Calcineurin inhibitor cyclosporine A activates renal Na-K-Cl cotransporters via local and systemic mechanisms.

Calcineurin dephosphorylates nuclear factor of activated T cells transcription factors, thereby facilitating T cell-mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation but may cause side effects, including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar rats, AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured thick ascending limb cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial action of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.

Expression of the Na-K-2Cl cotransporter by macula densa and thick ascending limb cells of rat and rabbit nephron.

Sodium and chloride transport by the macula densa and thick ascending limb of Henle's loop participates importantly in extracellular fluid volume homeostasis, urinary concentration and dilution, control of glomerular filtration, and control of renal hemodynamics. Transepithelial Na and Cl transport across the apical membrane of thick ascending limb (TALH) cells is mediated predominantly by a loop diuretic sensitive Na-K-2Cl cotransport pathway. The corresponding transport protein has recently been cloned. Functional studies suggest that the cotransporter is expressed by macula densa cells as well as by TALH cells. The current studies were designed to identify sites of Na-K-2Cl cotransporter expression along distal nephron in rabbit and rat. Non-isotopic high-resolution in situ hybridization, using an antisense probe for the apical form of the Na-K-2Cl cotransporter identified expression throughout the TALH, from the junction between inner and outer medulla to the transition to distal convoluted tubule. Expression by macula densa cells was confirmed by colocalization using markers specific for macula densa cells. First, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that did not stain with anti-Tamm-Horsfall protein antibodies. Second, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that show positive NADPH-diaphorase reaction, indicating high levels of constitutive nitric oxide synthase activity. In rat, levels of Na-K-2Cl cotransporter mRNA expression were similar in TALH and macula densa cells. In rabbit, expression levels were higher in macula densa cells than in surrounding TALH cells. The present data provide morphological support for a previously established functional concept that Na-K-2Cl cotransport at the TALH is accomplished by the expression of a well-defined cotransporter. At the macula densa, this transporter may establish a crucial link between tubular salt load and glomerular vascular regulation.

Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion.

We studied the effects of dietary NaCl intake on the renal distal tubule by feeding rats high or low NaCl chow or by chronically infusing furosemide. Furosemide-treated animals were offered saline as drinking fluid to replace urinary losses. Effects of naCl intake were evaluated using free-flow micropuncture, in vivo microperfusion, and morphometric techniques. Dietary NaCl restriction did not affect NaCl delivery to the early distal tubule but markedly increased the capacity of the distal convoluted tubule to transport Na and Cl. Chronic furosemide infusion increased NaCl delivery to the early distal tubule and also increased the rates of Na and Cl transport above the rates observed in low NaCl diet rats. When compared with high NaCl intake alone, chronic furosemide infusion with saline ingestion increased the fractional volume of distal convoluted tubule cells by nearly 100%, whereas dietary NaCl restriction had no effect. The results are consistent with the hypotheses that (a) chronic NaCl restriction increases the transport ability of the distal convoluted tubule independent of changes in tubule structure, (b) high rates of ion delivery to the distal nephron cause tubule hypertrophy, and (c) tubule hypertrophy is associated with increases in ion transport capacity. They indicate that the distal tubule adapts functionally and structurally to perturbations in dietary Na and Cl intake.

Expression of the thiazide-sensitive Na-Cl cotransporter by rabbit distal convoluted tubule cells.

