Béatrice L'Azou

In vitro effects of nanoparticles on renal cells.

BackgroundThe ability of nanoparticles to cross the lung-blood barrier suggests that they may translocate to blood and to targets distant from their portal of entry. Nevertheless, nanotoxicity in organs has received little attention. The purpose of this study was to evaluate nanotoxicity in renal cells using in vitro models. Various carbon black (CB) (FW2–13 nm, Printex60-21 nm and LB101-95 nm) and titanium dioxide (TiO2-15 and TiO2-50 nm) nanoparticles were characterized on size by electron microscopy. We evaluated theirs effects on glomerular mesangial (IP15) and epithelial proximal tubular (LLC-PK1) renal cells, using light microscopy, WST-1 assay, immunofluorescence labeling and DCFH-DA for reactive oxygen species (ROS) assay.ResultsNanoparticles induced a variety of cell responses. On both IP15 and LLC-PK1 cells, the smallest FW2 NP was found to be the most cytotoxic with classic dose-behavior. For the other NPs tested, different cytotoxic profiles were found, with LLC-PK1 cells being more sensitive than IP15 cells. Exposure to FW2 NPs, evidenced in our experiments as the most cytotoxic particle type, significantly enhanced production of ROS in both IP15 and LLC-PK1 cells. Immunofluorescence microscopy using latex beads indicated that depending on their size, the cells internalized particles, which accumulated in the cell cytoplasm. Additionally using transmission electronic microscope micrographs show nanoparticles inside the cells and trapped in vesicles.ConclusionThe present data constitute the first step towards determining in vitro dose effect of manufactured CB and TiO2 NPs in renal cells. Cytotoxicological assays using epithelial tubular and glomerular mesangial cell lines rapidly provide information and demonstrated that NP materials exhibit varying degrees of cytotoxicity. It seems clear that in vitro cellular systems will need to be further developed, standardized and validated (relative to in vivo effects) in order to provide useful screening data about the relative toxicity of nanoparticles.

Cytoskeletal Reorganization by Mycophenolic Acid Alters Mesangial Cell Migration and Contractility

Abstract—Cytoskeleton alterations are a hallmark of mesangial cell activation during glomerulosclerosis. The aim of this study was to investigate whether mycophenolic acid (MPA) affects cytoskeletal organization and motility of human mesangial cells. Using the IP15 cell line, we found that treatment with 1 &mgr;mol/L MPA inhibited both receptor-dependent (angiotensin II) and receptor-independent (KCl) contractile responses, as well as serum-induced migration activity, suggesting alterations in the intracellular mechanisms that control mesangial cell motility. Immunofluorescence studies of MPA-treated cells provided evidence for decreased membrane disassembly/reassembly of &agr;-smooth muscle actin and F-actin fibers, which was correlated with sustained quantitative and qualitative modifications of actin-associated proteins: calponin was overexpressed and became associated with actin fibers, whereas phosphorylation levels of cofilin and myosin light chain increased, suggesting both an activation of the mechanisms responsible for actin polymerization and an inhibition of actin-depolymerizing processes. These observations support a stabilizing effect of MPA on the mesangial actin cytoskeleton, which constitutes an additive action by which MPA, beyond its anti-inflammatory, antiproliferative and antifibrotic properties, might protect against excessive mesangial activation in the context of various glomerulopathies and kidney transplantation.

Circadian rhythms of renal hemodynamics in unanesthetized, unrestrained rats.

