Renal function tests as indicators of kidney injury in subacute toxicity studies

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

57,414-424

(1981)

Renal Function Tests as Indicators of Kidney in Subacute Toxicity Studies

Injury

M. KLUWE

WILLIAM

National Institute of Environmental Health Sciences, P. 0. Box 12233, Research Triangle Park, North Carolina 27709 Received June 2, 1980; accepted October 30, 1980 Renal Function Tests as Indicators of Kidney Injury in Subacute Toxicity Studies. KLUWE, M. (1981). Toxicol. Appl. Pharmacol. 57,414-424. Several tests of renal function were conducted on adult, male Fischer 344 rats treated for 15 days with biphenyl, carbon tetrachloride or mercuric chloride. Each of the test compounds was administered at four different doses, the lowest of which was subnephrotoxic while the highest was sublethal. Tests included measurements of urine specific gravity, pH and volume, urinary excretions of glucose, protein, electrolytes, and several enzymes, serum concentrations of urea nitrogen, creatinine, and electrolytes, creatinine clearance, and kidney weight. Ability to concentrate urine after water deprivation, kidney morphology, and accumulation of organic ions by renal cortical tissue in vitro were also examined. In general, in vitro accumulation of organic ions, urinary concentrating ability, and kidney weight were the most sensitive and consistent indicators of nephrotoxicity. Standard urinalyses, serum analyses, quantitative enzymuria, and pathological changes in renal morphology were less sensitive and less consistent indicators of injury. The most advantageous tests, therefore, appeared to be those that measured total. functional renal capacity. W.

Since the kidney is a common target of toxic chemicals (Foulkes and Hammond, 1973, a need exists for the inclusion of sensitive, yet simple and reliable, tests of renal function in general toxicity screens. State-ofthe-art methodologies commonly employ urinalysis, serum analysis, measurement of kidney weight, and histological examination to detect nephrotoxicity. The mammalian kidney in a nonstressed state, however, utilizes only a portion (-20-25%) of total renal capacity (Gottschalk and Lassiter, 1974) and standard tests may be insufficiently sensitive to detect subtle toxic effects because of this functional reserve. Changes in microscopic morphology can localize the lesion within the kidney, but the absence of such effects does not preclude injury (Peters, 1965; Striker et al., 1968). For these reasons, perhaps, nephrotoxicity 0041-008X/81/030414-1 1$02.00/O Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

414

is a relatively uncommon finding in the preclinical evaluation of therapeutic drugs, but a common side effect of their clinical use (Peters, 1965). Increased urinary excretions of kidneyderived enzymes have been proposed as early indicators of chemical nephropathies, particularly those resulting from heavy metal intoxications (Wright and Plummer, 1974; Cotrell et al., 1976; Ngaha and Plummer, 1977). Increased enzyme excretions, however, are generally transient (Nomiyama et al., 1974; Stroo and Hook, 1977) and may dissipate despite continued toxicant exposure (Prescott and Ansari, 1969). Thus, the utility of enzymuria as an index of kidney function following repeated chemical exposure has not been adequately demonstrated. Tests that measure the functional capabilities of the kidney, such as

RENAL TABLE DOSAGESOF

FUNCTION

1

EXPERIMENTAL

CHEMICALS

(Relative dose) 1

2

3

4

Chemical

Quantitative dose administered (mg/kg)

Biphenyl CCI, HgCI,,

33 50 0.20

100 150 0.60

300 450 1.80

900 1350 5.40

urinary concentrating ability and accumulation of organic ions in vitro, also appear to be sensitive indicators of chemical nephropathy (Balazs er al., 1963; Sharrat and Fraser, 1963; Bemdt, 1976; Hirsch, 1976: Phillips et al., 1977) and may be useful in routine screening procedures. The following experiments were conducted to compare the relative sensitivities of several tests to detect kidney injury following repeated, oral exposure to known nephrotoxic chemicals. Three toxicants with markedly differing effects on renal ultrastructure and function (Booth et al., 1961; Striker ef a/., 1968; Ganote et al., 1975) were used to provide a wide spectrum of chemical injuries. Each was administered in four different doses to assess the relative sensitivities of the tests. The oral route (gavage) was used because all three chemicals are absorbed from the gut and incorporations of the test compound into the diet or drinking water are the most common methods of administration in long-term rodent toxicity testing. The tests were selected on the basis of potential sensitivity, ease and precision of performance and current use in clinical and toxicology laboratories. METHODS Animals. Weanling, male, Fischer 344 rats’ were housed in a temperature, humidity, and light-cycle

’ Charles River, Portage, MI.

