Na+/H+ Exchanger Regulatory Factor 3 Is Critical for Multidrug Resistance Protein 4–Mediated Drug Efflux in the Kidney

Na⁺/H⁺ exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3^−/−) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3^−/− mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.Assembly of protein complexes by adaptor proteins with PSD-95/discs large/ZO-1 (PDZ) domains plays an important role in the regulation of many membrane proteins, especially in epithelial and neuronal tissues.^1,2 For example, PDZ-based adapter proteins in epithelia, such as Shank2, synaptic scaffolding molecule (S-SCAM), and Na⁺/H⁺ exchanger regulatory factors (NHERFs), are known to be involved in the regulation of cell surface expression and the activity of many membrane transporters and receptors in the respiratory, digestive, urinary, and reproductive organs.^3–6 The four members of the NHERF proteins (NHERF1 to 4) contain either two or four PDZ domains and were the first family of PDZ-containing proteins shown to be involved in epithelial transport. NHERF3, also known as PDZK1 or CAP70, has four PDZ domains and is normally localized to the apical microdomains of gastrointestinal and kidney epithelia. Each PDZ domain of NHERF3 has been proposed to bind independently to the PDZ binding motif of various membrane proteins, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the Na⁺/H⁺ exchanger 3, the urate transporter, and the organic anion transporter 4.^7–10 However, Nherf3 knockout (Nherf3^−/−) mice do not have a discernible phenotype except mild hypercholesterolemia,¹¹ and, consequently, the in vivo role of NHERF3 in transepithelial transport remains poorly understood.Transporters belonging to either the ATP binding cassette (ABC) or the solute-linked carrier superfamilies of membrane proteins play diverse roles in the pharmacokinetic and pharmacodynamic pathways of drugs and their metabolites.¹² Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the C subfamily of ABC transporters and mediates the efflux of a group of organic molecules using energy generated from the binding and hydrolysis of ATP.¹³ Cumulative evidence suggests that MRP4 pumps out purine compounds and plays an important role in the renal elimination of purine-based antiviral and antineoplastic agents.^14,15 However, the regulatory mechanisms for MRP4 expression and function have not been extensively studied.The COOH terminus of MRP4 contains a class 1 PDZ interaction motif (-S/T-X-Ф, where Ф is a hydrophobic amino acid),¹⁶ indicating that MRP4 may interact with PDZ-based adaptors in epithelia. In preliminary studies using a yeast two-hybrid assay, MRP4 was found to strongly interact with NHERF3 among several PDZ adaptor proteins expressed in epithelial tissues. The aim of the present study was to identify the physiologic and pharmacologic roles of the interaction of NHERF3 with MRP4 using a variety of in vitro and in vivo experimental approaches. The results obtained provide strong evidence that NHERF3 is a key regulator of organic transport in the kidney, particularly the MRP4-mediated clearance of purine-based drug molecules.     

Static Langer's line and wound contraction rates according to anatomical regions in a porcine model

This study investigated wound contraction rates according to anatomical regions and wound morphology according to skin tension line in a micropig porcine model. Of the four animals used, skin tension morphology was determined in one pig. In the remaining three pigs, six pairs of full‐thickness skin excisions were created on the dorsum (six square and six circular). The wounds were grouped, Wounds #1 through #5, according to the skin tension line and anatomical regions: Wounds #1 and #2, cephalic; Wounds #4 and #5, caudal; and Wound #3, center. Wound sizes and contraction rates were calculated for 28 days. A static tension topography of the micropig dorsum was obtained. Excisional wounds deformed along the local tension vector and healed in this fashion. Wound contraction rates were significantly higher for cephalic wounds (p = 0.004). No significant difference in wound contraction rates were observed between square and circular wounds. Final wound morphology was related to the local tension vector and initial wound shape. Cephalic wounds contracted more quickly. Further studies are needed to characterize scar formation after primarily closed surgical wounds in relation to the newly established skin tension topography and to elucidate the mechanism behind the variable wound contraction rates in the cephalocaudal gradient.     

Malignancy Risk Stratification in Thyroid Nodules with Benign Results on Cytology: Combination of Thyroid Imaging Reporting and Data System and Bethesda System

Background

The indications of repeat fine-needle aspiration (FNA) for thyroid nodules with benign results of the Bethesda system were investigated.

