Abstract
To ascertain the early pathophysiological features in canine renal papillary necrosis (RPN) caused by the neurotransmission enhancer nefiracetam, male beagle dogs were orally administered nefiracetam at 300 mg/kg/day for 4 to 7 weeks in comparison with ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), at 50 mg/kg/day for 5 weeks. During the dosing period, the animals were periodically subjected to laboratory tests, light-microscopic, immunohistochemical, and electron-microscopic examinations and/or cyclooxygenase (COX)-2 mRNA analysis. In laboratory tests, a decrease in urinary osmotic pressure and increases in urine volume and urinary lactate dehydrogenase (LDH) level were early biomarkers for detecting RPN. Light-microscopically, nefiracetam revealed epithelial swelling and degeneration in the papillary ducts in week 7, while ibuprofen displayed degeneration and necrosis in the papillary interstitium in week 5. In immunohistochemical staining with COX-2 antibody, nefiracetam elicited a positive reaction within interstitial cells around the affected epithelial cells in the papillary ducts (upper papilla) in week 7, and ibuprofen positively reacted within interstitial cells adjacent to the degenerative and/or necrotic lesions in week 5. Ultrastructurally, nefiracetam exhibited reductions of intracellular interdigitation and infoldings of epithelial cells in the papillary ducts, whereas ibuprofen showed no changes in the identical portions. Thus, the early morphological change in the papilla brought about by nefiracetam was quite different from that elicited by ibuprofen. By the renal papillary COX-2 mRNA expression analysis, nefiracetam exceedingly decreased its expression in week 4, but markedly increased it in week 7, suggesting an induction of COX-2 mRNA by renal papillary lesions. These results demonstrate that the epithelial cell in the papillary ducts is the primary target site for the onset of RPN evoked by nefiracetam.
Introduction
Renal papillary necrosis (RPN) is known to be caused by chronic use of mixed analgesics or NSAIDs (Alden and Frith, 1991; Bach and Thanh, 1998; William et al., 1996), 2-bromoethylamine hydrobromide (Bach et al., 1991; Sabatini, 1984) or D-ormaplatin (Kolaja et al., 1994). Nevertheless, advances in understanding the pathogenesis of RPN have still been slow. As for NSAID-induced RPN, although direct cytotoxic action and/or ischemic injury through inhibition of the vasodilatory effects of renal prostaglandins (PGs) as a result of cyclooxygenase (COX) inhibition have been reported as important contributing factors, the entire mechanism remains unclear (Alden and Frith, 1991; Khan and Silverman, 1999; Rocha et al., 2001; Sabatini, 1988; Whiting et al., 1999).
Nefiracetam (N -(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide, Figure 1) is a novel pyrrolidone derivative possessing nootropic effects. In pharmacological studies, nefiracetam has facilitated cognitive function in a wide variety of animal models. In brief, this compound has reduced amnesia produced by scopolamine (Sakurai et al., 1989) and apomorphine (Nabeshima et al., 1994). Nefiracetam has also been found to attenuate amnesia produced by the neurotoxin AF64A (Abe et al., 1994), electrolytic lesions of the basal forebrain (Nishizaki et al., 1998), and hypoxia (Hiramatsu et al., 1992), and further has improved the cognitive performance of aged rats (Nabeshima, 1994). In pharmacokinetic studies, nefiracetam was extensively metabolized, and more than twenty metabolites were observed in serum, urine, and tissues (Sellers et al., 2004). In previous long-term toxicological investigations with rats, dogs, and monkeys (Hooks et al., 1994; Jindo et al., 1994; Kajimura et al., 1994; Tsuchiya et al., 2003), degeneration and/or necrosis in the renal papilla was observed only in dogs given 300 mg/kg/day of nefiracetam for 8 to 11 weeks. This alteration was provoked by the inhibition of renal PG synthesis resulting from relatively high penetration and retention of the nefiracetam metabolite M-18 into the canine renal papilla. In laboratory tests, a decrease in urinary osmotic pressure and increases in urine volume and urinary lactate dehydrogenase (LDH) level were observed at the early phase (Tsuchiya et al., 2003).
