N-acetyl-β-D-glucosaminidase Research Articles

Sensitive Electrochemiluminescence Immunosensor for Detection of N-Acetyl-β-d-glucosaminidase Based on a "Light-Switch" Molecule Combined with DNA Dendrimer.

Here, a novel "light-switch" molecule of Ru (II) complex ([Ru(dcbpy)2dppz](2+)-DPEA) with self-enhanced electrochemiluminescence (ECL) property is proposed, which is almost nonemissive in aqueous solution but is brightly luminescent when it intercalates into DNA duplex. Owing to less energy loss and shorter electron-transfer distance, the intramolecular ECL reaction between the luminescent [Ru(dcbpy)2dppz](2+) and coreactive tertiary amine group in N,N-diisopropylethylenediamine (DPEA) makes the obtained "light-switch" molecule possess much higher light-switch efficiency compared with the traditional "light-switch" molecule. For increasing the loading amount and further enhancing the luminous efficiency of the "light-switch" molecule, biotin labeled DNA dendrimer (the fourth generation, G4) is prepared from Y-shape DNA by a step-by-step assembly strategy, which provides abundant intercalated sites for [Ru(dcbpy)2dppz](2+)-DPEA. Meanwhile, the obtained nanocomposite (G4-[Ru(dcbpy)2dppz](2+)-DPEA) could well bind with streptavidin labeled detection antibody (SA-Ab2) due to the existence of abundant biotin. Through sandwiched immunoreaction, an ECL immunosensor was fabricated for sensitive determination of N-acetyl-β-d-glucosaminidase (NAG), a typical biomarker for diabetic nephropathy (DN). The detemination linear range was 0.1 pg mL(-1) to 1 ng mL(-1), and the detection limit was 0.028 pg mL(-1). The developed strategy combining the ECL self-enhanced "light-switch" molecular and DNA nanotechnology offers an effective signal amplification mean and provides ample potential for further bioanalysis and clinical study.

Estimation of the reference level of urinary cadmium by using hybrid method in cadmium-polluted and non-polluted areas in China

ABSTRACTThis study investigated the reference level of urinary cadmium (UCd) of two Cd-polluted areas and one non-polluted area in China by applying the updated hybrid approach. Urinary N-acetyl-β-d-glucosaminidase (NAG) and β-2-microglobulin (β2MG) were considered as indicators of early renal dysfunction. UCd was regarded as an indicator of the total internal Cd exposure. We recruited 3285 inhabitants for this investigation. Among them, 2106 were from polluted areas, while the other 1179 were from non-polluted areas. BMDL10 (the lower 95% confidence limit of the benchmark dose (BMD)) of UCd for NAG was 1.55 μg/g creatine in men and 1.59 μg/g creatine in women. Using β2MG as the renal effect marker, BMDL10 of UCd was 1.58 μg/g and 1.53 μg/g in men and women, respectively. Our BMDL estimates accounting for age were lower than those reported in previous studies, suggesting that policies to control Cd exposure are warranted.

Evaluation of novel biomarkers of nephrotoxicity in Cynomolgus monkeys treated with gentamicin

Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days −7, −3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-β-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.

Seasonal patterns of nitrogen cycling in subtropical short-hydroperiod wetlands: Effects of precipitation and restoration

In the event of increased frequency of extreme wet or dry events resulting from climate change, it becomes more important to understand the temporal dynamics of soil nitrogen (N) processes in ecosystems. Here, seasonal patterns of N cycling were characterized in subtropical wetlands in Everglades National Park, Florida, USA. Two restored sites and one reference site with different nutrient status, soil depth, and vegetation communities, were selected. Soil available N, microbial biomass, potential N mineralization and denitrification rates, enzyme activities of leucine aminopeptidase (LAP) and N-acetyl-β-d-glucosaminidase (NAG) were measured across the wet and dry seasons from 2010 to 2011. In general, most N processes were significantly correlated with soil water contents (P<0.05) which reflected the precipitation regime. The lower elevation and shallower soil (2–3cm depth) at the restored site may contribute to their higher soil water contents compared to the reference site with ~10cm soil depth, which further led to the earlier peaks of microbial biomass at the two restored sites. Potential N mineralization was positively correlated with LAP at the restored sites whereas with NAG at the reference site (P<0.05), implying that different vegetation composition may provide varying substrates for soil microbes. The build-up of nitrate in the dry spring of 2011 induced a pulse of denitrification after rewetting by a sudden rainfall, implying the presence of a hot moment of denitrification during the dry-rewetting transition period. The decrease of MBC:MBN ratio from dry to wet season indicates a possible microbial composition shift from fungi to bacteria, shedding lights on the potential contribution of fugal groups to denitrification in the dry season. Our study highlight that even under the same climate regime, the small-scale variations could affect the seasonal patterns of N cycling.

