1 Laboratory for Research on Peptide Metabolism, Faculty of Medicine, 2 Department of Nephrology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium and 3 Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, Surrey, UK
Correspondence and offprint requests to: Catherine Lebeau, Laboratory for Research on Peptide Metabolism, Université Libre de Bruxelles, Campus Erasme CP 603, Route de Lennik 808, B-1070 Brussels, Belgium. Email: clebeau{at}ulb.ac.be
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Abstract |
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Methods. Using a rat model of AA nephropathy, the proximal tubular lesions induced by daily subcutaneous injections of AA for 35 or 5 days were characterized biochemically and histologically. Urinary excretion of proteins, albumin, low molecular weight proteins, N-acetyl-ß-D-glucosaminidase, -glutathione S-transferase, leucine aminopeptidase and neutral endopeptidase (NEP) was determined and related to histological conventional findings and immunostainings of NEP and megalin.
Results. In both protocols, an acute phase of release of urinary markers was observed within the first 3 days of AA treatment in parallel with a significant increase of specific AA-related DNA adducts reflecting early tubular intoxication. A dramatic loss of the proximal tubule brush border was histologically confirmed, while the expression of megalin decreased at the damaged apical epithelium (mainly of the S3 segment).
Conclusion. Proximal tubule injury occurs early after AA intoxication in rats, with a link between specific AADNA adduct formation, decreased megalin expression and inhibition of receptor-mediated endocytosis of low molecular weight proteins, bringing in vivo confirmation of previous in vitro studies.
Keywords: aristolochic acid nephropathy; low molecular weight proteins; megalin; neutral endopeptidase; proximal tubule injury; tubular proteinuria
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Introduction |
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Pathological examinations of kidneys from human cases showed extensive interstitial fibrosis with atrophy and loss of tubules. These lesions progressed from the superficial to the deep cortex [5]. Structural impairment of the proximal tubule epithelium in CHN patients was attested by a progressive decrease in urinary neutral endopeptidase (neprilysin, NEP) levels, reflecting the loss of the brush border and an abnormal urinary release of the lysosomal enzyme N-acetyl-ß-D-glucosaminidase (NAG), another marker of tubular damage [6]. These alterations led to a defect of tubular reabsorption of filtered microproteins such as Clara cell protein, retinol-binding protein, ß2-microglobulin and 1-microglobulin, indicating tubular dysfunction [6,7].
Experimentally, the toxicity of AA to the proximal tubule was confirmed by investigation on a well-established opossum kidney (OK) proximal tubule cell line. Following exposure of OK cell monolayers to AA, the reabsorption of albumin and ß2-microglobulin was significantly inhibited and the expression of megalin decreased, demonstrating alterations of the receptor-mediated endocytosis process [8].
An experimental rat model of AA-induced nephropathy has been developed in our laboratory [9,10]. Daily subcutaneous (s.c.) AA injections for 35 days resulted in renal failure, interstitial fibrosis and tubular atrophy. A reduction of urinary excretion of leucine aminopeptidase (LAP) was already observed at day 10, suggesting proximal tubular alterations caused by AA.
In numerous clinical and experimental situations, urinary excretion rates of enzymes and low molecular weight proteins have been studied as early signs of structural and/or functional insult of tubular epithelial cells [11]. Enzymes may be released from different cellular parts of the tubular epithelium: NAG is of lysosomal origin, -glutathione S-transferase (
-GST) is a cytosolic enzyme, and LAP and NEP are present at the apical brush border of proximal tubule. In physiological conditions, low molecular weight proteins and albumin are freely filtered across the glomerular capillary wall and almost completely reabsorbed by the proximal tubular cells. Their excretion level into urine is increased in the case of reduced reabsorption by these cells, which is due to an increased reabsorptive load, or to functional/structural damage. Increased excretion rates of urinary enzymes or proteins are typically observed before elevation of other parameters of renal function, such as plasma creatinine level, and are to be considered as subclinical markers of tubular insult.
According to this background, the experimental observations of AA effects prompted us to investigate in greater detail the early structural and functional impairments of the proximal tubule in the rat model of AA-induced nephropathy.
