mesenchymal transition (EMT) induced by TGF-β1 is one of well-recognized factors contributing to renal fibrosis. However, the underlying molecular mechanisms of EMT are not fully understood. Brachyury, an evolutionarily conserved transcription factor, was recently identified as an important factor promoting EMT in human carcinoma cell lines. There is no evidence that Brachyury is involved in renal tubular EMT.ResultsOur results demonstrated that Brachyury was prominently induced in TGF-β1-treated human proximal tubular epithelial (HK-2) cells and that this induction was accompanied by changes characteristic of EMT. Blockage of Brachyury expression by short interfering RNA (siRNA) in HK-2 cells effectively reversed the TGF-β1-induced EMT phenotype. Brachyury induction repressed E-cadherin transcription; the E-cadherin promoter contains a Brachyury binding site, and decreased expression of E-cadherin occurred in Brachyury-overexpressing cells when they were transfected with reporter constructs using the promoter. This effect was partially mediated by Slug and Snail, as knockdown of Snail and Slug by siRNA effectively reversed Brachyury-mediated EMT and partially restored E–cadherin expression. The expression of Brachyury also presented in a rat model of obstructive nephropathy and in tubulointerstitial fibrosis tissues of IgA nephropathy, suggesting that it may have a role in EMT and renal fibrosis in vivo.ConclusionOur results demonstrate for the first time that Brachyury plays an important role in regulating TGF-β1–mediated renal EMT and could be an attractive target for progression of renal disease therapies.

RUNX3 Mediates Suppression of Tumor Growth and Metastasis of Human CCRCC by Regulating Cyclin Related Proteins and TIMP-1

Here we presented that the expression of RUNX3 was significantly decreased in 75 cases of clear cell renal cell carcinoma (CCRCC) tissues (p<0.05). Enforced RUNX3 expression mediated 786-O cells to exhibit inhibition of growth, G1 cell-cycle arrest and metastasis in vitro, and to lost tumorigenicity in nude mouse model in vivo. RUNX3-induced growth suppression was found partially to regulate various proteins, including inhibition of cyclinD1, cyclinE, cdk2, cdk4 and p-Rb, but increase of p27Kip1, Rb and TIMP-1. Therefore, RUNX3 had the function of inhibiting the proliferative and metastatic abilities of CCRCC cells by regulating cyclins and TIMP1.

Twist Is a New Prognostic Marker for Renal Survival in Patients with Chronic Kidney Disease

Background/Aims: Our previous studies indicate that Twist plays important roles in hypoxia-induced tubular epithelial-mesenchymal transition and the development of kidney fibrosis in cellular and animal models. However, the expression and clinical significance of Twist in patients with chronic kidney disease are not clear. Methods: We analyzed the degree of expression and localization of Twist in renal biopsies from a wide variety of hypoxic kidney diseases and correlated their immunostaining scores with clinical and histologic parameters. In particular, we also retrospectively analyzed whether the degree of expression of Twist in the renal interstitium was correlated with renal survival. Results: Activated Twist was strongly expressed in tubular epithelial cell nuclei from the kidneys of patients with chronic kidney diseases, while little positive staining for Twist was found in the renal tubules of normal kidneys (p = 0.001). Twist protein in the tubulointerstitium was inversely correlated with estimated glomerular filtration rate (eGFR; r = –0.468, p = 0.029) and positively correlated with serum creatinine (r = 0.44, p = 0.045) and the percentage of tubulointerstitial fibrosis (r = 0.551, p = 0.000). Moreover, a high level of Twist was correlated with activation of HIF-1α expression and E-cadherin repression across all disease groups (p = 0.000, p = 0.000, respectively). By multivariate analysis, the experimental data show that the factors influencing renal survival were eGFR [relative risk (RR) 4.39 (95%CI 1.342, 14.393), p = 0.014] and the degree of expression of Twist [RR 3.43 (95% CI 1.098, 10.684), p = 0.034]. Conclusions: Our results raise the possibility that Twist activation is a common mechanism in the pathophysiology of a wide range of chronic hypoxic renal diseases and that Twist staining in renal biopsy specimens might provide a valuable histologic index of progression.