Apical Na ⁺ -H ⁺ exchange plays a critical role in the transcellular and paracellular reabsorption of NaCl by the PT. Furthermore, Na ⁺ -H ⁺ exchange plays a key role in the functional “absorption” of HCO ₃ ⁻ by the early PT (HCO ₃ ⁻ does not actually move across the apical membrane but is generated within cells with H ⁺ via carbonic anhydrase activity see Chapter 9 ). As the movement of Na ⁺ and HCO ₃ ⁻ drives osmotic water movement, it increases the luminal concentration of Cl ⁻ , driving passive paracellular transport of Cl ⁻ . Increases in luminal Cl ⁻ also help drive the apical uptake of Cl ⁻ during transcellular transport. Na ⁺ -H ⁺ exchange is predominantly mediated by the NHE proteins, encoded by the nine members of the SLC9 gene family; NHE3 in particular plays an important role in proximal tubular physiology. NHE3 is expressed at the apical membrane of S1, S2, and S3 segments, as are other Na ⁺ -dependent H ⁺ transporters including NHE8. ^, NHE8 predominates over NHE3 in the neonatal PT, with subsequent induction of NHE3 and downregulation of NHE8 in mature, adult nephrons. The primacy of NHE3 in mature PTs is illustrated by the renal phenotype of NHE3 knockout mice, which have a 62% reduction in proximal fluid absorption and a 54% reduction in baseline chloride absorption. ^, Although the severe salt-wasting phenotype seen in global NHE3 knockout mice results largely from its deletion in intestine, NHE3 disruption in the kidney showed that it plays a role in maintaining blood pressure at baseline and plasma Na ⁺ in response to increased or reduced dietary NaCl, with compensatory upregulation of the distal Na ⁺ reabsorptive pathways (NaCl transport, Na ⁺ -Cl-cotransporter [NCC], and epithelial Na ⁺ channel, ENaC).

Evidence for the involvement of an apical anion exchanger in transepithelial NaCl transport by the PT first came from the use of anion transport inhibitors. DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid), furosemide, and SITS all reduce fluid absorption from the lumen of PT (PT) segments perfused with solutions containing NaCl. Determining the identity of this anion exchanger has been challenging. Early studies using isolated PT membrane vesicles suggested the existence of a Cl ⁻ -OH ⁻ exchanger, but other evidence pointed toward a more dominant Cl ⁻ -formate exchanger activity in the absence of significant Cl ⁻ -OH ⁻ exchange. Formate transport stimulated by a pH gradient (H ⁺ -formate cotransport or formate-OH ⁻ exchange) is saturable, consistent with a carrier-mediated process rather than diffusion of formic acid across the apical membrane. Cl ⁻ -oxalate and SO ₄ ²⁻ -oxalate exchange mechanisms have also been detected in PT membrane vesicles. ^, On the basis of differences in the affinities and inhibitor sensitivity of the Cl ⁻ -oxalate and Cl ⁻ -formate exchange activities, two separate apical exchangers, a Cl ⁻ -formate exchanger and a Cl ⁻ –formate-oxalate exchanger, may exist ( Fig. 6.5 ). In isolated perfused rabbit, rat, and mouse PTs, both formate and oxalate significantly increased fluid transport. This increase in fluid transport was inhibited by DIDS, suggesting involvement of the DIDS-sensitive anion exchanger(s) detected in earlier vesicle studies. The oxalate- and formate-dependent anion transporters in the PT are likely coupled to distinct Na ⁺ entry pathways (Na ⁺ -SO ₄ ²⁻ cotransport and Na ⁺ -H ⁺ exchange, respectively). Coupling of Cl ⁻ -oxalate exchange and Na ⁺ -SO ₄ ²⁻ cotransport requires the additional presence of SO ₄ ²⁻ -oxalate exchange, which has been demonstrated in membrane vesicle studies. The formate effect on Cl ⁻ transport, but not the oxalate effect, is abolished in NHE3 knockout mice demonstrating the formate and Na ⁺ -H ⁺ connection. Finally, tubular perfusion data from superficial and juxtamedullary proximal convoluted tubules suggest heterogeneity in the dominant mode of anion exchange along the PT, such that Cl ⁻ -formate exchange is absent in juxtamedullary proximal convoluted tubule (PCTs), in which Cl ⁻ -OH ⁻ exchange may instead be dominant.

Transepithelial NaCl transport in the proximal tubule.

(A) In the simplest scheme, Cl ⁻ enters the apical membrane via a Cl ⁻ -OH ⁻ exchanger, coupled to Na ⁺ entry via NHE3. (B) Alternative apical anion exchange activities that couple to Na ⁺ -H ⁺ exchange and Na ⁺ -SO ₄ ²⁻ cotransport. (C) Alternative apical anion exchange activities that couple to Na ⁺ -H ⁺ and oxalate (Ox ⁻ ) or formate (HCOO ⁻ ) exchange. See text for details.

While the SLC26A4 anion exchanger, also known as pendrin, is capable of Cl ⁻ -formate exchange when expressed in Xenopus laevis oocytes, its expression in the PT is minimal or absent in several species. Furthermore, formate-stimulated NaCl transport in this nephron segment is unimpaired in Slc26A4 null mice. The role of SLC26A4 in Cl ⁻ transport by the distal nephron is reviewed later in this chapter (see the subhead “Cl ⁻ Transport” under “Connecting Tubules and the Cortical Collecting Duct” later). Another member of the SLC26 family, SLC26A6, is present at the apical membrane of PT cells. Murine SLC26A6 when expressed in Xenopus oocytes, mediates the multiple modes of anion exchange that have been implicated in transepithelial NaCl by the PT including Cl ⁻ -formate, Cl ⁻ -OH ⁻ , Cl ⁻ -SO ₄ ²⁻ , and SO ₄ ²⁻ -oxalate exchange. However, tubule perfusion experiments in SLC26A6 null mice did not reveal a reduction in baseline Cl ⁻ or fluid transport, and whole animal studies revealed no effects on NaCl homeostasis or blood pressure. SLC26A6 does appear to be the dominant Cl ⁻ -oxalate exchanger along the PT. Slc26a6 knockout mice display reduced fractional excretion of oxalate and loss of oxalate-induced tubular fluid transport and Cl ⁻ -oxalate exchange in membrane vesicles. ^, Other candidates for PT Cl ⁻ transport include SLC26A7 and SLC26A2. ^{, ,} SLC26A7 has been reported to act as a Cl ⁻ /HCO3 ⁻ exchanger, while SLC26A2 displays SO ₄ ²⁻ /Cl ⁻ exchange activity.

SLC26A6 mediates electrogenic Cl ⁻ -OH ⁻ and Cl ⁻ -HCO ₃ ⁻ exchange, and most if not all the members of this family are electrogenic in at least one mode of anion transport. ^{, , , ,} Electroneutrality of transcellular NaCl transport may be preserved by coexpression of two or more electrogenic SLC26 exchangers in the same membrane. Notably, the stoichiometry and electrophysiology of Cl ⁻ -base exchange differ for individual members of the family. For example, SLC26A6 exchanges one Cl ⁻ for two HCO ₃ ⁻ anions, whereas SLC26A3 exchanges two Cl ⁻ anions for one HCO ₃ ⁻ anion. ^, Apical K ⁺ channels in the PT may also stabilize membrane potential during NaCl absorption. One channel involved may be Kir4.2, recently identified as a major component of the basolateral K ⁺ conductance in PT, where it contributes to the driving force for HCO ₃ ⁻ exit through the Na ⁺ -HCO ₃ ^– cotransporter NBCe1.

