Urinary Enzyme Activity Research Articles

Study of some factors affecting stability of N-acetyl-β-D-glucosaminidase and aminopeptidase N in urine at 37°C

Little is known of conditions which influence the stability of urinary enzymes upon storage in the bladder at 37°C. Using a continuous flow system simulatingin vivo conditions, we studied the influence of the pH of urine on the stability of two renal parenchymal enzymes N—Acetyl—β—D—Glucosaminidase (2—acetamido—2—deoxy—β—D—glucoside acetamidodeoxy glucohydrolase, NAG, EC 3.2.1.30) and L—Alanine aminopeptidase (Aminopeptidase N, AAP, EC 3.4.11.2). This continuous flow model that we have described can be employed to study the influence of pH on the stability of any renal enzyme excreted in urine. We also studied thein vitro effects of varying concentrations of low molecular weight regulatory metabolites such as urea, creatinine and uric acid and of some drugs excreted in urine, on the assay of these two enzymes. Urinary pH, urea content and some antibiotics seem to influence measured urinary NAG and AAP activities and we therefore express the need for caution before diagnostic interpretation of the urinary enzyme activities are made.

Evidence of circadian variations in mercuric-chloride-induced proximal tubular necrosis by digital imaging microscopy

Among the many factors which are considered as important in the nephrotoxicity of xenobiotics such as dose, age, sex, genetic strain or nutritional status, the time of dosing has emerged, during the last decade, as a major determinant of the extent of the damage (see review by Cal et al. [I]). A large number of publications have demonstrated statistically significant circadian variations in numerous aspects of renal function, e.g. glomerular filtration rate, renal blood flow, electrolyte excretion or urinary enzyme activity [l]. Time-dependent rhythmic variations in renal susceptibility to heavy metals or antibiotics have been shown using urine-concentrating ability or enzymuria as markers of nephrotoxicity [2]. Temporal variations in such markers are undoubtedly the expression of temporal alterations in metabolic processes. Since metabolic processes are based on cellular functions, the question arises: To what extent are differential structural manifestations as a function of the time of dosing visible at the cellular level? This question remains unresolved to date. This is partly due to the lack of a suitable quantitative technique to detect such rhythms. Though stereological techniques appeared to provide an objective tool in this respect, one of their major disadvantages is that they are rather time-consuming to perform, expecially when it is necessary to consider a large number of parameters [3]. The recent considerable development of the compu-

Assessment of renal function and damage in animal species. A review of the current approach of the academic, governmental and industrial institutions represented by the animal clinical chemistry association

There are a wide variety of laboratory tests available to assess damage to and functional impairment of the kidneys, although the effectiveness of these tests varies greatly depending upon the site specificity of the damage and to a lesser extent upon the animal species involved. Several traditional tests of renal dysfunction and damage, including plasma creatinine and urea, and urinalysis (dipstick and/or quantitative protein), can be used in the first instance to detect nephrotoxicity. A second tier of specific, targetted indicators (concentration test, urinary enzymes, clearance of analytes, specific proteins, etc.) may then be applied to identify further the site of the lesion and the functional status of the kidneys. The glomerular filtration rate (GFR) may be estimated from the clearance of exogenous and endogenous substances. The difficulty in obtaining accurately timed urine samples limits the value of these tests in small animals, although methods that do not involve urine collection are available. The kidney is the origin of several enzymes found in urine that can be used to monitor the toxic effects of chemicals and therapeutic substances. Selective measurement of enzyme activities in urine can be used to detect the site of the renal lesion after traditional tests have established the presence of renal injury. Separation of proteins in urine by electrophoretic techniques may also be used to discriminate damage to different parts of the nephron. Renal cell excretion in urine is a sensitive but unreliable indicator of acute damage to the proximal tubule. The rate of cell excretion is not a good predictor of the severity of tubular injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymuria as an index of renal damage in sheep with induced aminoglycoside nephrotoxicosis