TESTS

415

(12 hr light, 12 hr dark) controlled room throughout the study. The treatments began when the animals were fully matured (approximately 200 g body weight, 8 weeks of age). Treatments. All test chemicals were given orally, by gavage. BiphenyP and carbon tetrachloride (CCI,1” were dissolved in corn oil’ and mercuric chloride (HgCl$ in distilled water. Four different doses of each test chemical were administered, plus an appropriate vehicle control (Table 1). The lowest doses (1) were without detectable effects on animal health. The highest doses (4) were greater than one-third of the LD,, (data not shown) and reduced body weights. but did not cause any deaths. The test compounds were administered on Days I. 2. 5-9, and 12-15 of the experiment. The animals were temporarily placed in stainless-steel metabolism cages designed for separate collection of urine and feces at 4 PM on Day 13 and urine was collected for the next 16 hr in glass tubes suspended in ice in insulated (polyurethane foam) containers. Water was removed at 4 PM on Day 14 to achieve a state of mild dehydration, the bladders were evacuated 16 hr later, and the rats transferred to metabolism cages and 4 hr urine samples collected for determination of specific gravity (test of urine concentrating ability ). The animals were anesthetized on Day 16 with 50 mg/kg of pentobarbita16 and approximately 2 ml of blood was obtained from the orbital sinus. The rats were then sacrificed by cervical dislocation, urine obtained by bladder puncture, and liver and kidneys recoved. Carcasses were examined grossly for signs of tissue injury. Urinalyses. Urinalyses conducted on 16 hr samples collected over ice were indistinguishable from those conducted on freshly voided urine in all cases and the former, therefore, were used routinely. The pH values of the 16 hr urines were acidic (6 or 6.51, indicating an absence of bacterial overgrowth. Urinary concentrations of protein, glucose, ketone, and blood (Hemoglobin) and urine pH were estimated with reagent strips’ and pH was also determined with a hydrogensensitive electrode.” Creatinine and glucose in the urine were measured with a centrifugal analyzer.!’ sodium and potassium by flame photometry.‘” and protein hv * Fisher Chemical Company, Fairlawn, NJ. 3 Spectrophotometric grade, Fisher Chemical Company. 4 Laboratory grade, Fisher Chemical Company. 5 Sigma Chemical Company, St. Louis, MO. 6 Nembutal, Abbott Laboratories, Chicago, IL. ’ Labstix, Miles Laboratories, Elkhart. IN. s Coming Instruments, Medfield, MA. 9 Centrifichem System 400, Union Carbide Corporation, Rye, NY. lo Instrumentation Laboratories, Lexington. MA.

416

WILLIAM

250

I

w&

M. KLUWE

Biphenyl

Cl

23

1lluI 4

c

I

2

3

4

FIG. 1. Body weights at sacrifice. Bars represent the mean ? SE of six rats. Open bars are significantly less than control (C), p < 0.05.

