Comparison of Hydrostatic Filtration Dialysis with Ultracentrifugation Methods for the Identification and Proteomic Profiling of Urinary Extracellular Vesicles.

BACKGROUND: Urinary extracellular vesicles (UEVs) carry rich markers of their parent cells, so they can serve as possible biomarkers of kidney diseases. METHODS: In this study, we isolated urinary extracellular vesicles from five individuals using a simple, clinically applicable method called hydrostatic filtration dialysis (HFD) and compared it to the gold-standard ultracentrifuga-tion (UC) with transmission electron microscopy (TEM). We also employed a proteomic approach using pooled human urine samples from the same five individuals to profile the protein composition of UEVs to evaluate the effectiveness of these two methods. RESULTS: Notably, using TEM, we found that all isolations contained 0 - 400 nm vesicles with the traditionally reported morphology, although the TEM results showed that the UEVs isolated from HFD compared to those from UC are larger and more extensive. We obtained a total of 2,564 UEV proteins in the two methods. We showed a large overlap (2,185 > 85%) between the proteins identified by both isolation methods. The result also showed that the obtained proteins in extracellular vesicles, which are isolated with these methods, are consistent with the results in currently available databases. However, in the associated gene ontologies, the enriched proteins found by the two methods showed some differences. CONCLUSIONS: The HFD method is clinically feasible and allows large-scale protein profiling of UEV biomarkers. The results of this study also provide valuable UEV protein data from the methodological comparison, which might be valuable to other researchers.

Associaton of Retinol Binding Protein 4 (RBP4) Levels With Hyperuricemia: A Cross-Sectional Study in a Chinese Population.

Background: There are few studies on predictive biomarkers for hyperuricemia, and the predictive value of these biomarkers tends to be poor. Additionally, no reports have described the predictive value of retinol binding protein 4 (RBP4) for hyperuricemia. Purpose: This study was performed to evaluate the value of RBP4 for predicting the risk of hyperuricemia in a general population, determine whether RBP4 could be used alone or in combination with other factors to predict the risk of hyperuricemia in the general population, and establish an optimum predictive model. Methods: We conducted a population-based cross-sectional survey in 2018, involving a questionnaire, physical examination, and laboratory testing. We enrolled 2303 individuals by stratified random sampling, and 2075 were included in the data analysis after applying the eligibility criteria. Results: Serum RBP4 level had a highly significant association with hyperuricemia (P<0.001). After adjusting for potential confounders, logistic regression indicated that the risk of hyperuricemia was highest in the highest RBP4 quartile (odds ratio: 7.9, 95% confidence interval [CI]: 4.18-14.84, compared to the lowest quartile). The area under the receiver operating characteristic (ROC) curve (AUC) for RBP4 was 0.749 (95% CI: 0.725-0.774, P<0.001), which was higher than that for all the other predictors assessed. The optimum model for predicting hyperuricemia in the general population consisted of RBP4, sex (male), body mass index, serum creatinine, high-sensitivity C-reactive protein, fasting blood glucose, insulin, and alcohol consumption. The AUC was 0.804 (95% CI: 0.782-0.826, P<0.001). Conclusions: RBP4 is strongly associated with hyperuricemia, and its predictive value was higher than that of traditional predictors.

Tissue Inhibitor of Matrix Metalloproteinase 2-Loaded Polylactic Acid-Polyethylene Glycol Nanoparticles Directly Regulates Renal Tubular Epithelial Cell Proliferation via Protein Tyrosine Kinase KIT.

TIMP2 has been previously reported to be associated with acute kidney injury (AKI); however, the underlying mechanism remains unclear. Therefore, the present study investigated the regulation of TIMP2 in human tubular epithelial cells HK-2 cells. Proliferation of HK-2 cells treated by TIMP2 at normal expression level was detected. GST pulldown and mass spectrometry were performed to investigate the interacting protein of TIMP2 and immunofluorescence test was used to determine the location of the protein in HK-2 cells. Cell viability as well as the expression level of CCND1, C-FOS, MAPK1 and P-MAPK1 were detected in the samples treated by overexpressed TIMP2 and the inhibitor of the interacting protein KIT. TIMP2 significantly inhibited cell proliferation compared with the control and BB-94-treated groups (P < 0.05). KIT was identified as the interacting protein of TIMP2, and was located in both the cytoplasm and membrane of HK-2 cells. Inhibited KIT and the overexpressed of TIMP2 both significantly suppressed cell proliferation and decreased the expression levels of CCND1, MAPK1, and P-MAPK1 compared with the control (P < 0.05). No significant difference was observed in cell proliferation and the expression level of aforementioned proteins between overexpressed TIMP2 and KIT-inhibited group. The results revealed that TIMP2 regulates cell proliferation by reducing the expression levels of CCND1, MAPK1, and P-MAPK1 via KIT, indicating that TIMP2 directly regulates cell proliferation without inhibiting matrix metalloproteinases in AKI. Furthermore, PLA-PEG nanoparticles successfully transported TIMP2 to the target with no significant effect on cell proliferation.