SUMMARY Acute nephrotoxicosis was induced in ewes by daily sc administration of gentamicin. Activity of 3 urine enzymes, γ-glutamyltransferase (ggt), β-N-acetylglucosaminidase (ags), and β-glucuronidase (grs), were measured during the development of aminoglycoside nephrotoxicosis. Measurements from timed, volume-measured urine samples were performed on days 0, 7, and 8. Measurements from urine samples obtained without volume measurement (spot samples) were performed daily. Urine ggt and ags activities were high 3 days prior to detection of high serum creatinine concentration and 1.5 days before the appearance of casts in the urine sediment; values consistently remained in the abnormal range until termination of the study. High urine grs activity was inconsistent and transient; serum ggt activity did not change during the course of the study. Urine ggt and ags activities expressed as total excretion per unit time and body weight, enzyme activity per unit volume, and as ratio of urine enzyme activity to urine creatinine concentration were strongly correlated. Urine ggt and ags, but not grs activities, are suitable indicators of renal tubular cell damage in sheep with aminoglycoside nephrotoxicosis. Urine ggt and ags activities indicate cellular changes occurring several days prior to the first indications of renal functional change.

Effects of High Energy Shock Wave Exposure on Renal Function during Extracorporeal Shock Wave Lithotripsy for Kidney Stones

In order to study the effects of high energy shock wave exposure on the kidney in extracorporeal shock wave lithotripsy (ESWL) using Dornier HM3, renal hemodynamics and renal function before and after ESWL were analyzed by 99mTc-DTPA renoscintigraphy. Various urinary enzyme activities (LDH, GOT, GPT, NAG, gamma-GTP) and low molecular protein concentrations (alpha 1-microglobulin, beta 2-microglobulin) before and after ESWL were also compared. In the early phase of the renoscinitgram obtained in the 1st min after injection of 99mTc-DTPA, the time required to reach maximum radioactivity was significantly prolonged after ESWL in both the affected and contralateral kidney. This indicated that renal blood flow decreased in both the affected and contralateral kidney immediately after ESWL. An analysis of the 30-min renoscintigram showed that urinary clearance was delayed in the affected kidney in spite of no overt obstruction due to stone fragments. As for urinary enzyme activities and low molecular protein concentrations, they were standardized by urinary creatinine concentration measured at the same time. Urinary LDH, GOT, GPT and NAG activities remarkably increased on the day of ESWL followed by a decrease close to pretreatment levels on the 4th day, though these levels were still significantly higher than pretreatment levels. Urinary gamma-GPT activity was significantly higher than the pretreatment level only on the day of ESWL. Urinary alpha 1-microglobulin and beta 2-microglobulin concentrations significantly increased on the day of ESWL and were still high on the 4th day.(ABSTRACT TRUNCATED AT 250 WORDS)

糖尿病における尿中酵素測定の臨床的意義

The aim of this study was to clarify the clinical significance of urinary enzyme activity in patients with diabetes mellitus. Patients were divided into two groups: group A - 102 outpatients, group B-23 inpatients. Spot urine samples before breakfast from group A and aliquots of 24-hours urine collections at 4 degrees C from group B were used. Urinary enzyme activities (N-acetyl- beta-D-glucosaminidase: NAG, alkaline phosphatase: ALP, leucine aminopeptidase: LAP, gamma-glutamyl transpeptidase: gamma-GTP) were determined by spectrophotometric assay, rate assay, Tuppy method and Orlowski method, respectively. 1) In group A, the percentage of the cases which showed higher than the normal range (NAG: 1.3-8.7, ALP: 4.2-17.7, LAP: 0-22.9 U/g. cer.) was 42.2% in NAG, 21.6% in ALP, and 8.8% in LAP. In a multiple regression analysis, the predictor variables which contributed to NAG were HbA1c, age, urinary protein and the one that contributed to ALP, LAP, gamma-GTP was urinary beta 2-microglobulin. 2) In group B, 87% of NAG was above the normal range (Mean +/- 2 SD; 4.8 +/- 3.9 U/day). There was no difference in the NAG activity between patients with and without nephropathy. The percent of high activities of ALP, LAP and gamma-GTP were 17%, 17%, 4%, respectively. Most of them were patients with nephropathy. There were correlations among ALP, LAP and gamma-GTP, though no correlation existed between NAG and the other three enzymes. These results suggested: 1) NAG reflects lysosomal dysfunction of both glomerular and proximal tubular epithelial cells which may be caused by poor glycemic control.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary enzyme activities, low molecular weight protein and microalbuminuria in gouty patients

