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Relationships between histological and functional indices of acute chemically induced nephrotoxicity
F U N D A M E N T A L A N D APPLIED T O X I C O L O G Y 3:543-551 (1983)
Relationships Between Histological and Functional Indices of Acute Chemically Induced Nephrotoxicity H I R O A K I M I Y A J I M A ' , W I L L I A M R. H E W I T T ? , M I C H E L G. C O T E , and G A B R I E L L. P L A A ' * D ~ p a r t e m e n t de P h a r m a c o l o g i c , Facult~ de M ~ d e c i n e , U n i v e r s i t 6 de M o n t r d a l , M o n t r d a l , Q u e b e c , C a n a d a H 3 C 3J7
ABSTRACT
Relationships Between Histological and Functional Indices of Acute Chemically Induced Nephrotoxicity. Miyajima, H., Hewitt, W.R., C~t~, M.G., and Plaa, G.L. (1983). Fundam. Appl. Toxicol. 3:543-551. Acute renal injury was produced in rats with K.zCr,~O7(5-40 mg/kg, sc) HgCi2 (0.5-5.0 rag/kg, sc) or cephaloridine (0.5-3.0 g/kg, sc). Histological (percentage of normal, degenerated or necrotic cells) and functional indices (relative kidney weight, renal cortical slice accumulation of organic ions, and blood urea nitrogen content) were evaluated 48 hours later. The relative sensitivity of each of these indices was determined for each nephrotoxicant. Renal cortical accumulation of organic ions appeared to be the most sensitive of the functional parameters. A quantitative histological evaluation was found to be as sensitive an indicator of nephrotoxicity as organic ion accumulation. Alterations in each of the functional indices were significantly correlated with changes in renal histology.
as have increases in blood urea nitrogen and creatinine content (Sharratt, 1970; Kluwe, 1981; Piperno, 1981; Berndt, 1981). Similarly, alterations in glomerular filtration rate, urine concentration and dilution capacity, various metabolic processes, and in rive and in vitro renal organic ion secretion have been utilized successfully as indices of nephrotoxicity (Sharratt, 1970; Phillips et al., 1977; Kluwe, 1981; Kacew and Hirsch, 1981). However the relative sensitivity and applicability of these and/or other parameters, such as histopathologieal alterations, as indices of chemically ir~duced kidney injury remain in question. Kluwe (1981 ) found -hat accumulation in vitro of organic ions by renal cortical tissue, altered kidney weight and changes in urinary concentrating ability were among the most sensitive and versatile indicators of injury, while changes in renal morphology were considered to be less sensitive and less consistent indicators. In contrast, Sharratt and Frazer (1963)suggested that histopathology was a more effective index of renal damage than urinalysis or a battery of renal function tests.
INTRODUCTION The kidney is a target organ for a host of toxic chemicals found in the environment or used for therapy of disease states. As such, reliable, sensitive and versatile tests are needed to screen for the nephrotoxic potential of drugs and cheraicals. Several urinary and renal functional parameters have been evaluated for their utility as probes of chemically induced nephrotoxicity. Analyses of urine for abnormally high concentrations of glucose, proteins, blood and enzymes of renal origin have been found useful for detecting and quantifying nephrotoxic insults
Unfortunately, relatively few studies have directly compared toxicant-induced alterations in renal fun.ctional tests with a quantitative histological analysis of kidney injury. Thus the primary objective of this investigation was to determine if a correlation existed between alterations in one or more of the functional parameters and the severity of the lesion as judged by histopathology. The focus of the investigation was narrowed by 1) selecting nephrotoxicants known to selectively damage the proximal tubule, and 2) selecting functional parameters currently employed in the assessment of chemically induced acute tubular necrosis. In addition, information on the relative sensitivities of the functional and histological indices of proximal tubular injury was derived.
*Visiting Scientist from Takeda Chemical Industries, Ltd,, Osaka, Japan, ~'Chemical Industry Institute of Toxicology Postdoctoral Fellow. Present .address: Smith Kline & French LaboratOries, Philadelphia. PA 19107. *"To whom reprint requests should be sent.
Functional Evaluation of Potassium
Varying degrees of kidney damage were produced with three known nephrotoxicants: potassium dichromate (Hirsch, 1973; Berndt, 1975; Kacew and Hirsch, 1981 ), mercuric chlo-
TABLE 1 Dichromate-induced
Nephrotoxicity
PAH S/M Ratio Dose (m~lkg) 0 5 I0 20 40
Kidney Weight X 100 Body Weight 0.78 0.74 0.82 0.94 1.50
-P + ± :t: :t:
0.03 (7) 0.03 (6) 0.03 (7) 0.04)'(8) 0.08 I' (6)
-Lactate 6.54 8.14 8.16 4.74 2.95
444-I4-
0.63 (7) 0.19J'(6) 0.78 j' (7) 0.41b(8) 0.32 j' (6)
TEA filM Ratio
+Lactate 21.80 23.38 13.93 5,08 3,20
in M a l e R a t s "
+ 1.35 (7) + 0,65 (6) + 1.30 h (7) + 0.32~'(8) --t- 0.3~4~' (6)
-Lactate 22.54 17.86 9.15 7.01 2.91
4-44::k 4-
1.27 (7) 1,01J'(6) 0.66 j' (7) 0.36~'(8) 0.47 b (6)
BUN
+Lactate 22.98 20.66 10.62 7.05 3.35
4- 1.14 (7) -t- 1.19 (6) + 0.61 t' (7) ::]: 0.46~'(8) -I- 0.62 b (6)
(mgla00 mL) 16 21 24 98 150
4- 1 (7) --1- 1 (6) 4- I (7) -P 10"(8) 4- 26 b (6)
"Rats were sacrificed 48 h r a f t e r receiving 0, 5, 10, 20 or 40 mg/kg K2CreOr, sc, Values represent the mean ::k SE d e t e r m i n e d in (n) rats. t'Values significantly different from the centre] (0 mglkg) group, p < 0.05. Copyright 1983, Societyel Toxicology
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ride (Hirsch, 1973; Ganote el al., 1975; Phillips el a/., 1977) and cephaloridine (CPH) (Atkinson el al., 1966; Wold, 1981). Nephrotoxicity was monitored by alterations in: 1) relative kidney weight, 2) renal cortical slice accumulation (resting and lactate-stimulated) of an organic anion (p-aminohippurate, PAH), 3) renal cortical slice accumulation (resting and lactatestimulated) of an organic cation ( t e t r a e t h y l a m m o n i u m ion, TEA), and 4) blood urea nitrogen (BUN) content.
TABLE 2 Histological Evaluation of Potassium Dichromate-induced N e p h r o t o x i c i t y in M a l e Rats" Dose (mglkg)
METHODS Male, Sprague-Dawley rats (150 to 300 g) were purchased from Bio-Breeding Laboratories and maintained on Purina Lab Chow and water ad/fbilum. Animals were used after a suitable period of acclimation in the animal quarters. The K.,Cr.,OT, HgCI= and CP}; were dissolved in water and administered subcutaneously in various dosages. Control animals received an equivalent volume of water subcutaneously. Forty-eight hours after administration of the toxicant, the animals were lightly anesthetized w i t h ether and blood removed via the abdominal aorta, rinsed in ice-cold 0.9% NaCI and weighed. Sections from one kidney were fixed in 10% buffered formalin for histological processing.
Degenerated Tubules
Necrotic Tubules
Total Abnormal Tubules
0 5
I.o ¢ 0.5 (7) 0.7 ~ 1.7h(o)
0.0 _+ 0.0 (7) 0.0 --- 0.0 (o)
1.o + 0.5 (7) a.7 +__ 1.7t'(o)
10 20 40
43.7 ± 4.1 u(7) 30.0 ~ 2.3" (14) 47.5 ± 3.o"(o)
40 -e 1.3 (7) 4o.1 ± 1.5 ~' (8) 52.5 2: 3.o"(o)
47.7 ± 4. °" (7) B5.8 ± 1.7 I' (g} tO0 ~ 0 {o,~
"Rats :'.'ere sacrificed 48 hr after receiving 0, 5, 10, 20 or 40 mglkg K~Cr~O:,sc. Values represent the mean percentage~ :t= S E d e t e r m i n e d
in ~nt rals. "Values ~ignificantly different from the control (0 mglkg) ~.,roup. p < 0.05.
D e t e r m i n a t i o n o f P A H a n d TEA uptake After being weighed, the kidneys were stored briefly in icecold 0.9% NaCI. Thin renal cortical slices were prepared freehand and placed in 3.0 mL of the phosphate-buffered (pH 7.4) incubation medium described by Cross and Taggart (1950). Sodium lactate (10 raM)was included in one-half of the incu-
t~
~J FIG. 1. P h o t o m i c r o g r a p h s o f k i d n e y s e c t i o n s o b t a i n e d f r o m r a t s treated subcutaneously with potassium dichromate. The rats were killed 48 h r a f t e r t r e a t m e n t : A- 5 m g / k g ; B- 10 m g l k g ; C - 20 m g / k g ; D 40 m g l k g . ( H P S , X120).
544
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INDICES OF ACUTE N E P H R O T O X I C I T Y
bation beakers as a metabolic substrate. Two beakers were incubated with and two without sodium lactate in a Dubnoffmetabolic shaker at 25°C under a gas phase of 100% 02. Accumulation of PAH and TEA was determined in slices incubated for 90 min in medium containing both 7.4 x 10 s M PAH and 1.72 x 10 s M [14C] TEA (~0.02 /iCi l~C/mL medium). Following incubation, slices were removed from the medium, blotted and weighed. Tissue and medium digests were prepared essentially as outlined by Cross and Taggart (1950). The The concentration of PAH in aliquots of the slice and medium digests was determined by the method of Smith et aL (1945). Radioactivity present in 1.0-mL aliquots of the tissue and medium supernatants was determined by liquid scintillation spectrometry following addition of 10 mL of Aquasol (New England Nuclear). Counting efficiency was determined by internal standardization with [~4C] toluene. Accumulation of PAH and TEA was expressed as the slice-to-medium ( S / M ) ratio where S = milligrams (dpm) of PAH(TEA) per gram wet weight of tissue and M = milligrams (dpm) of PAH(TEA) per milliliter of incubation medium.
fixative, dehydrated and embedded in paraffin. Sections (5/zm) from the paraffin-embedded tissue were cut and stained with hematoxylin-phloxin-safranin (HPS).
A n alys es The BUN content was determined by the Urease-Berthelot method (Sigma Chemical Co.).
