Markers of tubular dysfunction

Toxicology Letters, 46 (1989) 197-204

197

TXL 02131

Markers of tubular dysfunction M. Piscator Department of Environmental Hygiene, Karolinska Institute, Stockholm (Sweden)

SUMMARY Since the first description to diagnostic

means

in the excretion mination information insensitive

of tubular

for detecting

of low-molecular-weight

of total

protein

is an economic

on the type of damage. methods,

effects

proteins

enzymes released of nephrotoxic

in 1958, much in the function

has been made with regard

from damaged

phosphate

Small increases accuracy.

Deter-

but does not give specific

and amino acids are relatively

on diet and nutritional tubular

tubule.

with great

large populations

of glucose,

is also dependent agents,

progress

of the proximal

can now be determined

way of screening

Determinations

since their excretion

of high-molecular-weight as well as chronic

proteinuria

small changes

status.

Determination

cells may be of use for studies of acute

but more data are needed.

INTRODUCTION

During the first half of the century the methods used for detecting renal disease were based on proteinuria. Acute nephritis, chronic nephritis and nephrosis were the major renal diseases of clinical interest. Tubulopathies were described early in the century, but it was not until 1936 that the complete Fanconi syndrome was described [l]. The main features were glucosuria, phosphaturia and organic aciduria, later shown to be aminoaciduria. Proteinuria was also seen in such cases - mainly children with inherited disease - but it was not until 1958 that tubular proteinuria was described as a special protein pattern of the urine [2]. Separation of concentrated urine proteins by paper electrophoresis showed that patients with diseases such as the Fanconi syndrome, galactosaemia and hypokalaemia had a protein pattern quite different from patterns seen in glomerular disease. An unusual form of proteinuria in cadmium-exposed workers had been described in 1950 [3]. Free electrophoresis showed that proteins migrating like cu-globulins constituted a large part of the urine proteins. It was also shown that tests commonly used at that time for detecting proteinuria, i.e., the boiling test, the nitric acid test and Esbach’s test, had a low sensitivity for detecting the proteinuria, whereas

198

trichloracetic acid was shown to be a suitable reagent. In follow-up of these workers it was shown in 1962 that the proteinuria was of the tubular type [4]. Methods were developed urine

for quantitative

from

cadmium-exposed

determination workers

of urine

proteins

[5]. Further

led to the identification

studies

of several

on low-

molecular-weight proteins, such as /3z-microglobulin, lysozyme and ribonuclease [6], and later to the discovery of retinol-binding protein and cur-microglobulin [7,8]. The experience gained in studies on the tubular dysfunction of chronic cadmium poisoning was then used for studies on a major renal disease in south-eastern Europe, Balkan nephropathy [9]. It was shown that in the early stages there was mainly tubular damage, progressing to severe interstitial changes with glomerular involvement. The development of immunological methods for the determination of Pz-microglobulin [lO,ll] was a major breakthrough, which made it possible to screen for tubular dysfunction and was used for epidemiological studies of people exposed to cadmium both in industrial environments [ 121 and in cadmium-polluted areas in Japan [13] and Europe [14]. Retinol-binding protein and oli-microglobulin have also been suggested as markers for tubular dysfunction [15,16]. The tubular dysfunction caused by cadmium has been studied extensively, but &-microglobulin has also been used as a marker in studies of other industrial nephrotoxic agents, e.g., mercury and lead [17], uranium [18] and solvents [19,20]. During recent years, there has also been an interest in using urinary enzymes as markers of nephrotoxicity; N-acetyl-/3-D-glucosaminidase (NAG) has been studied extensively [2 1,221. The merits of some of the methods used for detecting tubular dysfunction are discussed below, with emphasis on the excretion of low-molecular-weight proteins. TUBULAR

