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      Combined in vivo and in vitro Approaches to Analysis of Renal Tubule Function

      Cardiorenal Medicine

      S. Karger AG

      Micropuncture, Microperfusion, Transepithelial transport, Urine acidification, Cell pH regulation

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          The study of renal tubule function obtained its first impulse with whole kidney approaches, such as the clearance technique, and is presently centered on cellular analysis involving intracellular ion activities and on molecular studies describing the structure of transporter molecules and the function of their subunits and constitutional building blocks as well as their distribution along the nephron and within individual cells. Between these extremes a number of successful techniques have dominated the field, including the micropuncture methods which were pioneered by Richards, Walker and others in the late 1920s and 1930s, and were revived and perfectioned in the following decades leading to complex in vivo and in vitro micromethods that for an important period of time have been the center of scientific progress in this area. In the present paper, some of the methods in this field are shortly reviewed, specifically from the point of view of acid-base physiology applied to the renal tubule.

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          Chloride conductive pathways which support electrogenic H+ pumping by Leishmania major promastigotes.

          The proton extrusion mechanisms of Leishmania promastigotes were studied in terms of electrogenic movements of protons and anions (Cl- and HCO3-). Changes in membrane potential (Vm) and intracellular pH (pHi) were monitored fluorimetrically with the potential sensitive dye bis-oxonol and the pH-sensitive dye tetraacethoxymethyl 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein, respectively. In nominal bicarbonate-free medium (pHe 7.4, 28 degrees C), Vm and pHi of Leishmania promastigotes were maintained at -113 +/- 4 mV and 6.75 +/- 0.02, respectively. In Cl- free (gluconate-based) medium, cells underwent a time-dependent acidification (0.3 pH units) and a long term membrane hyperpolarization (7-10 mV), both of which were greatly enhanced in the presence of the anion blocker, 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid (H2DIDS). Cells in Cl(-)-free medium underwent a marked depolarization upon treatment with the H(+)-ATPase inhibitor dicyclohexylcarbodiimide (DCCD), but hyperpolarized after repletion with Cl-. In Cl(-)-depleted cells, replenishment of Cl- led to a H2DIDS-sensitive cytoplasmic alkalinization and a small initial hyperpolarization. Cells exposed either to DCCD or to the H+ uncoupler carbonylcyanide chlorophenylhydrazone caused a marked cytoplasmic acidification and membrane depolarization. In the presence of 25 mM HCO3-, promastigotes maintained an almost neutral cytosol, irrespective of H+ pump action or ionic composition of the medium. The present observations provide evidence for the operation of a DCCD-sensitive electrogenic H(+)-ATPase which contributes to the maintenance of a highly hyperpolarized plasma membrane in Leishmania promastigotes. H+ pump activity required a parallel pathway of Cl- ions in order to dissipate the pump generated electrical potential. In nominally CO2-free media, the two electrogenic systems are implicated in the maintenance of cell pH and indirectly in electrochemically driven nutrient uptake. In physiological CO2/HCO3(-)-containing media, the H+ pump and Cl- channel play a role only secondary to that of HCO3- in pHi homeostasis.
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            Role of Cl − in Electrogenic H + Secretion by Cortical Distal Tubule


              Author and article information

              Nephron Exp Nephrol
              Cardiorenal Medicine
              S. Karger AG
              October 1998
              11 September 1998
              : 6
              : 5
              : 454-461
              Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil
              20555 Exp Nephrol 1998;6:454–461
              © 1998 S. Karger AG, Basel

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              Page count
              Figures: 5, References: 72, Pages: 8
              Self URI (application/pdf):
              In vivo, in vitro and Mathematical Analysis ofRenal Tubular Function


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