Probenecid prevents acute tubular necrosis in a mouse model of aristolochic acid nephropathy
Authors: Thomas E.R. Baudoux, Agnieszka A. Pozdzik, Volker M. Arlt, Eric G. De Prez, Marie-Hélène Antoine, Nathalie Quellard, Jean-Michel Goujon, Joëlle L. Nortier
Journal: Kidney International (2012)

Experimental aristolochic acid nephropathy is characterized by early tubulointerstitial injury followed by fibrosis, reproducing chronic lesions seen in humans. In vitro, probenecid inhibits aristolochic acid entry through organic anion transporters, reduces specific aristolochic acid-DNA adduct formation, and preserves cellular viability. To test this in vivo, we used a mouse model of aristolochic acid nephropathy displaying severe tubulointerstitial injuries consisting of proximal tubular epithelial cell necrosis associated to transient acute kidney injury followed by mononuclear cell infiltration, tubular atrophy, and interstitial fibrosis. Treatment with probenecid prevented increased plasma creatinine and tubulointerstitial injuries, and reduced both the extent and the severity of ultrastructural lesions induced by aristolochic acid, such as the loss of brush border, mitochondrial edema, and the disappearance of mitochondrial crests. Further, the number of proliferating cell nuclear antigen–positive cells and total aristolochic acid-DNA adducts were significantly reduced in mice receiving aristolochic acid plus probenecid compared with mice treated with aristolochic acid alone. Thus, we establish the nephroprotective effect of probenecid, an inhibitor of organic acid transporters, in vivo toward acute proximal tubular epithelial cell toxicity in a mouse model of aristolochic acid nephropathy. Experimental aristolochic acid nephropathy is characterized by early tubulointerstitial injury followed by fibrosis, reproducing chronic lesions seen in humans. In vitro, probenecid inhibits aristolochic acid entry through organic anion transporters, reduces specific aristolochic acid-DNA adduct formation, and preserves cellular viability. To test this in vivo, we used a mouse model of aristolochic acid nephropathy displaying severe tubulointerstitial injuries consisting of proximal tubular epithelial cell necrosis associated to transient acute kidney injury followed by mononuclear cell infiltration, tubular atrophy, and interstitial fibrosis. Treatment with probenecid prevented increased plasma creatinine and tubulointerstitial injuries, and reduced both the extent and the severity of ultrastructural lesions induced by aristolochic acid, such as the loss of brush border, mitochondrial edema, and the disappearance of mitochondrial crests. Further, the number of proliferating cell nuclear antigen–positive cells and total aristolochic acid-DNA adducts were significantly reduced in mice receiving aristolochic acid plus probenecid compared with mice treated with aristolochic acid alone. Thus, we establish the nephroprotective effect of probenecid, an inhibitor of organic acid transporters, in vivo toward acute proximal tubular epithelial cell toxicity in a mouse model of aristolochic acid nephropathy. Human aristolochic acid nephropathy (AAN) is a tubulointerstitial (TI) nephritis reported after intake of herbal remedies containing aristolochic acid (AA).1.Vanherweghem J.L. Depierreux M. Tielemans C. et al.Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs.Lancet. 1993; 341: 387-391Abstract PubMed Scopus (967) Google Scholar,2.Vanhaelen M. Vanhaelen-Fastre R. But P. et al.Identification of aristolochic acid in Chinese herbs.Lancet. 1994; 343: 174Abstract PubMed Scopus (310) Google Scholar It is histologically characterized by a typical corticomedullary gradient of interstitial fibrosis and the progressive atrophy of proximal tubules, resulting in the rapid deterioration of renal function to the end stage.3.Depierreux M. Van Damme B. Vanden Houte K. et al.Pathologic aspects of a newly described nephropathy related to the prolonged use of Chinese herbs.Am J Kidney Dis. 1994; 24: 172-180Abstract Full Text PDF PubMed Scopus (245) Google Scholar,4.Cosyns J.P. Jadoul M. Squifflet J.P. et al.Chinese herbs nephropathy: a clue to Balkan endemic nephropathy?.Kidney Int. 1994; 45: 1680-1688Abstract Full Text PDF PubMed Scopus (252) Google Scholar AA intoxication also leads to the formation of specific AA-DNA adducts, which are premutagenic lesions involved in the development of AAN-associated urothelial cancer, and their long-term presence in renal tissue is used as a biomarker of AA exposure.5.Schmeiser H.H. Bieler C.A. Wiessler M. et al.Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy.Cancer Res. 1996; 56: 2025-2028PubMed Google Scholar,6.Nortier J.L. Martinez M.C. Schmeiser H.H. et al.Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi).N Engl J Med. 2000; 342: 1686-1692Crossref PubMed Scopus (925) Google Scholar AA-induced TI nephritis was experimentally reproduced in rabbits, mice, and rats.7.Cosyns J.P. Dehoux J.P. Guiot Y. et al.Chronic aristolochic acid toxicity in rabbits: a model of Chinese herbs nephropathy?.Kidney Int. 2001; 59: 2164-2173Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8.Debelle F.D. Nortier J.L. De Prez E.G. et al.Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats.J Am Soc Nephrol. 2002; 13: 431-436PubMed Google Scholar, 9.Sato N. Takahashi D. Chen S.M. et al.Acute nephrotoxicity of aristolochic acids in mice.J Pharm Pharmacol. 2004; 56: 221-229Crossref PubMed Scopus (113) Google Scholar, 10.Shibutani S. Dong H. Suzuki N. et al.Selective toxicity of aristolochic acids I and II.Drug Metab Dispos. 2007; 35: 1217-1222Crossref PubMed Scopus (123) Google Scholar A biphasic evolution of TI lesions was identified in our Wistar rat model.11.Pozdzik A.A. Salmon I.J. Husson C.P. et al.Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy.Nephrol Dial Transplant. 2008; 23: 2480-2491Crossref PubMed Scopus (66) Google Scholar,12.Pozdzik A.A. Salmon I.J. Debelle F.D. et al.Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation.Kidney Int. 2008; 73: 595-607Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar In the early, so-called acute phase, transient tubular necrosis located in the S3 segment (proximal tubular epithelial cells (PTECs)) and mononuclear cell infiltration are observed; later, in the so-called chronic phase, tubular atrophy and interstitial fibrosis are clearly the prominent features. In this step-by-step model, inflammatory cells were proposed as the physiopathological link between both phases.11.Pozdzik A.A. Salmon I.J. Husson C.P. et al.Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy.Nephrol Dial Transplant. 2008; 23: 2480-2491Crossref PubMed Scopus (66) Google Scholar In vitro data early confirmed that PTECs were the target of AA,13.Lebeau C. Arlt V.M. Schmeiser H.H. et al.Aristolochic acid impedes endocytosis and induces DNA adducts in proximal tubule cells.Kidney Int. 2001; 60: 1332-1342Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar suggesting the presence of specific molecular mechanisms responsible for the accumulation of AA in PTECs. The excretion of numerous organic anions (OAs), including endogenous metabolites, through PTECs is actually achieved via unidirectional transcellular transport, involving the uptake of OAs from the blood across the basolateral membrane and their extrusion across the apical membrane into the tubular lumen. OA transporters (OATs) have a key role in this process. At least 11 isoforms of OATs have been identified; a majority of them was found in the kidney. OATs are exchangers linked to two other transporters, the sodium dicarboxylate cotransporter and the sodium-potassium ATPase. OAs are taken up by OAT 1 and/or 3 in the basolateral membrane of the proximal tubule. This uptake is processed in parallel to the countertransport of α-ketoglutarate. The drug then crosses the cell and is excreted in the lumen of the tubule.14.Ahn S.Y. Nigam S.K. Toward a systems level understanding of organic anion and other multispecific drug transporters: a remote sensing and signaling hypothesis.Mol Pharmacol. 2009; 76: 481-490Crossref PubMed Scopus (111) Google Scholar,15.Nigam S.K. Bush K.T. Bhatnagar V. Drug and toxicant handling by the OAT organic anion transporters in the kidney and other tissues.Nat Clin Pract Nephrol. 2007; 3: 443-448Crossref PubMed Scopus (67) Google Scholar The activity of OATs has been associated with proximal tubular injury due to the accumulation of toxics, such as uremic toxins, drugs, and mercuric species.14.Ahn S.Y. Nigam S.K. Toward a systems level understanding of organic anion and other multispecific drug transporters: a remote sensing and signaling hypothesis.Mol Pharmacol. 2009; 76: 481-490Crossref PubMed Scopus (111) Google Scholar, 15.Nigam S.K. Bush K.T. Bhatnagar V. Drug and toxicant handling by the OAT organic anion transporters in the kidney and other tissues.Nat Clin Pract Nephrol. 2007; 3: 443-448Crossref PubMed Scopus (67) Google Scholar, 16.Ho E.S. Lin D.C. Mendel D.B. et al.Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1.J Am Soc Nephrol. 2000; 11: 383-393PubMed Google Scholar, 17.Torres A.M. Dnyanmote A.V. Bush K.T. et al.Deletion of multispecific organic anion transporter Oat1/Slc22a6 protects against mercury-induced kidney injury.J Biol Chem. 2011; 286: 26391-26395Crossref PubMed Scopus (66) Google Scholar In human embryonic kidney cells (HEK293) and Xenopus laevis oocytes, three human isoforms (OAT1, OAT3, or OAT4) were reported to have a role in intracellular accumulation of AA.18.Babu E. Takeda M. Nishida R. et al.Interactions of human organic anion transporters with aristolochic acids.J Pharmacol Sci. 2010; 113: 192-196Crossref PubMed Scopus (26) Google Scholar,19.Bakhiya N. Arlt V.M. Bahn A. et al.Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy.Toxicology. 2009; 264: 74-79Crossref PubMed Scopus (62) Google Scholar Moreover, probenecid (PBN) blocked AA entry by inhibition of human OATs, reducing the formation of AA-DNA adduct,19.Bakhiya N. Arlt V.M. Bahn A. et al.Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy.Toxicology. 2009; 264: 74-79Crossref PubMed Scopus (62) Google Scholar and preserved cell viability.18.Babu E. Takeda M. Nishida R. et al.Interactions of human organic anion transporters with aristolochic acids.J Pharmacol Sci. 2010; 113: 192-196Crossref PubMed Scopus (26) Google Scholar We investigated this last aspect in vivo in a mouse model of AAN. We hypothesized that PBN, by reducing AA entry through OATs, could protect PTECs against lesions, preventing AA-DNA adduct formation, and thus preserve cell viability. Ninety-six mice C57BL/6 were randomly assigned to four groups of 24 animals each. According to group, mice were injected with AA, AA+PBN, or solvent (polyethylene glycol (PEG))+PBN. The control group was injected with PEG (Figure 1). AA (5mg/kg body weight) or PEG was injected once a day and PBN (150mg/kg body weight) twice a day. These dosing regimens of PBN have been shown to inhibit OAT.20.Emeigh Hart S.G. Wyand D.S. Khairallah E.A. et al.Acetaminophen nephrotoxicity in the CD-1 mouse. II. Protection by probenecid and AT-125 without diminution of renal covalent binding.Toxicol Appl Pharmacol. 1996; 136: 161-169Crossref PubMed Scopus (34) Google Scholar Plasma creatinine (PCr) level, TI lesions, DNA-repair processes (proliferating cell nuclear antigen tissue expression), and AA-DNA adduct formation were quantified in each group after 2, 4, 5, and 8 days of AA injections. A transient acute kidney injury, as reflected by a significant increase in PCr levels, was observed in mice receiving AA after 5 days of injections as compared with control animals (PCr (mg/dl), median (min–max): 0.353 (0.222–0.504) vs 0.135 (0.112–0.211); P<0.0022). The addition of PBN prevents PCr increase in AA animals (PCr (mg/dl), median (min–max): 0.125 (0.105–0.139) vs 0.353 (0.222–0.504); P<0.0022). No significant change in PCr levels was measured in the PEG+PBN group as compared with controls (Figure 2). As demonstrated in Figures 3 and 4a–h, the renal parenchyma from PEG and PEG+PBN groups remained normal in optical microscopy analyses at all studied time points of the protocol. In contrast, early histological lesions were present in the AA group (Figures 3 and 4i–l). As early as day 2, a swelling of PTECs was found in the medullary rays (Figure 4i). In the same areas, prominent PTEC necrosis was observed at days 4 and 5 (Figures 3 and 4j–k). After 8 days of AA treatment, tubular atrophy was clearly widespread as reflected by dilatation and flattening of PTECs, as well as tubular basement membrane thickening. In the surrounding interstitial areas, mononuclear cell infiltration was observed at day 4 and progressively extended to day 8. At that time point, this inflammatory process was associated with extracellular matrix deposition. In mice receiving AA+PBN, swelling and necrosis of PTECs was limited to few tubules located in the medullary rays only at day 4 without any interstitial inflammatory cell infiltration (Figures 3 and 4n). Moreover, proximal tubules, as well as the surrounding interstitial areas, appeared normal under optical microscopy analysis at days 5 and 8 (Figures 3 and 4o–p). Throughout the protocol, no abnormality was detected within the glomeruli from all groups under optical microscopy analysis.Figure 4Histological analysis of tubulointerstitial injury in aristolochic acid (AA)-treated mice compared with mice receiving AA+probenecid (PBN). Representative photomicrographs of renal cortex longitudinal sections at studied time points in each group. No lesions were observed in controls: (a–d) polyethylene glycol (PEG) and (e–h) PEG+PBN. (i–l) In the AA group, swelling of proximal tubular epithelial cells was observed after 2 days of injection ($), followed by tubular necrosis at days 4 and 5 (arrow). Tubular atrophy (star) and progressive interstitial fibrosis (arrowhead) were present after 8 days of injection. (m–p) In the AA+PBN group, tubular atrophy and interstitial fibrosis were absent on day 8. Original magnification × 400, Goldner's trichrome–stained kidney longitudinal sections.View Large Image Figure ViewerDownload (PPT) As compared with controls, the semiquantitative