In the present study, to ascertain the early pathophysiological features in RPN induced by nefiracetam, dogs were orally administered nefiracetam at 300 mg/kg/day for 4 to 7 weeks in comparison with ibuprofen, an NSAID, at 50 mg/kg/day for 5 weeks. Periodic laboratory tests including urine volume, urinary osmotic pressure, and urinary LDH were performed to reconfirm the biomarkers for the onset of RPN. At the same time, the renal morphological aspects were investigated by light- and electron-microscopic examinations and immunohistochemical staining with COX antibodies in conjunction with COX-2 mRNA expression analysis.
Materials and Methods
Chemicals
Nefiracetam was synthesized in the Akita Factory of Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). Ibuprofen was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents were the highest grade available from commercial sources.
Animal Treatment
A total of 22 male LRE strain or Nosan beagles (8.9–12.6 kg) at approximately 17 to 19 months of age were used. Dogs were housed individually under controlled conditions at a temperature of 21–25°C, a relative humidity of 40–80%, a lightning cycle of 12 hours (from 8:00 to 20:00 each day), and with 15 or more changes of filtered air/hour. Commercial diets were given ad libitum, and the animals were allowed free access to water except on the sampling day for urine or serum. They were divided into 6 groups as follows. Group 1, intact control group (Nos. 1, 2 and 3); Group 2, nefiracetam treatment group at 300 mg/kg/day for 4 weeks (Nos. 4, 5, 6, and 7); Group 3, nefiracetam treatment group at 300 mg/kg/day for 5 weeks (Nos. 8, 9, 10, and 11); Group 4, nefiracetam treatment group at 300 mg/kg/day for 6 weeks (Nos. 12, 13, 14, and 15); Group 5, nefiracetam treatment group at 300 mg/kg/day for 7 weeks (Nos. 16, 17, 18, and 19) and Group 6, ibuprofen treatment group at 50 mg/kg/day for 5 weeks (Nos. 20, 21, and 22). In this study, the first dosing week was designated as week 1. Periodic laboratory tests were performed in dogs from groups 1 (nontreated control), 5 (nefiracetam for 7 weeks), and 6 (ibuprofen for 5 weeks) before the commencement of treatment and weekly thereafter. At the end of the respective treatment periods, the dogs were killed by exsanguination from the axillary artery under pentobarbital anesthesia (25 mg/kg, iv, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan), and the bilateral kidneys were removed.
Laboratory Tests
Urinalysis and serum biochemistry were performed once before treatment and weekly during the treatment period. Urine was collected by using a collecting tray for approximately 17 hours (overnight). During collection, the dogs were denied food and water, and urine was kept cool on ice. After centrifugation (4°C, 1500 rpm, 10 minutes) of collected urine, approximately 5 mL of the supernatant was filtered through a membrane filter (0.8 μm, DISMIC-25cs, ADVANTEC, Tokyo, Japan). A 2.5-mL sample of the filtered supernatant was applied on the disposable column PD-10 (Sephadex G-25 M, Amersham Biosciences UK Ltd., Buckinghamshire, UK) and 3.5 mL of 0.0625 M Tris-HCl (pH 6.8) was used for elution. In fresh urine, osmotic pressure was measured with an osmometer (model 3C2, Advanced Instruments, Inc., Norwood, MA, USA). Creatinine (CRE) content in the filtered urine supernatant was measured with a CRE-KAINOS (KAINOS Laboratories, Inc., Tokyo, Japan). LDH in the eluted solution was determined with a LDH CII test (Wako Pure Chemical Industries, Ltd.). CRE values were used for the correction of LDH level. Approximately 3.5 mL of blood was collected from the cephalic vein into a tube containing polymer gel as a serum separating agent, and serum was separated by centrifugation (4°C, 3000 rpm, 15 minutes). Serum CRE and urea nitrogen (UN) were measured by a Hitachi 7350 auto-analyzer (Hitachi Ltd., Tokyo, Japan).