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Subjects and methods |
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After 1 week acclimatization (at day 0), weight-matched rats were randomly assigned to two groups. One group (n = 36) received s.c. injections of AA (10 mg/kg body weight) (mixture containing 40% AAI and 60% AAII, Acros Organics Co., Geel, Belgium) daily for 35 days. AA was dissolved in polyethylene glycol (PEG) 400 (Fluka Chemie, Buchs, Switzerland) to a final concentration of 10 mg/ml and diluted in distilled water before injection. The control group (n = 36) was treated with a v/v solution of PEG 400 and distilled water only. Body weights were measured weekly for adjustment of drug dosages.
Six rats per group were anaesthetized on days 3, 7, 10, 14, 18 and 35 with an intraperitoneal (i.p.) injection of a mixture containing ketamine (Imalgen, LEO Pharma Belgium, Wilrijk, Belgium) and xylazine (Rompun, Bayer, Brussels, Belgium). Rats were dissected to collect blood by intracardiac puncture with a syringe containing potassium-EDTA, and the kidneys were excised and immediately fixed. Before sacrifice, rats were placed in rodent metabolic cages for urine collection.
A second series of experiments performed as detailed above included the follow-up of urinary parameters during 35 days. Briefly, four male Wistar rats were injected daily with AA mixture (10 mg/kg body weight) and three control rats with PEG solution for 35 days. Urine collection was performed on days 0, 3, 5, 7, 10, 13, 15, 18, 22, 24, 27, 31 and 35 for each rat.
Short-term protocol (5 days)
With respect to the same experimental conditions as described above, 60 male Wistar rats were divided into two groups. One group (n = 30) was injected daily with AA mixture (10 mg/kg body weight) and another group (control, n = 30) with PEG solution for up to 5 days. Six rats per group were anaesthetized on days 1, 2, 3, 4 and 5 and dissected to collect blood from the heart, and the kidneys were excised. Before sacrifice, urine was collected at days 0, 1, 2, 3, 4 and 5.
All protocols were approved by the local Ethical Committee for Animal Care (Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium).
Urine, blood and kidney samples
After collection, urine samples were stored at 20°C. Blood samples were centrifugated at 1600 g for 15 min at 4°C and the plasma was stored at 20°C until assayed.
Immediately after bilateral nephrectomy, half of a kidney was fixed in 4% buffered formaldehyde for histological analyses of paraffin-embedded sections. Before freezing kidney tissues for cryostat sections, the remaining half of a kidney was fixed in 4% phosphate-buffered paraformaldehyde and then cryoprotected by successive immersion in 10, 20 and 30% sucrose.
Renal cortex was dissected, frozen in liquid nitrogen and stored at 80°C for further detection of specific AADNA adducts.
Plasma and urinary creatinine assays
Plasma creatinine levels were measured by a sensitive accurate high-performance liquid chromatography method adapted from Xue et al. as described previously [9] and expressed in µmol/l.
Urinary creatinine values were determined by a pseudokinetic Jaffé's method using a Creatinine Diagnostic Kit (Sigma-Aldrich, Bornem, Belgium) and expressed in mmol/l.
Urinary excretion of proteins and enzymes
Total proteinuria was quantified by the Bradford binding assay as detailed previously [9]. Briefly, Bradford reagent was added to standards or urine samples. The optical density was measured at 620 nm and reported to a bovine serum albumin standard curve (BSA; Sigma-Aldrich, Bornem, Belgium), and expressed in mg/mmol creatinine.
Urinary excretion of LAP was measured by a spectrofluorimetric assay as detailed previously [9] and expressed in µmol AMC (7-amino-4-methyl-coumarin) produced/mmol creatinine/h.
Urinary excretion of NEP was determined by a spectrofluorimetric assay as detailed previously [6] and expressed in µg/mmol creatinine.
Urinary excretion of lysosomal NAG was measured by a colorimetric assay following the manufacturer's protocol (Roche Diagnostics Belgium, Brussels, Belgium) and expressed in mU/mmol creatinine.
Urinary excretion of cytosolic -GST was measured by an enzyme immunoassay following the protocol of the manufacturer (Biotrin, Herman Biognostics, Wemmel, Belgium) and expressed in µg/mmol creatinine.