Catheters were placed in the jugular vein and femoral artery of male Sprague-Dawley rats and connected to a specially designed perfusor for continuous constant infusion of 0.9% NaCl and a syringe to perform simultaneous and intermittent blood collections. This permitted continuous 24-h study of renal hemodynamics, estimated by inulin (Cin) and p-amino-hippuric acid (CPAH) clearances; Cin represents glomerular filtration rate and CPAH renal plasma flow. Animals were individually housed in metabolism cages in a controlled environment with light/dark 12:12 h. Urine was collected every 4 h (12:00, 16:00, 20:00, 24:00, 04:00, and 08:00) and blood sampled at the midpoint of urine collection periods. Urine and plasma sodium, potassium, inulin, and PAH were spectrophotometrically assessed. During continuous infusion of isotonic saline, Cin exhibited circadian changes with large decrease between 12:00 and 20:00 h (0.9 +/- 0.2 ml/min) and acrophase at 00:30 h. Rhythmicity in CPAH was similar with the minimum between 16:00 and 20:00 h (2.5 +/- 0.3 ml/min) and peak between 00:00 and 04:00 h (acrophase at 00:25 h). Water and electrolyte excretion were also circadian rhythmic with a similar nighttime enhancement and daytime minimum. Such circadian changes persisted during continuous 0.9% NaCl infusion for several consecutive days. The unanesthetized, unrestrained rat model enables investigations in renal chronopharmacology and chronotoxicology.

Comparative cytotoxicity of cadmium forms (CdCl2, CdO, CdS micro- and nanoparticles) in renal cells

Cadmium (Cd) is a potent selective toxicant that preferentially accumulates in the kidneys where it is known to induce cellular injury. The cellular mechanisms explaining toxicity have been well documented by in vivo and in vitro studies. However, experiments have mainly been performed on cadmium chloride (CdCl2), while several other forms, such as cadmium oxide (CdO) and cadmium sulfide (CdS), are of interest regarding occupational exposure. CdS is mainly used in nanotechnology, primarily to construct particles known as quantum dots (QDs). The present study focuses on cadmium forms that differ compared to their solubility and their particle size (CdS micro- and uncoated nanoparticles (NPs)), and investigates their effects on mesangial and proximal tubular kidney cells. Cellular toxicity was evaluated by using Neutral Red, MTT and WST-1 assays. Intracellular Cd content was measured using inductively coupled optical emission spectrometry (ICP-OES) and cellular stress was investigated via the effects of Cd complexing bioligands using size exclusion chromatography ICP-MS (SEC-ICP-MS). Data indicated the variability of cytotoxicity responses after CdCl2, CdO, and CdS exposure that were strongly correlated to the cellular Cd content. Release of Cd2+ linked to solubility and particle degradation were considered to be the cause of these toxicities. The bioinduction of the Cd–MT also evidenced release of Cd2+. Our in vitro data identified heterogeneity of Cd toxicity that was dependent on the physico-chemical properties of the form studied and, when considering particle size, constitute an additional step toward the determination of nanoparticle effects in renal cells.

Cadmium induces direct morphological changes in mesangial cell culture

The cadmium produced by industrial and agricultural practice represents a major environmental pollutant which may induce severe damage, especially in the kidney where cadmium accumulates. While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets. The present study investigated the effects of cadmium on glomerular mesangial cell cultures after short- and long-term exposures, requiring for each endpoint specific culture conditions. After 30 min exposure to 1 microM CdCl(2), used as non-lethal concentration, 0.14 ng/microg proteins of cadmium was internalized by the cells as evaluated by atomic emision spectrometry and induced a significant, cell surface reduction (8.9+/-1.9%). These morphological changes could be correlated to smooth muscle alpha-actin disorganization, without quantitative change in its protein expression level as evaluated by Western-blot and Northern-blot analysis (SMAmRNA/28sRNA, 1.78 CdCl(2) vs. 1.42 control). For longer exposure times, in complex medium, cadmium uptake was efficient (0.36 ng/microg proteins) and induced changes in the actin cytoskeleton with no loss of cell membrane integrity. This study suggests that cultured mesangial cells provide an alternative model to study the effect of cadmium, and underlines the importance of using well-defined conditions to study further intracellular mechanisms.