score of TI injury obtained in AA-treated mice revealed tubular necrosis from day 4 to 8, with an evident peak at day 5 (Figure 5a), lymphocytic infiltration from day 5 (Figure 5b), and marked tubular atrophy at day 5 accompanied by progressive interstitial fibrosis (Figure 5c and d, respectively). In the AA+PBN group, a significant reduction of all the semiquantitative scores was found: of tubular necrosis on days 5 (P<0.0013) and 8 (P<0.0025), of lymphocytic infiltrate (P<0.0013), of tubular atrophy (P<0.0018) (day 8), as well as of interstitial fibrosis, on days 5 (P<0.0022) and 8 (P<0.0013) (Figure 5a–d). To further assess the distribution of necrotic tubules, an immunostaining of NEP was performed and evaluated. NEP is a specific marker for the brush border of S3 segment of the proximal tubule in rats.21.Ronco P. Pollard H. Galceran M. et al.Distribution of enkephalinase (membrane metalloendopeptidase, E.C. 3.4.24.11) in rat organs. Detection using a monoclonal antibody.Lab Invest. 1988; 58: 210-217PubMed Google Scholar As shown in Figure 6, immunostaining of NEP in control groups demonstrated that NEP-positive cells were mainly located in medullary rays and in the outer stripe of the outer medulla, reproducing the typical distribution of NEP-positive cells in pars recta of proximal tubule observed previously in our rat model.11.Pozdzik A.A. Salmon I.J. Husson C.P. et al.Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy.Nephrol Dial Transplant. 2008; 23: 2480-2491Crossref PubMed Scopus (66) Google Scholar,22.Lebeau C. Debelle F.D. Arlt V.M. et al.Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.Nephrol Dial Transplant. 2005; 20: 2321-2332Crossref PubMed Scopus (112) Google Scholar No disappearance of NEP immunostaining was observed in the PEG or PEG+PBN groups. AA administration led to a progressive necrosis of PTECs, especially NEP positive, as suggested by the progressive disappearance of NEP staining in the medullary rays and the presence of intratubular necrotic NEP-positive cells on day 5 (Figure 6c). At day 8, NEP-positive cells had completely disappeared from medullary rays (Figure 6d). On the contrary, NEP immunostaining was maintained in the AA+PBN group (Figure 6e and f). As PBN administration was effective in significantly preventing acute kidney injury and TI lesions induced by AA, we examined the possible effect of PBN on AA-DNA adduct formation in kidney cortex tissue samples. As shown in Figure 7a, the pattern of AA-DNA adducts 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). This pattern is identical to those observed previously in our rat model and in AA-exposed patients.6.Nortier J.L. Martinez M.C. Schmeiser H.H. et al.Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi).N Engl J Med. 2000; 342: 1686-1692Crossref PubMed Scopus (925) Google Scholar,22.Lebeau C. Debelle F.D. Arlt V.M. et al.Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.Nephrol Dial Transplant. 2005; 20: 2321-2332Crossref PubMed Scopus (112) Google Scholar In the AA+PBN group, as compared with the AA group, there were no significant changes in AA-DNA adducts at days 2, 4, and 5, whereas a significant reduction of the total AA-DNA adduct levels was observed at day 8 (Figure 7b). There was no correlation between the PCr levels or the TI scores and AA-DNA adduct formation (data not shown). Previous in vivo studies showed that DNA adduct formation by AA reaches a steady-state level, which is likely the result of a balance between adduct formation and their loss through either DNA-repair processes or apoptosis.22.Lebeau C. Debelle F.D. Arlt V.M. et al.Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.Nephrol Dial Transplant. 2005; 20: 2321-2332Crossref PubMed Scopus (112) Google Scholar,23.Arlt V.M. Zuo J. Trenz K. et al.Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice.Int J Cancer. 2011; 128: 21-32Crossref PubMed Scopus (28) Google Scholar Moreover, this level seems to be reached quickly, even 2 days after the first injection in a rat model.22.Lebeau C. Debelle F.D. Arlt V.M. et al.Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.Nephrol Dial Transplant. 