Effect of clofibrate on fatty acid metabolism in the kidney of puromycin-induced nephrotic rats.

Proteinuria plays an essential role in the progression of tubulointerstitial damage, which causes end-stage renal disease. An increased load of fatty acids bound to albumin reabsorbed into proximal tubular epithelial cells (PTECs) contributes to tubulointerstitial damage. Fibrates, agonists of peroxisome proliferator-activated receptor α (PPARα), have renoprotective effects against proteinuria whereas the effects of these compounds on fatty acid metabolism in the kidney are still unknown. Therefore, the present study examined whether the renoprotective effects of clofibrate were associated with improvement of fatty acid metabolism in puromycin aminonucleoside (PAN)-induced nephrotic rats. Rats were allocated to the control, PAN or clofibrate-treated PAN group. Biochemical parameters, renal injury and changes in fatty acid metabolism were studied on day14. PAN increased proteinuria, lipid accumulation in PTECs, excretions of N-acetyl-β-D-glucosaminidase (NAG) and 8-hydroxydeoxyguanosine (8OHdG) and the area of caspase 3-positive tubular cells. It decreased renal expressions of medium-chain acyl-CoA dehydrogenase (MCAD), cytochrome P450 (CYP)4A, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα) without change of the expression of PPARα. Clofibrate reduced proteinuria, lipid accumulation, NAG excretion and the area of caspase 3-positive tubular cells. However, albumin excretion was not reduced and 8OHdG excretion was increased. Clofibrate minimized changes in MCAD, CYP4A, PGC-1α and ERRα expressions with increased PPARα, very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain acyl-CoA dehydrogenase (LCAD) expressions. Clofibrate is protective against renal lipotoxicity in PAN nephrosis. This study indicates that clofibrate has renoprotective effects through maintaining fatty acid metabolism in the kidney of PAN-induced nephrotic rats.

The Toxicity of Aluminum Chloride on Kidney of Rats.

This study investigated the toxicity of aluminum chloride (AlCl3) exposure in the rat kidney. Forty male Wistar rats (5weeks old), weighing 110-120g, were randomly divided into four groups: control group (CG, 0g/L AlCl3), low dose group (LG, 0.4g/L AlCl3), mid dose group (MG, 0.8g/L AlCl3), and high dose group (HG, 1.6g/L AlCl3). Rats were administered AlCl3 in their drinking water for 120days. A variety of measurements were taken including superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities, malondialdehyde (MDA) concentration in the kidney and blood urea nitrogen (BUN), and cystatin C (Cys-C) concentrations in the serum. In addition, Al and β2-microglobulin (β2-MG) concentrations and the activity of N-acetyl-β-D-glucosaminidase (NAG) in the urine were determined. The results showed that in the AlCl3-treated groups SOD and GSH-PX activities were decreased, while NAG activity and Al, MDA, BUN, Cys-C, and β2-MG concentrations were increased, compared with the CG. This study indicates that AlCl3 exposure induces oxidative stress and suppresses kidney function.

Risk Assessment Study of Fluoride Salts: Probability-Impact Matrix of Renal and Hepatic Toxicity Markers.

The present risk assessment study of fluoride salts was conducted by oral administration of three different doses of sodium and potassium fluorides (NaF, KF) and zinc fluoride tetrahydrate (ZnF2 •4H2O) to male Wistar rats. The rats were divided into control and nine experimental groups, to which oral injections of 0.5mL distilled water and 0.5mL of fluoride solutions, respectively, were given. The dosage of fluoride compounds was adjusted to contain 2.1mg (low-dose group, LG), 4.3mg (mid-dose group, MG), and 5.4 mg fluoride per 200g rat body weight (high-dose group, HG) corresponding to 5, 10, and 12.5% of LD50 values for NaF. The 24-h urine volume, N-acetyl-β-D-glucosaminidase (NAG) and creatinine clearance (Ccr) were measured as markers of possible acute renal impact. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined in serum samples as markers of acute hepatic impact. The levels of serum and urinary fluoride were determined to evaluate fluoride bioavailability. The results reveal that higher doses of NaF, KF, and ZnF2 induced renal damage as indicated by higher urinary NAG (p<0.05 with ≥90th percentile of control). High doses of ZnF2 also induced a significant Ccr decrease (p<0.05 with ≤10th percentile of control). Low doses of NaF and mid-doses of ZnF2 induced polyuria (p<0.05 with ≥90th percentile of control) while medium doses of NaF and low doses of KF also induced liver damage, as indicated by a high level of AST (p<0.05 with ≥90th percentile of control). These findings suggest that oral administration of fluoride is a potential, dose-dependent risk factor of renal tubular damage.

