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In a study of the value of urinary enzyme activities as an indication of glomerular or tubular damage, the effects of two nephrotoxic agents (4-nitrophenylarsonic acid and rabbit anti-rat kidney serum) on the urinary excretion of four β-glycosidases were compared using fluorimetric assays with 4-methylumbelliferyl substrates. The electrophoretic mobilities on starch gel of urinary β-galactosidase, β-glucosidase, β-glucosaminidase and β-glucuronidase, at various times up to 26 days after the injection of the nephrotoxins into the rat, were compared with those of the corresponding enzymes present in normal rat urine, kidney and serum. 4-Nitrophenylarsonic acid causes tubular damage characterised by an immediate rise in the rates of excretion of the four β-glycosidases, followed by a return to normal values. In Masugi glomerulonephritis the immediate rise in enzyme excretion is much less marked but greater increases occur after 10 to 12 days. The increases in excretion rate correlate roughly with the stages of the disease. Both glomerular and tubular damage produce characteristic changes in the excretion of all of the enzymes studied, but β-glucosidase appears to be the most useful indicator of kidney tubular damage, as it is the least widely distributed of these enzymes and is not normally found in urine or serum at significant levels of activity.

The diurnal variation of urinary N- acetyl -β- glucosaminidase and β-galactosidase in man was examined by a fluorimetric assay method and it was concluded that normal and abnormal excretion rates can be distinguished by analysis of a single random sample. The changes in the rate of excretion of these enzymes following surgery to the kidney were investigated in 13 cases. Immediate rises in excretion were found when the kidney sustained severe damage. The rise was roughly proportional to the degree of damage suffered. Nephrectomy, or mobilisation of the kidney, produced no change in the excretion rate. It was found by starch gel electrophoresis that a form of N- acetyl -β- glucosaminidase not normally found in the urine or plasma, but present in the kidney, was excreted by patients having damaged kidneys. The results presented suggest that the study of the urinary excretion of β-glycosidases may be of value in assessing the presence and extent of kidney damage.

The labelling of pepsin-digested human glomerular basement membrane (pHGBM) with a newly developed fluorescent iodine acceptor 7-hydroxy-coumarin-3-acetic acid N-hydroxysucciniimydyl ester (CASE) is described. The binding of a monoclonal antibody to pHGBM was assessed by radiobinding assays, and when directly iodinated pHGBM was used there was no apparent binding. When CASE was conjugated to pHGBM prior to iodination 11 % binding was achieved. CASE acting as an iodine acceptor may be useful for proteins containing few or inaccessible tyrosine residues or which are destroyed by introduction of 125I. Since CASE is fluorescent, small amounts of material can be detected during isolation prior to iodination.IodinationProtein labellingHuman glomerular basement membrane7-Hydroxy-coumarin-3-acetic acid N-hydroxysucciniimydyl esterFluorescence

A new fluorescent protein labelling agent, 7-amino-4-methyl coumarin-3-acetic acid (AMCA), emits in the blue region (440-460 nm) on activation with UV light (350 nm). The active reagent is the N-hydroxysuccinimide ester which reacts with lysine residues under mild conditions to form photostable amide links. The Stokes shift of 100 nm compared to 30 nm for Fluorescein isothiocyanate (FITC) allows easy filter discrimination of exciting and emitting radiation. The agent has been demonstrated in use for fluorohistochemical examination of human kidney glomeruli, using the sandwich technique and compared with the same procedure using FITC-labelled antibodies. The good quantum yield coupled with convenient emission lines in the mercury spectrum allows photographic exposure time of fluorescent labelled sections to be reduced to a quarter of that required for a corresponding FITC conjugate. AMCA-immunoglobulin conjugates were not susceptible to photobleaching and have a storage life at -20 degrees C of more than two years.

1. 1. The A, B, I 1 and I 2 forms of N -acetyl-β-glucosaminidase present in urine, serum, kidney, liver and cerebral spinal fluid were separated on DEAE-cellulose and their presence confirmed by cellogel electrophoresis. The relative activities of each enzyme were determined by integrating the area under the elution peaks. 2. 2. Serum A-form was eluted at a lower molarity of chloride than liver A-form and this was designated the A s -form to distinguish it from the A-form of N -acetyl-β-glucosaminidase found in liver and kidney. 3. 3. The P-form of N -acetyl-β-glucosaminidase present in the serum of a group of pregnant women was not detectable in urine samples from the same women. 4. 4. Urinary NAG activities were found to be abnormally high in patients with impaired renal function. 5. 5. The activity of both N -acetyl-β-glucosaminidases A and B increased in pathological urines. The higher the total N -acetyl-β-glucosaminidase activity excreted the higher the % of activity of the B-form present. 6. 6. In a number of patients with haematuria an A-form similar to the serum A s -form was present in the urine.