Urinary enzyme activities, low molecular weight protein and microalbuminuria in gouty patients

Studies on the Nephrotoxicity of Aminoglycoside Antibiotics and Protection from These Effects (9): Protective Effect of Inositol Hexasulfate against Tobramycin-Induced Nephrotoxicity

We examined the protective effect of inositol hexasulfate (IS6) against tobramycin (TOB)-induced nephrotoxicity. In the electrophoretic analysis, TOB alone and IS6 alone were observed as single spots on the cathode and anode sides, respectively. However, in the mixture of TOB and IS6 preincubated at 37°C for 3 hr, the tailing of the spots of TOB and IS6 were observed from the origin to the cathode and the anode sides, respectively, and the overlapping of the spots of TOB and IS6 was recognized at the origin. These results indicated that TOB directly interacted with IS6 in vitro. Assay of TOB binding to rat kidney brush border membranes (BBMs) indicated that IS6 inhibited the binding of TOB to BBMs through an interaction of TOB and IS6. No significant reduction in intrarenal TOB level was observed in the rats given TOB (90 mg/kg, s.c.) and IS6 (153 or 610 mg/kg, S.C.). However, the treatment of rats with a combination of TOB and IS6 reduced the degree of necrosis of renal tubular cells and also suppressed the increases in urinary protein, urinary enzyme activities, blood urea nitrogen and plasma creatinine induced by TOB. Additionally, we detected a complex of TOB and IS6 in the urine of rats given both compounds simultaneously. These results indicate that IS6 protects against TOB-induced nephrotoxicity and that the protective action of IS6 may be due to the inhibition of TOB binding to BBMs through an interaction of TOB with IS6.

A Method for Determining Urinary Enzyme Activities as Nephrotoxic Indicators in Rats

A simple, useful method was developed for detecting drug nephrotoxicity in rats. Rat urine excreted by stimulation of the sacral part of the back was collected in a beaker, and enzymes in 0.2 ml of the urine were partially purified by centrifugal ultrafiltration using an Amicon MPS-1 kit. Activities of N-acetyl-β-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), γ-glutamyltranspeptidase (γGTP) and lactate dehydrogenase (LDH), and the protein concentration of the enzyme preparation suspended in phosphate-buffered saline were represented as creatinine ratios. A marked increase in these enzyme activities and protein concentration were observed in rats with kidneys damaged by treatment with cephaloridine, HgCI2, cisplatin or gentamicin. The patterns of increase of these indicators differed with the drug, but in general, LDH showed the highest response and NAG, the longest lasting one. These data agreed with the results reported by other researchers using dialyzed 24-hr urine. Thus, we concluded that this method is an efficient one for determining drug nephrotoxicity in rats.

Elevation of urinary N-acetyl-beta-D-glucosaminidase and beta-galactosidase activities in workers with long-term exposure to aromatic nitro-amino compounds.

Aromatic nitro-amino compounds are used as raw materials of dyes, rubber, pesticides and drugs. A common toxicological reaction to these compounds is the formation of methemoglobin followed by cyanosis and anemia. Little is known about renal damage in workers occupationally exposed to aromatic nitro-amino compounds. Recently, certain urinary enzyme activities have been measured as an index of renal damage. In animal experiments, the activity of urinary N-acetyl-{beta}-D-glucosaminidase (NAG) was increased in rats given p-aminophenol. The authors assay urinary enzyme activities of workers handling aromatic nitro-amino compounds, and discuss the nephrotoxic effects of these compounds used industrially.

Effects of prenatal methylmercury exposure on urinary proximal tubular enzyme excretion in neonatal rats

The basal developmental pattern of excretion of 3 proximal tubular enzymes was determined in 8-h urinary specimens from neonatal rats. Gammaglutamyltransferase (GGT), alkaline phosphatase (ALP), and N-acetyl-β-glucosaminidase (NAG) activities were measured at 3, 6, 9 and 12 days after birth. Subsequently, methylmercury chloride (CH 3HgCl), known to induce foetotoxic changes in the proximal tubule was administered on 8 days, 10 and 12 of gestation at 3 or 6 mg/kg and its effects on the enzyme activities were examined. Dose-related increases in the 3 enzyme activities occured at dose levels that produced no maternal or postnatal toxicity, nor overt morphological malformation of the kidney. The peak of enzyme activities averaged about 200% and 130% of the control values for GGT, ALP, and NAG respectively, and occured on days 3 and 6 in the treated groups. Urinary enzyme activities returned to the control levels from days 6 to 12. Our data point to the possibility of detecting CH 3HgCl-induced prenatal effect on the kidney by measuring the 8-h urinary excretion of enzymes by rats in the early postnatal period.

