Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump

The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain- sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular concentration of K+, ranging from approximately 10 to 15 mM at low K+ and increasing to approximately 30 mM as the intracellular K+ was increased. The Na pump had a K0.5 for extracellular K+ of 1.3 mM in the presence of saturating concentrations of intracellular Na+. Measurements of the Na,K-ATPase activity under comparable conditions rendered similar values for the K0.5 of Na+ and K+. The Na pump activity in the intact tubules saturated as a function of extracellular Na at approximately 80 mM Na, with a K0.5 of 30 mM. Since Na pump activity under these conditions could be further stimulated by increasing Na+ entry with the cationophore nystatin, these values pertain to the Na+ entry step and not to the Na+ dependence of the intracellular Na+ site. When tubules were exposed to different extracellular K+ concentrations and the intracellular Na+ concentration was subsaturating, the Na pump had an apparent K0.5 of 0.4 mM for extracellular K. Under normal physiological conditions, the Na pump is unsaturated with respect to intracellular Na+, and indirect analysis suggests that the proximal cell may have an intracellular Na+ concentration of approximately 35 mM.     

Effect of diabetes on natriuresis in the presence of ouabain and ethacrynic acid in perfused rat kidney

1. The effect of diabetes on renal sodium retention was investigated. 2. The technique involved retrograde perfusion from the renal veins via the kidneys, and then through the renal arteries and dorsal aorta. 3. Sodium retention by diabetic rat kidney was 58% lower than that in the normal rats. 4. Ouabain (15 mM) in perfusate increased sodium retention by 30% in normal rat kidney as compared to a 54% increase in diabetic rat kidney. 5. Ethacrynic acid (1 mM) in perfusate resulted in a 42% reduction in sodium retention in the normal rat kidney as compared to a 43% decrease in the diabetic rat kidney. 6. Control of hyperglycemia in diabetic rats with insulin therapy resulted in sodium retention that is not significantly different from that of normal rats. 7. The results suggest that diabetes has no effect on the peritubular ouabain-sensitive Na--K-ATPase pump, or the luminal ethacrynic acid-sensitive Na-K counter transport pump. Furthermore, the data suggest a reversible effect of diabetes on sodium retention during insulin therapy.     

Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity

Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.     

Rheogenic transport in the renal proximal tubule

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.     

Intracellular ion activities in Necturus proximal tubule

Ion-sensitive microelectrodes were used to measure the intracellular activities of Na, K, and Cl in proximal tubules of the perfused Necturus kidney. Cell Cl was 2-3 times higher than the value predicted for passive distribution during perfusion with normal Ringer; intracellular Na was far below the level for passive distribution. Cell Na and Cl fell to very low values when the lumen was NaCl-free. Cl entry into the tubule cell from the lumen required luminal Na. Na entered the cell across the luminal membrane both by diffusion and by coupled movement with Cl.     

Active ion transport in the renal proximal tubule. III. The ATP dependence of the Na pump

The dependence of the Na pump activity of intact renal tubules on the ATP concentration was investigated using a suspension of rabbit cortical tubules. Rotenone (an inhibitor of mitochondrial oxidative phosphorylation) was used in graded fashion to alter the cellular ATP, and the Na pump activity was measured when the pump was stimulated by adding KCl to tubules suspended in a K+-free medium. The K+ uptake into the tubule was measured using an extracellular K+ electrode, and the oxygen consumption (QO2) was measured using a Clark-type oxygen electrode. The Na pump activity was found to have a linear, nonsaturating dependence on the ATP concentration. However, the Na,K- ATPase hydrolytic activity (assayed biochemically) of lysed proximal tubule membranes demonstrated saturation and had a K0.5 value of 0.4 mM ATP. Presumably, unknown cytosolic factors present in the intact renal cell but not normally present in the biochemical assay accounted for the differences between the two measurements. The data suggest that an alteration in the intracellular ATP will result in a proportional change in active ion transport activity. Moreover, additional findings also suggest that the basal (non-transport-related) QO2 may be redirected to support the proximal Na pump activity when transport activity is stressed. Thus, basal respiration is not invariant under all conditions, and ion transport activity appears to be maintained foremost among cellular ATP-dependent processes.     

