The renal and blood pressure response to low sodium diet in P2X4 receptor knockout mice.

In the kidney, purinergic (P2) receptor-mediated ATP signaling has been shown to be an important local regulator of epithelial sodium transport. Appropriate sodium regulation is crucial for blood pressure (BP) control and disturbances in sodium balance can lead to hypo- or hypertension. Links have already been established between P2 receptor signaling and the development of hypertension, attributed mainly to vascular and/or inflammatory effects. A transgenic mouse model with deletion of the P2X4 receptor (P2X4-/- ) is known to have hypertension, which is thought to reflect endothelial dysfunction and impaired nitric oxide (NO) release. However, renal function in this model has not been characterized; moreover, studies in vitro have shown that the P2X4 receptor can regulate renal epithelial Na+ channel (ENaC) activity. Therefore, in the present study we investigated renal function and sodium handling in P2X4-/- mice, focusing on ENaC-mediated Na+ reabsorption. We confirmed an elevated BP in P2X4-/- mice compared with wild-type mice, but found that ENaC-mediated Na+ reabsorption is no different from wild-type and does not contribute to the raised BP observed in the knockout. However, when P2X4-/- mice were placed on a low sodium diet, BP normalized. Plasma aldosterone concentration tended to increase according to sodium restriction status in both genotypes; in contrast to wild-types, P2X4-/- mice did not show an increase in functional ENaC activity. Thus, although the increased BP in P2X4-/- mice has been attributed to endothelial dysfunction and impaired NO release, there is also a sodium-sensitive component.

Protease stimulation of renal sodium reabsorption in vivo by activation of the collecting duct epithelial sodium channel (ENaC).

BACKGROUND: Proteases can increase the activity of the epithelial sodium channel (ENaC) by cleaving its α- or γ-subunit. However, evidence so far comes only from studies in vitro in either heterologous expression systems or isolated nephron segments. The present study has tested whether exposure to a luminal protease can alter sodium reabsorption along the rat collecting duct in vivo. METHODS: Rats on normal laboratory chow were prepared for renal micropuncture. Late distal tubules of superficial nephrons were microinjected and perfused twice (3 nL min(-1) for 3-6 min) with a solution similar to native tubular fluid, but containing (14)[C]inulin and (22)Na. The first perfusion was either a control solution or solution containing amiloride 1 mM or hydrochlorothiazide (HCTZ ) 1 mM; the second perfusion was either a control solution (time control) or a solution containing chymotrypsin 2 µg mL(-1) ± aprotinin 100 µg mL(-1) or amiloride 1 mM or HCTZ 1 mM. Urinary recoveries of (14)[C]inulin and (22)Na were recorded. RESULTS: In time controls, the Na/In ratio did not change significantly (32.2 ± 3.4% versus 34.5 ± 3.1%). In contrast, chymotrypsin reduced the ratio from 33.3 ± 3.8% to 25.5 ± 2.5% (P < 0.05), indicating an increase in sodium reabsorption. When co-injected with chymotrypsin, the protease inhibitor aprotinin abolished the stimulatory effect of chymotrypsin on sodium reabsorption (31.7 ± 3.4% versus 32.1 ± 2.1%), while aprotinin alone had no effect. When chymotrypsin was co-injected with HCTZ, the Na/In ratio decreased from 36.8 ± 2.3% to 28.0 ± 3.4% (P < 0.05), whereas when given with amiloride, there was no change in the ratio (45.8 ± 3.4% versus 45.5 ± 2.3%), indicating that stimulation of sodium reabsorption by chymotrypsin was ENaC-dependent. CONCLUSIONS: These findings demonstrate proteolytic activation of ENaC in vivo, and suggest that changes in protease activity of the glomerular filtrate and tubular fluid in health or disease could affect net renal sodium excretion.

Pathophysiology and management of hypokalemia: a clinical perspective.