Quantitative analysis of the renal cortical necrosis and degeneration produced by the toxicants was performed by the method of Chalkley (1943) as described previously (Hewitt et aL, 1980). Briefly, one ocular was fitted with a micrometer eyepiece containing a grid on w h i c h 16 points of reference were chosen. The sections were examined at 400 X magnification. Each section was evaluated by scanning a series of microscopic fields chosen at random. In each field, the tissue element immediately beneath each of the points of reference was termed a " h i t " ; thus a total of 16 hits was examined per field. A single section from each animal was evaluated until hits on 100 tubules and 100 glomeruli were categorized (normal, degenerated, or necrotic). A series of 5 to 10 random fields for the tubules and 100 to 120 fields for the glomeruli were required to obtain the desired number. Both proximal and distal tubules were included in the scan. The number of normal, degenerated and necrotic tubules was expressed as a percentage of the total number of tubules examined in each section; the results for each group were expressed as the mean + SE. Similarly, the number of normal or degenerated gtomeruli was expressed as a percentage of the total examined.
Morphology Immediately after the animals were sacrificed, a section from one kidney was immersed in a 10% buffered-formalin
Materials Cephaloridine was generously supplied by Eli Lilly and Company. Tetra [ld4C] ethylammonium bromide (Spec. act. 3.0
TABLE 3 R e l a t i o n s h i p B e t w e e n F u n c t i o n a l a n d H i s t o l o g i c a l E v a l u a t i o n s of P o t a s s i u m D i c h r o m a t e - i n d u c e d N e p h r o t o x i c i t y in M a l e R a t s " Correlation
Coefficient (r) -0.95 ~' -0.95 b -0.92 b -0.91 h -0.87 t' -0.86 b -0.83 ~' 0.81 b 0.80 I' -0.78 ~' -0.77" -0.77 b 0.66 ~'
X-Axis
Regression Line (y = re(x) + (b))
Y-Axis
Abn T u b (%)~ Abn T u b (%) Abn T u b (%) Nec T u b (%) Deg T u b (%) Deg T u b (%) Nec T u b (%) AbnTub(%) Nec T u b (%) Nec T u b (%) DegTub(%) Nec T u b (%) Abn T u b (%)
PSR (+Lactate)" TSR (+Lactate) TSR (-Lactate) PSR (+Lactate) TSR (-Lactate) TSR (+Lactate) TSR (+Lactate) BUN BUN TSR (-Lactate) PSR(+Lactate) PSR (-Lactate} (Kidney Wt) X 100
y y y y y y y y y y y y Y
Nec T u b (%)
Body Wt (Kidney Wt)
X 100
Y = 0.004 (x) + (0.78)
0.62 h -0.59 h 0.51 t'
Deg T u b (%) Abn T u b (%) Deg T u b (%)
Body Wt BUN PSR (-Lactate) (Kidney Wt) . X 100
y = 1.85 (x) + (9.38) y = -0.04 (x) + (8.00) Y = 0.004 (x) + (0.76)
-0.21
Deg T u b (%)
Body Wt PSR (-Lactate)
y = -0.32 (x) + (22.61)
0.66 b
= -0.20 (x) -0.19 (x) = -0.17 (x) ": -0.31 (x) = -0.33 (x) = -0.34 (x) = -0.26 (x) = 1,20(x) = 1.91 (x) = -0.24 (x) = -0.33(x) = -0.08 (x) = 0.002 (x) =
+ (23.39) + (~.2.II) + (20.49) + (19,99) + (21.097 + (22.43) + (18.49) + (1.54) + (20.01) + (16.94) + (22.61) + (7.83) + (0.74)
"Functional and histological data from individual rats described in Tables 1 and 2 were analyzed, each line consisted of 34 points. hSignificant correlation coefficient, p < 0.05. ~Abn Tub (%) = Abnormal Tubules (%) Nec T u b (9o) = Necrotic T u b u l e s (%7 Deg T u b (%) = Degenerated T u b u l e s (%) PSR = PAH SIM Ratio TSR = TEA S/M Ratio
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MIYAIIMA, HEWITT, C.OTE' AND PLAA TABLE 4 Functional Evaluation of Mercuric Chloride-induced
Dose (mg/kg) 0 0.5 1.0 2.0 3.0 5.0
PAH S/M Ratio
Kidney Weight × 100 Body Weight 0.82 0.88 1.10 1.18 1.02 1.08
+ + ± -I± ±
0.02 (8) 0.03 (4) 0,04" (9) 0.08 t' (9) 0.4 (3) 0.02 (2)
N e p h r o t o x i c i t y in M a l e R a t s "
-Lactate 7.33 6.09 8.28 5.16 4.4o 4.43
-P 0.52 _-E 0.21 ± 0.56 -f- 0.40" ± 0.08 t' -t- 0.1 l
TEA S/M Ratio
+Lactate (5) (4) (9) (9) (3) (2)
20.38 20.o3 1o.57 12.36 4.77 4.26
+ ± ± ± -I±
-Lactate
1.35 (8) 0.21 (4) 0.62 (9) 1.38 t' (9) 0.26 t' (3) 0.o4" (2)
21.86 18.52 13.94 10.39 3.20 1.22
± ± ± + 4.-t-
BUN
+Lactate
0.75 (8) 1.22 (4) 1.20" (9) 0.77" (9) 1.40 I' (3) 0.19 h (2)
20.79 21.18 1o.24 11.99 3.02 1.20
-I- 0.26 (8) ± 0.44 {4) ::i: 0.89 h (9) -f- 1.01 t' (9) 4- 1.24 h (3) + 0.14 L' (2)
(mg/100 mL) 17 21 116 196 223 258
± I (8) ± 2 (4) -t- 14 I' (9) -t- 25 h (9) 4- 14 h (3) -t- 41' (2)
"Rats were sacrificed 48 hr after receiving O, 0.5, 1.0, 2.0, 3.0 or 5.0 mglkg HgClz, sc. Values represent the mean ± SE determined in (n) rats. t'Values significantly different from the control (0 mg/kg) g r o u p , p '< 0.05.
mCi/mmol) and [~"C] toluene were purchased from New England Nuclear. All other chemicals were obtained at the highest commercial purity available and used as supplied.
Dose-related increases in relative kidney weight and BUN content were observed in addition to a significant depression of slice PAH (both resting and lactate-stimulated) and TEA accumulation (Table 1). Quantitative histological evaluation of the kidney appeared to be as sensitive an index of injury as the functional parameters. A significant increase in the percentage of abnormal (degenerated and necrotic) tubules was observed at the lowest dosage (5 mg/kg) of K2Cr.)O7 administered (Table 2). Doserelated increases in the proportion of abnormal tubules were noted with increasing dosages of K2Cr,)OT. An appreciable proportion of necrotic tubules was observed in rats treated with 20 or 40 mg/kg; significant numbers of degenerated tubules were found with dosages of K2Cr.)O7 greater than 5 mg/kg (Table 2). Glomerular degeneration (about 1 to 6% of total/section) was observed in all treatment groups (data not shown). However, no treatment-related differences could be discerned. Representative photomicrographs are presented in Figure 1. The correlations between the functional and histological indices of renal injury are displayed in Table 3. Although alterations in each of the functional parameters were significantly correlated with changes in renal histology, the strength of the correlation varied appreciably depending on the particular functional and histological parameters studied, tn general, alterations in slice organic ion accumulation were highly correlated with K2Cr2OT-induced changes in renal histology. The most notable exception was resting slice PAH accumulation. Although this parameter was sensitive to K2Cr207 dama~l'e, its correlation with altered renal histology was relatively poor.
Statistical analyses Appropriate data were submitted to statistical analysis using a completely randomized design analysis of variance. Treatment means were tested using the Student-NewmanKeuls procedure (Sokal and Rohlf, 1969). The 0.05 level of probability was used as the criterion of significance. RES UL TS K2Cr207 Appreciable differences were observed in the sensitivity of the various indices of renal injury to K2Cr2OT-induced nepl~rotoxicity (Table 1 ). A statistically significant increase in resting (determined in lactate-free medium) slice PAH accumulation was observed in rats treated with 5 mg/kg K2Cr2OT. Slice TEA accumulation in a lactate-free medium was significantly depressed by the low dosage of K2Cr~Or, whereas slice TEA uptake in a lactate-supplemented medium was not altered by 5 mg/kg KzCr.~OT. Increasing the K2Cr207 dosage to 10 mg/kg significantly altered slice PAH and TEA accumulation regardless of the lactate content of the incubation medium. However, resting (lactate-free) slice PAH accumulation was increased at this dosage of K2Cr2OT, but lactate-stimulated slice PAH uptake was depressed. No changes in relative kidney weight or BUN content were noted at this dosage. Administration of 20 or 40 mg/kg K2Cr207 resulted in a marked degree of renal injury.
TABLE 5 Histological Evaluation of Mercuric Chloride-lnduced N e p h r o t o x i c i t y i n M a l e Rats" Dose (mg/kg) 0 0.5 1.0 2.0 3.0 5.0
Degenerated Tubules 2.1:1:0.4 13.0 ~ 3.4" 19.2 4- 1.9" 22.2 -b 3.6" 21.3 4- 1.2" 22.5 ::[: 1.S"
(8) (4) (9) (9) (3) (2)
Necrotic Tubules 0.0 1.8 42.1 52.9 66.7 67.5
-t- 0.0 (8) -I- 1.8 (4) ± 4.9" (9) 4- 5.1 b (9) -I-. 4.1 h (3) 4" 4.5" (2)
Total Abnormal Tubules 2.1 14.8 61.3 75.1 88.0 90.0
+ + -t-t-t-t-
0.4 (8) 4.1 b (4) 5,3 I' (9) 6.3" (9) 3.1 b (3) 3.0 h (2)
"Rats w e r e sacrificed 48 h r after receiving 0, 0.5, 1.0, 2.0, 3.0 or 5.0 m g / k g [-lgCI2, sc. Values r e p r e s e n t the m e a n percentages + SE d e t e r m i n e d in (n) rats. "Values significantly different f r o m the control (0 mgtkg) g r o u p , p < 0.05. 546
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INDICES OF ACUTE NEI'HROTOXICITY
BUN content and relative kidney weight were less sensitive indices of K=Cr2OT-induced renal inju~t~v a,nd were not highly correlated with the histological appeara,,~ce of the kidney.