Physicochemical characteristics and bronchial epithelial cell cytotoxicity of Folpan 80 WG® and Myco 500®, two commercial forms of folpet

BackgroundPesticides, in particular folpet, have been found in rural and urban air in France in the past few years. Folpet is a contact fungicide and has been widely used for the past 50 years in vineyards in France. Slightly water-soluble and mostly present as particles in the environment, it has been measured at average concentration of 40.1 μg/m3 during its spraying, 0.16–1.2 μg/m3 in rural air and around 0.01 μg/m3 in urban air, potentially exposing both the workers and the general population. However, no study on its penetration by inhalation and on its respiratory toxicity has been published. The objective of this study was to determine the physicochemical characteristics of folpet particles (morphology, granulometry, stability) in its commercial forms under their typical application conditions. Moreover, the cytotoxic effect of these particles and the generation of reactive oxygen species were assessed in vitro on respiratory cells.ResultsGranulometry of two commercial forms of folpet (Folpan 80WG® and Myco 500®) under their typical application conditions showed that the majority of the particles (>75%) had a size under 5 μm, and therefore could be inhaled by humans. These particles were relatively stable over time: more than 75% of folpet remained in the particle suspension after 30 days under the typical application conditions. The inhibitory concentration (IC50) on human bronchial epithelial cells (16HBE14o-) was found to be between 2.89 and 5.11 μg/cm2 for folpet commercial products after 24 h of exposure. Folpet degradation products and vehicles of Folpan 80 WG® did not show any cytotoxicity at tested concentrations. At non-cytotoxic and subtoxic concentrations, Folpan 80 WG® was found to increase DCFH-DA fluorescence.ConclusionThese results show that the particles of commercial forms of folpet are relatively stable over time. Particles could be easily inhaled by humans, could reach the conducting airways and are cytotoxic to respiratory cells in vitro. Folpet particles may mediate its toxicity directly or indirectly through ROS-mediated alterations. These data constitute the first step towards the risk assessment of folpet particles by inhalation for human health. This work confirms the need for further studies on the effect of environmental pesticides on the respiratory system.

Cytotoxic effects and cellular oxidative mechanisms of metallic nanoparticles on renal tubular cells: impact of particle solubility

Many uncertainties remain regarding the potential toxic effect of nanoparticles (NPs). NPs can cross biological barriers, be carried in blood to kidneys, and damage renal cells. Yet, there is little data regarding NPs’ nephrotoxicity. The aim of this study was to understand the cytotoxic mechanisms induced by metallic NPs with different solubility properties (TiO2, ZnO, and CdS). Studies were performed in vitro on human epithelial tubular cells (HK-2). Cellular and molecular mechanisms were investigated through the oxidative stress status. Using the WST-1 assay it was found that the cytotoxicity of NPs was dependent on the particle size and metal solubility. Exposure to soluble CdS and ZnO NPs led to cell death in a dose-dependent manner related to release of metallic cations (Cd2+ and Zn2+). Insoluble TiO2 NPs had no cytotoxic effect. An analysis of ROS production and lipid peroxidation clearly revealed the involvement of oxidative stress in cell toxicity. Soluble ZnO and CdS NPs caused lysosomal membrane destabilization (acridine orange) and nuclear condensation (DAPI). A molecular approach was used for signaling pathways. ZnO and CdS NPs induced the translocation of Nrf2 and NF-κB and induction of antioxidant enzymes. TiO2 NPs did not cause lysosomal membrane destabilization or nuclear condensation. TiO2 NPs slightly activated Nrf2 nuclear translocation, but no significant NF-κB nuclear translocation was observed. For TiO2, the oxidative stress was not sufficient to trigger any membrane disruption. This study provides additional knowledge about the renal toxicity of NPs. The release of metal ions represents an important factor for determining the toxicity. This in vitro assay constitutes an additional step in nanomaterial safety assessment.

ICP/OES Application for Assessing Cadmium Uptake (or Toxicity) in Glomerular Cells: Influence of Extracellular Calcium