Potassium (K(+)) ions are the predominant intracellular cations. K(+) homeostasis depends on external balance (dietary intake [typically 100 mmol per day] versus excretion [95% via the kidney; 5% via the colon]) and internal balance (the distribution of K(+) between intracellular and extracellular fluid compartments). The uneven distribution of K(+) across cell membranes means that a mere 1% shift in its distribution can cause a 50% change in plasma K(+) concentration. Hormonal mechanisms (involving insulin, ß-adrenergic agonists and aldosterone) modulate K(+) distribution by promoting rapid transfer of K(+) across the plasma membrane. Extrarenal K(+) losses from the body are usually small, but can be marked in individuals with chronic diarrhea, severe burns or prolonged sweating. Under normal circumstances, the kidney's distal nephron secretes K(+) and determines final urinary excretion. In patients with hypokalemia (plasma K(+) concentration <3.5 mmol/l), after the exclusion of extrarenal causes, alterations in sodium ion delivery to the distal nephron, mineralocorticoid status, or a specific inherited or acquired defect in distal nephron function (each of which affects distal nephron K(+) secretion), should be considered. Clinical management of hypokalemia should establish the underlying cause and alleviate the primary disorder. This Review aims to inform clinicians about the pathophysiology and appropriate treatment for hypokalemia.

Direct micropuncture evidence that matrix extracellular phosphoglycoprotein inhibits proximal tubular phosphate reabsorption.

BACKGROUND: Matrix extracellular phosphoglycoprotein (MEPE) is a putative phosphatonin that we have shown in previous studies to be phosphaturic in rats. Its site of action in the nephron remains to be confirmed. METHODS: We made micropuncture collections from late proximal convoluted tubules in anaesthetized rats to assess directly the effect of MEPE on phosphate reabsorption in the proximal tubule. RESULTS: MEPE had no effect on glomerular filtration rate or single-nephron filtration rate, but it increased phosphate excretion significantly. In animals infused with vehicle alone (time controls), no significant change was seen in either the proximal tubular fluid:plasma phosphate concentration ratio (TF/P(Pi)) or the fraction of filtered phosphate reaching the late proximal convoluted tubule (FD(Pi)); whereas in rats infused with MEPE, TF/P(Pi) increased from 0.49 ± 0.07 to 0.68 ± 0.04 (n = 22; P = 0.01) and FD(Pi) increased from 0.20 ± 0.03 to 0.33 ± 0.03 (n = 22; P < 0.01). CONCLUSIONS: The results confirm the phosphaturic effect of MEPE and indicate that much, if not all, of this effect is a result of reduced reabsorption of phosphate in the proximal convoluted tubule. This is consistent with the recent finding of MEPE-induced reductions in apically located NaPT2a in the proximal tubule.

Nucleotides downregulate aquaporin 2 via activation of apical P2 receptors.

Vasopressin regulates water reabsorption in the collecting duct, but extracellular nucleotides modulate this regulation through incompletely understood mechanisms. We investigated these mechanisms using immortalized mouse collecting duct (mpkCCD) cells. Basolateral exposure to dDAVP induced AQP2 localization to the apical membrane, but co-treatment with ATP internalized AQP2. Because plasma membrane-bound P2 receptors (P2R) mediate the effects of extracellular nucleotides, we examined the abundance and localization of P2R in mpkCCD cells. In the absence of dDAVP, P2Y(1) and P2Y(4) receptors localized to the apical membrane, whereas P2X(2), P2X(4), P2X(5), P2X(7), P2Y(2), P2Y(11), and P2Y(12) receptors localized to the cytoplasm. dDAVP induced gene expression of P2X(1), which localized to the apical domain, and led to translocation of P2X(2) and P2Y(2) to the apical and basolateral membranes, respectively. In co-expression experiments, P2R activation decreased membrane AQP2 and AQP2-mediated water permeability in Xenopus oocytes expressing P2X(2), P2Y(2,) or P2Y(4) receptors, but not in oocytes expressing other P2R subtypes. In summary, these data suggest that AQP2-mediated water transport is downregulated not only by basolateral nucleotides, mediated by P2Y(2) receptors, but also by luminal nucleotides, mediated by P2X(2) and/or P2Y(4) receptors.

Ectonucleotidases in the kidney.

Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-5'-nucleotidase and alkaline phosphatases, have been identified in the renal vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2 and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule. NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal segments. Ecto-5'-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high K (m) values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes' varied distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology and pathophysiology.

Effects of extracellular nucleotides on renal tubular solute transport.