A thiazide-sensitive Na-Cl cotransporter contributes importantly to mammalian salt homeostasis by mediating Na-Cl transport along the renal distal tubule. Although it has been accepted that thiazide-sensitive Na-Cl cotransport occurs predominantly along the distal convoluted tubule in rats and mice, sites of expression in the rabbit have been controversial. A commonly accepted model of rabbit distal nephron transport pathways identifies the connecting tubule, not the distal convoluted tubule, as the predominant site of thiazide-sensitive Na-Cl cotransport. The thiazide-sensitive Na-Cl cotransporter has been cloned recently. The present experiments were designed to localize sites of thiazide-sensitive Na-Cl cotransporter mRNA expression along the rabbit distal nephron. Nonradioactive in situ hybridization with a thiazide-sensitive Na-Cl cotransporter probe was combined with immunocytochemistry with an antibody that recognizes distal convoluted tubule cells and with a Na+/Ca2+ exchanger antibody that recognizes only connecting tubule cells. The results indicate that thiazide-sensitive Na-Cl cotransporter mRNA is highly expressed by cells of the distal convoluted tubule and not by connecting tubule cells. Segments that stain with the Na+/Ca2+ exchanger antibody (connecting tubules) do not demonstrate thiazide-sensitive Na-Cl cotransporter mRNA expression. We conclude that the predominant site of thiazide-sensitive Na-Cl cotransporter mRNA expression in rabbit distal nephron is the distal convoluted tubule and that sites of mRNA expression of electroneutral Na and Cl transport are similar in rabbits, rats, and mice.

The continuous erythropoietin receptor activator (C.E.R.A.) corrects anemia at extended administration intervals in patients with chronic kidney disease not on dialysis: results of a phase II study.

AIM: This study was designed to assess the potential of the continuous erythropoietin receptor activator (C.E.R.A.) to correct anemia at extended administration intervals in erythropoiesis-stimulating agent-naive patients with chronic kidney disease (CKD) not on dialysis and to determine its optimal starting dose. METHODS: Patients were assigned to subcutaneous C.E.R.A. at 3 doses: 0.15, 0.30 and 0.60 microg/kg/wk. During the first 6 weeks, dose adjustments for efficacy were not permitted in order to assess dose response. Within each of the 3 dose groups, patients were randomized to receive C.E.R.A. QW, Q2W or Q3W; the total dose during the first 6 weeks was the same for a particular dose group across the frequency subgroups. During the next 12 weeks, dose was adjusted according to predefined hemoglobin (Hb) criteria. The primary efficacy parameter was change in Hb over 6 weeks, estimated from regression analysis between baseline and the point at which the patient received a dose change or blood transfusion. It therefore provided an estimate of Hb increase based on starting dose. Other endpoints included Hb response rate (proportion of patients with a Hb increase > 1.0 g/dl on 2 consecutive occasions). A 1-year extension period investigated long term tolerability and efficacy. RESULTS: A dose-dependent relationship was noted in the mean change in Hb from baseline over 6 weeks (p < 0.0001), independent of administration schedule (p = 0.9201). There was also a significant relationship between Hb change and median serum C.E.R.A. concentration (p < 0.0001). Erythropoietic responses were sustained in all groups with mean changes from baseline in Hb > 1.2 g/dl observed at doses > or = 0.30 microg/kg/wk. Hb response rate increased with increasing dose: 67, 72 and 90% with C.E.R.A. 0.15, 0.30 and 0.60 microg/kg/wk, respectively. Generally, the median Hb response time was faster with increasing dose (89, 43 and 31 days, respectively). Response was unrelated to administration frequency. Stable Hb concentrations were maintained throughout the 1-year extension period. C.E.R.A. was generally well tolerated, and the most common adverse events were hypertension, urinary tract infection and renal failure. CONCLUSIONS: C.E.R.A. corrected anemia and maintained sustained and stable control of Hb over 1 year. These results suggest that 0.60 microg/kg subcutaneous C.E.R.A. given twice monthly is a suitable starting dose for further investigation in Phase III studies in patients with CKD not on dialysis.

Solubilization and partial purification of the thiazide diuretic receptor from rabbit renal cortex.

This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitive Na/Cl transport pathway of the renal distal tubule. In the current study, [3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compared with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25-35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current results indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.

Effects of calcium infusion on blood pressure in hypertensive and normotensive humans.

