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.

New in vitro insights on a cell death pathway induced by magnolol and honokiol in aristolochic acid tubulotoxicity.

Aristolochic acids (AA) are nephrotoxic agents found in Aristolochia species whose consumption leads to the onset of a progressive tubulointerstitial fibrosis. This AA-nephropathy was first reported during the Belgian outbreak of the 1990's in which more than a hundred patients consumed slimming pills containing an Aristolochia species and Magnolia officinalis. The patients developed an end-stage kidney disease requiring dialysis or transplantation. Magnolol and honokiol are bioactive compounds from M. officinalis known for their potent antioxidant activity. As they can alleviate oxidative stress, we investigated their respective effects on AA-mediated tubulotoxicity using HK-2 cells. Magnolol and honokiol were able to reduce the oxidative stress associated with AA-treatment. Cytotoxicity alleviation was further investigated and overall cell viability measurements unexpectedly revealed that both compounds worsened the survival of AA-treated cells. Flow cytometry analyses of annexin V/PI stained cells indicated that the lignans efficiently prevented AA-induced apoptosis; but favored necrosis. Microscopy observations highlighted extensive vacuolization; other types of cell death, including autophagy, paraptosis or accelerated senescence were excluded. Ki-67 index and cell cycle analysis indicated that both magnolol and honokiol inhibited proliferation by blocking the cell cycle at the G1 phase; they also prevented the AA-induced G2/M arrest.

In vitro effects of Panax ginseng in aristolochic acid-mediated renal tubulotoxicity: apoptosis versus regeneration.

This in vitro study aimed to determine the effects of a Panax ginseng extract on aristolochic acid-mediated toxicity in HK-2 cells. A methanolic extract of ginseng (50 µg/mL) was able to reduce cell survival after treatment with 50 µM aristolochic acid for 24, 48, and 72 h, as evidenced by a resazurin reduction assay. This result was confirmed by a flow cytometric evaluation of apoptosis using annexin V-PI staining, and indicated higher apoptosis rates in cells treated with aristolochic acid and P. ginseng extract compared with aristolochic acid alone. However, P. ginseng extract by itself (5 and 50 µg/mL) increased the Ki-67 index, indicating an enhancement in cellular proliferation. Cell cycle analysis excluded a P. ginseng extract-mediated induction of G2/M cell cycle arrest such as the one typically observed with aristolochic acid. Finally, ß-catenin acquisition was found to be accelerated when cells were treated with both doses of ginseng, suggesting that the epithelial phenotype of renal proximal tubular epithelial cells was maintained. Also, ginseng treatment (5 and 50 µg/mL) reduced the oxidative stress activity induced by aristolochic acid after 24 and 48 h. These results indicate that the ginseng extract has a protective activity towards the generation of cytotoxic reactive oxygen species induced by aristolochic acid. However, the ginseng-mediated alleviation of oxidative stress did not correlate with a decrease but rather with an increase in aristolochic acid-induced apoptosis and death. This deleterious herb-herb interaction could worsen aristolochic acid tubulotoxicity and reinforce the severity and duration of the injury. Nevertheless, increased cellular proliferation and migration, along with the improvement in the epithelial phenotype maintenance, indicate that ginseng could be useful for improving tubular regeneration and the recovery following drug-induced kidney injury. Such dual activities of ginseng certainly warrant further in vivo studies.

Substitution between Aristolochia and Bryonia genus in North-Eastern Morocco: toxicological implications.

ETHNOPHARMACOLOGICAL RELEVANCE: Although acknowledged as toxic herbs, Aristolochia species are still widely used worldwide. The aristolochic acids (AA) they contain can induce the so-called "aristolochic acid nephropathy", leading to renal fibrosis and upper urinary tract cancer. Traditional Moroccan medicine still often uses Aristolochia species under the vernacular name of Bereztem for the treatment of numerous ailments, notably cancer, diabetes or digestive tract disorders. As the botanical identity and renal toxicity of used species remain unexplored, the safety of patients may be threatened. MATERIAL AND METHODS: Ethnopharmacological data were collected from herbalists from the provinces of Oujda and Berkane, located in North-Eastern Morocco. Samples of Bereztem were collected at herbalist shops and checked for their content in AA using TLC and LC-MS methods. The toxicity of crude methanolic extracts of each herb was assessed on a HK-2 cell-based in vitro model by measurement of the cell survival to evaluate cytotoxicity and by assessment of renal-specific toxicity via (i) the evaluation of genes (E-cadherin and α-smooth muscle actin) expression by RT-qPCR; (ii) the quantities of ß-catenin and vimentin by immunofluorescence microscopy; (iii) the secretion of fibronectin; and (iv) the excretion of interleukin-6. RESULTS: The survey indicated that, among 42 herbalists visited, 33 were retailers of Bereztem, which was generally sold as a cancer treatment. Botanical investigations revealed that Aristolochia longa was frequently substituted by Bryonia dioica, which was associated with a higher cytotoxicity. Parameters specific to renal toxicity were also found to be enhanced, as compared to Aristolochia baetica and A. longa: down-regulation of ß-catenin and E-cadherin and up-regulation of vimentin and α-smooth muscle actin, and secretion of fibronectin and interleukin-6. CONCLUSION: In accordance with the Moroccan regulations, the use of so-called Aristolochia species should be discontinued. On one hand, the correctly identified aristolochia contain nephrotoxic aristolochic acids; on the other hand, aristolochia are massively substituted in North-Eastern Morocco and adulterated by a well-known toxic herb, B. dioica. Our data indicate that the bryony renal toxicity may be deleterious in shorter time periods than aristolochia. Reinforced on-site controls are needed to remind herbalists and harvesters that these herbs should be prohibited.

