The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro.

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(-/-) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography-mass spectrometry (GC-MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(-/-) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(-/-) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.

Severe myoglobinuric acute kidney injury in a kidney recipient: rapid recovery after hemodialysis with the super high-flux membrane Theralite®â€©.

Myoglobinuric acute kidney injury (AKI) is a severe condition requiring early therapeutic strategies. Early recognition and treatment are crucial to reduce morbidity and mortality rate. Here, we report a kidney recipient with severe rhabdomyolysis and AKI secondary to parvovirus B19 infection. Initiation of hemodialysis with the super high-flux filter Theralite® (Gambro, cut-off 45 kDa, 2.1 m2) resulted in the clearance of myoglobin from 61 to 71% after 3 hours. Elimination rates of IL-6 and ß2-microglobulin were ~ 30 - 64% and 55 - 71% after 3 hours, respectively. Renal graft function rapidly recovered. The place of this effective but expensive procedure still needs to be defined and validated in high-risk patients.
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Bacillus Calmette-Guerin therapy in non-muscle-invasive bladder carcinoma after renal transplantation for end-stage aristolochic acid nephropathy.

Intravesical instillation of bacillus Calmette-Guerin (BCG) is the treatment of choice for non-muscle-invasive bladder cancer (NMIBC) of high grade and/or carcinoma in situ. This study evaluated the feasibility, efficacy, and tolerance of BCG instillations in eight kidney recipients for end-stage aristolochic acid nephropathy (AAN), a condition at high risk of urothelial carcinoma, and diagnosed for NMIBC. Five of them had relapsed after mitomycin C treatment. Tolerance to BCG was evaluated clinically and regular follow-up with fluorescence cystoscopy was performed along with renal graft function monitoring. Immunosuppression doses were adjusted and prophylactic anti-tuberculous treatment given to reduce risks of graft rejection and infection. After a mean follow-up period of 50 months, seven of the eight patients are free of relapse and kidney graft function remained unchanged. Tolerance was good, except for one episode of fever and one early discontinuation because of subjective discomfort. No systemic tuberculous infection was observed. This is the first clinical observation of successful BCG therapy for NMIBC in patients given transplant for end-stage AAN. Under standardized conditions, immunotherapy based on intravesical BCG is feasible, effective, and well tolerated in renal transplantation.

Human bone morphogenetic protein-7 does not counteract aristolochic acid-induced renal toxicity.

Aristolochic acids (AA) are nephrotoxic and profibrotic agents, leading to chronic kidney disease. As some controversial studies have reported a nephroprotective effect of exogenous recombinant human bone morphogenetic protein (rhBMP)-7 in several models of renal fibrosis, we investigated the putative effect of rhBMP-7 to prevent progressive tubulointerstitial damage after AA intoxication in vitro and in vivo. In vitro, the toxicity of AA on renal tubular cells was demonstrated by an increase in vimentin as well as a decrease in ß-catenin expressions, reflecting a dedifferentiation process. Increased fibronectin and interleukin-6 levels were measured in the supernatants. Enhanced α-SMA mRNA levels associated to decreased E-cadherin mRNA levels were also measured. Incubation with rhBMP-7 only prevented the increase in vimentin and the decrease in ß-catenin expressions. In vivo, in a rat model of AA nephropathy, severe tubulointerstitial lesions induced by AA after 10 and 35 days (collagen IV deposition and tubular atrophy), were not prevented by the rhBMP-7 treatment. Similarly, rhBMP-7 did not ameliorate the significant increase in urinary concentrations of transforming growth factor-ß. In summary, our in vitro data demonstrated a poor beneficial effect of rhBMP-7 to reverse cell toxicity while, in vivo, there was no beneficial effect of rhBMP-7. Therefore, further investigations are needed to confirm the exact role of BMP-7 in progressive chronic kidney disease.

Exceptionally long-term persistence of DNA adducts formed by carcinogenic aristolochic acid I in renal tissue from patients with aristolochic acid nephropathy.

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA-AAI adduct induces AT to TA transversions in the tumor-suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin-layer chromatography 32P-postlabeling and mass spectrometric analysis we report the detection of dA-AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA-AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA-AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA-associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long-term persistence of dA-AAI provides a useful long-term biomarker of exposure and attests to the role of AA in human urothelial malignancy.