Preferential coupling of Cl ⁻ -formate exchange to Na ⁺ -H ⁺ exchange mediated by NHE3 (see Fig. 6.5 ) may involve the scaffolding proteins that serve to cluster different transporters to separate membrane microdomains. Notably, while both SCL26A6 and NHE3 bind to the scaffolding protein PDZK1, distribution of SLC26A6 is selectively impaired in PDZK1 knockout mice. Petrovic and colleagues have also reported a novel activation of proximal Na ⁺ -H ⁺ exchange by luminal formate, suggesting a direct effect of formate per se on NHE3 that may partly explain the preferential coupling of Cl ⁻ -formate exchange to NHE3. Despite these intriguing observations, the relative importance of transcellular vs passive paracellular Cl ^– reabsorption in the PT remains to be established with certainty.

Basolateral Na ⁺ -K ⁺ -ATPase activity establishes the Na ⁺ gradient for transcellular NaCl transport by the PT and provides the major exit pathway for Na ⁺ . To preserve the electroneutrality of transcellular NaCl transport, basolateral Na ⁺ exit must be balanced by an equal exit of Cl ⁻ . Several exit pathways for Cl ⁻ have been identified in PT including K ⁺ -Cl ⁻ cotransport, Cl ⁻ channels, and various modalities of Cl ⁻ -HCO ₃ ⁻ exchange (see Fig. 6.5 ).

Several lines of evidence support the existence of a swelling-activated basolateral K ⁺ -Cl ⁻ cotransporter (KCC) in the PT. KCC proteins are encoded by four members of the cation-chloride cotransporter gene family (KCCs 1–4). KCC1, KCC3, and KCC4 are all expressed in the kidney with strong expression of KCC3 and KCC4 at the basolateral membrane of the PT. Basolateral membrane vesicles isolated from the renal cortex reportedly contain K ⁺ -Cl ⁻ cotransport activity, and electroneutral K ⁺ -Cl ⁻ cotransport has been detected at the basolateral membrane of proximal straight tubules. ^, Cell swelling in response to apical Na ⁺ absorption is postulated to activate volume-sensitive basolateral K ⁺ -Cl ⁻ cotransporter activity that coordinates transepithelial absorption of NaCl. Activation of apical Na ⁺ -glucose transport in proximal tubular cells strongly activates a barium-resistant (Ba ²⁺ ) K ⁺ efflux pathway that is inhibited by 1 mmol/L furosemide, a concentration sufficient to inhibit activity of all KCC isoforms. In addition, a volume regulatory decrease (VRD) in Ba ²⁺ -blocked PTs swollen by hypotonic conditions is blocked by 1 mmol/L furosemide. Furthermore, targeted deletion of KCC3 or KCC4 in mice reduces VRD in the PT and perfused PTs from KCC3 knockout mice display lower transepithelial fluid transport. Together, these data suggest an important role for basolateral K ⁺ -Cl ⁻ cotransport in transcellular NaCl reabsorption.

The basolateral Cl ⁻ conductance of mammalian proximal tubular cells is relatively low, suggesting a lesser role for Cl ⁻ channels in transepithelial NaCl transport. Basolateral anion substitutions have significant effects on intracellular Cl ^– concentrations but minimal effects on the membrane potential; reciprocal changes in basolateral membrane potential do not affect intracellular Cl ⁻ . ^{, ,} Similar to basolateral K ⁺ -Cl ⁻ cotransport, basolateral Cl ⁻ channels in the PT may be relatively inactive in the absence of cell swelling. Cell swelling activates both K ⁺ and Cl ⁻ channels at the basolateral membranes of proximal tubular cells. ^{, ,} Seki and associates reported the presence of a basolateral Cl ⁻ channel in S3 segments of the rabbit nephron that is resistant to the KCC inhibitor H74. The molecular identity of these and other basolateral Cl ⁻ channels in the proximal nephron is not known with certainty, although S3 segments have been shown to express mRNA exclusively for the swelling-activated CLC-2 Cl ⁻ channel. Its role, however, in transcellular NaCl reabsorption is not yet clear.

Finally, there is functional evidence for Na ⁺ -dependent and Na ⁺ -independent Cl ⁻ -HCO ₃ ⁻ exchange at the basolateral membrane of proximal tubular cells. ^{, ,} The impact of Na ⁺ -independent Cl ⁻ -HCO ₃ ⁻ exchange on basolateral exit is thought to be minimal. In contrast, there is a 75% reduction in the rate of decrease in intracellular Cl ⁻ after the removal of basolateral Na ⁺ alone. The Na ⁺ -dependent Cl ⁻ -HCO ₃ ⁻ exchanger may thus play a considerable role in basolateral Cl ⁻ exit, with recycled exit of Na ⁺ and HCO ₃ ⁻ via the basolateral Na ⁺ -HCO ₃ ⁻ cotransporter NBC1 (see Fig. 6.5 ).

A fundamental property of the kidney is the phenomenon of glomerulotubular balance, whereby changes in the glomerular filtration rate (GFR) are paralleled by changes in tubular reabsorption, thus maintaining a constant fractional reabsorption of fluid and NaCl ( Fig. 6.6 ). Although the distal nephron is capable of adjusting reabsorption in response to changes in tubular flow, the impact of GFR on NaCl reabsorption by the PT is particularly pronounced ( Fig. 6.7 ). Glomerulotubular balance is independent of direct neuronal and systemic hormonal control and is thought to be mediated by the additive effects of luminal and peritubular factors.

Glomerulotubular balance.

The tubular fluid-to-plasma ratio of the nonreabsorbable marker, inulin (TF/P Inulin), at the end of the proximal tubule (PT), which is used as a measure of fractional water absorption by the PT, does not change as a function of single-nephron GFR (SNGFR). Measurements were done during antidiuresis (triangles) and water diuresis (circles).

From Schnermann J, Wahl M, Liebau G, Fischbach H. Balance between tubular flow rate and net fluid reabsorption in the proximal convolution of the rat kidney. I. Dependency of reabsorptive net fluid flux upon proximal tubular surface area at spontaneous variations of filtration rate. Pflugers Arch. 1968;304:90–103.

Glomerulotubular balance.

Shown is the linear increase in absolute fluid reabsorption by the late proximal tubule as a function of single-nephron GFR (SNGFR).

From Spitzer A, Brandis M. Functional and morphologic maturation of the superficial nephrons. Relationship to total kidney function. J Clin Invest. 1974;l53:279–287.

Fluid shear stress (FSS) within the lumen contributes to glomerulotubular balance by increasing solute and water absorption. Du and coworkers ^, reported linear flow dependence of fluid and HCO ₃ ⁻ transport in isolated perfused murine PTs ( Fig. 6.8 ), mediated by NHE3 and the H ⁺ -ATPase, as discussed later. The estimated microvillus torque, which is a function of tubular flow, exhibits a linear relationship with fluid and HCO ₃ ⁻ absorption. ^, Consistent with an effect of torque rather than flow per se, increasing viscosity of the perfusate by the addition of dextran increases the effect on fluid transport.

Glomerulotubular balance; flow-dependent increases in fluid (J _v ) and HCO ₃ ⁻ (J _HCO3 ) absorption by perfused mouse proximal tubules.

Absorption also increases when bath albumin concentration increases from 2.5 to 5 g/dL.

From Du Z, Yan Q, Duan Y, et al. Axial flow modulates PT NHE3 and H-ATPase activities by changing microvillus bending moments. Am J Physiol Renal Physiol. 2006;l290:F289–F296.

Pharmacologic inhibition or genetic ablation of NHE3 in mice reveals that tubular flow activates proximal HCO ₃ ⁻ reabsorption mediated by NHE3 and the apical H ⁺ -ATPase. ^, Inhibition of the actin cytoskeleton with cytochalasin D reduces the effect of FSS and HCO ₃ ⁻ transport by impairing movement of NHE3 and the Na ⁺ /K ⁺ -ATPase, but not the H ⁺ -ATPase, to the plasma membrane. This suggests flow-dependent movement of microvilli activates these transport proteins via their linkage to the cytoskeleton ( Fig. 6.9 for NHE3). Redistribution of peripheral actin bands and increased formation of tight junctions and adherens junctions, as observed in cultured tubule cells, is hypothesized to maximize flow-activated transcellular salt and water absorption.

