Rapid screening of purification strategies for the capture of a human recombinant F(ab')2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS.

The development of a capture step of a human recombinant F(ab')(2) produced and expressed in baculovirus-infected cells was investigated by screening three mixed-mode chromatography sorbents (HEA HyperCel, PPA HyperCel and MEP HyperCel) and two ion exchangers (Q Ceramic HyperD F, S Ceramic HyperD F) sorbents using a 96-well plate format and SELDI-MS. HEA HyperCel gave the best separation performance therefore the conditions tested in micro-plate were transferred to laboratory scale chromatographic experiments, confirming that the recombinant F(ab')(2) was effectively captured on the mixed-mode sorbent without any pre-treatment of the crude extract with a 82% recovery and a 39-fold purification.

Effects of cadmium and uranium on some in vitro renal targets.

Metals are major pollutants not only in occupational settings but also in the general environment. Chronic exposure of workers has been related to severe damage, especially at the renal level. While toxic compounds such as metals are well known to severely impair tubular functions, it is clear that nephrotoxicants can act on various other renal targets, i.e., vascular and glomerular ones. In vitro models are available to assess these toxicities and can be used to better understand the different cell targets. This paper summarizes data obtained in our laboratory after exposure of isolated renal structures such as glomeruli, and cell cultures such as glomerular mesangial and tubular epithelial cells, to cadmium and uranium. Morphometric studies by image analysis of isolated glomeruli and mesangial cultured cells showed that cadmium and uranium induced a dose- and time-dependent glomerular contraction accompanied by disorganization of the cytoskeleton. Classical viability tests demonstrated various factors influencing the metal toxicity. The important roles of pH, extracellular protein concentrations and the nature of the anion accompanying the metal were demonstrated. These data obtained in in vitro models provide better understanding of the cytotoxicity after metal uptake and accumulation in glomerular and tubular cells. Moreover, the glomerular and tubular cytotoxicity they induce may be correlated with severe renal hemodynamic changes in vivo. Finally, we briefly present eventual improvements for in vitro renal models by the use of new cell models such as immortalized human cell lines or by the introduction of porous supports and perifusion devices.

Influence of cadmium speciation for the evaluation of in vitro cadmium toxicity on LLC-PK(1) cells.

The main objective of the present work was to assess the potentiality of in vitro models to improve our understanding of cadmium-induced toxicity, especially on epithelial renal cells. Indeed cadmium, a potent toxic metal, poses a serious environmental threat and the mechanisms of its renal toxicity need to be clarified. Cytotoxicity studies presented here were performed in a tubular proximal original established porcine kidney cell line (LLC-PK(1)). We have compared cytotoxicity induced by different chemical cadmium forms in LLC-PK(1) cells as a function of media cell culture pH and protein content. Cadmium stock solutions were prepared either by dissolving cadmium chloride or cadmium sulphate with increasing protein concentrations in the media cell culture. Its pH was monitored during experiments. Cytotoxicity was measured by neutral red uptake after 24 h of exposure. Dose-dependent cytotoxicity curves, calculated with REGTOX, were systematically correlated with pH and protein content. Experiments in vitro revealed that cadmium was dose-dependently toxic for LLC-PK(1) for concentrations ranging from 10(-4) to 10(-6) M. We have noticed a lack of influence of the media cell culture pH on the cadmium cytotoxicity. REGTOX determines closely the EC(50) values but EC(50)CdCl(2)>EC(50)CdSO(4) and cadmium have been assayed with an inductively coupled atomic emission spectrometer (ICP/AES) directly in the media cell culture and the cellular pellet.

Cadmium nephrotoxicity assessed in isolated rat glomeruli and cultured mesangial cells: evidence for contraction of glomerular cells.