Electrophoresis of urinary proteins
Urinary proteins were precipitated with v/v 10% trichloracetic acid solution, centrifuged at 4000 g for 10 min, washed with 100% ethanol and dried in a Speed-Vac evaporator. The pellets were resuspended in Laemmli sample buffer (31.2 mM TrisHCl, 5% glycerol, 1% SDS, 0.5% ß-mercaptoethanol, bromophenol blue). Protein samples (20 µg) were heated at 95°C for 2 min and separated by ready gel TrisHCl 420% (Bio-Rad Laboratories, Nazareth-Eke, Belgium). The gels were coloured with Coomassie blue solution (30% methanol, 10% acetic acid, 60% H2O, 0.1% Coomassie blue), then incubated with a decoloration solution (40% methanol, 10% acetic acid, 50% H2O, 0.5% glycerol) and dried.
The relative urinary contents of low molecular weight proteins (LMWPs) were evaluated semi-quantitatively by scanning (CanoScan N676U, Canon Inc., Tokyo, Japan) followed by analysis of the electrophoretic gels with a computer-assisted image analyser system (Scion Image version b4.0.2, Scion Corporation, MA). The quantity of LMWPs was reported relative to total proteins levels and expressed in mg/mmol creatinine.
Albumin assay
Urinary and plasma albumin were measured by using the rat albumin enzyme-linked immunosorbent assay (ELISA) quantitation kit (Bethyl Laboratories, IMTEC Diagnostics, Antwerpen, Belgium). Briefly, plates were coated with a capture rabbit anti-rat albumin affinity-purified antibody (1:100). After blocking, standards, urine or plasma samples (dilution x1000 or x2 x 106, respectively) were added, then detected by a rabbit anti-rat albuminhorseradish peroxidase (HRP)-conjugated antibody (1:50 000) and revealed by the enzyme substrate 3,3',5,5'-tetramethylbenzidine, which reacted with peroxidase. The reaction was stopped with sulfuric acid (1 N) and the resulting yellow end-product was read at 450 nm. The data were related to a standard rat albumin curve and expressed in mg/mmol creatinine.
Histological studies
Conventional microscopy
For brush border integrity studies, fixed kidney tissues embedded in paraffin were cut at 5 µm and stained with periodic acidSchiff (PAS). Briefly, tissue sections were dewaxed, rehydrated, incubated for 10 min with 1% periodic acid, washed and incubated for 15 min with Schiff reagent. After washing, sections were counterstained with Mayer's haematoxylin.
Neutral endopeptidase immunostaining
Immunostaining of the brush border NEP (neprilysin) was performed on paraffin-embedded kidney sections. After dewaxing and hydration, endogenous peroxidase activity was inhibited by incubation of tissue sections in methanol containing 0.3% H2O2 for 30 min. Sections were blocked for 20 min with goat normal blocking serum (Vectastain Elite®ABC kit Rabbit IgG, Vector Laboratories, Labconsult, Brussels, Belgium), then with avidin D solution and with biotin solution (Avidin/biotin blocking kit, Vector Laboratories). After overnight incubation at room temperature with the primary rabbit anti-neprilysin polyclonal antibody (1:2000; Chemicon International, Biognost, Heule, Belgium), sections were treated for 30 min with biotinylated secondary goat anti-rabbit IgG antibody. Then, after 30 min incubation with avidin and biotinylated HRP complex, staining was revealed by incubation in 3,3'-diaminobenzidine (DAB) solution (DAB substrate kit for peroxidase, Vector Laboratories). Finally, sections were counterstained with Mayer's haematoxylin.
Semi-quantitation of NEP immunostaining was performed by two independent observers (C.L. and A.P.) counting the NEP-positive proximal tubules by field at 200x magnification. Ten fields were examined per kidney section, corresponding to a mean population of 85 proximal tubules per field. The final result for each time point was expressed as a percentage of total proximal tubules.
Megalin immunostaining
Immunostaining of megalin was performed on kidney cryostat sections of 8 µm. After incubation in Tris-buffered (10 mM, pH 7.6) saline (0.9%), the endogenous peroxidase activity was inhibited by incubation of tissue sections in methanol containing 0.3% H2O2 for 30 min. Sections were blocked for 20 min with horse normal blocking serum (Vectastain Elite®ABC kit Mouse, Vector Laboratories), then with avidin D solution and with biotin solution. After overnight incubation at room temperature with the primary mouse monoclonal anti-megalin antibody (1:1000; kindly provided by Dr V. Marshansky, Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA), sections were treated for 30 min with biotinylated secondary horse anti-mouse antibody. Then, after 30 min incubation with avidin and biotinylated HRP complex, staining was revealed by incubation in DAB solution. Finally, sections were counterstained with Mayer's haematoxylin.
