Lumen-positive Transepithelial Voltage Research Articles

Distinct cell types along thick ascending limb express pathways for monovalent and divalent cation transport.

Kidney thick ascending limb cells reabsorb sodium, potassium, calcium, and magnesium and contribute to urinary concentration. These cells are typically viewed as of a single type that recycles potassium across the apical membrane and generates a lumen-positive transepithelial voltage driving calcium and magnesium reabsorption, although variability in potassium channel expression has been reported. Additionally, recent transcriptomic analyses suggest that different cell types exist along this segment, but classifications have varied and have not led to a new consensus model. We used immunolocalization, electrophysiology and enriched single nucleus RNA-Seq to identify thick ascending limb cell types in rat, mouse and human. We identified three major TAL cell types defined by expression of potassium channels and claudins. One has apical potassium channels, low basolateral potassium conductance, and is bordered by a sodium-permeable claudin. A second lacks apical potassium channels, has high basolateral potassium conductance and is bordered by calcium- and magnesium-permeable claudins. A third type also lacks apical potassium channels and has a high basolateral potassium conductance, but these cells are ringed by sodium-permeable claudins. The recognition of diverse cell types resolves longstanding questions about how solute transport can be modulated selectively and how disruption of these cells leads to human disease.

Magnesium biology.

Magnesium (Mg2+) is essential for energy metabolism, muscle contraction and neurotransmission. As part of the Mg-ATP complex, it is involved in over 600 enzymatic reactions. Serum Mg2+ levels are tightly regulated between 0.7 and 1.1mmol/L by interplay of intestinal absorption and renal excretion. In the small intestine, Mg2+ is absorbed paracellularly via claudin-2 and -12. In the colon, transcellular absorption of Mg2+ is facilitated by TRPM6/7 and CNNM4. In the kidney, the proximal tubule reabsorbs only 20% of the filtered Mg2+. The majority of the filtered Mg2+ is reabsorbed in the thick ascending limb, where the lumen-positive transepithelial voltage drives paracellular transport via claudin-16/-19. Fine-tuning of Mg2+ reabsorption is achieved in the distal convoluted tubule (DCT). Here, TRPM6/7 tetramers facilitate apical Mg2+ uptake, which is hormonally regulated by insulin and epidermal growth factor. Basolateral Mg2+ extrusion is Na+ dependent and achieved by CNNM2 and/or SLC41A3. Hypomagnesemia (serum Mg2+ <0.7mmol/L) develops when intestinal and/or renal Mg2+ (re)absorption is disturbed. Common causes include alcoholism, type 2 diabetes mellitus and the use of pharmacological drugs, such as proton-pump inhibitors, calcineurin inhibitors and thiazide diuretics. Over the last decade, research on rare genetic and acquired Mg2+ disorders have identified Mg2+ channel and transporter activity, DCT length, mitochondrial function and autoimmunity as mechanisms explaining hypomagnesemia. Classically, treatment of hypomagnesemia depended on oral or intravenous Mg2+ supplementation. Recently, prebiotic dietary fibers and sodium-glucose cotransporter 2 inhibitors have been proposed as promising new therapeutic pathways to treat hypomagnesemia.

Increased expression of renal TRPM6 compensates for Mg(2+) wasting during furosemide treatment.