A range of P2 receptor subtypes has been identified along the renal tubule, in both apical and basolateral membranes. Furthermore, it has been shown that nucleotides are released from renal tubular cells, and that ectonucleotidases are present in several nephron segments. These findings suggest an autocrine/paracrine role for nucleotides in regulating tubular function. The present review catalogues the known actions of extracellular nucleotides on tubular solute transport. In the proximal tubule, there is firm evidence that stimulation of apical P2Y(1) receptors inhibits bicarbonate reabsorption, whilst basolaterally applied ATP has the opposite effect. Clearance studies suggest that systemic diadenosine polyphosphates profoundly reduce proximal tubular fluid transport, through as yet unidentified P2 receptors. To date, only circumstantial evidence is available for an action of nucleotides on transport in the loop of Henle; and no studies have been made on native distal tubules, though observations in cell lines suggest an inhibitory effect on sodium, calcium and magnesium transport. The nephron segment most studied is the collecting duct. Apically applied nucleotides inhibit the activity of small-conductance K(+) channels in mouse collecting duct, apparently through stimulation of P2Y(2) receptors. There is also evidence, from cell lines and native tissue, that apically (and in some cases basolaterally) applied nucleotides inhibit sodium reabsorption. In mice pharmacological profiling implicates P2Y(2) receptors; but in rats, the receptor subtype(s) responsible is/are unclear. Recent patch-clamp studies in rat collecting ducts implicate apical P2Y and P2X subtypes, with evidence for both inhibitory and stimulatory effects. Despite considerable progress, clarification of the physiological role of the tubular P2 receptor system remains some way off.

Sodium-dependent regulation of renal amiloride-sensitive currents by apical P2 receptors.

The epithelial sodium channel (ENaC) plays a major role in the regulation of sodium balance and BP by controlling Na(+) reabsorption along the renal distal tubule and collecting duct (CD). ENaC activity is affected by extracellular nucleotides acting on P2 receptors (P2R); however, there remain uncertainties over the P2R subtype(s) involved, the molecular mechanism(s) responsible, and their physiologic role. This study investigated the relationship between apical P2R and ENaC activity by assessing the effects of P2R agonists on amiloride-sensitive current in the rat CD. Using whole-cell patch clamp of principal cells of split-open CD from Na(+)-restricted rats, in combination with immunohistochemistry and real-time PCR, we found that activation of metabotropic P2R (most likely the P2Y(2) and/or (4) subtype), via phospholipase C, inhibited ENaC activity. In addition, activation of ionotropic P2R (most likely the P2X(4) and/or (4/6) subtype), via phosphatidylinositol-3 kinase, either inhibited or potentiated ENaC activity, depending on the extracellular Na(+) concentration; therefore, it is proposed that P2X(4) and/or (4/6) receptors might function as apical Na(+) sensors responsible for local regulation of ENaC activity in the CD and could thereby help to regulate Na(+) balance and systemic BP.

Matrix extracellular phosphoglycoprotein causes phosphaturia in rats by inhibiting tubular phosphate reabsorption.

BACKGROUND: Matrix extracellular phosphoglycoprotein (MEPE), first isolated from tumour-derived tissue from a patient with oncogenic hypophosphataemia, is a putative phosphatonin that has received much less attention than fibroblast growth factor-23. To date, its effect on renal tubular phosphate reabsorption remains undefined. METHODS: A renal clearance study was performed in anaesthetized rats infused intravenously with a range of doses of MEPE. RESULTS: MEPE had no effect on glomerular filtration rate (inulin clearance) but caused rapid, dose-dependent increases in absolute and fractional phosphate excretion, wholly attributable to reduced phosphate reabsorption. At a maximal dose, MEPE increased fractional phosphate excretion more than 2-fold, whereas no change was observed in time controls. CONCLUSION: The results lend support to the hypothesis that MEPE contributes to the phosphaturia of oncogenic hypophosphataemia and of hypophosphataemic rickets.

Servo-control of water and sodium homeostasis during renal clearance measurements in conscious rats.

Servo-controlled fluid and sodium replacement during clearance studies is used in order to prevent loss of body fluid and sodium following diuretic/natriuretic procedures. However, even under control conditions, the use of this technique is sometimes associated with increases in proximal tubular fluid output (assessed by lithium clearance) and excretion rates. The present study examined the reason for these increases. The first series of experiments showed that one cause is volume overloading. This can occur if the servo system is activated from the start, i.e., during the establishment of a suitably high urine flow rate by constant infusion of hypotonic glucose solution. The second series of experiments showed that replacement of blood samples with donor blood can also lead to increases in fractional lithium excretion and accompanying increases in water and sodium excretion, a problem not seen when blood samples are replaced with the animal's own red blood cells resuspended in isotonic saline. When these pitfalls are avoided, servo-controlled sodium and fluid replacement is a reliable technique that makes it possible to study the effects of natriuretic and/or diuretic stimuli without interference from unwanted changes in extracellular volume.