Light Microscopy
The left kidney was removed, fixed in 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd.), embedded in paraffin wax, sectioned at 5-μm thickness, stained with hematoxylin and eosin (H&E) and alcian-blue, and examined microscopically (Table 1). The renal papilla included the upper papilla and papillary tip.
Immunohistochemical Staining of COX-1 or COX-2 in the Renal Papilla
A section from the left kidney was deparaffinized, and an antigen was retrieved with Dako Proteinase K solution (Dako Cytomation Co., Ltd., Kyoto, Japan). After incubation with 3% hydrogen peroxide for 5 minutes, the sections were pre-blocked with Nonspecific Staining Blocking Reagent containing 0.25% casein and carrier protein (Dako Cytomation Co., Ltd.), and then were incubated with a primary antibody overnight at 4°C. As COX-1 and COX-2 primary antibodies, ovine COX-1 polyclonal rabbit antiserum (No. 160108, Cayman Chemical Co., Ann Arbor, MI, USA) and murine COX-2 polyclonal rabbit antibody (No. 160106, Cayman Chemical Co.), respectively, were used. These antibodies were diluted 1:400 with 0.1% Tween-Tris buffer. Immunore-active complexes were detected via an LSAB 2 Systems-HRP (Dako Cytomation Co., Ltd.) and visualized with diaminobenzidine (DAB, Dako Cytomation Co., Ltd.), which reacts with peroxidase to give a brown reaction product (Table 1). The sections were counterstained briefly with hematoxylin. All control sections were treated with no primary antibody.
Electron Microscopy
In the present study, since apparent morphological alterations were light-microscopically noted in week 7 for nefiracetam and in week 5 for ibuprofen, electron-microscopic examination was performed only for these points. A tiny portion from the left kidney (papillary section) was removed, fixed in 3% glutaraldehyde and 2% osmic acid, dehydrated with alcohol, and embedded in epoxy resin. Thick sections were made and stained with 1% toluidine blue for light microscopic survey. Ultrathin sections stained with uranyl acetate and lead citrate were examined with a transmission electron microscope (H-500, Hitachi Ltd., Table 1).
Quantitative Real-Time RT-PCR Analyses of Papillary COX-2 Gene Expression
In the present work, inasmuch as the early morphological features in RPN brought about by nefiracetam were quite different from those by ibuprofen, serial changes in the renal papillary COX-2 mRNA expression were assessed only in nefiracetam-treated groups. Total RNA was extracted from an approximately 40 mg portion of the papilla from the right kidney with an Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA, USA), which includes DNase treatment to remove genomic DNA. Extracted total RNA was converted to cDNA with cocktails consisting of 1× PCR buffer (Applied Biosystems, Foster City, CA, USA), 5 mM MgCl2, 1 mM dNTP mixture (Toyobo Co., Ltd., Osaka, Japan), 1U/μL RNase inhibitor (Toyobo Co., Ltd.), 2.5 U/μL Random Primer (Takara Bio Inc., Shiga, Japan), and 0.125 U/μL AMV Reverse Transcriptase XL (Takara Bio Inc.). The following primers and TaqMan probes were designed by using a Primer Express (Applied Biosystems) for the detection of each target gene sequence. Real-time PCR data and analyses were collected in Applied Biosystems 7700 Sequence Detection System instruments. The amount of each gene target is normalized to an endogenous control (18S rRNA, 20 × Pre-Developed TaqMan Assay Reagents, Applied Biosystems), and then expressed as the ratio to the mean control values (each value in a treated dog/the mean value of 3 control dogs).