Scaffolding protein NHERF (Na ⁺ -H ⁺ exchanger regulatory factor) links the Na ⁺ -H ⁺ exchanger NHE3 to the cytoskeleton and signaling proteins.

NHERF binds to ezrin, which in turn links to protein kinase A (PKA) and the actin cytoskeleton. NHERF also binds to SGK1 (serum- and glucocorticoid-regulated kinase 1), which activates NHE3. C, Catalytic subunit of PKA; PDZ, domain named for the PSD95, Drosophila disk large (Drosophila), and ZO-1 proteins; R, regulatory subunit of PKA.

The roles of dopamine, angiotensin II (AngII), and calcium on FSS-induced sodium and bicarbonate transport have been examined. Luminal dopamine completely inhibited the flow-induced increase in Na ⁺ transport, mainly through the D _1A receptor. Deletion of AT _1A receptors in mice also abrogated flow-induced increments in Na ⁺ transport, but these effects may be related to the profound basal reductions in NHE3 activity. When AT1 receptor blockers are employed, flow-induced Na ⁺ transport remained. ^, Intracellular activation of IP3, through a local calcium signal, may mediate the effects of flow on NHE3 activity, although increased calcium influx does not appear to play a role. Flow and torque do not appear to stimulate convective flow of chloride through the paracellular pathway.

Another mechanism for glomerulotubular balance operating from the luminal side involves limiting solute concentration. Solutes, such as bicarbonate, amino acids, and glucose, that are reabsorbed coupled to sodium will be depleted earlier along the PT when flow is low, thereby limiting reabsorption rates along the segment as a whole.

Peritubular factors also play an important additive role in glomerulotubular balance. Increases in GFR increase filtration fraction, increasing postglomerular protein concentration and thus peritubular oncotic pressure. Changes in peritubular protein concentration have important effects on proximal tubular NaCl reabsorption that have been seen in not only combined capillary and tubular perfusion experiments ^, but also isolated perfused PT segments where the effect of hydrostatic pressure is abolished. Increases in peritubular protein concentration have an additive effect on the flow-dependent activation of proximal fluid and HCO ₃ ⁻ absorption, which appears to predominantly affect transcellular transport, though the mechanism for this is still not completely clear. There are also changes in absorption that correlate with changes in peritubular hydrostatic pressure, as occurs during expansion or contraction of the extracellular fluid volume.

Fluid and NaCl reabsorption by the PT are affected by a number of hormones and neurotransmitters. The major hormonal influences on renal NaCl transport along the nephron are shown in Fig. 6.9 . Renal sympathetic tone exerts a particularly important stimulatory influence in the PT, as does AngII; dopamine is a major inhibitor of proximal tubular NaCl reabsorption.

Unilateral denervation of the rat kidney causes a marked natriuresis and reduction in proximal NaCl reabsorption, while low-frequency electrical stimulation of renal sympathetic nerves increases proximal tubular fluid absorption. Basolateral epinephrine and/or norepinephrine stimulate proximal NaCl reabsorption via both α- and β-adrenergic receptors. Several lines of evidence suggest that α ₁ -adrenergic receptors exert a stimulatory effect on proximal NaCl transport via activation of basolateral Na ⁺ -K ⁺ -ATPase and apical Na ⁺ -H ⁺ exchange; the role of α ₂ -adrenergic receptors is more controversial. Ligand-dependent recruitment of the scaffolding protein NHERF-1 by β ₂ -adrenergic receptors results in direct activation of apical NHE3, bypassing the otherwise negative effect of downstream cyclic AMP (cAMP; see later). ^,

AngII has potent effects on proximal NaCl reabsorption and therefore on blood pressure. Genetic deletion of AT _1A receptors from PT cells reduced proximal fluid reabsorption, lowered basal blood pressure, shifted pressure natriuresis, and attenuated the hypertensive response to AngII infused chronically. Although basal abundances of NHE3 and NaPi2 were similar in PTs from control and knockout mice, the abundance of NHE3 and NaPi2 was lower following AngII infusion in mice lacking AT _1A , suggesting that these effects are mediated, at least in part, through these two prominent Na ⁺ transport pathways.

Despite this clear stimulatory effect, AngII also has a bimodal (sometimes called “biphasic”) effect on PT Na ⁺ transport in rats, rabbits, and mice. Stimulation of NaCl reabsorption occurs at 10 ⁻¹² to 10 ⁻¹⁰ M, whereas inhibition of NaCl reabsorption occurs at concentrations greater than 10 ⁻⁷ M ( Fig. 6.10 ). However, plasma AngII concentrations typically do not exceed 10 ^-9 M, even during pathologic states, such as 2-kidney/1-clip Goldblatt hypertension. Furthermore, this biphasic role of AngII may not hold true for all species, and in human PT obtained from nephrectomy, concentrations up to 10 ⁻⁶ M AngII stimulate NaCl reabsorption, primarily owing to a stimulatory effect of the nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway on extracellular signal–regulated kinase (ERK) phosphorylation. Although the plasma AngII concentration is typically below the inhibitory concentration, it should be noted that the luminal AngII concentration in the PT frequently exceeds that in plasma, and thus plasma concentrations may not be the sole determining factor. Given the substantial effects of proximal AT _1A receptor deletion to reduce blood pressure and shift pressure natriuresis, AngII is likely to be stimulatory along the PT under most physiologic conditions.

Neurohumoral influences on NaCl absorption by the proximal tubule, thick ascending limb, and collecting duct.

Factors that stimulate (→) and inhibit (−|) sodium reabsorption are as follows: α1 adr, α ₁ -Adrenergic agonist; AII, angiotensin II (low and high referring to picomolar and micromolar concentrations, respectively); ANP/Urod, atrial natriuretic peptide and urodilatin; AVP, arginine vasopressin; β adr, β-adrenergic agonist; BK, bradykinin; CCD, cortical collecting duct; CTAL, cortical thick ascending limb; ET, endothelin; GC, glucocorticoids; IMCD, inner medullary collecting duct; MC, mineralocorticoids; MTAL, medullary thick ascending limb of Henle’s loop; OMCD, outer medullary collecting duct; PTH, parathyroid hormone; PAF, platelet-activating factor; PCT, proximal convoluted tubule; PGE ₂ , prostaglandin E ₂ ; PST, proximal straight tubule.

From Feraille E, Doucet A. Sodium-potassium-adenosine triphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev. 2001;81:345–418.

Further complexity of AngII signaling arises from the presence of AT ₁ receptors at both the luminal and basolateral membranes in the PT. AngII application to the luminal or peritubular side of perfused tubules has a similar bimodal effect on fluid transport, albeit with more potent effects at the luminal side. Experiments using receptor antagonists and knockout mice have indicated that the stimulatory and inhibitory effects of AngII are both mediated via AT ₁ receptors due to signaling at the luminal and basolateral membranes. However, other work has identified that AT ₂ receptors working through NO-cGMP pathway downregulate NHE3 and Na ⁺ -K ⁺ -ATPase, leading to natriuresis and reduced blood pressure. Finally, AngII is also synthesized and secreted by the PT, exerting a potent autocrine effect on proximal tubular NaCl reabsorption. Though evidence suggests most angiotensinogen is derived from liver, proximal tubular cells express mRNA for angiotensinogen, renin, and angiotensin-converting enzyme, allowing for local generation of AngII. Indeed, luminal concentrations of AngII can be 100- to 1000-fold higher than plasma levels. Evidence suggests that AngII generated by PT cells may exert direct intracellular effects. Mitochondrial-specific delivery of AngII increased NHE3 expression through mitochondrial AT _1A receptor activation, an effect countered by activation of mitochondrial AT ₂ receptor-mediated NO-cGMP activity. ^, Proximal tubular and systemic synthesis of AngII may be subject to different control. In fact, intrarenal AngII appears to stimulate proximal NaCl and fluid reabsorption even when dietary salt intake is high, thereby helping to prevent rises in glomerular filtration from increasing late proximal flow. It should be noted that in this study, as in many salt-loading studies in rodents, animals received 1% saline as drinking solution. Thus the high-salt load was accompanied by free water deprivation, a circumstance that leads to stress and inflammation.