DYSFUNCTION

Tubular proteinuria This type of proteinuria is the result of decreased reabsorption of proteins from the glomerular filtrate [6,10,23], which means that a large number of small proteins, which constitute only a small part of the total protein in plasma, are much more prominent among the urinary proteins. The concentration of &-microglobulin in plasma is normally about 1.5 mg/l which can be compared to an albumin concentration of about 45 000 mg/l, i.e., a ratio of about 30 000. In normal urine, the ratio is about 50, but it may be even smaller when tubular dysfunction is present. In a typical case of tubular dysfunction, the majority of the urinary proteins have molecular weights below 70 000. Albumin may constitute only about 20% of the total protein, which explains why it is difficult to determine total urinary protein quantitatively by standard methods in such cases [4,5]. It was also shown that dipsticks were unsuitable for detecting tubular proteinuria. A dye-binding method was studied, but deemed to be less suitable for quantitative determinations than the

199

biuret

method

after precipitation

with Tsuchiya’s

reagent

[5]. In clinical

Coomassie Brilliant Blue method has been shown to be unsuitable determination of drug-induced tubular proteinuria [24].

work,

the

for quantitative

The determination of total protein does not, of course, differentiate between glomerular and tubular proteinuria, but has been shown to be very useful for repeated monitoring of cadmium workers [4,6,25,26]. However, if combined with electrophoresis of urine proteins, an accurate diagnosis can be obtained, and the total protein may indicate the magnitude of the dysfunction. Since, generally, spot samples are examined, adjustment should be made for specific density or creatinine [25,26]. The quantitative determination of specific proteins, e.g., albumin and &-microglobulin, has been successfully applied in many studies [lo, 13,27-291 and has made it possible to establish dose-response relationships for renal effects of cadmium. One advantage of /3z-microglobulin is that the relative clearance, i.e., in relation to creatinine clearance, can be estimated if the protein is also determined in serum. If the pH is controlled, this is a very sensitive method for detecting small changes in tubular function [30]. Normally, about 99.95% of filtered &-microglobulin is reabsorbed, and a decrease of 0.1% in reabsorption capacity thus results in a three-fold increase in /3z-microglobulin excretion in urine. /3z-Microglobulin can be determined accurately in urine and serum by several methods. One disadvantage is that this protein may degrade at a urinary pH below 5.5, which can be avoided by giving oral doses of bicarbonate at least 8 h before urine collection [23]. Retinol-binding protein and olr-microglobulin are less susceptible to degradation and have been recommended as better markers for tubular proteinuria [15,16]. Especially in people with chronic tubular dysfunction, there seems to be a relationship between the excretion of Pz-microglobulin and urinary pH at pH above 5.5. The excretion increased by a factor of about five in the pH range 5.5-6.5 in three cadmium workers whose urines were sampled repeatedly [31]. Similar studies are lacking for other low-molecular-weight proteins, but some data indicate that in cadmium-exposed workers retinol-binding protein seems to vary with &-microglobulin [32]. In normal people, there is not the same dependence on pH above 5.5 [33]. Relative clearance of µglobulin was studied before employment in a battery factory and after six months of exposure to low levels of cadmium (M. Piscator, unpublished data). It was found that in 13 people with similar urinary pH on both occasions, there was no change in clearance (mean values of 0.032% and 0.031%, respectively) and a positive correlation coefficient (r=0.53). In seven people with larger variations in urinary pH, the relative clearance varied more (mean values of 0.030% and 0.037%, respectively), and there was no correlation (r= -0.05). Fig. 1 shows that there were also two outliers, each with one normal and one clearly abnormal clearance. In a group of 20 people without chronic tubular impairment it is not surprising if one in 20 has an abnormal value, since many factors, e.g., fever

200

II

0.15-

. .

PH <- 0.3

”

>

0.3

O.lo-

0.05-

”

o

z .

Fig.

1. Relative clearance

.

l

.* . t-0

0

of six months.

l

2. c

0.05

ai0

of /3zmicroglobulin

(M. Piscator,

unplublished

determined

I

0.15

in 22 people

on two occasions

at an interval

data.)

and drugs, can cause minor transient elevations in the excretion of /&-microglobulin. In an earlier report [30], a group of people was followed for 28 months after cessation of exposure to cadmium. During the study, several determinations were made of ,&-microglobulin, creatinine and cadmium in urine and Pz-microglobulin and creatinine in serum. A dose-effect relationship of the hockey-stick type could be shown by relating the median values of the relative clearance of &-microglobulin to the mean concentration of cadmium in urine (Fig. 2). When the maximum

t

E

1 0 Fig. 2. Relative

clearance

I

a5

Ref. 30.)

