Comamonas flocculans sp. nov., a Floc-Forming Bacterium Isolated from Livestock Wastewater.

A Gram-negative, aerobic, non-motile, rod-shaped, floc-forming, and non-spore-forming bacterium, designated as NLF-7-7T, was isolated from the biofilm of a sample collected from a livestock wastewater treatment plant in Nonsan, Republic of Korea. Strain NLF-7-7T, forms a visible floc and grows in the flocculated state. Cells of strain NLF-7-7T grew optimally at pH 6.5 and 30 °C and in the presence of 0.5% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NLF-7-7T belonged to the family Comamonadaceae, and was most closely related to Comamonas badia DSM 17552T (95.8% similarity) and Comamonas nitrativorans 23310T (94.0% similarity). The phylogenetic and phenotypic data indicate strain NLF-7-7T is clearly distinguished from the Comamonas lineage. The major cellular fatty acids were C10:0 3OH, C16:0, and summed feature 3 (C16:1 ω6c/C16:1 ω7c). The respiratory quinone was Q-8. The polar lipids were composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified aminolipid. The DNA G+C content of strain NLF-7-7 was 68.0 mol%. Based on the phenotypic, chemotaxonomic, and phylogenetic properties, strain NLF-7-7T represents a novel species of the genus Comamonas, for which the name Comamonas flocculans sp. nov. is proposed. The type strain is C. flocculans NLF-7-7T (=KCTC 62943T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Comamonas flocculans NLF-7-7T is MN527436. The whole-genome shotgun BioProject Number is PRJNA555370 with the Accession Number CP042344.

Noncovalent tethering of nucleic acid aptamer on DNA nanostructure for targeted photo/chemo/gene therapies.

Here, we report various therapeutic cargo-loadable DNA nanostructures that are shelled in polydopamine and noncovalently tethered with cancer cell-targeting DNA aptamers. Initial DNA nanostructure was formed by rolling-circle amplification and condensation with Mu peptides. This DNA nanostructure was loaded with an antisense oligonucleotide, a photosensitizer, or an anticancer chemotherapeutic drug. Each therapeutic agent-loaded DNA nanostructure was then shelled with polydopamine (PDA), and noncovalently decorated with a poly adenine-tailed nucleic acid aptamer (PA) specific for PTK7 receptor, resulting in PA-tethered and PDA-shelled DNA nanostructure (PA/PDN). PDA coating shell enabled photothermal therapy. In the cells overexpressing PTK7 receptor, photosensitizer-loaded PA/PDN showed greater photodynamic activity. Doxorubicin-loaded PA/PDN exerted higher anticancer activity than the other groups. Antisense oligonucleotide-loaded PA/PDN provided selective reduction of target proteins compared with other groups. Our results suggest that the PA-tethered and PDA-shelled DNA nanostructures could enable the specific receptor-targeted phototherapy, chemotherapy, and gene therapy against cancer cells.

Activation of AMPK by berberine induces hepatic lipid accumulation by upregulation of fatty acid translocase CD36 in mice.

Emerging evidence has shown that berberine has a protective effect against metabolic syndrome such as obesity and type II diabetes mellitus by activating AMP-activated protein kinase (AMPK). AMPK induces CD36 trafficking to the sarcolemma for fatty acid uptake and oxidation in contracting muscle. However, little is known about the effects of AMPK on CD36 regulation in the liver. We investigated whether AMPK activation by berberine affects CD36 expression and fatty acid uptake in hepatocytes and whether it is linked to hepatic lipid accumulation. Activation of AMPK by berberine or transduction with adenoviral vectors encoding constitutively active AMPK in HepG2 and mouse primary hepatocytes increased the expression and membrane translocation of CD36, resulting in enhanced fatty acid uptake and lipid accumulation as determined by BODIPY-C16 and Nile red fluorescence, respectively. Activation of AMPK by berberine induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and subsequently induced CCAAT/enhancer-binding protein ß (C/EBPß) binding to the C/EBP-response element in the CD36 promoter in hepatocytes. In addition, hepatic CD36 expression and triglyceride levels were increased in normal diet-fed mice treated with berberine, but completely prevented when hepatic CD36 was silenced with adenovirus containing CD36-specific shRNA. Taken together, prolonged activation of AMPK by berberine increased CD36 expression in hepatocytes, resulting in fatty acid uptake via processes linked to hepatocellular lipid accumulation and fatty liver.