Effect of catecholamines on electrolyte and water transport in the nephron of lower vertebrates

Micropuncture technique and electron microprobe analysis have been used to investigate the role of noradrenalin in ion and water balance in the renal tubules of the lamprey Lampetra fluviatilis and newt Triturus vulgaris. Noradrenalin decreased Na, K, and Ca concentrations in the proximal lumen of the lamprey increasing the value of (TF/P)in from 1.1 +/- 0.1 to 1.3 +/- 0.1 (p less than 0.001). Regitin blocked these effects. Noradrenalin perfusion of the peritubular capillaries in newt kidney increased ion and water reabsorption in the proximal segment of the nephron and resulted in differential changes of ion transport in the distal tubule, increasing reabsorption of Na, Cl and K and inhibiting that of Ca and Mg. The rate of glomerular filtration in the nephron remained practically unaffected. The data obtained reveal direct effect of noradrenalin on the renal tubular function in lower vertebrates, this effect being realized presumably via alpha-adrenoreceptors.     

Kinetics of Na(+) transport in necturus proximal tubule

The dependence of proximal tubular sodium and fluid readsorption on the Na(+) concentration of the luminal and peritubular fluid was studied in the perfused necturus kidney. Fluid droplets, separated by oil from the tubular contents and identical in composition to the vascular perfusate, were introduced into proximal tubules, reaspirated, and analyzed for Na(+) and [(14)C]mannitol. In addition, fluid transport was measured in short-circuited fluid samples by observing the rate of change in length of the split droplets in the tubular lumen. Both reabsorptive fluid and calculated Na fluxes were simple, storable functions of the perfusate Na(+) concentration (K(m) = 35-39 mM/liter, V(max) = 1.37 control value). Intracellular Na(+), determined by tissue analysis, and open-circuit transepithelial electrical potential differences were also saturable functions of extracellular Na(+). In contrast, net reabsorptive fluid and Na(+) fluxes were linearly dependent on intracellular Na(+) and showed no saturation, even at sharply elevated cellular sodium concentrations. These concentrations were achieved by addition of amphotericin B to the luminal perfusate, a maneuver which increased the rate of Na(+) entry into the tubule cells and caused a proportionate rise in net Na(+) flux. It is concluded that active peritubular sodium transport in proximal tubule cells of necturus is normally unsaturated and remains so even after amphotericin-induced enhancement of luminal Na(+) entry. Transepithelial movement of NaCl may be described by a model with a saturable luminal entry step of Na(+) or NaCl into the cell and a second, unsaturated active transport step of Na(+) across the peritubular cell boundary.     

Ion exchangers mediating NaCl transport in the renal proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.     

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.     

Transcellular mechanisms of amino acid uptake by distal rat ileum in situ

A 10 cm distal ileal intestinal perfusion technique was employed in Sprague-Dawley rats in situ. The perfused segment was removed, weighed, its surface area measured, homogenized, digested in HNO3 and assayed for L(1-14C)alanine and L-phenyl (1-14C)alanine. Steady state for L-alanine and L-phenylalanine absorption by the intact intestinal segment was observed at 10 and 15 min respectively. Exposure of the intestinal mucosa to 1 mM ouabain showed no effect on amino acid absorption. Preloading the intestinal epithelium with ouabain resulted in approximately 66% and 48% reduction in L-alanine and L-phenylalanine absorption respectively. Removal of Na from the buffer with and without exposure of the mucosa to 1 mM ouabain decreased absorption of L-alanine and L-phenylalanine by approximately 77% and 52% respectively. Removal of Na from the buffer and preloading the intestinal epithelium with ouabain resulted in approximately 85% and 81% reduction in L-alanine and L-phenylalanine absorption respectively. A 5, 10 and 25 fold increase in luminal L-alanine and L-phenylalanine concentration in Na-free choline Krebs Ringer after preloading with ouabain resulted in increase of amino acid absorption of approximately the same order of magnitude. Both an amino acid-carrier mediated transport process and a ouabain resistant Na-dependent-amino acid pump exist at the mucosal side. Both an ouabain sensitive Na-dependent-amino acid pump and an ouabain resistant Na-independent amino acid pump exist at the serosal side. Approximately 15-20% of absorbed amino acids are passively translocated.     