Use of urinary enzymes as markers of nephrotoxicity

Urinary excretion of enzymes can be monitored continuously at low cost, with commonly available laboratory equipment; there is little discomfort for the animal or person investigated. Under controlled conditions, the time-course and dose-dependence of some pathophysiological events in the kidney can be followed by measuring urinary enzymes. The activities of enzymes in urine are, however, affected by a large number of parameters, even under physiological conditions. Furthermore, the heterogeneity of the kidney renders application of urinary enzymes for diagnostic purposes difficult. Assessment of renal injury by urinary analysis may be possible only if the pathophysiological mechanisms and the time sequence of the nephrotoxicity are known.

Evaluation of the nephrotoxicity of aromatic nitro-amino compounds by urinary enzyme activities.

Nephrotoxicity of some aromatic nitro-amino compounds were evaluated by urinary enzyme activities and renal histopathological changes. Male Fischer 344 rats were intraperitoneally injected with aniline, p-aminophenol, acetyl-p-aminophenol, p-chloroaniline, p-chloronitrobenzene, p-anisidine, or p-nitroaniline at 1.0 mmol/kg. In the rats injected with p-aminophenol, necrosis of renal tubular epithelial cells and remarkable elevation of urinary N-acetyl-beta-D-glucosaminidase (NAG) and gamma-glutamyltranspeptidase (gamma-GTP) activities were observed. Injection with p-chloroaniline caused significant elevation of the urinary NAG and gamma-GTP activities. p-Anisidine and p-nitroaniline induced swelling of the tubular epithelial cells and a significant elevation in urinary NAG activities in rats, which was also caused by p-chloronitrobenzene. However, administration of aniline or acetyl-p-aminophenol did not change either the urinary enzymes or renal histopathology. These results indicate that p-aminophenol is a highly nephrotoxic substance, and that nephrotoxicity of p-chloroaniline, p-chloronitrobenzene, p-anisidine and p-nitroaniline exceed that of acetyl-p-aminophenol which has been known to cause a renal damage.

Urinary excretion of N-acetyl-β- d-glucosaminidase and alanine aminopeptidase during pregnancy

The assay of enzyme activity in urine seems a reliable and safe method to monitor different kidney diseases. However, its use in pregnant patients might be limited by the modifications of kidney function during pregnancy. The aim of the present study was to evaluate the trend of excretion of the lysosomal enzyme N-acetyl-β- d-glucosaminidase (NAG) and the brush border enzyme alanine aminopeptidase (AAP) during uncomplicated pregnancies. NAG excretion showed a significant increase (P < 0.001) throughout pregnancy, while no significant modification of AAP levels was demonstrated. These data support the hypothesis that the two enzymes are excreted into the urine through different mechanisms and might constitute markers for different pathological events. As the increase of NAG excretion may be related to the kidney functional adaptation to pregnancy, different cut-off limits must be established in this period.

Therapeutic effects and influence on the urinary enzyme activity of human urinary trypsin inhibitor (ulinastatin) in cases with acute renal failure-Changes in the urinary activities of NAG and AAP

Therapeutic effects and influence on the urinary enzyme activity of human urinary trypsin inhibitor (ulinastatin) in cases with acute renal failure-Changes in the urinary activities of NAG and AAP

Quantitative morphologic changes in nephron structures and urinary enzyme activity pattern in sodium-maleate-induced renal injury.

The morphologic changes in sodium-maleate-induced acute renal injury in the rat were quantified by a stereologic analysis. The major changes were confined to an increase in endocytic vacuoles and a decrease in mitochondrial inner membrane surface area. These results were found to be linked to significantly increased urinary activities of the cytosolic of the cytosolic enzymes fructose-1,6-bisphosphatase (FBP) and lactate dehydrogenase, the lysosomal enzyme N-acetyl-beta-glucosaminidase (NAG) and the NAD-dependent mitochondrial isocitrate dehydrogenase (ICDH). The highest increase was found for NAG, followed by FBP and ICDH.

