Toxicology. 69 (19913 121-132 Elsevier Scientific Publishers Ireland Ltd.
121
Distal nephrotoxicity of cisplatin demonstrated by urinary kallikrein excretion and morphological study in rats G u y Bompart, Claudine Orfila and Jean-Pierre Girolami lnstitut National de la Sant~ et de la Recherche M~dicale. Unit~ 133, Dkpartement de Physiologie et Pathologie R~nales, 133 route de Narbonne, Facult~ de M~decine Rangueil, F-31062 Toulouse (France) (Received March 15th, 1991; accepted June 2rd, 19913
Summary The nephrotoxic effect of cisplatin (4 mg/kg body wt, i.p. injection) was specifically evaluated on the distal tubule. We measured both the tissue concentration and the urinary excretion of kallikrein (UKE), a serine protease mainly synthesized and secreted in the distal connecting tubular cells. In a parallel morphological study, we evaluated the tissue lesions. On the basis of UKE, the three distinct phases of nephrotoxicity were observed. The induction phase, 1 day after cisplatin injection, was associated with a transient increase in UKE. During the maintenance phase, the kallikrein concentration was significantly decreased both in renal cortex and urine for up to 10 days, suggesting an alteration in the biosynthesis with a decrease in the activation of inactive kallikrein. The recovery phase, 21 days after cisplatin injection, was suggested by the incomplete but significant tendency to return towards control values of active UKE. Histological examinations of cisplatin-treated rats showed early lesions of proximal tubules on day 1. The injuries worsened and tubular necrosis was frequently observed on the following days. Distal tubular changes were less marked but vacuolization and desquamation of epithelial cells and swollen and disrupted mitochondria were demonstrated. This study adds new evidence that UKE is a useful and reliable non-invasive index to assess possible nephrotoxlc effects in the distal tubule which are also directly visualized by histological lesions. Key words." Cisplatin; Rat; Distal tubule; Electron microscopy; Renal kallikrein
Introduction Cisplatin (cis-diamminedichloroplatinum II) is a widely used antineoplastic drug which exhibits therapeutic activity against tumors of the bladder, testis, ovary and other solid tumors [1,2]. It is recognized that the specific site responsible for the pharmacological activity of cisplatin is DNA [3,4] and that binding between the platinum complex and DNA is very tight or irreversible [5,6]. However, besides its good antitumor activity, cisplatin induces serious side effects such as haematological, Correspondence to." G. Bompart, Inserm U133, 133 route de Narbonne, Facult6 de M6decine, Rangueil 31062, Toulouse, France. 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.
122 renal, neurological and hearing toxicity [7-9]. The kidney is considered to be the main target organ for cisplatin-induced toxicity. Acute and chronic nephrotoxicity of cisplatin occurs in humans and animals especially after repeated administration. Morphological damages have been mainly described in the P3 segment of the proximal tubule [101. Uraemia, creatininaemia and urinary parameters such as polyuria, natriuresis, kaliuresis, osmolality and urinary excretion of glucose, protein, N-acetylB-glucosaminidase (NAG), gamma-glutamyl transpeptidase (GGT) and alanine aminopeptidase (AAP) are the most widely used non-invasive markers for the early detection of tubular damage caused by cisplatin treatment [10-13]. These biochemical parameters provide a reliable index to estimate the proximal tubular damage and are in agreement with the histological changes which show that the proximal tubule is the most severely damaged segment [ 10,12]. However, Daugaard et al. [121 showed that the distal tubule was also affected as revealed by histological examinations. Studies using an immunohistochemical technique, the stop-flow method and microdissection have established that kallikrein is synthesized and secreted into the urine in the distal tubule [14,15]. Thus, this restrictive localization of the renal kallikrein could be particularly important to validate urinary kallikrein excretion (UKE) as a reliable index of distal tubular damage. In a series of studies, we have established the use of UKE as a non-invasive marker of distal nephron degeneration during exposure to heavy metals such as cadmium [ 16], sodium chromate [17], mercuric chloride [18] and molybdenum [19]. Therefore, in the present work we investigated further the possible distal effect of cisplatin by measuring UKE. In the protocol used, we studied the time-course evolution of UKE in parallel with the tissue content of this enzyme, the glomerular filtration rate estimated by the creatinine clearance and the distal morphological changes after a single i.p. injection of cisplatin in normal rats. Materials and methods
Chemicals S-2266 chromogenic substrate for glandular kallikreins was obtained from Kabivitrum (S 11287, Stockolm, Sweden). Cisplatin was kindly provided by R. Bellon Laboratory (Neuilly, Seine, France). A creatinine reagent kit was purchased from Beckman Instruments (Gagny, France 93220). Deoxycholic acid sodium salt was provided by Sigma Chemical Co. (St Louis, MO) All other reagents of analytical grade were obtained from Merck (Darmstadt, Germany).
