Christelle Monteil

Cell lines with extended in vitro growth potential from human renal proximal tubule: Characterization, response to inducers, and comparison with established cell lines

Few model systems exist for the study of injury to human renal proximal tubule epithelium. Optimized differentiated human renal epithelial cell lines with extended in vitro growth potential would provide an alternative model system to primary culture or other available non-human mammalian kidney cell lines. For this purpose, human renal tubule epithelial cells were isolated from normal kidney cortex and exposed in culture to a hybrid immortalizing virus, adenovirus 12-SV40. Cell lines were developed by limiting dilution, and three selected cell lines were screened for growth pattern, production of immortalizing virus, tumorigenicity, and ploidy. Cell lines were also monitored for response to inducer agents and matrix factors and were screened for expression of biochemical properties and differentiation markers of renal epithelium. All three are nonproducers of the immortalizing virus and are nontumorigenic. They grow in monolayer, have intermediate growth kinetics, and express markers of renal proximal tubular epithelium by immunohistochemistry. They also express biochemical properties comparable to other widely used proximal tubular cell lines including LLC-RK1, OK, and HK-2 and comparable to human tubular cells in stable culture. Growth medium containing low levels of fetal calf serum, or epidermal growth factor combined with parathyroid hormone, produced optimal growth characteristics, brush border enzyme expression, biochemical properties, and glucose transport in a selected cell line. The addition of dimethyl sulfoxide allows maintenance in morphologically intact monolayers for prolonged periods. These cell lines should be useful model systems for the study of human renal proximal tubular injury or disease.

Role of reactive oxygen species in cocaine-induced cardiac dysfunction.

OBJECTIVE Contractility alterations and LV hypertrophy after chronic cocaine administration have been shown to be accompanied by an increase in oxidative stress. This study was carried out to investigate whether the production of reactive oxygen species is an early event of primary importance in cocaine-induced myocardial injury or simply occurs as a consequence of the ventricular dysfunction itself. METHODS AND RESULTS After 2 days of cocaine administration to rats, no differences were observed in echocardiographic parameters between the cocaine-treated group and the control group. However, an increase in oxidative stress in the myocardium was indicated by an increase in lipid peroxidation (+35%, cocaine vs. control), an increase in antioxidant enzymes (catalase +110%, glutathione peroxidase +40% and superoxide dismutase +38%) and of NADPH-driven superoxide production (assessed by chemiluminescence). Furthermore, higher gp91phox and p22phox mRNA expression, measured by quantitative real-time RT-PCR, was found in the cocaine group. On day 8, cocaine administration induced a cardiac dysfunction, characterized by a decrease in cardiac index (-30%, cocaine vs. controls) and left ventricular (LV) fractional shortening (-23%, cocaine vs. controls). This LV dysfunction was prevented by antioxidant treatment (100 mg/kg/day vitamin C and 100 U/kg/day vitamin E). Moreover, in these animals, antioxidant treatment decreased lipid peroxides and decreased the activity of NADPH oxidase, associated with the downregulation of gp91phox. CONCLUSION These data indicate that cocaine administration induces early NADPH-driven O2-. release which may play an important role in the development and progression of the LV dysfunction observed after chronic cocaine abuse.

Reduced cardiac remodelling and prevention of glutathione deficiency after omega‐3 supplementation in chronic heart failure

n‐3 polyunsaturated fatty acids (omega‐3) supplementation is associated with reduced cardiovascular mortality and post‐infarction death. However, the impact of omega‐3 supplementation in congestive heart failure (CHF) is still unknown. This study assesses the effects of omega‐3 supplementation on left ventricular (LV) function and remodelling. We assessed, in rats with CHF induced by left coronary ligation, the effects of a 1‐week and a 12‐week supplementation with omega‐3 (450 mg/kg per day) on LV hemodynamics, function and structure. Chronic omega‐3 reduces total peripheral resistance due to an increase in cardiac output without modification of arterial pressure. Only chronic omega‐3 reduces LV end‐diastolic pressure and LV relaxation constant. Moreover, chronic omega‐3 decreases LV systolic and diastolic diameters, LV weight and collagen density. Acute and chronic omega‐3 increase LV γ‐glutamyl‐cysteine synthetase and oppose glutathione deficiency resulting in a reduction of myocardial oxidized glutathione. In experimental CHF, long‐term omega‐3 supplementation improves LV hemodynamics and function and prevents LV remodelling and glutathione deficiency. The latter might be one of the mechanisms involved, but whether other mechanism, independent of myocardial redox ‘status’, such as reduced inflammation, are implicated remains to be confirmed.

