Hyperosmolarity decreases Lcn2 expression in mIMCD3 cells and rat primary IMCD cells. a Expression levels of Lcn2 mRNA by qPCR in mIMCD3 cells exposed to 300 or 600 mosmol/l for 24-72 h. The data obtained were normalized to the expression of the reference genes glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), and β2-microglobulin (B2m). Means ± SEM of 4-5 experiments are shown. Statistical analysis compares the two osmotic conditions by unpaired t-test. b Expression of Lcn2 in mIMCD3 cells exposed to norm- or hyperosmotic media for 72 h was detected by immunofluorescence microscopy of permeabilized cells. Nuclei were counterstained with Hoechst 33342. Statistical analysis shows means ± SEM of 3 experiments. Arbitrary units (a.u.) are normalized to normosmotic media (5.0 ± 1.1 a.u.), and the two osmotic conditions are compared by unpaired t-test. c Kinetics of Lcn2 release from mIMCD3 cells exposed to norm- or hyperosmotic media for 72 h. Lcn2 release was determined by immunoblotting. The upper blot is representative of 4 different experiments. To quantify experiments, Lcn2 standards were immunoblotted (lower blot). All immunoblot bands were analyzed by densitometry using ImageJ software and normalized to cell numbers. Lcn2 release (nmol/l) is plotted as means ± SEM of 4 experiments. Statistical analysis compares the two osmotic conditions by unpaired t-test. d Lcn2 gene expression in rat primary IMCD cells cultured in norm- or hyperosmotic media for 7 days. Relative Lcn2 gene expression was determined by qPCR. Means ± SD of 3 experiments are plotted. Data are normalized to 300 mosmol/l media and compares the two osmotic conditions by unpaired t-test. e Lcn2 release from rat primary IMCD cells one week after exposure to norm- or hyperosmotic media. Lcn2 in cell culture medium was assayed by ELISA. Means ± SEM of 7 experiments are shown and statistical analysis by unpaired t-test compares the two experimental conditions.