Intraluminal ATP concentrations in rat renal tubules.

It is becoming increasingly recognized that stimulation of apical P2 receptors can influence solute transport in the nephron, but, to date, no information is available on endogenous intraluminal nucleotide concentrations in vivo. This study measured intraluminal ATP concentrations in the renal tubules of anesthetized rats. Proximal tubular concentrations were found to be in the range of 100 to 300 nmol/L, with no significant variation along the S2 segment, whereas concentrations in the early distal tubule were markedly lower. Using collections of varying duration, the half-life of ATP in collected proximal tubular fluid was found to be 3.4 min, indicating significant breakdown by soluble nucleotidases. For assessment of whether proximal tubular ATP was filtered or secreted, experiments were performed in Munich-Wistar rats. The ATP concentration in midproximal tubules (142 +/- 23 nmol/L) was more than four-fold higher than in Bowman's space (32 +/- 7 nmol/L; P < 0.001), whereas fractional water reabsorption between the two sites was modest. In experiments that were designed to determine the effects of (patho)physiologic disturbances on intraluminal ATP, rats were either volume expanded or subjected to hypotensive hemorrhage. Neither maneuver affected proximal tubular luminal ATP concentrations significantly; rapid degradation of secreted ATP by ecto- and soluble nucleotidases is a possible explanation. It is concluded that the proximal tubule secretes ATP into the lumen, where it may have an autocrine/paracrine regulatory role.

A hypothesis linking sodium and lithium reabsorption in the distal nephron.

BACKGROUND: A hypothesis is proposed linking Na(+) and Li(+) reabsorption in the distal nephron. The handling of these two ions in the distal nephron is related because they share the same apical membrane entry mechanism: the amiloride-sensitive Na(+) channel (ENaC). However, the two ions exit the cell through different transport mechanisms: Na(+) via the Na(+)-K(+)-ATPase and Li(+) via the Na(+)/H(+) exchanger. Studies in rats have shown that under normal circumstances hardly any Li(+) is reabsorbed in the distal nephron, so that the urinary excretion of Li(+), expressed as a fraction of the delivery to the early distal tubule (FE(Li dist)), amounts to approximately 0.97. In contrast, during severe dietary Na(+) restriction, FE(Li dist) decreases to 0.50-0.60. Our hypothesis is that the absence of distal Li(+) reabsorption during intake of a normal diet can be explained by a negative driving force for Li(+) entrance across the apical membrane in those segments in which ENaC is active. METHOD: We propose a model that incorporates this concept. RESULTS: The model indicates that the lowering of FE(Li dist) during dietary Na(+) restriction can be explained by activation of apical ENaC in extra sub-segments further downstream. In these extra sub-segments the driving force for Li(+) reabsorption is positive, leading to significant Li(+) reabsorption. During dietary K(+) restriction, FE(Li dist) is reduced to 0.35-0.55. The model shows that this reduction in FE(Li dist) can be explained by hyperpolarization of the apical membrane in ENaC-containing sub-segments, which is known to occur in this condition. CONCLUSION: We conclude that the model may improve current understanding of both Na(+) and Li(+) handling in the distal nephron.

Immunolocalization of ectonucleotidases along the rat nephron.

Evidence is accumulating that extracellular nucleotides act as autocrine/paracrine agents in most tissues, including the kidneys. Several families of surface-located enzymes, collectively known as ectonucleotidases, can degrade nucleotides. Using immunohistochemistry, we have examined the segmental distribution of five ectonucleotidases along the rat nephron. Perfusion-fixed kidneys were obtained from anesthetized male Sprague-Dawley rats. Cryostat sections of cortical and medullary regions were incubated with antibodies specific to the following enzymes: ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1, NTPDase2, NTPDase3, ectonucleotide pyrophosphatase phosphodiesterase 3 (NPP3), and ecto-5'-nucleotidase. Sections were then costained with Phaseolus vulgaris erythroagglutinin (for identification of proximal tubules) and antibodies against Tamm-Horsfall protein (for identification of thick ascending limb), calbindin-D(28k) (for identification of distal tubule), and aquaporin-2 (for identification of collecting duct). The tyramide signal amplification method was used when the ectonucleotidase and marker antibody were raised in the same species. The glomerulus expressed NTPDase1 and NPP3. The proximal tubule showed prominent expression of NPP3 and ecto-5'-nucleotidase in most, but not all, segments. NTPDase2 and NTPDase3, but not NPP3 or ecto-5'-nucleotidase, were expressed in the thick ascending limb and distal tubule. NTPDase3, with some low-level expression of ecto-5'-nucleotidase, was also found in cortical and outer medullary collecting ducts. Inner medullary collecting ducts displayed low-level staining for NTPDase1, NTPDase2, NTPDase3, and ecto-5'-nucleotidase. We conclude that these ectonucleotidases are differentially expressed along the nephron and may play a key role in activation of purinoceptors by nucleotides and nucleosides.

