Age related changes of N-acetyl-beta-D-glucosaminidase and L-alanine aminopeptidase in mouse kidney, urine and plasma

Age and sex dependent differences of N-acetyl-beta-D-glucosaminidase (NAG) and L-alanine aminopeptidase (AAP) activities in kidney, urine and plasma of male and female mice were studied. The sex difference in NAG activity appeared between 27 and 38 days of age with the manifestation of significant differences in body weight and kidney growth. NAG activity in male kidneys was 3-fold that in females and its urinary level in mature males was over 10-fold higher. Androgenic regulation was found not only in the NAG contents in the kidneys and in the urinary excretion but also in the plasma NAG level, which showed higher in females. On the other hand, AAP activity in kidney, urine and plasma did not show much sex differences. Age related changes in AAP activity were not found except in the kidney and marked androgenic regulation was also not found in AAP. These results indicate that NAG and AAP, which are both urinary enzymes used as indicators of renal lesions, may be regulated differently.     

How can storage time and temperature affect enzymic activities in urines?

We measured a tubular brush-border enzyme (alanine aminopeptidase, AAP) and a lysosomal hydrolase (N-acetyl-beta-D-glucosaminidase, NAG) in morning urines from 15 healthy normal subjects to check if different storage times and temperatures could modify enzyme concentrations. Short-term (24 h) storage time at room temperature or 4 degrees C does not affect AAP and NAG activities. Both enzymes are well preserved at -70 degrees C. AAP dramatically falls after 1 month at -20 degrees C, lowering to about 8% of the initial value after only 4 days of storage. On the contrary, NAG is well preserved at these storage conditions. Centrifugation has revealed not critical for measurement of these two enzymes.     

Zinc level in the blood of children with nephrotic syndrome and correction of its deficiency with animal blood preparation Livex]

Serum and erythrocyte zinc levels have been assayed in in 45 children with steroid-dependent nephrotic syndrome in both acute phase and remission. Out of these children a group of 22 has been distinguished. These patients have been treated with Livex--animal blood preparation containing amino acids and trace elements, including zinc. It was found, that serum zinc has been significantly lowered in children with the acute phase of nephrotic syndrome who were not treated with Livex. Erythrocyte zinc levels have been normal in these patients. A significant increase in both serum and erythrocyte zinc levels has been noted during remission, but they have still been lower that in healthy children. A three-month cure with Livex produced statistically significant increase in zinc levels in children during remission.     

Plasminogen activator in nephrotic syndrome

Plasminogen activators were studied in blood urine in 207 patients with nephrotic syndrome of different etiological forms. The blood plasminogen activator activity was decreased in chronic glomerulonephritis, SLE, systemic vasculities as result of great level of inhibitors (L2M), penetration of enzymes to abdominal and pleural transudates, excretion to urine. The blood plasminogen activator activity and urokinase level in chronic glomerulonephritis was dependent on the degree of nephrotic syndrome. The plasminogen activator in amyloidosis was sharply elevated because of permanent irritability of endothelial wall by amyloid mass. Venous occlusion caused the release of plasminogen activator to blood only in more favourable clinical course of nephrotic syndrome.     

Studies on urinary gamma-glutamyl transpeptidase in nephrotic syndrome patients

(1) Variations in the levels of GGT were measured in urine specimens taken in the early morning in control and in 20 consecutive adult patients with uncomplicated nephrotic syndrome. (2) The urinary GGT activity was increased in all cases of nephrotic syndrome patients investigated with different etiology. (3) A significant correlation was found between urinary GGT activity and serum albumin (r = 0.727) but not with serum cholesterol (r = 0.129). (4) These findings suggest that enhanced excretion of urinary GGT may be stimulated by decreased albumin concentration or oncotic pressure but does not appear to be due to leakage from plasma. (5) A systematic study on urinary GGT showed that GGT activity was decreased to the upper limit of normal control values in nephrotic syndrome patients after remission.     

More on effects of storage time and temperature on urinary enzymes: a 1-year study.

Results of our conclusive study on urinary enzyme stability during sample storage are reported. We measured alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) in morning urines from 9 healthy normal subjects immediately after collection and throughout a 1-year storage at -70 and -20 degrees C. AAP proved to be quite stable at -70 degrees C (99.2% of the basal value at the end of the year). NAG is partially preserved (84.1% of the basal value) at -70 degrees C, but significantly decreased (50.4%) at -20 degrees C.     