BackgroundFurosemide is a loop diuretic, which blocks the Na+, K+, 2Cl− cotransporter (NKCC2) in the thick ascending limb of Henle (TAL). By diminishing sodium (Na+) reabsorption, loop diuretics reduce the lumen-positive transepithelial voltage and consequently diminish paracellular transport of magnesium (Mg2+) and calcium (Ca2+) in TAL. Indeed, furosemide promotes urinary Mg2+ excretion; however, it is unclear whether this leads, especially during prolonged treatment, to hypomagnesaemia. The aim of the present study was, therefore, to determine the effect of chronic furosemide application on renal Mg2+ handling in mice.MethodsTwo groups of 10 mice received an osmotic minipump subcutaneously for 7 days with vehicle or 30 mg/kg/day furosemide. Serum and urine electrolyte concentrations were determined. Next, renal mRNA levels of the epithelial Mg2+ channel (TRPM6), the Na+, Cl− cotransporter (NCC), the epithelial Ca2+ channel (TRPV5), the cytosolic Ca2+-binding protein calbindin-D28K, as well parvalbumin (PV), claudin-7 (CLDN7) and claudin-8 (CLDN8), the epithelial Na+ channel (ENaC) and the Na+–H+ exchanger 3 (NHE3) were determined by real-time quantitative polymerase chain reaction. Renal protein levels of NCC, TRPV5, calbindin-D28K and ENaC were also measured using semi-quantitative immunohistochemistry and immunoblotting.ResultsThe mice chronically treated with 30 mg/kg/day furosemide displayed a significant polyuria (2.1 ± 0.3 and 1.3 ± 0.2 mL/24 h, furosemide versus control respectively, P < 0.05). Furosemide treatment resulted in increased serum concentrations of Na+ [158 ± 3 (treated) and 147 ± 1 mmol/L (control), P < 0.01], whereas serum K+, Ca2+ and Mg2+ values were not significantly altered in mice treated with furosemide. Urinary excretion of Na+, K+, Ca2+ and Mg2+ was not affected by chronic furosemide treatment. The present study shows specific renal upregulation of TRPM6, NCC, TRPV5 and calbindin-D28K.ConclusionsDuring chronic furosemide treatment, enhanced active reabsorption of Mg2+ via the epithelial channel TRPM6 in DCT compensates for the reduced reabsorption of Mg2+ in TAL.

Proteinase-activated Receptor 2 Stimulates Na,K-ATPase and Sodium Reabsorption in Native Kidney Epithelium

Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.

An antidiuretic peptide (Tenmo-ADFb) with kinin-like diuretic activity on Malpighian tubules of the house cricket, Acheta domesticus (L.)

Acheta domesticus is reported to have an antidiuretic hormone that reduces Malpighian tubule secretion. Identified peptides known to work in this way (Tenmo-ADFa and ADFb, and Manse-CAP(2b)) were tested as candidates for the unidentified hormone, along with their second messenger, cyclic GMP. Only Tenmo-ADFb was active, but was diuretic, as was 8-bromo cyclic GMP. The activity of Tenmo-ADFb is comparable to that of the cricket kinin neuropeptide, Achdo-KII, but it is much less potent. Its activity was unaffected by deleting either the six N-terminal residues or the C-terminal phenylalanine. At high concentrations, tubule secretion is doubled by Tenmo-ADFb and Achdo-KII, but their actions are non-additive, suggesting they have a similar mode of action. Both stimulate a non-selective KCl and NaCl diuresis, which is consistent with the opening of a transepithelial Cl(-) conductance. In support of this, the diuretic response to Tenmo-ADFb and Achdo-KII is prevented by a ten-fold reduction in bathing fluid chloride concentration, and both peptides cause the lumen-positive transepithelial voltage to collapse. The Cl(-) conductance pathway appears likely to be transcellular, because the Cl(-) channel blocker DPC reduces both basal and peptide-stimulated rates of secretion. The effects of 8-bromo cyclic GMP on transepithelial voltage and composition of the secreted fluid are markedly different from those of Tenmo-ADFb. This is the first report of the antidiuretic factor Tenmo-ADFb stimulating tubule secretion. Although the actions of Tenmo-ADFb are indistinguishable from those of Achdo-KII, it is unlikely to act at a kinin receptor, because the core sequence (residues 7-12) lacks the Phe and Trp residues that are critical for kinin activity.

Dose-dependent effects of CRF-like diuretic peptide on transcellular and paracellular transport pathways.

The mechanism of action of synthetic Culex corticotropin-releasing factor (CRF)-like diuretic peptide (CCRF-DP) was investigated in isolated, perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Low concentrations of CCRF-DP (10(-10) and 10(-9) M) caused depolarizing oscillations of the lumen-positive transepithelial voltage (Vt) in Malpighian tubules, whereas high concentrations (10(-8) and 10(-7) M) first depolarized and then transiently hyperpolarized Vt; CCRF-DP always lowered transepithelial resistance (Rt), regardless of voltage depolarization or hyperpolarization. The short-circuit current (Isc), an electrical estimate of active transepithelial transport of Na and K, remained unchanged at low concentrations of CCRF-DP, but Isc more than doubled at high concentrations. These effects of CCRF-DP suggest dose-dependent sites of action: low concentrations of CCRF-DP affect the paracellular pathway, and high concentrations affect both paracellular and transcellular pathways.