DNA adduct analysis from renal cortex of AA-treated rats
DNA isolation from kidney samples was performed by a standard phenol extraction method. 32P-Post-labelling analysis using nuclease P1 enrichment, chromatography on polyethyleneimine-cellulose thin-layer plates (Machery and Nagel, Düren, Germany), autoradiography using a Packard Instant Imager (Canberra Co., Dowers Grove, IL) and quantitation were performed as described previously [12]. Results were expressed as DNA adducts per 108 normal nucleotides.
Statistical analysis
Data are presented as mean±SEM. Comparisons between controls and AA-treated rats for each time point and between AA-treated rats from consecutive days were performed by the non-parametric MannWhitney test. Correlations between structural and functional markers were evaluated by the Spearman non-parametric test. Statistical significance was set at P<0.05.
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Results |
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Using individual biochemical data for each time-point, relationships between structural and functional impairment of the proximal tubule were found: NAG enzymuria correlated tightly with proteinuria (r = 0.80, n = 36, P<0.0001), with albuminuria (r = 0.54, n = 36, P = 0.0007) and with plasma creatinine levels (r = 0.65, n = 36, P<0.0001).
Bearing these data in mind, it was interesting to perform sequential urinary measurements. Urinary parameters of three control rats and four AA-treated rats were analysed after 0, 3, 5, 7, 10, 13, 15, 18, 22, 24, 27, 31 and 35 days. Total urinary protein, urinary albumin, NAG, -GST, LAP and NEP were not different from basal values during 35 days in control rats (data not shown). For all the four AA-treated rats, a rapid and transient increase of proteinuria and albuminuria was detected around day 5 (from day 3 to day 7) and a second one around day 27 (from day 25 to day 30) (Figure 1A). In parallel, urinary NAG and
-GST increased dramatically during the first days of AA intoxication, with a maximum at days 5 or 7, then decreased and finally increased to a lesser extent during the last days (Figure 1B). A similar pattern of excretion was observed for urinary LAP and NEP, two membrane-bound brush border enzymes, with the earliest release at days 3 and 7 (Figure 1C).
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Total proteinuria increased significantly in AA-treated rats relative to controls as early as day 1, and extended to day 5 (Figure 2A). Albuminuria significantly increased from day 2 (73.2±43.3 vs 3.6±1.0 mg/mmol creatinine; P<0.01) to day 5 (213.5±46.6 vs 3.0±0.3 mg/mmol creatinine; P<0.01). Plasma albumin levels did not change with time (data not shown). In parallel with total proteinuria, NAG enzymuria increased significantly from day 2 to day 5 (Figure 2B). The time course of NAG enzymuria followed the progressive increase of plasma creatinine, reflecting acute renal failure (Figure 2C).
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Detection of AADNA adducts
Specific AADNA adducts were detected only in renal tissue samples from AA-treated rats for each day using the short-term protocol. As described previously, the typical AADNA adduct pattern on thin-layer chromatography plates consisted of three major adduct spots: 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI, spot 1), 7-(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI, spot 2) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII, spot 3) [12].
Quantitative analysis of these adducts revealed an almost linear increase in DNA binding for each of the individual AADNA adducts during the first 2 days, reaching a steady-state level after that time (Figure 3). The long-term protocol showed that the steady-state level persisted to day 35 (total AADNA binding at day 35: 1049±377 adducts per 108 nucleotides, with mean values of individual AADNA adducts of 280, 405 and 365 adducts per 108 nucleotides for spots 1, 2 and 3, respectively).
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Discussion |
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The relevance of the animal model as well as the dose of AA used have already been described in the initial experimental study [9]. The choice of AA dose was based on our clinical experience in CHN patients [13]. The total amount of AA ingested by some patients was 10 mg/kg body weight, which represents a daily dose of 0.29 mg AA/kg body weight for 35 days. The dose of AA used in the experimental model was then 30 times higher than that for CHN patients. The impact of the dose seems to be crucial to develop nephrotoxicity, as we previously showed that 1 mg/kg body weight of AA instead of 10 mg/kg body weight injected into rats did not result in the onset of renal failure and interstitial fibrosis [9].