Chloride reabsorption by renal proximal tubules of necturus

Summary Movement of Cl from the lumen ofNecturus proximal tubule into the cells is mediated and dependent on the presence of luminal Na. Intracellular Cl activity was monitored with ion selective microelectrodes. In Cl Ringer's perfused kidneys, cell Cl activity was 24.5±1.1mm, 2 to 3 times higher than that predicted for passive distribution. When luminal NaCl was partially replaced by mannitol (capillaries perfused with Cl Ringer's) cell Cl decreased showing a sigmoidal dependence on luminal NaCl. Peritubular membrane potential was unaltered. Sulfate Ringer's perfusion of the kidneys washed out all cell Cl but did not alter peritubular membrane potential. Chloride did not enter the cell when the tubule lumen was perfused with 100mm KCl, LiCl, or tetramethylammonium Cl. Luminal perfusion of NaCl caused cell Cl to rise rapidly to the same value as the controls in the Cl Ringer's experiments. Perfusion of the tubule lumen with mixtures of NaCl and Na₂SO₄, while the capillaries contained sulfate Ringer's yielded a sigmoidal dependence of cell Cl on luminal NaCl activity. Chloride movement from the lumen into the proximal tubule cells required approximately equal concentrations of Na and Cl. Current clamp experiments indicated that intracellular chloride activity was insensitive to alterations in liminal membrane potential, suggesting that chloride entry was electrically neutral. The transcellular chloride flux was calculated to constitute about one half of the normal chloride reabsorption rate. We conclude that the cell Cl activity is primarily determined by the NaCl concentration in the tubule lumen and that Cl entry across the luminal membrane is mediated.     

Pleural surface fluorescence measurement of Na+ and Cl- transport across the air space-capillary barrier.

We developed a pleural surface fluorescence method to measure Na(+) and Cl(-) transport in perfused mouse lungs. The air space was filled with aqueous fluid containing membrane-impermeant fluorescent indicators of Cl(-) (lucigenin) or Na(+) (Sodium Green). After instillation of a Cl(-)-free solution into the air space, an increase in perfusate Cl(-) concentration from 0 to 30 mM produced a decrease in surface lucigenin fluorescence (6.5%/min) corresponding to Cl(-) influx of 1.0 mM/min. Cl(-) influx was increased to 2.1 +/- 0.3 mM/min by forskolin, and the increase was inhibited by glibenclamide. cAMP-stimulated Cl(-) influx was decreased by 57% in CFTR null mice. After instillation of a Na(+)-free solution into the air space, an increase in perfusate Na(+) concentration from 0 to 30 mM gave increased Sodium Green fluorescence (Na(+) influx of 1.2 mM/min), which increased approximately fivefold after cAMP agonists. Cl(-) and Na(+) transport were not affected in lungs from mice lacking aquaporins AQP1 or AQP5. Our results establish a pleural surface fluorescence method to measure unidirectional Cl(-) and Na(+) flux in intact lung and provide evidence for cAMP-stimulated transcellular Cl(-) and Na(+) transport.     

Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C.

This review updates our current knowledge on the regulation of Na+/H+ exchanger, Na+,K+,Cl- cotransporter, Na+,Pi cotransporter, and Na+,K+ pump in isolated epithelial cells from mammalian kidney by protein kinase C (PKC). In cells derived from different tubule segments, an activator of PKC, 4beta-phorbol 12-myristate 13-acetate (PMA), inhibits apical Na+/H+ exchanger (NHE3), Na+,Pi cotransport, and basolateral Na+,K+ cotransport (NKCCl) and augments Na+,K+ pump. In PMA-treated proximal tubules, activation of Na+,K+ pump probably plays a major role in increased reabsorption of salt and osmotically obliged water. In Madin-Darby canine kidney (MDCK) cells, which are highly abundant with intercalated cells from the collecting duct, PMA completely blocks Na+,K+,Cl- cotransport and decreases the activity of Na+,Pi cotransport by 30-40%. In these cells, agonists of P2 purinoceptors inhibit Na+,K+,Cl- and Na+,Pi cotransport by 50-70% via a PKC-independent pathway. In contrast with MDCK cells, in epithelial cells derived from proximal and distal tubules of the rabbit kidney, Na+,K+,Cl- cotransport is inhibited by PMA but is insensitive to P2 receptor activation. In proximal tubules, PKC-induced inhibition of NHE3 and Na+,Pi cotransporter can be triggered by parathyroid hormone. Both PKC and cAMP signaling contribute to dopaminergic inhibition of NHE3 and Na+,K+ pump. The receptors triggering PKC-mediated activation of Na+,K+ pump remain unknown. Recent data suggest that the PKC signaling system is involved in abnormalities of dopaminergic regulation of renal ion transport in hypertension and in the development of diabetic complications. The physiological and pathophysiological implications of PKC-independent regulation of renal ion transporters by P2 purinoceptors has not yet been examined.     

Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')- bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.     

Is there a role for renal alpha 2-adrenoceptors in the pathogenesis of hypertension?

Current information suggests that alpha 2-adrenoceptors do not directly influence vascular resistance or Na reabsorption in the rat kidney. To reexamine the effects of alpha 2-agonists we used isolated rat kidneys perfused at 37.5 degrees C with precise measurement of renal artery pressure and flow. The recirculating perfusate contained pyruvate as the sole metabolic substrate which enabled us to use gluconeogenesis as an index of proximal tubular alpha 1-responses. Clonidine and guanfacine in 100 nM concentrations decreased phosphate excretion without altering Na, Cl, or K reabsorption or gluconeogenesis; 500 nM concentrations increased vascular resistance and decreased glomerular filtration rate and Na, Cl, and K excretion with no significant effect on gluconeogenesis. Prior thyroparathyroidectomy prevented the antiphosphaturic but not the antinatriuretic or vascular responses. Clonidine, an alpha 2-agonist with some alpha 1-activity, was a more potent vasoconstrictor than methoxamine or guanfacine. In the presence of prazosin (1 microM), norepinephrine (60 nM) stimulated phosphate reabsorption; norepinephrine alone did not stimulate phosphate reabsorption which indicates alpha 1-antagonism of this alpha 2-response to NE. These results and a literature review suggest that increased renal alpha 2-adrenoceptors could raise renal vascular resistance, reduce renin secretion, and antagonize parathyroid hormone effects on Pi, Ca, HCO3, and Na reabsorption to produce a low renin type of hypertension with increased proximal Na reabsorption and abnormal Ca and Pi excretion.     

Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways.

The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity.     

Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s?1) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.     

Toxicity of adenine nucleotides in the isolated perfused kidney: selective destruction of the S2 segment of the proximal tubule.

In an attempt to ameliorate the morphological abnormalities and decreased renal function produced by hypoxia in the isolated perfused rat kidney, adenosine triphosphate (ATP) was added to the perfusate medium. No improvement was noted in the histological changes or renal function. Paradoxically, however, in oxygenated control kidneys, ATP (2.5-10 mM), caused a severe injury remarkably limited to the S2 segments of proximal tubule. This injury was more destructive than that observed with complete ischemia for the same period of time or with inhibitors of glycolysis, intermediary metabolism, or respiratory chain function. Tubular damage produced by ATP was paradoxically prevented by hypoxia and mitochondrial inhibition. The mechanism of this selective toxic injury to the proximal tubule remains unclear and may depend upon intact transport metabolism of the cell.     

Na:K pump abundance and function in MDCK cells: effect of low ambient potassium.

Side-specific expression and activity of Na:K pump was studied in Madin-Darby canine kidney (MDCK) cells, a tissue culture model of distal renal tubular epithelium, exposed to low ambient potassium. Confluent monolayers grown on teflon filters in dual chambers were treated with a low K+ medium from 45 min to 72 h. After both acute (45 min) and longer-term (24-72 h) exposure to low K+ (0.7 mM), cation cycling rate of existing pump units increased substantially, while there was no significant change in total cell Na-K-ATPase activity or in basolateral surface pump density. Although a small quantity of Na:K pumps (less than 10%) was consistently present apically, it also did not increase after exposure to low K+, or when the monolayers were provided K+ only from the apical side. In MDCK monolayers low K+ enhances the rate of K+ uptake by the existing pump units but does not increase the total number of pumps or their deployment on either cell surface.