Early urinary markers of target nephron segments as studied in cadmium toxicity.

A number of chemicals may adversely affect one or more of the anatomical structures of the kidney, such as the glomerulus, the tubular apparatus, the medullary, or interstitial cells. To recognize subclinical renal dysfunction, a battery of new, non-invasive tests was applied in comparison to established ones. The study on cadmium exposed subjects, performed within the framework of a collaborative European research project, exemplifies the concept of target selectivity within a nephron. One hundred seventy-two subjects were classified according to urinary cadmium excretion as controls (< 1.5 micrograms/g creatinine), or subjects with moderate or high cadmium body burden (1.5 to 5 micrograms/g creatinine, > 5 micrograms/g creatinine). Twenty-six urinary analytes (such as serum derived proteins, tubular enzymes, eicosanoids) and four plasma markers, related to the function or integrity of specific nephron segments, were investigated in a cross-sectional study. The group with the moderate cadmium body burden showed alterations of proximal tubular integrity, that is, increased excretion of tubular brush-border antigens. The group with higher cadmium body burden revealed an involvement of the whole nephron. The most prominent quantitative changes were found for the glomerular markers high molecular weight proteins, and thromboxane B2 and for the proximal tubular markers retinol binding protein, alpha 1-microglobulin, N-acetyl-beta-D-glucosaminidase, and the intestinal alkaline phosphatase. A diagnostic approach to screen for nephrotoxicity due to environmental hazards like cadmium should include proximal tubular markers (alpha 1-microglobulin and tubular enzymes, that is, intestinal alkaline phosphatase) but the measurement of glomerular markers is also advisable.

Changed excretion of urinary proteins and enzymes by chronic exposure to lead.

Fifteen various serum and urine parameters were evaluated as indicators of renal alterations induced by lead in 82 male workers of a battery plant chronically exposed to lead (median of blood lead concentration: 2.03 mumol/l). The control group comprised 44 non-exposed healthy volunteers (0.34 mumol/l). High-molecular-mass proteins (transferrin, immunoglobulin G (IgG), (albumin)) were determined in urine as markers of glomerular integrity; low-molecular-weight proteins and parenchymal enzymes (alpha 1-microglobulin, beta 2-microglobulin, retinol-binding protein, lysozyme, ribonuclease, N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), alkaline phosphatase (AP), gamma-glutamyltransferase (GGT)) as indicators of changes in the proximal tubule; Tamm-Horsfall glycoprotein and kallikrein as markers of the distal tubule. There was a positive correlation between tubular indicators and blood lead concentration as well as the erythrocyte protoporphyrin (EPP). About 30% of the lead-exposed workers showed an increased excretion of alpha 1-microglobulin, NAG, ribonuclease, and/or Tamm-Horsfall protein, whereas the glomerular indicators remained unchanged. The combined determination of NAG and alpha 1-microglobulin in urine could be helpful in the early detection of lead-induced changes in the nephron.

Nephron target sites in chronic exposure to lead.

With established urinary markers of kidney integrity the early renal effects of lead have previously been considered to be mainly tubular or tubulointerstitial. In a cross-sectional study on 81 male lead-exposed workers and 45 age-matched controls (median blood lead concentrations 2.03 and 0.34 mumol/l respectively) not only well-established but also new urinary markers of renal integrity preferentially or exclusively located along the different nephron segments were analysed. Markers related to the glomerulus were 6-keto-prostaglandin 1 alpha, thromboxane B2, mainly produced in the glomerulus, and the extracellular matrix protein fibronectin. Markers of the proximal tubule were the brush-border antigens BBA, BB50, and HF5 and the intestinal alkaline phosphatase. Prostaglandin E2 and F2 alpha, preferentially synthesized in the collecting duct and medullary interstitial cells, served as markers of these more distal nephron segments. In contrast to previous studies on the early phase of lead nephrotoxicity, not only tubular but also glomerular involvement could be shown in the study presented here by increases in the median values of 6-keto-prostaglandin 1 alpha and decreases in fibronectin. The proximal tubular markers intestinal alkaline phosphatase and BBA confirmed that this particular segment of the nephron is affected by lead. Effects on the collecting tubule or medullary interstitial cells could also be observed. It is concluded that lead affects both the glomerulus and the tubular apparatus and that combinations of new and established markers could be valuable for a better definition and early detection of lead nephropathy.

Rapid screening of low molecular mass proteinuria: evaluation of the first immunochemical test strip for the detection of alpha 1-microglobulin in urine.