HgC/~ Administration of HgCI2 to rats produced a different pattern of results than that obtained with K2Cr2OT.The lowest dosage of HgCb administered did not produce appreciabt'e changes in the functional indices of renal damage (Table 4). |n contrast, 1.0 mg/kg HgCI,) significantly elevated relative kidney weight and BUN content and depressed slice TEA uptake. However, neither resting nor lactate-stimulated slice PAH accumulation were altered by this dosage of HgCI,,. Dosages greater than 1.0 mg/kg produced significant, dose-related decreases in s!ice TEA and lactate-stimulated slice PAH accumulation. T~he depression in transport capacity was accompanied by dose. related elevation in BUN content. Although resting slice PAH uptake was significantly reduced by treating rats with 2.0 and 3.0 mg/kg, the reduction observed with 5.0 mg/kg was not statistically significant. This observation was likely due to the small number of animals surviving at 5.0 mg/kg rather than to an insensitivity of the parameter to damage. A similar explana-
[,
tion would appear to account for the failure to observe a statistically significant elevation in relative kidney weight in rats given 3.0 or 5.0 mg/kg (Table 4). The quantitative histological analysis of HgCl.,-induced renal injury offered a greater degree of sensitivity than did the functional indices of damage (Table 5). The proportion of degenerated and abnormal tubules was elevated in rats receiving 0.5 mg/kg HgCI,,, a dosage that did not alter any functional index of injury. Dosages greater than 0.5 mg/kg were associated with significant increases in the number of degenerated and necrotic tubules and, consequently, the proportion of abnormal tubules (Table 5). No treatment-related glomerular changes were observed. Representative photomicrographs are presented in Figure 2. Alterations in each of the functional parameters of renal damage were significantly correlated with alterations in the histologic appearance of the kidneys (Table 6). In contrast to K,)Cr,)O~-induced nephrotoxicity (Table 3), an elevated BUN content was the functional parameter most highly correlated with HgCl,~-induced histological damage (Table 6). For the most part, slice organic ion accumulation was also highly correlated with histologic alterations in HgCb-poisoned rats. However, as
.
FIG. 2. Photon~icrographs of kidney sections obtained from rats treated subcutaneously with mercuric chloride. The rats were killed 48 hr after treatment: A- 0.5 mg/kg; 13- 1.0 mglkg; C- 2.0 mglkg; D3.0 mglkg. (HPS, X120). Fundamental and Applied Toxicology
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eOTE A N D
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PLAA
TABLE 6 Relationship Between Functional and Histological Evaluation of
Mercuric Chloride-induced Nephrotoxicity in Male Rats" Correlation Coefficient (rJ
X-Axis
Regression Line (y = re(X) + (b))
Y-Axis
0.94 b 0.93 b -0,87 ~' -0.87" -0.83" -0.82 ~' -0.81 t' -0.80 I' 0.73 t' 0.71 t'
AhnTub(%)' NecTub(%) Abn T u b (%) Nec "rub (%) Nec Tub (%) Abn "rub (%) NecTob(%) AbnTub(%) DegTub(%) Nec T u b (%)
BUN' BUN TSR (-L,'lctate) "I'SR (-Lactate) "I'SR (+Lactate) TSR (+Lactate) PSR(+Lactate) PSR(+Lactate) BUN (Kidney Wt) X 100
y y y y y y y y y Y
0.70 t'
Abn T u b (%)
Body Wt (Kidney Wt)
Y = 0.004 (X) + (0.8,1)
-0.o7 b -0.59 I' -0.57" 0.5o"
Deg T u b (%) DegTub(%) DegTub(%) DegTub(%)
Body Wt TSR (-Lactate) TSR(+Lactate) PSR(+Lactate) (Kidney Wt) X 100
y y y Y
-0.44 I' -0,40 t' -0.37 ~'
AbnTub(%) NecTub(%) Deg T u b (%)
Body Wt PSR(-Lactate) PSR(-Lactate) PSR (-Lactate)
y = -0.02(X) + (7.05) y = -0.O3(X) + (7.41) y = -0.07 (X) + (7.50)
,'< IO0
= = = = r.: = = = = =
= = = =
2.51 (X) + (-3.37) 3.16(X) + (12.30) -0.17 (X) + (21.87) -0,21 (X) + (20.90) -0,20 (X) + (21.,It~) -0.15 (X) + (22.18) -0.18(X) * (21.11) -0.14 (X) + (21.79) o.81 (X) + (13.03) 0.005 (X) + 10.85)
-0,44 (x) -0.38(x) -0.34(X) 0,011 (X)
+ + + +
(20.70) (20.08) (20.44) (0.85)
"Functional and histological data f r o m individual rats described in Tables 4 and 5 w e r e analyzed. Each line consisted of 35 points. bSignificant correlation coefficient, p < 0.05. ' A b n T u b ( % ) = A b n o r m a l T u b u l e s (%) N e c T u b ( % ) = Necrotic T u b u l e s (%) D e g T u b ( % ) = Degenerated T u b u l e s (%) PSR : P A H S I M R a t i o TSR = T E A S / M Ratio
observed with K,)Cr.,OT, resting slice PAH accumulation was relatively poorly correlated with renal histological changes (Table 6).
Cephaloridine Resting slice PAH uptake was the most sensitive indicator of CPH-induced renal injury (Table 7). Slices from rats treated with 1.0 g/kg CPH had significantly lower resting PAH S / M ratios than did slices from control animals; this observation could be related to CPH-induced injury or simply to competitive inhibition of PAH accumulation by CPH, a substrate of the organic anion transport system. No other functional parameter was significantly altered at this dosage. CPH dosages above
1.0 g/kg significantly increased relative kidney weight and depressed slice organic ion accumulation. Statistically significant alterations in BUN content were not observed at CPH dosages less than 2.5 g/kg (Table 7). Appreciable histological alterations were not found in kidneys from rats receiving 0.5 or 1.0 g/kg CPH (Table 8). Significant increases in the proportion of tubules classified as degenerated or necrotic were observed with 2.0, 2.5 and 3.0 g/kg (Table 8). No treatment-related glomerular changes were observed. Representative photomicrographs are presented in Figure 3. As observed previously (Tables 3, 6), alterations in functional and histological indices of renal injury were, in general,
TABLE 7
Functional E v a l u a t i o n o f C e p h a l o r i d i n e - i n d u c e d Dose (glkg) 0 0.5 1.O 2.0 2.5 3.0
PAH SIM Ratio
Kidney Weight X 10o Body Weight 0.87 0.81 0.88 1.04 1.07 1.32
4:i: + :t: 4±
0.02 ( l l ) 0.03 (4) 0.04 (10) 0.03" (10) 0.05 b (11) 0.04 t' (5)
Nephrotoxicity
-Lactate 7.78 8.58 6.09 4.61 4.74 3.83
:t: 0.46 (11) d: 0.73 (4) -I- 0.27 b (10) -_i: 0.33 b (10) -4" 0.31 b (11) -I- 0.25 t' (5)
TEA SIM Ratio
* Lactate 20.74 5 : 1 . 2 4 (11) 17.43:5= 1.85 (4) 17.84 4" 0.37 (10) 13.62 d: 1.33 h (10) 14.40 q'- 1.72 b (11) 6.93 4" 1.72 t' (5)
in M a l e Rats"
-Lactate 20.52 20.43 17.70 12.16 12.33 6.76
+ :i: :t: :t: 4" 4"
0.75 (11) 1.20 (4) 0.51 (10) 0.64 b (10) 1.38 t' (11) 1.52 t' (5)
BUN
+Lactate 20.64 20,30 19.42 15.10 13.37 8.17
5:0.80 (It) 5= 1.05 (4) :t: 0.73 (10) :J: 0.99 I' (I0) 4" 1.58 b (11) :t: 2.15 t' (5)
(mg/100 mL) 18 16 16 4,6 62 90
± 1 (11) 4" 2 (4) 4- I (10) :t: 8 (I0) 4" 16 h (11) q- 26 h (5)
"Rats w e r e sacrificed 48 hr after receiving 0, 0.5, 1.0, 2.0, 2.5 or 3.0 glkg CPH, sc. Values r e p r e s e n t the mean 5= SE determined in (n) rats. bValues significantly different f r o m the control (0 g/kg) g r o u p , p < 0.05.
548
Fundam. Appl. To.tiroL (3)
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INDICES OF ACUTE N E P H R O T O X I C I T Y "FABLE 8 H i s t o l o g i c a l E v a l u a t i o n of C e p h a l o r i d i n e - i n d u c e d N e p h r o t o x i c i t y in Male Rats" Dose (g/kg,~ 0 0.5 1.0 2.0 2.5 30
Degenerated Tubules 1,8 2.2 2.o 1o.4 14.2 1o.2
:.t 0.4 (If) ± 0.2 (-11 ± 0.7 (to) -_+ 2. l)'(JO) ± 2,4)'(11) :L 3.4)'(5)
Total Abnormal Tubules
Necrotic Tubules 0.0 0.0 0.0 18.1 23.0 55..I
3_ :z. _t 3.: 32 ~
0.0 ( I I ) 0,0 (J) 0.0 (I0) 4.2t'(10)
0.5"(11) 13.1~'(5)
1.8 2,2 2.0 34.5 37.2 71.4
~c ~ at_ i 3. 3=
0,.I ( I I ) 0.2 (4) 0,7 {I0) 4,7b(10) 8.4h(11) 13.5)'(5)
"Rats were ~acrificcd 48 hr a~ter receiving O, 0.5, 1.0, 2.0, 2.5 or 3.0 gtkg C'['I I, ~c. \~a)ues represent the mean perct, nt,tges =L SI[ deter/r~int.'d m in) rat.,,. "Value,,, .,,igniticant~y dift'erenl frt)m the control (0 glkg) group,
p <2 0.05. well correlated (Tabte 9), In addition, the relationships between functional and histological parameters appeared similar to those found in HgCb-treated rats. Elevated BUN content was the functional parameter most highly correlated with CPHreduced histological damage. Depression of slice organic ion accumulation was also well correlated with histological altera-
tions in CPH-treated animals. However, the distinction between resting slice PAH uptake and the other indices of organic ion accumulation was less clear in CPH-treated rats than in rats given K,~Cr,~O;(Table 3) or HgCI2 (Table 6).