Renal transplant recipients were divided into five categories according to their clinical course from transplantation to their discharge from hospital. Total N-acetyl-beta-D-glucosaminidase (NAG) activity in urine was determined using a chromogenic substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The isoenzyme composition of the urine of each patient was determined by semi-automated DEAE-cellulose chromatography. Although raised NAG activity was found in stable transplant patients compared to controls, the level of activity was constant and no change in the isoenzyme profile was found. In reversible rejection there was a marked increase in the intermediate forms, particularly I2 and a concomitant fall in the relative amount of the A-form present but the profile became normal when the patient stabilised. Much more complex patterns were observed in patients who did not respond to treatment. Both the B and I forms were elevated with a fall in the A-form and in one case excretion of the serum As form was observed. The intermediate forms were always increased in rejection.

N-Acetyl-beta-glucosaminidase, beta-galactosidase, beta-glucosidase, acid and alkaline phosphatase were monitored in urine kidney homogenates and serum of rats with papillary damage induced with ethyleneimine. Serum urea levels, total protein in the urine and urine volume were monitored throughout the study. Histological studies showed that the injection of ethyleneimine caused immediate papillary necrosis, followed later by secondary cortical involvement. Minor papillary necrosis induced by a low dose (0.5 mul/kg) of ethyleneimine was characterised by a rise in urinary N-acetyl-beta-glucosaminidase activity which was followed later by an increase in the activity of the other enzymes monitored. More severe papillary necrosis induced with a higher dose of ethyleneimine (5.0 mul/kg) resulted in an immediate rise in the activities of all the urinary enzymes which then decreased only to rise again when cortical involvement occurred. Serum urea was unaltered but urine volume and protein were increased coincidentally with the urinary enzyme activities. The value of the assay of urinary enzymes in distinguishing papillary from glomerular and tubular damage is assessed. The possible relevance of the ethyleneimine model to the etiology of papillary nephropathy is discussed.

The nephrotoxicity of three different dose levels of propyleneimine (10, 20 and 30 microliter/kg body wt) administered intraperitoneally to rats was studied and 20 microliters/kg body weight was found to be the most appropriate sublethal dose. Injection of propyleneimine (10 microliters/kg body wt) produced a small rise in N-acetyl-beta-D-glucosaminidase (NAG) activity, minor histological damage but no change in urine volume. Six rats were injected with 20 microliters/kg body weight, and urine was collected over the following 16 days. An immediate increase in urine volume, osmolality together with a concomitant decrease in specific gravity, was accompanied by a small increase in creatinine excretion and a more marked increase in the sodium and potassium content of urine after the administration of the nephrotoxin. NAG activity increased immediately and peaked on day 3, the activity remained elevated until day 12 when it fell to near normal levels. The activity of both beta-D-galactosidase and beta-D-glucosidase increased 9 days after administration of the nephrotoxin. In contrast, no consistent change was found in the excretion of the brush border marker enzymes, leucine aminopeptidase (LAP), alanine aminopeptidase (AAP) or alkaline phosphatase (ALP). Proteinuria increased sharply the day after injection and remained abnormal. Increased urinary albumin excretion and the predominance of low molecular weight proteins was demonstrated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Evidence is presented that propyleneimine exerts its early toxic effect on the renal papilla.

Glomerular basement membrane (GBM) was prepared from 2- to 3-month-old Sprague-Dawley rats by differential sieving and sonication. 80% of the membrane was soluble in 1% sodium dodecyl sulphate (SDS) and 1% 2-mercaptoethanol. The soluble fraction was resolved into 15 bands in the molecular weight range 30,000 to 300,000 by SDS-polyacrylamide gel electrophoresis. The major bands present had apparent molecular weights of 100,000 and 148,000. Treatment of GBM with SDS alone solubilised mainly low molecular weight components (45,000-150,000) but when the residue was treated with SDS and 2-mercaptoethanol higher molecular weight material was solubilised. Partial solubilisation of GBM was also achieved with pepsin. Digestion for 18 h at 4 degrees C resulted in 20% of the membrane being solubilised but this was increased to 55% at 10 degrees C. The amino-acid composition of pepsin-soluble GBM was more collagen-like than the residue remaining after enzyme digestion. Although the residue was more polar than whole GBM it still contained significant amounts of glycine and hydroxyproline and could be further subfractionated with SDS into a soluble fraction, the amino acid content of which was similar to whole GBM and a collagenous residue containing 317 residues/1,000 of glycine. When pepsin solubilised GBM was subjected to horizontal electrophoresis in SDS-polyacrylamide slab gels the principal bands migrated in the pro-alpha-chain region. This material was heterogeneous and in addition to the principal components, 6 components with apparent molecular weights less than 95,000 were present together with high molecular weight material in the gamma and beta regions of the gel. The band pattern of the pepsin-insoluble material was similar although the intensities of some individual bands varied significantly from that of the pepsin-soluble material. Rat GBM can therefore be fractionated by treatment with SDS alone, SDS together with 2-mercaptoethanol and pepsin digestion. The data reported is compatible with the presence of a mixture of collagen-like and polar regions rather than a major single collagenous component (type IV collagen).

