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Characteristics of cells isolated from the medullary thick ascending limb of henle's loop
Biochemical
aspects
of kidney
function
V
Na-K-ATPase ALONGTHE MAMMALIAN NEPHRON College
Alain de France,
Doucet, Francois Morel and Adrian I. Katz Paris, France and University of Chicago, Chicago,
USA
To define the nephron sites where changes in Na-K-ATPase activity occur’during physiologic adaptations we developed a simple micromethod sensitive enough to deterIndividual tubule segments were mine ATPase activity in single nephron segments. dissected understereomicroscopicobservation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 ~1 assay medium. Enzymatic activity was determined by direct hydrolysis of [y-32P]ATP and was expressed as a function of tubule length. The Na-K-ATPase activity profile was similar along the rat, mouse and rabbit nephron, with absolute levels of the enzyme decreasing in this order. Enzyme activity per mm tubule length was highest in the distal convoluted tubule and the thick ascending limb of Henle’s loop, intermediate in the proximal convoluted tubule and lower in the pars recta and collecting tubule. The activity measured in the thin limbs of Henle’s loop was very low, and close to the sensitivity limit of the method. In mice chronically loaded with potassium, basal Na-K-ATPase activity was modified in specific segments of the nephron: enzyme activity increased by 200% in the cortical and medullary collecting tubule over that measured in the same structures in control mice, while it remained unaffected in other segments of the nephron. These results suggest that the collecting tubule is the major site of renal potassium adaptation in this species. Such correlations of Na-K-ATPase activity in single tubules with functional measurements obtained with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.
CHARACTERISTICSOF CELLS ISOLATEDFROMTHE MEDULLARY THICK ASCENDINGLIMB OF HENLE’S LOOP Jill Eveloff, E. Bayerdgrffer and Rolf Kinne Max-Planck-Institut fur Biophysik, Heinrich Hoffman Str. 6000 Frankfurt (am Main) 71, Germany.
7,
The thick ascending limb of Henle’s loop (TALH) is integrally involved in the countercurrent mechanism for urinary concentration and dilution. It is the site of action of many diuretic drugs, such as furosemide, which act by inhibiting the active transport of chloride out of the tubular lumen. In order to further characterize the physiology of this tubular segment, cells from the TALH were isolated from rabbit renal medulla. Tubules were obtained by incubating medullary fragments in 0.2% collagenase-0.25% hyaluronidase for lh at 37’C. Then single cells were prepared by 8 successive digestions with 0.25% trypsin for 20 min at 20°C. The medullary cells were placed on a continuous ficoll gradient (2.6-30.1%) and centrifuged at 1400 xg for 45 min. A dense cell fraction was obtained (d = 1.124), which compared to the starting cell fraction was enriched 5.8 times in calcitonin-stimulated adenylate cyclase, but showed low arginine vasopressin-stimulated adenylate cyclase (0.3 times enriched). Na-K-ATPase was enriched 4.2 times, whereas alkaline phosphatase activity was decreased. Oxygen consumption (QO2) of the cells was inhibited up to 50% by the diuretics furosemide and bumetanide in a dose dependent manner and was also sensitive to the Na and Cl concentration in the incubation medium. Thus, the data support the thesis that the cells in this dense fraction are derived from the TALH. Despite the long digestion period with proteolytic enzymes, the cells were intact as judged by several viability tests: Q02 was 33.6 Pl 02/mg protein.h and only stimulated 1.3% by 1mMsuccinate but 88% by 0.05 mM 2,4 dinitrophenol. The Na and K levels of the cells were 9 mM/L cell H20 and 139 mM/L cell H20, respectively and ouabain inhibited Q02 by 51%, indicating that in these cells the plasma membranes were intact and that metabolism and active ion transport processes were tightly coupled. Thus, this cell preparation might serve to elucidate the cellular metabolism, the molecular mechanisms of chloride transport and the action of diuretics in the medullary TALH. This project was partially supported by USPHS Grant AM 05841.