Methods

A total of 1,398 nodules were classified according to the Thyroid Imaging Reporting and Data System (TIRADS). TIRADS category 3 included nodules without solidity, hypoechogenicity or marked hypoechogenicity, microlobulated or irregular margins, microcalcifications, and taller-than-wide shape on ultrasonography (US). Categories 4a, 4b, 4c, and 5 included nodules with one, two, three or four, or five suspicious US features, respectively. The malignancy risks, and odds ratio (OR) with 95 % confidence interval (CI) were calculated. Analyses were performed for all nodules, nodules >10 mm, and nodules ≤10 mm.

Results

Of 1.398 nodules, 43 (3.1 %) were malignanct. The malignancy risks of benign nodules with categories 3, 4a, and 4b were 0.7, 1.2, and 0.7 %, respectively, whereas those for nodules with categories 4c and 5 were 9.8 and 22.2 %, respectively. The ORs of nodules with categories 4c and 5 were 19.4 (95 % CI 5.0–76.2) and 50.6 (95 % CI 10.4–245.0), respectively. In nodules >10 mm, the malignancy risks of categories 4c and 5 were 2.7 and 20 %, respectively, and the ORs were 10.7 (95 % CI 1.2–93.7) and 236.1 (95 % CI 12.6–4426.4), respectively. In nodules ≤ 10 mm, the malignancy risks of categories 4c and 5 were 12.6 and 22.6 %, respectively, and the ORs were 10.1 (95 % CI 1.3–78.0) and 18.9 (95 % CI 2.1–168.9), respectively.

Conclusions

Repeat US-guided FNA should be considered in benign thyroid nodules with three or more suspicious US features regardless of size.     

External Validation of Online Predictive Models for Prediction of Cancer-specific Mortality and All-cause Mortality in Patients with Urothelial Carcinoma of the Urinary Bladder

Purpose

The objective of the study was to validate the previously reported lookup Table and Bladder Cancer Research Consortium (BCRC) nomogram in predicting cancer-specific mortality (CSM) and all-cause mortality (ACM) after radical cystectomy using an external cohort from South Korea.

Methods

The study comprised 409 patients. Discrimination was quantified with the concordance index. The relationship between the model-derived and actual CSM and ACM was graphically explored within calibration plots. Clinical net benefit was evaluated by decision curve analysis.

Results

Of the 409 patients, 147 (35.9 %) had died from various causes. One hundred two deaths were attributable to bladder cancer. For CSM at 5 years, the bootstrap-corrected concordance indices of the American Joint Committee on Cancer (AJCC) staging system, lookup Table, and BCRC nomogram were 71.8 % (95 % confidence interval [CI] 66.9–76.5), 73.0 % (95 % CI 67.9–78.0), and 76.2 % (95 % CI 71.6–80.9), respectively. For ACM at the same time point, the discrimination accuracies of these models were 70.7 % (95 % CI 66.7–74.6), 72.8 % (95 % CI 68.5–76.9), and 76.2 % (95 % CI 72.3–80.2), respectively. The calibration plots tended to exaggerate both survival outcomes in all models. When compared to the lookup Table as well as the AJCC staging system, the BCRC nomogram performed well across a wide range of threshold probabilities using decision curve analysis.

Conclusions

The BCRC nomogram was characterized by higher accuracy and larger potential clinical benefit compared to the lookup Table. However, there is a great need for additional models that consider outcomes of patients for whom the existing models do not apply.     

Interaction of citrate‐coated silver nanoparticles with earthworm coelomic fluid and related cytotoxicity in Eisenia andrei

Understanding the interaction of nanoparticles with biological fluid is important for predicting the behavior and toxicity of nanoparticles in living systems. The earthworm Eisenia andrei was exposed to citrate‐coated silver nanoparticles (cAgNPs), and the interaction of cAgNPs with earthworm coelomic fluid (ECF), the cytotoxicity of cAgNPs in earthworm coelomocytes was assessed. The neutral red retention assay showed a reduction in lysosomal stability after exposure. The toxicity of silver ions dissolved from cAgNPs in the soil medium was not significant. The aggregation and dissolution of cAgNPs increased in ECF, which contains various electrolytes that alter the properties of nanoparticles, and their subsequent toxicity. Microscopic and dissolution studies demonstrated that the aggregation of cAgNPs rapidly increased, and readily dissolved in ECF. The bioavailability of cAgNPs to earthworms induced lysosomal cytotoxicity. This is the first report to test the interaction and lysosomal cytotoxicity of nanoparticles in earthworm biofluids. Copyright © 2014 John Wiley & Sons, Ltd.     