Claudin 4-targeted nanographene phototherapy using a Clostridium perfringens enterotoxin peptide-photosensitizer conjugate.

In this study we designed a claudin 4-directed dual photodynamic and photothermal system, in which a 30-amino acid claudin 4-binding peptide, Clostridium perfringens enterotoxin (CPE), was linked to a photodynamic agent chlorin e6 (Ce6) through a polyethylene glycol spacer (CPC) and anchored onto reduced graphene oxide (rGO) nanosheets to form CPC/rGO nanosheets. For comparison, a conjugate of polyethylene glycol and Ce6 (PC) was anchored onto the rGO nanosheets to generate PC/rGO. Both PC and CPC generated reactive oxygen species upon irradiation at 660 nm. Application of CPC/rGO to claudin 4-overexpressing U87 glioblastoma cells in vitro resulted in a significantly higher cellular uptake compared to application of PC/rGO. Upon irradiation at 660 and 808 nm, the CPC/rGO-treated U87 cells generated significantly higher reactive oxygen species and caused significantly higher temperature increase, and showed most potent anticancer effect compared to the other groups. Taken together, these results suggest that CPC/rGO is potentially useful as a tumor-specific combined phototherapy.

Comparative analysis of reverse-transcription-polymerase chain reaction for Aichivirus detection.

Aichivirus-A (AiV-A), a member of the Kobuvirus genus of the family Picornaviridae, was first reported in stool samples of patients with non-bacterial gastroenteritis in Aichi Prefecture, Japan, in 1989. AiV has been reported from in various aquatic environments, such as surface water and sewage, can be transmitted via the fecal-oral route through contaminated water. As AiV is known to acute gastroenteritis worldwide, developing methods for AiV detection from contaminated environments and food is required. In the present study, we established an effective polymerase chain reaction (PCR) method to detect AiV. Various real-time reverse transcription (RT)-PCR and conventional PCR methods for AiV detection were compared, and the limit of detection was confirmed by comparing the sensitivity at varied primer concentrations and PCR conditions. The final detection limits were 102 copy/µL in conventional PCR, and 101 copy/µL in the real-time RT-PCR. The optimized method used in this study might aid in detecting AiV contamination.

Initial preclinical safety of non-replicating human endogenous retrovirus envelope protein-coated baculovirus vector-based vaccines against human papillomavirus.

Human endogenous retrovirus (HERV) envelope protein-coated, baculovirus vector-based HPV 16 L1 (AcHERV-HPV16L1) is a non-replicating recombinant baculoviral vaccine. Here, we report an initial evaluation of the preclinical safety of AcHERV-HPV16L1 vaccine. In an acute toxicity study, a single administration of AcHERV-HPV16L1 DNA vaccine given intramuscularly (i.m.) to mice at a dose of 1 × 10(8) plaque-forming units (PFU) did not cause significant changes in body weight compared with vehicle-treated controls. It did cause a brief increase in the weights of some organs on day 15 post-treatment, but by day 30, all organ weights were not significantly different from those in the vehicle-treated control group. No hematological changes were observed on day 30 post-treatment. In a range-finding toxicity study with three doses of 1 × 10(7) , 2 × 10(7) and 5 × 10(7) PFU once daily for 5 days, the group treated with 5 × 10(7) PFU showed a transient decrease in the body weights from day 5 to day 15 post-treatment, but recovery to the levels similar to those in the vehicle-treated control group by post-treatment day 20. Organ weights were slightly higher for lymph nodes, spleen, thymus and liver after repeated dosing with 5 × 10(7) PFU on day 15, but had normalized by day 30. Moreover, repeated administration of AcHERV-HPV16L1 did not induce myosin-specific autoantibody in serum, and did not cause immune complex deposition or tissue damage at injection sites. Taken together, these results provide preliminary evidence of the preclinical safety of AcHERV-based HPV16L1 DNA vaccines in mice.