The following primers and probes were used for PCR. Forward primer, 5′-CTG TGG GCC AGG AGG TCT T-3′; Reverse primer, 5′-CAC TCT GTT ATG CTC CCG CAG-3′; TaqMan probe, 5′-TGC CTG GTC TGA TGA TGT ATG CCA CC-3′.
Statistical Analyses
The quantitative laboratory data in groups 1 (intact control), 5 (nefiracetam: 7-week treatment) and 6 (ibuprofen: 5-week treatment) are represented as the group mean ± standard deviation (SD) at each sampling point, and following analyses were carried out: (1) A paired t-test compared with the predose value in each group was applied, and (2) A 2-group comparison between the intact control group and each treatment group was carried out as follows. The homogeneity of the variance between two groups was analyzed by F-test at each sampling point. If the variance was homogeneous, Student’s t-test was subsequently used. If the variance between the groups was not homogeneous, then Aspin-Welch’s t-test was used.
The COX-2 mRNA expression data in the group 1 and nefiracetam-treated groups 2, 3, 4, and 5 (corresponding to 4-, 5-, 6-, and 7-week treatment, respectively) are represented as the group mean ± SD. Then, the aforementioned 2-group comparison between the intact control group and each treatment group was performed. A 2-tailed p-value less than 5% was considered to be statistically significant. EXSAS ver. 6.10 (Arm Corporation, Osaka, Japan), a software package, was used for these analyses.
Animal Welfare
All experimental procedures were performed in accordance with the in-house guidelines of the Institutional Animal Care and Use Committee of Daiichi Pharmaceutical Co., Ltd.
Results
Laboratory Tests
In dogs receiving nefiracetam, decreased urinary osmotic pressure and a tendency of increasing urine volume were observed in weeks 6 to 7. Urinary LDH tended to be higher from week 5. Serum UN showed somewhat higher values than that in the predose or control group throughout the study period without any remarkable increase in serum CRE. In dogs given ibuprofen, a decrease in urinary osmotic pressure, increases in urine volume and urinary LDH and a slight increase in serum UN were sporadically noted in weeks 1 to 5 (Figure 2). All alterations seen in ibuprofen were earlier and severer than those in nefiracetam.
Light Microscopy
In dogs given nefiracetam, no treatment-related changes were observed by week 6. In week 7, however, epithelial swelling of the papillary ducts (upper papilla) was seen in 3 of 4 dogs with increased eosinophilic-staining intensity in the cytoplasm of epithelial cells (Table 2, Figure 3C). Degeneration and desquamation of epithelial cells was sometimes noted (Figure 3C, inset). The alcian-blue staining intensity was comparable to the control throughout the dosing periods (Figure 3D). Mineralization of the papilla without detectable change in interstitial cells was noted in 2 of 4 dogs (Table 2). In dogs receiving ibuprofen, degeneration and necrosis of the interstitium in the papillary tip were noted in 2 of 3 dogs (Table 2 and Figure 3E); however, no remarkable changes were noted in the remaining papillary ducts. The alcian-blue staining intensity was markedly decreased in the degenerative and/or necrotic areas of the papillary interstitium (Figure 3F). Mineralization of the papilla was seen in 1 of 3 dogs (Table 2).
Immunohistochemical Staining of COX-1 or COX-2 in the Renal Papilla
In the COX-1 staining, there were no significant differences among the control, nefiracetam, and ibuprofen groups (Table 2). In the COX-2 staining, the immunoreaction in control dogs was seen in the cytoplasm of interstitial cells in the papillary tip (Table 2 and Figures 4A and 5). In dogs receiving nefiracetam, though the immunoreaction was within the cytoplasm of interstitial cells in the papillary tip by week 6, similarly as in the control, interstitial cells around affected epithelial cells (upper papilla) were positively reacted in week 7 (Table 2 and Figures 4B and 5). In dogs given ibuprofen, a marked positive immunoreaction was seen within interstitial cells adjacent to the degenerative and/or necrotic areas of interstitium in the papilla (Table 2 and Figures 4C and 5).
