Human glomerular mesangial IP15 cell line as a suitable model for in vitro cadmium cytotoxicity studies.

Cadmium represents a major environmental pollutant that 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. Owing to their contractile properties, glomerular mesangial cells play a major role in the control of glomerular hemodynamics and influence the ultrafiltration coefficient. Cell cultures provide alternative and fruitful models for study of in vitro toxicology. However, the use of primary human mesangial cell cultures is hampered by their limited survival span and their rapid dedifferentiation during passages. This study presents a human stable immortalized mesangial cell line, designated IP15. Cell characteristics were investigated by the detection of known mesangial markers, as well as their ability to contract in response to angiotensin II. IP15 cells were used to investigate cadmium uptake and morphological changes such as cell contraction and cytoskeleton protein expression. The IC(50) cytotoxicity index was obtained with 3.55 micromol/L using neutral red assay for 24 h. After cadmium exposure (1 micromol/L, determined as nonlethal concentration), 0.38 microg Cd/mg protein was internalized by the cells as evaluated by inductively coupled plasma optical emission spectrometry (ICP/OES). Cadmium induced a significant cell surface reduction that correlated with smooth-muscle alpha-actin disorganization. Thus, the IP15 cell line is a suitable model for study of in vitro cadmium cytotoxicity in mesangial cells and allows sufficient material to be obtained for future studies of the intracellular effects of cadmium exposure.

Role of the sodium-dependent phosphate co-transporters and of the phosphate complexes of uranyl in the cytotoxicity of uranium in LLC-PK1 cells.

Although uranium is a well-characterized nephrotoxic agent, very little is known at the cellular and molecular level about the mechanisms underlying the uptake and toxicity of this element in proximal tubule cells. The aim of this study was thus to characterize the species of uranium that are responsible for its cytotoxicity and define the mechanism which is involved in the uptake of the cytotoxic fraction of uranium using two cell lines derived from kidney proximal (LLC-PK(1)) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK(1) cells with colchicine, cytochalasin D, concanavalin A and PMA increased the sodium-dependent phosphate co-transport and the cytotoxicity of uranium. On the contrary, replacement of the extra-cellular sodium with N-methyl-D-glucamine highly reduced the transport of phosphate and the cytotoxic effect of uranium. Uranium cytotoxicity was also dependent upon the extra-cellular concentration of phosphate and decreased in a concentration-dependent manner by 0.1-10 mM phosphonoformic acid, a competitive inhibitor of phosphate uptake. Consistent with these observations, over-expression of the rat proximal tubule sodium-dependent phosphate co-transporter NaPi-IIa in stably transfected MDCK cells significantly increased the cytotoxicity of uranium, and computer modeling of uranium speciation showed that uranium cytotoxicity was directly dependent on the presence of the phosphate complexes of uranyl UO(2)(PO(4))(-) and UO(2)(HPO(4))(aq). Taken together, these data suggest that the cytotoxic fraction of uranium is a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate co-transporter system.

In vitro endothelial cell susceptibility to xenobiotics: comparison of three cell types.

In three different endothelial cell (EC) cultures (primary human umbilical cord vein, so-called HUVEC; and immortalized cell lines HBMEC and EA-hy-926), the effects of different xenobiotics were studied in order to standardize vascular EC models for in vitro pharmacotoxicological studies. Cell characteristics were first investigated by the production and the mRNA levels of known endothelial markers in the three EC culture models. EC secretory products, tissue plasminogen activator (tPA) and von Willebrand factor (vWF), were present in the supernatant of the immortalized cell lines. The mRNA levels of vWF, tPA, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and beta -integrin subunit, which are involved in the control of platelet function, coagulation, and fibrinolysis as well as in cell-matrix interactions, were investigated in all EC types. For at least three parameters, cultured cells provided marked characteristics of EC phenotype, in HUVEC and in immortalized cell lines, regardless of their origin from the macro- or microcirculation. Toxicity experiments were assessed after 24 h exposure to cadmium, cyclosporin A and cisplatin by MTT assay. These experiments show nonsignificant difference in susceptibility to cyclosporin A and cadmium on HUVEC, HBMEC, and EA-hy-926. However, HBMEC, seems to be highly susceptible to cisplatin compared to HUVEC, the latter being more sensitive than EA-hy-926. For experiments conducted with cyclosporin and cadmium, cell lines could constitute an alternative material for routine cytotoxicity studies.

Characterization of mapacalcine-sensitive Ca(2+) channels in rat kidney.