Disorders of calcium and parathyroid hormone homeostasis have been reported in subjects with essential hypertension. In many of these studies, dietary intakes of sodium and calcium were not carefully controlled. The present study was designed to compare calcium and parathyroid hormone homeostasis in normal and hypertensive subjects on controlled dietary sodium and calcium intakes and to examine the impact of dietary sodium loading on hemodynamic and metabolic responses to infused calcium. Seven subjects with essential hypertension and seven age-matched and sex-matched controls were studied while consuming a standard diet containing 600 mg of elemental calcium. Each subject was studied while consuming 10, 160, and 510 mEq of sodium per day, before, during, and after a 3-hour calcium infusion (3.75 mg/kg/hr). Before calcium infusion, hypertensive subjects had increased urinary cyclic adenosine 3',5'-monophosphate excretion independent of sodium intake (p less than 0.05). Urinary potassium excretion was greater in normotensive than in hypertensive subjects (p = 0.002). At baseline, dietary sodium intake had no effect on systolic, diastolic, or mean arterial pressure. During calcium infusion, systolic pressure increased in both groups, whereas diastolic pressure increased only when dietary sodium content was high and mean arterial pressure increased only in hypertensive subjects (p = 0.007). Together, these data provide evidence for interactions between dietary sodium intake and the cardiovascular response to calcium. They confirm that hypertensive subjects exhibit enhanced parathyroid gland function even when dietary factors are controlled, and they suggest that these subjects are more sensitive to the cardiovascular effects of short-term calcium infusion.

A new class of cardiotonic agents: structure-activity correlations for natural and synthetic analogues of the alkaloid A new class of A new class of cardiotonic agents: structure-activity correlations for natural and synthetic analogues of the alkaloid pumiliotoxin B (8-hydroxy-8-methyl-6-alkylidene-1-azabicyclo[4.3.0]nonanes).

Pumiliotoxin B (PTX-B, 6-(6',7'-dihydroxy-2',5'-dimethyl-(E)-4'-octenylidene)-8-hydroxy-8 -methyl-1- azabicyclo-[4.3.0] nonane) increases the force of contractures of spontaneously beating guinea pig atrial strips by 3- to 5-fold with half-maximal effects at about 3 microM and increases rates of atrial contractions by 2- to 3-fold with half-maximal effects at about 6 microM. The presence of an axial 7-hydroxy substituent (PTX 339A) decreases the efficacy but not the potency of PTX-B as a positive inotropic agent while having only slight effects on activity as a positive chronotropic agent. The presence of an equatorial 7-hydroxy substituent (PTX 339B) greatly decreases efficacy and potency of PTX-B as a positive chronotropic and inotropic agent. Pumiliotoxin A which lacks the side-chain 7'-hydroxy group of PTX-B causes only a 2-fold increase in force of contracture at 54 microM while having minimal effects on rate. The presence of an axial 7-hydroxy substituent (PTX 323B' and 323B", epimeric at the 6'-hydroxy) markedly enhances positive inotropic and chronotropic effects of PTX-A. Another congener, PTX 251D with a 6-(2'-methylhexylidene) side chain, and a synthetic analogue with a 6-(6'-heptenylidene) side chain are cardiac depressants. Both lack hydroxyl groups in the side chain. The presence of an omega-1 hydroxy group in the side chain of PTX 251D yields an alkaloid (267C) with weak positive inotropic effects and minimal chronotropic effects. The presence of an axial 7-hydroxy group in the indolizidine ring of PTX 251D results in a compound (PTX 267A) with very weak positive inotropic effects while retaining the negative chronotropic effects of PTX 251D. A synthetic analogue with a 6-(7'-hydroxyheptylidene) side chain is a cardiac depressant even though it contains a side-chain hydroxyl corresponding in position to the 7'-hydroxyl of the side chain of PTX-B. The positive chronotropic and inotropic effects of pumiliotoxin B are reversed only by relatively high concentrations of the calcium channel blockers nifedipine and verapamil, suggesting that pumiliotoxin B may owe its cardiotonic activities to effects on internal mobilization of calcium.

