Application of urinary N-acetyl-β-D-glucosaminidase combined with serum retinol-binding protein in early detection of diabetic nephropathy

BACKGROUND Diabetic nephropathy(DN)is a microangiopathy of type 2 diabetes mellitus(T2DM),which can damage the kidney through various ways and mechanisms due to the nature of the disease,involving the renal interstitium and glomeruli.However,in the early stage of the disease,patients only showed kidney volume increase and glomerular hyperthyroidism,and typical symptoms that are difficult to arouse individual attention were noticed.AIM To observe the expression of serum retinol-binding protein(RBP)and urinary Nacetyl-β-D-glucosaminidase(NAG)in patients with DN,and to analyze their value in disease prediction,so as to provide new targets for early diagnosis and treatment of DN.METHODS The baseline data of 50 T2DM patients treated in our hospital between January 2021 and December 2022 were retrospectively reviewed and included in group A.The baseline data of 50 patients with type 2 DN admitted to our hospital during the same period were collected and included in group B.The baseline data and serum RBP and urine NAG expression were compared between the two groups to analyze their value in the early prediction of DN.RESULTS There was no significant difference in age,gender,duration of diabetes,combined hyperlipidemia and combined hypertension between the two groups(P>0.05);the expression of urinary NAG and serum RBP in group B was higher than that in group A,and the difference was statistically significant(P<0.05);a multiple logistic regression model was established,and the results showed that urinary NAG and serum RBP were related to the presence or absence of injury in diabetic patients,and overexpression of urinary NAG and serum RBP may be risk factors for renal injury in T2DM patients(OR>1,P<0.05);receiver operating curve curve was plotted,and the results showed that the area under the curve of urinary NAG and serum RBP expression alone and in combination for predicting DN was>0.80,and the predictive value was satisfactory;bivariate Spearman linear correlation analysis showed that there was a positive correlation between urinary NAG and serum RBP expression in patients with DN(r=0.566,P=0.000).CONCLUSION The increased expression of urinary NAG and serum RBP may be the risk factors leading to the progression of T2DM to DN.The possibility of DN can be considered in patients with urinary NAG and serum RBP overexpression by examining the expression of urinary NAG and serum RBP in patients with T2DM in clinical practice.

Denaturation Kinetics of N-Acetyl-β-D-glucosaminidase from Scylla serrata in Urea Solution

N-Acetyl-β-D-glucosaminidase（NAGase, EC 3.2.1.52）, which catalyzes the cleavage of N-acetylgluco- samine polymers, plays important roles in the molting, digestion of chitinous foods in green crab. In the study, the efforts of urea on the activity of NAGase purified from the viscera of green crab（Scylla serrata） have been studied. The results show that appropriate concentrations of urea can lead to reversible inactivation of the enzyme, and the value of the inhibitor concentration leading to 50% of enzyme activity lost（IC50） is estimated to be 0.63 mol/L. The inactivation kinetics has been studied via the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The value of k＋0 is larger than that of k′＋0, indicating the free enzyme molecule is more fragile than the enzyme-substrate complex in urea solution. It is suggested that the presence of the substrate offers the marked protection of this enzyme against inactivation by urea.

Early renal injury indicators can help evaluate renal injury in patients with chronic hepatitis B with long-term nucleos(t)ide therapy

BACKGROUND Patients with chronic hepatitis B(CHB)with long-term nucleos(t)ide therapy may experience renal insufficiency.Traditional renal function indicators,such as urine protein,serum urea nitrogen(BUN),and serum creatinine,are normal when early mild lesions occur.Therefore,more sensitive renal function indicators are needed.AIM To investigate the significance of early renal injury indicators in evaluating renal injury in patients with CHB with long-term nucleos(t)ide therapy.METHODS We collected the clinical data of 69 outpatients with CHB at Peking University First Hospital from March 2018 to January 2020 who had been treated with longterm nucleos(t)ide therapy and analyzed the results of early renal injury indicators.Continuous normal distribution data were analyzed by the t-test to determine the difference between two groups.Continuous non-normally distributed data were analyzed by the Mann-Whitney U-test between two groups.The Kruskal-Wallis H test was used to determine the differences among multiple groups.Enumeration data were analyzed by the chi-square test.The related factors of early renal injury indicators were analyzed by logistic regression analysis.RESULTS The average treatment duration with nucleos(t)ide analogs of the 69 patients with CHB was 99.7±28.7 mo.The cases of patients with elevated BUN and hypophosphatemia were 6(8.7%)and 13(18.8%),respectively;31(44.9%)patients had abnormal early renal injury indicators,including 9 patients with abnormal urine microalbumin,7 patients with abnormal urine immunoglobulin,6 patients with abnormal urine transferrin,and 19 patients with abnormalα1 microglobulin.There were no significant differences in the mean values of age,sex,BUN,estimated glomerular filtration rate(eGFR),serum uric acid,serum calcium,or serum phosphorus between the two groups of patients with and without early renal injury indicators.However,the mean levels of serum creatinine and urine creatinine,N-acetyl-β-D-glucosidase enzyme,α1 microglobulin,and urine immunoglobulin in the former group of patients were significantly higher than those in the latter group of patients(P<0.05).The incidence of early renal injury in patients with eGFR≥90,60-89,and 30-59 mL/(min·1.73 m2)was 36.4%(8/22),47.6%(20/42),and 60%(3/5),respectively.Logistic regression analysis results showed that gamma-glutamyl transpeptidase[odds ratio(OR)=1.05(1.008-1.093),P=0.020],direct bilirubin[OR=1.548(1.111-2.159),P=0.010],serum creatinine[OR=1.079(1.022-1.139),P=0.006],and age[OR=0.981(0.942-1.022),P=0.357]were independent predictors of early renal injury.CONCLUSION Patients with CHB treated with long-term nucleos(t)ide analog therapy had a high probability of early renal injury,and early renal injury indicators were highly sensitive and could be used to monitor early renal impairment.