Regulation of lipid accumulation by AMP-activated kinase [corrected] in high fat diet-induced kidney injury.

AMP-activated protein kinase (AMPK) is an important energy sensor that may be critical in regulating renal lipid accumulation. To evaluate the role of AMPK in mediating renal lipid accumulation, C57BL/6J mice were randomized to a standard diet, a high-fat diet, or a high-fat diet plus AICAR (an AMPK activator) for 14 weeks. Renal functional and structural studies along with electron microscopy were performed. Mice given the high-fat diet had proximal tubule injury with the presence of enlarged clear vacuoles, and multilaminar inclusions concurrent with an increase of tissue lipid and overloading of the lysosomal system. The margins of the clear vacuoles were positive for the endolysosomal marker, LAMP1, suggesting lysosome accumulation. Characterization of vesicles by special stains (Oil Red O, Nile Red, Luxol Fast Blue) and by electron microscopy showed they contained onion skin-like accumulations consistent with phospholipids. Moreover, cholesteryl esters and phosphatidylcholine-containing phospholipids were significantly increased in the kidneys of mice on a high-fat diet. AMPK activation with chronic AICAR treatment prevented the clinical and structural effects of high-fat diet. Thus, high-fat diet contributes to a dysfunction of the lysosomal system and altered lipid metabolism characterized by cholesterol and phospholipid accumulation in the kidney. AMPK activation normalizes the changes in renal lipid content despite chronic exposure to lipid challenge.

Results of the HepZero study comparing heparin-grafted membrane and standard care show that heparin-grafted dialyzer is safe and easy to use for heparin-free dialysis.

Heparin is used to prevent clotting during hemodialysis, but heparin-free hemodialysis is sometimes needed to decrease the risk of bleeding. The HepZero study is a randomized, multicenter international controlled open-label trial comparing no-heparin hemodialysis strategies designed to assess non-inferiority of a heparin grafted dialyzer (NCT01318486). A total of 251 maintenance hemodialysis patients at increased risk of hemorrhage were randomly allocated for up to three heparin-free hemodialysis sessions using a heparin-grafted dialyzer or the center standard-of-care consisting of regular saline flushes or pre-dilution. The first heparin-free hemodialysis session was considered successful when there was neither complete occlusion of air traps or dialyzer, nor additional saline flushes, changes of dialyzer or bloodlines, or premature termination. The current standard-of-care resulted in high failure rates (50%). The success rate in the heparin-grafted membrane arm was significantly higher than in the control group (68.5% versus 50.4%), which was consistent for both standard-of-care modalities. The absolute difference between the heparin-grafted membrane and the controls was 18.2%, with a lower bound of the 90% confidence interval equal to plus 7.9%. The hypothesis of the non-inferiority at the minus 15% level was accepted, although superiority at the plus 15% level was not reached. Thus, use of a heparin-grafted membrane is a safe, helpful, and easy-to-use method for heparin-free hemodialysis in patients at increased risk of hemorrhage.

The epidemiology, diagnosis, and management of aristolochic acid nephropathy: a narrative review.

It has been 20 years since the first description of a rapidly progressive renal disease that is associated with the consumption of Chinese herbs containing aristolochic acid (AA) and is now termed aristolochic acid nephropathy (AAN). Recent data have shown that AA is also the primary causative agent in Balkan endemic nephropathy and associated urothelial cancer. Aristolochic acid nephropathy is associated with a high long-term risk for renal failure and urothelial cancer, and the potential worldwide population exposure is enormous. This evidence-based review of the diagnostic approach to and management of AAN draws on the authors' experience with the largest and longest-studied combined cohort of patients with this condition. It is hoped that a better understanding of the importance of this underrecognized and severe condition will improve epidemiologic, preventive, and therapeutic strategies to reduce the global burden of this disease.

Prevention of tunneled cuffed hemodialysis catheter-related dysfunction and bacteremia by a neutral-valve closed-system connector: a single-center randomized controlled trial.