The Product of Red Blood Cells and Hematocrit Can Be Used as a Novel Indicator of Impaired Fasting Blood Glucose Status.

OBJECTIVE: To explore whether the red blood cell count multiplied by hematocrit index (RBCHct) in blood routine parameters can indicate the risk of impaired fasting blood glucose (IFG), and whether it is related to insulin resistance and inflammation. METHODS: In this cross-sectional study, previous history of diabetes was excluded, and people with normal and impaired IFG were included. We use Spearman analysis to evaluate the correlation between RBCHct index and fasting plasma glucose, insulin resistance homeostasis model assessment (HOMA-IR), and hypersensitive C-reactive protein (hs-CRP). Binary logistic regression analysis was used to evaluate the RBCHct index for assessing the potential risk of IFG, and the receiver operating characteristic (ROC) curve was used to evaluate the RBCHct index for diagnosing insulin resistance and chronic low-grade inflammatory efficacy among those with IFG. RESULTS: Correlation analysis showed that the RBCHct index and fasting plasma glucose (r=0.088, P=0.003); HOMA-IR (r=0.199, P<0.001); and hs-CRP (r=0.097, P=0.001) were positively correlated. After adjusting for confounding factors, the risk of IFG in the third and fourth quartiles of the RBCHct index increased to 1.889 and 3.048 times. The area under the ROC curve of the RBCHct index for diagnosis of insulin resistance state (HOMA-IR) was 0.695 (p<0.001), and the area under the ROC curve of the RBCHct index for the diagnosis of chronic low-inflammatory state (hs-CRP) was 0.641 (P=0.010). CONCLUSION: The RBCHct index may be a potential indicator for assessing the risk of prediabetes and is closely related to whether the body is in a state of insulin resistance and inflammation under IFG.

High-Sensitivity C-Reactive Protein Leads to Increased Incident Metabolic Syndrome in Women but Not in Men: A Five-Year Follow-Up Study in a Chinese Population.

PURPOSE: Metabolic syndrome (MetS), characterized by a constellation of insulin resistance, central obesity, hypertension, and hyperlipidemia, is a global health threat. High-sensitivity C-reactive protein (hs-CRP) has been shown to be associated with type 2 diabetes and cardiovascular disease; however, its association with incident MetS is less known. Therefore, the aim of this study was to examine the prospective association between hs-CRP and MetS among a Chinese population in a 5-year follow-up study. PATIENTS AND METHODS: The levels of hs-CRP were measured using serum samples collected at baseline recruitment in 2012 from 886 participants without MetS. Follow-up interviews were conducted in 2018, and MetS was diagnosed by 2017 criteria from the Chinese Diabetes Society. Multivariate logistic regression models were used to assess the overall and sex-specific associations between hs-CRP and incident MetS. The odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were computed with adjustment for demographic, socioeconomic, clinical, and lifestyle factors. RESULTS: After a mean follow-up duration of 5.40 ± 0.56 years, 116 (13.3%) participants developed MetS. In the total study population, increased hs-CRP levels were associated with a higher risk of MetS (OR comparing extreme quartiles of hs-CRP: 4.06 [95% CI: 1.91-8.65]) in the fully-adjusted model. When stratified by sex, the positive association was only observed in women (OR: 4.82 [1.89-12.3]) but not in men (OR: 3.15 [0.82-12.1]; P-interaction = 0.039). CONCLUSION: In this study of a Chinese population, a positive association between hs-CRP and incident MetS was found only in women and not in men. Sex-specific prediction and intervention of MetS using hs-CRP as a target should be further evaluated.

Association of Lipid Parameters with the Risk of Chronic Kidney Disease: A Longitudinal Study Based on Populations in Southern China.