Effect of polyaspartic acid on CdCl2-induced nephrotoxicity in the rat

We produced an animal model of CdCl₂ nephrotoxicity in rats, and treated them with polyaspartic acid (PAA) to prevent renal damage. Male Sprague-Dawley (SD) rats (190–200 g) were used to induce proximal renal tubular damage by daily injection of CdCl₂ 3.0 mg/1,000 g body wt for 2 wk. CdCl₂-exposed SD rats exhibited significant increases in urine volume, urinary excretion ofN-acetyl-β-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and fractional excretion of sodium (FENa) and a decrease in the percentage of tubular reabsorption of phosphate (%TRP). Of these indicators of proximal tubular function, AAP and %TRP are more sensitive than NAG or FENa. No glycosuria or aminoaciduria, however, were observed. PAA markedly improved these indicators of proximal tubular function. Daily urinary protein excretion and creatinine clearance, on the other hand, did not change after administration of PAA. Cd concentrations in the cortex were 3 times higher than in the medulla, however, there were no differences between Cd-treated rats and PAA-treated rats. Our animal model is an excellent one for determining the effect of cadmium on renal proximal tubule damage. PAA appears to be useful in the treatment of CdCl₂ nephrotoxicity.     

肾病综合征患儿行为问卷调查及相关影响因素分析

目的:分析肾病综合征(NS)患儿的行为调查和相关影响因素。方法:选择2015年1月~2016年3月我院住院或者门诊治疗的患儿60例作为观察组,并选择同期同年龄组健康体检儿童50例作为对照组。采用自编人口学调查问卷表调查研究对象家庭状况、患病情况、活动情况和激素使用情况,采用Rutter儿童行为问卷(父母问卷)进行儿童行为调查。采用x~2检验进行单因素分析,采用logistic回归分析进行多因素分析。结果:观察组NS患儿A行为发生率(18.33%)、N行为发生率(20.00%)和总行为问题发生率(38.33%)均高于对照组,差异有统计学意义(P0.05)。Logistic回归分析结果显示,发病持续时间、复发次数、总住院天数、日均户外活动时间、激素持续时间和激素使用总量是影响NS患儿总行为问题的因素,日均户外活动时间是NS患儿总行为问题的保护因素。结论:NS患儿较健康儿童存在更严重的行为问题,其行为问题受发病持续时间、复发次数、总住院天数、日均户外活动时间、激素持续时间和激素使用总量等因素的影响。     

The protective role of selenium on the toxicity of cisplatin-contained chemotherapy regimen in cancer patients

The effect of selenium (Se) in reducing the toxicity of cisplatin in cancer patients was studied. Forty-one patients were randomized into group A (20 patients with Se administration in first cycle of chemotherapy as study cases and without Se in second cycle of chemotherapy as control) and group B (21 patients without Se in first cycle of chemotherapy and with Se in second cycle of chemotherapy). The 400 μg per day of Se as Seleno-Kappacarrageenan were administered from 4 before to 4 d after chemotherapy for study cases. The serum Se increased from 70.4±22.86 to 157.04±60.23 ng/mL (P<0.001) in patients received Se. The cisplatin dosage was iv administration in 60–80 mg/m² on the first day. The results showed that the peripheral WBC counts on day 14 after initiation of chemotherapy in study cases was significantly higher than the controls (3.35±2.01 vs 2.31±1.38 [×10⁹L])/L,p<0.05). On the other hand, the consumption of GCSF for the cases was significantly less than the controls (110.1±82.2 vs 723.6±192.6 IU,p<0.05). The volumes of blood transfusion for the study group were also significantly less than the controls (0 vs 62±38mL,p<0.05). The nephrotoxicity of cisplatin was measured by urine enzymes (NAG, GGT, AAP, LAP, and ALP) were determined prior to and at 2, 24, 48, and 72h after initiation of chemotherapy. The urine enzymes NAG, GGT, AAP, and ALP after chemotherapy for cases were significantly lower than the controls. No toxicity of Seleno-Kappacarrageenan was noted. The above results suggest that the Se can be used as an agent for reducing the nephrotoxicity and bone marrow suppression induced by cisplatin.     

Urinary output modulation of alanine aminopeptidase, gamma-glutamyl transpeptidase and N-acetyl-beta-D-glucosaminidase by castration and testosterone in male normal rat

The urinary excretion of alanine aminopeptidase (AAP), gamma-glutamyl transpeptidase (gamma-GT) and of N-acetyl-beta-D-glucosaminidase (NAG) was studies in normal and castrated rats receiving either testosterone for 5 post-operative weeks or no hormone. In castrated rats the urinary output of AAP and gamma-GT was significantly lower than in sham control or in castrated rats receiving testosterone. In addition, an excess of exogenously given testosterone had no effect on enzymuria of normal rats. The urinary excretion of NAG was influenced neither by castration nor by testosterone. These results suggest that endogenous testosterone is responsible for a permanent positive control on the urinary excretion of AAP and gamma-GT.     