DISCUSSION If the ability to detect injury at the lowest effective dosage of a toxicam is accepted as the criterion for sensitivity of a toxicity screening test (Kluwe, 1981), it is clear (Table 10) that histological evaluation of renal injury can be as sensitive an indicator of nephrotoxicity as the functional parameters evaluated in this study, tn terms of sensitivity, the functional parameters exhibited variability, but this is understandable since the nephrotoxicants employed have different initial sites of action. Although the most sensitive functional indicator of nephrotoxicity varied with the toxicant administered, some general trends were apparent. Renal cortical slice accumulation of one or both organic indices and appeared to be the most consistently sensitive of the functional indices. Addition of lactate to the incubation medium did not increase the sensitivity of slice PAH accumulation as an index of injury (Table 10), but did facilitate interpretation since toxicant-induced elevations in slice PAH accumulation were not observed. Given that slice
,
i
(
FIG, 3. P h o t o m i c r o g r a p h s o f k i d n e y s e c t i o n s o b t a i n e d f r o m r a t s I r e a t e d s u b c u t a n e o u s l y w i t h c e p h a l o r i d i n e . T h e r a t s w e r e killed 48 h r a f t e r t r e a t m e n t : A- 1.0 glkg; B- 2.0 glkg; C- 2,5 glkg; D - 3 . 0 g/kg. (HPS, X120). Fundamental and Applied Toxicology
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MI"¢AIIMA, HEWITT, ~ O T E A N D I'LAA TABLE 9 R e l a t i o n s h i p B e t w e e n F u n c t i o n a l a n d H i s t o l o g i c a l E v a l u a t i o n of C e p h a l o r i d i n e - i n d u c e d N e p h r o t o x i c i t y in M a l e R a t s " Correlation Coefficient (r)
X-Axis
Regression Line (y = re(X) + (b))
Y-Axis BUN"
y : 1.32(X) + (8.ol) y = 1.e,o(X} + (15.03) y = -0.17 (X) ÷ (20.38)
0.o3 t' 0.o2 b -0,8o" -0.85" -0.84 t' -0.811' -0.7C' 0.78 h
AbnTub(%) Net" Tub (%} Abn Tub (%) Nec Tub (%) NecTub(%) Abn T u b (%) Nec Tub (%) AbnTub(%)
TSR (+Lactate) "I'SR (+Lactate) TSR(-Lactate) I'SR (+Lactate) I'SR (+Lactate) (Kidney Wt) ,z 100
y y y y Y
-0.77" 0.75"
Deg T u b (%) NecTub(%)
Body WI "I'SIC, (-Lactate) (Kidnev Wt)
y = -0.50 (X) + (19.o0) v = 0.00o(X} * (0.O0)
-0.74" -0.72 b -0.70 t' -O.oC' -0.o2" -0.c,2" 0.o0 h 0.34 ~'
BUN
= -0.20 iX) + (19.3o)
= -0.20(X) + (18.08) = -0.1o iX) * (19,55) = -0.20 (X) + (18.O1) = 0.005(X) + (0.87)
AbnTub(%) Deg Tub (%) AbnTub(%) DegTub(%) NecTub(%) l.)eg "l'ub (%) D e g T u b (%)
" "" 100 Body WI TSR(-Lactate) TSR(+Lactate) PSR(-Lactate) l"SR(-Lactate) I'SR(-Lactate) I'SR (+Lactate) (Kidney Wt) ,K 100
y y y y y y y
DegTub(%)
Body Wt BUN
y = 0.75(X) + (31.10/
"
= = : = = = :
-0.14 (X) -0.4oiX) -0.05(X) -0.1o(X) -0.05(X) -0.42 (X) 0.014 (X)
+ (18.74) * (20,5o) + (o.9,1) *" (7.25) + (o.o0) + (19.53) + (0.87)
"Functional and histological data from individual rats described in Tables 7 and 8 were analyzed. Each line consisted of 51 points. t'Significant correlation coefficient, p < 0.05. ' A b n T u b (%) = Abnormal T u b u l e s (%) N e c T u b { % ) = Necrotic T u b u l e s (%) D e g T u b ( % ) = Degenerated T u b u l e s (%) I'SR = I'AH S/IVl Ratio "FSR = TEA SIM l;',atio
TEA accumulation can be 1) as sensitive an indicator of nephrotoxicity as slice PAH uptake (Table 10), 2) determined simultaneously with PAH transport, and 3) determined in the presence or absence of a metabolic substrate without appreciable alterations in sensitivity (Table 10), it appears reasonable to suggest that the combination of lactate-simulated PAH and TEA accumulation by renal cortical slices would be an appropriate screening test for the ability of toxicants to injure the proximal tubule. It is also noteworthy that alterations in each of the functional indices were significantly correlated with changes in renal histology (Tables 3, 6, 9). With the exception of PAH accumulation by resting slices, organic ion accumulation was highly correlated with renal damage as determined by histopathology, thus supporting the earlier conclusion that lactatestimulated organic ion accumulation was a more appropriate measure of proximal tubule injury than resting slice organic ion uptake. Similarly, the close correlation between BUN content and histological evidence of kidney injury in HgCI.,- and CPH-treated rats, coupled with its relative ease of performance, suggests that BUN remains a useful index of kidney damage despite its inability to detect a minimal degree of renal injury (Sharratt and Fraser, 1963; Sharratt, 1970; Kluwe, 1981.; Berndt, 1981). It was not surprising to observe a significant correlation between structural and functional indices of nephrotoxicity. Wachsmuth (1982) demonstrated a correlation between his550
tochemically quantified renal damage and the urinary excretion of various enzymes, in particular lactic dehydrogenase. However, other investigators (Sharratt and Frazer, 1963; Kluwe, 1981 ; Berndt, 1981 ) have found a relatively poor correlation between various functional indices of damage and morphological changes in renal morphology. In particular, 8erndt (1981) has presented several examples where organic ion accumulation by renal cortical slices in vilro was reduced when kidneys from these animals were judged to be normal, or at worst mildly affected, by light microscopy. Although these
TABLE 10 Relative Sensitivity of Indices of Nephrotoxicity" Lowest Relative Dosage (1-37 at which a Significant Alteration was O b s e r v e d Parameter
K.~Cr.~O~
HgClz
Cephaloridine
3 1 2 I 2 3 1 3 I
2 3 3 2 2 2 ! 2 l
2 I 2 2 2 3 2 2 2
Relative Kidney Weight I'AH S/M Ratio (-Lactate) I'AH S/M Ratio (+Lactate) TEA S/M Ratio C-Lactate) TEA S/M Ratio (+Lactate)
BUN Degenerated T u b u l e s (%) Necrotic T u b u l e s (%) A b n o r m a l T u b u l e s (%)
"Format adapted from Kluwe (1981).
Fundarn. AppL Toxicol. (3)
November/December, 1983
INDICES OF ACUTE NEI'FIROTOXICITY observations appear to conflict with the results of the present investigation, histological assessment may miss a nephrotoxic lesion if timed incorrectly (Piperno, 1981 ). In this study, sufficient time (48 hr) was allowed to elapse for a histologically detectable lesion to appear. In the investigations cited by 8erndt (1981 ), kidneys were examined histologically at earlier times (12-24 hr following toxicant administration). Thus the apparent discrepancy between these studies is likely to arise from a difference in experimental design rather than a lack of a renal correlation between structural and functional parameters of kidney injury. However, these observations do suggest that evaluations of the relative sensitivity of toxicity tests based solely on dose-response considerations may not be completely valid; the time-response relationship should also be evaluated. In summary, organic ion (PAH and TEA) accumulation by renal cortical slices incubated in a Jactate-supplemented ir~cubation medium was considered to be the best functional indicator of renal injury used in this study due to its relative sensitivity, ease of interpretation and good correlation with a quantitative histological evaluation of damage. The quantitative histological procedure used was found to be as sensitive an indicator of nephrotoxicity as organic ion accumulation under the conditions of the study. However, additional investigation is required to document the relative sensitivities of the histological and functional indices at different time points in the progression of the lesion.
A CKNO WLEDGEMENT We gratefully acknowledge the expert technical assistance of Johanne Couture-Mdnard and Monique Morisset. This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES Atkinson, R.M., Currie, J,P., Davis, B., Pratt, D.A.H., Sharpe, H.M., and Tomich, E.G. (] 96b). Acute Toxicity of Cephaloridine, an Antibiotic Derived from Cephalosporin C. To.tiroL AppL Phurmacol. 8:398-406. Berndt, W.O. (1975). The Effect of Potassium Dichromate on Renal Tubular Transport Processes. ToxicoL Appl. PharmacoL 32:40-52. Berndt, W.O. (198l). Use of Renal Function Tests in the Evaluation of Nephrotoxic Effects. In. Toxicolog3"of the Kidmp" J.H. Hook, ed., pp. 1-29, Raven Press, New York.
Fundamental and Applied Toxicology
(3) i!.12/83
Chalkley, H.IN. (]943). Method for the Quantitative Morphologic Analysis of Tissues. d. Natl. Cancer hist. 4:47-53. Cross, R.J. and Taggarl, J.V. (1950). Renal Tubular Transport: Accumulation of p-Aminohippurate by Rabbit Kidney Slices. A met..I, l'hysioL J 6 ] : 1,8] - 190. Ganote, C.E., Reimer, K.A., and Jennings, R.B. (1975), Acute Mercuric Chloride Nephrotoxicity: An Electron Microscopic and Metabolic Study. lab. hlvest. 31:633-647. /4ewitt, W.R., Miyayima, H., C8t6, M.G,, and Plaa, G.L. (1980). Acute Alterations of Chloroform-induced Hepato- and Nephrotoxicity by n-Hexane, Methyl n-Butyl Ketone and 2,5Hexanedione. To.rh'oL AppL PhartnacoL 53:230*248. Hirsch, G.H. () 973). Differential Effects of Nephrotoxic Agents on Renal Organic Ion Transport and Metabolism. J. Pharmacol. F.~TJ. 77wr. ] 80:593-599. Kacew, S. and Hirsch, G.H. (/981}. Evaluation of Nephrotoxicity of Various Compounds by Means of hi Iqtro Techniques and Compa risen to ht Vivo Met hods. In: Toxicology of'the Kidney, J.B. Hook, ed., pp. 77-98, Raven Press, New York. Kluwe, W.M. (1981 ). Renal Function Tests as Indicators of Kidney Injury in Subacute Toxicity Studies. ToxicoL ,,lppl. PharmacoL 57:414-42.4. Phillips, R., Yamauchi, M., C8tl, M.G., and Plaa, G.L. (1977). Assessment of Mercuric Chloride-induced Nephrotoxicity b), p-Aminohippuric Acid Uptake and the Activity of Four Gluconeogenic Enzymes in Rat Renal Cortex. Toxicol. AppL l"harmacol. 41:407-422. Piperno, E. (1981). Detection of Drug-induced Nephrotoxicity with Urinalysis and Enzymuria Assessment: In: Toxicology o f the Kidno', ].B. Hook, ed., pp. 31-35, Raven Press, New York. Sharratt, M. (~970). Renal Function Tests in Laboratory Animals. In: Metabolic A.ffJects o f Food Safi, ty, F.J. Roe, ed., pp. 1] 9-167, Academic Press, New York. Sharratt, M. and Frazer, A.C. (1963). The Sensitivity of Function Tests in Detecting Reqal Damage in the Rat. ToxicoL AppL PharmacoL 5:36-48. Smith, H.W., Finkelstein, N., Aliminosa, L., Crawford, B., and Graber, M. (]945). The Renal Clearhnces of Substituted Hippuric Acid Derivatives and Other A,'omatic Acids in Dog and Man. d. Clin. htvest. 24:388-404. Sokal, R.R. and Rohif, F.J. (1969). Biometry, Freeman, San Francisco. Wachsmuth, E.D. (1982). Quantification of Acute Cephaloridine Nephrotoxicity in Rats: Correlation of Serum and 24-hr Urine Analyses with Proximal Tubule Injuries. To.tirol. Appl. PharmacoL 63:429-445. Wold, I.S. (1981}. Antibiotic Nephropathies. In: Toxicology o f the Kidnc~', J.B. Hook, ed., pp. 251-266, R,~ven Press, New York.
