Toxicology Letters, 53 (1990) 169-172
169
Elsevier
TOXLET
02408
Evidence of circadian variations in mercuricchloride-induced proximal tubular necrosis by digital imaging microscopy
Jean-Charles Call, Jacques SaureP and Jean Cambarj ‘Department of Pharmacy, University of Manchester, Manchester (U.K.), Pathologie. Le Bouscat (France)
and Goupe
21nsrirur d’Histologie et Gyro-
d’Etude de Physiologie et Physiopathologie R&ales, FaculiP
de Pharmacie, Bordeaux (France)
Key words: Image analysis;
Chrononephrotoxicity;
Histology;
Mercuric
chloride;
Rat
INTRODUCTION
Among the many factors which are considered as important in the nephrotoxicity of xenobiotics such as dose, age, sex, genetic strain or nutritional status, the time of dosing has emerged, during the last decade, as a major determinant of the extent of the damage (see review by Cal et al. [I]). A large number of publications have demonstrated statistically significant circadian variations in numerous aspects of renal function, e.g. glomerular filtration rate, renal blood flow, electrolyte excretion or urinary enzyme activity [l]. Time-dependent rhythmic variations in renal susceptibility to heavy metals or antibiotics have been shown using urine-concentrating ability or enzymuria as markers of nephrotoxicity [2]. Temporal variations in such markers are undoubtedly the expression of temporal alterations in metabolic processes. Since metabolic processes are based on cellular functions, the question arises: To what extent are differential structural manifestations as a function of the time of dosing visible at the cellular level? This question remains unresolved to date. This is partly due to the lack of a suitable quantitative technique to detect such rhythms. Though stereological techniques appeared to provide an objective tool in this respect, one of their major disadvantages is that they are rather time-consuming to perform, expecially when it is necessary to consider a large number of parameters [3]. The recent considerable development of the compuAddress for correspondence:
J.-C. Cal, Department
of Pharmacy,
University
of Manchester,
Ml3 9PL. U.K. 0378-4274/90/$3.50
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
Manchester
170
terized image cell processing of digitized, microscopic cell images, which first appeared 15 years ago, prompted us to use this powerful new tool in conjunction with the routinely used staining procedures of histology. The present study was designed to provide for the first time a quantitative account of the differential changes that take place in the rat kidney 24 h after injection of a single dose of mercuric chloride as a well-known model of acute tubular necrosis, according to the time of dosing. In addition, as several hormones and particularly sex hormones are believed to play an important role in the resistance of the kidney to the nephrotoxic insult [4], the experiments were conducted in both male and female animals. METHODS
Animals Twenty-four male and 24 female Sprague-Dawley rats were maintained for 2 weeks under constant environmental conditions in a temperature (22 + IV) and humidity (50% + 5%) controlled room. They were allowed free access to food and water and exposed to a 12-h light/dark cycle (0800/2000). Male (250 If: 10 g) and female (200 & IO g) 8-week-old animals received a single i.p. injection of 1 mg/kg of mercury as mercuric chloride or of saline (control groups) at one of the 4 following equispaced circadian stages: 0500 h, 1100 h, 1700 h or 2300 h (in June). Tissue sampling and processing Twenty-four hours after dosing, the animals were killed with an overdose of pentobarbital and the left kidney removed immediately and placed in neutral buffered formol-saline. After fixation for at least I week at room temperature, the tissue was processed for routine histology and embedded in paraffin wax. Longitudinal sections (6 pm) were cut and stained with Harris’ Haematoxylin and Eosin. Quantitative ~~tolag~cai analysis Slides were analysed by digital imaging microscopy with a Biocom system consisting of a Compaq@ microcomputer (640 Kb of RAM) and of the specific Biocom@ software, ImalTM for image analysis coupled with