A new semiquantitative immunochemical test strip for urinary alpha 1-microglobulin, a marker protein for tubular proteinuria, was assessed. This test strip has four colour zones, reflecting alpha 1-microglobulin concentrations of ca. 10, 25, 50, and 80 mg/l. alpha 1-Microglobulin concentrations were measured by means of the test strip and an immunonephelometric method in 330 samples collected as the second voided morning urine. The reading time of the test strip must be strictly observed. Reading one minute earlier or later than the 5 min stated in the instructions led to misclassification of over 70% of the results. Correlation between both methods was highly significant, with a Spearman rank correlation coefficient of rs = 0.84 (P < 0.001). There was a partial overlap of the test strip results in different concentration ranges. An elevation of alpha 1-microglobulin was defined as > 25 mg/l, calculated as the upper limit of the central 95% interval of alpha 1-microglobulin concentration in urine samples measured in a previous study of 304 healthy adults. Using this definition of alpha 1-microglobulin elevation, a sensitivity of 97.5%, specificity of 73.6%, a false-positive rate of 16.6%, and a false-negative rate of 0.9% of the test strip results were obtained. A fraction of 82.4% of the 330 samples investigated was correctly classified as having increased alpha 1-microglobulin concentration or not. Methodical improvements of the test strip are necessary to reduce overlapping results, in order to make the test suitable for screening purposes.

Sandwich enzyme immunoassay of cystatin C in serum with commercially available antibodies.

We developed a sandwich enzyme immunoassay for determining cystatin C in serum by using commercially available antibodies. We optimized each assay step (e.g., concentrations of coating rabbit anti-human cystatin C antibodies and horseradish peroxidase-conjugated antibodies) and studied the binding kinetics of antigen and antibodies. The within-assay CV was < 5%, the between-assay CV was 8.8%, the detection limit was 0.9 microgram/L, and the assay can be performed within 2 h. Cystatin C concentrations in sera from men were significantly higher than in women (mean and SD: 2.14 +/- 0.31 vs 1.78 +/- 0.26 mg/L). We studied the cystatin C concentrations in sera of 31 outpatients with suspected kidney damages to characterize the behavior of this low-M(r) protein as a possible indicator for estimating the glomerular filtration rate. The correlation with the values obtained by a standard isotopic method involving 99mTc-diethylenetriaminopentaacetic acid was rs = -0.89. The diagnostic sensitivity of cystatin C was 88.2% of that of the standard isotope clearance method and better than those of the conventional serum indicators of reduced kidney function, beta 2-microglobulin (64.7%) and creatinine (52.9%).

Urinary proteins and enzymes as early indicators of renal dysfunction in chronic exposure to cadmium.

We tested the diagnostic sensitivity of various urinary analytes for detecting cadmium-induced nephropathy at an early stage. We investigated 73 healthy persons (control group 1) and individuals exposed to cadmium, either environmentally (n = 36, risk group 2) or occupationally (n = 62, exposed group 3). All data were related to limits of the central 95% reference intervals of the control group. The serum creatinine and ribonuclease values, indicators of the glomerular filtration rate, were not different in the three groups. In the exposed persons (group 3), proximal tubular indicators (low-M(r) proteins lysozyme, ribonuclease, retinol-binding protein, and alpha 1-microglobulin) were more often increased than the glomerular indices (higher-M(r) proteins transferrin, IgG, and albumin). Both the low-M(r) proteins and tubular enzymes were differently altered in their excretion rates. Alanine aminopeptidase, alkaline phosphatase, and N-acetyl-beta-D-glucosaminidase increased even in the risk group 2. alpha 1-Microglobulin was increased in the exposed persons whose cadmium excretion was < 5 mumol/mol creatinine. The combined determination of alpha 1-microglobulin and N-acetyl-beta-D-glucosaminidase exceeded the corresponding upper reference limits in 30% of group 2 and 39% of group 3. We recommend screening for these two analytes to detect cadmium-induced renal dysfunction at an early stage.

Development of an immunoenzymometric assay for alpha 1-microglobulin and measurement of its serum concentration in normal and HIV-infected persons.

alpha 1-Microglobulin was purified from urine to a purity of 97.7% in a yield of 25.8%, and was used to produce antibodies in sheep. These antibodies, purified by affinity chromatography, were used to develop a rapid one-step and a two-step immunoenzymometric assay (IEMA). The equilibrium in the reaction between solid phase-adsorbed antibodies and antigen and between the antigen and enzyme-labelled antibodies was attained within 30 and 100 min, respectively. The one-step IEMA permits a good differentiation of low alpha 1-microglobulin concentrations after 30 min reaction time. Its detection limit is 0.35 micrograms/l, and its measurement range is between 0.5 and 100 micrograms/l. The IEMA correlates well with radial immunodiffusion (r = 0.973). The mean alpha 1-microglobulin serum concentration in women is insignificantly lower (33.2 mg/l) than in men (36.1 mg/l). In both sexes the alpha 1-microglobulin concentration increases with age. HIV-infected symptomless men have a significantly lower (15.9 mg/l) alpha 1-microglobulin concentration in serum than normal persons, whereas in AIDS patients it is significantly higher (45.5 mg/l).