The ingredients in Saengshik,a formulated health food,inhibited the activity of α-amylase and α-glucosidase as anti-diabetic function

BACKGROUND/OBJECTIVES

We investigated total 26 ingredients of Saengshik which will be commercially produced as an anti-diabetic dietary supplement.

SUBJECTS/METHODS

Thirteen vegetables, nine cereals, three legumes and one seed were extracted with aqueous ethanol for 2 h at 60℃, and evaluated for their inhibitory effects against α-amylase and α-glucosidase and for total phenolic and flavonoid contents.

RESULTS

All ingredients inhibited α-amylase activity except cabbage. Strong inhibitory activity of α-amylase was observed in leek, black rice, angelica and barley compared with acarbose as a positive control. Stronger inhibition of α-glucosidase activity was found in small water dropwort, radish leaves, sorghum and cabbage than acarbose. All Saengshik ingredients suppressed α-glucosidase activity in the range of 0.3-60.5%. Most ingredients contained total phenols which were in the range of 1.2-229.4 mg gallic acid equivalent/g dried extract. But, total phenolic contents were not observed in carrot, pumpkin and radish. All ingredients contained flavonoid in the range of 11.6-380.7 mg catechin equivalent/g dried extract.

CONCLUSIONS

Our results demonstrate that Saengshik containing these ingredients would be an effective dietary supplement for diabetes.     

Deep Neural Network-Based Prediction of the Risk of Advanced Colorectal Neoplasia

Background/AimsRisk prediction models using a deep neural network (DNN) have not been reported to predict the risk of advanced colorectal neoplasia (ACRN). The aim of this study was to compare DNN models with simple clinical score models to predict the risk of ACRN in colorectal cancer screening.MethodsDatabases of screening colonoscopy from Kangbuk Samsung Hospital (n=121,794) and Kyung Hee University Hospital at Gangdong (n=3,728) were used to develop DNN-based prediction models. Two DNN models, the Asian-Pacific Colorectal Screening (APCS) model and the Korean Colorectal Screening (KCS) model, were developed and compared with two simple score models using logistic regression methods to predict the risk of ACRN. The areas under the receiver operating characteristic curves (AUCs) of the models were compared in internal and external validation databases.ResultsIn the internal validation set, the AUCs of DNN model 1 and the APCS score model were 0.713 and 0.662 (p<0.001), respectively, and the AUCs of DNN model 2 and the KCS score model were 0.730 and 0.667 (p<0.001), respectively. However, in the external validation set, the prediction performances were not significantly different between the two DNN models and the corresponding APCS and KCS score models (both p>0.1).ConclusionsSimple score models for the risk prediction of ACRN are as useful as DNN-based models when input variables are limited. However, further studies on this issue are warranted to predict the risk of ACRN in colorectal cancer screening because DNN-based models are currently under improvement.     

Characterization of PLA/PBSeT Blends Prepared with Various Hexamethylene Diisocyanate Contents

Poly (lactic acid) (PLA) is the most widely available commercial bioplastic that is used in various medical and packaging applications and three-dimensional filaments. However, because neat PLA is brittle, it conventionally has been blended with ductile polymers and plasticizers. In this study, PLA was blended with the high-ductility biopolymer poly (butylene-sebacate–co–terephthalate) (PBSeT), and hexamethylene diisocyanate (HDI) was applied as a crosslinking compatibilizer to increase the miscibility between the two polymers. PLA (80%) and PBSeT (20%) were combined with various HDI contents in the range 0.1–1.0 parts-per-hundred rubber (phr) to prepare blends, and the resulting physical, thermal, and hydrolysis properties were analyzed. Fourier-transform infrared analysis confirmed that –NH–C=OO⁻ bonds had formed between the HDI and the other polymers and that the chemical bonding had influenced the thermal behavior. All the HDI-treated specimens showed tensile strengths and elongations higher than those of the control. In particular, the 0.3-phr-HDI specimen showed the highest elongation (exceeding 150%) and tensile strength. In addition, all the specimens were hydrolyzed under alkaline conditions, and all the HDI-treated specimens degraded faster than the neat PLA one.