a Leitz@ microscope and a x 40 objective. The optical density of the tubular lumen or of the lumen due to vacuoles, blebs and other translucent spaces specifically enlarged or constituted during the necrotic process was determined on a scale of 256 grey levels. A set of macroinst~ctions was written to allow automatic calculation of the area of these spaces. For each field observed, this area was recorded as a percentage of the total area of the field. At least 5 fields at the cortico-medullary junction per slide were examined. Statistical analyst All data are reported as mean It standard error. Multiway analysis of variance
171
was used when app~~ble for testing the effect of HgClz, time and sex on the size of the lumen in the cortico-medullary zone. The overall variability of the data was assessed by the single cosinor method [5] using a program specifically designed by J.-C. Cal for the Macintosh@ microcomputer. RESULTS AND DISCUSSION
Routine histology coupled with digital imaging microscopy appeared as a powerful quantitative and non-subjective tool for the study of differential renal damages according to the time of dosing or obviously to the drug. It made it possible to highlight small though non-significant circadian changes in the size of the tubular lumen in control rats as a function of the time of sampling since it ranged from 2.47 kO.38 at 1700 to 4.85kO.71 at 2300 in male rats and from 2.32kO.42 at 2300 to 6.14kO.69 at 0500 in female rats. This might be viewed as the result of a circadian variation in the height of the microvilli of the brush border as it was already shown for rat ileum [6], and could be further investigated using electron microscopy. Twenty-four hours after injection of mercuric chloride, the quantitative analysis showed a lo-fold increase in the translucent area since it ranged from 23.4f3.4 at 0500 to 48.79 f 4.11 at 1700 in male rats and from 25.8 1 f 2.25 at 0500 to 34.95 rt: 1.94 at 1700 in female rats. This significant but gradual increase supports the rationale on which we based the use of digital imaging microscopy for this application. Indeed, as was previously described [7], HgCl*-indu~d renal tubular necrosis is a~ompanied at every stage by a gradual increase in translucent areas from an apical vacuolation of tubular cell cytoplasm within 4 h, which rapidly develops to lead to bleb formation within 8 h, and to disruption of brush border followed by cell breakdown, fragmentation and dissolution by 24-48 h. Thus, the present procedure allowed us to gain a rapid and almost automatic quantitative account of these changes assessing the differential level of translucent areas in kidneys from various groups of rats. Furthermore, from these results, a statistical analysis is now possible contrary to usual qualitative histology. Using the cosinor method, circadian rhythms were detected 24 h after HgClz, either in male (PC 0.0001) or in female (P< 0.001) rats. The peak of nephrotoxicity occurred in both sexes for rats injected at the end of the rest period (at 1736 f 0030 in male rats and at 1824 + 0054 in female rats). These results are in close agreement with previous reports based on enzymuria data [2]. Using the above-described indices, the mean level of necrosis observed in all the male groups (3 1.23 f 1.4) appeared slightly higher than in the female ones (29.38 &-0.89) which is consistent with the findings by Harber and Jennings [4] using classical histology, In contrast, the amplitude of the rhythms (half the difference between the trough and the crest of the variations) was highly different between male (14.62f2.43) and female rats (5.12 f 1.33; PC 0.0001). This sex-related difference suggests that male hormones play a significant role in the resistance of the kidney to nephrotoxic drugs which varies as a function of the time of day. According to Schiebler and Danner
172