Associations among environmental exposure to manganese, neuropsychological performance, oxidative damage and kidney biomarkers in children

Environmental exposure to manganese (Mn) results in several toxic effects, mainly neurotoxicity. This study investigated associations among Mn exposure, neuropsychological performance, biomarkers of oxidative damage and early kidney dysfunction in children aged 6–12 years old. Sixty-three children were enrolled in this study, being 43 from a rural area and 20 from an urban area. Manganese was quantified in blood (B-Mn), hair (H-Mn) and drinking water using inductively coupled plasma mass spectrometry (ICP-MS). The neuropsychological functions assessed were attention, perception, working memory, phonological awareness and executive functions – inhibition. The Intelligence quotient (IQ) was also evaluated. The biomarkers malondialdehyde (MDA), protein carbonyls (PCO), δ-aminolevulinate dehydratase (ALA-D), reactivation indexes with dithiothreitol (ALA-RE/DTT) and ZnCl2 (ALA-RE/ZnCl2), non-protein thiol groups, as well as microalbuminuria (mALB) level and N-acetyl-β-D-glucosaminidase (NAG) activity were assessed. The results demonstrated that Mn levels in blood, hair and drinking water were higher in rural children than in urban children (p<0.01). Adjusted for potential confounding factors, IQ, age, gender and parents' education, significant associations were observed mainly between B-Mn and visual attention (β=0.649; p<0.001). Moreover, B-Mn was negatively associated with visual perception and phonological awareness. H-Mn was inversely associated with working memory, and Mn levels from drinking water with written language and executive functions – inhibition. Rural children showed a significant increase in oxidative damage to proteins and lipids, as well as alteration in kidney function biomarkers (p<0.05). Moreover, significant associations were found between B-Mn, H-Mn and Mn levels in drinking water and biomarkers of oxidative damage and kidney function, besides between some oxidative stress biomarkers and neuropsychological tasks (p<0.05). The findings of this study suggest an important association between environmental exposure to Mn and toxic effects on neuropsychological function, oxidative damage and kidney function in children.

Associations of Low Environmental Exposure to Multiple Metals with Renal Tubular Impairment in Korean Adults.

Recently several studies reported that the renal toxicity of lead (Pb) and cadmium (Cd) may exist in even a low level exposure. In terms of the deterioration of tubular function, it affects the loss of divalent metals and leads to other complications, so renal tubular effect of heavy metals should be well managed. Considering the exposure to heavy metals in reality, it is hard to find the case that human is exposed to only one heavy metal. We designed a cross-sectional study using Korean Research Project on the Integrated Exposure Assessment (KRIEFS) data to investigate the renal effects of multiple metal exposure in general population. We used blood Pb and urinary Cd as exposure measures, and urinary N-acetyl-β-D-glucosaminidase (NAG) and β2-microglobulin (β2-MG) as renal tubular impairment outcome. We conducted linear regression to identify the association between each heavy metal and urinary NAG and β2-MG. And then, we conducted linear regression including the interaction term. Of 1953 adults in KRIEFS (2010~2011), the geometric mean of blood Pb and urinary Cd concentration was 2.21 μg/dL (geometric SD = 1.49 μg/dL) and 1.08 μg/g cr (geometric SD = 1.98 μg/g cr), respectively. In urinary Cd, the strength of the association was also high after adjusting (urinary NAG: β = 0.44, p < 0.001; urinary β2-MG: β = 0.13, p = 0.002). Finally, we identified the positive interactions for the two renal biomarkers. The interaction effect of the two heavy metals of β2-MG was greater than that of NAG. It is very important in public health perspective if the low level exposure to multiple heavy metals has an interaction effect on kidney. More epidemiological studies for the interaction and toxicological studies on the mechanism are needed.

Self-Enhanced Electrochemiluminescence Nanorods of Tris(bipyridine) Ruthenium(II) Derivative and Its Sensing Application for Detection of N-Acetyl-β-d-glucosaminidase.

