Stimulation of an N-ethylmaleimide-sensitive ATPase in the collecting duct segments of the rat nephron by metabolic acidosis

A plasma membrane ATPase sensitive to inhibition by N-ethylmaleimide (NEM) and insensitive to inhibition by oligomycin and ouabain has been shown to be involved in acidification of urine in the turtle bladder. The activity of this NEM-sensitive ATPase was determined in four types of distal nephron segments of normal rats and in rats treated with ammonium chloride. The enzyme activity was determined by a fluorometric micromethod in which ATP hydrolysis was coupled to NADH oxidation. Significant activities (10-35 pmol ADP X min-1 X mm-1) of NEM-sensitive ATPase were present in the distal convoluted tubule (DCT) and in the cortical and outer and inner medullary collecting duct segments of normal rats. In metabolic acidosis produced by ammonium chloride treatment (plasma CO2 content = 15.3 +/- 0.8 mequiv./L), the NEM-sensitive ATPase activity was increased significantly (60-100%) in the collecting duct segments without showing a significant change in the enzyme activity in the DCT. Our data are consistent with the hypothesis that a plasma membrane H+-ATPase (inhibited by NEM but not by oligomycin or ouabain) is involved in H+ secretion in the mammalian collecting duct.     

Basolateral glucose transport in distal segments of the dog nephron

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.     

Characterization of N-ethylmaleimide-sensitive proton pump in the rat kidney. Localization along the nephron

This study is aimed both at characterizing an ATPase activity in rat kidney equivalent to the proton pump described in bovine kidney medulla and at localizing this enzyme along the nephron. Membrane fractions isolated from kidney homogenates by differential and density gradient centrifugations were enriched 7-fold in ATPase activity sensitive to N-ethylmaleimide (NEM). These fractions also displayed ATP-dependent proton transport. ATPase activity and proton transport in vesicles had similar pharmacological properties as both were insensitive to vanadate and ouabain and had similar sensitivities toward NEM (apparent Ki = 20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 50 microM). Proton transport was dependent on chloride availability as chloride addition to the extravesicular medium stimulated proton transport in a dose-dependent fashion (apparent K 1/2 = 7 mM). NEM-sensitive ATPase activity displaying similar pharmacological properties as proton transport in vesicles was also found in single segments of nephron. It was insensitive to vanadate and ouabain, was inhibited by similar concentrations of NEM (apparent Ki = 15-20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 30 microM), and is therefore likely to be a proton pump. NEM-sensitive ATPase was localized in all the segments of the rat nephron; its activity was highest in proximal convoluted tubules; intermediate in proximal straight tubules, thick ascending limbs, and cortical collecting tubules; and lowest in outer medullary collecting tubules.     

Differential expression of E-cadherin,N-cadherin and beta-catenin in proximal and distal segments of the rat nephron.

Background  

The classical cadherins such as E- and N-cadherin are Ca²⁺-dependent cell adhesion molecules that play important roles in the development and maintenance of renal epithelial polarity. Recent studies have shown that a variety of cadherins are present in the kidney and are differentially expressed in various segments of the nephron. However, the interpretation of these findings has been complicated by the fact that the various studies focused on different panels of cadherins and utilized different species. Moreover, since only a few of the previous studies focused on the rat, information regarding the expression and localization of renal cadherins in this important species is lacking. In the present study, we have employed dual immunofluorescent labeling procedures that utilized specific antibodies against either E- or N-cadherin, along with antibodies that target markers for specific nephron segments, to characterize the patterns of cadherin expression in frozen sections of adult rat kidney.     

Differential regulation of ROMK (Kir1.1) in distal nephron segments by dietary potassium

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.     

Purification of N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

An N-ethylmaleimide-sensitive ATPase was purified 100-fold from chromaffin granule membranes. The purification procedure included solubilization with polyoxyethylene 9 lauryl ether, chromatography on hydroxylapatite and DEAE-cellulose columns, and glycerol gradient centrifugations. Inclusion of phosphatidylserine and a mixture of protease inhibitors during the purification procedure was necessary to maintain the activity of the preparation. The purified preparation contained four major polypeptides with molecular masses of about 115, 72, 57, and 39 kDa, which were copurified with the ATPase activity. The 115-kDa subunit binds [14C]dicyclohexylcarbodiimide and the subunits of 115 and 39 kDa bind [14C]N-ethylmaleimide. The ATP-dependent proton uptake activity of chromaffin granule membranes is inhibited 50% with about 20 microM N-ethylmaleimide, while over 5 mM concentrations of the inhibitor were required to block the ATPase activity of the membranes. The ATPase activity of the purified enzyme was inhibited via two different affinities: a high affinity site with a Ki in the microM range and a low affinity site in the mM range, each contributing to about 50% inhibition of the enzyme. It is concluded that the proton-ATPase of chromaffin granule membranes contains at least four subunits with the 115-kDa polypeptide being the main subunit having the active site for the ATPase activity of the enzyme.     