351
Relationships Between Histological and Functional Indices of Acute Chemically Induced Nephrotoxicity H I R O A K I M I Y A J I M A ' , W I L L I A M R. H E W I T T ? , M I C H E L G. C O T E , and G A B R I E L L. P L A A ' * D ~ p a r t e m e n t de P h a r m a c o l o g i c , Facult~ de M ~ d e c i n e , U n i v e r s i t 6 de M o n t r d a l , M o n t r d a l , Q u e b e c , C a n a d a H 3 C 3J7
ABSTRACT
Relationships Between Histological and Functional Indices of Acute Chemically Induced Nephrotoxicity. Miyajima, H., Hewitt, W.R., C~t~, M.G., and Plaa, G.L. (1983). Fundam. Appl. Toxicol. 3:543-551. Acute renal injury was produced in rats with K.zCr,~O7(5-40 mg/kg, sc) HgCi2 (0.5-5.0 rag/kg, sc) or cephaloridine (0.5-3.0 g/kg, sc). Histological (percentage of normal, degenerated or necrotic cells) and functional indices (relative kidney weight, renal cortical slice accumulation of organic ions, and blood urea nitrogen content) were evaluated 48 hours later. The relative sensitivity of each of these indices was determined for each nephrotoxicant. Renal cortical accumulation of organic ions appeared to be the most sensitive of the functional parameters. A quantitative histological evaluation was found to be as sensitive an indicator of nephrotoxicity as organic ion accumulation. Alterations in each of the functional indices were significantly correlated with changes in renal histology.
as have increases in blood urea nitrogen and creatinine content (Sharratt, 1970; Kluwe, 1981; Piperno, 1981; Berndt, 1981). Similarly, alterations in glomerular filtration rate, urine concentration and dilution capacity, various metabolic processes, and in rive and in vitro renal organic ion secretion have been utilized successfully as indices of nephrotoxicity (Sharratt, 1970; Phillips et al., 1977; Kluwe, 1981; Kacew and Hirsch, 1981). However the relative sensitivity and applicability of these and/or other parameters, such as histopathologieal alterations, as indices of chemically ir~duced kidney injury remain in question. Kluwe (1981 ) found -hat accumulation in vitro of organic ions by renal cortical tissue, altered kidney weight and changes in urinary concentrating ability were among the most sensitive and versatile indicators of injury, while changes in renal morphology were considered to be less sensitive and less consistent indicators. In contrast, Sharratt and Frazer (1963)suggested that histopathology was a more effective index of renal damage than urinalysis or a battery of renal function tests.
INTRODUCTION The kidney is a target organ for a host of toxic chemicals found in the environment or used for therapy of disease states. As such, reliable, sensitive and versatile tests are needed to screen for the nephrotoxic potential of drugs and cheraicals. Several urinary and renal functional parameters have been evaluated for their utility as probes of chemically induced nephrotoxicity. Analyses of urine for abnormally high concentrations of glucose, proteins, blood and enzymes of renal origin have been found useful for detecting and quantifying nephrotoxic insults
Unfortunately, relatively few studies have directly compared toxicant-induced alterations in renal fun.ctional tests with a quantitative histological analysis of kidney injury. Thus the primary objective of this investigation was to determine if a correlation existed between alterations in one or more of the functional parameters and the severity of the lesion as judged by histopathology. The focus of the investigation was narrowed by 1) selecting nephrotoxicants known to selectively damage the proximal tubule, and 2) selecting functional parameters currently employed in the assessment of chemically induced acute tubular necrosis. In addition, information on the relative sensitivities of the functional and histological indices of proximal tubular injury was derived.
*Visiting Scientist from Takeda Chemical Industries, Ltd,, Osaka, Japan, ~'Chemical Industry Institute of Toxicology Postdoctoral Fellow. Present .address: Smith Kline & French LaboratOries, Philadelphia. PA 19107. *"To whom reprint requests should be sent.
Functional Evaluation of Potassium
Varying degrees of kidney damage were produced with three known nephrotoxicants: potassium dichromate (Hirsch, 1973; Berndt, 1975; Kacew and Hirsch, 1981 ), mercuric chlo-
TABLE 1 Dichromate-induced
Nephrotoxicity
PAH S/M Ratio Dose (m~lkg) 0 5 I0 20 40
Kidney Weight X 100 Body Weight 0.78 0.74 0.82 0.94 1.50
-P + ± :t: :t:
0.03 (7) 0.03 (6) 0.03 (7) 0.04)'(8) 0.08 I' (6)
-Lactate 6.54 8.14 8.16 4.74 2.95
444-I4-
0.63 (7) 0.19J'(6) 0.78 j' (7) 0.41b(8) 0.32 j' (6)
TEA filM Ratio
+Lactate 21.80 23.38 13.93 5,08 3,20
in M a l e R a t s "
+ 1.35 (7) + 0,65 (6) + 1.30 h (7) + 0.32~'(8) --t- 0.3~4~' (6)
-Lactate 22.54 17.86 9.15 7.01 2.91
4-44::k 4-
1.27 (7) 1,01J'(6) 0.66 j' (7) 0.36~'(8) 0.47 b (6)
BUN
+Lactate 22.98 20.66 10.62 7.05 3.35
4- 1.14 (7) -t- 1.19 (6) + 0.61 t' (7) ::]: 0.46~'(8) -I- 0.62 b (6)
(mgla00 mL) 16 21 24 98 150
4- 1 (7) --1- 1 (6) 4- I (7) -P 10"(8) 4- 26 b (6)
"Rats were sacrificed 48 h r a f t e r receiving 0, 5, 10, 20 or 40 mg/kg K2CreOr, sc, Values represent the mean ::k SE d e t e r m i n e d in (n) rats. t'Values significantly different from the centre] (0 mglkg) group, p < 0.05. Copyright 1983, Societyel Toxicology
Fundamentaland Applied Toxicology
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MIYAJIMA, HEWITT, ~OTE A N D PLAA
ride (Hirsch, 1973; Ganote el al., 1975; Phillips el a/., 1977) and cephaloridine (CPH) (Atkinson el al., 1966; Wold, 1981). Nephrotoxicity was monitored by alterations in: 1) relative kidney weight, 2) renal cortical slice accumulation (resting and lactate-stimulated) of an organic anion (p-aminohippurate, PAH), 3) renal cortical slice accumulation (resting and lactatestimulated) of an organic cation ( t e t r a e t h y l a m m o n i u m ion, TEA), and 4) blood urea nitrogen (BUN) content.
TABLE 2 Histological Evaluation of Potassium Dichromate-induced N e p h r o t o x i c i t y in M a l e Rats" Dose (mglkg)
METHODS Male, Sprague-Dawley rats (150 to 300 g) were purchased from Bio-Breeding Laboratories and maintained on Purina Lab Chow and water ad/fbilum. Animals were used after a suitable period of acclimation in the animal quarters. The K.,Cr.,OT, HgCI= and CP}; were dissolved in water and administered subcutaneously in various dosages. Control animals received an equivalent volume of water subcutaneously. Forty-eight hours after administration of the toxicant, the animals were lightly anesthetized w i t h ether and blood removed via the abdominal aorta, rinsed in ice-cold 0.9% NaCI and weighed. Sections from one kidney were fixed in 10% buffered formalin for histological processing.
Degenerated Tubules
Necrotic Tubules
Total Abnormal Tubules
0 5
I.o ¢ 0.5 (7) 0.7 ~ 1.7h(o)
0.0 _+ 0.0 (7) 0.0 --- 0.0 (o)
1.o + 0.5 (7) a.7 +__ 1.7t'(o)
10 20 40
43.7 ± 4.1 u(7) 30.0 ~ 2.3" (14) 47.5 ± 3.o"(o)
40 -e 1.3 (7) 4o.1 ± 1.5 ~' (8) 52.5 2: 3.o"(o)
47.7 ± 4. °" (7) B5.8 ± 1.7 I' (g} tO0 ~ 0 {o,~
"Rats :'.'ere sacrificed 48 hr after receiving 0, 5, 10, 20 or 40 mglkg K~Cr~O:,sc. Values represent the mean percentage~ :t= S E d e t e r m i n e d
in ~nt rals. "Values ~ignificantly different from the control (0 mglkg) ~.,roup. p < 0.05.
D e t e r m i n a t i o n o f P A H a n d TEA uptake After being weighed, the kidneys were stored briefly in icecold 0.9% NaCI. Thin renal cortical slices were prepared freehand and placed in 3.0 mL of the phosphate-buffered (pH 7.4) incubation medium described by Cross and Taggart (1950). Sodium lactate (10 raM)was included in one-half of the incu-
t~
~J FIG. 1. P h o t o m i c r o g r a p h s o f k i d n e y s e c t i o n s o b t a i n e d f r o m r a t s treated subcutaneously with potassium dichromate. The rats were killed 48 h r a f t e r t r e a t m e n t : A- 5 m g / k g ; B- 10 m g l k g ; C - 20 m g / k g ; D 40 m g l k g . ( H P S , X120).