Optimization of RT-PCR methods for enterovirus detection in groundwater.

Enteroviruses (EVs), which belong to the Picornaviridae family, infect individuals asymptomatically or cause mild symptoms (fever, runny nose, cough, skin rash, sneezing, mouth blister). Severe cases can cause various diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, or myocarditis, especially in infants. These viruses can be transmitted via the fecal-oral route via contaminated water. In this study, we established a polymerase chain reaction (PCR) method for detecting EVs in water sample using Coxsackievirus B5 (CV-B5) and Echovirus 30 (E-30), which belong to species B of the four species of EVs (EV-A to D). Several methods have been investigated and compared for the detection of EVs, including real-time reverse transcription (RT) polymerase chain reaction and conventional RT-PCR. The most sensitive primer sets were selected, and the PCR conditions were modified to increase sensitivity. We also quantified the detection limits of real-time and conventional RT-PCR. The detection limits of conventional RT-PCR were detected in 105-106 copy/mL for CV-B5 and 106-107 copy/mL for E-30, respectively. This optimized method for detecting EVs is expected to contribute substantially to the investigation of EV outbreaks in water samples.

Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation.

Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1-PRK2 signaling pathway.

Preclinical pharmacokinetics and biodistribution of human papillomavirus DNA vaccine delivered in human endogenous retrovirus envelope-coated baculovirus vector.

PURPOSE: Test pharmacokinetics and biodistribution of a human papillomavirus(HPV)16L1 DNA vaccine delivered in human endogenous retrovirus envelope protein (HERV)-expressing baculovirus (AcHERV) and those of naked plasmid vaccine. METHOD: HPV16L1 gene was administrated as a naked plasmid or in AcHERV to mice via intravenous and intramuscular routes. HPV16L1 gene was extracted and assayed by quantitative real-time polymerase chain reaction, which was determined to have a detection limit of 50 copies/µg genomic DNA.. RESULTS: Mean residence times of HPV16L1 in AcHERV were 4.8- and 272.2-fold higher than naked HPV16L1 DNA vaccines after intramuscular and intravenous administration, respectively. Naked HPV16L1 DNA levels 1 month after injection were >3 orders of magnitude lower in each tissue tested than AcHERV-delivered HPV16L1, which was retained in most tissues without specific tissue tropism. AcHERV-delivered HPV16L1 administered intramuscularly persisted at the injection sites. However, the levels of copy numbers in muscle were low (1,800/µg genomic DNA) after 1 month, and undetectable after 6 months. CONCLUSIONS: HPV16L1 delivered via AcHERV resides longer in the body than HPV16L1 in naked form. The lack of tissue tropism ensures the safety of AcHERV vectors for further development.

Comparison of enrichment methods for isolating Enterohemorrhagic Escherichia coli in kimchi.

This study was conducted to compare the efficiency of four enrichment methods of Enterohemorrhagic Escherichia coli by using the 16S rRNA amplicon sequencing and a predictive model. Four different methods (US FDA, ISO, Japan Food Hygiene Association and Korea Ministry of Food and Drug Safety) were used to enrich EHEC in kimchi inoculated with cocktails of EHEC strains (NCCP 13720, NCCP 13721, and NCCP 14134). The maximum growth rate (µmax) and lag phase duration (LPD) were compared using the Baranyi model, and 16S rRNA targeted sequencing was performed with samples at the end of the exponential phase. As a result, the µmax and LPD values of Baranyi model developed for the four enriched media ranged from 0.82 to 0.92 and from 2.35 to 2.68, respectively, suggesting that the growth of EHEC was similar in all four enrichment media. As for the relative abundance of the bacterial composition at the family level, Enterobacteriaceae was identified as the major component (>50%) in all four enriched media. The relative abundance of Enterobacteriaceae was highest (>90%) in the two enriched media with 20 mg/L novobiocin, demonstrating that significant growth of non-targeted bacteria takes place in enrichment broths utilizing <20 mg/L novobiocin or different antibiotics. In conclusion, this study suggests that all four enrichment broth are suitable for growing EHEC in kimchi and the use and concentration of antibiotics such as novobiocin in enrichment media may have a critical role in species diversity.