The PT is also a target for natriuretic hormones; in particular, dopamine synthesized in the PT has negative autocrine effects on proximal NaCl reabsorption. Proximal tubular cells have the requisite enzymatic machinery for the synthesis of dopamine, using l -dopa reabsorbed from the glomerular ultrafiltrate. Dopamine synthesis by proximal tubular cells and release into the tubular lumen is increased after volume expansion or a high-salt diet, resulting in a considerable natriuresis. ^, Luminal dopamine antagonizes the stimulatory effect of epinephrine on volume absorption in perfused proximal convoluted tubules, consistent with an autocrine effect of dopamine released into the tubular lumen. ^, Dopamine primarily exerts its natriuretic effect via D ₁ -like dopamine receptors. D ₁ receptors are expressed at the apical and luminal membranes of PTs. ^, Targeted deletion of the D _1A and D ₅ receptors in mice leads to hypertension by mechanisms that include reduced proximal tubular natriuresis. ^, PT-specific deletion of aromatic amino acid decarboxylase (AADC), which produces dopamine, clearly demonstrates the role of intrarenal dopamine, leading to upregulation of sodium transporters along the nephron, upregulation of the intrarenal renin-angiotensin axis, decreased natriuresis in response to l -dopa, and reduced medullary cyclo-oxygenase-2 (COX-2) expression, with reduced urinary prostaglandin levels. These mice also exhibit salt-sensitive hypertension and ultimately a significantly shorter life span compared with wild-type mice.

The natriuretic effect of dopamine in the PT is modulated by atrial natriuretic peptide (ANP), which inhibits apical Na ⁺ -H ⁺ exchange via a dopamine-dependent mechanism ( Fig. 6.12 ). ANP appears to induce recruitment of the D ₁ dopamine receptor to the plasma membrane of PT cells, thus sensitizing the tubule to the effect of dopamine. The inhibitory effect of ANP on basolateral Na ⁺ -K ⁺ -ATPase occurs via a D ₁ -dependent mechanism, with a synergistic inhibition of Na ⁺ -K ⁺ -ATPase by the two hormones. Furthermore, dopamine and D ₁ receptors appear to play critical permissive roles in the in vivo natriuretic effect of ANP.

Effect of dopamine on trafficking of the Na ⁺ -H ⁺ exchanger NHE3 in the proximal tubule.

Microdissected proximal convoluted tubules were perfused for 30 minutes with 10 ⁻⁵ mol/L dopamine (DA) , in the lumen or bath, inducing a retraction of immunoreactive NHE3 protein from the apical membrane.

From Bacic D, Kaissling B, McLeroy P, et al. Dopamine acutely decreases apical membrane Na/H exchanger NHE3 protein in mouse renal PT. Kidney Int. 2003;64:2133–2141.

Finally, there is considerable crosstalk between the major antinatriuretic and natriuretic influences on the PT. For example, ANP inhibits AngII–dependent stimulation of proximal tubular fluid absorption, presumably via the dopamine-dependent mechanisms discussed earlier. ^, Dopamine also decreases expression of AT ₁ receptors for AngII in cultured proximal tubular cells. Furthermore, the provision of l -dopa in the drinking water of rats decreases AT ₁ receptor expression in the PT, suggesting that dopamine synthesis in the PT resets the sensitivity to AngII. AngII signaling through AT ₁ receptors decreases expression of the D ₅ dopamine receptor, whereas renal cortical expression of AT ₁ receptors is in turn increased in knockout mice deficient in the D ₅ receptor. Similar interactions have been found between proximal tubular AT ₁ receptors and the D ₂ -like D ₃ receptor.

The apical Na ⁺ -H ⁺ exchanger NHE3 and the basolateral Na ⁺ -K ⁺ -ATPase are primary targets for the antinatriuretic and natriuretic pathways discussed earlier. Since NHE3 mediates the rate-limiting step in transepithelial NaCl absorption, it is the dominant target for regulatory pathways. NHE3 is regulated by the combined effects of direct phosphorylation and dynamic, C-terminal interaction with scaffolding proteins and signal transduction proteins that primarily regulate NHE3 trafficking to and from the brush border membrane ( Fig. 6.11 and Fig. 6.12 ). ^, Up to 40% of basal activity of NHE3 depends on phosphorylation of serine 719 by C-terminal binding of casein kinase 2; this phosphorylation mediates formation of NHE3-containing protein complexes and alters its distribution to lipid rafts, lowering NHE3 activity. ^,

Biphasic effect of AngII on Na ⁺ reabsorption in microperfused proximal tubules (PTs).

The steady-state transepithelial Na ⁺ concentration gradient (peritubular-luminal), ΔC _Na , that developed in a stationary split droplet is used as an indication of the rate of active Na ⁺ reabsorption. This is plotted as a function of peritubular AngII concentration; low concentrations activate Na ⁺ absorption by the PT, whereas higher concentrations inhibit it.

From Harris PJ, Navar LG. Tubular transport responses to angiotensin. Am J Physiol. 1985;248:F621–F630.

Increases in cAMP have a profound inhibitory effect on apical Na ⁺ -H ⁺ exchange in the PT. Intracellular cAMP is increased in response to dopamine signaling via D ₁ -like receptors and/or parathyroid hormone (PTH)–dependent signaling via the PTH receptor, whereas AngII–dependent activation of NHE3 is associated with a reduction in cAMP. PTH potently inhibits NHE3, presumably to promote distal delivery of Na ⁺ -HCO ₃ ⁻ that promotes distal calcium reabsorption. Activation of protein kinase A (PKA) by increased cAMP results in direct phosphorylation of NHE3; although several sites in NHE3 are phosphorylated by PKA, serines 552 (S552) and 605 (S605) are the specific residues connected to cAMP-mediated NHE3 inhibition. Phospho-specific antibodies, which specifically recognize the phosphorylated forms of S552 and S605, have demonstrated dopamine-dependent increases in the phosphorylation of both residues. Moreover, immunostaining of rat kidneys has revealed that S552-phosphorylated NHE3 localizes at the coated pit region of the brush border membrane, where the oligomerized inactive form of NHE3 predominates. ^, The cAMP-stimulated phosphorylation of NHE3 by PKA thus causes the transporter to redistribute to an inactive submicrovillar population, similar to the effect dopamine (see Fig. 6.11 ). A number of other regulators of NHE3, including gastrin and uroguanylin, have been found to exert a functional effect through phosphorylation of S552 and/or S605. ^,

Regulation of NHE3 by cAMP also requires scaffolding proteins that contain PDZ domains (see Fig. 6.12 ). NHE regulatory factor-1 (NHERF-1) was purified as a cellular factor required for the inhibition of NHE3 by PKA. NHERF-2 also interacts with the C-terminus of NHE3. NHERF-1 and NHERF-2 have similar effects on NHE3 in cultured cells. The related protein PDZK1 interacts with NHE3 and a number of other epithelial transporters and is also required for expression of the anion exchanger SLC26A6 at brush border membranes of the PT.