BACKGROUND: Hemodialysis (HD) tunneled cuffed catheters may be fitted with neutral-valve closed-system connectors. Such connectors, which are flushed with saline solution and used for 3 consecutive HD sessions, provide a mechanically closed positive-pressure barrier and potentially may be useful to prevent catheter-related bacteremia and dysfunction. STUDY DESIGN: Single-center randomized controlled trial. SETTING & PARTICIPANTS: 66 adult HD patients with a tunneled cuffed catheter. INTERVENTION: Neutral-valve closed-system connector (Tego Needlefree Hemodialysis Connector) versus trisodium citrate, 46.7%, locking solution (Citra-Lock; control group). OUTCOMES: Primary composite outcome was the incidence rate of catheter-related dysfunction or bacteremia. Secondary outcomes were the separate incidence rates of catheter-related dysfunction and bacteremia and the cost of both procedures. MEASUREMENTS: Catheter dysfunction was defined as the requirement of urokinase and/or a mean blood flow ≤250 mL/min during 2 consecutive HD sessions. Catheter-related bacteremia was defined as ≥2 positive blood cultures. Time of catheter use was calculated and the incidence rate of complications was expressed per 100 person-years. RESULTS: 66 patients were followed up for a median of 86 (IQR, 29-200) days. The composite primary outcome was not significantly reduced in the closed-system-connector intervention group versus the citrate-locking-solution control group (63.56 vs 71.51 per 100 person-years; P = 0.3). Catheter dysfunction in the intervention group was not decreased versus controls (59.59 vs 51.64 per 100-person-years; P = 0.9). Only 6 catheter-related bacteremia events were identified, one in the intervention group (3.97 vs 19.86 per 100 person-years; P = 0.06). LIMITATIONS: Small size of the patient population and single-center study. CONCLUSIONS: Superiority of the closed-system connector in terms of prevention of the primary efficacy end point compared to the standard locking solution was not observed. Further evaluation in a larger study is suggested.

Probenecid prevents acute tubular necrosis 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.

Experimental Aristolochic Acid Nephropathy: A Relevant Model to Study AKI-to-CKD Transition.

Aristolochic acid nephropathy (AAN) is a progressive tubulointerstitial nephritis caused by the intake of aristolochic acids (AA) contained in Chinese herbal remedies or contaminated food. AAN is characterized by tubular atrophy and interstitial fibrosis, characterizing advanced kidney disease. It is established that sustained or recurrent acute kidney injury (AKI) episodes contribute to the progression of CKD. Therefore, the study of underlying mechanisms of AA-induced nephrotoxicity could be useful in understanding the complex AKI-to-CKD transition. We developed a translational approach of AKI-to-CKD transition by reproducing human AAN in rodent models. Indeed, in such models, an early phase of acute tubular necrosis was rapidly followed by a massive interstitial recruitment of activated monocytes/macrophages followed by cytotoxic T lymphocytes, resulting in a transient AKI episode. A later chronic phase was then observed with progressive tubular atrophy related to dedifferentiation and necrosis of tubular epithelial cells. The accumulation of vimentin and αSMA-positive cells expressing TGFß in interstitial areas suggested an increase in resident fibroblasts and their activation into myofibroblasts resulting in collagen deposition and CKD. In addition, we identified 4 major actors in the AKI-to-CKD transition: (1) the tubular epithelial cells, (2) the endothelial cells of the interstitial capillary network, (3) the inflammatory infiltrate, and (4) the myofibroblasts. This review provides the most comprehensive and informative data we were able to collect and examines the pending questions.

Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice.

Aristolochic acid (AA) is the causative agent of urothelial tumors associated with AA nephropathy and is also implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. These tumors contain AA-characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock-in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and nontarget (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI-induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nfκb, aryl hydrocarbon receptor, Tp53 and cell cycle signaling as the most important pathways modulated in kidney. Expression of Nfκb1 and other Nfκb-target genes was confirmed by quantitative real-time PCR (qRT-PCR) and was consistent with the induction of Nfκb1 protein. Myc oncogene, frequently overexpressed in urothelial tumors, was upregulated by AAI on the microarrays and confirmed by qRT-PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue-specific responses in AAI-induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies.

Aristolochic acid nephropathy revisited: a place for innate and adaptive immunity?