544
Fundnm. Applo Toxicol. (3)
November/December, 1983
INDICES OF ACUTE N E P H R O T O X I C I T Y
bation beakers as a metabolic substrate. Two beakers were incubated with and two without sodium lactate in a Dubnoffmetabolic shaker at 25°C under a gas phase of 100% 02. Accumulation of PAH and TEA was determined in slices incubated for 90 min in medium containing both 7.4 x 10 s M PAH and 1.72 x 10 s M [14C] TEA (~0.02 /iCi l~C/mL medium). Following incubation, slices were removed from the medium, blotted and weighed. Tissue and medium digests were prepared essentially as outlined by Cross and Taggart (1950). The The concentration of PAH in aliquots of the slice and medium digests was determined by the method of Smith et aL (1945). Radioactivity present in 1.0-mL aliquots of the tissue and medium supernatants was determined by liquid scintillation spectrometry following addition of 10 mL of Aquasol (New England Nuclear). Counting efficiency was determined by internal standardization with [~4C] toluene. Accumulation of PAH and TEA was expressed as the slice-to-medium ( S / M ) ratio where S = milligrams (dpm) of PAH(TEA) per gram wet weight of tissue and M = milligrams (dpm) of PAH(TEA) per milliliter of incubation medium.
fixative, dehydrated and embedded in paraffin. Sections (5/zm) from the paraffin-embedded tissue were cut and stained with hematoxylin-phloxin-safranin (HPS).
A n alys es The BUN content was determined by the Urease-Berthelot method (Sigma Chemical Co.).
Quantitative analysis of the renal cortical necrosis and degeneration produced by the toxicants was performed by the method of Chalkley (1943) as described previously (Hewitt et aL, 1980). Briefly, one ocular was fitted with a micrometer eyepiece containing a grid on w h i c h 16 points of reference were chosen. The sections were examined at 400 X magnification. Each section was evaluated by scanning a series of microscopic fields chosen at random. In each field, the tissue element immediately beneath each of the points of reference was termed a " h i t " ; thus a total of 16 hits was examined per field. A single section from each animal was evaluated until hits on 100 tubules and 100 glomeruli were categorized (normal, degenerated, or necrotic). A series of 5 to 10 random fields for the tubules and 100 to 120 fields for the glomeruli were required to obtain the desired number. Both proximal and distal tubules were included in the scan. The number of normal, degenerated and necrotic tubules was expressed as a percentage of the total number of tubules examined in each section; the results for each group were expressed as the mean + SE. Similarly, the number of normal or degenerated gtomeruli was expressed as a percentage of the total examined.
Morphology Immediately after the animals were sacrificed, a section from one kidney was immersed in a 10% buffered-formalin
Materials Cephaloridine was generously supplied by Eli Lilly and Company. Tetra [ld4C] ethylammonium bromide (Spec. act. 3.0
TABLE 3 R e l a t i o n s h i p B e t w e e n F u n c t i o n a l a n d H i s t o l o g i c a l E v a l u a t i o n s of P o t a s s i u m D i c h r o m a t e - i n d u c e d N e p h r o t o x i c i t y in M a l e R a t s " Correlation
Coefficient (r) -0.95 ~' -0.95 b -0.92 b -0.91 h -0.87 t' -0.86 b -0.83 ~' 0.81 b 0.80 I' -0.78 ~' -0.77" -0.77 b 0.66 ~'
X-Axis
Regression Line (y = re(x) + (b))
Y-Axis
Abn T u b (%)~ Abn T u b (%) Abn T u b (%) Nec T u b (%) Deg T u b (%) Deg T u b (%) Nec T u b (%) AbnTub(%) Nec T u b (%) Nec T u b (%) DegTub(%) Nec T u b (%) Abn T u b (%)
PSR (+Lactate)" TSR (+Lactate) TSR (-Lactate) PSR (+Lactate) TSR (-Lactate) TSR (+Lactate) TSR (+Lactate) BUN BUN TSR (-Lactate) PSR(+Lactate) PSR (-Lactate} (Kidney Wt) X 100
y y y y y y y y y y y y Y
Nec T u b (%)
Body Wt (Kidney Wt)
X 100
Y = 0.004 (x) + (0.78)
0.62 h -0.59 h 0.51 t'
Deg T u b (%) Abn T u b (%) Deg T u b (%)
Body Wt BUN PSR (-Lactate) (Kidney Wt) . X 100
y = 1.85 (x) + (9.38) y = -0.04 (x) + (8.00) Y = 0.004 (x) + (0.76)
-0.21
Deg T u b (%)
Body Wt PSR (-Lactate)
y = -0.32 (x) + (22.61)
0.66 b
= -0.20 (x) -0.19 (x) = -0.17 (x) ": -0.31 (x) = -0.33 (x) = -0.34 (x) = -0.26 (x) = 1,20(x) = 1.91 (x) = -0.24 (x) = -0.33(x) = -0.08 (x) = 0.002 (x) =
+ (23.39) + (~.2.II) + (20.49) + (19,99) + (21.097 + (22.43) + (18.49) + (1.54) + (20.01) + (16.94) + (22.61) + (7.83) + (0.74)
"Functional and histological data from individual rats described in Tables 1 and 2 were analyzed, each line consisted of 34 points. hSignificant correlation coefficient, p < 0.05. ~Abn Tub (%) = Abnormal Tubules (%) Nec T u b (9o) = Necrotic T u b u l e s (%7 Deg T u b (%) = Degenerated T u b u l e s (%) PSR = PAH SIM Ratio TSR = TEA S/M Ratio
Fundamental and Applied Toxicology
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MIYAIIMA, HEWITT, C.OTE' AND PLAA TABLE 4 Functional Evaluation of Mercuric Chloride-induced
Dose (mg/kg) 0 0.5 1.0 2.0 3.0 5.0
PAH S/M Ratio
Kidney Weight × 100 Body Weight 0.82 0.88 1.10 1.18 1.02 1.08
+ + ± -I± ±
0.02 (8) 0.03 (4) 0,04" (9) 0.08 t' (9) 0.4 (3) 0.02 (2)
N e p h r o t o x i c i t y in M a l e R a t s "
-Lactate 7.33 6.09 8.28 5.16 4.4o 4.43
-P 0.52 _-E 0.21 ± 0.56 -f- 0.40" ± 0.08 t' -t- 0.1 l
TEA S/M Ratio
+Lactate (5) (4) (9) (9) (3) (2)
20.38 20.o3 1o.57 12.36 4.77 4.26
+ ± ± ± -I±
-Lactate
1.35 (8) 0.21 (4) 0.62 (9) 1.38 t' (9) 0.26 t' (3) 0.o4" (2)
21.86 18.52 13.94 10.39 3.20 1.22
± ± ± + 4.-t-
BUN
+Lactate
0.75 (8) 1.22 (4) 1.20" (9) 0.77" (9) 1.40 I' (3) 0.19 h (2)
20.79 21.18 1o.24 11.99 3.02 1.20
-I- 0.26 (8) ± 0.44 {4) ::i: 0.89 h (9) -f- 1.01 t' (9) 4- 1.24 h (3) + 0.14 L' (2)
(mg/100 mL) 17 21 116 196 223 258
± I (8) ± 2 (4) -t- 14 I' (9) -t- 25 h (9) 4- 14 h (3) -t- 41' (2)
"Rats were sacrificed 48 hr after receiving O, 0.5, 1.0, 2.0, 3.0 or 5.0 mglkg HgClz, sc. Values represent the mean ± SE determined in (n) rats. t'Values significantly different from the control (0 mg/kg) g r o u p , p '< 0.05.
mCi/mmol) and [~"C] toluene were purchased from New England Nuclear. All other chemicals were obtained at the highest commercial purity available and used as supplied.
Dose-related increases in relative kidney weight and BUN content were observed in addition to a significant depression of slice PAH (both resting and lactate-stimulated) and TEA accumulation (Table 1). Quantitative histological evaluation of the kidney appeared to be as sensitive an index of injury as the functional parameters. A significant increase in the percentage of abnormal (degenerated and necrotic) tubules was observed at the lowest dosage (5 mg/kg) of K2Cr.)O7 administered (Table 2). Doserelated increases in the proportion of abnormal tubules were noted with increasing dosages of K2Cr,)OT. An appreciable proportion of necrotic tubules was observed in rats treated with 20 or 40 mg/kg; significant numbers of degenerated tubules were found with dosages of K2Cr.)O7 greater than 5 mg/kg (Table 2). Glomerular degeneration (about 1 to 6% of total/section) was observed in all treatment groups (data not shown). However, no treatment-related differences could be discerned. Representative photomicrographs are presented in Figure 1. The correlations between the functional and histological indices of renal injury are displayed in Table 3. Although alterations in each of the functional parameters were significantly correlated with changes in renal histology, the strength of the correlation varied appreciably depending on the particular functional and histological parameters studied, tn general, alterations in slice organic ion accumulation were highly correlated with K2Cr2OT-induced changes in renal histology. The most notable exception was resting slice PAH accumulation. Although this parameter was sensitive to K2Cr207 dama~l'e, its correlation with altered renal histology was relatively poor.
Statistical analyses Appropriate data were submitted to statistical analysis using a completely randomized design analysis of variance. Treatment means were tested using the Student-NewmanKeuls procedure (Sokal and Rohlf, 1969). The 0.05 level of probability was used as the criterion of significance. RES UL TS K2Cr207 Appreciable differences were observed in the sensitivity of the various indices of renal injury to K2Cr2OT-induced nepl~rotoxicity (Table 1 ). A statistically significant increase in resting (determined in lactate-free medium) slice PAH accumulation was observed in rats treated with 5 mg/kg K2Cr2OT. Slice TEA accumulation in a lactate-free medium was significantly depressed by the low dosage of K2Cr~Or, whereas slice TEA uptake in a lactate-supplemented medium was not altered by 5 mg/kg KzCr.~OT. Increasing the K2Cr207 dosage to 10 mg/kg significantly altered slice PAH and TEA accumulation regardless of the lactate content of the incubation medium. However, resting (lactate-free) slice PAH accumulation was increased at this dosage of K2Cr2OT, but lactate-stimulated slice PAH uptake was depressed. No changes in relative kidney weight or BUN content were noted at this dosage. Administration of 20 or 40 mg/kg K2Cr207 resulted in a marked degree of renal injury.
