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.

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.

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.

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.

Early detection of acute cisplatin nephrotoxicity: interest of urinary monitoring of proximal tubular biomarkers.

BACKGROUND: Renal toxicity induced by cisplatin (CisPt) is a clinical issue in patients with or without chronic kidney disease (CKD). Proximal tubular injury can result in acute kidney injury (AKI), which may compromise the course of chemotherapy and the prognosis. The purpose of this study was to investigate the time course of urinary markers of acute tubulotoxicity and to assess the usefulness of such monitoring in a routine clinical setting. METHODS: This work is an open prospective pilot study carried out among 23 patients receiving a platinum-based chemotherapy. Individual comorbidities, plasma parameters of kidney function (urea, creatinine) and estimated glomerular filtration rate were registered. Urinary excretion of leucine aminopeptidase, neutrophil gelatinase-associated lipocalin, cystatin C, liver fatty acid-binding protein and interleukin-18 were monitored during successive chemotherapy cycles. Episodes of AKI were identified according to KDIGO (Kidney Disease Improving Global Outcomes) 2012 guidelines. RESULTS: A total of 28 patients were recruited; among them 23 agreed to be part of the study, of whom 18 received CisPt and 5 carbo- or oxaliplatin. Of the 18 CisPt patients, 12 had a preexisting CKD. Sixteen AKI episodes were observed in 13 patients receiving CisPt with a pejorative evolution in seven cases (partial recovery of the renal function); a transient but dramatic increase in urinary biomarkers was observed 3 h after chemotherapy initiation, whereas plasma creatinine rise appeared 72 h after the end of CisPt treatment. Identified precipitating factors included: dehydration due to lack of fluid intake or diuretic use, exposure to high CisPt doses, regular use of nonsteroidal anti-inflammatory drugs and/or iodinated contrast agents and sepsis. CONCLUSION: Even if numerous precipitating factors could be avoided, the monitoring of urinary markers seemed helpful for the early detection of subclinical AKI induced during CisPt chemotherapy.

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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Protective Effects of Pistacia lentiscus L. fruit extract against calcium oxalate monohydrate induced proximal tubular injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The world prevalence of kidney stones is increasing and plants are frequently used to treat urolithiasis. Pistacia lentiscus L, a plant which freely grows around the Mediterranean basin areas, is widely used for various pathologies. P. lentiscus has an important impact as it has economical value on top of its pharmacological interest. Decoctions of its aerial parts and/or resin are used to treat kidney stones. AIM OF THE STUDY: To in vitro assess the potential nephroprotective effect of Pistacia lentiscus ethanolic fruit extract (PLEF) on proximal tubular cells in response to the adhesion of calcium oxalate monohydrate (COM) crystals. MATERIALS AND METHODS: Human Kidney [HK]-2 cells were incubated with and without COM in the presence or absence of PLEF. Cell viability was measured by the resazurin assay. The expression of E-cadherin was analyzed by PCR. The extracellular production of H2O2 was measured by Amplex® Red H2O2 Assay. The numbers of detached or non-adherent COM crystals in the presence of PLEF were microscopically captured and counted using ImageJ software. The interaction of PLEF with COM and the effect of PLEF on crystal size were analyzed by flow cytometry. The spectrophotometric measurement of turbidity was performed for assessing the COM concentration. RESULTS: PLEF incubated with COM was able to increase the cell viability. The decrease of E-cadherin expression after incubation with COM was counteracted by PLEF. Overproduction of H2O2 induced by COM was also inhibited by PLEF. Observations using flow cytometry showed that interactions between PLEF and the COM crystals occurred. PLEF was also effective in reducing the particles size and in lowering COM concentration. CONCLUSION: Our data show that COM tubulotoxicity can be significantly reversed by PLEF -at least in part- via an inhibition of COM crystals adhesion onto the apical membrane. This early beneficial effect of PLEF needs to be further investigated as a useful strategy in nephrolithiasis prevention.

Blocking TGF-ß Signaling Pathway Preserves Mitochondrial Proteostasis and Reduces Early Activation of PDGFRß+ Pericytes in Aristolochic Acid Induced Acute Kidney Injury in Wistar Male Rats.

BACKGROUND: The platelet-derived growth factor receptor ß (PDGFRß)+ perivascular cell activation becomes increasingly recognized as a main source of scar-associated kidney myofibroblasts and recently emerged as a new cellular therapeutic target. AIMS: In this regard, we first confirmed the presence of PDGFRß+ perivascular cells in a human case of end-stage aristolochic acid nephropathy (AAN) and thereafter we focused on the early fibrosis events of transforming growth factor ß (TGFß) inhibition in a rat model of AAN. MATERIALS AND METHODS: Neutralizing anti-TGFß antibody (1D11) and its control isotype (13C4) were administered (5 mg/kg, i.p.) at Days -1, 0, 2 and 4; AA (15 mg/kg, sc) was injected daily. RESULTS: At Day 5, 1D11 significantly suppressed p-Smad2/3 signaling pathway improving renal function impairment, reduced the score of acute tubular necrosis, peritubular capillaritis, interstitial inflammation and neoangiogenesis. 1D11 markedly decreased interstitial edema, disruption of tubular basement membrane loss of brush border, cytoplasmic edema and organelle ultrastructure alterations (mitochondrial disruption and endoplasmic reticulum edema) in proximal tubular epithelial cells. Moreover, 1D11 significantly inhibited p-PERK activation and attenuated dysregulation of unfolded protein response (UPR) pathways, endoplasmic reticulum and mitochondrial proteostasis in vivo and in vitro. CONCLUSIONS: The early inhibition of p-Smad2/3 signaling pathway improved acute renal function impairment, partially prevented epithelial-endothelial axis activation by maintaining PTEC proteostasis and reduced early PDGFRß+ pericytes-derived myofibroblasts accumulation.