A self-enhanced electrochemiluminescence (ECL) reagent, synthesized by covalently linking bis(2,2'-bipyridyl)(4'-methyl-[2,2']bipyridinyl-4-carboxylicacid) ruthenium(II) (Ru(bpy)2(mcbpy)(2+)) with tris(3-aminopropyl)amine (TAPA), has been chosen as precursor to prepare nanorods ([Ru(bpy)2(mcbpy)(2+)-TAPA]NRs) with high luminous efficiency via a solvent-evaporation-induced self-assembly procedure. Due to the shorter electron-transfer path and less energy loss, the intramolecular reaction between the luminescent Ru(bpy)2(mcbpy)(2+) and coreactive tertiary amine group in TAPA has shown improved luminous efficiency compared with the common intermolecular ECL reactions. Moreover, using the electrochemiluminescent Ru(II)-based complex as precursor to directly prepare a nanostructure with high electro-active surface area is a more effective and convenient method for enhancing the immobilized amount of Ru(II)-based complex in the construction of biosensors compared with the traditional immobilized methods. Meanwhile, the obtained nanorods could be further functionalized easily, owing to their positive electrical property and the amino group on the surface. Here, Pt nanoparticles functionalized [Ru(bpy)2(mcbpy)(2+)-TAPA]NRs are used to load the detection antibody (Ab2). In addition, the Au/Pd dendrimers (DRs) with hierarchically branched structures are synthesized to immobilize capture antibody (Ab1) with increased amount. Based on sandwiched immunoreactions, a simple and sensitive "signal-on" immunosensor is constructed for the detection of N-acetyl-β-d-glucosaminidase (NAG), a biomarker of diabetic nephropathy, with excellent linearity in concentrations from 1 ng mL(-1) to 0.5 pg mL(-1) and a detection limit of 0.17 pg mL(-1).

Prognostic Value of Tubulointerstitial Lesions, Urinary N-Acetyl-β-d-Glucosaminidase, and Urinary β2-Microglobulin in Patients with Type 2 Diabetes and Biopsy-Proven Diabetic Nephropathy.

Some biomarkers of renal tubular injury are reported to be useful for predicting renal prognosis in the early stage of diabetic nephropathy (DN). Our study compared predictions of the renal prognosis by such biomarkers and by histologic tubulointerstitial damage. Among 210 patients with type 2 diabetes and biopsy-proven DN managed from 1985 to 2011, 149 patients with urinary N-acetyl-β-d-glucosaminidase (NAG) and urinary β2-microglobulin (β2-MG) data at the time of renal biopsy were enrolled. The primary outcome was a decline in eGFR of ≥50% from baseline or commencement of dialysis for ESRD. The median follow-up period was 2.3 years (interquartile range, 1.1-5.3), and the primary outcome was noted in 94 patients. Mean eGFR was 46.3±23.2 ml/min per 1.73 m(2), and 132 patients (89%) had overt proteinuria at baseline. Cox proportional hazards analysis revealed that the association of urinary NAG and β2-MG with the outcome was attenuated after adjustment for known promoters of progression (+1 SD for log NAG: hazard ratio [HR], 1.14; 95% confidence interval [95% CI], 0.84 to 1.55; +1 SD for log β2-MG: HR, 1.23; 95% CI, 0.94 to 1.62). In contrast, the interstitial fibrosis and tubular atrophy (IFTA) score was still significantly correlated with the outcome after adjustment for the same covariates (+1 for IFTA score: HR, 2.31; 95% CI, 1.56 to 3.43). Moreover, adding the IFTA score to a model containing known progression indicators improved prediction of the outcome (increase of concordance index by 0.02; 95% CI, 0.00 to 0.05; category-free net reclassification improvement by 0.54; 95% CI, 0.03 to 1.05; and relative integrated discrimination improvement by 0.07; 95% CI, -0.08 to 0.22). Adding urinary NAG and β2-MG excretion to known promoters of progression did not improve prognostication, whereas adding the IFTA score did. The IFTA score may be superior to these tubulointerstitial markers for predicting the renal prognosis in advanced DN.

Use of urine biomarker-derived clusters to predict the risk of chronic kidney disease and all-cause mortality in HIV-infected women.