Evaluation of Hygiene Indicators and Sampling Plan for Detecting Microbial Contamination in Health Functional Foods.

ABSTRACT: This study aimed to monitor microbial contamination levels in a variety of health functional foods and to establish new microbial criteria. Indicator organisms (i.e., aerobic bacteria, coliform bacteria, and Escherichia coli) were monitored in 10 health functional food categories (743 items, 3,715 samples). The mean total aerobic counts of ginseng and Korean red ginseng were -0.35 and -0.74 log CFU/g; and the mean total coliform counts were -1.4 and -1.39 log CFU/g, respectively. In addition, the mean total coliform counts of fiber and protein products were -1.34 and -1.22 log CFU/g, respectively. However, no aerobic or coliform cells were detected in any other health functional food products (vitamins, minerals, probiotics, milk thistle extract, propolis, eicosapentaenoic acid, docosahexaenoic acid, or lutein products), and no E. coli was detected in any of the categories. These results can potentially be used to update the microbial criteria of the Health Functional Food Code.

Development and validation of a predictive model for pathogenic Escherichia coli in fresh-cut produce.

This study was performed to develop and validate a predictive growth model of pathogenic Escherichia coli to ensure the safety of fresh-cut produce. Samples were inoculated with a cocktail of seven E. coli strains of five pathotypes (EHEC, Enterohemorrhagic E. coli; ETEC, Enterotoxigenic E. coli; EPEC, Enteropathogenic E. coli; EIEC, Enteroinvasive E. coli, and EAEC, Enteroaggregative E. coli) and stored at 4, 10, 12, 15, 25, 30, and 37°C. Growth of pathogenic E. coli was observed above 12°C. The primary growth model for pathogenic E. coli in fresh-cut produce was developed based on the Baranyi model. The secondary model was developed as a function of temperature for lag phase duration (LPD) and maximum specific growth rate (µmax) based on the polynomial second-order model. The primary and secondary models for pathogenic E. coli were fitted with a high degree of goodness of fit (R2 ≥ 0.99). The bias factor (Bf), accuracy factor (Af), and root mean square error (RMSE) were 0.995, 1.011, and 0.084, respectively. The growth model we developed can provide useful data for assessing the quantitative microbial risk of pathogenic E. coli in fresh-cut produce intended for human consumption. In addition, it is thought to be widely available in industries that produce, process, distribute, and sell fresh-cut produce.

Quantitative microbial risk assessment of pathogenic Escherichia coli in commercial kimchi in South Korea.

Owing to convenience, ease of preparation, and price, the consumption of commercial kimchi is gradually rising in South Korea. Here, we estimated the risk level posed by pathogenic Escherichia coli in commercial kimchi products using the quantitative microbial risk assessment (QMRA) approach to develop measures for preventing potential foodborne outbreaks from kimchi consumption. We collected 610 samples of commercial kimchi products produced in Korea, 267 kimchi samples from foreign countries imported to Korea, and 187 raw materials used in kimchi preparation, and analyzed them for contamination with pathogenic E. coli. A Predictive model was developed to observe the survival characteristics of pathogenic E. coli. A dose-response model was selected, and the risk level was estimated using @RISK software. Although a prior epidemiological study indicated the frequent occurrence of foodborne outbreaks arising from contaminated kimchi products consumed in food service facilities, we found a low probability of foodborne illness caused by pathogenic E. coli in commercial kimchi products.

Biosynthesis of (R)-(-)-1-Octen-3-ol in Recombinant Saccharomyces cerevisiae with Lipoxygenase-1 and Hydroperoxide Lyase Genes from Tricholoma matsutake.