Mapacalcine receptors have been found to be associated with a Ca(2+) permeability insensitive to all known calcium blockers. Recently, high densities of mapacalcine receptors have been detected in the choroid plexus of rat brain. To determine a possible role for these channels, we have investigated their presence on other structures which, like choroid plexus, are involved in the secretion of biological fluids. Our data demonstrate that there are specific mapacalcine receptors on kidney membranes and glomeruli preparations. The mapacalcine receptors were present in all structures of the kidney. However, autoradiographic data demonstrated that superficial part of the cortex was more labeled than the other part of the kidney. These data would suggest that mapacalcine receptors could play a role in calcium homeostasis.

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.

In vitro effects of cadmium on two different animal cell models.

The aim of this study was to assess comparatively the effects of cadmium on two different in vitro cell models, a cell line derived from proximal tubule renal cells (LLC-PK1) and haemocytes or blood cells of mussels (Mytilus galloprovincialis). Cells were seeded in 96-well microplates and exposed in vitro to different concentrations of cadmium (CdCl(2)) ranging from 10 to 2000 microM for haemocytes and from 1 to 100 microM for LLC-PK1 cells, added to the culture medium. After 24 h of exposure, different assays were performed on haemocytes: neutral red uptake, phagocytosis of neutral red-stained zymosan, XTT assay, activity of lysosomal acid phosphatase and demonstration of the actin cytoskeleton using TRITC-labeled phalloidin. Cell viability expressed as LC50 was 750 microM when using the neutral red assay and 400 microM with the XTT assay. The phagocytic ability and the activity of acid phosphatase increased significantly in cells treated with Cd in a non dose-dependent manner. Doses of Cd above 100 microM caused disruption of the actin cytoskeleton. In LLC-PK1 cells, cell viability expressed as LC50 was found to be around 40 microM when using the neutral red assay and 50-60 microM with MTT and LDH assays, respectively. These results show that mussel haemocytes are in general more resistant to Cd exposure than LLC-PK1 cells. Furthermore, Cd appears to stimulate phagocytic and lysosomal activities in haemocytes in vitro.

Potential use of isolated glomeruli and cultured mesangial cells as in vitro models to assess nephrotoxicity.

The purpose of this short review is to present the potential of using isolated glomeruli and cultured mesangial cells as two different in vitro models to assess the glomerular effect of molecules with nephrotoxic properties. The advantage of using isolated renal glomeruli is that they conserve the architecture of this anatomical region of the kidney; moreover, they are free of any vascular, nervous or humoral influences derived from other regions of the kidney. Mesangial cells are perivascular pericytes located within the central portion of the glomerular tuft between capillary loops. Mesangial cells have a variety of functions including synthesis and assembly of the mesangial matrix, endocytosis and processing of plasma macromolecules, and control of glomerular hemodynamics, mainly the ultrafiltration coefficient Kf, via mesangial cell contraction or release of vasoactive hormones. Most authors agree that mesangial cells play a major role in glomerular contraction, filtration surface area, and Kf regulation. One of the major effects of toxicants on glomerular structures is contraction. We can assess quantitatively the degree of toxicant-induced mesangial cell contraction or glomerular contraction by measuring the changes in planar cell surface area or apparent glomerular cross-sectional area after exposition to the toxicant. These in vitro models can also reveal glomerular effects of xenobiotics that are difficult or impossible to observe in vivo. In addition, these studies permit a fundamental examination of the mechanism of action of xenobiotics on glomerular cells, including the possibility that at least a part of their effects are mediated by local mediators released by glomerular cells. We review the effects and the mechanisms of action of several toxicants such as gentamicin, cyclosporin, cisplatin, and cadmium on isolated glomeruli or cultured mesangial cells. As such in vitro results confirm in vivo renal hemodynamic changes caused by toxicants, we conclude that these models are fruitful tools for the study of renal toxicity. These in vitro systems might also serve as a predictive tool in the evaluation of drugs inducing changes in glomerular filtration rate and as a way to propose protective agents against these dramatic hemodynamic effects.

In vitro models to study mechanisms involved in cyclosporine A-mediated glomerular contraction.