Experimental protocols Ninety-six male Sprague-Dawley rats (IFFA CREDO, 300 + 4 g mean body weight) housed eight per cage with free access to food (diet UAR A-40, normal sodium: 113 mequiv/kg) and tap water were randomized into two groups: a vehicletreated control group (n = 48) and a cisplatin-treated group (4 mg/kg body wt, i.p., n = 48). Room temperature and humidity were maintained at 20°C and 70'¼,, respectively. Lighting was controlled by an automatic timer on a 12 h light/12 h dark cycle. Cisplatin treatment consisted of a single i.p. administration of cisplatin solution in
123
a sterile isotonic vehicle-containing mannitol (l"/,,). Control group and cisplatintreated rats were divided into six sub-groups (DO, Dl, D3, D6, DI0, D21; n = 8 each). After a 24-h acclimatization period in individual metabolic cages, 24-h urine was collected 1 day before (DO) and 1 (DI), 3 (D3), 6 (D6), 10 (D10) and 21 (D21) days after cisplatin injection in control and cisplatin-treated sub-groups. The control and cisplatin-treated rats were weighed then killed by decapitation 1 day before and 1, 3, 6, 10 and 21 days after cisplatin injection, then the kidneys were quickly removed, weighed and stored at -80°C for kallikrein determination. Tissue homogenate Kallikrein measurements in cortex require homogenization of the tissue and solubilisation of the membrane-bound enzyme with deoxycholate according to our previously described protocol after manual dissection of the cortex [20]. In brief, preweighed cortex was homogenized with a Potter Elvehjem in 0.1 M Tris-HC! buffer, pH 8.2 (1 ml/100 mg of fresh tissue) in the presence of 1 mM EDTA and 1 mM 1,10-phenanthroline as kininase inhibitors, as recently described for quantification of renal kininogen except that kailikrein inhibitors were omitted [21]. After 30 s of homogenization in a refrigerated bath, the deoxycholic acid concentration of the medium was adjusted to 0.5%. Incubation, ! h at 4°C with gentle agitation was allowed for solubilization of membrane-bound kallikrein. The extract was centrifuged at 45 000 x g for 30 min at 4°C and the supernatant was used to measure the kallikrein. Protein concentration in the supernatant was measured according to Lowry's method [22]. Biochemical assays Urinary and plasma creatinine were determined by the colorimetric method of Jaff~ using the Beckman creatinine no. 2 kit and an automated centrifugal analyzer (Cobas Bio Roche). Because kallikrein is excreted both as an active and an inactive form, kallikrein activity was estimated before and after trypsin activation by the amidolytic method measuring the hydrolysis of the synthetic substrate Val-Leu-Arg-p-nitroaniline as previously described [23]. The liberation of p-nitroaniline was measured photometrically at 405 nm. Results were expressed in micromoles of substrate hydrolyzed per min per 24 h urinary volume (#mol/min per 24 h) or in nanomoles per rain per mg of protein (nmol/min per mg prot) when measured in tissue. Trypsin activation allows the determination of the active form which is expressed as a percentage of the total activatable form. Kallikrein immunoreactive concentration was measured with a specific radioimmunoassay [20] using rabbit antiserum raised against purified rat urinary kallikrein [21]. Results were calculated using the LogitLog linearization of the standard curve and expressed in/~g of kallikrein excreted per 24 h urinary volume (/~g kal/24 h) or in ng per mg of protein when measured in cortex (ng kal/mg prot). In a previous study [20] we demonstrated that the antibody binds both the active and inactive forms of kallikrein since trypsinisation of the samples did not induce any changes in the immunoreactive concentration of kallikrein.