Molecular forms of beta-hexosaminidase and cathepsin D in serum and urine of healthy subjects and patients with elevated activity of lysosomal enzymes.

A procedure is described that allows the characterization of the molecular forms of beta-hexosaminidase and cathepsin D in controls and pathological specimens of human serum and human urine. The following observations were made. (1) In human serum, beta-hexosaminidase (alpha- and beta-chain) and cathepsin D are present predominantly in their high-molecular-weight precursor forms. In human urine, these enzymes exist as both precursor and mature forms. (2) Cathepsin D precursor from serum and urine differs in the number of oligosaccharides that are sensitive to endo-beta-N-acetylglucosaminidase H. Therefore the urine enzyme is not likely to originate from the serum. (3) The presence exclusively of precursors of beta-hexosaminidase and of cathepsin D in the sera of patients with hepatitis suggests that in hepatitis secretion of lysosomal enzymes is elevated, rather than the enzymes leaking from damaged cells. (4) In the urine of patients with nephrotic syndrome, beta-hexosaminidase and cathepsin D are present in grossly elevated amounts, but do not differ in the polypeptide patterns from controls. (5) In urine from a patient with mucolipidosis II, the elevated activity of beta-hexosaminidase is accounted for mainly by the precursor forms. Mature beta-chain of beta-hexosaminidase is lacking, and incompletely processed beta-hexosaminidase polypeptides are present. Both the precursor and the mature forms of cathepsin D are increased. They contain only complex oligosaccharides.     

Urinary zinc excretion and zinc status of patients with Β-thalassemia major

In this study, zinc status and urinary zinc excretion with and without desferrioxamine (DFO) infusion and the relationship between urinary zinc excretion and renal tubular dysfunction in thalassemia major (TM) patients were investigated. Forty TM patients were given four DFO infusions on alternate days over a 1-wk period prior to the transfusion. On each day that DFO was given, a 24-h urine collection initiated. DFO was omitted for 1-wk before the following transfusion and during the period four 24-h urine collections were performed. Twenty healthy children provided 24-h urine collection as controls. Blood samples were taken on each of two consecutive transfusion days of the patients and from the controls. Urinary zinc excretion was measured and plasma and red blood cell (RBC) zinc analysis were performed by inductively coupled plasma-atomic emission spectrophotometry. UrinaryN-acetyl-Β-D-glucosaminidase (NAG) activity and creatinine were determined in morning urine specimens. The mean plasma zinc concentration was significantly lower in the patients not given DFO compared to the values of the patients given DFO and the control group. The mean RBC zinc concentration (Μmol/g Hb) in the patients (with and without DFO) and the control group were similar. Urinary zinc excretion was significantly higher in the patients receiving DFO compared to the control group, whereas urinary zinc excretion in the patients not given DFO was not different from the controls. Urinary NAG indices (U/g Cr) were significantly higher in the patients compared to controls. Urinary zinc excretion was correlated with the urinary NAG indices.     

Serum malondialdehyde levels,myeloperoxidase and catalase activities in patients with nephrotic syndrome

Abstract

Objectives

Some studies have indicated the pathophysiological importance of reactive oxygen species (ROS) in patients with nephrotic syndrome. Myeloperoxidase (MPO) is a leukocyte-derived enzyme-generating ROS that has been proposed to exert a wide array of pro-atherogenic effects throughout all stages of the atherosclerotic process. The aim of this study was to investigate the serum malondialdehyde (MDA) levels, MPO and catalase activities in patients with adult nephrotic syndrome.

Patients and Methods

Twenty-four patients with nephrotic syndrome and 24 healthy controls were enrolled. Serum MPO activity, catalase activity, and MDA levels were assessed.

Results

Serum MPO activity and MDA levels were signi?cantly higher in patients with nephrotic syndrome than controls (both, P < 0.001), while catalase activity was signi?cantly lower (P < 0.001). Serum catalase activity was found to be significantly correlated with MPO activity (r = ?0.417, P = 0.003) and MDA levels (r = ?0.532, P = 0.007). The serum MDA levels were also found to be significantly correlated with MPO activity (r = 0.419, P = 0.003).

