Reagents Anti HO antibody was purchased
Anti-HO-1 antibody was purchased from Assay Design and R&D systems respectively. Anti-β-actin antibody, heme (hemin) and desferoxamine (DFO), were obtained from Sigma-Aldrich. Hemopexin deficient (HPX−) serum was a kind gift from Dr. EmanualaTolosano, Molecular Biotechnology Center, University of Torino, Italy.
Rats Adult male Sprague-Dawley rats, 300g in body weight, were employed in this study. Animals were reared in accordance to the European Union Directive for care and use of laboratory animals and all procedures were approved by the Hellenic Veterinary Administration and the ethical committee of ‘Evangelismos’ Hospital.
Isolation and treatment of glomeruli Glomeruli were isolated from kidneys of wild type (WT) rats by an established differential sieving method  and incubated at 37˚C in a 5% CO2 environment in Dulbecco’s modified Eagle׳s medium (DMEM) containing 10% complete (HPX+) serum or defined amounts (v/v) of HPX−serum. Glomeruli were incubated with defined concentrations of hemin dissolved in dimethylsulfoxide (DMSO) in the presence of HPX+ or HPX−serum. Negative control samples consisted of glomeruli incubated with vehicle (DMSO) only. Protein extracts were prepared using lysis buffer (150mM NaCl, 50mM Tris D-Luciferin 8.0, and 1% Triton X containing a protease inhibitors cocktail) and concentration was determined by the Bradford assay. RNA was extracted by an established Trizol-based method.
Cell culture Primary rat GEC were a kind gift of Dr. B.S. Kasinath, Nephrology Division, (University of Texas at San Antonio). Cells were routinely cultured in Dulbecco׳s Modified Eagle׳s Medium (DMEM) containing 10% Fetal Bovine serum (FBS) in a humidified incubator with 95% air and 5% CO2.
Reverse transcription reaction and Real-time PCR amplification Glomerular RNA concentration was determined by spectrophotometry. Reverse transcription reactions were performed using TaqMan Reverse Transcription Reagents kit (Applied Biosystems). Real-time PCR was carried out at the following conditions: 25°C for 10min, 48°C for 30min and 95°C for 5min. Each reaction consisted of 2μl primer-probe assay mix (IDT), 10μl Master Mix (Applied Biosystems) and 8μl cDNA. Values were analysed by the ΔΔCT method.
Statistical Analyses Values are presented as mean±SE (standard error). Statistical analyses were performed with either t-test, where applicable, or analysis of variance (ANOVA) for more than two group comparisons. When significant, post hoc analysis was performed, with the least significant difference (LSD) test. A p value<0.05 was chosen as statistically significant.
Acknowledgments We would like to thank Ms Zoe Kolia for technical assistance on animal work. This study was co-financed by European Union (European Social Fund – ESF) and Greek National Funds through the Operational Program ‘‘Education and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF)-Research Funding Program: Aristeia I to E. A. L. and supported by a Research and Experimental center of ELPEN Pharmaceutical company scholarship to M. G. D.
Data The data of an animal experiment on the effects of methacholine (MCh) infusion on desflurane (DES) uptake and elimination are shared with this data article. It comprises baseline hemodynamics, gas exchange and ventilation data in healthy piglets, and cardiopulmonary data during MCh infusion. The mean values of hemodynamics, ventilation, and gas exchange prior to and during MCh infusion are displayed in Tables 1 and 2, respectively. The experimental protocol is presented in Fig. 1. Multiple Inert Gas Elimination Technique (MIGET) was used to determine alveolar ventilation and pulmonary perfusion distribution (V.A/Q.) before and during MCh infusion. The median values of MIGET variables and IQR are given in Table 3. The fractional alveolar ventilation and pulmonary perfusion in relation to the V.A/Q. compartments is displayed in Fig. 2.