aspects
of kidney
function
V
Na-K-ATPase ALONGTHE MAMMALIAN NEPHRON College
Alain de France,
Doucet, Francois Morel and Adrian I. Katz Paris, France and University of Chicago, Chicago,
USA
To define the nephron sites where changes in Na-K-ATPase activity occur’during physiologic adaptations we developed a simple micromethod sensitive enough to deterIndividual tubule segments were mine ATPase activity in single nephron segments. dissected understereomicroscopicobservation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 ~1 assay medium. Enzymatic activity was determined by direct hydrolysis of [y-32P]ATP and was expressed as a function of tubule length. The Na-K-ATPase activity profile was similar along the rat, mouse and rabbit nephron, with absolute levels of the enzyme decreasing in this order. Enzyme activity per mm tubule length was highest in the distal convoluted tubule and the thick ascending limb of Henle’s loop, intermediate in the proximal convoluted tubule and lower in the pars recta and collecting tubule. The activity measured in the thin limbs of Henle’s loop was very low, and close to the sensitivity limit of the method. In mice chronically loaded with potassium, basal Na-K-ATPase activity was modified in specific segments of the nephron: enzyme activity increased by 200% in the cortical and medullary collecting tubule over that measured in the same structures in control mice, while it remained unaffected in other segments of the nephron. These results suggest that the collecting tubule is the major site of renal potassium adaptation in this species. Such correlations of Na-K-ATPase activity in single tubules with functional measurements obtained with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.
CHARACTERISTICSOF CELLS ISOLATEDFROMTHE MEDULLARY THICK ASCENDINGLIMB OF HENLE’S LOOP Jill Eveloff, E. Bayerdgrffer and Rolf Kinne Max-Planck-Institut fur Biophysik, Heinrich Hoffman Str. 6000 Frankfurt (am Main) 71, Germany.
7,
The thick ascending limb of Henle’s loop (TALH) is integrally involved in the countercurrent mechanism for urinary concentration and dilution. It is the site of action of many diuretic drugs, such as furosemide, which act by inhibiting the active transport of chloride out of the tubular lumen. In order to further characterize the physiology of this tubular segment, cells from the TALH were isolated from rabbit renal medulla. Tubules were obtained by incubating medullary fragments in 0.2% collagenase-0.25% hyaluronidase for lh at 37’C. Then single cells were prepared by 8 successive digestions with 0.25% trypsin for 20 min at 20°C. The medullary cells were placed on a continuous ficoll gradient (2.6-30.1%) and centrifuged at 1400 xg for 45 min. A dense cell fraction was obtained (d = 1.124), which compared to the starting cell fraction was enriched 5.8 times in calcitonin-stimulated adenylate cyclase, but showed low arginine vasopressin-stimulated adenylate cyclase (0.3 times enriched). Na-K-ATPase was enriched 4.2 times, whereas alkaline phosphatase activity was decreased. Oxygen consumption (QO2) of the cells was inhibited up to 50% by the diuretics furosemide and bumetanide in a dose dependent manner and was also sensitive to the Na and Cl concentration in the incubation medium. Thus, the data support the thesis that the cells in this dense fraction are derived from the TALH. Despite the long digestion period with proteolytic enzymes, the cells were intact as judged by several viability tests: Q02 was 33.6 Pl 02/mg protein.h and only stimulated 1.3% by 1mMsuccinate but 88% by 0.05 mM 2,4 dinitrophenol. The Na and K levels of the cells were 9 mM/L cell H20 and 139 mM/L cell H20, respectively and ouabain inhibited Q02 by 51%, indicating that in these cells the plasma membranes were intact and that metabolism and active ion transport processes were tightly coupled. Thus, this cell preparation might serve to elucidate the cellular metabolism, the molecular mechanisms of chloride transport and the action of diuretics in the medullary TALH. This project was partially supported by USPHS Grant AM 05841.