Conclusions

We concluded that serum MPO activity and oxidative stress were increased and that serum catalase activity was decreased in patients with adult nephrotic syndrome. In addition, these results indicate that increased MPO activity is associated with an oxidant–antioxidant imbalance that may contribute to atherosclerosis in patients with adult nephrotic syndrome.     

Involvement of IL-6 in mesangial proliferative glomerulonephritis

In this study, we demonstrated that IL-6 was a possible autocrine growth factor for rat mesangial cells (MC). rIL-6 induced in vitro growth of rat MC at a concentration of 2 to 200 ng/ml and IL-6 activity was found in the supernatant of cultured rat MC. Northern blot analysis as well as in situ hybridization revealed that IL-6 mRNA was expressed in the cultured MC. Of urine samples from patients with mesangial proliferative glomerulonephritis (PGN) 50% were found to contain significant IL-6 activity (ranging from 30 to 126 pg/ml). Urine samples from other type of primary glomerular diseases such as minimal change nephrotic syndrome or healthy volunteers contained no detectable IL-6 activity. Only 2 of 27 urine samples from membranous nephropathy contained detectable amount of IL-6. Furthermore, there was some relationship between the levels of urine IL-6 and the progressive stage of PGN. Finally, by immunohistochemical staining using an anti-IL-6 mAb, it was shown that MC in the affected glomeruli of PGN patients produced IL-6, whereas MC obtained from the patients with membranous nephropathy, minimal change nephrotic syndrome or normal kidney were not found to produce IL-6. These data suggest that deregulated production of IL-6 is involved in PGN and the measurement of urine IL-6 is helpful for the differential diagnosis of PGN as well as for monitoring the progression of PGN.     

联合标记物检测在重症感染中合并急性肾损伤的诊断价值

目的:探讨尿中性粒细胞明胶酶相关载脂蛋白(neutrophil gelatinase-associated lipocalin,NGAL)、尿肾损伤分子-1(kidneyinjury molecule-1,Kim-1)、尿N-乙酰-β-D-氨基葡萄糖苷酶(N-acecyl-β-D-glucosaminidase,NAG)、尿微量白蛋白(mALB)在重症感染中合并急性肾损伤的敏感性及临床价值。方法:回顾分析60例在新疆自治区人民医院ICU住院的重症感染合并急性肾损伤(AKI)患者的尿NGAL、Kim-1、NAG及mALB的变化情况。健康体检者20例为对照组。尿NGAL、Kim-1、mALB测定采用酶联免疫法(ELISA)检测,尿NAG测定采用对硝基苯酚(PNP)比色法检测,并以ROC曲线分析其敏感性。结果:AKI组患者尿液中的NGAL、Kim-1、NAG、mALB的测定浓度明显高于对照组,差异具有统计学意义(P<0.001),通过ROC曲线、诊断试验结果显示:尿NGAL、Kim-1曲线下面积分别为0.986、0.956,95%可信区间分别是0.968～1.004、0.910～1.001,较尿NAG、mALB更具有敏感性(P<0.001)。结论:尿NGAL、尿Kim-1的浓度检测对重症感染合并急性肾损伤的诊断更具有敏感性,与NAG、mALB联合检测有助于急性肾损伤的早期监测,对预防急性肾损伤的发生、发展具有重要的临床价值。     

Enzyme immunoassay of beta-hexosaminidase isoenzymes in human urine and renal cortex with monoclonal antibodies

Enzyme immunoassay (EIA) methods with monoclonal antibodies specific for N-acetyl-beta-D-glucosaminidase (NAG) isoenzymes A and B in human urine are presented. The proportion of NAG B obtained with the EIA methods was similar to that found with ion-exchange chromatography. In fresh human control urines, NAG B was found to be approximately 20% of the total NAG activity. A significant correlation was obtained between total NAG activity in human urine assayed with a conventional enzyme substrate method and the total NAG activity obtained as the sum of NAG A and NAG B analyzed with the EIA methods. Total NAG activity with the latter (EIA) methods showed about 30% higher values than found by the enzyme substrate method, which probably was due to inhibitors of NAG activity present in urine did not interfere with the EIA methods. The content of NAG A and NAG B in renal cortex was determined with the EIA methods. NAG B accounted for about 20% of the total NAG activity, which was similar to that found in fresh human urines.     