Although individual urine biomarkers are associated with chronic kidney disease (CKD) incidence and all-cause mortality in the setting of HIV infection, their combined utility for prediction remains unknown. We measured eight urine biomarkers shown previously to be associated with incident CKD and mortality risk among 902 HIV-infected women in the Women's Interagency HIV Study: N-acetyl-β-d-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), alpha-1 microglobulin (α1m), interleukin 18, neutrophil gelatinase-associated lipocalin, albumin-to-creatinine ratio, liver fatty acid-binding protein and α-1-acid-glycoprotein. A group-based cluster method classified participants into three distinct clusters using the three most distinguishing biomarkers (NAG, KIM-1 and α1m), independent of the study outcomes. We then evaluated associations of each cluster with incident CKD (estimated glomerular filtration rate <60 mL/min/1.73 m(2) by cystatin C) and all-cause mortality, adjusting for traditional and HIV-related risk factors. Over 8 years of follow-up, 177 CKD events and 128 deaths occurred. The first set of clusters partitioned women into three groups, containing 301 (Cluster 1), 470 (Cluster 2) and 131 (Cluster 3) participants. The rate of CKD incidence was 13, 21 and 50% across the three clusters; mortality rates were 7.3, 13 and 34%. After multivariable adjustment, Cluster 3 remained associated with a nearly 3-fold increased risk of both CKD and mortality, relative to Cluster 1 (both P < 0.001). The addition of the multi-biomarker cluster to the multivariable model improved discrimination for CKD (c-statistic = 0.72-0.76, P = 0.0029), but only modestly for mortality (c = 0.79-0.80, P = 0.099). Clusters derived with all eight markers were no better for discrimination than the three-biomarker clusters. For predicting incident CKD in HIV-infected women, clusters developed from three urine-based kidney disease biomarkers were as effective as an eight-marker panel in improving risk discrimination.

Urinary Lysosomal Enzyme Activities and Albuminuria in Ghanaian Patients with Type 2 Diabetes Mellitus.

Renal tubular lysosomal enzyme activities like alanine aminopeptidase (AAP) and N-acetyl-β-D-glucosaminidase (NAG) have been shown to increase in patients developing diabetic nephropathy and nephrosclerosis. This study aimed to determine the activities of N-acetyl-β-D-glucosaminidase and alanine aminopeptidase and albumin concentration in urine samples of patients with type 2 diabetes. One hundred and thirty (65 type 2 diabetic and 65 nondiabetic) subjects participated in this study. Blood samples were drawn for measurements of fasting blood glucose, albumin (Alb), lipids, and creatinine (Cr). Early morning spot urine samples were also collected for activities of alanine aminopeptidase (AAP), N-acetyl-β-D-glucosaminidase (NAG), and concentration of albumin (U-Alb) and creatinine (U-Cr). Both NAG/Cr and AAP/Cr were significantly increased in diabetic subjects compared to controls (p < 0.001). There was positive correlation between NAG/Cr and Alb/Cr (r = 0.49, p < 0.001) and between NAG/Cr and serum creatinine (r = 0.441, p < 0.001). A negative correlation was found between NAG/Cr and eGFR (r = −0.432, p < 0.05). 9.3% and 12% of diabetics with normoalbuminuria had elevated levels of AAP/Cr and NAG/Cr, respectively. We conclude that measuring the urinary enzymes activities (NAG/Cr and AAP/Cr) could be useful as a biomarker of early renal involvement in diabetic complications.

Do teas rich in antioxidants reduce the physicochemical and peroxidative risk factors for calcium oxalate nephrolithiasis in humans? Pilot studies with Rooibos herbal tea and Japanese green tea.

Several experimental and animal studies have demonstrated that substances rich in antioxidants can reduce the physicochemical and peroxidative risk factors for calcium oxalate (CaOx) renal stone formation in urine and blood. However, there are very few such investigations in humans. In the present pilot study, two varieties of tea, a green one from Japan (JGT) and a herbal one from South Africa (Rooibos) (RT), both rich in antioxidants, were administered to a group of CaOx stone formers (SF) (n=8) for 30days. Both teas were analysed for polyphenols by high-performance liquid chromatography and for minerals by plasma atomic and optical emission spectroscopy. 24h urines (baseline and day 30) were analysed for lithogenic factors. CaOx metastable limits and crystal nucleation and growth kinetics were also determined in each urine sample. Deposited crystals were inspected by scanning electron microscopy. Blood samples were collected (baseline and day 30). Biomarkers of oxidative stress including plasma and urinary thiobarbituric acid reactive substances (TBARS) and urinary N-acetyl-β-D-glucosaminidase (NAG) were also determined. Urinary physicochemical risk factors were also investigated after ingestion of RT for 30days in two control groups (CG1 and CG2), the latter one of which consisted of habitual JGT drinkers. Statistical analyses were performed using Wilcoxon signed rank tests and Mann-Whitney tests for paired and independent measurements, respectively. Several flavonoids and catechins were quantified in RT and JGT, respectively, confirming that both teas are rich sources of antioxidants. Mineral content was found to be far below dietary reference intakes. There were no significant changes in any of the urinary physicochemical or peroxidative risk factors in the control groups or in SF, except for the supersaturation (SS) of brushite (Bru) which decreased in the latter group after ingestion of JGT. Crystal morphology showed a tendency to change from mixed CaOx mono- and di-hydrate to monohydrate after ingestion of each tea. Since the latter form has a stronger binding affinity for epithelial cells, this effect is not protective. Analysis of the physicochemical and peroxidative risk factors in CG1 and CG2 did not reveal any evidence of a synergistic effect between the two teas. Paradoxically, baseline risk factors in the habitual JGT control group were significantly raised relative to those in CG1. Our preliminary results suggest that ingestion of RT and JGT does not reduce the risk factors for CaOx stone formation in humans, but these findings need to be tested in further studies involving much larger sample sizes.