5

Cadmium

of µglobulin

mium in urine. The clearance values represent a period of 28 months with urine pH >6.0. (From

I

I

1.0

In urine (pa/g

in ten men (0)

10

I

20

I

30

Creatinine)

and three women

(0)

in relation

to cad-

median values based on three to eight determinations over The cadmium values are means during the same period.

201

clearance values were plotted against the cadmium concentrations, the dose-effect relationship was as shown in Fig. 3. This indicates the need for repeated examinations, especially when the effects are very small. Enzymuria The validity of urinary enzymes as markers has to be examined in detail. NAG seems to have been studied the most. An increased excretion was seen in workers exposed to lead, mercury or solvents [22], but no comparison was made with the excretion of other proteins. In studies on cadmium-exposed people, NAG and pgalactosidase excretion was not as great as that of /3z-microglobulin [28,35]. NAG is a high-molecular-weight lysosomal enzyme which is released from damaged tubular cells and is probably a better marker for acute effects than for chronic effects. Thus, salicylates caused a marked increase in NAG excretion [36]. In a study of patients treated with an aminoglycoside (gentamicin), NAG was found to be a better marker of tubular injury than Pz-microglobulin [37]. Recently, trehalase has been suggested as a marker for tubular dysfunction [38]. Other markers In addition to low-molecular-weight proteins and renal enzymes, many other substances have been determined in urines from people with tubular disorders. Glucose, phosphate and amino acid nitrogen were determined in urine from cadmium workers [39], but marked increases in excretion were seen only when the excretion of total protein was quite high: i.e., a complete Fanconi syndrome was not a common finding. These substances are furthermore more dependent on dietary in-

g t

0

03

l

ii =

E

Pe = 2

Q2-

l

9

l

k _; E k Q 6

0 0

O.l-

0 0

fi E B 0

l

:

l

0

, a5

5

1

10

20

30

Cadmium in urine @g/g creatlnlne)

Fig. 3. Maximal same group

values for relative clearance

as in Fig. 2. (From

Ref. 30.)

of Pz-microglobulin

in relation

to cadmium

excretion

in the

202

take and nutritional status than the serum proteins; therefore, they must be studied under more carefully controlled conditions than generally can be achieved when screening in the field. Examples are studies on hypokalaemia. In a study of obese patients during starvation, hypokalaemia caused moderate tubular dysfunction with tubular proteinuria and aminoaciduria [40]. Total protein excretion increased, as did the excretion of ribonuclease. When potassium was given there was a change to normal conditions. In a more recent study on patients with hypokalaemia [41], there was increased excretion of /3z-microglobulin, NAG and alanine aminopeptidase. People with hypokalaemia seem to be useful for studies of markers of tubular dysfunction, especially since the effects can be reversed by potassium treatment. Although the proximal tubule is the part mainly affected by nephrotoxic agents, the distal part may sometimes be the target. This is especially true for lithium, but many of the substances mentioned earlier may also cause disturbances in concentrating and acidifying capacity. Function tests are easy to perform, but in order to obtain a complete picture of how the tubules handle water and electrolytes, a complicated analytical programme must be used. CONCLUSIONS

A number of tests are available for studies of tubular function. The most sensitive one for detecting small changes in the proximal tubule is the quantitative determination of low-molecular-weight proteins. Determination of total protein may be useful in the screening of large populations but must be combined with electrophoretic separation of urinary proteins or quantitative determination of some high- and lowmolecular-weight proteins to distinguish tubular from glomerular proteinuria. Determination of other urinary constituents should be reserved for special studies. Reference is made to a recent paper in which early detection of renal disease is discussed [42]. REFERENCES 1 Chesney,

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