The risks of metals for health are highlighted by their chemical stability and their persistence in the environment. Chronic exposure to low cadmium (Cd) concentrations results in renal dysfunction mainly. Cd has been regarded primarily as a renal tubular toxicant, but glomerular structures may also be affected. Since the cellular environment may influence metal toxicity, differences concerning Cd uptake and toxicity were evaluated according to calcium (Ca) medium concentrations. An optimized inductively coupled plasma emission spectrometry method (ICP/OES) was developed under defined conditions, as a selective analytical tool to determine cadmium uptake in glomerular mesangial cells. The performance characteristics of the analytical system were evaluated for both Cd and Ca by calibration (50 to 250 μg/L and 1 to 5 mg/L), linearity (r 2 .9968 and .9943), limits of detection (1 μg/L and 0.1 mg/L) and quantitation (3 μg/L and 0.3 mg/L), accuracy with spiking, and repeatability (1.2 and 2.9%) with matrix matched standards. Total intracellular Cd content was significantly threefold lower in 0.175 mM Ca medium (Ca-free Eagles minumum essential medium [EMEM] medium with 5% fetal bovine serum [FBS]) than in EMEM medium (1.8 mM Ca) with respectively 0.16 and 0.37 μg/mg proteins after 24 h of Cd (1 μM) exposure. Similar differences were obtained in cytotoxicity studies with a fourfold reduction in the mortality index (IC50). Complementary assays using Ca-spiked medium reinforced that Cd cytotoxicity and uptake were significantly dependent on the concentration of extracellular Ca. These findings suggest direct link between Cd uptake and toxicity, underlining the relevance of the analytical method.

Monitoring biological effects of 20 nm versus 100 nm silica nanoparticles induced on a human renal cell line using Fourier transform infrared spectroscopy

Due to their small size, nanoparticles (NPs) and particularly silica NPs (SiO2-NPs) exhibit unique properties that confer them especially great biological reactivity. Thus, intensive research studies are performed to elucidate their potential toxicity and/or their interaction with biological systems. Standard assays focus on one specific biological event which require the use of several tests to determine all potential effects induced by SiO2-NPs. Moreover, because of the interaction between the reagent and SiO2-NPs, the reliability of colorimetric or fluorometric methods is questioned when assessing nanomaterials. Therefore, Fourier transform infrared (FTIR) spectroscopy was used as a reagent-free and time-saving tool, combined with Principal Component Analysis (PCA), to reveal biochemical effects induced by 100 nm versus 20 nm SiO2-NPs on human kidney cells (HK-2) at two subtoxic concentrations (10 and 25 μg ml−1) during 24 h. This technique allows revealing dose-dependent responses and differences in biological effects between 20 nm and 100 nm SiO2-NPs with a greater impact of 20 nm SiO2-NPs. Moreover, SiO2-NP cell uptake has been highlighted. Toxic biochemical effects induced by 20 nm SiO2-NPs are detected as hallmarks of lipid peroxidation (1746 cm−1) and apoptosis (1654, 1746 and 2922 cm−1). This study demonstrates that this technique is more sensitive than cytotoxic assays and may be appropriate as a simple screening tool to quickly monitor cellular biochemical changes induced by NPs.

Protective effect of Verapamil in cyclosporin-incubated human and murine isolated glomeruli

Cyclosporin A (CsA)-induced nephrotoxicity is characterized by a decrease in the glomerular filtration rate (GFR) which is associated with a large increase in renal vascular resistance (RVR). Using a video image analyzer, we have demonstrated CsA-induced glomerular vasoconstriction in rat isolated glomeruli as assessed by a significant reduction of glomerular area. This vasoactive response explains in part the renal hemodynamic changes and the development of CsA-induced reversible decline in renal function. To confirm the direct vasoactive effect of CsA on glomeruli and to determine if calcium-blocking agents modified this response, we compared the changes in area of isolated rat and human glomeruli incubated either with CsA alone or with CsA plus verapamil. The area of the isolated glomeruli was quantitatively evaluated by a camera video image analyzer; each glomerulus served as its own control. Area kinetics were studied at 5 min intervals over 30 min. CsA-induced glomerular size reduction is dose dependent (-4.2% for 10(-10) M and -10.2% for 10(-6) M) and time dependent (-2.3% at 5 min, -4.7% at 10 min, and -12.1% at 30 min for 10(-6) M). With verapamil pretreatment, CsA-induced reduction in glomerular size was reduced (-0.6% and -3.6%, respectively, for 10(-6) M and 10(-7) M verapamil). Thus, verapamil can be considered as a protective agent against CsA-induced vasoconstriction in rat and human isolated glomeruli.