NHERF-1 colocalizes with NHE3 in microvilli of the brush border of the PT, whereas NHERF-2 is predominantly expressed at the base of microvilli in the vesicle-rich domain. The NHERFs assemble a multiprotein, dynamically regulated signaling complex that includes NHE3 and several other transport proteins. They also bind to the actin-associated protein ezrin, thus linking NHE3 to the cytoskeleton. This may be particularly important for the mechanical activation of NHE3 by microvillar bending implicated in glomerulotubular balance (see earlier). ^{, ,} Ezrin also interacts directly with NHE3, binding to a separate binding site within the C-terminus. Ezrin anchors PKA, bringing it into close proximity with NHE3 and facilitating its phosphorylation (see Fig. 6.12 ). Analysis of knockout mice for NHERF-1 has revealed it is not required for baseline NHE3 activity but is required for cAMP-dependent regulation by PTH. One long-standing paradox has been that β-adrenergic receptors, which increase cAMP in the PT, activate apical Na ⁺ -H ⁺ exchange. This was resolved by the observation that the first PDZ domain of NHERF-1 interacts with the β ₂ -adrenergic receptor in an agonist-dependent fashion; this interaction disrupts the interaction between the second PDZ domain and NHE3, resulting in NHE3 activation despite the catecholamine-dependent increase in cAMP.

As discussed earlier, at concentrations higher than 10 ⁻⁷ M (see Fig. 6.10 ), AngII inhibits PT NaCl absorption. This inhibition is dependent on the activation of brush border phospholipase A ₂ , which results in arachidonic acid production. Metabolism of arachidonic acid by cytochrome P450 mono-oxygenases in turn generates 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrioenoic acids (EETs), compounds that inhibit NHE3 and the basolateral Na ⁺ -K ⁺ -ATPase. ^, EETs and 20-HETE have also been implicated in the reduction in proximal NaCl absorption that occurs during pressure natriuresis, inhibiting Na ⁺ -K ⁺ -ATPase and removing NHE3 from the brush border membrane.

Antinatriuretic stimuli such as AngII acutely increase NHE3 expression at the apical membrane, partly by inhibiting cAMP generation. Low-dose AngII (10 ⁻¹⁰ M) also increases insertion of NHE3 into the plasma membrane via a mechanism that requires phosphatidylinositol-3-kinase (PI3K). Treatment of rats with captopril thus results in removal of NHE3 and associated proteins from the brush border of PT cells. Glucocorticoids also increase NHE3 activity by inducing NHE3 gene transcription and acutely stimulating the protein into the plasma membrane. Glucorticoid-dependent exocytosis of NHE3 appears to require NHERF-2, which acts in this context as a scaffolding protein for the glucocorticoid-induced serine-threonine kinase SGK1 (see later, “Regulation of NaCl Transport in the Connecting Tubule and Cortical Collecting Duct: Aldosterone”). The acute effect of dexamethasone has thus been shown to require direct phosphorylation of serine 663 in the NHE3 protein by SGK1.

Finally, many of the natriuretic and antinatriuretic pathways that influence NHE3 have parallel effects on the basolateral Na ⁺ -K ⁺ -ATPase (see Feraille and Doucet for a detailed review). Inhibition by dopamine is associated with removal of active Na ⁺ -K ⁺ -ATPase from the basolateral membrane, somewhat analogous to the effect on NHE3 expression at the apical membrane. This inhibitory effect is primarily mediated by protein kinase C (PKC), which directly phosphorylates serine 18 of the α ₁ -subunit of Na ⁺ -K ⁺ -ATPase, the predominant α-subunit in the kidney. This phosphorylation does not affect enzymatic activity of the Na ⁺ -K ⁺ -ATPase but induces a conformational change that enhances binding of PI3K to an adjacent, proline-rich domain. The recruited PI3K then stimulates dynamin-dependent endocytosis of the Na ⁺ -K ⁺ -ATPase complex via clathrin-coated pits. Evidence suggests that in addition to its pump function, the Na ⁺ -K ⁺ -ATPase has a signaling function involving Src kinase. Along the PT, this is proposed to tonically suppress sodium reabsorption through effects on NHE3 and NBCe1A and may explain the natriuretic effects of endogenous cardiotonic steroids, which activate Na ⁺ -K ⁺ -ATPase signaling function.

The TAL reabsorbs about 30% of filtered NaCl (see Fig. 6.1 ). In addition to an important role in maintaining extracellular fluid volume, NaCl reabsorption by the water-impermeable TAL is a critical component of the renal countercurrent multiplication system that permits production of either dilute or concentrated urine. In concert with the countercurrent mechanism, NaCl reabsorption by the thin and TALs increases medullary tonicity, facilitating water absorption by the collecting duct (see Chapter 10 ).

Notwithstanding the morphologic heterogeneity described earlier, cells of the medullary TAL, cortical TAL, and macula densa share the same basic transport mechanisms ( Fig. 6.14 ). NaCl reabsorption by the TAL is thus a secondarily active process, driven by the favorable electrochemical gradient for Na ⁺ established by the basolateral Na ⁺ -K ⁺ -ATPase. ^, Apical Na ⁺ -K ⁺ -2Cl ⁻ cotransport is mediated by the furosemide-sensitive cation-chloride cotransporter NKCC2, encoded by SLC12A1 . This is a member of the cation-chloride cotransporter family of proteins that includes the thiazide-sensitive transporter NCC and the potassium chloride cotransporters. Functional expression of NKCC2 in Xenopus oocytes yields Cl ⁻ – and Na ⁺ -dependent uptake of Rb ⁺ (a radioactive substitute for K ⁺ ) and Cl ⁻ – and K ⁺ -dependent uptake of ²² Na ⁺ . ^, As expected, NKCC2 is sensitive to micromolar concentrations of furosemide, bumetanide, and other loop diuretics.

Transepithelial NaCl transport pathways in the thick ascending limb (TAL).

CLCNKB, Cl ⁻ channel; KCC4, K ⁺ -Cl ⁻ cotransporter-4; NKCC2, Na ⁺ -K ⁺ -2Cl ⁻ cotransporter-2; ROMK, renal outer medullary K ⁺ channel.

Immunofluorescence indicates expression of NKCC2 protein along the entire length of the TAL. In particular, immunoelectron microscopy reveals expression in both rough and smooth cells of the TAL. NKCC2 expression in subapical vesicles is particularly prominent in smooth cells, suggesting a role for vesicular trafficking in the regulation of NKCC2 (see later, “Regulation of NaCl Transport by the Thick Ascending Limb”). NKCC2 expression and activity is also present in macula densa cells. ^, This is significant given the role of the macula densa in tubuloglomerular feedback (TGF) and renal renin secretion; luminal loop diuretics block TGF and the suppression of renin release by luminal Cl ⁻ .

Alternative splicing of exon 4 of the SLC12A1 gene yields three variant NKCC2 proteins (NKCC2-A, NKCC2-B, and NKCC2-F) that differ within transmembrane domain 2 and the adjacent intracellular loop. ^, NKCC2-F has a low affinity for Cl ⁻ (K _m = 113 mmol/L), and NKCC2-B has a high affinity (K _m = 8.9 mmol/L), whereas NKCC2-A has an intermediate affinity (K _m = 44.7 mmol/L). ^, These isoforms differ in axial distribution along the tubule, with NKCC2-F expressed in the inner stripe of the outer medulla, NKCC2-A in the outer stripe, and NKCC2-B in cortical TAL. There is thus an axial distribution of the anion affinity of NKCC2 along the TAL, from a low-affinity, high-capacity transporter (NKCC2-F) to a high-affinity, low-capacity transporter (NKCC2-B). In situ hybridization suggests that rabbit macula densa exclusively expresses the NKCC2-B isoform. However, selective knockout of NKCC2-B does not eliminate NKCC2 expression in the murine macula densa, which also seems to express NKCC2-A. The comparative phenotypes of NKCC2-A and NKCC2-B knockout mice are consistent with the relative Cl ⁻ affinity of each isoform. Thus targeted deletion of NKCC2-A selectively reduces TGF responses at the higher range of tubular flow rates (a low-affinity, high-capacity situation), whereas NKCC2-B deletion reduces responses at low flow rates. Loss of NKCC2-A almost abolishes the suppression of plasma renin activity by isotonic saline infusion, which appears more robust in NKCC2-B knockout mice than wild-type littermates.