1. 1. Some practical aspects of the assay of urinary N -acetyl-β-glucosaminidase activity were studied in patients with renal disease. 2. 2. Centrifugation and dialysis of urine were not necessary prior to assay. 3. 3. When enzyme activity was related to urinary creatinine random urine samples could be used. 4. 4. Since preservatives used to prevent bacterial growth may inhibit enzyme activities, samples should be assayed immediately after collection or stored at 4° Cor-20° C. 5. 5. Mild visible haemoglobinuria, eg. derived from lysed red blood cells did not interfere with the enzyme assay.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were assayed in every urine void throughout 24 hours in 17 normal people and in four patients with renal disease. The variation in NAG activity due to changing rates of urine flow was almost eliminated by factoring enzyme activity by the urinary creatinine concentration. Random samples of urine may thus be used for assay. The results of NAG assay in 36 patients with acute and chronic renal diseases showed that NAG was a sensitive indicator of renal damage. This simple test may be valuable in detecting or monitoring renal disease.

1. (1) N -Acetyl-β- d -glucosaminidase (NAG), β- d -galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described. 2. (2) Thirty-five of the hypertensive patients were considered to have primary renal disease. The urinary activity of NAG was increased in 27 (77%) of these patients and the detection of primary renal disease was not enhanced by measurements of the other urinary enzymes. Testing the urine both for NAG activity and protein, led to the detection of 91% of these patients. 3. (3) The assay procedures described are simple to perform and can be carried out in outpatient clinics. The measurement of urinary NAG activity is a cheap and reliable method for detecting renal disease in hypertensive patients but maximum diagnostic yield is achieved when proteinuria is determined as well.

& Winzler, 1966). The composition of the insoluble residue of glomerular basement membrane resembled that of fibrillar collagen. In contrast, the soluble material contained more polar amino acids and was characterized by the presence of the sugars found in the heteropolysaccharide unit (Spiro, 1967). When solubilized glomerular basement membrane was subjected to electrophoresis in sodium dodecyl sulphate, 12 major and ten minor protein bands were separated. The mol.wt. range of the bands was 25000-240000. The best separation of subunits was obtained on 5 % gels. The principal bands were isolated and analysed for amino acid composition. The amounts of each component amino acid varied considerably between the bands, but in most bands there was relatively more of the polar amino acids than were present in whole basement membrane or the insoluble residue. However, in some of the isolated bands, recovered glycine values were similar to those found in the insoluble residue. This suggests that some collagen-like components are present. Lower than expected values for hydroxyproline were found in some bands on amino acid analysis and confirmed with the Woessner (1961) method. There was therefore a wider range of hydroxypro1ine:glycine ratio than was reported by Sat0 & Spiro (1976) for subunits of cow glomerular basement membrane. These studies have demonstrated the heterogeneous nature of the subunit composition of rat glomerular basement membrane and provide a useful starting point for future studies on the normal and pathological composition of glomerular basement membrane. These studies were supported by grants from the Wellcome Trust and National Kidney Research Fund (U.K.).

1. The yield of GBM from rat kidney increased with rat age and the membrane became more collagen-like, as judged by the glycine, hydroxyproline, hydroxylysine content and the degree of glycosylation. 2. GBM became progressively less soluble in sodium dodecyl sulphate (SDS) and 2-mercaptoethanol with age. An increase in the lower molecular weight subunits of membranes prepared from older rats was demonstrated by polyacrylamide gel electrophoresis.