Regulatory interdependence of cloned epithelial Na+ channels and P2X receptors.

Epithelial Na+ channels (ENaC) coexist with a family of ATP-gated ion channels known as P2X receptors in the renal collecting duct. Although ENaC is itself insensitive to extracellular ATP, tubular perfusion of ATP can modify the activity of ENaC. To investigate a possible regulatory relationship between P2X receptors and ENaC, coexpression studies were performed in Xenopus oocytes. ENaC generated a persistent inward Na+ current that was sensitive to the channel blocker amiloride (I(am-s)). Exogenous ATP transiently activated all cloned isoforms of P2X receptors, which in some cases irreversibly inhibited I(am-s). The degree of inhibition depended on the P2X receptor subtype present. Activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptor subtypes inhibited I(am-s), whereas activation of P2X1, P2X3, and P2X5 receptors had no significant effect. The degree of inhibition of I(am-s) correlated positively with the amount of ionic charge conducted by P2X receptor subtypes. ENaC inhibition required Na+ influx through I(am-s)-inhibiting P2X ion channels but also Ca2+ influx through P2X4 and P2X(4/6) ion channels. P2X-mediated inhibition of I(am-s) was found to be due to retrieval of ENaC from the plasma membrane. Maximum amplitudes of ATP-evoked P2X-mediated currents (I(ATP)) were significantly increased for P2X2, P2X(2/6), and P2X5 receptor subtypes after coexpression of ENaC. The increase in I(ATP) was due to increased levels of plasma membrane-bound P2X receptor protein, suggesting that ENaC modulates protein trafficking. In summary, ENaC was downregulated by the activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptors. Conversely, ENaC increased the plasma membrane expression of P2X2, P2X(2/6), and P2X5 receptors.

Simultaneous determination of anions in nanoliter volumes.

BACKGROUND: The study of renal tubular transport requires the ability to accurately measure ion concentrations in samples taken from single tubules. Sample collection and analysis are laborious, so methods allowing determination of multiple ion species in a small volume sample are advantageous. This article describes a method for the simultaneous analysis of anions at physiologic concentrations in nanoliter volumes of tubular fluid. METHOD: The analysis is performed using capillary zone electrophoresis. Diluted samples are moved along a capillary by bulk transport and separated according to charge and size. Peaks corresponding to anions are obtained by ultraviolet (UV) detection; peak area is proportional to ion concentration. RESULTS: The anions chloride, nitrate, citrate, phosphate, and bicarbonate were separated in less than 4 minutes, and iothalamate in less than 5 minutes. Simultaneous quantitative analysis was performed for chloride, phosphate, and bicarbonate, demonstrating detection limits of 12 fmol for chloride, 12 fmol for phosphate, and 72 fmol for bicarbonate. A comparison between this method and a flow-through microfluorimeter analysis of chloride showed good agreement between the two micro-methods. Illustrative data from proximal and distal tubular fluid samples obtained by micropuncture (volume 30-70 nL) are given, as are results from urine samples. RESULTS: Results for chloride, phosphate, and bicarbonate in control material are in close agreement with the certified values, while values in tubular fluid are in accordance with previously published results. CONCLUSION: This method provides a straightforward means of analyzing multiple anions in small volume biological samples.

Renal aluminium handling in the rat: a micropuncture assessment.

