EPR study of lipid phase in renal cortical membrane organelles from intact and cadmium-intoxicated ratsIntroductionMaterials and methodsAnimals and treatmentIsolation of membrane organelles from the kidney cortexSpin labelingESR line shape evaluationFluorometric assay of ion conductances in BBM vesiclesPresentation of data and statistical analysisResultsComparison of dynamics and ordering in the membrane organelles from rat kidney cortexEPR comparison of BBM from untreated and Cd-treated ratsIon conductance in isolated BBM from control and Cd-treated ratsDiscussionAcknowledgementsReferencesEPR study of lipid phase in renal cortical membrane organelles from intactand cadmium-intoxicated ratsMarta Zˇuvic´-Butoraca, Carol M. Herak-Krambergerb, Dubravka Krilovc,Ivan Sabolic´b, Janko N. Herakd,*aSchool of Medicine, University of Rijeka, Rijeka, CroatiabUnit of Molecular Toxicology, Institute for Medical Research and Occupational Health, Zagreb, CroatiacDepartment of Physics, Medical School, University of Zagreb, Zagreb, CroatiadDivision of Biophysics, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇic´a 1, 10001 Zagreb, CroatiaReceived 15 July 2005; received in revised form 23 September 2005; accepted 27 September 2005Available online 26 October 2005AbstractNumerous studies have demonstrated various structure/function correlations at the level of transport proteins in the kidney cell membranes andvarious intracellular organelles. However, characterization of the lipid phase of these membranes is rare. Here, we report the differences in lipidorganization and dynamics of the brush-border membranes (BBM), basolateral membranes (BLM) and endocytotic vesicles (EV), isolated fromthe kidney cortex of intact rats, studied with the EPR spectroscopy of the spin-labeled membrane lipids. The EPR spectra were analyzed bycomparing experimentally observed line shapes with the line shapes calculated according to the theoretical model developed for liquid crystals. Inthe fitting procedure, three different lipid domains were assumed, which revealed clear differences in the lipid ordering and rotational correlationtimes, as well as in the lipid partition of these domains in each of the three types of membranes. A similar approach, used to compare thespectroscopic characteristics of BBM from control and cadmium-intoxicated rats, showed significantly changed ordering and increased molecularmobility in the lipid phase of BBM from Cd-treated animals. As tested by an established fluorescence assay, the Cd-induced changes in the lipidmobility co localized with ¨5-fold higher conductance of BBM for potassium, with unchanged conductance for protons.D 2005 Elsevier B.V. All rights reserved.Keywords: Basolateral membrane; Brush border membrane; Endocytic vesicle; EPR spectroscopy; Heavy metal nephrotoxicity; Ion conductance; Membranefluidity1. IntroductionCadmium is an occupational and environmental toxic metalwith potent nephrotoxic effects in humans and experimentalanimals. Nephrotoxicity induced by chronic exposition tocadmium is manifested by various structural and functionaldamages, largely in proximal tubules, that result in an acquiredFancony syndrome with a variety of urinary symptoms of thepoor nephron functions, that include proteinuria, glucosuria,aminoaciduria, hyperosmolar polyuria, impaired secretion oforganic anions, and increased fractional excretion of monova-lent and divalent cations and anions [1–6]. Studies have shownthat Cd-induced tubular disfunctions largely result from theloss of brush-border (BBM) and basolateral (BLM) membranesand their transporters and/or from direct or indirect inhibitionof the activity of these transporters [2–14]. In our recentstudies, we provided evidence that Cd can directly damage theintegrity of plasma membranes and intracellular organelles, anddecrease endocytosis and intracellular vesicle recycling ofBBM transporters by damaging the activity of the V-ATPaseand arrangement of cytoskeleton, thus leading to the overallloss of BBM structural and functional integrity [6,14 –16].Although the detailed mechanisms of Cd toxicity inmammalian kidneys is a subject of continuous investigation,it is notorious that most of the studies in this field so far havebeen conducted at the level of membrane transporters andvarious intracellular proteins, whereas lipid phase in the renalcell membranes and intracellular organelles has been seldom0005-2736/$ - see front matter D 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamem.2005.09.022* Corresponding author. Fax: +385 1 4856 201.E-mail address: [email protected] (J.N. Herak).Biochimica et Biophysica Acta 1718 (2005) 44 – 52http://www.elsevier.com/locate/bbaaddressed [17]. Some biochemical studies of lipids inmembranes of renal cortical brush border membranes(BBM), basolateral membranes (BLM), lysosomes, andendocytic vesicles (EV; endosomes), isolated from intact rats,wereperformednearly30yearsago[18] .Thatstudyexhibited clear differences in the relative content of various(phospho)lipids in the limiting membrane of these organelles.As shown in recent studies, in the mammalian kidney Cdinduces oxidative stress and increases production of reactiveoxidative species (ROS) that enhance peroxidation of lipids,however, in unspecified membranes and/or organelles [19 –22]. These findi ngs indicate that the lipid phase may play animportant, but poorly defined role in intracellular onset anddevelopment of Cd (nephro)toxicity.In order to provide information on structure and dynam icsof the membrane lipid phase in renal membranes, which maybe organelle-specific and compromised by Cd treatment, in thisstudy, we first characterized the spin labeled lipid phase by theelectron paramagnetic resonance (EPR) spectroscopy in vari-ous organelles from the kidney cortex of control rats.Thereafter, using optimal parameters, the EPR spectroscop icdata for the renal cortical BBM from control and Cd-treatedrats were compared and correlat ed with the mem branepermeability for potassium and protons, determined by theacridine orange fluorescence quench method.2. Materials and methods2.1. Animals and treatmentThree-month-old male Wistar rats from the breeding colony at the Institutein Zagreb were used. Animals were bred and maintained according to the Guidefor Care and Use of Laboratory Animals (Washington DC, Academic Press,1996). Before and during experiments, animals had free access to standardlaboratory food and