C. Barlet-Bas

Mechanism of increased tubular Na-K-ATPase during streptozotocin-induced diabetes

Since the mechanisms responsible for stimulation of kidney Na-K-ATPase during streptozotocin-induced diabetes are unknown, we studied the possible role(s) of kidney hyperfiltration and hypertrophy and of hyperaldosteronism on Na-K-ATPase induction. For this purpose, we studied the relationship between Na-K-ATPase activity in individual nephron segments and alterations of glomerular filtration rate during the early phase of diabetes. Within 2 days after streptozotocin administration, Na-K-ATPase activity markedly increased in the proximal convoluted tubule, medullary thick ascending limb and cortical and outer medullary collecting tubule, but not in the proximal straight tubule, cortical thick ascending limb and distal convoluted tubule. Streptozotocin administration also markedly enhanced the glomerular filtration rate but only after 4 days following initiation of treatment. Changes in Na-K-ATPase were specific since the activity of adenylate cyclase, another marker of basolateral membranes, was not altered. Finally, when animals were adrenalectomized prior to streptozotocin treatment, Na-K-ATPase stimulation was curtailed in the collecting tubule but not in more proximal segments. These results suggest that diabetes alters Na-K-ATPase activity in specific nephron segments independent of alterations of glomerular filtration rate and of kidney hypertrophy, and that the stimulation of collecting tubule Na-K-ATPase is secondary to hyperaldosteronism.

Presence of two isoforms of Na, K-ATPase with different pharmacological and immunological properties in the rat kidney

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the α1 isoform of the catalytic subunit, whereas the collecting duct expresses an α3-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC50 ≈ 5.10−6 M) which is recognized by an anti-α3 antibody and another moiety of lower affinity for ouabain (IC50 ≈ 5.10−4 M) which is recognized by an anti-α1 antibody. Whether these two subpopulations correspond to different isoforms of the α subunit of Na,K-ATPase (α1 and α3-like) remains to be determined.

REGULATION OF RENAL NA+,K+-ATPASE IN RAT THICK ASCENDING LIMB DURING K+ DEPLETION : EVIDENCE FOR MODULATION OF NA+ AFFINITY

1. NaCl reabsorption along the loop of Henle is reduced in K(+)‐depleted rats. Because Na+,K(+)‐ATPase energizes this transport and because K+ depletion is known to induce an upregulation of Na+,K(+)‐ATPase in most tissues, the regulation of this enzyme was investigated at the level of single thick ascending limbs of the loop of Henle freshly microdissected from rats fed either a normal (control rats) or a low‐K+ diet (LK rats). 2. Within 2 weeks of K+ depletion, Na+,K(+)‐ATPase activity and [3H]ouabain binding were increased by 30‐50% in the medullary portion of the thick ascending limb (MTAL). 3. Despite this increase in the number of Na+,K(+)‐ATPase units, the transport capacity of the Na+,K+ pump, determined by ouabain‐sensitive Rb+ uptake in the presence of an extracellular concentration of Rb+ mimicking the kalaemia determined in control (4.0 mM Rb+) and LK rats (2.3 mM Rb+), was reduced in MTAL from LK rats. 4. Inhibition of the Na+,K+ pump was not accounted for by changes in either extracellular K+ or intracellular Na+ concentrations, but by a decrease in the pump affinity for Na+. 5. Because this change in the apparent affinity of the Na+,K+ pump for Na+ was detectable in intact but not in permeabilized MTAL cells, it is probably induced by a rapidly reversible cytosolic factor.

Characterization and localization of ouabain-insensitive Na-dependent ATPase activities along the rat nephron

Single segments of rat nephron contain two distinct ouabain-insensitive, K-independent, Na-dependent ATPase activities: a Na-stimulated ATPase and a Na-inhibited ATPase. Na-inhibited ATPase activity is found in the proximal tubule and the thick ascending limb of Henles loop but is absent in the collecting tubule whereas Na-stimulated ATPase is exclusively located in the proximal convoluted tubule. Na-inhibited ATPase, but not Na-stimulated ATPase, is totally abolished in the presence of 100 microM Ca2+. Conversely, Na-stimulated ATPase, but not Na-inhibited ATPase, is curtailed when nephron segments are preincubated at pH 7.2 whereas it is activated at pH 7.8. Finally, Na-stimulated ATPase displays an apparent Km for Na+ of approximately 10 mM, and is dose-dependently inhibited by the diuretic triflocin (IC50 approximately 6 x 10(-6) M).

Chapter 10 Involvement of Na+,K+-ATPase in Antinatriuretic Action of Mineralocorticoids in Mammalian Kidney

Publisher Summary Although mineralocorticoids have long been known to enhance the reabsorption of sodium and the secretion of potassium and protons in the kidney, the exact sites and mechanisms of these actions in the nephron are not clearly established as yet. Indeed, most of our knowledge on the mechanism of control of sodium transport by mineralocorticoids derives from studies on frog skin or toad bladder. These studies have been very fruitful, especially as these epithelia can be mounted in Ussing chambers, allowing electrophysiological and flux measurements. However, the mechanism of action of aldosterone might not be similar in these epithelia and in the kidney, as the latter has a much higher capacity of sodium transport. This problem was circumvented in part by the development of established cell lines (A6 cells) derived from the kidney of Xenopus laewis. When grown on collagen-coated filters mounted on plastic rings, these cells develop as a polarized monolayer that can be studied like a natural planar epithelium.

Effects of cold exposure and hibernation on renal Na,K-ATPase of the jerboa Jaculus orientalis

Changes in activity and abundance of renal Na,K-ATPase were evaluated during cold exposure and hibernation of the jerboa Jaculus orientalis by measuring the hydrolytic activity, the number of units and the transport activity of Na,K-ATPase in isolated nephron segments. As compared to controls, jerboas exposed to cold (6 °C) for 4–5 weeks displayed mild diuresis, decreased urinary osmolality and increased kaliuresis. In cold-exposed jerboas, Na,K-ATPase hydrolytic activity was reduced in the medullary thick ascending limb and enhanced in the cortical and outer medullary collecting duct, whereas it was not altered in other nephron segments. The number of Na,K-ATPase units and the activity of Na,K-pump, determined by [3H]-ouabain binding and by ouabain-sensitive rubidium uptake respectively, changed in parallel with the hydrolytic activity in the medullary thick ascending limb and cortical collecting duct. The maximal rate of activity (Vmax) of Na,K-ATPase was not modified further during hibernation. Thus, cold exposure, but not the onset of hibernation, induces segment-specific changes in the abundance and activity of Na,K-ATPase units which are likely to be related to the entry into hibernation, but not to the maintenance of some renal functions during deep hibernation.

Characterization of the molecular isoforms of Na+/K+-ATPase submits along the rat nephron by polymerase chain reaction

It is commonly admitted that the kidney only expresses the α1 and β1 isoforms of Na+/K+-ATPase subunits. However, previous results from our laboratory indicate an heterogeneity of Na+/K+-ATPase along the successive segments constituting the rabbit nephron: The collecting tubule displays a higher affinity for ouabain (4) and for sodium (2) than more proximal segments such as the proximal tubule and the thick ascending limb of Henle’s loop. Furthermore, Na+/K+-ATPase from rabbit collecting duct is recognized by an anti-α3 antibody but not by an anti-α1 antibody, whereas the opposite is observed in nephron segments located upstream (1). These findings suggest that the rabbit collecting duct preferentially expresses an α3-like isoform of Na+/K+-ATPase catalytic subunit, whereas other nephron segments would express the α1 isoform.