At the time of the set-up of the rat model of AA nephropathy [9], preliminary experiments had already shown progressive increase of urinary parameters as well as plasma creatinine in control rats. This observation could be related to the growth of the animals rather than to a possible toxicity of the solution used to dissolve AA. Indeed, rats injected with water or PEG solution did not demonstrate any alteration of biochemical parameters and renal tissue samples were histologically normal.
The present study was thus carried out to characterize in the rat model of AA nephropathy early proximal tubular injury induced by AA intoxication, in particular the respective structural and functional impairments. We demonstrated a biphasic evolution of renal function and morphological alterations. Early proximal tubule dysfunction and structural abnormalities occurred within the first 3 days, reflecting an acute phase of proximal tubular injury. Indeed, biochemical measurements showed that tubular proteinuria, NAG, -GST, LAP and NEP enzymuria, already increased within the first 3 days of AA intoxication, returned to basal levels, and finally were enhanced again, indicating that tubular impairment occurred in two phases and presented an acute phase during the first days of AA treatment. Differences in the design of the two long-term protocols should be considered to explain the absence of biphasic evolution in this first long-term protocol. In the first series of experiments, urine samples were collected at fixed time points before the sacrifice of the animals (days 3, 7, 10, 14, 18 and 35). In contrast, in the second series of experiments, individual urine collections were analysed more regularly all throughout the protocol (days 0, 3, 5, 7, 10, 13, 15, 18, 22, 24, 27, 31 and 35). In addition to the heterogeneity between samples from different animals from one time point to another, the probability of missing the peaks of urinary enzymes and proteins is far greater in the fixed time points' long-term protocol than in the longitudinal follow-up.
In a recent study in rats, acute renal failure was noticed after 5 days of AA treatment (4 mg/day) but recovery of renal function was observed by day 14 after withdrawal of AA [16]. Signs of regeneration of tubular epithelium were present but tubular dilatations still remained severe. In another study using FVB mice, daily i.p. AA injections (5 mg/kg body weight) for 2 weeks induced tubular degeneration and renal failure. Regeneration of tubular epithelium with moderate peritubular fibrosis was seen 4 weeks after cessation of AA treatment [17]. In the present model of AA intoxication, the progression to chronic renal failure and interstitial fibrosis after an early acute phase can probably be related to repeated s.c. AA injections to rats for 35 days. Interestingly, similar observations of acute tubular necrosis with consecutive renal failure were reported recently in a patient after ingestion of AA for 10 days [18].
It should be noted that early signs of impairment can be related to a rapid intense intoxication of the cells. Indeed, AA-related DNA adducts were detected as early as day 1 and reached a steady-state level at day 2, which remained almost constant up to day 35, as previously observed [12]. In these first days of intoxication, a significant increase of plasma creatinine was observed, suggesting early renal dysfunction.
From a histological point of view, structural lesions of proximal tubule epithelium associated with acute renal failure are known to be of ischaemic or toxic origin. Epithelial cells of the proximal tubule S3 segment typically show early morphological changes, including the formation of blebs in the apical membrane, with loss of brush border [19]. Our histological observations are in agreement with these descriptions: cell swelling after the first day of AA injection followed by necrosis 3 days later, progressive loss of tubular brush border from the second day, and reduction of NEP immunostaining along the S3 segment of the proximal tubule. This enzyme is located in the pars recta of that proximal tubule [20]. Furthermore, the expression of megalin, a receptor involved in receptor-mediated endocytosis, was also reduced early (on day 2) at the apical epithelium of this segment. However, the progression of the tubular atrophy process was so severe that megalin immunostaining studies were not justified beyond day 10.
Interestingly, the early reduction of megalin expression paralleled previous in vitro observations in OK cells: after 24 h intoxication with AA, as reflected by the presence of specific AA-related DNA adducts, the receptor-mediated endocytosis process of LMWP reabsorption was inhibited and the expression of megalin significantly decreased [8].
In conclusion, structural and functional impairments of the S3 segment proximal tubules occur rapidly in experimental AA nephropathy. These data thus confirm in vivo that AA administration induces a very early proximal tubular dysfunction. They might explain the Fanconi syndrome described in some forms of human AA intoxication [21]. A more pronounced intoxication leads, on the other hand, to severe tubular atrophy and interstitial fibrosis.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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