Urinary Kidney Injury Molecules in Children with Iron-Deficiency Anemia.

BackgroundThe aim of this study was to investigate the urine levels of human kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl-β-D-glucosaminidase (NAG), and liver-type fatty acid-binding protein (L-FABP) in children with iron-deficiency anemia (IDA).Material/MethodsThirty-five children with IDA and 32 matched healthy controls were recruited. We assessed complete blood count, serum iron, iron-binding capacity, ferritin, serum levels of urea, creatinine (Cr), sodium (Na), potassium (K), calcium (Ca), and glucose levels. Estimated glomerular filtration rate (eGFR) was calculated. Urinary NAG, NGAL, KIM-1, and L-FABP were measured and divided by urine creatinine for comparisons.ResultsThere were no significant differences in serum urea, Cr, or eGFR between the IDA group and the control group (p>0.05, for all). IDA patients had significantly higher urine NGAL/Cr, L-FABP/Cr, KIM-1/Cr, and NAG/Cr compared with the control group (p<0.05). There were significant negative correlations between hemoglobin, hematocrit, red blood cell count, and urine NGAL/Cr, NAG/Cr, L-FABP/Cr, KIM-1/Cr levels (p<0.05).ConclusionsHigher urinary kidney injury molecule levels in IDA patients suggest a possible subclinical renal injury in pediatric IDA patients whose renal functions and serum electrolytes were normal.

Ulinastatin Protects against Acute Kidney Injury in Infant Piglets Model Undergoing Surgery on Hypothermic Low-Flow Cardiopulmonary Bypass.

ObjectiveInfants are more vulnerable to kidney injuries induced by inflammatory response syndrome and ischemia-reperfusion injury following cardiopulmonary bypass especially with prolonged hypothermic low-flow (HLF). This study aims to evaluate the protective role of ulinastatin, an anti-inflammatory agent, against acute kidney injuries in infant piglets model undergoing surgery on HLF cardiopulmonary bypass.MethodsEighteen general-type infant piglets were randomly separated into the ulinastatin group (Group U, n = 6), the control group (Group C, n = 6), and the sham operation group (Group S, n = 6), and anaesthetized. The groups U and C received following experimental procedure: median thoracotomy, routine CPB and HLF, and finally weaned from CPB. The group S only underwent sham median thoracotomy. Ulinastatin at a dose of 5,000 units/kg body weight and a certain volume of saline were administrated to animals of the groups U and C at the beginning of CPB and at aortic declamping, respectively. Venous blood samples were collected at 3 different time points: after anesthesia induction in all experimental groups, 5 minutes, and 120 minutes after CPB in the Groups U and C. Markers for inflammation and acute kidney injury were tested in the collected plasma. N-acetyl-β-D-glucosaminidase (NAG) from urine, markers of oxidative stress injury and TUNEL-positive cells in kidney tissues were also detected.ResultsThe expressions of plasma inflammatory markers and acute kidney injury markers increased both in Group U and Group C at 5 min and 120 min after CPB. Also, numbers of TUNEL-positive cells and oxidative stress markers in kidney rose in both groups. At the time point of 120-min after CPB, compared with the Group C, some plasma inflammatory and acute kidney injury markers as well as TUNEL-positive cells and oxidative stress markers in kidney were significantly reduced in the Group U. Histologic analyses showed that HLF promoted acute tubular necrosis and dilatation.ConclusionsHLF cardiopulmonary bypass surgery could intensify systemic inflammatory responses and oxidative stress on infant piglets, thus causing acute kidney injury. Ulinastatin might reduce such inflammatory response and oxidative stress and the extent of kidney injury.