[8], the number of pinocytic vesicles at the proximal tubule would be under the control of sex hormones. That could lead to a differential absorption of mercuric chloride during the nycthemeron as a function of plasma sex hormones and thereby give a clue to the origin of the circadian rhythm in HgCi~-indu~d tubular necrosis. This study demonstrated, therefore, the value of digital imaging microscopy as a general tool for nephrotoxicity screening which could replace the somewhat subjective and the difficult-to-reproduce rating of histological lesions. In addition, the findings of this study showed that a significantly different degree of necrosis may be induced by the same drug when varying only the time of its administration. That could be of particular significance for the therapeutic use of nephrotoxic drugs at a time when drugs are increasingly given once daily. ACKNOWLEDGEMENTS
We wish to thank Professor B. Bloch and Miss C. Lemoine from the Faculty of Medicine, University of Bordeaux II, for allowing us to use image analysis system and for technical advice. REFERENCES 1 Cal, J.-C., Dorian, C., Catroux, P. and Cambar, J. (1989) Nephrotoxicity of heavy metals and antibiotics. In: B. Lemmer (Ed.), Chronopharmacology, Cellular and Biochemical Interactions. Marcel Dekker, Inc., New York, pp. 65.5-681. 2 Cal, J.-C., Pariat, C., Piriou, A., Courtois; P. and Cambar, J. (1985) Chronobiological approach of nephrotoxicity: time dependent acute renal failure induced by aminoglycoside antibiotics and heavy metals in rodents. In: P.H. Bach and E.A. Lock (Eds.), Renal Heterogeneity and Target Ceil Toxicity. John Wiley and Sons, Chichester, pp. 381-384. 3 Vollrath, L. (1985) Chronomorphology - today. Chronobiologia 12,307-322. 4 Harber, M.H. and Jennings, R.B. (1965) Renal response of the rat to mercury. The effect of sex and sex hormones. Arch. Pathol. 79,218222. 5 Nelson, W., Tong, Y.L., Lee, J.K. and Halberg, F. (1979) Methods for cosinor rhythmometry. Chronobiologia 6,305-323. 6 Stevenson, N.R., Day, SE. and Sitren, H. (1979) Circadian rhythmicity in rat intestinai villus length and cell number. Int. J. Chronobiol. 6, l-12. 7 Haagsma, B.H. and Pound, A.W. (1979) Mercuric chloride-induced renal tubular necrosis in the rat. Br. J. Exp. Pathol. 60,341-352. 8 Schiebler, T.H. and Danner, K.G. (1978) The effect of sex hormones on the proximal tubules in the rat kidney. Cell Tissue Res. 192,527-549.
169
Elsevier
TOXLET
02408
Evidence of circadian variations in mercuricchloride-induced proximal tubular necrosis by digital imaging microscopy
Jean-Charles Call, Jacques SaureP and Jean Cambarj ‘Department of Pharmacy, University of Manchester, Manchester (U.K.), Pathologie. Le Bouscat (France)
and Goupe
21nsrirur d’Histologie et Gyro-
d’Etude de Physiologie et Physiopathologie R&ales, FaculiP
de Pharmacie, Bordeaux (France)
Key words: Image analysis;
Chrononephrotoxicity;
Histology;
Mercuric
chloride;
Rat
INTRODUCTION
Among the many factors which are considered as important in the nephrotoxicity of xenobiotics such as dose, age, sex, genetic strain or nutritional status, the time of dosing has emerged, during the last decade, as a major determinant of the extent of the damage (see review by Cal et al. [I]). A large number of publications have demonstrated statistically significant circadian variations in numerous aspects of renal function, e.g. glomerular filtration rate, renal blood flow, electrolyte excretion or urinary enzyme activity [l]. Time-dependent rhythmic variations in renal susceptibility to heavy metals or antibiotics have been shown using urine-concentrating ability or enzymuria as markers of nephrotoxicity [2]. Temporal variations in such markers are undoubtedly the expression of temporal alterations in metabolic processes. Since metabolic processes are based on cellular functions, the question arises: To what extent are differential structural manifestations as a function of the time of dosing visible at the cellular level? This question remains unresolved to date. This is partly due to the lack of a suitable quantitative technique to detect such rhythms. Though stereological techniques appeared to provide an objective tool in this respect, one of their major disadvantages is that they are rather time-consuming to perform, expecially when it is necessary to consider a large number of parameters [3]. The recent considerable development of the compuAddress for correspondence:
J.-C. Cal, Department
of Pharmacy,
University