124
Morphological observations Portions of rat renal cortex were examined 1, 3, 6, 10 and 21 days after cisplatin administration. For electron microscopy, tissue was fixed in 2.5'¼, gluteraldehyde in 0.1M cacodylate-HCl buffer for 1 h. The tissue was then washed in c a c o d y l a t e - H C l buffer, post-fixed in 2% osmium tetroxide and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate and examined in a Hitachi electron microscope. Semi-thin sections were stained with periodic acid-Schiff methenamine silver and observed by light microscopy under a Dialux microscope.
Statistical analysis Data were expressed by mean values ± S.D. and analysed by Students t-test, P values of _ 0.05 were considered statistically significant. Results
Effect on urinary parameters, body and kidney weight Creatinine clearance was significantly reduced at only 3 days after cisplatin treatment with a minimum at day 10 and remained depressed up to day 21 (Fig. IA). The urinary volume was significantly increased from day 1-6 after cisplatin treatment reaching a maximum value at day 1 with a progressive return to base line level (Fig. 1B). At-the end of the experimentation, cisplatin-treated rats gained less body weight than those of the control group (Table I). The relative kidney weight was significant-
Z
2,0
Cisplatin •
1,0 ~
Control
0,5 I
0,0
I
I
I
ti e
B
50 ----la------
40
•
Cisplatin Control
~ 30 ~
20
----k a. I
I
i
i
15 20 DAYS Fig. 1. Time-course-dependent effect of cisplatin (4 mg/kg body wt) on creatinine clearance (A) and diuresis (B). ** and *** significantly different from control at P < 0.01 and 0.001 by Student's t-test, respectively. 5
10
121
Distal nephrotoxicity of cisplatin demonstrated by urinary kallikrein excretion and morphological study in rats G u y Bompart, Claudine Orfila and Jean-Pierre Girolami lnstitut National de la Sant~ et de la Recherche M~dicale. Unit~ 133, Dkpartement de Physiologie et Pathologie R~nales, 133 route de Narbonne, Facult~ de M~decine Rangueil, F-31062 Toulouse (France) (Received March 15th, 1991; accepted June 2rd, 19913
Summary The nephrotoxic effect of cisplatin (4 mg/kg body wt, i.p. injection) was specifically evaluated on the distal tubule. We measured both the tissue concentration and the urinary excretion of kallikrein (UKE), a serine protease mainly synthesized and secreted in the distal connecting tubular cells. In a parallel morphological study, we evaluated the tissue lesions. On the basis of UKE, the three distinct phases of nephrotoxicity were observed. The induction phase, 1 day after cisplatin injection, was associated with a transient increase in UKE. During the maintenance phase, the kallikrein concentration was significantly decreased both in renal cortex and urine for up to 10 days, suggesting an alteration in the biosynthesis with a decrease in the activation of inactive kallikrein. The recovery phase, 21 days after cisplatin injection, was suggested by the incomplete but significant tendency to return towards control values of active UKE. Histological examinations of cisplatin-treated rats showed early lesions of proximal tubules on day 1. The injuries worsened and tubular necrosis was frequently observed on the following days. Distal tubular changes were less marked but vacuolization and desquamation of epithelial cells and swollen and disrupted mitochondria were demonstrated. This study adds new evidence that UKE is a useful and reliable non-invasive index to assess possible nephrotoxlc effects in the distal tubule which are also directly visualized by histological lesions. Key words." Cisplatin; Rat; Distal tubule; Electron microscopy; Renal kallikrein
Introduction Cisplatin (cis-diamminedichloroplatinum II) is a widely used antineoplastic drug which exhibits therapeutic activity against tumors of the bladder, testis, ovary and other solid tumors [1,2]. It is recognized that the specific site responsible for the pharmacological activity of cisplatin is DNA [3,4] and that binding between the platinum complex and DNA is very tight or irreversible [5,6]. However, besides its good antitumor activity, cisplatin induces serious side effects such as haematological, Correspondence to." G. Bompart, Inserm U133, 133 route de Narbonne, Facult6 de M6decine, Rangueil 31062, Toulouse, France. 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.