The use of xanthine oxidase for a specific and sensitive fluorimetric determination of 6-mercaptopurine in serum

Excretion of urinary N-acetyl-beta-D-glucosaminidase has been found to be elevated in diabetic humans and rats. This urinary glycosidase may reflect blood sugar control over time, since it has been significantly and positively correlated with hemoglobin A1 in children with insulin-dependent diabetes. Other studies have suggested that urinary NAG may predict diabetic nephropathy. In order to more carefully define the relationship between urinary NAG excretion and blood and urine sugars, hemoglobin A1, and microalbuminuria, 48 rats were made diabetic by the use of streptozotocin. All rats were uninephrectomized at 3 weeks. Of these, 23 were treated with daily insulin injections, 25 were untreated, and both groups were compared to 13 control, nondiabetic rats. Urine volume, glucose, albumin, and blood sugar were all significantly (P less than 0.05) elevated in the untreated rats compared to the treated and control groups. Urinary NAG:UCr was significantly (P less than 0.01) elevated in the untreated group with lower but still elevated levels (P less than 0.05) in the treated rats. To further define the time course of the increase in UNAG:UCr 12 rats were followed serially at 12-hr intervals for 92 hr after streptozotocin. Urinary NAG increased significantly (P less than 0.05) at 12 hr after streptozotocin injection and reached a plateau at 36 hr while hemoglobin A1 did not rise until 2 weeks after onset of hyperglycemia. Urinary NAG increases more rapidly than hemoglobin A1 after onset of hyperglycemia and glycosuria.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-acetyl-beta-glucosaminidase and albuminuria in acromegaly

Significantly increased albuminuria and N-acetyl-beta-glucosaminidase activity in serum and urine have been observed in acromegalic patients in comparison with healthy persons (P less than 0.001). No relationship between these biochemical variables and serum growth hormone or insulin concentration was found in our group of patients. Significant correlation was determined between urinary NAG activity and albuminuria of acromegalic patients (r = 0.84).     

Characterization of glycosaminoglycans in urine from patients with nephrotic syndrome and control subjects, and their effects on lipoprotein in lipase

Previously we found that the α₁-acid glycoprotein fraction from urine of patients with the nephrotic syndrome stimulated the lipoprotein lipase reaction in vivo and in vitro. The activator was separated from the α₁-acid glycoprotein and identified as a glycosaminoglycan. The studies reported here were undertaken to characterize and quantify the glycosaminoglycans contained in urine of patients with the nephrotic syndrome and to compare these to the glycosaminoglycans in urine of control subjects. We found that free low molecular weight glycosaminoglycans, heparan sulfate and chondroitin 4-sulfate, are excreted in both patients with the nephrotic syndrome and controls, however, patients with the nephrotic syndrome excreted much less of both glycosaminoglycans. The free form of heparan sulfate was found to be the activator which stimulated the lipoprotein lipase reaction in vitro in the presence of apolipoprotein C_II. In addition, the urine from patients with the nephrotic syndrome contained a protein-glycosaminoglycan complex which was absent in control urine. Glycosaminoglycans in the complex could be released by papain digestion or by trichloroacetic acid. Our evidence indicates that this glycosaminoglycan fraction is a low charge form of chondroitin sulfate.     

Quantitative enzymatic, immunologic and histochemical studies of clinically relevant human kidney alterations using image analysis

Tissue sections of kidneys from 172 patients with various pathologic conditions, such as hydronephrosis, interstitial nephropathies, ischemia, chronic graft rejection and renal cancer, were evaluated by an image analysis technique. Structurally defined kidney alterations were monitored for enzymatic, immunologic and other histochemical changes. Indicator enzymes of the proximal tubule, alanine-aminopeptidase (AAP), alkaline phosphatase (AP), beta-glucoronidase (beta-Gl) and gamma-glutamyltranspeptidase (GGTP), were used as parameters for screening. Enzyme concentrations were found to be significantly decreased in kidney sections of patients with various renal diseases (AP less than 15%, AAP less than 55% and beta-Gl less than 60%) as compared to normal kidney tissues (100%). AAP concentration was measured quantitatively by specific immunofluorescence using an antienzyme antibody. Immunofluorescence of AAP was comparable to that of AAP calculated by the colorimetric technique (substrate: DL-alanine-beta-naphthylamide-HCl) and decreased to less than 50% in altered kidney tissues. Furthermore, kidney cancer (less than 20%) and kidney tissue adjacent to tumours (less than 65%) displayed significantly decreased levels of kidney marker enzyme activity. This study suggests that (1) the diseased kidney is characterized by a defined change in key enzymes of the cell surface and (2) renal cancer exhibits partial depletion of these constituents. Image analysis of the pattern of enzyme activity appears to be a useful tool in the analysis of renal pathology.