the method of Pesce and Strande (1973); all were expressed as the amount excreted in urine per 16 hr. One-milliliter aliquots of urine were dialyzed in cellulose tubing” for 4 hr against deionized water at 4°C to remove interfering substances and analyzed for alkaline phosphatase (AP), y-glutamyl transpeptidase (GGT), and lactate dehydrogenase (LDH) activities with a centrifugal analyzer using Union Carbide reagents. The activity of N-acetylglucosaminidase (NAGA) in dialyzed urine was measured by the method of Maruhn (1976) using extraneous 4-nitrophenyl-P-d-glucosaminide5 as substrate. AP, GGT, and LDH were expressed as IU excreted per 16 hr and NAGA as units excreted per 16 hr, where one unit is equal to the formation of 1 nmol of product (p-nitrophenol) per 15 min. Specific gravity of the urine was determined with a refractometer.‘* Serum analyses. Whole blood was allowed to clot for 1 hr at room temperature and the serum withdrawn after centrifugation. Creatinine and urea nitrogen concentrations and AP, GGT, LDH, and glutamic pyruvic transaminase (GPT, on C&treated rats only) activities in serum were measured with a centrifugal analyzer using Union Carbide reagents. Serum sodium and potassium concentrations were measured by flame photometry. Accumulation of organic ions in vitro. A strip of cortex from the left kidney of each experimental animal was removed and cut into thin (30 pm) slices with an automated tissue slicer.13 The slices were incubated in a phosphate-buffered medium (Cross and Taggart, 1950) containing 1 x 10d3 M lactate, 6.7 x 10e5 M [3Hlp-aminohippurate14 (PAH), and ‘I 8,000-12,000 MW cutoff, Union Carbide Corporation, Chicago, Il. I* American Optical, Buffalo, NY. I3 Brinkman Instruments, Westbury, NY. I4 New England Nuclear Corporation, Boston, MA.

1 x lO-3 M [14C]tetraethylammonium (TEA) as described previously (Kluwe and Hook, 1978), except that PAH and TEA were determined by liquid scintillation spectrometry. Accumulation of organic ions in vitro was expressed as a slice-to-medium ratio (S/M), where S = mg PAH or TEA per g tissue and M = mg PAH or TEA per ml medium. Histology. Samples of kidney and liver were fixed in 10% neutral buffered formalin (pH 7.4), embedded in paraftin, sectioned at 6 pm, and stained with hematoxylin and eosin. Statistics. The quantitative data were analyzed by two-way analysis of variance (to distinguish variance due to different examination dates from that due to experimental error) and mean values for each treatment group were compared to the appropriate control using Student’s t test (Sokal and Rohlf, 1%9). Nonparametric data (from reagent strips) were compared to control with the Mann-Whitney U test (Sokal and Rohlf, 1%9).

RESULTS All rats receiving the highest doses (4) exhibited overt signs of toxicity (unthrifty appearance, diarrhea, dried blood around the nose and mouth) by Day 16, but only the biphenyl-treated animals appeared near death. Body weights were significantly lower in treated animals than in controls for doses 3 and 4 of biphenyl and CCI, and for dose 4 of HgCl, (Fig. 1). In general, doses 3 and 4 of all three chemicals retarded body weight gains early (Day 2) in the study, while the highest doses caused actual losses in body weight by the end of the experiment.

RENAL

_ 7 :: F % ; g il C Y

*---=

5 s

‘.‘OO

r--------L

. . ..__.

4 _____..

r ______.

*

I

15

IO60

1060 I040 I020

&--.&---a i.

I

C

IO 5

__-_ -&--a

=

L

I

2 Relative

-

*

3

i

4

I

dose

FIG. 2. Urine volume and specific gravity. Circles represent urine volume, squares represent urine specific gravity in animals supplied water ad lib., triangles represent urine specific gravity in waterdeprived animals (mean 2 SE of six rats). Open symbols are significantly different than control (C), p < 0.05.

Sixteen hour urine volumes averaged 4-5 ml in all control groups and were unaffected by the administration of the test compounds, except for an increase with the high dose of biphenyl (Fig. 2). Water deprivation increased the specific gravity of urine threeto fourfold in control rats, but rats receiving TABLE URINALYSIS