It should be mentioned in this context that NHE3 functions as an alternative mechanism for apical Na ⁺ absorption by the TAL. There is also evidence in mouse cortical TAL for HCO ₃ ⁻ absorption mediated via parallel Na ⁺ -H ⁺ and Cl ⁻ -HCO ₃ ⁻ exchange, though this mechanism seems less important for transepithelial NaCl transport than in the PT. There is considerable upregulation of both apical Na ⁺ -H ⁺ exchange and NHE3 protein in the TAL of acidotic animals, paired with induction of AE2, a basolateral Cl ⁻ -HCO ₃ ⁻ exchanger. ^, A recently generated TAL-specific NHE3 knockout mouse model revealed that under normal conditions, TAL NHE3 has a minor contribution for acid-base balance or can be compensated for by distal segments. However, it does appear to play a role in sustained urinary acidification in response to furosemide. NHE3 in the TAL is also upregulated by increased flow, not via shear stress as in the PT, but by the production of endogenous O ₂ ⁻ and activation of PKC.

The development of a lumen-positive PD plays a critical role in driving paracellular transport of Na ⁺ , Ca ²⁺ , and Mg ²⁺ along the TAL (see Fig. 6.14 ). This lumen-positive transepithelial PD is generated by the combination of luminal recycling of K ⁺ via Na ⁺ -K ⁺ -2Cl ⁻ cotransport and apical K ⁺ channels, along with basolateral depolarization due to Cl ⁻ exit through Cl ⁻ channels. ^,

Apical K ⁺ channels are required for transepithelial NaCl transport by the TAL. ^, Three types of apical K ⁺ channels have been identified in the TAL, a channel with a conductance of 30 picoSiemen (30 pS), a channel with a conductance of 70 pS, and a high-conductance, calcium-activated maxi-K ⁺ channel (see Fig. 6.14 ). The higher open probability (P _o ) and greater density of the 30 pS and 70 pS channels versus the maxi-K ⁺ channel suggest that these are the primary routes for K ⁺ recycling across the apical membrane. The 70 pS channel appears to mediate about 80% of the apical K ⁺ conductance of TAL cells. The low-conductance, 30 pS channel has been identified as the renal outer medullary K ⁺ channel, ROMK, the prototypic inward-rectifying K ⁺ channel, since the 30 pS channel is absent from the apical membrane of ROMK knockout mice. In humans, three isoforms of ROMK (ROMK1, 2, and 3) are generated by alternative splicing of the KCNJ1 gene; ROMK3 has not been detected in rats or mice. ROMK2 has the shortest amino-terminus, ROMK1 has an additional 16 residues, and ROMK3 an additional 22 residues (compared with ROMK2). ROMK1 mRNA is expressed in the mid and late distal tubule and the cortical collecting duct (CCD), as well as in the outer medullary collecting duct (OMCD), but not along the TAL. Consistent with its absence along the TAL, ROMK1-deficient mice do not display a Bartter syndrome phenotype.

ROMK2 mRNA is expressed from the medullary TAL through the CCD but is absent from the OMCD. ROMK3 is expressed from the medullary TAL through the distal convoluted tubule (DCT). ROMK protein has been detected at the apical membrane of medullary TAL, cortical TAL, and macula densa, but not all cells in the TAL are labeled with ROMK antibody, suggesting that ROMK might be absent in TAL cells with high basolateral Cl ⁻ conductance and low apical K ⁺ conductance (see earlier). ^, These cells are thought to correspond to the smooth-surfaced morphologic subtype of TAL cells (S cells) ; however, distribution of ROMK protein by immunoelectron microscopy has not yet been reported.

ROMK clearly plays a critical role in NaCl absorption by the TAL, given that loss-of-function mutations in its gene, KCNJ1, are associated with Bartter syndrome. Moreover, pharmacologic ROMK inhibitors function as potent diuretics in vivo, primarily due to inhibition of TAL NaCl transport. ^, However, these observations were difficult to reconcile with the observation that the heterologous expression of ROMK in Xenopus oocytes yielded a channel with a conductance of about 30 pS, yet a 70 pS K ⁺ channel is the dominant conductance at the apical membrane of TAL cells. ^, This paradox was resolved by the observation that the 70 pS channel is absent from the TAL of ROMK knockout mice, indicating that ROMK also functions as a subunit of the 70 pS channel.

ROMK activity in the TAL is modulated by association with other proteins that likely generate the 70 pS. ROMK interacts with scaffolding proteins NHERF-1 and NHERF-2 (see earlier, “Regulation of Proximal Tubular NaCl Transport: Neurohumoral Influences”) via the C-terminal PDZ-binding motif of ROMK; NHERF-2 is coexpressed with ROMK in the TAL. The association of ROMK with NHERFs brings ROMK into closer proximity to the cystic fibrosis transmembrane regulator protein (CFTR). This ROMK-CFTR interaction is required for the native ATP and glibenclamide sensitivity of apical K ⁺ channels in the TAL.

The reported transepithelial resistance in the TAL is between 10 and 50 Ω.cm ² , but water permeability of the TAL is extremely low, less than 1% that of the PT, yet the TAL is not considered a tight epithelium. ^, These hybrid characteristics of relatively low electrical resistance and very low water permeability allow the TAL to generate and sustain NaCl gradients of up to 120 mmol/L. ^, The particular repertoire of claudins expressed in the TAL determines the resistance and ion selectivity and the lack of aquaporins lowers water permeability.

TALs perfused with NaCl develop a lumen-positive, transepithelial PD ^{, ,} that plays a critical role in the paracellular reabsorption of Na ⁺ , Ca ²⁺ , and Mg ²⁺ (see Fig. 6.14 ). In the transepithelial transport of Na ⁺ , the stoichiometry of NKCC2 (1Na ⁺ :1K ⁺ :2Cl ⁻ ) is such that other mechanisms are necessary to balance the exit of Cl ⁻ at the basolateral membrane; consistent with this requirement, data from mouse TAL have indicated that about 50% of transepithelial Na ⁺ transport occurs via the paracellular pathway. ^, In the absence of vasopressin, apical NaCl cotransport is not K ⁺ dependent (see “Regulation of NaCl Transport by the Thick Ascending Limb”), reducing the lumen-positive PD; switching to K ⁺ -dependent Na ⁺ -K ⁺ -2Cl ⁻ cotransport in the presence of vasopressin results in a doubling of NaCl reabsorption, without an effect on oxygen consumption likely due to passive reabsorption through the cation-permeable paracellular pathway. ^,

Unlike in the PT, the voltage-positive PD in the TAL is generated almost entirely by transcellular transport, rather than by paracellular diffusion. Mouse TAL segments primarily express claudin-14, claudin-16, claudin-19, claudin-3, and the “b” splice form of claudin-10. ^, In vasopressin-stimulated mouse TAL, with a lumen-positive PD of 10 mV, the maximal increase in NaCl in the lateral interspace is about 10 mmol/L. Tight junctions in the TAL are cation selective, ^, but P _Na /P _Cl ratios can be highly variable in individual tubules, ranging from 2 to 5 in a single study of perfused mouse TAL. The claudin profile of cells along the TAL is mosaic, with some cell interfaces expressing the Na ⁺ -selective claudin 10b, whereas others express claudins 3/16/19. This mosaic pattern suggests the existence of spatially separated paracellular routes for Na ⁺ and Ca ²⁺ /Mg ²⁺ . Regardless, assuming a net P _Na /P _Cl ratio of about 3, the maximal dilution potential in the mouse TAL is between 0.7 and 1.1 mV, consistent with a dominant effect of transcellular processes on the lumen-positive PD.