TABLE 5 Histological Evaluation of Mercuric Chloride-lnduced N e p h r o t o x i c i t y i n M a l e Rats" Dose (mg/kg) 0 0.5 1.0 2.0 3.0 5.0
Degenerated Tubules 2.1:1:0.4 13.0 ~ 3.4" 19.2 4- 1.9" 22.2 -b 3.6" 21.3 4- 1.2" 22.5 ::[: 1.S"
(8) (4) (9) (9) (3) (2)
Necrotic Tubules 0.0 1.8 42.1 52.9 66.7 67.5
-t- 0.0 (8) -I- 1.8 (4) ± 4.9" (9) 4- 5.1 b (9) -I-. 4.1 h (3) 4" 4.5" (2)
Total Abnormal Tubules 2.1 14.8 61.3 75.1 88.0 90.0
+ + -t-t-t-t-
0.4 (8) 4.1 b (4) 5,3 I' (9) 6.3" (9) 3.1 b (3) 3.0 h (2)
"Rats w e r e sacrificed 48 h r after receiving 0, 0.5, 1.0, 2.0, 3.0 or 5.0 m g / k g [-lgCI2, sc. Values r e p r e s e n t the m e a n percentages + SE d e t e r m i n e d in (n) rats. "Values significantly different f r o m the control (0 mgtkg) g r o u p , p < 0.05. 546
Fundam. AppL ToxicoL (3)
November/December, 1983
INDICES OF ACUTE NEI'HROTOXICITY
BUN content and relative kidney weight were less sensitive indices of K=Cr2OT-induced renal inju~t~v a,nd were not highly correlated with the histological appeara,,~ce of the kidney.
HgC/~ Administration of HgCI2 to rats produced a different pattern of results than that obtained with K2Cr2OT.The lowest dosage of HgCb administered did not produce appreciabt'e changes in the functional indices of renal damage (Table 4). |n contrast, 1.0 mg/kg HgCI,) significantly elevated relative kidney weight and BUN content and depressed slice TEA uptake. However, neither resting nor lactate-stimulated slice PAH accumulation were altered by this dosage of HgCI,,. Dosages greater than 1.0 mg/kg produced significant, dose-related decreases in s!ice TEA and lactate-stimulated slice PAH accumulation. T~he depression in transport capacity was accompanied by dose. related elevation in BUN content. Although resting slice PAH uptake was significantly reduced by treating rats with 2.0 and 3.0 mg/kg, the reduction observed with 5.0 mg/kg was not statistically significant. This observation was likely due to the small number of animals surviving at 5.0 mg/kg rather than to an insensitivity of the parameter to damage. A similar explana-
[,
tion would appear to account for the failure to observe a statistically significant elevation in relative kidney weight in rats given 3.0 or 5.0 mg/kg (Table 4). The quantitative histological analysis of HgCl.,-induced renal injury offered a greater degree of sensitivity than did the functional indices of damage (Table 5). The proportion of degenerated and abnormal tubules was elevated in rats receiving 0.5 mg/kg HgCI,,, a dosage that did not alter any functional index of injury. Dosages greater than 0.5 mg/kg were associated with significant increases in the number of degenerated and necrotic tubules and, consequently, the proportion of abnormal tubules (Table 5). No treatment-related glomerular changes were observed. Representative photomicrographs are presented in Figure 2. Alterations in each of the functional parameters of renal damage were significantly correlated with alterations in the histologic appearance of the kidneys (Table 6). In contrast to K,)Cr,)O~-induced nephrotoxicity (Table 3), an elevated BUN content was the functional parameter most highly correlated with HgCl,~-induced histological damage (Table 6). For the most part, slice organic ion accumulation was also highly correlated with histologic alterations in HgCb-poisoned rats. However, as
.
FIG. 2. Photon~icrographs of kidney sections obtained from rats treated subcutaneously with mercuric chloride. The rats were killed 48 hr after treatment: A- 0.5 mg/kg; 13- 1.0 mglkg; C- 2.0 mglkg; D3.0 mglkg. (HPS, X120). Fundamental and Applied Toxicology
(3) 11-12/83
547
eOTE A N D
MIYAJIMA, HEWITT,
PLAA
TABLE 6 Relationship Between Functional and Histological Evaluation of
Mercuric Chloride-induced Nephrotoxicity in Male Rats" Correlation Coefficient (rJ
X-Axis
Regression Line (y = re(X) + (b))
Y-Axis
0.94 b 0.93 b -0,87 ~' -0.87" -0.83" -0.82 ~' -0.81 t' -0.80 I' 0.73 t' 0.71 t'
AhnTub(%)' NecTub(%) Abn T u b (%) Nec "rub (%) Nec Tub (%) Abn "rub (%) NecTob(%) AbnTub(%) DegTub(%) Nec T u b (%)
BUN' BUN TSR (-L,'lctate) "I'SR (-Lactate) "I'SR (+Lactate) TSR (+Lactate) PSR(+Lactate) PSR(+Lactate) BUN (Kidney Wt) X 100
y y y y y y y y y Y
0.70 t'
Abn T u b (%)
Body Wt (Kidney Wt)
Y = 0.004 (X) + (0.8,1)
-0.o7 b -0.59 I' -0.57" 0.5o"
Deg T u b (%) DegTub(%) DegTub(%) DegTub(%)
Body Wt TSR (-Lactate) TSR(+Lactate) PSR(+Lactate) (Kidney Wt) X 100
y y y Y
-0.44 I' -0,40 t' -0.37 ~'
AbnTub(%) NecTub(%) Deg T u b (%)
Body Wt PSR(-Lactate) PSR(-Lactate) PSR (-Lactate)
y = -0.02(X) + (7.05) y = -0.O3(X) + (7.41) y = -0.07 (X) + (7.50)
,'< IO0
= = = = r.: = = = = =
= = = =
2.51 (X) + (-3.37) 3.16(X) + (12.30) -0.17 (X) + (21.87) -0,21 (X) + (20.90) -0,20 (X) + (21.,It~) -0.15 (X) + (22.18) -0.18(X) * (21.11) -0.14 (X) + (21.79) o.81 (X) + (13.03) 0.005 (X) + 10.85)
-0,44 (x) -0.38(x) -0.34(X) 0,011 (X)
+ + + +
(20.70) (20.08) (20.44) (0.85)
"Functional and histological data f r o m individual rats described in Tables 4 and 5 w e r e analyzed. Each line consisted of 35 points. bSignificant correlation coefficient, p < 0.05. ' A b n T u b ( % ) = A b n o r m a l T u b u l e s (%) N e c T u b ( % ) = Necrotic T u b u l e s (%) D e g T u b ( % ) = Degenerated T u b u l e s (%) PSR : P A H S I M R a t i o TSR = T E A S / M Ratio
observed with K,)Cr.,OT, resting slice PAH accumulation was relatively poorly correlated with renal histological changes (Table 6).
Cephaloridine Resting slice PAH uptake was the most sensitive indicator of CPH-induced renal injury (Table 7). Slices from rats treated with 1.0 g/kg CPH had significantly lower resting PAH S / M ratios than did slices from control animals; this observation could be related to CPH-induced injury or simply to competitive inhibition of PAH accumulation by CPH, a substrate of the organic anion transport system. No other functional parameter was significantly altered at this dosage. CPH dosages above
1.0 g/kg significantly increased relative kidney weight and depressed slice organic ion accumulation. Statistically significant alterations in BUN content were not observed at CPH dosages less than 2.5 g/kg (Table 7). Appreciable histological alterations were not found in kidneys from rats receiving 0.5 or 1.0 g/kg CPH (Table 8). Significant increases in the proportion of tubules classified as degenerated or necrotic were observed with 2.0, 2.5 and 3.0 g/kg (Table 8). No treatment-related glomerular changes were observed. Representative photomicrographs are presented in Figure 3. As observed previously (Tables 3, 6), alterations in functional and histological indices of renal injury were, in general,
TABLE 7
Functional E v a l u a t i o n o f C e p h a l o r i d i n e - i n d u c e d Dose (glkg) 0 0.5 1.O 2.0 2.5 3.0
PAH SIM Ratio
Kidney Weight X 10o Body Weight 0.87 0.81 0.88 1.04 1.07 1.32
4:i: + :t: 4±
0.02 ( l l ) 0.03 (4) 0.04 (10) 0.03" (10) 0.05 b (11) 0.04 t' (5)
Nephrotoxicity
-Lactate 7.78 8.58 6.09 4.61 4.74 3.83
:t: 0.46 (11) d: 0.73 (4) -I- 0.27 b (10) -_i: 0.33 b (10) -4" 0.31 b (11) -I- 0.25 t' (5)
TEA SIM Ratio
* Lactate 20.74 5 : 1 . 2 4 (11) 17.43:5= 1.85 (4) 17.84 4" 0.37 (10) 13.62 d: 1.33 h (10) 14.40 q'- 1.72 b (11) 6.93 4" 1.72 t' (5)
in M a l e Rats"
-Lactate 20.52 20.43 17.70 12.16 12.33 6.76
+ :i: :t: :t: 4" 4"
0.75 (11) 1.20 (4) 0.51 (10) 0.64 b (10) 1.38 t' (11) 1.52 t' (5)
BUN
+Lactate 20.64 20,30 19.42 15.10 13.37 8.17
5:0.80 (It) 5= 1.05 (4) :t: 0.73 (10) :J: 0.99 I' (I0) 4" 1.58 b (11) :t: 2.15 t' (5)
(mg/100 mL) 18 16 16 4,6 62 90
± 1 (11) 4" 2 (4) 4- I (10) :t: 8 (I0) 4" 16 h (11) q- 26 h (5)
"Rats w e r e sacrificed 48 hr after receiving 0, 0.5, 1.0, 2.0, 2.5 or 3.0 glkg CPH, sc. Values r e p r e s e n t the mean 5= SE determined in (n) rats. bValues significantly different f r o m the control (0 g/kg) g r o u p , p < 0.05.