6.0 or 6.5

+ I(-30 Negative Negative Negative

2

REAGENT

Normal results

Test

PH Protein Glucose Ketones Blood

WITH

417

TESTS

doses 3 and 4 of biphenyl and Ccl, did not achieve as high a urine specific gravity as did controls (Fig. 2). The magnitudes of the effects of these two compounds were quite different; rats treated with dose 4 of biphenyl were completely unable to increase urine specific gravity while rats treated with dose 4 of CCL, increased urine specific gravity significantly but not to the same extent as did controls. Sixteen hour urine samples were generally a light yellow color, except for urine from rats receiving dose 4 of biphenyl. These were opalescent and contained visible amounts of blood and a white, alkali-soluble precipitate that was probably p-hydroxybiphenyl (Booth et N/. , 1961). pH values, protein, glucose, and ketone concentrations were all normal as determined by reagent strip analyses (Table 2). Blood (hemoglobin) was detected in urine from biphenyl-treated animals at dose 4. in CC&-treated animals at doses 3 and 4. and in HgCl,-treated animals at doses 2, 3, and 4. As shown in Fig. 3, none of the test chemicals increased urinary protein excretion (mg/16 hr). However, Ccl, and HgCl, slightly decreased urinary protein excretion. Urine glucose excretion was unaffected except for a twofold increase in rats treated with dose 4 of biphenyl (Fig. 3).

x k z

CCI, I IOO10001060IWO1020-

FUNCTION

mg%) or +2 (-100 mg%) (less than 100 mg%) (less than 10 mg% acetoacetic acid) (less than 0.015% hemoglobin)

STRIPS

Experimental

results

All normal All normal All normal All normal Biphenyl: 2 of 6 moderate and 4 of 6 large at dose 4 (significant): all normal at lower doses Ccl, : 3 of 6 small and 3 of 6 moderate at dose 4 (significant): 2 of 6 small at dose 3 (not significant): all normal at lower doses HgClz : 4 of 6 small at dose 4 (significant); 2 of6 small at dose 3 (not significant): 1 of 6 small at dose 2 (not significant); all normal at lower doses

418

WILLIAM

M. KLUWE 4

l NAGA rnLOH

g 12.0 2 $

8.0

; e P ? E 5

80-

rAP .GGT

I

3

4.0 E g e P 12.0-

b

Biphenyl

t

,’

40-

1208.0 4.0 -

2

None of the test chemicals increased serum glucose concentrations (CC& significantly reduced serum glucose, Table 3). Thus, the increased urinary glucose excretion in biphenyl-treated rats could not be attributed to an increase in serum glucose. Expression of urinary protein and glucose as excretions per 100 g body weight (mgI16 hr/lOO g body weight), to normalize for chemical-induced changes in body weights, provided similar results (data not shown). Urinary excretions of sodium and potassium (mEq/l6 hr) were unaffected by the test chemicals except for the highest dose of biphenyl, which caused a twofold increase in urinary sodium excretion (data not

WA t

FIG. 3. Urinary excretion of protein and glucose. Values are the mean rt SE of six rats. Open symbols are significantly different than control (C), p < 0.05.

1 C

T

I

2 Relative

3

4

I

dose

FIG. 4. Urinary enzyme excretion. Values are the mean k SE of six rats, expressed relative to controls. Normal (control) values: 0.68 2 0.11 IU/16 hr AP, 6.37 k 0.62 IU/16 hr GGT, 0.069 f 0.012 IU/16 hr LDH, 1113 k 160 U/16 hr NAGA (N = 18 rats). Open symbols are significantly different than control (C), p < 0.05.

shown), though serum sodium concentration was unaltered. The relative effects of the test chemicals on the urinary excretions of several enzymes are shown in Fig. 4. The highest dose of biphenyl increased AP excretion nearly threefold, while dose 4 of CC& increased LDH excretion by a factor of two.

TABLE

3

SERUM GLUCOSE CONCENTRATIONS Glucose (mg%) .? k SE Control

1

Chemical Biphenyl ccl, Hi&

2

3

4

140 k 21 116 k 10” 140% 6”

137 k 13 119 2 13” 15Ok 3

(Relative dose) 142 + 9 144 2 6 150 k 7

134 2 14 138 ? 23 143k 5

142 2 14 131 + 9” 148k 4

a Significantly less than control, p < 0.05, N = 6 rats.