Nephroprotective effects of ferulic acid, Z-ligustilide and E-ligustilide isolated from Angelica sinensis against cisplatin toxicity in vitro.

Cisplatin (CisPt), a chemotherapeutic drug applied against solid tumors, is highly detrimental to the kidney. The risk of acute kidney injury implies adequate patient hydration to ensure sufficient diuresis; this strategy, now implemented in clinical practice, remains however incompletely satisfactory. New pharmacological approaches relying on the discovery of bioactive compounds need to be developed. Based on previous studies reporting renoprotective activities for extracts of Angelica sinensis (Oliv.) Diels roots, three of its major active compounds, ferulic acid, Z-ligustilide and E-ligustilide, were investigated for possible alleviation of CisPt-induced nephrotoxicity. Five phenomena involved in acute kidney injury and subsequent fibrosis were investigated: (i) modulation of cell survival via reduction of the apoptosis rate; (ii) reduction of oxidative stress; (iii) improvement of tubular regeneration capacities through proliferation and migration; (iv) limitation of extracellular matrix and collagen deposition; and (v) prevention of the dedifferentiation processes via the ß-catenin pathway. Ferulic acid emerged as the most potent compound for alleviating cell death and collagen deposition, and for enhancing cell regeneration capacities. It also partially inhibited the ß-catenin pathway, but was ineffective in lowering oxidative stress. Z- and E-ligustilides, however, were effective for limiting the oxidative stress, but only moderately affected other parameters. Ferulic acid appears to be a promising nephroprotective drug lead deserving further preclinical investigation.

Potential nephroprotective effects of the Chinese herb Angelica sinensis against cisplatin tubulotoxicity.

CONTEXT: Acute kidney injury (AKI) is often encountered in patients receiving cisplatin (CisPt), a chemotherapeutic drug that induces numerous toxic side effects. Techniques used to limit nephrotoxicity during CisPt treatment are not fully effective; about a third of patients experience AKI. New nephroprotective strategies, including pharmacological approaches, must be developed. OBJECTIVE: The present study investigated the nephroprotective potential of Angelica sinensis (Oliv.) Diels (Apiaceae) root towards CisPt tubulotoxicity. MATERIALS AND METHODS: HK-2 cells were incubated with CisPt (10 µM) and/or with a methanolic extract of A. sinensis (AS). Nephroprotective capacity was evaluated by means of cellular viability (resazurin assay) and apoptosis (annexin-V/PI staining), oxidative stress generation (H2DCF-DA oxidation), Ki-67 index (immunofluorescence), cell cycle analysis (DNA staining), cell migration rate (scratch assay), extracellular matrix deposition (collagen determination), and ß-catenin relocalization. RESULTS: CisPt decreased cell viability [76% versus Ctrl], which was associated with an increased apoptosis. Simultaneous treatment with 50 µg/ml AS enhanced cell survival [84% versus Ctrl] and decreased the apoptosis rate. AS could not alleviate CisPt-induced oxidative stress; but doses of 5 and 50 µg/ml raised the Ki-67 index [135 and 244% versus Ctrl] and cell migration rates [1.2 and 1.3-fold versus Ctrl]. Finally, both doses of AS limited the amount of collagen deposition [121.6 and 119.6% for 5 and 50 µg/ml, respectively, versus 131.0% for CisPt-treated cells] and prevented the relocalization of ß-catenin from the membrane to the nucleus. CONCLUSION: These results confirm the nephroprotective potential of A. sinensis and require further investigations aiming at identifying its active compounds.

Methods applied to the in vitro primary toxicology testing of natural products: state of the art, strengths, and limits.

The present review attempts to build up a comprehensive picture of the major primary techniques used to screen and assess the cytotoxicity of plant complex mixtures. These can be based on metabolic activity, on membrane integrity, on morphological features, on cell growth; the type of cell death can also be established from more or less specific events (e.g., apoptosis, autophagy, DNA damage detection, reactive oxygen species involvement). This review will discuss the benefits, the difficulties, and the challenges that may occur along cytotoxicity testing of raw extracts and isolated natural compounds.

Protective effects of schizandrin and schizandrin B towards cisplatin nephrotoxicity in vitro.

Renal proximal tubular epithelial cells are the main targets of toxic drugs such as cisplatin (CisPt), an alkylating agent indicated for the treatment of solid organ tumors. Current techniques aiming at reducing nephrotoxicity in patients receiving CisPt are still not satisfactory as they can only partially prevent acute kidney injury. New nephroprotective strategies remain to be developed. In the present in vitro study, schizandrin (Schi) and schizandrin B (Schi B), major phytochemicals from Schisandra chinensis (Turcz.) Baill. fruits, were tested on HK-2 cells along four processes that could help alleviate CisPt toxicity. Results indicated that: (i) both Schi and Schi B enhanced cell survival via reducing apoptosis rate; (ii) only Schi showed moderate effects towards modulation of regeneration capacities of healthy cells; (iii) both Schi and Schi B limited extracellular matrix deposition; and (iv) both compounds could help preventing dedifferentiation processes via the ß-catenin pathway. Schi and Schi B present promising activities for future development of protective agents against CisPt nephrotoxicity.