Ca2+, Mg2+ and K+ transport in the cortical and medullary thick ascending limb of the rat nephron: influence of transepithelial voltage

Isolated segments of rat cortical (cTAL) and medullary (mTAL) thick ascending limbs were microperfused and the transepithelial net fluxes (JX) were determined by measuring the composition of the collected fluid with an electron microprobe. When perfused with symmetrical solutions both segments showed similar JNa and JCl and lumen-positive transepithelial voltage (Vte = 7-8 mV). JMg, JCa and JK were not significantly different from zero. When perfused with asymmetrical solutions (lumen 50 mM, bath 150 mM NaCl), the mean Vte were 23 mV and 17 mV in the cTAL and mTAL respectively; this rise was accompanied by significant increases in JMg and JCa in the cTAL, but not in the mTAL, and a marked increase in JK in both segments. It is concluded that, in the rat, divalent cations can be reabsorbed in the cTAL, and K+ can be reabsorbed in the cTAL and mTAL. The transport is voltage-dependent. The mTAL can reabsorb neither Mg2+ nor Ca2+, whatever Vte.

Diluting segment in kidney of dogfish shark. I. Localization and characterization of chloride absorption

Single tubules, dissected from the peritubular sheath of the dorsal bundle zone of kidney of the dogfish shark, Squalus acanthias, were perfused in vitro at 17-18 degrees C. This segment is the largest of the five in the peritubular sheath and had average inner and outer diameters of 46.9 +/- 1.2 and 74.4 +/- 2.1 microns, respectively (n = 32). These values suggest that this is the intermediate IV segment. When perfused with symmetrical buffered elasmobranch saline, intermediate IV segments exhibited high rates of Cl- absorption (JCl, pmol.s-1.cm-2): 1,696 at an average perfusion rate (Vo) of 8.2 nl/min. Cl- absorption was highly flow dependent [1/JCl = 57.95(1/Vo) + 1.75; r = 0.71, P less than 0.01]. Maximal rates of Cl-absorption, calculated from reciprocal transformation of the flow dependence of JCl, yielded a value of 5,714 pmol.s-1.cm-2. In the presence of a 200-mosmol/kg transepithelial osmotic gradient, fluid absorption was negligible. The spontaneous transepithelial voltage (Vte, mucosal with respect to serosal compartment) averaged 8.0 +/- 1.0 mV (n = 26). Such active transport of Cl- in the absence of fluid movement and in the presence of a lumen-positive transepithelial voltage is characteristic of amphibian and mammalian diluting segments. Na(+)-to-Cl- permeability ratios (PNa/PCl) averaged 2.5 +/- 0.5, indicating that, as in mammalian thick ascending limbs, this segment is Na+ (cation) permselective. Vte was dependent on the presence of Na+ and Cl- in the external solutions and was reversibly abolished by isosmotic replacement with N-methyl-D-glucamine or with isethionate, respectively. Ouabain inhibited Vte but was not reversible within the time course of these experiments. Furosemide (10(-4) M), but not equimolar concentrations of amiloride or hydrochlorothiazide, added to the luminal perfusate inhibited both Vte and JCl. These results suggest that apical membrane Na+ entry in intermediate IV segments is mediated by Na(+)-K(+)-Cl- cotransport and is consistent with the existence of a functional role of urinary dilution in the reabsorption of urea in the elasmobranch kidney.

Ca2+ absorption in the pars recta of cortical S2 rabbit proximal tubules: role of diffusion.

