EPR study of lipid phase in renal cortical membrane organelles from intact and cadmium-intoxicated rats

Numerous studies have demonstrated various structure/function correlations at the level of transport proteins in the kidney cell membranes and various intracellular organelles. However, characterization of the lipid phase of these membranes is rare. Here, we report the differences in lipid organization and dynamics of the brush-border membranes (BBM), basolateral membranes (BLM) and endocytotic vesicles (EV), isolated from the kidney cortex of intact rats, studied with the EPR spectroscopy of the spin-labeled membrane lipids. The EPR spectra were analyzed by comparing experimentally observed line shapes with the line shapes calculated according to the theoretical model developed for liquid crystals. In the fitting procedure, three different lipid domains were assumed, which revealed clear differences in the lipid ordering and rotational correlation times, as well as in the lipid partition of these domains in each of the three types of membranes. A similar approach, used to compare the spectroscopic characteristics of BBM from control and cadmium-intoxicated rats, showed significantly changed ordering and increased molecular mobility in the lipid phase of BBM from Cd-treated animals. As tested by an established fluorescence assay, the Cd-induced changes in the lipid mobility co localized with approximately 5-fold higher conductance of BBM for potassium, with unchanged conductance for protons

Expression and immunolocalization of metallothioneins MT1, MT2 and MT3 in rat nephron

Rodent kidneys exhibit three isoforms of metallothioneins (MTs), MT1, MT2 and MT3, with poorly characterized localization along the nephron. Here we studied in adult male Wistar rats the renal expression of MTs mRNA by end-point RT-PCR and MT proteins by immunochemical methods The expression pattern of MT1 mRNA was cortex (CO)>outer stripe (OS)=inner stripe (IS)=inner medulla (IM), of MT2 mRNA was IM>CO>IS=OS, and of MT3 mRNA was IM>CO=OS=IM. MT1/2-antibody stained with heterogeneous intensity the cell cytoplasm and nuclei in proximal tubule (PT) and thin ascending limb, whereas MT3-antibody stained weakly the cell cytoplasm in various cortical tubules and strongly the nuclei in all nephron segments. However, the isolated nuclei exhibited an absence of MT1/2 and presence of MT3 protein. In MT1/2-positive PT cells, the intracellular staining appeared diffuse or bipolar, but the isolated brush-border, basolateral and endosomal membranes were devoid of MT1/2 proteins. In the lumen of some PT profiles, the heterogeneously sized MT1/2-rich vesicles were observed, with the limiting membrane positive for NHE3, but negative for V-ATPase, CAIV, and megalin, whereas their interior was positive for CAII and negative for cytoskeleton. They seem to be pinched off from the luminal membrane of MT1/2-rich cells, as also indicated by transmission electron microscopy. We conclude that in male rats, MTs are heterogeneously abundant in the cell cytoplasm and/or nuclei along the nephron. The MT1/2-rich vesicles in the tubule lumen may represent a source of urine MT and membranous material, whereas MT3 in nuclei may handle zink and locally-produced reactive oxygen species

Spolno-neovisna ekspresija izmjenjivača klora i mravlje kiseline Cfex (Slc26a6) u gušterači, tankom crijevu i jetri štakora i povišena ekspresija u bubrezima mužjaka

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2 jejunum > ileum) i kanalikularnoj membrani hepatocita. U bubrezima je a) prijenosnik rCfex imunolokaliziran u četkastoj membrani proksimalnih kanalića sa segment-specifičnim obrascem (S1=S2 ženke) zbog stimulacijskoga učinka androgena nakon puberteta. Međutim, izlučivanje oksalata urinom nije bilo sukladno ekspresiji bubrežnoga prijenosnika rCfex. Dakle, nejasan je učinak povišene ekspresije prijenosnika rCfex u proksimalnim kanalićima mužjaka na izlučivanje oksalata, a postojanje prijenosnika u kanalikularnoj membrani hepatocita mogući je put izlučivanja oksalata putem žuči

Formation of free radicals in oxidation of human low-density lipoprotein. An EPR spin trapping study

The EPR spin trapping method with PBN as a spin trap was used to study the formation of free radicals in human low-density lipoprotein (LDL) upon ageing at 37 °C. The PBN associated radicals could be observed after 10 - 20 hours of incubation, depending on the pretreatment of LDL and the individual donor. The radicals observed in the first period of time (until about 20 - 25 hours of incubation) are related to the degradation products of PBN. After that, additional radicals associated with lipids are observed. The presence of EDTA does not prevent the process. The present experiments prove that some relatively fast oxidation process takes place in LDL incubated at physiological temperature. The process is mild, as judged from the essentially unchanged concentration of thiobarbituric acid-reactive substances (TBARS). An active role of the spin trap is not excluded

Cadmium inhibits vacuolar H(+)ATPase-mediated acidification in the rat epididymis

In rats, an acidic luminal pH maintains sperm quiescence during storage in the epididymis. We recently showed that vacuolar H(+)ATPase-rich cells in the epididymis and vas deferens are involved in the acidification of these segments. Treatment of rats with cadmium (Cd) leads to alkalinization of this fluid by an unknown mechanism. Because Cd may affect H(+)ATPase function, we examined 1) the in vivo effect of Cd poisoning on H(+)ATPase-rich cell morphology and on the abundance and distribution of the 31-kDa H(+)ATPase subunit in cells along the rat epididymis, and 2) the in vitro effect of Cd on H(+)ATPase activity and function in the isolated vas deferens. Immunofluorescence and immunoblotting data from rats treated with Cd for 14-15 days (2 mg Cd/kg body mass/day) showed that 1) H(+)ATPase-positive cells regressed to a prepubertal phenotype, and 2) H(+)ATPase was lost from the apical pole of the cell and was redistributed into an intracellular compartment. In experiments in vitro, Cd inhibited bafilomycin-sensitive ATPase activity in isolated total cell membranes and, as measured using a proton-selective extracellular microelectrode, inhibited proton secretion in isolated vas deferens. We conclude that alkalinization of the tubule fluid in the epididymis and vas deferens of Cd-treated rats may result from the loss of functional H(+)ATPase enzyme in the cell apical domain as well as from a direct inhibition of H(+)ATPase function by Cd