of Manchester,
Ml3 9PL. U.K. 0378-4274/90/$3.50
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
Manchester
170
terized image cell processing of digitized, microscopic cell images, which first appeared 15 years ago, prompted us to use this powerful new tool in conjunction with the routinely used staining procedures of histology. The present study was designed to provide for the first time a quantitative account of the differential changes that take place in the rat kidney 24 h after injection of a single dose of mercuric chloride as a well-known model of acute tubular necrosis, according to the time of dosing. In addition, as several hormones and particularly sex hormones are believed to play an important role in the resistance of the kidney to the nephrotoxic insult [4], the experiments were conducted in both male and female animals. METHODS
Animals Twenty-four male and 24 female Sprague-Dawley rats were maintained for 2 weeks under constant environmental conditions in a temperature (22 + IV) and humidity (50% + 5%) controlled room. They were allowed free access to food and water and exposed to a 12-h light/dark cycle (0800/2000). Male (250 If: 10 g) and female (200 & IO g) 8-week-old animals received a single i.p. injection of 1 mg/kg of mercury as mercuric chloride or of saline (control groups) at one of the 4 following equispaced circadian stages: 0500 h, 1100 h, 1700 h or 2300 h (in June). Tissue sampling and processing Twenty-four hours after dosing, the animals were killed with an overdose of pentobarbital and the left kidney removed immediately and placed in neutral buffered formol-saline. After fixation for at least I week at room temperature, the tissue was processed for routine histology and embedded in paraffin wax. Longitudinal sections (6 pm) were cut and stained with Harris’ Haematoxylin and Eosin. Quantitative ~~tolag~cai analysis Slides were analysed by digital imaging microscopy with a Biocom system consisting of a Compaq@ microcomputer (640 Kb of RAM) and of the specific Biocom@ software, ImalTM for image analysis coupled with a Leitz@ microscope and a x 40 objective. The optical density of the tubular lumen or of the lumen due to vacuoles, blebs and other translucent spaces specifically enlarged or constituted during the necrotic process was determined on a scale of 256 grey levels. A set of macroinst~ctions was written to allow automatic calculation of the area of these spaces. For each field observed, this area was recorded as a percentage of the total area of the field. At least 5 fields at the cortico-medullary junction per slide were examined. Statistical analyst All data are reported as mean It standard error. Multiway analysis of variance
171
was used when app~~ble for testing the effect of HgClz, time and sex on the size of the lumen in the cortico-medullary zone. The overall variability of the data was assessed by the single cosinor method [5] using a program specifically designed by J.-C. Cal for the Macintosh@ microcomputer. RESULTS AND DISCUSSION
Routine histology coupled with digital imaging microscopy appeared as a powerful quantitative and non-subjective tool for the study of differential renal damages according to the time of dosing or obviously to the drug. It made it possible to highlight small though non-significant circadian changes in the size of the tubular lumen in control rats as a function of the time of sampling since it ranged from 2.47 kO.38 at 1700 to 4.85kO.71 at 2300 in male rats and from 2.32kO.42 at 2300 to 6.14kO.69 at 0500 in female rats. This might be viewed as the result of a circadian variation in the height of the microvilli of the brush border as it was already shown for rat ileum [6], and could be further investigated using electron microscopy. Twenty-four hours after injection of mercuric chloride, the quantitative analysis showed a lo-fold increase in the translucent area since it ranged from 23.4f3.4 at 0500 to 48.79 f 4.11 at 1700 in male rats and from 25.8 1 f 2.25 at 0500 to 34.95 rt: 1.94 at 1700 in female rats. This significant but gradual increase supports the rationale on which we based the use of digital imaging microscopy for this application. Indeed, as