Individual partes rectae of cortical S2 proximal tubules were dissected from rabbit kidneys and perfused in vitro. The bath was a protein-free, plasmalike solution, whereas the perfusate simulated "late" proximal tubular fluid (high [Cl-], low [HCO3-], absence of Na+-cotransported solutes). Transepithelial voltages at the perfusion and collection ends were measured simultaneously in doubly cannulated tubules. Ionized calcium concentrations in perfusates, collectates, and the bath were calculated from measurements of calcium ion activities made in situ with Ca2+-selective microelectrodes. Epithelial calcium permeability was estimated from the bath-to-lumen movement of 45Ca. Total calcium concentrations of perfused and collected fluids were measured by continuous-flow microcolorimetry. Under these conditions there was a lumen-positive transepithelial voltage that was equal at both ends of the tubules. Luminal [Ca2+] [corrected], which decreased along the length of the tubule, was greater than that of the bath. Using the measured calcium permeability, we calculated the diffusional component of the net calcium absorptive flux from the mean transepithelial delta [Ca2+] and the measured transepithelial voltage. We found that ionic diffusion accounted for the net flux, suggesting that diffusion is the primary mechanism of calcium absorption in this segment of the rabbit nephron.

Electrophysiology of collecting duct H+ secretion: effect of inhibitors.

Segments of the outer medullary collecting duct were isolated from the inner stripe of the rabbit kidney (OMCDi), perfused in vitro, and impaled across their basolateral membranes with voltage-recording microelectrodes. The disulfonic stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-4) M) and the carbonic anhydrase inhibitor acetazolamide (10(-4) M) depolarized the lumen-positive transepithelial voltage (VT) toward 0 mV when added to the bath solution. Concurrently, the basolateral membrane voltage (Vbl) hyperpolarized. The hyperpolarization of Vbl, which averaged 19.3 +/- 2.9 mV (n = 11) for SITS and 22.7 +/- 3.5 mV (n = 11) for acetazolamide, was not due to an alteration in the ionic selectivity of the basolateral membrane, which was highly Cl- selective. The hyperpolarization of Vbl could best be explained by a decrease in the intracellular [Cl-], and the associated shift in the emf for Cl- (ECl) across the basolateral membrane. The decrease in intracellular [Cl-] could be attributed to inhibition of a Cl-HCO3 antiporter in the basolateral membrane. SITS appeared to inhibit this antiporter directly, whereas the effect of acetazolamide was indirect, probably secondary to a decrease in the intracellular [HCO3-]. Finally, both SITS and acetazolamide induced or unmasked an electroneutral K+-coupled transport system in the basolateral membrane.

Active absorption of NH4+ by rat medullary thick ascending limb: inhibition by potassium.

These experiments were designed to determine the relative contributions of active NH4+ transport and voltage-driven NH4+ diffusion to direct NH4+ absorption by the medullary thick ascending limb of the rat. Medullary thick ascending limbs were perfused in vitro with solutions containing 25 mM HCO3 and 4 mM total ammonia. Under steady-state conditions, the lumen-positive transepithelial voltage (VT) was not sufficient to account for the observed decrease in lumen NH4+ concentration, consistent with active absorption of NH4+. Flux calculations based on VT and measured NH4+ permeability (6 x 10(-5) cm/s) indicate that the majority (at least 65%) of total ammonia absorption is due to active transport of NH4+. The remainder of NH4+ absorption can be accounted for by voltage-driven diffusion. Increasing the potassium concentration from 4 to 24 mM in perfusate and bath markedly inhibited total ammonia absorption but did not affect VT, NH4+ permeability, or HCO3 absorption. These results are consistent with inhibition of the active component of NH4+ absorption by potassium. The active NH4+ absorption is likely mediated by cotransport of Na+, NH4+, and Cl- across the apical cell membrane. Inhibition of active NH4+ absorption by an increase in potassium concentration may be due, in part, to competition between NH4+ and K+ for a common binding site on the Na+ -K+ -2Cl- cotransport system.

Ca2+ transport in diluting segment of frog kidney.