Tricholoma matsutake is an ectomycorrhizal fungus, related with the host of Pinus densiflora. Most of studies on T. matsutake have focused on mycelial growth, genes and genomics, phylogenetics, symbiosis, and immune activity of this strain. T. matsutake is known for its unique fragrance in Eastern Asia. The most major component of its scent is (R)-(-)-1-octen-3-ol and is biosynthesized from the substrate linoleic acid by the sequential reaction of lipoxygenase and peroxide lyase. Here, we report for the first time the biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake using the yeast Saccharomyces cerevisiae as a host. In this study, cDNA genes correlated with these reactions were cloned from T. matsutake, and expression studies of theses genes were carried out in the yeast Saccharomyces cerevisiae. The product of these genes expression study was carried out with Western blotting. The biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake in recombinant Saccharomyces cerevisiae was subsequently identified with GC-MS chromatography analysis. The biosynthesis of (R)-(-)-1-octen-3-ol with S. cerevisiae represents a significant step forward.

Marked individual variation in isoflavone metabolism after a soy challenge can modulate the skeletal effect of isoflavones in premenopausal women.

Soy-isoflavones may act as estrogenic agonists or antagonists depending on the endogenous hormone status. These clinical effects can be exerted variably in individuals by the metabolic ability to produce a more potent metabolite than precursors. The objective of this randomized, double-blind, placebo-controlled study was to investigate the skeletal effect of isoflavones according to their metabolic variability in premenopausal women. Volunteers were randomly assigned to receive either soy-extract isoflavones (n=32) or lactose (n=21) once a day for three menstrual cycles. After intervention, the urinary excretions of isoflavones and their metabolites were significantly higher in the soy group than in the placebo group and showed a large inter-individual variation. Women in the soy group were divided into subgroups according to their ability to excrete more potent metabolites. Serum osteocalcin and urine deoxypyridinoline showed a tendency to increase after a challenge in equol high-excretors. Serum osteocalcin concentration in the genistein high-excretors increased significantly after a challenge (P=0.04) but did not increase in either the placebo or genistein low-excretors. An estrogenic antagonistic effect of isoflavones on bone turnover was observed in premenopausal women who are able to produce more potent metabolites.

Progress of Middle East respiratory syndrome coronavirus vaccines: a patent review.

INTRODUCTION: Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged as a new pathogen, causing severe complications and a high case fatality rate. No direct treatments are available as yet, highlighting the importance of prevention through suitable vaccination regimes. The viral spike (S) protein has been characterized as a key target antigen for vaccines. In particular, S protein domains have been utilized to produce high titers of neutralizing antibodies. Areas covered: Since the first report of MERS-CoV infection, a limited number of MERS-CoV-specific patents have been filed. Patents related to MERS-CoV are categorized into three areas: treatments, antibodies, and vaccines (receptor-related). This review mainly focuses on the types and efficacies of vaccines, briefly covering treatments and antibodies against the virus. MERS-CoV vaccine forms and delivery systems, together with comparable development strategies against SARS-CoV are additionally addressed. Expert opinion: Vaccines must be combined with delivery systems, administration routes, and adjuvants to maximize T-cell responses as well as neutralizing antibody production. High immune responses require further study in animal models, such as human receptor-expressing mice, non-human primates, and camels. Such a consideration of integrated actions should contribute to the rapid development of vaccines against MERS-CoV and related coronaviruses.

Selective Activation of Anticancer Chemotherapy by Cancer-Associated Fibroblasts in the Tumor Microenvironment.