Research progress on markers of urine tubular damage in idiopathic membranous nephropathy

Idiopathic membranous nephropathy is a common primary glomerular disease.Recently,it has been found that the progression and prognosis of glomerular diseases are not only related to the glomeruli itself,but also to the severity of renal tubular injury.Although blood creatinine and urinary protein are recognized biomarkers,they appear late,are insensitive,lack specificity,and are difficult to respond to IMN conditions in a timely and accurate manner.Markers such as N-acetyl-β-D-glucosaminidase,kidney injury molecule 1,neutrophil gelatinase-associated lipoprotein in urine can dynamically reflect the progress of kidney injury in the early stage,and are detected as noninvasive,so It has been applied in IMN,but such research is lacking,and the scope needs to be further expanded and discussed in depth.

Thiazolylhydrazone dervatives as inhibitors for insect N-acetyl-β-D-hexosaminidase and chitinase

Insect chitinase and N-acetyl-β-D-hexosaminidases(Hex)are potential targets for developing new pesticides.Here,a series of thiazolylhydrazonesⅠ(with substituted group R1 at N3)andⅡ(with substituted group R1 at N2)were designed,synthesised and evaluated as competitive inhibitors of OfHexl and OfChi-h,from the agricultural pest Ostrinia furnacalis,DerivativesⅠ-3 d andⅡ-3 d,with phenoxyethyl group at R1,demonstrated the best inhibitory activities against OfHexl and OfChi-h.Molecular docking analysis indicated that the branched conformation compound II-3 d(Ki=1.5μmol/L)formed more hydrogen bonds with OfHexl than the stretched conformation compound I-3 d(Ki=5.9μmol/L).The differences in compounds’binding conformations with OfChi-h explained differences in inhibitory activity of compounds I-3 d(Ki=1.9μmol/L)and II-3 d(Ki=4.1μmol/L).This work suggests a novel scaffold for developing specific Hex and Chi-h inhibitors.

Cloning, expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis

Chitinase catalyzes β,4-glycosidic linkages in chitin and has attracted re- search interest due to it being a potential pesticide target and an enzymatic tool for preparation of N-acetyl-β D-glucosamine. An individual insect contains multiple genes encoding chitinases, which vary in domain architectures, expression patterns, physiological roles and biochemical properties. Herein, Of ChtS, the glycoside hydrolase family 18 chiti- nase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that Of CHT5 consisted of an open reading frame of 1 665-bp nucleotides. Phy- logenic analysis suggested Of Cht5 belongs to the Group I insect chitinases. Expression of Of Cht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate pre- cipitation and metal chelating chromatography, about 7 mg of the recombinant Of Cht5 was purified to homogeneity from 1 L culture supernatant. Of Cht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward a-chitin. When chi- tooligosaccharides [（GlcNAc）n, n = 3-6] were used as substrates, Of Cht5 was observed to possess the highest catalytic efficiency parameter toward （GlcNAc）4 and predominan- tely hydrolyzed the second glycosidic bond from the non-reducing end. Together with fl-N-acetyl-D-hexosaminidase OfHexl, Of Cht5 achieved its highest efficiency in chitin degradation that yielded N-acetyl-β D-glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of Of Cht5 versus Of Hexl in the range of 9 ： 1-15 ： 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.

Clinical Observation on Diabetic Nephropathy Treated with Alcohol Extraction of Flos Abelmoschus Manihot

Sixty-eight cases of Non-Insulin Dependent Diabetes Mellitus complicated with Nephropathywere randomly divided into two groups : the treated group, 35 cases treated with alcohol extraction of Flos A-belmoschus manihot, Gliclazide and Captopril Tablets; and the control group, 33 cases treated with Gli-clazide and Captopril Tablets, over a period of 8 weeks. The total effective rate in the treated and controlgroup were 83.87% and 31.03% respectively (P< 0 . 01 ) , urinary micro-albumin were 31 . 7 and 76.3mg/L(P < 0. 05) , proteinuria were 0. 41 and 0 . 77g/24hr ( P < 0. 01 ) , blood 2-microglobulin were 3317. 8 and3473. 1 hg/ml ( P < 0. 05) , urinary 2-microglobulin were 367. 2 and 641 . 5ng/ml ( P < 0. 01 ) , urinary N-acetyl- -glucosaminidase (NAG) were 26. 3 and 66. 7u/L ( P < 0. 01 ) , plasma lipid peroxide (LPO) were6. 13 and 8. 78 nmol/L ( P<0. 05) , and plasma superoxide anion were 8. 36 and 10. 42 kcpm respectively( P< 0. 05) . It was suggested that alcohol extraction of Abelmoschus manihot could eliminate oxygen freeradicals, alleviate renal tubular-interstitial diseases, improve renal function, and reduce proteinuria.