Urinary mercury levels and early changes in kidney function in dentists and dental assistants.

Mercury exposure and renal function parameters were examined in 68 dentists and 64 dental assistants. The levels of mercury in urine were low: only three individuals exceeded 20 micrograms/l. Increased excretion of urinary proteins and increased activity of urinary enzymes were observed. This enhanced prevalence of renal function changes appeared not to be related to the mercury urine level, age, sex, or smoking and drinking habits. Only for men was a positive relation between the level of mercury in urine and the activity of beta-galactosidase found. The proteinuria may be due to one or more potential nephrotoxic agents used in dental practice.

Urinary enzyme excretion after donor nephrectomy.

A number of recent studies of long-term kidney donors have reviewed glomerular function and blood pressure. Little attention has been paid to the potentially damaging effects of compensatory hyperfiltration on renal tubular cells after donor nephrectomy. The urinary excretion of high-molecular-weight enzymes is a sensitive indicator of renal tubular cell damage. This study compares the urinary excretion of four enzymes (alanine aminopeptidase, alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase) in a group of 77 subjects who had undergone unilateral nephrectomy up to 21 years previously with 52 healthy non-nephrectomized controls. The urinary excretion for all four enzymes by the remaining kidney after contralateral nephrectomy in the kidney donors was significantly greater than the enzyme excretion per single kidney in the control group (p less than 0.001). No correlation was found between the degree of enzymuria and either glomerular filtration rate or time since nephrectomy. The elevated activity of urinary enzymes in kidney donors may be related to increased metabolism by the renal tubular cells after contralateral nephrectomy. This study suggests that long-term compensatory hyperfiltration does not damage tubular cells, at least over this time scale.

Urinary enzyme activities in patients treated with gold and other antirheumatic drugs

We have studied 48 rheumatoid arthritis (RA) patients treated with nonsteroidal antiinflammatory drugs (NSAIDs), except salicylates, in 31 of whom parenteral gold was associated as therapeutic agent. In order to assess initial tubular involvement, the activities of some urinary enzymes were measured: N-acetylglucosaminidase (NAG, EC 3.2.1.30), microsomal amino-peptidase (MAP, EC 3.4.11.2) and gamma-glutamyltransferase (GGT; EC 2.3.2.2). Results were compared with a control group of 51 subjects of similar age, with no rheumatic symptoms and normal renal function. Both groups of patients (31 with gold therapy and 17 without) showed a significantly increased activity of NAG in urine, but the increase was greater in those treated with gold. MAP and GGT were not elevated significantly in either group. There was no correlation, however, between the increase of NAG and the cumulative dose of gold. NAG, MAP and GGT activities in serum yielded no relevant information. All the usual tests of renal function were also normal. Determination of NAG in urine may be regarded as a sensitive test, capable of detecting selective involvement of renal tubular cells, whose final diagnostic and prognostic significance merits further evaluation.

Protective effect of verapamil on ischaemic renal injury in dogs.

Three groups of female mongrel dogs were used. In each dog right nephrectomy was performed. In the first group, 60-min complete occlusion of the left renal artery was followed by 50.4% reduction in RBF (P less than 0.05), 53.4% reduction in GFR (P less than 0.01) and 46.7% reduction in urine flow rate (UV) (P less than 0.05). These haemodynamic changes were accompanied by a significant (P less than 0.01) increase in urinary enzyme activity. In the second group, following Verapamil 0.5 mg/kg i.v. bolus, there was diuresis and natriuresis, urine flow rate (UV) increased by 39% and UNaV by 60% (P greater than 0.05); there were no changes in RBF or GFR. In the third group, pretreatment with Verapamil prior to the arterial occlusion modified the deleterious effects of the 60-min renal ischaemia. RBF decreased by 9.4%, GFR by 28.5% and UV by 11.1% (P greater than 0.05); there was no change in the urinary enzyme activity. It is concluded that Verapamil can protect against ischaemic renal damage.