122 renal, neurological and hearing toxicity [7-9]. The kidney is considered to be the main target organ for cisplatin-induced toxicity. Acute and chronic nephrotoxicity of cisplatin occurs in humans and animals especially after repeated administration. Morphological damages have been mainly described in the P3 segment of the proximal tubule [101. Uraemia, creatininaemia and urinary parameters such as polyuria, natriuresis, kaliuresis, osmolality and urinary excretion of glucose, protein, N-acetylB-glucosaminidase (NAG), gamma-glutamyl transpeptidase (GGT) and alanine aminopeptidase (AAP) are the most widely used non-invasive markers for the early detection of tubular damage caused by cisplatin treatment [10-13]. These biochemical parameters provide a reliable index to estimate the proximal tubular damage and are in agreement with the histological changes which show that the proximal tubule is the most severely damaged segment [ 10,12]. However, Daugaard et al. [121 showed that the distal tubule was also affected as revealed by histological examinations. Studies using an immunohistochemical technique, the stop-flow method and microdissection have established that kallikrein is synthesized and secreted into the urine in the distal tubule [14,15]. Thus, this restrictive localization of the renal kallikrein could be particularly important to validate urinary kallikrein excretion (UKE) as a reliable index of distal tubular damage. In a series of studies, we have established the use of UKE as a non-invasive marker of distal nephron degeneration during exposure to heavy metals such as cadmium [ 16], sodium chromate [17], mercuric chloride [18] and molybdenum [19]. Therefore, in the present work we investigated further the possible distal effect of cisplatin by measuring UKE. In the protocol used, we studied the time-course evolution of UKE in parallel with the tissue content of this enzyme, the glomerular filtration rate estimated by the creatinine clearance and the distal morphological changes after a single i.p. injection of cisplatin in normal rats. Materials and methods
Chemicals S-2266 chromogenic substrate for glandular kallikreins was obtained from Kabivitrum (S 11287, Stockolm, Sweden). Cisplatin was kindly provided by R. Bellon Laboratory (Neuilly, Seine, France). A creatinine reagent kit was purchased from Beckman Instruments (Gagny, France 93220). Deoxycholic acid sodium salt was provided by Sigma Chemical Co. (St Louis, MO) All other reagents of analytical grade were obtained from Merck (Darmstadt, Germany).
Experimental protocols Ninety-six male Sprague-Dawley rats (IFFA CREDO, 300 + 4 g mean body weight) housed eight per cage with free access to food (diet UAR A-40, normal sodium: 113 mequiv/kg) and tap water were randomized into two groups: a vehicletreated control group (n = 48) and a cisplatin-treated group (4 mg/kg body wt, i.p., n = 48). Room temperature and humidity were maintained at 20°C and 70'¼,, respectively. Lighting was controlled by an automatic timer on a 12 h light/12 h dark cycle. Cisplatin treatment consisted of a single i.p. administration of cisplatin solution in
123
a sterile isotonic vehicle-containing mannitol (l"/,,). Control group and cisplatintreated rats were divided into six sub-groups (DO, Dl, D3, D6, DI0, D21; n = 8 each). After a 24-h acclimatization period in individual metabolic cages, 24-h urine was collected 1 day before (DO) and 1 (DI), 3 (D3), 6 (D6), 10 (D10) and 21 (D21) days after cisplatin injection in control and cisplatin-treated sub-groups. The control and cisplatin-treated rats were weighed then killed by decapitation 1 day before and 1, 3, 6, 10 and 21 days after cisplatin injection, then the kidneys were quickly removed, weighed and stored at -80°C for kallikrein determination. Tissue homogenate Kallikrein measurements in cortex require homogenization of the tissue and solubilisation of the membrane-bound enzyme with deoxycholate according to our previously