AIMS: The histological features of aristolochic acid nephropathy (AAN) consist of paucicellular interstitial fibrosis, severe tubular atrophy, and almost intact glomeruli with media lesions of interlobular arteries. As an early phase of interstitial inflammation preceded peritubular fibrosis in the rat model of AAN, the aim was to investigate the presence of inflammatory cells in human AAN. METHODS AND RESULTS: Reports of confirmed cases and case series of AAN were reviewed in terms of interstitial inflammation and found to have very conflicting results. This prompted us to search for and characterize inflammatory cells within the native kidneys provided from four end-stage AAN patients. Prior aristolochic acid exposure was attested by the intrarenal presence of the typical aristolactam I-derived DNA adduct. Besides the tubulointerstitial lesions usually seen in the cortex, a massive infiltration of macrophages, T and B lymphocytes was detected by immunohistochemistry in the medullary rays and in the outer medullae with some extension to the upper cortical labyrinth. CONCLUSIONS: In parallel with histological findings reported in the rat model, inflammatory cells are present preferentially in the interstitium of the medullary rays and of the outer medulllae in renal interstitium from human AAN cases, even in the terminal stages. Further studies must be undertaken to determine the respective roles of innate and adaptive immunity in the progression of AAN.

Revisiting nephrogenic systemic fibrosis in 6 kidney transplant recipients: a single-center experience.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) is a fibrotic disorder occurring in patients with renal dysfunction. Exposure to gadolinium (Gd)-based contrast agents (GBCAs) during renal impairment is associated with development of NSF. METHODS: A cross-referenced search of kidney transplantation and radiology databases at a single institution revealed the prevalence of NSF in the transplant population. Clinical records and skin biopsy specimens from 6 patients with kidney transplant given a diagnosis of NSF were reviewed to identify contributing factors. RESULTS: Between January 1999 and December 2006, NSF was diagnosed in 6 of 705 patients with kidney transplant (0.9%). Renal function was impaired in all patients. Of 33 patients with kidney transplant exposed to GBCAs, 5 (15.2%) developed NSF. Disease onset ranged from 7 days to 11 months after exposure to GBCAs. All 5 patients exposed to GBCAs who developed NSF were also treated with a beta-blocker and clinical improvement was observed with discontinuation. The sixth case NSF appeared unrelated to Gd, without a known exposure, and testing of tissue via mass spectrometry revealed no Gd. Symptoms of NSF in this patient disappeared after administration of darbepoetin was switched from subcutaneous to intravenous injection. One patient with NSF who manifested the highest Gd level in tissue died 22 months after disease onset. LIMITATIONS: The study represents the retrospective experience of only a single center. CONCLUSIONS: NSF can develop in kidney transplant recipients with altered graft function. In these patients, exposure to GBCAs appears associated with development of NSF. The role of beta-blockers in the course of the disease merits further investigation.

Balkan Endemic Nephropathy and the Causative Role of Aristolochic Acid.

Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA) contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion) in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies.

Aristolochic acid nephropathy: a worldwide problem.

Aristolochic acid nephropathy (AAN), a progressive renal interstitial fibrosis frequently associated with urothelial malignancies, was initially reported in a Belgian cohort of more than 100 patients after the intake of slimming pills containing a Chinese herb, Aristolochia fangchi. Although botanicals known or suspected to contain aristolochic acid (AA) were no longer permitted in many countries, several AAN cases were regularly observed all around the world. The incidence of AAN is probably much higher than initially thought, especially in Asia and the Balkans. In Asian countries, where traditional medicines are very popular, the complexity of the pharmacopoeia represents a high risk for AAN because of the frequent substitution of the botanical products by AA-containing herbs. In the Balkan regions, the exposure to AA found in flour obtained from wheat contaminated with seeds of Aristolochia clematitis could be responsible for the so-called Balkan-endemic nephropathy. Finally, despite the Food and Drug Administration's warnings concerning the safety of botanical remedies containing AA, these herbs are still sold via the Internet.

Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy.