Electron Microscopy
In control dogs (animal Nos. 1 and 3), epithelial cells of the papillary ducts showed a cuboidal appearance with the nucleus located in the center of the cell, and short microvilli were seen on the luminal surface. The lateral plasma membrane was interdigitated, and the basal membrane displayed infoldings. Small numbers of mitochondria and vacuoles were noted in the cytoplasm (Figure 6A). In dogs receiving nefiracetam (animal Nos. 18 and 19), epithelial cells of the papillary ducts showed swelling and the microvilli were decreased or absent. Membranous blebs were noted in the luminal surface. Intracellular interdigitation and infoldings in both lateral and basal membranes were inconspicuous. These epithelial cells revealed sparse cytoplasm containing large vacuoles (Figure 6B). In dogs given ibuprofen (animal No. 21), no remarkable changes in the epithelial cells of the papillary ducts were noted.
COX-2 mRNA Expression in the Renal Papilla
Nefiracetam exceedingly decreased COX-2 mRNA expression in week 4, but it recovered to the basal control level in weeks 5 to 6. Afterward, COX-2 mRNA markedly increased in week 7 (Figure 7).
Discussion
In our previous study (Tsuchiya et al., 2003), nefiracetam caused degeneration and/or necrosis in the renal papilla only in dogs administered nefiracetam at 300 mg/kg/day for 8 to 11 weeks; namely, decreases in urinary osmotic pressure were observed from week 5, followed by increases in urine volume and urinary LDH from week 8. The initial renal morphological change was necrosis of ductal epithelial cells in the papilla in week 8. In the present work, to ascertain the earlier pathophysiological features in RPN, male beagle dogs were given orally 300 mg/kg/day of nefiracetam for 4 to 7 weeks in comparison with 50 mg/kg/day of ibuprofen, an NSAID, for 5 weeks, and laboratory tests, light-microscopic, immunohistochemical, and electron-microscopic examinations, and/or COX-2 mRNA analysis were periodically performed.
In laboratory tests, decreased urinary osmotic pressure and increased urine volume in weeks 6 to 7 of nefiracetam treatment were essentially consistent with those obtained in week 8 of the previous study (Tsuchiya et al., 2003), but an increased urinary LDH level was observed earlier (week 5). Alternatively, these parameters were reconfirmed to be useful early biomarkers to detect the onset of RPN evoked by nefiracetam. Ibuprofen also elicited decreased urinary osmotic pressure, increased urine volume and increased urinary LDH, although there was a great interindividual variance. The onset and severity of these changes by ibuprofen were earlier and stronger than those by nefiracetam, which would be closely linked with necrosis of the interstitium in the papillary tip observed only in the ibuprofen group as mentioned below.
Light-microscopically, in dogs receiving nefiracetam, no changes were seen in the kidney by week 6. In week 7, however, epithelial swelling and degeneration of the papillary ducts appeared accompanied with a positive immunoreaction to COX-2 within interstitial cells around the affected epithelial cells in the upper papilla. Ultrastructurally, epithelial swelling, impaired microvilli, and blebbing on the luminal surface were noted with reductions of intracellular interdigitation and infoldings in both lateral and basal membranes. In contrast, treatment with ibuprofen resulted in degeneration and necrosis of the interstitium in the papillary tip in week 5; however, no remarkable changes were observed in the remaining papillary ducts. In the COX-2 staining, unlike the case with nefiracetam, ibuprofen showed a positive reaction within interstitial cells adjacent to degenerative and/or necrotic areas in the papillary tip. Ultrastructurally, no changes in epithelial cells of the papillary ducts were noted. Thus, these data implied that the early morphological alteration in the papilla brought about by nefiracetam was quite different from that by ibuprofen. Mineralization in the papilla observed in both nefiracetam and ibuprofen treatment may suggest the preexistence of damaged regions. Cellular swelling, loss of microvilli, and blebs seen in the electron-microscopic examination of nefiracetam-treated animals may be indicative of reversible injuries under the hypoxic or ischemic conditions (Cotran et al., 1999). Since dogs given nefiracetam showed severe hypoactivity, of which some lay on their side (Kashida et al., 1996), the continued hypoxic states due to repeated treatment seemed to be a trigger to evoke papillary epithelial swelling.