RENAL

FUNCTION TABLE

SERUM

419

TESTS

4

ENZYME

ACTIVITIES

Activity” (2 2 SE) Control

1

2

3

4

Enzyme

Chemical

(Relative dose)

LDH

Biphenyl CCI, HgCl~

824 ‘- 169 573 k 94 571 _f 67

721 2 113” 547 + 85 537 c 79

702 c 124O 785 2 301’ 538 2 60

659 + 163” 606 r 158 442 2 57”

547 k 19oh 325 i: 49” 481 2 27”

AP

Biphenyl CCI, I-WI,

263 r 26 289 t 39 302 + 9

268 t 2902 274 f

263 i 23 341 2 68C 289 k 18

232 + 18” 392 t 92’ 265 + 21°

234 _c 23O 501 IC 84 232 IT 38”

Gm-

CCI,

238 +

452 2 131

244 -c 52’

32 AZ 22

17 13 18

63 -+ 74

Y

a W/liter. * Significantly less than control, p < 0.05, N = 6 rats. ’ Significantly greater than control, p < 0.05, N = 6 rats.

The remainder of the treatment regimens failed to increase enzyme excretion, but in some instances reduced enzymuria below control levels. When expressed as units of enzyme activity excreted per 16 hr per 100 g body weight (body weights declined at high doses), small but significant increases of NAGA and GGT in the high dose biphenyl group and of LDH in the high dose HgCl, group were apparent (data not shown). Biphenyl treatment decreased serum LDH activities, Ccl, increased serum GPT and AP activities but decreased serum LDH activities at high doses, while HgCl, decreased serum LDH activity (Table 4). Increased excretion of AP occurred despite a decrease in serum AP activity at dose 4 of biphenyl, while increased excretion of LDH occurred despite a decrease in serum LDH activity at dose 4 of Ccl, (Fig. 4 and Table 4). Thus, increased enzyme excretion was not dependent on increased serum enzyme activities. GGT and NAGA activities in serum were below the detection limit of the assay procedures (2 IU/liter and 15 units/ml, respectively). Serum concentrations of sodium, potassium, and urea nitrogen (BUN) were unaltered by treatment with biphenyl, CCII,

or HgClz (data not shown). The concentration of creatinine in the serum was not elevated by any of the test chemicals, but declined following treatments with biphenyl and CC& (Fig. 5). Accordingly, small increases in creatinine clearance were detected in some groups of rats treated with biphenyl or CC&, but only the highest dose

-

5 oz-

I

06 04 021

I

JO4

112

L--2---+---L,

,

C

I

-3

, 2 Relative

^

. 2

,

,

3

4

06 104

dose

FIG. 5. Serum creatinine concentration and creatinine clearance. Values are the mean + SE of six rats. Open symbols are significantly different than controls (C), p < 0.05.

420

WILLIAM Elphenyl 12 IO 8 6

I I4

I6 14 12 IO

CCI,

I

I 18 5 I6 ;i 14 z I2 + IO

& 12 I ‘0 20 6

I4

I6 16 I4 I2 IO

I2 IO a 6 Cl234

Cl234 Relative

dose

FIG. 6. vifro. The

Maximum accumulation of organic ions in abilities of renal cortical slices to accumulate and maintain a concentration gradient for the anion PAH and the cation TEA are expressed as slice-tomedium ratios (S/M). Values are the mean -c SE of six rats. Open bars are significantly less than controls (C). p < 0.05.

of HgCl, reduced the creatinine clearance (Fig. 5), and this effect disappeared when the clearance was expressed per 100 g body weight (data not shown). The effects of the various treatments on in vitro accumulation of p-aminohippurate (PAH S/M) and tetraethylammonium (TEA S/M) are illustrated in Fig. 6. Doses 2, 3, and 4 of biphenyl and dose 4 of Ccl, and