Cadmium (Cd), an important pollutant, causes severe damage at the renal tubular level. Numerous previous studies have focused upon Cd tubular nephrotoxicity. The present study of Cd-induced glomerular damage examined the vasoactive effect of Cd in freshly isolated glomeruli and mesangial cells. Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by culture for outgrowth of cells. Quantitative evaluation of glomerular and cellular contractions was performed by morphometric measurement of the area with an automatized image analyzer following different incubation times with Hanks' balanced salt solution or Cd2+. Each glomerulus or mesangial cell served as its own control. Cd lethality was measured with microassay methods (neutral red, MTT uptake, and lactate dehydrogenase release), allowing the determination of an IC50. This ranged from 35 to 60 microM. CdCl2 induced a time-dependent contractile effect on isolated glomeruli; planar surface area decreases were 6.9% (1 microM), 7.5% (0.1 microM), and 7% (0.01 microM). The decrease started as soon as Cd was in contact with glomeruli and ended 40 min later: T5 (2%), T10 (3.5%), T20 (4. 2%), T30 (6.3%), T40 (7%). Cell size reduction was 19% (1 microM), 14% (0.1 microM), and 18% (0.01 microM) and was also time-dependent. To confirm that contractile events occurred during the cell shape changes, examination of the mesangial alpha-actin network was performed concurrently. These results indicate that Cd contracts glomerular structures. This may, in part, explain the reduction in glomerular filtration seen in Cd nephrotoxicity.

Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells.

Very few data are available concerning the in vitro toxicity of uranium. In this work, we have determined the experimental chemical conditions permitting the observation of uranium(VI) cytotoxicity on LLC-PK1 cells. Uranium solutions made either by dissolving uranyl acetate or nitrate crystals, or by complexing uranium with bicarbonate, phosphate or citrate ligands, were prepared and tested. Experiments demonstrated that only uranium solutions containing citrate and bicarbonate ligands concentrations tenfold higher than the metal, were soluble in the cell culture medium. Cytotoxicity studies of all these uranium compounds were performed on LLC-PK1 cells and compared using LDH release, neutral red uptake and MTT assays. Dose dependent cytotoxicity curves were only obtained with uranium-bicarbonate medium. This study has revealed a toxicity of uranium-bicarbonate complexes for 24 h expositions and for concentrations ranging from 7 x 10(-4)-10(-3) M, under these conditions, the CI50 (cytotoxicity index) was evaluated between 8.5 and 9 x 10(-4) M. In contrast, we noticed a lack of cytotoxicity response for uranium(VI)-citrate complexes. Electron transmission microscopy studies revealed, when LLC-PK1 cells were exposed to the uranium-bicarbonate system, that uranium penetrated and precipitated within the cytoplasmic compartment. Morphological studies conducted with citrate complexes did not show any cellular intake of uranium.

Protective effects of polyphenols against cadmium-induced glomerular mesangial cell myocontracture.

The main objective of this work was to determine the ability of polyphenols (procyanidoloic oligomers; PCO) to diminish the contracture of CdCl2-induced mesangial cells (smooth muscle cell type). Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by a culture step for outgrowth of cells. PCO lethality was measured by microassay (Neutral Red uptake). This study has revealed an absence of PCO toxicity during exposure for 24 h and for concentrations ranging from 0.031 to 1% (w/v) on rat renal mesangial cells. We observed a lack of cytotoxicity response for the PCO mixture dissolved in medium. Quantitative assessments of the planar cell surface area (PCSA) were performed with an accurate automatized image analyser. The use of isolated cultured mesangial cells permits us to evaluate by quantitative morphometric analysis the contracture elicited either with CdCl2 salts alone or by previous incubation with a non-lethal dose of PCO. When renal mesangial cells were exposed for 10 min to the PCO mixture, the Cd-mediated myocontracturant response of the mesangial cells was totally abolished. These results suggest that polyphenols could be effective against renal damages induced by cadmium.

Uranium-induced Vasoreactivity in Isolated Glomeruli and Cultured Rat Mesangial Cells.

The present study is aimed at investigating direct vasocontractive effects of uranium on two glomerular in vitro models (isolated rat glomeruli and cultured mesangial cells). For this study, sublethal doses of the uranium(VI)-bicarbonate complex were determined by cytotoxicity experiments on confluent cultured mesangial cells. Afterwards, using a morphoquantitative approach (computerized image analyser) the variations of planar surface areas (PSA) of glomeruli and mesangial cells exposed to uranium(VI)-bicarbonate have been assessed. Our results showed a significant reduction of PSA for glomeruli (-9% after 40 minutes of exposure to uranium: [U]=100 mum) and for rat mesangial cells (-14% after 40 minutes), while PSA values for control glomeruli and mesangial cells remain stable (ranging from 2.5 to 3.5% after 40 minutes of exposure to HBSS medium). These findings suggest that uranium renal target is not only the proximal tubule, but demonstrate, for the first time, that non- lethal uranium doses can induce direct vasoactive effects on glomerular structures and especially on mesangial cells.