Ultrastructure and functions of the cells of proximal and distal segments of the nephron of anadromous and freshwater fishes]

In the carp kidney, the proximal tubule cells have no infoldings of the basilar plasmatic membrane. In the Salmonidae nephrons the numerous parallel membranes of proximal tubule cell are formed by foldings of the basilar plasmatic membrane with numerous mitochondria in between. In O. nerka and S. malma experiments with MgCl2 injection showed that their kidneys secrete magnesium in contrast to the carp kidney. The numerous invaginations of proximal tubule cells membrane are suggested to be a morphological equivalent of the Na/Mg exchange mechanism.     

Decrease in N-ethylmaleimide-sensitive ATPase activity in collecting duct by metabolic alkalosis

Changes in systemic acid-base balance are known to influence acidification in the collecting duct. The H+ secretion in the collecting duct has been shown to be an electrogenic process and it has been suggested that an H-ATPase sensitive to inhibition by N-ethylmaleimide (NEM) is responsible for H+ secretion. This study was designed to determine the effect of metabolic alkalosis on NEM-sensitive ATPase activity in the microdissected segments of the distal nephron. Metabolic alkalosis was produced by giving NaHCO3 to normal rats for 7 days. The plasma total CO2 concentration in the experimental group was 31.5 +/- 1.8 mM compared with 23.4 +/- 1.0 mM in the control group. NEM-sensitive ATPase activity was significantly lower in the cortical collecting duct and in the outer and inner medullary collecting ducts of alkali-loaded rats than those of control rats. There was no significant difference in the enzyme activity between the two groups of animals in the other nephron segments examined. Our results suggest that NEM-sensitive H-APTase activity in all three segments of the collecting duct is modulated by the acid-base status of the animal.     

Cytochemical observations on the transport of horseradish peroxidase in different segments of the nephron

Synopsis Kidney slices from rats injected with horseradish peroxidase (HRP) 5–10 min before sacrifice were fixed with formaldehyde vapour for 4 hr at 37°C and compared with tissue fixed by perfusion or by immersion. Much more of the injected protein was retained in extracellular and vascular spaces of the nephron in vapour-fixed than in perfusion-fixed or immersion-fixed tissue. The extracellular localization of HRP in the lateral intercellular spaces and in the infoldings of the basal cell membranes showed characteristic differences in different segments of the nephron. The high concentration of HRP in the lateral intercellular spaces of the collecting tubules, as well as the early location of small phagosomes containing HRP in the apical, lateral, and basal cell regions suggested that HRP was reabsorbed through the cytoplasm into the intercellular spaces or excreted in the opposite direction. The intercellular spaces in the terminal segments of the proximal tubules also showed high concentrations of HRP which suggests participation of these spaces in protein transport between the lumen and the peritubular capillaries. The extracellular concentration of HRP early after injection was found, by colorimetric assays of homogenates, to be several times higher in the papilla than in the cortex.     

ATP directly enhances calcium channels in the luminal membrane of the distal nephron.

Calcium (Ca(2+)) transport by the distal tubule (DT) luminal membrane is regulated by the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers, protein kinases, and ATP as the phosphate donor. In this study, we questioned whether ATP itself, when directly applied to the cytosolic surface of the membrane could influence the Ca(2+) channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca(2+) uptake by these vesicles loaded with ATP or the carrier. The presence of 100 microM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca(2+) uptake from 0.57 +/- 0.02 to 0.71 +/- 0.02 pmol/microg per 10 sec (P < 0. 01) in the absence of Na(+) and from 0.36 +/- 0.03 to 0.59 +/- 0.01 pmol/microg per 10 sec (P < 0.01) in the presence of 100 mM Na(+). This effect was dose dependent with an EC(50) value of approximately 40 microM. ATP action involved the high-affinity component of Ca(2+) transport, decreasing the Km from 0.08 +/- 0.01 to 0.04 +/- 0.01 mM (P< 0.02). Replacement of the nucleotide by the nonhydrolyzable ATPgammas abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, we also investigated the effect of intravesicular cytochalasin on Ca(2+) transport. Inclusion of 20 microM cytochalasin B decreased 0.5 mM Ca(2+) uptake from 0.33 +/- 0.01 to 0.15 +/- 0.01 pmol/microg per 10 sec (P< 0.01). However, when both 100 microM ATP and 20 microM cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca(2+) uptake by the PT luminal membrane. We conclude that the high-affinity Ca(2+) channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca(2+) channels within this membrane.     

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 Henle's 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).