The value of urinary enzymes as non-invasive tests of renal integrity in toxicology and medicine is reviewed. Urinary enzymes provide very sensitive indicators of renal damage and although a wide variety of enzymes have been assayed, only a few have proved useful because of practical considerations. The assay of NAG and AAP have proved to be most valuable, while other enzymes, including LDH, lysozyme, kallikrein, carbonic anhydrase C, arylsulphatase A, alkaline phosphatase and γGT have proved to be useful in particular situations. The diagnostic potential of urinary enzymes is often enhanced by the simultaneous assay of more than one enzyme, particularly if the activities of the enzymes used are high in different regions of the nephron. Isoenzyme studies provide additional diagnostic information. Urinary enzyme assays are most useful when carried out in conjunction with simple non-invasive test of renal function, e.g. osmolality, and protein selectivity. The marked susceptibility of the kidney to toxic damage and the effect of drugs on subcellular organelles are discussed. Further, the problems of assaying enzymes in urine are considered and the various ways in which they can be overcome is described. Recent improvements in techniques, including automation, miniphotometers and “dipstick” tests, are described. The importance of age is emphasised, and different ways of expressing enzyme activities, which allow for varying urine flow, is discussed. Urinary enzymes can play a role in the detection of nephrotoxicity, monitoring the level of safe doses in patients, the evaluation of new drugs and in the control of industrial workers using hazardous chemicals. The most important nephrotoxins are certain antibiotics, heavy metals, herbicides and anti-inflammatory drugs. Urinary enzymes have considerable potential value for the early warning of rejection in transplant patients, screening for renal disease and for the detection of renal damage secondary to hypertension, diabetes and rheumatoid arthritis. The review concludes by considering how urinary enzymes will be used in the future as an aid to the toxicologist and clinician.

Publisher Summary The chapter deals with the possible effects of dietary sucrose on kidney. Long-term feeding of sucrose results in diffuse intercapillary glomerulosclerosis. An early observation that the kidney weight in sucrose-fed rats was elevated indicated a possibility of renal damage as a result of sucrose feeding. The results indicated pathological change in the rat kidney following administration of sucrose-rich diets, and the observed changes in rat glomerularbasement membrane (G.B.M.) were reported. The changes resemble those found in diabetic microangiopathy. Prominent renal changes were deduced in rats fed high sucrose diets. Nephrocalcinosis in the cortico–medullary region was also present in sucrose-fed rats. The long-term feeding of sucrose-rich diets results in abnormality of the structure, composition, and metabolism of rat G.B.M.

1. 1. Two forms of acid p -nitrophenyl phosphatase activity are present in rat kidney separable from subcellular fractions of renal cortical homogenates by gel filtration on Sephadex G-100. 2. 2. The lysosomal and the microsomal enzymes had molecular weight (mol. wt) of greater than 100,000 and had similar kinetic and inhibitory properties. 3. 3. In contrast, the soluble fraction contained not only this high molecular weight form but also a lower molecular weight form (mol. wt 20,000). 4. 4. This latter form differed in its response to inhibitors, had different K m value but had the same acid pH optimum.

Some of the biochemical changes in rat kidney following the administration of mercuric chloride have been determined. Mercuric chloride had an immediate effect on the renal brush border resulting in rapid loss of the microvilli. Plasma membranes were isolated and characterised at various stages in the necrotic process, mircovilli were absent from these preparations and the activities of marker enzymes for the brush border were significantly decreased. In contrast the basal plasma membranes were unaffected by the nephrotoxin during the early stages and no change occurred in the activity of (Na+ + K+)-ATPase, a marker enzyme for the basal membranes. The change in the pattern of urinary enzyme excertion closely paralleled the ultrastructural changes in the tubular cells. The sequence of subcellular change following the administration of mercuric chloride is discussed in relation to the known mechanism of action of this agent.

1. 1. The hexosaminidase activity of normal human serum can be resolved into at least four components by DEAE-cellulose chromatography. 2. 2. The major part of the activity is in a form having similar properties to the hexosaminidase A of human tissues. 3. 3. Two new forms designated I 1 and I 2 , intermediate in electrophoretic mobility between hexosaminidases A and B, have been separated. The I 2 form could not be distinguished from the P form present in sera from pregnant women. The concentration of this component in serum increased on storage. A serum sample from a diabetic patient also contained a high proportion of the I 2 form. 4. 4. There was a small proportion of hexosaminidase B in all sera examined but this component was only present in trace amounts in freshly drawn plasma. 5. 5. Application of the heat denaturation treatment commonly used in the detection of Tay-Sachs heterozygotes showed that the B and I forms had similar heat stability and that the heat labile A form can be converted to both I 1 , I 2 and B forms by this procedure. 6. 6. The single heat-stable component found in the serum of a child with Tay-Sachs disease was an intermediate form and not hexosaminidase B.

The distribution of lactase in rat kidney cortex was found to be typical of a lysosomal enzyme, while maltase was associated with the plasma membranes. Trehalase, invertase and cellobiase activities were not detected.