In the isolated-perfused frog (Rana pipiens) kidney the question of whether transepithelial transport of Ca2+ is a passive voltage driven process or involves active mechanisms was investigated. With conventional and ion-sensitive microelectrodes transepithelial electrical and electrochemical potential differences were measured. Luminal activities and transepithelial net fluxes of Ca2+ and Cl- were evaluated. Different transepithelial electrical voltages in a wide range (+20 to -4 mV) were generated by "chemical voltage clamping" and the dependence of Ca2+ net fluxes on these voltages investigated. The hormonal control of both Cl- and Ca2+ transport was studied by evaluating the effect of the cell-permeable cAMP analogue, db-cAMP and of the adenylate cyclase stimulator, forskolin. The experiments reveal that: (a) Ca2+ is reabsorbed along the diluting segment of frog kidney. (b) Ca2+ reabsorption is inhibited by furosemide because of the elimination of the transepithelial voltage. (c) There is a direct relationship between transepithelial voltage and Ca2+ reabsorption. (d) Neither Cl- nor Ca2+ reabsorption are affected by db-cAMP or forskolin. We conclude that Ca2+ reabsorption is passive, driven by the lumen-positive transepithelial voltage. It most likely occurs via the paracellular shunt pathway.

Diluting segment in avian kidney. II. Water and chloride transport.

The mammalian-type nephrons of avian kidneys contain a Henle's loop that runs parallel to the collecting ducts and the vasa recta. Thus we examined whether the thick ascending limb (TAL) of Henle's loop of the avian kidney acts as a diluting segment by measuring water and Cl transport in the isolated and perfused TAL of the quail, Coturnix coturnix. The TAL showed a lumen-positive transepithelial voltage (Vt) (+9.4 +/- 0.4 mV, n = 28). Net water flux (Jv) was nearly zero when the TAL was perfused and bathed with isosmotic solution. When the osmotic gradient was imposed, Jv increased only slightly, and thus the osmotic water permeability (Lp) was low. Arginine vasotocin (AVT) added to the hyperosmotic bath did not alter either Jv, Lp, or Vt. Cl efflux (lumen to bath, 370.4 +/- 27.7 peq X mm-1 X min-1) was higher than Cl influx (bath to lumen, 98.6 +/- 14.3 peq X mm-1 X min-1) when measured in the different tubules. AVT showed no effect on Cl efflux. These results indicate that in the TAL of the quail osmotic water permeability is low while net Cl reabsorption is present, suggesting that the TAL functions as a diluting segment.

PGE2, forskolin, and cholera toxin interactions in modulating NaCl transport in mouse mTALH.

Prostaglandin E2 (PGE2) inhibits the ADH-stimulated components of the lumen-positive transepithelial voltage (Ve) and of net chloride absorption (JnetCl) in the isolated microperfused mouse medullary thick ascending limb of Henle (mTALH), presumably by interfering with the ADH-dependent intracellular accumulation of cAMP. These experiments examined the interactions of PGE2 with two nonhormonal stimulators of adenylate cyclase--cholera toxin and forskolin--in an attempt to evaluate the means by which PGE2 inhibits ADH-stimulated transport in these mTALH segments. Forskolin (FSK) stimulated Ve in the mTALH with half-maximal stimulation at 1.4 X 10(-7) M FSK. PGE2 had no effect on FSK stimulation of Ve; 10(-6) M FSK reversed completely the PGE2 inhibition of ADH-stimulated Ve. A low concentration of cholera toxin, 5 X 10(-13) M, stimulated Ve and JnetCl in the mTALH; 10(-6) M PGE2 inhibited the stimulation by cholera toxin; and 10(-6) M FSK reversed the PGE2 inhibition of both Ve and JnetCl in cholera toxin-stimulated mTALH. A higher concentration of cholera toxin, 10(-10) M, stimulated Ve and JnetCl to values identical to those seen with maximal concentrations of ADH, but PGE2 did not inhibit the increments in either Ve or JnetCl produced by 10(-10) M cholera toxin. PGE2 appears to inhibit ADH stimulation of NaCl transport in mTALH by an action distal to hormone-receptor interactions yet proximal to the catalytic subunit of adenylate cyclase.

Effect of ouabain on K secretion in cortical collecting tubules from adrenalectomized rabbits.