A microalbuminuria assay using bromphenol blue.

A simple microalbuminuria assay using bromphenol blue/glycine reagent is described. Urine samples were prepared using gel filtration on Sephadex G-50 minicolumns and absorbance was measured at 610 nm 20 s after mixing 10 parts of eluate and 1 part of reagent. The detection limit of this method was 3 mg/l; within-run and between-run precision was between 0.5 and 4.1% for borderline and raised albumin concentrations. The recovery of albumin added to samples was 98.7 +/- 2.5%. Results obtained by this method correlated closely with values obtained by radial immunodiffusion (r = 0.987). The test is cheap (reagent costs about 5 cents) and suitable for the non-specialist laboratory.

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.

Measurement of lysozyme in human body fluids: comparison of various enzyme immunoassay techniques and their diagnostic application.

Three variants of the immunoenzymometric assay of human lysozyme with HRP-labeled antibodies were compared. The highest sensitivity (with a detection limit of 0.2 micrograms lysozyme/L) was achieved by a one-step assay lasting 2 h. Between-batch precision for the techniques was 6-11%. Lysozyme reference values were determined in serum, cerebrospinal fluid and urine. In serum they are age-dependent and in urine sex-dependent when related to creatinine excretion. Serum lysozyme is increased in only 57% of the patients with active rheumatoid arthritis and is also unreliable for indicating remission. In Crohn's disease the serum lysozyme reflects activity better, but it does not exceed the diagnostic value of alpha-1-acidic glycoprotein (orosomucoid). The lysozyme quantification in cerebrospinal fluid is useful in distinguishing between viral or bacterial meningitis.

Sialidase from different sources compared for electrophoretically separating serum alkaline phosphatase fractions from liver and bone.

We compared sialidase (neuraminidase; EC 3.2.1.18) from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens, seeking to improve the electrophoretic separation of the liver and bone isoenzymes of alkaline phosphatase (EC 3.1.3.1) on cellulose acetate membranes. Resolution is decisively determined by the type and activity of sialidase used in the preincubation of serum sample. Sialidase from Arthrobacter ureafaciens is not suited for this method. For optimal separation of the two isoenzymes we recommend the use of sialidase from Vibrio cholerae, determination of its activity with a standard procedure such as described here (mucin or sialyl lactose as substrates), and a final concentration of sialidase activity of 2.0 or 2.9 U/L (measured with mucin or sialyl lactose) in the incubation mixture.

Further evidence for tubular dysfunction in insulin dependent diabetes.

There is evidence that increased excretion of urinary enzymes and low-molecular mass proteins indicate impaired tubular function. The excretion of N-acetyl-beta-D-glucosaminidase (NAG), lysozyme, and ribonuclease in Type I diabetic patients with (n = 19) and without (n = 17) persistent proteinuria (urinary protein excretion greater than 0.5 g/day) was investigated and compared with this excretion in 30 weight- and gender-matched nondiabetic subjects without renal disease. Urinary NAG excretion was significantly higher in diabetic patients with and without persistent proteinuria (1.16 +/- 0.09 and 3.19 +/- 1.2 Umol/L creatinine, respectively) compared to controls (0.37 +/- 0.03 Umol/L creatinine p less than 0.01). In addition, the urinary excretion of lysozyme and ribonuclease was significantly increased in diabetic patients. Urinary NAG was found to correlate positively with albuminuria and proteinuria (r = 0.95 and 0.93, respectively), as well as with ribonuclease and lysozyme (r = 0.93 and 0.60; p less than 0.01) in patients with persistent proteinuria. Furthermore, NAG excretion was significantly related to the duration of diabetes (r = 0.36; p less than 0.05). No relationship existed between urinary NAG and serum creatinine, beta-2-microglobulin, and degree of metabolic control (HbA7). The lysozyme excretion, but not NAG excretion, was significantly related to hypertension in patients with clinical proteinuria. In conclusion, our results suggest a relationship between the development of tubular dysfunction and the impairment of glomerular function in diabetic nephropathy. An increased excretion of NAG and low-molecular mass proteins may indicate early nephropathy

[Urine enzymes and low molecular weight proteins as indicators of diabetic nephropathy]. / Harnenzyme und niedermolekulare Proteine als Indikatoren der diabetischen Nephropathie.

The urinary enzymes alanine amino-peptidase, alkaline phosphatase, gamma-glutamyltransferase and N-acetyl-beta-D-glucosaminidase and the two urine low-molecular mass proteins lysozyme and ribonuclease were measured in 30 healthy men and 36 insulin-dependent diabetics. 17 diabetics had "clinical proteinuria" (greater than 7.5 g/mol creatinine) and were defined as patients with manifest diabetic nephropathy. The remaining 19 diabetics were without proteinuria. The excretion rates of the two urine proteins and all enzymes except for gamma-glutamyltransferase were the highest in patients suffering from diabetic nephropathy. The excretion rates in both diabetic groups exceeded those of the control group. N-Acetyl-beta-D-glucosaminidase was more often increased than albumin in diabetics without manifest diabetic nephropathy. It is concluded that the tubular dysfunction is an early indicator of the incipient diabetic nephropathy. Thus, tubular parameters, especially the lysosomal enzyme N-acetyl-beta-D-glucosaminidase may be used in follow-up studies of diabetics.