2005; 20: 2321-2332Crossref PubMed Scopus (112) Google Scholar This could explain the only small differences in DNA adduct levels between the two groups after 8 days of treatment. Therefore, we conducted an additional experiment to investigate the early time course and kinetics of AA-DNA adduct formation. Thirty-two mice were injected with AA or AA+PBN as described previously, and four mice per group were killed after 6, 12, 18, and 24h. At these time points, differences in AA-specific DNA adduct levels between the two groups were clearly observed (Figure 7a), confirming that PBN significantly inhibits AA-DNA adduct formation. PCNA is a polymerase cofactor, involved in DNA-damage-repair processes and in the stability of the DNA microsatellite region.24.Moldovan G.L. Pfander B. Jentsch S. PCNA, the maestro of the replication fork.Cell. 2007; 129: 665-679Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar Only few tubular cells expressed PCNA in controls (PEG and PEG+PBN groups) (Figure 8a–h). In AA-treated mice, typical nuclear patterns of PCNA immunostaining were predominantly seen in PTECs and less frequently in interstitial cells from the corticomedullary junction as soon as day 2 and still increased until day 8 (Figure 8i–l). PBN administration resulted in a reduction of PCNA expression induced by AA in mice as soon as day 4 (Figure 8m–p). As compared with controls, the proportion of PCNA positively stained areas per field was higher in AA-treated mice from day 5 (P<0.0043) to day 8 (P<0.0152) (Figure 8q). This proportion was significantly decreased in the AA+PBN group at day 5 (P<0.0043) and day 8 (P<0.0022) (Figure 8q). Control groups (PEG and PEG+PBN) exhibited only mild mitochondrial swelling in few PTECs at day 8 (Figure 9a and b). In kidneys from AA-treated mice, considerable variation in the degree of cellular damage may occur. Normal tubules were frequently admixed with injured nephron showing extensive mitochondria disruption and altered brush borders (Figure 9c and e). PBN administration reduced both the extent and the severity of cellular damage induced by AA (Figure 9d and f). Since the cluster outbreak of the so-called Chinese herbs nephropathy in 1993, AAN is now recognized as a public health problem worldwide:25.Debelle F.D. Vanherweghem J.L. Nortier J.L. Aristolochic acid nephropathy: a worldwide problem.Kidney Int. 2008; 74: 158-169Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar it is identified as an environmental kidney disease in the Balkan region and probably underestimated in Asian countries where traditional Chinese medicine is widely used, as suggested by two recent studies.26.Yang L. Su T. Li X.M. et al.Aristolochic acid nephropathy: variation in presentation and prognosis.Nephrol Dial Transplant. 2012; 27: 292-298Crossref PubMed Scopus (77) Google Scholar,27.Chen C.H. Dickman K.G. Moriya M. et al.Aristolochic acid-associated urothelial cancer in Taiwan.Proc Natl Acad Sci USA. 2012; 109: 8241-8246Crossref PubMed Scopus (307) Google Scholar Understanding its physiopathology may lead to effective therapies, preventing the progression of chronic kidney disease. In the present study, we reproduced histopathological features of human AAN (tubular necrosis, inflammatory interstitial infiltrate, tubular atrophy, and interstitial fibrosis) in a short-term mouse model. Male C57BL/6 mice were injected daily with a mixture of AAI and AAII, the same as the one present in Aristolochia sp. and ingested by our patients. After 4 days of injection, a massive necrosis of PTECs from the medullary rays was observed, resulting in an acute kidney injury on day 5. This ‘acute’ phase was followed by a prominent atrophy and fibrosis on day 8. The normalization of creatinine on day 8 is consistent with our observations of the acute phase in the AAN rat model, in which a transient creatinine increase on day 5 was followed by a normalization of creatinine on day 8, contrasting with persistent histological lesions.22.Lebeau C. Debelle F.D. Arlt V.M. et al.Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.Nephrol Dial Transplant. 2005; 20: 2321-2332Crossref PubMed Scopus (112) Google Scholar In addition, such dissociation between PCr and histology during the recovery phase has been described in other models of acute kidney injury such as ischemia and reperfusion.28.Kwon O. Wang W.W. Miller S. Renal organic anion transporter 1 is maldistributed and diminishes in proximal tubule cells but increases in vasculature after ischemia and reperfusion.Am J Physiol Renal Physiol. 2008; 295: F1807-F1816Crossref PubMed Scopus (12) Google Scholar The addition of PBN prevented acute kidney injury and significantly reduced tubular necrosis, lymphocytic infiltrate, atrophy, and fibrosis. Moreover, immunohistochemical study using PCNA staining confirmed the protective effect of PBN from AA. Increase in PCNA staining reflects a proliferation process of PTECs secondary to necrosis, which is in accordance with previous histological findings obtained in our rat model using Ki67 immunostaining.12.Pozdzik A.A. Salmon I.J. Debelle F.D. et al.Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation.Kidney Int. 2008; 73: 595-607Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Finally, a reduction of AA-DNA adduct formation was found in mice receiving AA+PBN as compared with mice treated with AA alone. Recently, two in vitro studies demonstrated that PBN inhibits AA entry in human OAT-transfected HEK293 kidney cell lines19.Bakhiya N. Arlt V.M. Bahn A. et al.Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy.Toxicology. 2009; 264: 74-79Crossref PubMed Scopus (62) Google Scholar and human OAT-transfected cell lines derived from the second portion of the proximal tubule.18.Babu E. Takeda M. Nishida R. et al.Interactions of human organic anion transporters with aristolochic acids.J Pharmacol Sci. 2010; 113: 192-196Crossref PubMed Scopus (26) Google Scholar Further, the former study indicated that PBN can reduce AA-DNA adduct formation and that addition of PBN to AA preserved cellular viability. The present work brings significant in vivo results confirming the protective effects of PBN against AA-induced TI lesions by blocking AA entry into PTECs via OATs. Actually, these histomorphometric data can be related to a recent pharmacological study, focusing on the effects of PBN on AA liver and kidney metabolism.29.Xue X. Gong L.K. Maeda K. et al.Critical role of organic anion transporters 1 and 3 in kidney accumulation and toxicity of aristolochic acid I.Mol Pharm. 2011; 8: 2183-2192Crossref PubMed Scopus (59) Google Scholar These authors reported a significantly reduced accumulation of renal AAI in mice exposed to AA and PBN, as well as an increase in AAI liver content and biliary clearance. With regard to the evident protective effect of PBN in terms of TI AA-induced lesions, it could be surprising to measure a only slight difference of AA-DNA adduct level between the AA and AA+PBN groups. However, this discrepancy could be easily interpreted. First of all, in the AA+PBN group, AA may enter PTECs independently of OAT. Other endogenous transmembrane transporters or passive diffusion may also be involved in the uptake of AA, as suggested by a only partial blockade of AA entry by PBN in an in vitro model of human OAT-transfected HEK293 cells.19.Bakhiya N. Arlt V.M. Bahn A. et al.Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy.Toxicology. 2009; 264: 74-79Crossref PubMed Scopus (62) Google Scholar Second, DNA adduct formation by AA often quickly reaches a steady-state level as seen in previous in vivo studies and our present results. In addition, our data suggest that although AA-DNA adducts are a clue biomarker of AA exposure, there is no correlation between AA-specific DNA adduct levels and nephrotoxicity. Dissociation between AA-mediated nephrotoxicity and adduct formation was first suggested by a clinical case report of AA-induced tumor development without renal impairment.30.Nortier J.L. Schmeiser H.H. Muniz Martinez M.C. et al.Invasive urothelial carcinoma after exposure to Chinese herbal medicine containing aristolochic acid may occur without severe renal failure.Nephrol Dial Transplant. 2003; 18: 426-428Crossref PubMed Scopus (70) Google Scholar This observation was followed by two rodent studies showing that AA-DNA adducts were the basis for the carcinogenic effect of AA but were unrelated to nephrotoxic insult. Indeed, both AAI and AAII could cause similar types of DNA damage (i.e., bulky DNA adducts), whereas only AAI is nephrotoxic in vivo.9.Sato N. Takahashi D. Chen S.M. et al.Acute nephrotoxicity of aristolochic acids in mice.J Pharm Pharmacol. 2004; 56: 221-229Crossref PubMed Scopus (113) Google Scholar,10.Shibutani S. Dong H. Suzuki N. et al.Selective toxicity of aristolochic acids I and II.Drug Metab Dispos. 2007; 35: 1217-1222Crossref PubMed Scopus (123) Google Scholar In vitro studies confirmed that AAI is much more cytotoxic compared with AAII because of the presence of a methoxy group in position 8.31.Dickman K.G. Sweet D.H. Bonala R. et al.Physiological and molecular characterization of aristolochic acid transport by the kidney.J Pharmacol Exp Ther. 2011; 338: 588-597Crossref PubMed Scopus (54) Google Scholar,32.Balachandran P. Wei F. Lin R.C. et al.Structure activity relationships of aristolochic acid analogues: toxicity in cultured renal epithelial cells.Kidney Int. 2005; 67: 1797-1805Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar On the other hand, the carboxyl group rather than the nitro group is important to facilitate AA entry into tubular cells via OATs. Finally, nitroreduction results in N-hydroxy-aristolactam formation, and these metabolites bind covalently to the exocyclic amino groups of adenine or guanine forming AA-specific DNA adducts.33.Stiborova M. Frei E. Schmeiser H.H. Biotransformation enzymes in development of renal injury and urothelial cancer caused by aristolochic acid.Kidney Int. 2008; 73: 1209-1211Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,34.Schmeiser H.H. Stiborova M. Arlt V.M. Chemical and molecular basis of the carcinogenicity of aristolochia plants.Curr Opin Drug Discov Devel. 