M. KLUWE

HgCl, reduced PAH S/MS. TEA S/MS were reduced by doses 2, 3, and 4 of biphenyl, dose 4 of CC&, and doses 1, 2, 3, and 4 of HgCl,. (Reduced TEA S/MS in rats treated with doses 1, 2, and 3 of HgCl, were not considered toxicologically significantthough they were statistically significantbecause the changes were small and not dose related.) Absolute kidney weight was greater than control in rats receiving doses 3 and 4 of biphenyl (Fig. 7) and the tissues appeared pale and swollen upon gross examination. Diffuse dilatation of the distal convoluted tubules and collecting ducts was detected by light microscopy in half of the animals at the highest biphenyl dose, but not at any of the lower doses. Absolute kidney weight was lower in rats treated with doses 3 and 4 of Ccl, than in controls (Fig. 7), but kidney weight-to-body weight ratios were unaffected (data not shown). Kidneys from rats receiving Ccl, did not exhibit histological lesions. The highest dose of HgCl, increased absolute kidney weight (Fig. 7). The kidneys were visibly swollen on gross examination and diffusely scattered areas of epithelial cell swelling and vacuolation were detected histologically in the proximal convoluted tubules. Many of the affected cells contained dark, shrunken (pyknotic) nuclei and some tubules contained clumps of sloughed necrotic epithelial cells. Histo-

MCI, Al .

Blphenyl

2.5 t

CCI,

I

FIG. 7. Kidney weights at sacrifice. Bars represent the mean + SE of six rats. Open bars are significantly different than control (C), p < 0.05.

RENAL TABLE RELATIVE

SENSITIVITY

FUNCTION

DISCUSSION

5

OF RENAL

FUNCTION

TESTS

Lowest relative dose (1, 2, 3. 4) at which pathological change detected” Test

Biphenyl

CCI,

-

Urinalysis, direct Glucose excretion Protein excretion Specific gravity Volume

n n ” ”

4 ” ” ”

Urmalysis, reagent strips” PH Glucose concentration Protein concentration Ketone concentration Blood concentration

n n n ” 4

” n ” n 4

Enzymuria AP excretion GGT excretion LDH excretion NAGA excretion

n ” 4 ”

” ” ” ”

Serum analyses Urea nitrogen concentration Creatinine concentration Sodium concentration Potaswm concentration

” ” ” ”

” ” ” ”

3 ‘t

n ”

4 4

”

n 3

4 n

2

4

4

Pathology Kidney Kidney

weight (absolute) morphology tmicroscopicl

Special tests Creatinine clearance Maximum urine concentrating ability Accumulation of organic ions by slices of renal cortex in vitro (PAH and TEA S/M) ” Total body weight lower in biphenyldose 3. m HgCI,-treated at dose 4 (in n. No pathological changes detected. ’ Nonparametric data.

421

TESTS

and CCI,.treated rats at comparison to controls).

logical lesions were not detected at lower doses of HgCl,. The lowest nephrotoxicant dose, if any, that produced an alteration indicative of renal damage is listed in Table 5 for each experimental chemical and test. Only changes for which documented toxicological precedence exists were judged to be signs of injury (e.g., increased urinary glucose, enzyme excretion, and absolute kidney weight were considered signs of kidney injury, but decreases in these same parameters were not).

Three desirable characteristics for renal function tests in toxicity screens are listed below: (1) Sensitivity-ability to detect injury at the lowest effective dose. (2) Versatility-ability to detect injury from a wide variety of chemicals. (3) Ease and precision of performance. With these characteristics in mind, the data in Table 5 can be analyzed in two manners: within columns to compare the sensitivities of the various tests to detect kidney injury caused by a specific toxicant, and across columns to compare the abilities of individual tests to detect injury from a spectrum of nephrotoxicants. Comparisons within columns indicate that most of the test procedures were unable to detect kidney injury or did so only at the highest (near-lethal) doses. Test insensitivities may be due to the large functional reserve of the kidney or to its ability to repair tubular damage, though regeneration was not detected histologically. In addition, repeated treatment may stimulate adaptation of the kidney to the presence of the toxicant (Prescott and Ansari, 1969; W. M. Kluwe, unpublished information). Since adequate renal function and the maintenance of fluid and electrolyte homeostasis are essential for survival of the animal, changes in the standard function tests (e.g., blood urea nitrogen, massive glucosuria, serum electrolytes) are due to frank renal failure and occur only at lethal or nearlethal doses. In vitro accumulation of organic ions (PAH and TEA S/MS) and urine concentrating ability were among the more sensitive tests in this study. The former indirectly measures the capacity for energy-dependent transport of organic anions and cations by proximal tubular cells, the renal cells most commonly injured by toxic chemicals (Schreiner and Maher, 1965), while the