Morphological Retrospective Study of Peritoneal Biopsies from Patients with Encapsulating Peritoneal Sclerosis: Underestimated Role of Adipocytes as New Fibroblasts Lineage?

Background. Encapsulating peritoneal sclerosis (EPS) is a rare but serious complication of peritoneal dialysis (PD). Besides the endothelial-to-mesenchymal transition (EMT), recently peritoneal adipocytes emerged as a potential source of fibrosis. We performed immunohistochemistry to approach EMT and to localize peritoneal adipocytes in peritoneal biopsies from PD-related EPS patients. Material and Methods. We investigated tissue expression of podoplanin, cytokeratin AE1/AE3 (mesothelium), calretinin (adipocytes), alpha-smooth muscle actin [α-SMA] (mesenchymal cells), interstitial mononuclear cell inflammation, and neoangiogenesis (CD3, CD4, CD8, CD20, CD68, and CD31 immunostainings, resp.). Results. Three patients (1 man/2 women; 17, 64, and 39 years old, resp.) developed EPS after 21, 90, and 164 months of PD therapy. In patients with EPS, we observed (1) loss of AE1/AE3 cytokeratin+ mesothelial cells without any evidence of migration into the interstitium, (2) disappearance of adipose tissue, (3) diffuse infiltration of calretinin+ cells in the areas of submesothelial fibrosis with a huge number of α-SMA and calretinin+ fusiform cells, and (4) increased vascular density. Conclusion. We report that the involvement of EMT in peritoneal fibrosis is difficult to demonstrate and that the calretinin+ adipocytes might be an underestimated component and a new source of myofibroblasts in peritoneal remodeling during PD-related EPS.

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.

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.

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.

Mycobacterium fortuitum and Polymicrobial Peritoneal Dialysis-Related Peritonitis: A Case Report and Review of the Literature.

Mycobacterium fortuitum is a ubiquitous, rapidly growing nontuberculous mycobacterium (NTM). It is the most commonly reported NTM in peritoneal dialysis (PD) associated peritonitis. We report a case of a 52-year-old man on PD, who developed refractory polymicrobial peritonitis necessitating PD catheter removal and shift to hemodialysis. Thereafter, M. fortuitum was identified in the PD catheter culture and in successive cultures of initial peritoneal effluent and patient was treated with amikacin and ciprofloxacin for six months with a good and sustained clinical response. Months after completion of the course of antibiotics, the patient successfully returned to PD. To our knowledge, this is the first reported case of M. fortuitum peritonitis in the field of polymicrobial PD peritonitis. It demonstrates the diagnostic yield of pursuing further investigations in cases of refractory PD peritonitis. In a systematic review of the literature, only 20 reports of M. fortuitum PD peritonitis were identified. Similar to our case, a delay in microbiological diagnosis was frequently noted and the Tenckhoff catheter was commonly removed. However, the type and duration of antibiotic therapy varied widely making the optimal treatment unclear.

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.

Evaluation of diagnostic criteria for endemic nephropathy.

Diagnosis of endemic nephropathy (EN) is based on the combination of several clinical and laboratory criteria. Despite extensive research no specific diagnostic biomarker for EN has yet been identified. The aim of the study was to evaluate the diagnostic significance of the variables previously proposed as diagnostic criteria, but also new ones. After an extended questionnaire, the clinical and laboratory examination population in EN villages was classified according to the modified WHO criteria. The urinary active form of TGF-ß was measured with a bioassay using a cell line which expresses luciferase activity. In the study we used ROC analysis to examine the predictive value of the tested variables. In the study there was no difference in haemoglobin level between the study subgroups. Leucine aminopeptidase (LAP) in urine and active urinary TGF-ß levels were increased in the EN diseased group when compared to other subgroups, but they did not fulfil the statistical criteria needed for differentiating a diseased form from other study subgroups. Both kidney length and parenchima thickness, alfa1-microglobulinuria, and kidney function assessed by MDRD formula were the variables that differentiated the study subgroups well. Based on our results the cut-off value of alfa1-microglobulin for screening should be 23.5 mg/g creatinine instead of 15 mg/g creatinine in the present criteria, and for making a diagnosis of EN 31,5 mg/g creatinine. Persons with a positive family history for EN had a 5.8 times greater risk of developing EN when compared to a negative one. Taken together, the above-mentioned variables should be implemented in new uniform diagnostic criteria for EN.

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.