548
Fundam. Appl. To.tiroL (3)
November/Dece,~lber, 1983
INDICES OF ACUTE N E P H R O T O X I C I T Y "FABLE 8 H i s t o l o g i c a l E v a l u a t i o n of C e p h a l o r i d i n e - i n d u c e d N e p h r o t o x i c i t y in Male Rats" Dose (g/kg,~ 0 0.5 1.0 2.0 2.5 30
Degenerated Tubules 1,8 2.2 2.o 1o.4 14.2 1o.2
:.t 0.4 (If) ± 0.2 (-11 ± 0.7 (to) -_+ 2. l)'(JO) ± 2,4)'(11) :L 3.4)'(5)
Total Abnormal Tubules
Necrotic Tubules 0.0 0.0 0.0 18.1 23.0 55..I
3_ :z. _t 3.: 32 ~
0.0 ( I I ) 0,0 (J) 0.0 (I0) 4.2t'(10)
0.5"(11) 13.1~'(5)
1.8 2,2 2.0 34.5 37.2 71.4
~c ~ at_ i 3. 3=
0,.I ( I I ) 0.2 (4) 0,7 {I0) 4,7b(10) 8.4h(11) 13.5)'(5)
"Rats were ~acrificcd 48 hr a~ter receiving O, 0.5, 1.0, 2.0, 2.5 or 3.0 gtkg C'['I I, ~c. \~a)ues represent the mean perct, nt,tges =L SI[ deter/r~int.'d m in) rat.,,. "Value,,, .,,igniticant~y dift'erenl frt)m the control (0 glkg) group,
p <2 0.05. well correlated (Tabte 9), In addition, the relationships between functional and histological parameters appeared similar to those found in HgCb-treated rats. Elevated BUN content was the functional parameter most highly correlated with CPHreduced histological damage. Depression of slice organic ion accumulation was also well correlated with histological altera-
tions in CPH-treated animals. However, the distinction between resting slice PAH uptake and the other indices of organic ion accumulation was less clear in CPH-treated rats than in rats given K,~Cr,~O;(Table 3) or HgCI2 (Table 6).
DISCUSSION If the ability to detect injury at the lowest effective dosage of a toxicam is accepted as the criterion for sensitivity of a toxicity screening test (Kluwe, 1981), it is clear (Table 10) that histological evaluation of renal injury can be as sensitive an indicator of nephrotoxicity as the functional parameters evaluated in this study, tn terms of sensitivity, the functional parameters exhibited variability, but this is understandable since the nephrotoxicants employed have different initial sites of action. Although the most sensitive functional indicator of nephrotoxicity varied with the toxicant administered, some general trends were apparent. Renal cortical slice accumulation of one or both organic indices and appeared to be the most consistently sensitive of the functional indices. Addition of lactate to the incubation medium did not increase the sensitivity of slice PAH accumulation as an index of injury (Table 10), but did facilitate interpretation since toxicant-induced elevations in slice PAH accumulation were not observed. Given that slice
,
i
(
FIG, 3. P h o t o m i c r o g r a p h s o f k i d n e y s e c t i o n s o b t a i n e d f r o m r a t s I r e a t e d s u b c u t a n e o u s l y w i t h c e p h a l o r i d i n e . T h e r a t s w e r e killed 48 h r a f t e r t r e a t m e n t : A- 1.0 glkg; B- 2.0 glkg; C- 2,5 glkg; D - 3 . 0 g/kg. (HPS, X120). Fundamental and Applied Toxicology
(3) 11-12/83
5,49
MI"¢AIIMA, HEWITT, ~ O T E A N D I'LAA TABLE 9 R e l a t i o n s h i p B e t w e e n F u n c t i o n a l a n d H i s t o l o g i c a l E v a l u a t i o n of C e p h a l o r i d i n e - i n d u c e d N e p h r o t o x i c i t y in M a l e R a t s " Correlation Coefficient (r)
X-Axis
Regression Line (y = re(X) + (b))
Y-Axis BUN"
y : 1.32(X) + (8.ol) y = 1.e,o(X} + (15.03) y = -0.17 (X) ÷ (20.38)
0.o3 t' 0.o2 b -0,8o" -0.85" -0.84 t' -0.811' -0.7C' 0.78 h
AbnTub(%) Net" Tub (%} Abn Tub (%) Nec Tub (%) NecTub(%) Abn T u b (%) Nec Tub (%) AbnTub(%)
TSR (+Lactate) "I'SR (+Lactate) TSR(-Lactate) I'SR (+Lactate) I'SR (+Lactate) (Kidney Wt) ,z 100
y y y y Y
-0.77" 0.75"
Deg T u b (%) NecTub(%)
Body WI "I'SIC, (-Lactate) (Kidnev Wt)
y = -0.50 (X) + (19.o0) v = 0.00o(X} * (0.O0)
-0.74" -0.72 b -0.70 t' -O.oC' -0.o2" -0.c,2" 0.o0 h 0.34 ~'
BUN
= -0.20 iX) + (19.3o)
= -0.20(X) + (18.08) = -0.1o iX) * (19,55) = -0.20 (X) + (18.O1) = 0.005(X) + (0.87)
AbnTub(%) Deg Tub (%) AbnTub(%) DegTub(%) NecTub(%) l.)eg "l'ub (%) D e g T u b (%)
" "" 100 Body WI TSR(-Lactate) TSR(+Lactate) PSR(-Lactate) l"SR(-Lactate) I'SR(-Lactate) I'SR (+Lactate) (Kidney Wt) ,K 100
y y y y y y y
DegTub(%)
Body Wt BUN
y = 0.75(X) + (31.10/
"
= = : = = = :
-0.14 (X) -0.4oiX) -0.05(X) -0.1o(X) -0.05(X) -0.42 (X) 0.014 (X)
+ (18.74) * (20,5o) + (o.9,1) *" (7.25) + (o.o0) + (19.53) + (0.87)
"Functional and histological data from individual rats described in Tables 7 and 8 were analyzed. Each line consisted of 51 points. t'Significant correlation coefficient, p < 0.05. ' A b n T u b (%) = Abnormal T u b u l e s (%) N e c T u b { % ) = Necrotic T u b u l e s (%) D e g T u b ( % ) = Degenerated T u b u l e s (%) I'SR = I'AH S/IVl Ratio "FSR = TEA SIM l;',atio
TEA accumulation can be 1) as sensitive an indicator of nephrotoxicity as slice PAH uptake (Table 10), 2) determined simultaneously with PAH transport, and 3) determined in the presence or absence of a metabolic substrate without appreciable alterations in sensitivity (Table 10), it appears reasonable to suggest that the combination of lactate-simulated PAH and TEA accumulation by renal cortical slices would be an appropriate screening test for the ability of toxicants to injure the proximal tubule. It is also noteworthy that alterations in each of the functional indices were significantly correlated with changes in renal histology (Tables 3, 6, 9). With the exception of PAH accumulation by resting slices, organic ion accumulation was highly correlated with renal damage as determined by histopathology, thus supporting the earlier conclusion that lactatestimulated organic ion accumulation was a more appropriate measure of proximal tubule injury than resting slice organic ion uptake. Similarly, the close correlation between BUN content and histological evidence of kidney injury in HgCI.,- and CPH-treated rats, coupled with its relative ease of performance, suggests that BUN remains a useful index of kidney damage despite its inability to detect a minimal degree of renal injury (Sharratt and Fraser, 1963; Sharratt, 1970; Kluwe, 1981.; Berndt, 1981). It was not surprising to observe a significant correlation between structural and functional indices of nephrotoxicity. Wachsmuth (1982) demonstrated a correlation between his550
tochemically quantified renal damage and the urinary excretion of various enzymes, in particular lactic dehydrogenase. However, other investigators (Sharratt and Frazer, 1963; Kluwe, 1981 ; Berndt, 1981 ) have found a relatively poor correlation between various functional indices of damage and morphological changes in renal morphology. In particular, 8erndt (1981) has presented several examples where organic ion accumulation by renal cortical slices in vilro was reduced when kidneys from these animals were judged to be normal, or at worst mildly affected, by light microscopy. Although these
TABLE 10 Relative Sensitivity of Indices of Nephrotoxicity" Lowest Relative Dosage (1-37 at which a Significant Alteration was O b s e r v e d Parameter
K.~Cr.~O~
HgClz
Cephaloridine
3 1 2 I 2 3 1 3 I
2 3 3 2 2 2 ! 2 l
2 I 2 2 2 3 2 2 2
Relative Kidney Weight I'AH S/M Ratio (-Lactate) I'AH S/M Ratio (+Lactate) TEA S/M Ratio C-Lactate) TEA S/M Ratio (+Lactate)
BUN Degenerated T u b u l e s (%) Necrotic T u b u l e s (%) A b n o r m a l T u b u l e s (%)
"Format adapted from Kluwe (1981).
Fundarn. AppL Toxicol. (3)
November/December, 1983
INDICES OF ACUTE NEI'FIROTOXICITY observations appear to conflict with the results of the present investigation, histological assessment may miss a nephrotoxic lesion if timed incorrectly (Piperno, 1981 ). In this study, sufficient time (48 hr) was allowed to elapse for a histologically detectable lesion to appear. In the investigations cited by 8erndt (1981 ), kidneys were examined histologically at earlier times (12-24 hr following toxicant administration). Thus the apparent discrepancy between these studies is likely to arise from a difference in experimental design rather than a lack of a renal correlation between structural and functional parameters of kidney injury. However, these observations do suggest that evaluations of the relative sensitivity of toxicity tests based solely on dose-response considerations may not be completely valid; the time-response relationship should also be evaluated. In summary, organic ion (PAH and TEA) accumulation by renal cortical slices incubated in a Jactate-supplemented ir~cubation medium was considered to be the best functional indicator of renal injury used in this study due to its relative sensitivity, ease of interpretation and good correlation with a quantitative histological evaluation of damage. The quantitative histological procedure used was found to be as sensitive an indicator of nephrotoxicity as organic ion accumulation under the conditions of the study. However, additional investigation is required to document the relative sensitivities of the histological and functional indices at different time points in the progression of the lesion.
A CKNO WLEDGEMENT We gratefully acknowledge the expert technical assistance of Johanne Couture-Mdnard and Monique Morisset. This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES Atkinson, R.M., Currie, J,P., Davis, B., Pratt, D.A.H., Sharpe, H.M., and Tomich, E.G. (] 96b). Acute Toxicity of Cephaloridine, an Antibiotic Derived from Cephalosporin C. To.tiroL AppL Phurmacol. 8:398-406. Berndt, W.O. (1975). The Effect of Potassium Dichromate on Renal Tubular Transport Processes. ToxicoL Appl. PharmacoL 32:40-52. Berndt, W.O. (198l). Use of Renal Function Tests in the Evaluation of Nephrotoxic Effects. In. Toxicolog3"of the Kidmp" J.H. Hook, ed., pp. 1-29, Raven Press, New York.
Fundamental and Applied Toxicology
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