A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse

The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H+-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2′,7′-bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na+-dependent and -independent intracellular pH (pHi) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na+-dependent pHi-recovery was 0.144±0.008 (PT), 0.182±0.013 (CCD), and 0.112±0.010pH units/min. (ISOMCD). Na+-independent pHi recovery was found in all three segments (PT: 0.021±0.002, CCD: 0.037±0.002, ISOMCD: 0.033±0.002pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H+-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animal

Functional and pharmacological characterization of human Na+-carnitine cotransporter hOCTN2

L-Carnitin ist notwendig für der Translokation langkettiger Fettsäuren über die innere Mitochondrienmembran und somit für die anschließende Energiegewinnung durch b-Oxidation. Carnitin selbst wird über den kürzlich klonierten plasmamembranständigen hochaffinen Na+/Carnitin-Cotransporter hOCTN2 in die Zelle transportiert. Dieser Transporter gehört zu der Familie der organischen Kationentransporter (OCT). Ziel der vorliegenden Arbeit war eine funktionelle und pharmakologische Charakterisierung von hOCTN2 mittels Two-Electrodes-Voltage-Clamp und Flux-Messungen an hOCTN2-injizierten Xenopus laevis Oozyten. Der L-Carnitintransport durch hOCTN2 war Na+-abhängig, elektrogen und sättigbar. Die Halbsättigungskonzentration für Natrium betrug 0.3 ± 0.1 mM bei -60 mV. Es zeigte sich eine leichte Stereospezifität für L-Carnitin mit einem Km-Wert von 4.9 ± 0.3 µM für L-Carnitin und einem Km-Wert von 98.3 ± 38.0 für D-Carnitin. Weiterhin wurde der Einfluß des extra- und intrazellulären pH-Wertes auf die L-Carnitin-induzierten Ströme untersucht. Es fand sich eine Abnahme der L-Carnitin-induzierten Ströme bei Azidifizierung des extrazellulären Mediums. Eine intrazelluläre Ansäuerung mit Bicarbonat bewirkte eine signifikante Hemmung des Carnitinstromes um 22.3 ± 4.5 Im Gegensatz zu anderen Mitgliedern der OCT-Familie scheint hOCTN2 also ein hochaffiner Na+/Carnitin-Cotransporter zu sein, ohne Beteiligung eines Symport- oder Antiportmechanismus für andere Kationen als Natrium. hOCTN2 zeigte nur kleine Ströme für klassische Substrate anderer OCTs wie Cholin oder TEA, während sich bei rOCT1 und hOCT2 keine Ströme durch Carnitin induzieren ließen. Der hOCTN2 vermittelte L-Carnitintransport ließ sich durch einige Pharmaka hemmen, die ein sekundäres Carnitinmangelsyndrom auslösen können. Die stärksten Hemmstoffe waren Emetin, Verapamil, Quinidin, Betain und Cystein. Das Antiepileptikum Valproinsäure zeigte keinen nennenswerten inhibitorischen Effekt, was auf einen anderen HemmechL-Carnitine is essential for the translocation of acyl-carnitine into the mitochondria for β-oxidation of long-chain fatty acids. It is taken up into the cells by the recently cloned Na+-driven carnitine organic cation transporter OCTN2. Here we expressed hOCTN2 in Xenopus laevis oocytes and investigated with two-electrode voltage-clamp and flux measurements its functional and pharmacological properties as a Na+-carnitine cotransporter. L-carnitine transport was electrogenic. The L-carnitine-induced currents were voltage and Na+ dependent, with half-maximal currents at 0.3 ± 0.1 mM Na+ at -60 mV. Furthermore, L-carnitine-induced currents were pH dependent, decreasing with acidification. In contrast to other members of the organic cation transporter family, hOCTN2 functions as a Na+ - coupled carnitine transporter. Carnitine transport was stereoselective, with an apparent Michaelis-Menten constant (Km) of 4.8 ± 0.3 μM for L-carnitine and 98.3 ± 38.0 μM for D-carnitine. The substrate specificity of hOCTN2 differs from rOCT-1 and hOCT-2 as hOCTN2 showed only small currents with classic OCT substrates such as choline or tetraethylammonium; by contrast hOCTN2 mediated transport of betaine. hOCTN2 was inhibited by several drugs known to induce secondary carnitine deficiency. Most potent blockers were the antibiotic emetine and the ion channel blockers quinidine and verapamil. The apparent IC50 for emetine was 4.2 ± 1.2 μM. The anticonvulsant valproic acid did not induce a significant inhibition of carnitine transport, to a different mode of action. In summary, hOCTN2 mediates electrogenic Na+-dependent stereoselective high-affinity transport of L-carnitine and Na. HOCTN2 displays transport properties distinct from other members of the OCT family and is directly inhibited by several substances known to induce systemic carnitine deficiency

A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse

The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H+-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2′,7′-bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na+-dependent and -independent intracellular pH (pHi) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na+-dependent pHi-recovery was 0.144±0.008 (PT), 0.182±0.013 (CCD), and 0.112±0.010pH units/min. (ISOMCD). Na+-independent pHi recovery was found in all three segments (PT: 0.021±0.002, CCD: 0.037±0.002, ISOMCD: 0.033±0.002pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H+-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animal