BACKGROUND: Interstitial inflammation is a prominent feature associated with the severity of renal injury and progressive kidney failure. We utilized an animal model of aristolochic acid (AA)-induced nephropathy (AAN) to assess patterns of infiltration and inflammation during the evolution of tubulointerstitial damage and to relate them to the development of fibrosis. METHODS: Male Wistar rats receiving sc daily AA or vehicle were sacrificed between Days 1 and 35. Infiltrating mononuclear cells were characterized by immunohistochemistry. The kidney infiltrating T lymphocytes were phenotyped by flow cytometry. Urinary levels of Th-1/ Th-2 cytokines, of monocyte chemoattractant protein-1 and of active transforming growth factor-beta (TGF-beta) were measured. Tissue expression of phosphorylated smad 2/3 protein was used to examine the TGF-beta signalling pathway. RESULTS: In AA rats, monocytes/macrophages and T lymphocytes predominantly infiltrated areas of necrotic proximal tubular cells. The coexpressions of ED1 and/or Ki-67/MHCII by infiltrating cells reflected monocyte/macrophage proliferation and their activation, respectively. The accumulation of cytotoxic T lymphocytes was attested by severe signs of CD8+ cell tubulitis. The CD8/E-cadherin costaining confirmed intrarenal homing of CD8+CD103+ cells. Urinary levels of proinflammatory cytokines and of active TGF-beta significantly increased at Days 10 and 35. An early and persistent nuclear overexpression of phosphorylated smad 2/3 protein was detected in tubular and interstitial compartments. CONCLUSION: An early and massive interstitial inflammation characterized by activated monocytes/macrophages and cytotoxic CD8+CD103+ T lymphocytes is demonstrated for the first time during the progression of experimental AAN. The involvement in an interstitial fibrosis onset of active TGF-beta is highly suggested, at least via the psmad 2/3 intracellular signalling pathway.

CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy.

Experimental aristolochic acid nephropathy is characterized by transient acute proximal tubule necrosis and inflammatory cell infiltrates followed by interstitial fibrosis and tubular atrophy. The respective role of T-cell subpopulations has never been studied in the acute phase of the mouse model, and was heretofore exclusively investigated by the use of several depletion protocols. As compared to mice injected with aristolochic acids alone, more severe acute kidney injury was observed after CD4+ or CD8+ T-cells depletion. TNF-alpha and MCP-1 mRNA renal expressions were also increased. In contrast, regulatory T-cells depletion did not modify the severity of the aristolochic acids induced acute kidney injury, suggesting an independent mechanism. Aristolochic acids nephropathy was also associated with an increased proportion of myeloid CD11bhighF4/80mid and a decreased proportion of their counterpart CD11blowF4/80high population. After CD4+ T-cell depletion the increase in the CD11bhighF4/80mid population was even higher whereas the decrease in the CD11blowF4/80high population was more marked after CD8+ T cells depletion. Our results suggest that CD4+ and CD8+ T-cells provide protection against AA-induced acute tubular necrosis. Interestingly, T-cell depletion was associated with an imbalance of the CD11bhighF4/80mid and CD11blowF4/80high populations.

Aristolochic acid mutagenesis: molecular clues to the aetiology of Balkan endemic nephropathy-associated urothelial cancer.

Balkan endemic nephropathy (BEN) is found in certain rural areas of the Balkans and affects at least 25,000 inhabitants. Of the many hypotheses on BEN, the Aristolochia hypothesis has recently gained ground substantiated by the investigations on aristolochic acid nephropathy (AAN). On both clinical and morphological grounds, AAN is very similar to BEN. That exposure to aristolochic acid (AA) of individuals living in endemic areas through consumption of bread made with flour contaminated with seeds of Aristolochia clematitis is responsible for BEN is an old hypothesis, but one which is fully consistent with the unique epidemiologic features of BEN. Here, we propose an approach to investigate AA-induced mutagenesis in BEN that can provide molecular clues to the aetiology of its associated urothelial cancer. The molecular mechanism of AA-induced carcinogenesis demonstrates a strong association between DNA adduct formation, mutation pattern and tumour development. A clear link between urothelial tumours, p53 mutations and AA exposure should emerge as more tumour DNA from BEN patients from different endemic areas becomes available for mutation analysis. We predict that the observed p53 mutation spectrum will be dominated by AT --> TA transversion mutations as has already been demonstrated in the human p53 gene of immortalized cells after exposure to AAI and urothelial tumours from BEN patients in Croatia. Moreover, the detection of AA-specific DNA adducts in renal tissue of a number of BEN patients and individuals living in areas endemic for BEN in Croatia provides new evidence that chronic exposure to AA is a risk factor for BEN and its associated cancer.