422

WILLIAM

latter indirectly measures the capacity for active reabsorption of inorganic ions (principally Na+ and Cl-) and water by tubular cells (Peters, 1965). These tests measure functional capacities, rather than unstressed, steady-state function, and may be the most sensitive types of kidney function tests. The analysis of enzymuria for early diagnosis of kidney injury has been considered by several investigators (Raab, 1972; Stroo and Hook, 1977; Lockwood and Bosmann, 1979; Diezi and Biollaz, 1979), generally following a single toxicant exposure, and can be a sensitive indicator of certain types of acute renal damage. However, quantitative changes in urinary enzyme excretions after repeated treatments with biphenyl, CC&, and HgCl, were small (if present at ail), occurred only at high doses, and often involved decreases that were of unknown pathological significance. Therefore, enzymuria may be a poor indicator of kidney injury in subacute or long-term toxicity studies, though it may be of use for determining the site of tubular injury or the mechanism of the nephrotoxic effect (Ellis and Price, 1975; Price and Ellis, 1976; Kempson, et al., 1977). Absolute kidney weight was a relatively sensitive test in this study with known nephrotoxicants, but to be of significance in screening studies, a pathological mechanism for the increased weight (e.g., edema) should be ascertained. An alteration in kidney morphology, in contrast, is generally indicative of nephrotoxicity, but the absence of histological effects, as with CC&, does not preclude renal injury nor its presence, as with biphenyl, at doses lower than can be detected histologically. The most versatile tests in this study were hematuria (blood in urine) and the in vitro accumulation of organic ions by renal cortex. It is necessary to determine the site of the injury causing the hematuriaerythrocyte casts in urinary sediment indicate bleeding in the kidney, while the

M. KLUWE

presence of red blood cells without casts localize the injury to the bladder or other segments below the kidney (Bradley and Benson, 1969). Red blood cells, but not casts, were present in sediment from biphenyl-treated rats, while neither erythrocytes nor casts were detected in large quantities in sediment from Ccl, and HgC&treated rats (data not shown). Thus, biphenyl may have induced hematuria by an extrarenal effect. Since Ccl, and HgC& selectively damage the proximal tubular portion of the nephron (Striker et al., 1968; Ganote et al., 1973, the basolateral membrane of which contains the organic ion transport systems, sensitivity of PAH and TEA S/MS to CC& and HgCl, was not unexpected. In contrast, sensitivity of these tests to biphenyl injury was not expected since this compound appears to affect distal tubular structure and function (Ambrose et al., 1960; Booth et al., 1961). Although the reason for the biphenylinduced decline in S/M values is not apparent, the in vitro accumulation of organic ions would seem to be a versatile indicator of nephrotoxicity because of sensitivity to both proximal and distal tubular toxicants. Ease and precision of performance are necessary test attributes to ensure comparability of experimental results between different testing laboratories and, since the number of experimental animals involved in toxicity screens can be quite large, to assure that the procedures can be accomplished by personnel with minimal training. Most tests in this study, with specific reservaCommercial tions , meet these criteria. reagent strips commonly used in animal studies are often insensitive (e.g., for glucose) and provide only semiquantitative data. Furthermore, results are dependent on the commercial source of the product and the ability to match color intensities between the sample-treated reagent strip and printed standards (Hinberg et al., 1978; James and Bee, 1979). Thus, reagent strip assays are imprecise and experimental