The immunosuppressive drug, cyclosporin A (CsA), which is successfully used to prevent rejection in organ transplantation, induces renal side-effects as shown by a decrease in glomerular filtration rate and ultrafiltration coefficient regulated by the tone of mesangial cells.The aim of the present study was to investigate the effect of CsA on isolated glomeruli and mesangial cells, which constitute appropriate in vitro models for renal vasoreactivity studies. The roles of different intracellular and extracellular mediators such as calcium, endothelin-1 (ET-1), prostaglandins (TXA(2 )and PGI(2)) and reactive oxygen intermediates (ROIs) were analysed. CsA caused a concentration- and time-dependent decrease in the planar cross-sectional areas of isolated glomeruli and mesangial cells as determined by image analysis. Intracytosolic free calcium concentration determined by fluorimetric analysis was significantly increased after 30 min CsA (10 microM) incubation. In the contraction experiment, the calcium antagonist verapamil inhibited the CsA response. ET-1, TXB(2) and keto-PGF(1alpha) were determined directly, however no changes were found statistically significantly different from respective controls. In contrast to these results, the ET-1 specific antibody was able to reduce CsA-mediated cell contraction. In the presence of a prostacyclin agonist iloprost, CsA-induced contraction was also modified. The role of ROIs using a 2'7'-dichlorofluorescein diacetate (DCFdAc) fluorimetric method was directly determined by observing, with 10 microM CsA, a significant production of hydrogen peroxide (H(2)O(2)), which was able alone to induce mesangial cell contraction. Coincubation with the antioxidants led to a significant inhibition of mesangial cell contraction. These results suggest that CsA caused an imbalance in the normal level of all investigated vasoconstrictive and vasodilator mediators, which shifted towards the advantage of vasoconstrictive action.

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.

Intracellular behaviour of uranium(VI) on renal epithelial cell in culture (LLC-PK1): influence of uranium speciation.

The main objective of this work was to assess the potentiality of in vitro models to study and understand the uranium-induced cytotoxicity on renal cells. Cytotoxicity and morphological studies were performed in a tubular proximal original established cell line (LLC-PK1 cell line). Dose-dependent cytotoxicity response was obtained with the uranium bicarbonate complex. In vitro experiments revealed a toxicity of uranium-bicarbonate complexes after a 24-h exposition and for concentrations ranging from 7 x 10(-4) M to 10(-3) M. In contrast, a lack of cytotoxicity of uranium(VI) citrate complexes studied using the same experimental conditions was noticed. Furthermore, electron transmission microscopy and X-ray microanalysis studies, after exposition of LLC-PK1 cells to the uranium-bicarbonate system ([U] = 8 x 10(-4) M) revealed that uranium entered into the cells and it was precipitated within the cytoplasmic compartment as uranyl phosphate needles. Similar morphological studies conducted with citrate complexes did not show any intake of uranium by LLC-PK1 cells. Experiments conducted in phosphate free culture medium showed that uranium was incorporated as a soluble material and that the association of the metal with phosphate ions occurred in the cytoplasmic compartment of LLC-PK1 cells.

MR imaging of intrarenal macrophage infiltration in an experimental model of nephrotic syndrome.

The objective of this study was to use MR imaging to detect macrophage infiltration of the kidney after injection of ultrasmall superparamagnetic iron oxide (USPIO) particles in a rat model of experimental nephropathy. Ninety micromol of USPIO were injected intravenously in 10 rats with nephropathy secondary to intravenous injection of 5 mg of puromycin aminonucleoside (PAN), and in 10 control rats. The signal intensity was measured in each kidney compartment before and 24 h after injection of the contrast agent. FLASH sequences were performed on a spectrometer operating at 4.7 T. MR findings were compared with histological data. Twenty-four hours after injection of USPIO, a significant decrease (P < 0.0001) was observed in signal intensity in each kidney compartment in the PAN group. There was no variation in the control group. In the diseased kidneys, histological data revealed the presence of macrophages with iron oxide particles within their cytoplasm and lysosomes. Using USPIO, MR imaging can evidence infiltration of the rat kidney by macrophages.

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.

Comparative study of cyclosporin A, cyclosporin G, and the novel cyclosporin derivative IMM 125 in isolated glomeruli and cultured rat mesangial cells: a morphometric analysis.