The critical role of claudin-10b in paracellular Na ⁺ permeability is clearly illustrated by mutations in the CLDN10 gene that cause loss of function specifically of the TAL-specific isoform, claudin-10b. ^, Patients with these mutations present with H ypohidrosis- E lectrolyte imbalance- L acrimal gland dysfunction- I chthyosis- X erostomia (HELIX) syndrome. Loss of claudin-10b function leads to hypermagnesemia, hypokalemia with secondary hyperaldosteronism, low blood pressure, and hypocalciuria. The development of hypokalemia is consistent with reduced paracellular Na ⁺ reabsorption along the TAL increasing distal Na+ delivery (see later, “K ⁺ Secretion by the Distal Convoluted Tubule, Connecting Tubule, and Cortical Collecting Duct”), while hypermagnesemia and hypocalciuria likely arise from compensatory increases in expression of claudin-16 and claudin-19, which are routes predominantly for paracellular Mg ²⁺ and Ca ²⁺ reabsorption. ^, The role of claudin-10b is corroborated by the phenotype of claudin-10 knockout mice, which exhibit impaired TAL paracellular Na ⁺ transport with increased permeability to Mg ²⁺ and Ca ²⁺ . ^,

The Na ⁺ -K ⁺ -ATPase is the primary exit pathway for Na ⁺ at the basolateral membrane of TAL cells. The Na ⁺ gradient generated by Na ⁺ -K ⁺ -ATPase activity likely drives apical entry of Na ⁺ , K ⁺ , and Cl ⁻ through NKCC2 because inhibition of Na ⁺ -K ⁺ -ATPase with ouabain collapses the lumen-positive PD and abolishes transepithelial NaCl transport in the TAL. ^{, ,} Basolateral exit of Cl ⁻ from TAL cells is primarily but not exclusively electrogenic, mediated by an approximately 10 pS Cl ⁻ channel. ^{, ,} Reductions in basolateral Cl ⁻ depolarize the basolateral membrane, whereas decreases in intracellular Cl ⁻ induced by luminal furosemide have a hyperpolarizing effect. Intracellular Cl ⁻ activity during transepithelial NaCl transport is above its electrochemical equilibrium, with an intracellular negative voltage of −40 to −70 mV that drives basolateral Cl ⁻ exit. ^,

While at least two CLC chloride channels, CLC-K1 and CLC-K2 (called CLC-Ka and CLC-Kb in humans), are coexpressed in this nephron segment, ^, evidence suggests the dominant Cl ⁻ channel in the TAL is CLC-K2. First, CLC-K1 is highly expressed at the apical and basolateral membranes of the thin ascending limb, and CLC-K1 knockout mice display primary dysfunction of thin ascending limbs, rather than the TAL (see NaCl transport in the thin ascending limb). ^{, , ,} Second, loss-of-function mutations in CLC-Kb are associated with Bartter syndrome, indicating a dominant role of this channel in TAL NaCl transport. A common T481S polymorphism in human CLCNKB that increases channel activity 20-fold has been associated with hypertension, suggesting that gain of function in CLC-Kb increases NaCl transport by the TAL and/or other segments of the distal nephron. Finally, CLC-K2 protein is highly expressed at the basolateral membrane of mouse TAL, with further expression in DCT, connecting tubule (CNT), and type A intercalated cells. Clcnk2 knockout in mice ablates the ∼10 pS chloride channel in TAL cells and leads to salt-wasting, resembling Bartter syndrome. These mice also failed to respond to furosemide, indicating the central role of CLC-K2 in transepithelial NaCl transport. Furthermore, immunolabeling of CLC-K2 knockout kidney with antibodies that cross-react with CLC-K1 and CLC-K2 revealed signal extending beyond the thin ascending limb into the medullary TAL, suggesting that CLC-K1 plays a role there. More on Bartter syndrome is discussed in Chapter 71 .

A key advance was the characterization of the barttin subunit of CLC-K channels, which is coexpressed with CLC-K1 and CLC-K2 in several nephron segments including TAL. ^, Unlike rat CLC-K1, the rat CLC-K2, human CLC-Ka, and human CLC-Kb paralogs are not functional in the absence of barttin coexpression. ^, CLC-Kb coexpressed with barttin is highly selective for Cl ⁻ . ^{, ,} CLC-Kb/barttin channels are activated by increases in extracellular Ca ²⁺ and an alkaline extracellular pH but inhibited at an acidic pH. CLC-Ka/barttin channels have similar pH and calcium sensitivities but exhibit higher permeability to Br ⁻ . Despite the considerable homology between CLC-Ka and CLC-Kb, they also differ considerably in pharmacologic sensitivity to various Cl ⁻ channel blockers, potential lead compounds for the development of paralog-specific inhibitors.

Correlation between functional characteristics of CLC-K proteins with native Cl ⁻ channels in TAL has been problematic, but studies in knockout mice discussed earlier have begun to provide clarity. Wide variation in single-channel conductance has been reported for basolateral Cl ⁻ channels in the TAL, most likely due to experimental conditions. Single-channel conductance has not been reported for CLC-Kb/barttin channels because of poor expression in heterologous systems, complicating the comparison of CLC-Kb/barttin to native Cl ⁻ channels. Single-channel conductance has, however, been reported for the rat CLC-K1 mutant V166E mutant, which alters channel gating without expected effects on single-channel amplitude—coexpression with barttin increases the single-channel conductance of V166E CLC-K1 from about 7 pS to 20 pS. Therefore part of the reported variability in native single-channel conductance may reflect heterogeneity in CLC-Kb and/or CLC-Ka interactions with barttin. Regardless, a study using whole-cell recording suggests that CLC-K2 (CLC-Kb in humans) is the dominant Cl ⁻ channel in TAL and other segments of the distal nephron. Like CLC-Kb/barttin, this native channel is highly Cl ⁻ -selective. ^{, , ,} TAL cells from wild-type mice exhibited a dominant basolateral chloride conductance of 8 pS, which was entirely absent in Clcnk2 knockout mice. Coupled with the strong Bartter syndrome phenotype, these results support the key role of CLC-K2 in driving transepithelial NaCl transport along the TAL.