was previously described [7], HgCl*-indu~d renal tubular necrosis is a~ompanied at every stage by a gradual increase in translucent areas from an apical vacuolation of tubular cell cytoplasm within 4 h, which rapidly develops to lead to bleb formation within 8 h, and to disruption of brush border followed by cell breakdown, fragmentation and dissolution by 24-48 h. Thus, the present procedure allowed us to gain a rapid and almost automatic quantitative account of these changes assessing the differential level of translucent areas in kidneys from various groups of rats. Furthermore, from these results, a statistical analysis is now possible contrary to usual qualitative histology. Using the cosinor method, circadian rhythms were detected 24 h after HgClz, either in male (PC 0.0001) or in female (P< 0.001) rats. The peak of nephrotoxicity occurred in both sexes for rats injected at the end of the rest period (at 1736 f 0030 in male rats and at 1824 + 0054 in female rats). These results are in close agreement with previous reports based on enzymuria data [2]. Using the above-described indices, the mean level of necrosis observed in all the male groups (3 1.23 f 1.4) appeared slightly higher than in the female ones (29.38 &-0.89) which is consistent with the findings by Harber and Jennings [4] using classical histology, In contrast, the amplitude of the rhythms (half the difference between the trough and the crest of the variations) was highly different between male (14.62f2.43) and female rats (5.12 f 1.33; PC 0.0001). This sex-related difference suggests that male hormones play a significant role in the resistance of the kidney to nephrotoxic drugs which varies as a function of the time of day. According to Schiebler and Danner
172
[8], the number of pinocytic vesicles at the proximal tubule would be under the control of sex hormones. That could lead to a differential absorption of mercuric chloride during the nycthemeron as a function of plasma sex hormones and thereby give a clue to the origin of the circadian rhythm in HgCi~-indu~d tubular necrosis. This study demonstrated, therefore, the value of digital imaging microscopy as a general tool for nephrotoxicity screening which could replace the somewhat subjective and the difficult-to-reproduce rating of histological lesions. In addition, the findings of this study showed that a significantly different degree of necrosis may be induced by the same drug when varying only the time of its administration. That could be of particular significance for the therapeutic use of nephrotoxic drugs at a time when drugs are increasingly given once daily. ACKNOWLEDGEMENTS
We wish to thank Professor B. Bloch and Miss C. Lemoine from the Faculty of Medicine, University of Bordeaux II, for allowing us to use image analysis system and for technical advice. REFERENCES 1 Cal, J.-C., Dorian, C., Catroux, P. and Cambar, J. (1989) Nephrotoxicity of heavy metals and antibiotics. In: B. Lemmer (Ed.), Chronopharmacology, Cellular and Biochemical Interactions. Marcel Dekker, Inc., New York, pp. 65.5-681. 2 Cal, J.-C., Pariat, C., Piriou, A., Courtois; P. and Cambar, J. (1985) Chronobiological approach of nephrotoxicity: time dependent acute renal failure induced by aminoglycoside antibiotics and heavy metals in rodents. In: P.H. Bach and E.A. Lock (Eds.), Renal Heterogeneity and Target Ceil Toxicity. John Wiley and Sons, Chichester, pp. 381-384. 3 Vollrath, L. (1985) Chronomorphology - today. Chronobiologia 12,307-322. 4 Harber, M.H. and Jennings, R.B. (1965) Renal response of the rat to mercury. The effect of sex and sex hormones. Arch. Pathol. 79,218222. 5 Nelson, W., Tong, Y.L., Lee, J.K. and Halberg, F. (1979) Methods for cosinor rhythmometry. Chronobiologia 6,305-323. 6 Stevenson, N.R., Day, SE. and Sitren, H. (1979) Circadian rhythmicity in rat intestinai villus length and cell number. Int. J. Chronobiol. 6, l-12. 7 Haagsma, B.H. and Pound, A.W. (1979) Mercuric chloride-induced renal tubular necrosis in the rat. Br. J. Exp. Pathol. 60,341-352. 8 Schiebler, T.H. and Danner, K.G. (1978) The effect of sex hormones on the proximal tubules in the rat kidney. Cell Tissue Res. 192,527-549.