OBJECTIVE: To investigate which plasma lipid parameters are useful for detecting chronic kidney disease (CKD) in a Chinese population without known CKD or renal impairment. METHODS: This was a prospective study. In southern Chinese cities from 2012 to 2013, a total of 1037 subjects aged ≥ 18 years old received a survey. Logistic regression and multiple linear regression analyses were performed. The lipid parameters studied included total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), non-high-density lipoprotein cholesterol (nHDL-C), TG/HDL-C ratio, TC/HDL-C ratio and nHDL-C/HDL-C ratio. RESULTS: After adjusting for confounding factors, the fourth percentile of logTG/HDL-C was observed to be an independent risk factor for CKD (OR = 2.453, P < 0.001), and the highest quantile of the logTG/HDL-C ratio was associated with a higher prevalence of CKD (P < 0.05). This risk was reduced when the model was adjusted with Insulin resistance (IR) (OR = 2.034, P < 0.05). In the group of women, glucose metabolism disorders, high uric acid, and obesity, this risk was increased. Multiple regression models showed that log TG and nonHDL-C/HDL-C were negatively correlated with eGFR (P < 0.05), while log TG and TC were positively correlated with logACR (P < 0.05). The area under the curve (ROC) of lgTG/HDL was 0.623 (p < 0.001). CONCLUSION: The serum logTG/HDL-C ratio is the only suitable predictor of CKD, and IR may be the mechanism. This risk needs to be controlled in a specific population. Log TG and nonHDL-C/HDL-C were negatively correlated with eGFR, while log TG and TC were positively correlated with logACR.

Visceral Adiposity Index and Chronic Kidney Disease in a Non-Diabetic Population: A Cross-Sectional Study.

PURPOSE: To investigate the correlation between visceral obesity and pathogenesis of chronic kidney disease (CKD) among non-diabetic individuals, and to evaluate the potential of visceral adiposity index (VAI) as a predictor of CKD. PATIENTS AND METHODS: From December 2017 to March 2018, 1877 non-diabetic participants (male n=699, female n=1208) in southern China were recruited for a cross-sectional survey. Males and females were divided into four groups according to gender-specific quartiles of VAI scores. A logistic regression model was established to analyze the correlation between visceral adiposity index and CKD. RESULTS: Visceral adiposity index was positively correlated with CKD and was negatively associated with estimated glomerular filtration rate (eGFR). Using group one as the control, odds ratios (ORs) were calculated to determine the risk of developing CKD as VAI increased (male: group four 2.73 [P<0.005]; female: Group three 1.76 [P<0.05], Group four 2.88 [P<0.005]). When related factors such as history of hypertension, smoking, alcohol use, and physical inactivity were normalized in the logistic model before calculation, ORs became 2.73 (male: P<0.05), and 2.18 (female: P<0.05), respectively. The results differed after normalizing further for systolic blood pressure (SBP), diastolic blood pressure (DBP), hypersensitive c-reactive protein (hsCRP), interleukin-6 (IL-6), homocysteine (Hcy), superoxide dismutase (SOD), and retinol-binding protein (RBP). There were no significant differences in ORs among the female groups. CONCLUSION: Visceral adiposity index was significantly associated with CKD in non-diabetic individuals. It may be a good predictor of the pathogenesis of CKD and was dependent on hsCRP, IL-6, Hcy, SOD, RBP, and blood pressure levels in females and males with VAI scores of 1.41 and higher. Visceral adiposity index may be used to predict CKD in males with VAI less than 0.983.

Lymphocyte To High-Density Lipoprotein Ratio As A New Indicator Of Inflammation And Metabolic Syndrome.

PURPOSE: Metabolic syndrome (MetS), which is a global public health problem, is a state of chronic low-grade inflammation. This study looked at the changes in hematological parameters and the predictive value of the lymphocyte to high-density lipoprotein cholesterol (HDL-C) ratio (LHR) as a new index in subjects with and without MetS in coastal cities in southern China. PATIENTS AND METHODS: In this cross-sectional study, there were 852 participants (n = 598 with MetS and n = 254 without MetS). MetS was defined in accordance with the National Cholesterol Education Program, Adult Treatment Panel III (NCEP-ATP III) criteria. RESULTS: MetS was positively correlated with white blood cell count, total lymphocyte count, neutrophil count, red blood cell count, hematocrit, hemoglobin, and high-sensitivity C-reactive protein levels (p<0.05). In addition, there was a positive correlation between LHR and the number of metabolic risk factors for MetS. In a logistic regression analysis, LHR (odds ratio: 4.117; 95% CI: 2.766-6.309; p<0.001) was an independent predictor of MetS. When a receiver operating characteristic (ROC) curve analysis was used to assess the value of LHR for predicting MetS, the area under the curve yielded a cut-off value of 1.657, with a sensitivity of 65% and a specificity of 64% (p<0.0001). CONCLUSION: In summary, MetS can involve changes in blood parameters, and LHR may be a useful marker of inflammation to assess the presence and severity of MetS.