Diuretic resistance: physiology and therapeutics.

Diuretic drugs are usually effective treatment for edema when used judiciously. However, some patients become resistant to their effects. Adaptation to diuretic drugs and diuretic resistance may be caused by similar mechanisms. Diuretic adaptations can be classified as those that occur during diuretic action, those that cause sodium retention in the short term (causing 'post-diuretic NaCl retention'), and those that increase sodium retention chronically (the 'braking phenomenon'). Recent experimental work has indicated ways in which kidneys adapt to chronic diuretic treatment. First, nephron segments downstream from the site of diuretic action increase NaCl reabsorption during diuretic administration because delivered NaCl load is increased. Second, when diuretic concentrations in the tubule decline, the kidney tubules act to retain Na until the next dose of diuretic is administered. Third, the ability of the diuretic to increase renal NaCl excretion declines over time, an effect that results both from depletion of the extracellular fluid volume and from structural and functional changes of kidney tubules themselves. These adaptations all increase the rate of NaCl reabsorption and blunt the effectiveness of diuretic therapy. Many times, a second diuretic drug is effective treatment for diuretic resistance. Recent experimental results suggest that a second drug may act synergistically because it blocks the adaptive processes limiting the effectiveness of the first diuretic. Based on an understanding of the mechanisms of diuretic adaptation and resistance, treatment regimens can be designed to block specific adaptive mechanisms and improve diuretic effectiveness.

Sodium transport-related proteins in the mammalian distal nephron - distribution, ontogeny and functional aspects.

The mammalian distal nephron plays a pivotal role in adjusting urinary sodium excretion. Successive portions of the renal tubule are formed to adapt to this function, and an axial heterogeneity of the distal segments has been defined. The specific transport properties of these epithelia are accomplished by the expression of proteins (cotransporters, exchangers, channels) governing the movement of ions on either cell side. Molecular cloning of these proteins has had a marked impact on the study of their localization and function in the healthy and diseased kidney. Electroneutral cation-chloride cotransporters [Na(K)CC] have been localized to the thick ascending limb and the distal convoluted tubule using specific probes. Proteins implicated in the function of aldosterone target cells, such as the epithelial Na(+) channel (ENaC), the mineralocorticoid receptor (MR) and 11beta-hydroxysteroid dehydrogenase type 2 (11HSD2), an enzyme that confers mineralocorticoid specificity, have been found in the terminal portion of the nephron and the collecting duct. A mineralocorticoid-sensitive component of thiazide-sensitive NaCl transport has been identified in the distal convoluted tubule. Analysis of the ontogeny of these proteins in the maturing kidney has provided a detailed picture of epithelial differentiation and morphological specialization of the renal tubule. The study of mutations of the proteins related with NaCl transport has led to the identification of the molecular causes of inherited human diseases associated with hypo- or hypertension, and the respective sites of an impaired ion transport could be mapped to the renal tubule.

WNK kinases regulate sodium chloride and potassium transport by the aldosterone-sensitive distal nephron.

With-No-Lysine [K] (WNKs) are a recently discovered family of serine/threonine protein kinases that contain a uniquely structured catalytic domain. Mutations in the genes encoding two family members, WNK1 and WNK4, cause a chloride-dependent, thiazide-sensitive inherited syndrome of hypertension and hyperkalemia. Over the past 5 years, physiologic studies have demonstrated that these proteins regulate transcellular and paracellular epithelial ion flux. In this mini review, we discuss WNK1 and WNK4 gene products and their regulatory effects on sodium chloride and potassium handling in the aldosterone-sensitive distal nephron. Experimental observations regarding the effects of these proteins on transport processes mediated by the thiazide-sensitive Na-Cl co-transporter, the epithelial sodium channel, the renal outer medullary potassium channel, and the paracellular pathway integrate into a model that suggests an essential role for WNKs in coordinating renal Na-Cl reabsorption and K(+) secretion.