BACKGROUND: One adverse side-effect of the immunosuppressive drug cyclosporin A (CsA) is a decrease in glomerular filtration rate (GFR). This effect might be the result of increased glomerular contractions. The present study compared the contractile effects of CsA, cyclosporin G (CsG) and the novel cyclosporin derivative IMM 125 in isolated rat glomeruli and primary cultures of rat mesangial cells. METHODS: Interactive image analysis was used to measure glomerular and mesangial cell contraction. RESULTS: CsA, CsG, and IMM 125 at concentrations of 0, 10(-9), 10(-8), 10(-7), 10(-6) and 10(-5) M caused a time-dependent and a concentration-dependent contraction of isolated glomeruli and mesangial cells 30 min after incubation. In glomeruli, CsA was more potent than CsG and IMM 125. In mesangial cells, IMM 125 also exhibited the lowest contractile activity, while CsA and CsG were almost equally myoreactive. The absolute degree of the glomerular contraction was proportional to the number of contracting mesangial cells in one glomeruli. The number of responding cells after incubation with IMM 125 and CsG were lower compared to CsA, which might explain the different response with CsG and CsA in both models. CONCLUSIONS: Since the concentrations used in these experiments were close to that reached in rat serum after treatment with CsA, the present results suggest that the contractile effects of IMM 125 and CsG in isolated glomeruli were clearly smaller compared to CsA, which might reflect the cyclosporins induced GFR changes in vivo.

Comparative cytotoxicity of low- and high-osmolar contrast media to human fibroblasts and rat mesangial cells in culture.

RATIONALE AND OBJECTIVES: The authors investigate the relative sensitivity of rat mesangial cells to iodinated contrast media (CM) and control solutions versus less differentiated cells (ie, human fibroblasts) and compare the effects of low-osmolar ionic (ioxaglate) and nonionic (iopamidol) and high-osmolar ionic (diatrizoate) CM on rat mesangial cells. METHODS: The cytotoxic effects of ioxaglate and control solutions of sodium chloride and mannitol were assessed by neutral red uptake in isolated rat mesangial cells and human fibroblasts. In a second series of studies, the cytotoxic effects of ioxaglate, iopamidol, and diatrizoate (0 to 100 mg I/mL) on rat mesangial cells were compared. RESULTS: Rat mesangial cells were more sensitive to the cytotoxic effects of ioxaglate than the less differentiated human fibroblasts between 70 and 100 mg I/mL. A similar discrepancy was observed in the case of control solutions, sodium chloride, and mannitol. Ioxaglate and iopamidol induced a similar level of cytotoxicity in rat mesangial cells whereas the high-osmolar agent diatrizoate was significantly more cytotoxic. However, the calculated inhibitory concentrations of 50% of all three CM were associated with similar osmolalities, suggesting a major role for this parameter in the case of such media. CONCLUSIONS: Rat mesangial cells are more sensitive to the cytotoxic effects of CM and hyperosmolar solutions than the less differentiated human fibroblasts. High-osmolar CM are more cytotoxic than ionic and nonionic low-osmolar CM to rat mesangial cells. Ionicity seems to play no deleterious role at similar iodine concentrations because ioxaglate and iopamidol had equivalent cytotoxic effects on mesangial cells.

Cyclosporin-A- and angiotensin-II-induced vasoreactivity in isolated glomeruli and cultured mesangial cells, 4 and 24 h after renal mass reduction: role of vasodilatory prostaglandins.

Subtotal nephrectomy in the rat is followed by early hypertrophy and functional adaptation of the remnant glomeruli in which mesangial cells seem to play a leading part. Using a morphoquantitative approach, we assessed the vasoconstrictive response to 1 microM cyclosporin A (CsA) or 0.1 microM angiotensin II (AII) of isolated glomeruli and cultured mesangial cells obtained from normal rats and from rats after five-sixths nephrectomy. In normal rats, a significant reduction of glomerular size was observed after the administration of vasoactive agents (-14% after 30 min exposure to CsA or AII). After five-sixths nephrectomy the vasoconstrictive response, which was preserved at the 4th hour (-12% at the 30th minute), disappeared at the 24th hour (-1.5% at the 30th minute), but was maintained in animals pretreated with indomethacin (-10% at the 30th minute). Similar responses were observed with cultured mesangial cells at the first passage. Inhibition of prostaglandin synthesis abolishes the adaptative adjustments observed in partially nephrectomized rats during the first days after surgery. These findings suggest that renal vasodilatory prostaglandins may participate in the compensatory hemodynamic adjustments following subtotal nephrectomy.

Protective effect of three xanthine derivatives (theophylline, caffeine and pentoxifylline) against the cyclosporin A-induced glomerular contraction in isolated glomeruli and cultured mesangial cells.