Preparation of colloidal gold immunochromatography strip for detection of methamidophos residue.

Methamidophos (Met) is a broad spectrum organophosphorus insecticide and acaricide. Even a trace of its residue is harmful to humans and many animals. In this study, the synthesis and identification of colloidal gold particles and antibody-colloidal gold conjugates were performed, and the preparation of colloidal gold immunochromatography strip was conducted for detection of Met residue. The size of colloidal gold particles was checked using a transmission electron microscope (TEM). The formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. The preparation of colloidal gold immunochromatography strips, analysis of Met standard solutions, and other four pesticides and vegetable samples were operated with common methods and principals. The TEM images showed the average diameter of colloidal gold particles was almost the same size: approximately 40.0 nm in diameter. For the conjugation of colloidal gold and monoclonal antibody (MAb), 0.03 mg/ml of MAb was confirmed to be the minimum amount for stabilization of colloidal gold. With the prepared colloidal gold immunochromatography test strip to determine the standard Met solution, the results demonstrated a detection limit of approximately 1.0 mg/ml. Cross-reaction indicated that the strip had a high specificity to Met. The results of 10 green vegetable sample tests confirmed that one sample was positive by HPLC analysis. There was evidence to suggest that colloidal gold particles and antibody-colloidal gold conjugates were synthesized successfully. The prepared colloidal gold immunochromatography strip was applicable for preliminary screening of Met residue.

[Investigation on food contamination with parasites in Shanghai market].

OBJECTIVE: To understand the contamination status of food with parasites in Shanghai market, so as to provide the evidence for formulating the surveillance strategy in parasitic diseases and provide the technical support in the food safety. METHODS: The freshwater fish products, marine products, vegetables, snails and frogs were sampled with the cluster random method in the farmer-trades and supermarkets of the 18 districts in Shanghai City during 2005-2010 period, and all the specimen were screened by the digestion method, or crushing method or dissection method or saline floatation method to check the parasite metacercaria or larvae or eggs. RESULTS: A total of 5 185 specimen in 23 species of fishes were screened in fresh-water products, with parasite infection rate of 1.93%. About 4 033 specimens of 20 species of fishes and shrimps were screened and 1.76% of samples were infected with Clonorchis sinensis. Among all kind of fishes, the highest infection rate was 7.83% (48/613) in Pseudorasbora parve. No any infected specimen was found among 1 152 specimen of fresh water crustacean screened. Anisakis spp. were found in 12.7% of 433 specimens of 23 species of seawater products, among them, the higher infection rate of Anisakis spp. was found in Pneumatophorus japonicas and Trichiurus haumela with their infection rates of 50.00% (13/26) and 23.46% (42/179), respectively, which much higher than those found in other seawater products (P < 0.01). In 37 kinds of vegetables, the parasite eggs were found in one of 428 specimens with its infection rate of 0.47%, while no any parasite egg was found in 103 specimens of 10 kinds of fruits. No any Angiostrongylus cantonensis larvae were found in 330 snails, 31.37% of 102 frogs were found infected with Spirometra mansoni spargana. No any contamination with parasites was found in 116 meat specimens of pigs and cattle. In the same time, the intestinal parasite infection rate of residents was 0.42% (131/31 239). CONCLUSIONS: It is found that some of foods in Shanghai markets are contaminated with parasites. Therefore, it is necessary to enforce the activities in health education as well as to take integrated prevention measures in order to ensure the food safety.

[The effects of TCM Yishen Decoction on the expression of MMP-9 and TIMP-1 in kidney of mouse with IgA nephropathy].

AIM: To explore effects of Yishen Decoction on the expression of MMP-9 and TIMP-1 in kidney of mice with IgA nephropathy (IgAN). METHODS: The IgAN model was established by oral administration of bovine serum albumin (BSA) together with the injection of Staphylococcus enterotoxin B (SEB) through caudal vein. The mRNA and protein expression of MMP-9 and TIMP-1 were measured by fluorescent quantitative RT-PCR and immunohistochemistry respectively. RESULTS: No significant differences of mRNA and protein expression of MMP-9 and TIMP-1 were found between the low and high concentration Yishen Decoction treated group. But the expression of TIMP-1 in the Yishen Decoction treated group was lower than that in the IgAN group, while that of MMP-9 was higher than that in the control group. CONCLUSION: The TCM Yishen Decoction can inhibit the abnormal expression of TIMP-1, and strengthen the expression of MMP-9 in kidney of mouse with IgA nephropathy.