Previous studies have shown that Na-K-ATPase activity in the cortical collecting tubule of the rabbit increases significantly with mineralocorticoid stimulation and decreases significantly with adrenalectomy. The present study examined the effects of 10(-4) M ouabain on K secretion in the isolated perfused cortical collecting tubules from normal, adrenalectomized, and mineralocorticoid-stimulated animals. Potassium secretion was similar in the tubules from adrenalectomized (3.42 +/- 0.53 pmol X mm-1 X min-1) and from normal rabbits (3.38 +/- 0.36 pmol X mm-1 X min-1). K secretion was greater (15.1 +/- 3.0 pmol X mm-1 X min-1) in tubules from animals receiving 11-deoxycorticosterone acetate (DOCA). Ouabain inhibition of K secretion was 74% for the adrenalectomized group, 86% for the normal group, and 98% for the DOCA-treated group. The degree of inhibition was statistically equivalent among the three groups and not statistically different from complete inhibition of K secretion. Ouabain had no effect on the lumen-positive transepithelial voltage of the cortical collecting tubules from adrenalectomized rabbits but reduced the lumen-negative voltage in tubules from normal rabbits and reversed the polarity of the transepithelial voltage in cortical collecting tubules from DOCA-treated animals. Thus adrenal mineralocorticoids may be necessary for maximal K secretion by the cortical collecting tubule, but they are not essential to maintain K secretion during "normal" K intake. In all groups K secretion is totally dependent on Na-K-ATPase. The lumen-positive transepithelial voltage from DOCA-treated animals after addition of ouabain suggests an additional effect of mineralocorticoid to stimulate secretion of cations (protons) or reabsorption of anions.

Effects of arginine vasopressin on the thin ascending limb of Henle's loop of hamsters.

Arginine vasopressin (AVP) has been shown to stimulate active Cl transport across the medullary thick ascending limb of Henle's loop (MAL) in association with an increase in adenylate cyclase activity. To determine whether the failure to demonstrate active Cl transport across the thin ascending limb of Henle's loop (TAL) in previous in vitro perfusion studies was due to the absence of AVP in the preparation, we examined the effect of AVP on adenylate cyclase activity and Cl transport in the hamsters TAL. AVP (1 mU/ml) increased adenylate cyclase activity in the hamster TAL (20.7 +/- 5.2 control vs. 46.2 +/- 10.1 fmol . mm-1 . 30 min-1, n = 6, P less than 0.05) but not in the descending limb (27.8 +/- 7.0 control vs. 20.4 +/- 2.7, n = 4, P less than 0.05). When both MAL and TAL were perfused, a lumen-positive transepithelial voltage (Vt) was observed. The Vt was increased by adding 1 or 10 mU/ml AVP to the bath. When only the TAL was perfused, the Vt was not different from zero. Similar results were obtained in mouse renal tubules. In other experiments, AVP did not affect the diffusion potential generated when a transepithelial NaCl gradient was present. AVP or dibutyryl cAMP caused little or no change in efflux of radioactive chloride across the hamster TAL. These findings suggest that electrogenic chloride transport is not demonstrable in the TAL even in the presence of AVP. The physiologic role of AVP-sensitive adenylate cyclase in the TAL remains to be established.

New pathways for potassium transport in the kidney.

This review focuses on the hypothesis that potassium is recycled in the medulla by secretion into the pars recta or descending limb of long-looped nephrons and reabsorption from the ascending limb and/or medullary collecting duct. Evidence supporting the recycling hypothesis is summarized and the process is analyzed quantitatively by an examination of the mass flow of potassium reaching different sites along superficial and juxtamedullary nephrons and collecting tubules. From differences in potassium mass flow between sites, we have estimated the amount of potassium that must be secreted or absorbed by individual segments of the renal tubule. These rates of secretion and absorption are compared with the potassium transport characteristics of the respective segments, as assessed by isolated tubule perfusion in vitro and micropuncture in vivo. It is apparent that potassium secretion can occur passively in the pars recta and descending limb of long-looped nephrons as a consequence of the elevated potassium concentration in the medullary interstitium. At present, no active potassium absorptive mechanism has been demonstrated in any segment of the ascending limb. Due to the very high ionic permeability of the thin ascending segment and the lumen-positive transepithelial voltage in the thick ascending segment, however, considerable passive absorption likely occurs, although net potassium secretion has also been demonstrated in the cortical thick ascending limb. The high potassium concentration in the inner medullary interstitium and the difference in mass flow of potassium between the end of superficial nephrons in the cortex and the collecting ducts in the papilla, at least under certain circumstances, are best accounted for by net potassium reabsorption in the medullary collecting duct.