Uncertainties exist over the glomerular filtration of aluminium and virtually nothing is known about its segmental handling along the nephron. The present study has used micropuncture, combined with electrothermal atomic absorption spectroscopy, to determine directly the aluminium content of glomerular filtrate and of late PCTs (proximal convoluted tubules) and early distal tubules in anaesthetized Munich-Wistar rats infused with three different doses of aluminium citrate (plasma aluminium concentrations, 2.9+/-0.1, 5.2+/-0.4 and 10.0+/-0.9 microg.ml(-1) respectively). Aluminium filtration into Bowman's space was found to be considerably greater than that predicted by an in vitro filtration system: in all three groups it was essentially filtered freely. No significant aluminium reabsorption took place along the PCT, but with every dose the FD(Al) (fractional delivery of aluminium; tubular fluid:plasma aluminium/inulin concentration ratio) was lower at the early distal site than at the late PCT (P<0.001 in each case), indicating net aluminium reabsorption in the loop of Henle. This reabsorption amounted to 19-26% of the filtered aluminium load. In the low- and medium-dose groups, there was no significant difference between FD(Al) at the early distal site and that in the final urine; however, in the high-dose group, FD(Al) in the urine (1.02+/-0.06) exceeded that at the early distal tubule (0.75+/-0.04; P<0.001), suggesting aluminium secretion in the distal nephron. The results indicate that aluminium loads, when complexed with citrate, are excreted efficiently owing to a combination of glomerular filtration and minimal reabsorption.

The natriuretic effect of glibenclamide: evidence for a non-luminal site of action.

Inhibition of sodium reabsorption in the loop of Henle (LOH) contributes to the natriuretic effect of systemically administered glibenclamide. Although it has been suggested that the underlying mechanism involves inhibition of low-conductance potassium channels in the apical membrane of the thick ascending limb, these channels are relatively insensitive to glibenclamide ( K(i) ~200 micro M). In the present study we used capillary electrophoresis techniques to determine plasma and tubular fluid concentrations of glibenclamide in anaesthetised, glibenclamide-infused rats during maximal natriuresis. The plasma glibenclamide concentration was 158+/-29 micro M, whereas that in the tubular fluid entering the LOH was below detectable limits (10 micro M). In additional experiments, rats were infused intravenously with either glibenclamide or vehicle alone, while the LOH was perfused with a standard, glibenclamide-free solution. Loop sodium reabsorption ( J(Na)) was significantly reduced in the rats receiving the drug (vehicle: J(Na) 1.65+/-0.05 nmol/min, n=23; glibenclamide: J(Na) 1.34+/-0.07 nmol/min, n=36; P<0.01). In a further group of rats, glibenclamide was introduced directly into the LOH at a concentration known to inhibit the low-conductance potassium channel in vitro (250 micro M). However, J(Na) was unaffected. These data confirm that systemic glibenclamide inhibits sodium reabsorption in the LOH but argue strongly that it does not act from the luminal site.

Urinary acidification and distal renal tubular acidosis.

Historically, renal tubular acidosis (RTA) has been classified on a clinical basis, without any reference to the underlying disorder. Here we review the normal mechanisms of renal acidification and we identify disorders of specific transporters (genetic, disease-related or drug-induced) that lead to the main categories of distal RTA. We also describe the approach to diagnosis and the current treatment of distal RTA.

Amiloride inhibits proximal tubular reabsorption in conscious euvolemic rats.

Based on the results of micropuncture studies, it is generally assumed that amiloride inhibits Na+ (and Li+) reabsorption in the distal nephron, without affecting proximal tubular reabsorption. This is the basis for the use of amiloride to test for distal nephron Li+ reabsorption. We have examined the validity of this assumption by administering amiloride in doses of 0, 0.02, 0.07, 0.2 and 2.0 mg x kg(-1) x h(-1) to conscious, chronically instrumented rats fed a diet with a normal Na+ and K+ content. Na+ and water homeostasis was maintained by servo-controlled replacement in order to avoid any effect of volume depletion on proximal tubular reabsorption. The effects of the two highest doses of amiloride were also examined without Na+ and water replacement. In the servo-controlled rats, the two highest doses of amiloride increased the fractional excretion of both Na+ (FE(Na)) and Li+ (FE(Li)), whereas the two lowest doses affected only FE(Na). In the rats without servo-control, FE(Li) also rose in response to amiloride infusion, but the increase was significantly lower than that observed in the servo-controlled animals. Since distal Li+ reabsorption is absent or negligible in rats fed a diet with a normal Na+ and K+ content, the large increase in FE(Li) following the highest doses of amiloride (15-18% of the filtered load in servo-controlled rats) indicates inhibition of proximal tubular reabsorption. We conclude that amiloride, in doses usually employed to detect distal Li+ reabsorption, inhibits proximal tubular reabsorption in conscious euvolemic rats.