The physiologic basis of diuretic synergism: its role in treating diuretic resistance.

Diuretic drugs usually improve edema when used judiciously. Some patients, however, become resistant to their effects. Diuretic resistance may result from dietary indiscretion, poor compliance, impaired bioavailability, imparied diuretic secretion into the lumen of the renal tubule, or because other drugs interfere with diuretic activity. When easily treatable causes of diuretic resistance have been excluded, resistance often reflects the intensity of the stimuli to sodium retention. Recent experimental work has indicated ways in which the kidney adapts to chronic diuretic treatment and has indicated how these adaptations may limit diuretic effectiveness. First, nephron segments downstream from the site of diuretic action increase sodium-chloride (NaCl) reabsorption because the delivered NaCl load increases. Second, diuretic-induced contraction of the extracellular fluid volume stimulates kidney tubules to retain NaCl until the next dose of diuretic is administered. Third, kidney tubules themselves may become hypertrophic because they are chronically stimulated by diuretic-induced increases in NaCl delivery. These adaptations all increase the rate of NaCl reabsorption and blunt the effectiveness of diuretic therapy. When diuretic resistance is present, using a second diuretic drug that acts in a different nephron segment is often effective. Recent experimental results suggest that a second class of drug may act synergistically with the first by blocking the adaptive processes that limit diuretic effectiveness. On the basis of an understanding of the mechanisms of diuretic adaptation and resistance, treatment regimens can be designed to block specific adaptive mechanisms and to improve diuretic therapy.

Diuretic drugs and the treatment of edema: from clinic to bench and back again.

Despite wide variations in dietary NaCl intake, homeostatic mechanisms ensure that renal NaCl excretion matches intake at steady state. This does not imply, however, that extracellular fluid volume is maintained within narrow limits. In contrast with blood pressure, which appears to be tightly controlled, extracellular fluid volume varies significantly, even in normal individuals, when dietary NaCl intake changes. Cardiac, liver, or renal disease can perturb the relationship between NaCl intake and extracellular fluid volume and lead to symptomatic edema. All major classes of diuretic drugs in use today were developed between 1950 and 1970. These drugs were developed empirically, without knowledge of specific ion transport pathways, but experimental work during the past 15 years has shown that each major class of diuretic inhibits a specific ion transport protein in the kidney. These transport proteins have been characterized physiologically and the mechanisms by which each diuretic drug inhibits ion transport have been defined. Antibodies directed against these transport proteins have localized ion transport pathways to specific cell types along the nephron. Most recently, isoforms of each class of diuretic-sensitive Na transport pathway have been cloned. Ongoing experimental work is aimed at exploring relationships between families of transporters, determining the structural prerequisites for ion transport, and studying molecular mechanisms of transport regulation. Treatment of edema with diuretics is often straightforward, but can lead to adaptive changes in nephron structure and function. These adaptations can limit the effectiveness of diuretic drugs; maneuvers aimed at blocking these processes can be effective approaches to patients who are resistant to diuretic drugs.

Diuretic therapy and resistance in congestive heart failure.

Treatment of congestive heart failure has changed dramatically during the past 20 years, but diuretic drugs remain an essential component. Diuretics are essential despite the fact that these drugs stimulate the renin-angiotensin-aldosterone (RAA) axis and lead to adaptive responses that may be counterproductive. In this paper, new diuretic drugs and new uses of older drugs are discussed. These approaches emphasize low-dose combination therapy and may prove superior to traditional approaches that rely exclusively on loop diuretics. Such approaches aim to prevent adverse compensatory processes that appear to result from chronic diuretic treatment. These include acute and chronic increases in plasma renin activity and stimulation of the sympathetic nervous system, both of which increase afterload and may tend to increase mortality. They also include adaptive changes in nephron structure and function resulting from diuretic-induced increases in distal sodium load and diuretic-induced neurohormonal stimulation. These adaptations blunt the effectiveness of diuretic therapy. Diuretic strategies that rely on combinations of diuretics are emphasized as a method to prevent resistance. If diuretic resistance does develop, higher-dose combination regimens, continuous diuretic infusions and mechanical ultrafiltration can be used to overcome diuretic adaptations and restore diuretic efficacy. The goal of reducing the extracellular fluid volume with the least stimulation of the RAA axis and minimal changes in nephron architecture can be achieved in many patients.