As for the COX-1 staining, there were no significant differences among the control, nefiracetam, and ibuprofen groups. Generally, COX-1 has been reported as a constitutive isoform having housekeeping functions, whereas COX-2 is an inducible isoform induced by inflammatory stimuli or growth factors (Hao et al., 2000). More recent studies have suggested that COX-2 is also expressed in the renal medulla and is the major isoform contributing to medullary PG generation in vivo, especially in dehydrated subjects (Khan et al., 1998; Neuhofer et al., 2004). Sellers et al. (2004) have further shown that COX-1 is the main isoform involved in the synthesis of renal PGs in non-human primates and humans while both COX-1 and COX-2 contribute to the synthesis of renal PGs under basal conditions in rats and dogs. Hence, COX-2 was confirmed to play more important roles than COX-1 in the progression of RPN in dogs given nefiracetam.
The renal papillary COX-2 expression in nefiracetam-treated dogs decreased exceedingly in week 4, but returned to the basal control level in weeks 5 to 6. In week 7, however, COX-2 mRNA markedly increased as compared with the control. Therefore, it was considered that nefiracetam depressed COX-2 mRNA expression at the early phase, followed by a compensating increase, and then marked upregulation was seen in response to the progression of renal injuries with continuous treatment. This assumption was partly supported by the fact that the positive immunoreaction to COX-2 was increased in the interstitial cells around the affected epithelial cells of the upper papilla in week 7, suggesting an induction of COX-2 mRNA.
It has been reported that the epithelia promote organ homeostasis by restricting the flow of ions and solutes between cells across the epithelial cells, and the intercellular junctional complexes such as the tight junction, adherens junction, and desmosomes are important to maintain the cell-cell contact (Collares-Buzato et al., 1998; George et al., 2004; Johnson, 2005). Bachmann and Kriz (1982) have demonstrated that the epithelia (especially when salt secretion is stimulated) exhibit an elaborate “intracellular membranous labyrinth” created by cellular interdigitation and infoldings of the basolateral membrane. Goto et al. (2003) have previously shown that the nefiracetam metabolite M-18 reduces transepithelial electric resistance (TER) in the primary cultured uroepithelial cells of canine urinary bladder, and M-10 and M-18 displayed a deformation of uroepithelial cells and a slight reduction of the ZO-1 band, which is an essential protein associating with the tight junction. Likewise, we have reported that the concentration ratio of M-18 in the papilla to medulla or cortex is remarkably higher in dogs than in rats and monkeys, and that M-18 possessed inhibitory effects on PG synthesis in canine renal papillary slices (Tsuchiya et al., 2003). By taking into account these previous findings (Kashida et al., 1996; Tsuchiya et al., 2003), we propose the following mechanism underlying RPN evoked by nefiracetam: the reductions of intracellular interdigitation and infoldings at both lateral and basal membranes in epithelial cells of the papillary ducts may initially inactivate the paracellular transport. This is followed by decreased cellular integrity, and then M-18 penetration and retention into the renal papilla occur. M-18 inhibits renal papillary PG synthesis, and certain dogs finally incur ischemic injury due to decreased PG synthesis. Further investigation after 7-week treatment will be required to support the above suggestion.
In conclusion, the earliest morphological event in RPN evoked by nefiracetam is quite different from that by ibuprofen. The epithelial cells in the papillary ducts are considered to be the primary target site for the onset of RPN evoked by nefiracetam.