Plasma kidney injury molecule-1 in heart failure: renal mechanisms and clinical outcome.

Urinary kidney injury molecule-1 (KIM-1) is a marker of tubular damage and associated with worse outcome in heart failure (HF). Plasma KIM-1 has not been described in HF. In a renal mechanistic cohort of 120 chronic HF patients, we established the association between plasma KIM-1, renal invasive haemodynamic parameters {renal blood flow ([(131) I]hippuran clearance) and measured glomerular filtration rate (GFR; [(125) I]iothalamate)} and urinary tubular damage markers. The association between plasma KIM-1, plasma creatinine, and clinical outcome was further explored in a cohort of 2033 acute HF patients. Median plasma KIM-1 was 171.5 pg/mL (122.8-325.7) in chronic (n = 99) and 295.1 pg/mL (182.2-484.2) in acute HF (n = 1588). In chronic HF, plasma KIM-1 was associated with GFR (P < 0.001), creatinine, and cystatin C. Plasma KIM-1 was associated with urinary N-acetyl-β-d-glucosaminidase (NAG), but not with other urinary tubular damage markers. Log plasma KIM-1 predicted adverse clinical outcome after adjustment for age, gender, and GFR [hazard ratio (HR) 1.94, 95% confidence interval (CI) 1.07-3.53, P = 0.030]. Statistical significance was lost after correction for NT-proBNP (HR 1.61, 95% CI 0.81-3.20, P = 0.175). In acute HF, higher plasma KIM-1 levels were associated with higher creatinine, lower albumin, and presence of diabetes. Log plasma KIM-1 predicted 60-day HF rehospitalization (HR 1.27, 95% CI 1.03-1.55, P = 0.024), but not 180-day mortality or 60-day death or renal or cardiovascular rehospitalization. Plasma KIM-1 is associated with glomerular filtration and urinary NAG, but not with other urinary tubular damage markers. Plasma KIM-1 does not predict outcome in chronic HF after correction for NT-proBNP. In acute HF, plasma KIM-1 predicts HF rehospitalization in multivariable analysis.

Biomarkers of Kidney Injury Among Nicaraguan Sugarcane Workers

In Central America, an epidemic of chronic kidney disease of unknown cause disproportionately affects young male agricultural workers. Longitudinal cohort study. 284 sugarcane workers in 7 jobs were recruited from one company in northwestern Nicaragua. Blood and urine samples were collected before and near the end of the 6-month harvest season. Job category (cane cutter, seeder, seed cutter, agrichemical applicator, irrigator, driver, and factory worker); self-reported water and electrolyte solution intake. Changes in levels of urinary kidney injury biomarkers normalized to urine creatinine level, including neutrophil gelatinase-associated lipocalin (NGAL), interleukin 18 (IL-18), N-acetyl-β-d-glucosaminidase (NAG), and albumin; serum creatinine-based estimated glomerular filtration rate (eGFR). Mean eGFR was 113 mL/min/1.73 m(2) and<5% of workers had albuminuria. Field workers had increases in NGAL and IL-18 levels that were 1.49 (95% CI, 1.06 to 2.09) and 1.61 (95% CI, 1.12 to 2.31) times as high, respectively, as in non-field workers. Cane cutters and irrigators had the greatest increases in NGAL levels during the harvest, whereas cane cutters and seeders had the greatest increases in IL-18 levels. Electrolyte solution consumption was associated with lower mean NGAL and NAG levels among cane cutters and lower mean IL-18 and NAG levels among seed cutters; however, there was no overall effect of hydration among all workers. On average, workers with the largest increases in NGAL and NAG levels during the harvest had declines in eGFRs of 4.6 (95% CI,1.0to8.2) and 3.1 (95% CI,-0.6 to 6.7) mL/min/1.73 m(2), respectively. Surrogate exposure measure, loss to follow-up. Results are consistent with the hypothesis that occupational heat stress and volume depletion may be associated with the development of kidney disease, and future studies should directly measure these occupational factors. The presence of urine tubular injury markers supports a tubulointerstitial disease that could occur with repeated tubular injury.