Cyclosporin A (CyA), an immunosuppressive agent, induces in vivo a severe nephrotoxicity with large decrease in renal hemodynamics correlated with in vitro glomerular contraction. The aim of this study is to show the ability of three xanthine derivatives, caffeine, theophylline and pentoxifylline, to diminish the CyA-induced in vitro glomerular contraction. The use of isolated glomeruli and cultured rat mesangial cells permits us to evaluate by quantitative and qualitative morphometric analysis the contraction elicited either with CyA alone or with previous treatment with nontoxic concentrations of xanthine derivatives. Indirect immunofluorescence of actin filaments makes it possible to appreciate qualitative morphometric changes in mesangial cells. A 10-min pretreatment with caffeine, theophylline or pentoxifylline (10(-4) to 10(-9) M) abolishes the contraction elicited with 10(-6) M CyA. CyA alone induces -13.9% compared to CyA with 10(-6) M pentoxifylline which induces only -3.2% of reduction of planar glomerulus surface area after 30 min. Similar results were provided with cultured rat mesangial cells. As shown by indirect immunofluorescence xanthine derivatives prevent the cytoskeletal reorganization (alpha-actin) of cultured mesangial cells which occurs with CyA. The marked constriction induced by CyA in isolated glomeruli and mesangial cells can be prevented by xanthine derivatives.

Isolated glomeruli and cultured mesangial cells as in vitro models to study immunosuppressive agents.

Immunosuppressive agents, such as cyclosporin A (CsA), by their vasoconstrictive properties, induce in vivo in patients and rodents a dramatic fall in renal hemodynamics. The aim of this study is to review the ability of some physiological and/or pharmacological agents which are supposed to be involved in the renal physiopathology of CsA to prevent the contraction induced by CsA in two in vitro glomerular models. Isolated glomeruli are obtained by a sieving method from male Sprague-Dawley rat superficial cortex. Mesangial cells from these isolated glomeruli are cultured in RPM1 1640 medium with 20% FCS in 5% CO2 atmosphere. The area of isolated glomeruli and cultured mesangial cells is assessed by an image analyzer with a video camera. Each glomerulus and cell is its own control and is photographed before incubation with any drug (T0) and then during incubation at 5, 10, 20, and 30 min. Incubations are performed during 30 min with 10(-6) mol/L CsA either with a 10 min pretreatment with the vasoactive agent or without pretreatment. CsA alone induces a time- and dose-dependent decrease in glomerular structure area (-4.7% at 10 min, -10.3% at 20 min, and -12.0% at 30 min for isolated glomeruli); Cremophore excipient or control solute does not induce any significant decrease in surface area. CsA with 10(-6) mol/L verapamil pretreatment induces only a slight decrease: -1.5% at 10 min, -3.0% at 20 min, and -4.8% at 30 min. Calcium blockers nifedipine and felodipine produce similar results. Likewise, with 10(-8) mol/L prostacyclin analog (iloprost), only a slight area decrease in mesangial cells is noted: -1.3% at 5 min, -1.8% at 10 min, and -3.3% at 20 min; with 10(-6) mol/L TXA2 synthesis inhibitor (CGS 12970) the results are -2.0% at 10 min, -3.6% at 20 min, and -4.3% at 30 min. Finally, a similar protective effect can be noted with 10(-5) mol/L theophylline: -0.4; -1.5 and -1.9% at 10, 20, and 30 min. In conclusion, CsA-induced contraction in two in vitro glomerular models can be partially or even totally prevented by pretreatment with various pharmacological agents.

Nitric oxide (NO) donor 3-morpholinosydnonimine antagonizes cyclosporin A-induced contraction in two in vitro glomerular models.

Cyclosporin A induces in vivo a severe nephrotoxicity characterized by a large decrease in renal hemodynamics. The aim of this study is to establish the ability of the known NO donor 3-morpholinosydnonimine (SIN-1) to prevent the cyclosporin A-induced contraction by using rat isolated glomeruli and cultured glomerular mesangial cells. Isolated rat glomeruli are obtained from the renal superficial cortex by a sieving method. Mesangial cells are cultured in RPMI 1640 with 15% fetal calf serum. The planar surface area (PSA) of either isolated glomeruli or mesangial cells is assessed using an image analyzer. Each glomerulus or mesangial cell serves as its own control through calculation of the area before any drug incubation and after incubation for 10, 20 and 30 min either in control solution or in control solution with cyclosporin A alone or cyclosporin A and SIN-1. Cyclosporin A (10(-6) mol/L) induces an important time-dependent contraction of either glomerulus or mesangial cell. When pretreated with different concentrations of SIN-1 (10(-4) to 10(-9) mol/L), only a slight size decrease is noted. In conclusion, a direct constrictive effect of cyclosporin A in isolated glomeruli and mesangial cells can be prevented by the NO donor SIN-1, suggesting an important involvement of the nitric oxide pathway in the cyclosporin A-induced nephrotoxicity.