Divalent cation transport by the distal nephron: insights from Bartter's and Gitelman's syndromes.

Elucidation of the gene defects responsible for many disorders of renal fluid and electrolyte homeostasis has provided new insights into normal and abnormal physiology. Identifying the causes of Gitelman's and Bartter's syndromes has greatly enhanced our understanding of ion transport by thick ascending limb and distal convoluted tubule cells. Despite this information, several phenotypic features of these diseases remain confusing, even in the face of molecular insight. Paramount among these are disorders of divalent cation homeostasis. Bartter's syndrome is caused by dysfunction of thick ascending limb cells. It is associated with calcium wasting, but magnesium wasting is usually mild. Loop diuretics, which inhibit ion transport by thick ascending limb cells, markedly increase urinary excretion of both calcium and magnesium. In contrast, Gitelman's syndrome is caused by dysfunction of the distal convoluted tubule. Hypocalciuria and hypomagnesemia are universal parts of this disorder. Yet although thiazide diuretics, which inhibit ion transport by distal convoluted tubule cells, reduce urinary calcium excretion, they have minimal effects on urinary magnesium excretion, when given acutely. This review proposes mechanisms that may account for the differences between the effects of diuretic drugs and the phenotypic features of Gitelman's and Bartter's syndromes. These mechanisms are based on recent insights from another inherited disease of ion transport, inherited magnesium wasting, and from a review of the chronic effects of diuretic drugs in animals and people.

Infusion of bovine parathyroid hormone1-34 attenuates the pressor response to angiotensin II in spontaneously hypertensive rats.

Parathyroid hormone, long known to be important for calcium homeostasis, also has potent vascular effects. In the past, pharmacologic doses of parathyroid hormone or its active amino-terminal fragment (PTH1-34) were necessary to demonstrate vasoactivity. We assessed the vascular effects of physiologic doses of infused synthetic bovine PTH1-34 (15 U/hr or 1.5 micrograms/hr) on the pressor response to angiotensin II. PTH1-34 attenuated the pressor response (p less than 0.005) to one to 100 nanograms of angiotensin II in both Aoki-Okamoto spontaneously hypertensive and normotensive Wistar Kyoto rats. There was no difference in response to either PTH or angiotensin II between strains, and at the lower doses of angiotensin II, PTH1-34 dampened the pressor response by as much as 33%. These results suggest that endogenous PTH may modulate systemic blood pressure and regional vascular resistance. In various states of parathyroid gland stimulation, the peptide may exert physiologically important vascular effects.

Mammalian distal tubule: physiology, pathophysiology, and molecular anatomy.

The distal tubule of the mammalian kidney, defined as the region between the macula densa and the collecting duct, is morphologically and functionally heterogeneous. This heterogeneity has stymied attempts to define functional properties of individual cell types and has led to controversy concerning mechanisms and regulation of ion transport. Recently, molecular techniques have been used to identify and localize ion transport pathways along the distal tubule and to identify human diseases that result from abnormal distal tubule function. Results of these studies have clarified the roles of individual distal cell types. They suggest that the basic molecular architecture of the distal nephron is surprisingly similar in mammalian species investigated to date. The results have also reemphasized the role played by the distal tubule in regulating urinary potassium excretion. They have clarified how both peptide and steroid hormones, including aldosterone and estrogen, regulate ion transport by distal convoluted tubule cells. Furthermore, they highlight the central role that the distal tubule plays in systemic calcium homeostasis. Disorders of distal nephron function, such as Gitelman's syndrome, nephrolithiasis, and adaptation to diuretic drug administration, emphasize the importance of this relatively short nephron segment to human physiology. This review integrates molecular and functional results to provide a contemporary picture of distal tubule function in mammals.