RENAL

FUNCTION

results may vary considerably between laboratories, decreasing their utility as tools in toxicological screens. More quantitative methods should be employed whenever feasible. Many urinary enzyme activities can be measured by rapid, automated methods, but dialysis of the urine is generally required to remove endogenous (Werner et al., 1969) or treatment-related (e.g., HgC&) inhibiting substances and this contributes substantially to the time required. Moreover, the relative insensitivity of enzymuria in detecting kidney injury in this study suggests that urinary enzyme analyses may not be generally useful in subacute toxicity screens. Measurement of PAH and TEA S/MS requires significant amounts of time (for medium preparation, tissue incubation, and analyses) and immediate use of kidney tissue (can be stored in 0.9% saline at 4°C for 2 or 3 hr) after animal sacrifice. Nonetheless, S/M determinations are sensitive and versatile tests of kidney function and the experimental results are quite precise. In summary, PAH and TEA S/M determinations and urine concentrating ability appear to be the most sensitive and versatile tests of renal function. Absolute kidney weight and hematuria require additional procedures to be of diagnostic value while reagent strip assays are of limited usefulness because of imprecision and the semiquantitative nature of the results. Enzymuria does not appear to be a sensitive indicator of kidney injury following prolonged exposure to nephrotoxic chemicals and the absence of histological abnormalities does not preclude renal injury. Tests recommended for detecting kidney injury in repeated dose studies, based on the results of this investigation, would include determinations of urine concentrating ability and accumutation of organic ions by renal tissue in vitro, measurement of kidney weight, histological assessment of kidney morphology, and examination for hematuria.

423

TESTS

ACKNOWLEDGMENTS The author acknowledgesthe technical assistanceof Sam Cooper, Frank Harrington, and Ralph Wilson. Dr. Ernest E. McConnell (National Institute of Environmental Health Sciences) performed the histological examinations.

REFERENCES AMBROSE, A. M., BOOTH, A. N., AND DEEDS, F. (1960). A toxicological study of biphenyl, a citrus fungistat. Food Res. 25, 328-336. BALAZS, T., HATCH, A., ZAWIDZKA, Z., AND C&ICE, H. C. (1963). Renal tests in toxicity studies on rats. Toxicol.

Appl.

Pharmacol.

5,661-674.

BERNDT, W. 0. (1976). Renal function tests: What do they mean? A review of renal anatomy, biochemistry and physiology. Environ. Health Persp. 15, 55-71. BOOTH, A. N., AMBROSE, A. M., DEEDS, F., AND Cox, A. J., JR. (1961). The reversible nephrotoxic effects of biphenyl. Toxicol. Appl. Pharmacnl. 3, 560-567.

BRADLEY, G. M., AND BENSON, E. S. (1969). Examination of the urine. In Todd-Sanford Clinical Diagnosis by Laboratory Methods (I. Davidson and J. B. Henry, eds.), p. 35. W. B. Saunders, Philadelphia. COTTRELL, R. C., AGRELO, C. E., GANGOLLI, S. D., AND GRASSO, P. (1976). Histochemical and biochemical studies of chemically induced acute kidney damage in the rat. Food Cosmet. Toxicol. 14,593-598.

CROSS, R. J., AND TAGGART, J. V. (1950). Renal tubular transport: Accumulation of p-aminohippurate by rabbit kidney slices. Amer. J. Phvsiol. 161, 181- 190. DIEZI, J., AND BIOLLAZ, J. (1979). Renal function tests in experimental toxicity studies. Pharmacol. Ther. 5, 135-145. ELLIS, B. G., AND PRICE, R. G. (1975). Urinary enzyme excretion during renal papillary necrosis induced in rats with ethyleneimine. Chem. -Rio/. Interact.

11, 473-482.

FOULKES, E. C., AND HAMMOND, P. B. (1975). Toxicology of the kidney. In Toxicology, the Basic Science of Poisons (L. S. Cassarett and J. Doull, eds.). Macmillan, New York. GANOTE, C. E., REIMER, K. A., AND JENNINGS, R. B. (1975). Acute mercuric chloride nephrotoxicity. An electron microscopic and metabolic study. Lnh. Invest.

31, 633-647.

GOTTSCHALK, C. W., AND LASSITER, W. E. (1974). Urine formation in the diseased kidney. In Medical Physiology (V. B. Mountcastle, ed.), Vol. il. 13th ed. C. V. Mosby Co., St. Louis.

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