Selenium diet-supplementation improves cocaine-induced myocardial oxidative stress and prevents cardiac dysfunction in rats

Chronic cocaine abuse causes cardiac dysfunction and induces oxidative stress. The goal of this study was to evaluate whether an enhanced antioxidant pool, induced by the administration of selenium, may prevent the myocardial dysfunction induced by cocaine. Cocaine was administered for 7 days (15 mg/kg/day, i.p.) to rats pretreated for 4 weeks with selenium (1.16 mg/L/day, p.o.). Cardiac function was evaluated by cardiac index and left ventricular (LV) fractional shortening (FS) measured by echocardiography. The redox ratio and enzymatic activities of glutathione peroxidase (GPX) and superoxide dismutase (SOD) were measured in the LV myocardium. Cocaine administration induced a cardiac dysfunction, as evidenced by a decrease in cardiac index and LV FS as well as by an increase in LV diameters. Moreover, antioxidant markers and redox ratio were altered in rats after cocaine exposure. Selenite supplementation induced a significant limitation of cardiac index and FS alterations observed after cocaine administration. This improvement in cardiac function was associated with a redox ratio recovery while SOD and GPX activities remained unchanged. Thus, selenite reversed both the oxidative stress and the contractile dysfunction induced by cocaine administration. These results suggest a major role of oxidative stress in the cocaine‐induced cardiotoxicity.

Insulin and Glucose Impact on Functional and Morphological Differentiation of Rabbit Proximal Tubule Cells in Primary Culture

Glucose and insulin impact on cellular phenotype expression was assessed on rabbit proximal tubule cells in primary culture. Glycolysis, gluconeogenesis pathways and lysosomal, mitochondrial, detoxication system, apical and basolateral membrane marker enzyme activities were assessed. Both insulin and glucose deprivation partially prevented the rise in glycolysis and delayed the drop of gluconeogenesis pathways. Scanning electron microscopy analysis of the apical surface of the monolayer revealed a much higher density of microvilli in glucose-free cultures compared to cultures developed with glucose. In conclusion, culture medium deprivation in both glucose and insulin allowed closer functional, biochemical and morphological properties to those which exist in the in vivo situation for proximal tubule cells.

Characterization of Gentamicin-Induced Dysfunctions In Vitro: The Use of Optimized Primary Cultures of Rabbit Proximal Tubule Cells

Compared to prior studies which frequently pinpoint the impairment of one parameter or function, this paper reports for the first time an extensive characterization of the toxic effects of gentamicin in a single model of primary cultured rabbit proximal tubule cells developed without insulin and glucose. Biochemical, functional and morphological approaches were used. Cellular response pattern was examined after a 72-h exposure during either the exponential growth phase or the stationary confluency phase of the culture to 0.2, 1, and 2.5 mM gentamicin. The biochemical study after gentamicin exposure showed increased activities for N-acetyl-beta-D-glucosaminidase and alkaline phosphatase, decreased activities for sphingomyelinase, cathepsin B, Na+/K(+)-ATPase, lactate dehydrogenase and NADPH cytochrome C reductase. Functional evaluation revealed decreased protein synthesis and alpha-methylglucose transport after gentamicin exposure. Morphometric study made it possible to show that the density of lysosomes, the cell fractional volume of the lysosomal compartment, and the mean size of the lysosomal profiles are increased in the cells. Intracellular accumulation of gentamicin in proximal tubular cells was dose dependent and reached high levels in cultured cells. In conclusion, this model compared to others in the literature allowed us to demonstrate in vitro a close response pattern to the in vivo situation after gentamicin exposure.