[The low molecular weight proteins ribonuclease, beta 2 microglobulin and lysozyme in the serum and urine of patients with chronic kidney diseases]. / Die niedermolekularen Proteine Ribonuclease, beta 2-Mikroglobulin und Lysozym im Serum und Urin von Patienten mit chronischen Nierenerkrankungen.

The diagnostical relevance of the low-molecular proteins ribonuclease, beta 2-microglobulin and lysozyme in serum and urine to detect a reduced glomerular filtration rate was examined in 52 patients with chronic renal diseases. The radioisotope clearance using 99mTc-DTPA was the base reference; the reference values of the low-molecular proteins were estimated in a control group. Ribonuclease was increased above the upper borderline value, if the glomerular filtration rate was lower than 1.24 ml s-1. Creatinine, beta 2-microglobulin and lysozyme remain yet in part in the normal range. The estimation of the ribonuclease in serum is suitable to detect an impaired glomerular filtration rate if the creatinine value is still not increased. Thereby, the diagnostics in renal diseases may be improved in the creatinine-blind area.

Urinary enzymes and low-molecular-mass proteins as indicators of diabetic nephropathy.

We measured the excretion rates of six urinary enzymes that either originate from the proximal renal tubule, like alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), and N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), or that are typical low-molecular-mass proteins, like lysozyme (EC 3.2.1.17) and pancreatic ribonuclease (EC 3.1.27.5). These rates were compared with those of total protein and albumin in urine of 36 insulin-dependent diabetic men and 30 healthy men. Seventeen of the diabetics had "clinical proteinuria," defined as excretion of more than 7.5 g of protein per mole of urinary creatinine (group B). Group A comprised the 19 diabetics without proteinuria. Except for gamma-glutamyltransferase, the excretions of enzymes and proteins were significantly higher in diabetics than in controls and were greater in group B than in group A. N-Acetyl-beta-D-glucosaminidase was the analyte most often increased in group A (89%), followed by albumin and alkaline phosphatase (each 32%). All patients in group B showed increased excretion of N-acetyl-beta-D-glucosaminidase. We conclude from the comparative data that this enzyme may be useful as an early predictor of diabetic nephropathy.

Diuresis-dependent excretions of low-molecular mass proteins in urine: beta 2-microglobulin, lysozyme, and ribonuclease.

An investigation was carried out into how the low-molecular mass proteins beta 2-microglobulin, lysozyme, and ribonuclease were excreted over 8 h after high fluid intake (22 ml/kg of body weight in 15 min). With increasing urine flow rate the amount of lysozyme excreted per hour or per millimole creatinine increased more markedly than that of beta 2-microglobulin while at the same time the excretion rate of ribonuclease decreased. The effect of urinary flow upon the excretion rates of the various low-molecular mass proteins has to be considered as a preanalytical factor when these proteins are used as indicators of tubular dysfunction.

A rapid and sensitive enzyme immunoassay for Cu/Zn superoxide dismutase with polyclonal and monoclonal antibodies.

An enzyme immunoassay for the quantification of human Cu/Zn SOD in serum, urine and erythrocytes was developed applying monoclonal and polyclonal antibodies. The one-step assay is completed within 30 min and enables the detection of 0.3 microgram Cu/Zn SOD per litre. A Cu/Zn SOD concentration of 46 +/- 21.5 micrograms/l and of 1 +/- 0.6 micrograms/mmol creatinine was determined in the serum and the urine, respectively, of healthy individuals. A content of 15 +/- 1.7 ng Cu/Zn SOD was found in 10(6) erythrocytes. Patients with Down's syndrome exhibited a 3.8-fold, a 2-fold and a 1.6-fold higher concentration of Cu/Zn SOD in their serum, urine and erythrocytes.

Different susceptibility of cortical and medullary rat kidney mitochondria to ischemic injury.

Rat kidneys were exposed for different times to in vitro ischemia at 37 degrees C. Then the mitochondria of cortex and medulla were isolated and their respiratory qualities were determined. In dependence on the ischemic time, the active respiration (state 3) and the respiratory control index decreased whereas the resting state respiration (state 4) increased. The cortical mitochondria were affected more strongly by ischemia than medullary mitochondria. Therefore, the different susceptibility of cortical and medullary mitochondria to ischemia does not explain the suggested high risk of medulla for ischemia.