Urinary Markers for Early Detection of Diabetic Nephropathy in Type 1 Diabetes Mellitus

Background: diabetic nephropathy (DN) is a serious complication of diabetic mellitus associated with increased risk of morbidity and mortality. Diagnostic markers to detect DN at early stage are important as early intervention can slow loss of kidney functions and improve patient outcomes. N-acetyl-β-d-glucosaminidase (NAG) is a lysosomal enzyme, present in high concentrations in renal proximal tubular cells, Gamma-glutamyltransferase (GGT) is an enzyme which located along the proximal tubular brush border, Malondialdehyde (MDA) is a highly toxic product, formed in part by lipid oxidation derived free radicals, Reactive carbonyl derivatives (RCDS) is an oxidative stress marker in urine, as a measure of the oxidative modification of proteins and bete-2-microglobulin is filtered by the glomerulus, absorbed and catabolized by the proximal tubules. The aim of this study is to investigate the urinary outcome of these markers as early detectors of diabetic nephropathy in type 1 diabetic children. Subjects and methods: This case-control study included 67 children with type 1 diabetes mellitus (33 male; 34 female), age (11.03±1.05 years) and thirty one age (10.58±1.11 years) and sex (13 male; 18 female) matched healthy children (13 male; 18 female). Type 1 diabetic children were further subdivided into microalbuminuric and normoalbuminuric subgroups according to microalbuminuria concentration (30 mg/ g creatinine). Age, sex, diabetic duration and the current daily insulin dose, and family history of diabetes, weight, height, body mass index, systolic and diastolic blood pressure were recorded. Fasting plasma glucose, glycated hemoglobin, blood urea nitrogen, plasma creatinine, urinary creatinine, micoalbumin, N-acetyl-B-D glucosaminidase (NAG), Gama glutamyl transferase (GGT), Beta-2-microglobulin, Malondialdehyde (MDA) and Reactive carbonyl groups (RCDSS) were measured in all subjects. Results: a significant increase in tubular injury markers of diabetes (NAG, GGT, beta-2-microglobulin) and oxidative stress parameters (MDA, RCDSS) as compared to control subjects was found. Microalbuminuric subjects showed a significant elevatation in the urinary markers including NAG, GGT, beta-2-microglobulin, MDA, RCDSS as compared to normoalbuminuric subjects. The studied urinary tubular enzymes (NAG, GGT), oxidative stress markers (MDA, RCDSS) and Beta-2- microglobulin showed positive correlations with one another. Conclusion: The results of this study introduced the possibility of depending on tubular enzymes (NAG, GGT), oxidative stress markers (MDA, RCDSS) and β2 microglobulin as early, reliable, and sensitive predictors for diabetic nephropathy. The NAG activity index proved to be the most sensitive biomarker, then beta-2- microglobulin for early discovering the tubule cells damage.

Effect of salvianolic acid A and C compatibility on inflammatory cytokines in rats with unilateral ureteral obstruction

ObjectiveTo investigate the effect of salvianolic acid A and C component molecules, which are involved in drug compatibility, on inflammatory cytokine expression that affects human chemokine ligand 5 (CCL5) and chemokine ligand 10 (CXCL10) levels in rats with unilateral ureteral obstruction (UUO). MethodsFifty Sprague Dawley rats were randomly divided into five groups: normal, model, salvianolic acid A, salvianolic acid C and salvianolic acid A and C groups. The normal group was used as the control, and the other groups of rats had a UUO model established. The control group had free access to food and water, and the other groups received the corresponding drugs for 2 weeks. After the last administration, urine β2-microglobulin (β2-MG) and N-acetyl-β-D-glucosaminidase (NAG) levels were analyzed. After 24 h, all rats were sacrificed and the serum was analyzed for creatinine (Cr) and blood urea nitrogen (BUN) levels. Rat kidneys were removed, and CCL5 and CXCL10 inflammatory cytokine mRNA expression was measured using real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). Kidney fibrosis was observed using hematoxylin-eosin (HE) staining and Masson trichrome staining. ResultsIn the salvianolic acid A and salvianolic acid C treatment groups, serum Cr and urine NAG levels were significantly lower than in the model group (both P < 0.05). In all treatment groups, urine β2-MG levels were significantly lower than in the model group (all P < 0.05). Compared with model group, the pathological changes and collagen deposition improved to varying degrees (both P < 0.05). CCL5 and CXCL10 mRNA expression decreased to different degrees compared with the model group (both P < 0.05). ConclusionSalvianolic acid A and C are component molecules of drug compatibility, and they may protect renal function and improve tubular function and renal pathology to a certain degree in UUO. This improvement may be related to a reduction in inflammatory cytokines CCL5 and CXCL10 secretion in the UUO rat kidney.