described protocol after manual dissection of the cortex [20]. In brief, preweighed cortex was homogenized with a Potter Elvehjem in 0.1 M Tris-HC! buffer, pH 8.2 (1 ml/100 mg of fresh tissue) in the presence of 1 mM EDTA and 1 mM 1,10-phenanthroline as kininase inhibitors, as recently described for quantification of renal kininogen except that kailikrein inhibitors were omitted [21]. After 30 s of homogenization in a refrigerated bath, the deoxycholic acid concentration of the medium was adjusted to 0.5%. Incubation, ! h at 4°C with gentle agitation was allowed for solubilization of membrane-bound kallikrein. The extract was centrifuged at 45 000 x g for 30 min at 4°C and the supernatant was used to measure the kallikrein. Protein concentration in the supernatant was measured according to Lowry's method [22]. Biochemical assays Urinary and plasma creatinine were determined by the colorimetric method of Jaff~ using the Beckman creatinine no. 2 kit and an automated centrifugal analyzer (Cobas Bio Roche). Because kallikrein is excreted both as an active and an inactive form, kallikrein activity was estimated before and after trypsin activation by the amidolytic method measuring the hydrolysis of the synthetic substrate Val-Leu-Arg-p-nitroaniline as previously described [23]. The liberation of p-nitroaniline was measured photometrically at 405 nm. Results were expressed in micromoles of substrate hydrolyzed per min per 24 h urinary volume (#mol/min per 24 h) or in nanomoles per rain per mg of protein (nmol/min per mg prot) when measured in tissue. Trypsin activation allows the determination of the active form which is expressed as a percentage of the total activatable form. Kallikrein immunoreactive concentration was measured with a specific radioimmunoassay [20] using rabbit antiserum raised against purified rat urinary kallikrein [21]. Results were calculated using the LogitLog linearization of the standard curve and expressed in/~g of kallikrein excreted per 24 h urinary volume (/~g kal/24 h) or in ng per mg of protein when measured in cortex (ng kal/mg prot). In a previous study [20] we demonstrated that the antibody binds both the active and inactive forms of kallikrein since trypsinisation of the samples did not induce any changes in the immunoreactive concentration of kallikrein.
124
Morphological observations Portions of rat renal cortex were examined 1, 3, 6, 10 and 21 days after cisplatin administration. For electron microscopy, tissue was fixed in 2.5'¼, gluteraldehyde in 0.1M cacodylate-HCl buffer for 1 h. The tissue was then washed in c a c o d y l a t e - H C l buffer, post-fixed in 2% osmium tetroxide and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate and examined in a Hitachi electron microscope. Semi-thin sections were stained with periodic acid-Schiff methenamine silver and observed by light microscopy under a Dialux microscope.
Statistical analysis Data were expressed by mean values ± S.D. and analysed by Students t-test, P values of _ 0.05 were considered statistically significant. Results
Effect on urinary parameters, body and kidney weight Creatinine clearance was significantly reduced at only 3 days after cisplatin treatment with a minimum at day 10 and remained depressed up to day 21 (Fig. IA). The urinary volume was significantly increased from day 1-6 after cisplatin treatment reaching a maximum value at day 1 with a progressive return to base line level (Fig. 1B). At-the end of the experimentation, cisplatin-treated rats gained less body weight than those of the control group (Table I). The relative kidney weight was significant-
Z
2,0
Cisplatin •
1,0 ~
Control
0,5 I
0,0
I
I
I
ti e
B
50 ----la------
40
•
Cisplatin Control
~ 30 ~
20
----k a. I
I
i
i
15 20 DAYS Fig. 1. Time-course-dependent effect of cisplatin (4 mg/kg body wt) on creatinine clearance (A) and diuresis (B). ** and *** significantly different from control at P < 0.01 and 0.001 by Student's t-test, respectively. 5
10