Background: The tumor microenvironment has recently emerged as a new target of anticancer chemotherapy. Selective activation of anticancer chemotherapy in the tumor microenvironment would further reduce the toxicity of anticancer drugs toward normal tissues. Fibroblast activation protein (FAP) is known to be selectively overexpressed on cancer-associated fibroblasts (CAFs) in the tumor microenvironment. Here, we designed an anticancer chemotherapeutic system based on promelittin, a peptide toxin that is selectively converted from an inactive form to the pore-forming melittin upon cleavage by FAP in the tumor microenvironment. Methods: We conjugated promelittin-containing FAP-cleavable sequences to pegylated phospholipids and anchored them to reduced graphene oxide (rGO) nanosheets. The resulting nanosheets, PL-rGO, were tested for hemolysis and used for doxorubicin delivery. In vitro cocultures and in vivo tumor growth (n = 5 mice per group) with tissue immunostaining were used to test the selective activation of anticancer chemotherapy by FAP expressed on CAFs. Results: FAP-specific hemolytic activity of PL-rGO was observed in cocultures of CAFs and HT29 cells but not in HT29 cells alone. Doxorubicin-loaded PL-rGO (Dox/PL-rGO) showed 3.4-fold greater cell-killing efficacy (compared with free Dox in the CAF/HT29 coculture system, effects that were not observed in HT29 cells alone). Intravenously administered Dox/PL-rGO reduced the growth of HT29 tumors more effectively than other treatments (Dox/PL-rGO: mean = 200.6 mm(3), 95% confidence interval [CI] = 148.7 to 252.5 mm(3); free Dox: mean = 697.0 mm(3), 95% CI = 646.9 to 747.1 mm(3), PL: mean = 565.0 mm(3), 95% CI = 550.5 to 579.6 mm(3); Dox/rGO: mean = 637.6 mm(3), 95% CI = 619.5 to 655.7 mm(3); PL-rGO: mean = 464.4 mm(3), 95% CI = 433.0 to 495.8 mm(3)). Immunostaining of tumor tissues revealed that survival of CAFs and HT29 cells was lowest in the group treated with Dox/PL-rGO. Conclusions: The demonstration of selective activation of PL-rGO by FAP on CAFs suggests that PL-rGO may serve as a tumor microenvironment-responsive anticancer chemotherapy system.

Nanoformulation-based sequential combination cancer therapy.

Although combining two or more treatments is regarded as an indispensable approach for effectively treating cancer, the traditional cocktail-based combination therapies are seriously limited by coordination issues that fail to account for differences in the pharmacokinetics and action sites of each drug. The careful manipulation of dosing regimens, such as by the sequential application of combination treatments, may satisfy the temporal and spatial needs of each drug and achieve successful combination antitumor therapy. Nanotechnology-based carriers might be the best tools for sequential combination therapy, as they can be loaded with multiple cargos and may provide targeted and sustained delivery to target tumor cells. Single nanoformulations capable of sequentially releasing drugs have shown synergistic anticancer activity, such as by sensitizing tumor cells through cascaded drug delivery or remodeling the tumor vasculature and microenvironment to enhance the tumor distribution of nanotherapeutics. This review highlights the use of nanotechnology-based multistage drug delivery for cancer treatment, focusing on the ability of such formulations to enhance antitumor efficacy by applying sequential treatment and modulating dosing regimens, which are challenges currently being faced in the clinic.

Wiryeongtang regulates hypertonicity-induced expression of aquaporin-2 water channels in mIMCD-3 cells.

The kidneys have a key role in the homeostasis of water excretion and reabsorption. Water channels, particularly aquaporin-2 (AQP2), are important proteins in water homeostasis in the body through the short­term and long-term regulation of water permeability. Wiryeongtang (WRT) is a well-known traditional oriental medicine, which is used for the treatment of chronic edema and dysuresia. The aim of the present study was to evaluate the inhibitory effect of WRT on the hypertonicity-induced expression of AQP2 in the inner medullary collecting duct cell line (IMCD­3). Western blotting, reverse transcription­polymerase chain reaction and immunofluorescence analysis were performed to determine the effect of WRT under hypertonic stress. WRT attenuated the 175 mM NaCl hypertonic stress­induced increases in protein and mRNA levels of AQP2 and apical membrane insertion in a concentration­dependent manner. However, no differences were observed in the levels of AQP1, AQP3 or AQP4 between the hypertonic stress and WRT groups. WRT attenuated the hypertonicity-induced phosphorylation of glucocorticoid-inducible protein kinase 1. In addition, the mRNA expression of tonicity­responsive enhancer binding protein was attenuated by WRT under hypertonic stress. Pretreatment with WRT also decreased the hypertonic stress­induced expression of AQP2, as with KT5720, a protein kinase A inhibitor. These results provided evidence of the beneficial effect of the traditional formula WRT in regulating water balance in hypertonic stress of the renal collecting ducts.