Structural prerequisites for the hypotensive action of parathyroid hormone.

We assessed the vascular, phosphaturic, and calcemic responses to several synthetic parathyroid hormone (PTH) analogues. Bovine (b) PTH (1-34), human (h) PTH (1-34), hPTH (53-84), [ Nle8 , Nle18 , Tyr34 ]bPTH (1-34), and [ Nle8 , Nle18 , Tyr34 ]bPTH (3-34) were administered in doses between 1 and 500 micrograms/kg as bolus intravenous injections to male Wistar-Kyoto rats aged 18-26 wk. Antagonism of the action of PTH was assessed in rats pretreated with 10 or 100 micrograms/kg [ Nle8 , Nle18 , Tyr34 ]bPTH (3-34) followed by 10 micrograms/kg of bPTH (1-34), or with 10 micrograms/kg hPTH (53-84) followed by 10 micrograms/kg hPTH (1-34). Bovine PTH (1-34), hPTH (1-34), and [ Nle8 , Nle18 , Tyr34 ]bPTH (1-34) produced virtually identical log dose-dependent hypotension, with 100 micrograms/kg of each analogue producing a 56% reduction in mean arterial pressure. Neither hPTH (53-84) nor [ Nle8 , Nle18 , Tyr34 ]bPTH (3-34) demonstrated any effect on mean arterial pressure at doses up to 500 micrograms/kg. Pretreatment with the inactive analogues failed to antagonize the vasodilating response to either bPTH (1-34) or hPTH (1-34). The vasoactive analogues significantly increased urinary phosphorus excretion while the inactive analogues did not modify it. hPTH (1-34) produced a modest decrease in serum Ca2+ at 1 min after injection. The results document that the vasodilating effect of PTH is a specific action of the peptide. Deletion of the first two amino acid residues abolishes both the phosphaturic and hypotensive effects of the peptide. Acute changes in serum Ca2+ do not appear to be a prerequisite for the vasodilatory response. Inactive analogues of PTH do not antagonize the vascular actions of the peptide.

Unidirectional potassium fluxes in renal distal tubule: effects of chloride and barium.

Low luminal concentrations of chloride stimulate net potassium secretion by the renal distal tubule, independent of changes in transepithelial voltage. These effects are not prevented by the luminal application of the potassium channel blocking agent barium. Because net potassium secretion comprises secretory and absorptive components, we sought to evaluate the effects of chloride and barium on unidirectional potassium fluxes in the renal distal tubule. In vivo microperfusion methods were used in anesthetized Sprague-Dawley rats. Perfusion solutions contained either 42K or 86Rb as tracers for potassium. Tracer efflux coefficients, indicating apparent potassium permeability, were similar when measured using either isotope. Net potassium flux was determined as the difference between perfusion and collected rate, and unidirectional absorptive potassium flux was calculated as the product of the mean luminal potassium concentration and the tracer efflux coefficient. During perfusion with a solution that resembled fluid normally arriving at the early distal tubule, the absorptive potassium flux was approximately 25% of the unidirectional secretory flux. Reducing lumen chloride concentration increased net potassium secretion, because blood-to-lumen potassium flux increased from 61 +/- 12.7 to 96 +/- 14.6 pmol/min. Barium reduced both absorptive and secretory fluxes but did not prevent the stimulation of net potassium secretion that occurs when luminal chloride concentration is reduced. Apparent potassium permeability during perfusion with a solution that resembled fluid normally arriving at the early distal tubule was 800 nm/s when corrected for voltage. Together with the results of previous experiments, these results are consistent with the presence of a secretory pathway linking potassium with chloride in the luminal membrane of cells of the distal tubule.