2009; 12: 141-148PubMed Google Scholar On the other hand, AAI seems to directly cause renal injury by activating mitochondrial permeability transition,35.Qi X. Cai Y. Gong L. et al.Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid.Toxicol Appl Pharmacol. 2007; 222: 105-110Crossref PubMed Scopus (61) Google Scholar and reticulum endoplasmic stress.36.Hsin Y.H. Cheng C.H. Tzen J.T. et al.Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells.Apoptosis. 2006; 11: 2167-2177Crossref PubMed Scopus (66) Google Scholar Further, AA is responsible for increased oxidative stress–related DNA lesions due to glutathione depletions,37.Yu F.Y. Wu T.S. Chen T.W. et al.Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells.Toxicol In Vitro. 2011; 25: 810-816Crossref PubMed Scopus (56) Google Scholar and AA can block DNA replication causing cycle arrest and/or apoptosis in renal epithelial cells in vitro and in vivo.38.Li Y. Liu Z. Guo X. et al.Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro.Arch Toxicol. 2006; 80: 524-532Crossref PubMed Scopus (58) Google Scholar, 39.Chang H.R. Lian J.D. Lo C.W. et al.Aristolochic acid-induced cell cycle G1 arrest in human urothelium SV-HUC-1 cells.Food Chem Toxicol. 2007; 45: 396-402Crossref PubMed Scopus (21) Google Scholar, 40.Yang L. Besschetnova T.Y. Brooks C.R. et al.Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.Nat Med. 2010; 16 (531p following 143): 535-543Crossref PubMed Scopus (906) Google Scholar In conclusion, we developed an in vivo model of AAN characterized by an early episode of acute kidney injury induced by daily injections of AA. Despite highly nephrotoxic effects of AA, we were able to demonstrate a sustained protective effect of PBN by blocking AA entry into PTECs and then preventing acute tubular necrosis. All procedures were in accordance with the Ethical Committee for Animal Care (Faculty of Medicine, Université Libre de Bruxelles). After 1 week of acclimatization, 10-week-old C57BL/6 male mice, n=96 (Elevage Janvier, Le Genest Saint-Isle, France), were randomly assigned to four groups of 24 mice each and were injected intraperitoneally with solvent (PEG group) alone, PEG+PBN (4-((dipropylamino)sulfonyl)benzoic acid) (PEG group), AA (AA group), or AA+PBN (AA+PBN group). AA (Acros Organics, Geel, Belgium; 40% AAI and 60% AAII) was dissolved in PEG (Fluka Chemie, Buchs, Switzerland). PBN (Sigma-Aldrich, Bornem, Belgium) was solubilized in 0.5mol/l NaOH at 45°C for 10min and then diluted with phosphate-buffered saline and buffered to pH 7.4 with HCl. AA (5mg/kg body weight) or an equivalent volume of PEG was given once a day and PBN (150mg/kg body weight) twice a day. AA was given once a day intraperitoneally in 150μl of solvent, and PBN (150mg/kg body weight) was injected with 150μl of phosphate-buffered saline twice a day for a total of 8 days maximum. After 2, 4, 5, or 8 days of injection, six mice per group were killed. After intraperitoneal anesthesia with ketamine-HCl (Merial, Brussels, Belgium) and 2% xylazine (Bayer, Brussels, Belgium), a blood specimen was obtained by cardiac puncture and kidneys were harvested for analysis. Different samples of kidneys were fixed: one part in alcohol–formalin–acetic acid for optical microscopy, one in 4% buffered formaldehyde for immunohistochemistry, one in glutaraldehyde sodium cacodylate buffer for electron microscopy analysis, and one frozen in liquid nitrogen and stored at -80°C for subsequent DNA adduct analysis. TI injury semiquantification was evaluated on hematoxylin/eosin and Masson's trichrome–stained paraffin-embedded sections. Complete kidney sections were analyzed with a light microscope (Carl Zeiss, Oberkochen, Germany) using a × 20 magnification lens by two investigators (AAP and TB) blinded to the group origin of the mice. The scoring systems were defined as previously described:11.Pozdzik A.A. Salmon I.J. Husson C.P. et al.Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy.Nephrol Dial Transplant. 2008; 23: 2480-2491Crossref PubMed Scopus (66) Google Scholar,12.Pozdzik A.A. Salmon I.J. Debelle F.D. et al.Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation.Kidney Int. 2008; 73: 595-607Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar tubular necrosis: 0, normal tubules; 1, rare single necrotic tubule; 2, several clusters of necrotic tubules; 3, confluence of necrotic clusters; tubular atrophy: 0, normal tubules; 1, rare single atrophic tubule; 2, several clusters of atrophic tubules; 3, confluence of atrophic tubular clusters; lymphocytic infiltrate: 0, absent; 1, few scattered cells; 2, group of lymphocytes; 3, widespread infiltrate; interstitial fibrosis: 0, absent; 1, minimal fibrosis; 2, moderate fibrosis; 3, severe fibrosis. If differences in grading occurred, the appropriate sections were reexamined until a consensus was obtained. PCr excretion levels were determined as previously described using an high-performance liquid chromatography technique.11.Pozdzik A.A. Salmon I.J. Husson C.P. et al.Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy.Nephrol Dial Transplant. 2008; 23: 2480-2491Crossref PubMed Scopus (66) Google Scholar,12.Pozdzik A.A. Salmon I.J. Debelle F.D. et al.Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation.Kidney Int. 2008; 73: 595-607Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar The formalin fixed paraffin embedded sections (4μm) were attached to poly-L-lysine-pretreated slides (Sigma-Aldrich). After air-drying the paraffin from FFPE, tissue sections were removed (xylene solution). The sections were rehydrated and immersed in a retrieval solution, sodium citrate buffer (pH 6.0); the microwave oven technique was used (650W, 1 × 5min). Phosphate-buffered saline was used for all washing steps. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in a methanol solution (30min). Nonspecific protein-binding sites (background staining due to the Fc receptor) were blocked with 20% normal serum (Vectastain Elite ABC kit IgG; Vector Laboratories, Labconsult, Brussels, Belgium) and then with avidin D and biotin solution (avidin/biotin blocking kit; Vector Laboratories, Labconsult). Subsequently, the sections were incubated overnight with rabbit anti-mouse PCNA (1/4000) monoclonal primary antibody (Abcam, Cambridge, England, UK, ab2426) or with rat monoclonal antibody anti-NEP (1/4000) (Santa Cruz Biotechnology, Boechout, Belgium, sc-80021) and diluted in the blocking buffer. Slides were then incubated with specific biotinylated secondary antibody (Vectastain Elite ABC kit; Vector Laboratories, Labconsult). The extent of the specifically bound primary antibodies was visualized by means of the avidin–biotin peroxidase complex method. The diaminobenzidine/hydrogen peroxide was used as the chromogene substrate, producing a brown end product. Counterstaining with hematoxylin completed the processing. The specificity of antibodies used was established by the producer. Normal serum (5% solution) instead of the primary antibody (used in order to exclude nonspecific staining of kit reagents) showed no staining. Quantifications were performed by one investigator (TB) blinded to the group origin of the mice using ImageJ, a public domain Java image processing program (US NIH) software (available at http://rsb.info.nih.gov/ij), as detailed in Figure 10. Thresholding conditions were set identically for all images. Finally, the percentage of 3,3′-diaminobenzidine-positive surface corresponding to 3,3′-diaminobenzidine-positive cells was counted using the ImageJ analyse particle command. Analysis of cellular ultrastructure using transmission electron microscopy was performed in the same period. Small pieces of renal tissue were fixed in 3% glutaraldehyde in 0.1mol/l phosphate buffer, pH 7.2. Fixation was performed with the microwave oven technique. After rinsing, samples were postfixed in 1% osmium tetroxide in 0.1mol/l phosphate buffer for 1h at 4°C, processed through a graded acetone series, embedded in Araldite (TAAB Laboratories, Aldermaston, England, UK), and polymerized overnight at 60°C. Sections (50nm) were then stained with uranyl acetate and lead citrate and examined with a 10-10 JEOL electron microscope (JEOL, Tokyo, Japan). DNA was extracted from frozen tissues using a standard phenol–chloroform extraction method. 32P-postlabeling analysis,41.Phillips D.H. Arlt V.M. The 32P-postlabeling assay for DNA adducts.Nat Protoc. 2007; 2: 2772-2781Crossref PubMed Scopus (205) Google Scholar nuclease P1 enrichment, chromatography on polyethyleneimine-cellulose thin-layer plates (Macherey and Nagel, Düren, Germany), autoradiography using a Packard Instant Imager (Canberra, Downers Grove, IL), and quantification were essentially performed as described.42.Arlt V.M. Zuo J. Trenz K. et al.Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice.Int J Cancer. 2011; 128: 21-32Crossref PubMed Scopus (48) Google Scholar Results were expressed as DNA adducts per 108 normal nucleotides. All the scores and data obtained from AA and control groups were compared for each corresponding time point with the Kruskall–Wallis test followed by Mann–Whitney U-test and Bonferroni post-hoc test. Part of the study was supported by Cancer Research, UK, and the Fonds Erasme pour la Recherche Médicale, Brussels, Belgium. The authors are deeply grateful to J.L. Vanherweghem for fruitful discussions in each step of this work.