Electroneutral K ⁺ -Cl ⁻ cotransport has also been implicated in transepithelial NaCl transport in the TAL (see Fig. 6.14 ), functioning in K ⁺ -dependent Cl ⁻ exit at the basolateral membrane. The K ⁺ -Cl ⁻ cotransporter KCC4 is expressed at the basolateral membrane of medullary and cortical TAL and in macula densa. ^, TAL cells have been reported to contain a Cl ⁻ -dependent NH ₄ ⁺ transport mechanism that is sensitive to millimolar concentrations of furosemide and barium (Ba ²⁺ ). NH ₄ ⁺ ions have the same ionic radius as K ⁺ and are transported by KCC4 and other KCCs; KCC4 is also sensitive to Ba ²⁺ and millimolar furosemide, consistent with the pharmacology of NH ₄ ⁺ -Cl ⁻ cotransport in the TAL. Furthermore, consistent with basolateral membrane KCC4 expression, the basolateral membrane of TAL also contains a Ba ²⁺ -sensitive K ⁺ -Cl ⁻ transporter, accounting for the effects of basolateral Ba ²⁺ and/or increased K ⁺ on the transmembrane PD. ^{, , ,}

There is thus considerable evidence for basolateral K ⁺ -Cl ⁻ cotransport in the TAL, mediated by KCC4. ^, However, direct confirmation of a role for basolateral K ⁺ -Cl ⁻ cotransport in transepithelial NaCl transport is lacking. Indeed, KCC4 knockout mice do not have a prominent defect in function of the TAL but exhibit instead a renal tubular acidosis. This has been attributed to defects in acid extrusion by H ⁺ -ATPase in type A intercalated cells but could also result from reduced medullary NH ₄ ⁺ reabsorption by the TAL due to the loss of basolateral NH ₄ ⁺ exit through KCC4. ^{, ,}

Finally, there is also evidence for the existence of Ba ²⁺ -sensitive K ⁺ channel activity at the basolateral membrane of the TAL, providing an alternative exit pathway for K ⁺ to that mediated by KCC4. This may help stabilize the basolateral membrane potential above the equilibrium potential for Cl ⁻ , thus maintaining a continuous driving force for Cl ⁻ exit via CLC-Kb/barttin Cl ⁻ channels. Patch-clamp experiments have identified two types of K ⁺ channels in the basolateral membrane of the TAL: a 40-pS inwardly rectifying K ⁺ channel and a Na ⁺ – and Cl ^– -activated, 80 to 150 pS K ⁺ channel (KCa4.1 or slo2.2). ^, The 40 pS K ⁺ channel was absent in the TAL of Kcnj 10 ^-/- mice, suggesting that the 40 pS K ⁺ channel is a Kir4.1 and Kir4.5 heterotetramer. Although Kir4.1 is also detected in human TAL, loss-of-function KCNJ10 mutations do not cause Bartter syndrome, suggesting Kir4.1 disruption has no significant effect on transport function in the TAL. This may reflect secondary activation of alternative K ⁺ channels along TAL, as Kcnj10 ^-/- mice demonstrate vasopressin-induced stimulation of a 80 to 150 pS K ⁺ channel. Basolateral K ⁺ channels may also attenuate increases in intracellular K ⁺ that are generated by the basolateral Na ⁺ -K ⁺ -ATPase, thus maintaining transepithelial NaCl transport.

Transepithelial NaCl transport by the TAL is regulated by a complex blend of competing neurohumoral influences, which are required to maintain the urinary concentrating capacity and modulate salt balance. In particular, increases in intracellular cAMP tonically stimulate ion transport in the TAL; the stimulatory hormones and mediators that increase cAMP in this segment include vasopressin, PTH, glucagon, calcitonin, and β-adrenergic activation (see Fig. 6.10 ). These overlapping cAMP-dependent stimuli are thought to result in maximal baseline stimulation of transepithelial NaCl transport. For example, to characterize the vivo effect of these hormones requires prior suppression or absence of circulating vasopressin, PTH, calcitonin, and glucagon. This baseline activation is, in turn, modulated by a number of negative influences, most prominently prostaglandin E ₂ (PGE ₂ ) and extracellular Ca ²⁺ (see Fig. 6.10 ). Other hormones and autacoids working through cGMP-dependent signaling, including NO, have potent negative effects on NaCl transport within the TAL. In contrast, AngII has a stimulatory effect on NaCl transport within the TAL.

Vasopressin is perhaps the most extensively studied positive modulator of transepithelial NaCl transport in the TAL. The TAL, with the exception of macula densa cells, expresses type 2 vasopressin receptors (V ₂ Rs), and microdissected TALs respond to the hormone with an increase in intracellular cAMP. Vasopressin activates apical NKCC2 within minutes in perfused mouse TAL segments and also exerts a longer-term influence on NKCC2 expression and function. The acute activation of NKCC2 is achieved partly through insertion of NKCC2 from subapical vesicles to the plasma membrane. This trafficking-dependent activation is abrogated by treatment of perfused tubules with tetanus toxin, which cleaves the vesicle-associated membrane proteins VAMP-2 and VAMP-3. A rat model expressing a dominant-negative V ₂ R mutant under the control of the uromodulin promoter (active in TAL and the early part of the DCT ) has provided some insights into NKCC2 activation by vasopressin. These rats demonstrated polyuria and defective urinary concentration, as well as hypercalciuria, reminiscent of mild Bartter syndrome. The absence of V ₂ R expression in macula densa cells was suggested to maintain TGF independent of vasopressin signaling.

Activation of NKCC2 by vasopressin is also associated with phosphorylation of several of N-terminal threonines in the transporter protein. Treatment of rats with the V ₂ agonist desmopressin (DDAVP) induces phosphorylation of these residues in vivo. These threonine residues are substrates for SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress–responsive kinase 1), first identified as key regulatory kinases for NKCC1 and other cation-chloride cotransporters. Phosphorylation of these sites is required to maximally activate NKCC1 and NKCC2. ^, SPAK and OSR1 are in turn activated by upstream WNK ( W ith N o lysine [ K ]) kinases, such that SPAK or OSR1 require coexpression with WNK4 to activate NKCC1 fully, at least in in vitro systems. Consistent with a role for WNK4 in activation of NKCC2, WNK4 knockout mice display lower levels of phosphorylated NKCC2, a blunted natriuretic response to furosemide, hypokalemia, and normocalciuria.

Similarly, mice expressing a mutant SPAK that cannot be activated by upstream WNK kinases have a marked reduction in N-terminal phosphorylation of both NKCC2 and the thiazide-sensitive NaCl cotransporter (NCC), with associated salt-sensitive hypotension. SPAK also requires the sorting receptor SORLA (sorting protein-related receptor with A-type repeats) for proper trafficking within TAL cells, such that targeted deletion of SORLA results in a marked reduction in N-terminal NKCC2 phosphorylation. The WNK kinases appear to regulate SPAK and NKCC2 in a chloride-dependent fashion, phosphorylating and activating SPAK and the transporter in response to a reduction in intracellular chloride concentration (see “Integrated NaCl and K ⁺ Transport in the Distal Nephron”).

Of the two kinases, SPAK and OSR1, OSR1 appears more critical for NKCC2 function in the TAL, since nephron-specific deletion of OSR1 leads to decreased N-terminal phosphorylation of NKCC2 and a Bartter syndrome–like phenotype. Several groups reported an increase in NKCC2 N-terminal phosphorylation and an increased response to furosemide in SPAK knockout mice, suggesting overcompensation by OSR1. However, it has been determined that antibodies against phosphorylated NKCC2 also bind to phosphorylated NCC in the C57BL/6 mouse background used in these earlier studies. ^, Reanalysis of several mouse models with a specific antibody revealed disruption of activation of WNK-SPAK/OSR1 has little effect on baseline NKCC2 phosphorylation. However, baseline Na ⁺ absorption by isolated perfused TAL segments from SPAK-deficient mice is profoundly impaired, though this may reflect an absence of activating factors present in vivo. However, the SPAK/OSR1 pathway may be important for NKCC2 activation in response to hormonal signaling. Further complexity arises from the influence of the adaptor protein, calcium-binding protein 39 (CAB39, also called mouse protein-25, MO25 ), which can both increase SPAK/OSR1-driven phosphorylation of NKCC2 and activate SPAK/OSR1 independently of WNK kinases by promoting kinase dimerization. ^, WNK4 is also capable of direct interaction with CAB39, promoting activation of NKCC2 in the absence of SPAK or OSR1 expression. In support of SPAK/OSR1-independent activation of NKCC2, mice in which both SPAK and OSR1 were disrupted in the kidney still retain significant N-terminal phosphorylation of NKCC2. While this could reflect a direct effect of WNK4 on NKCC2, it could also result from direct phosphorylation by other pathways, such as vasopressin-protein kinase A or an as-yet unidentified kinase. Therefore there appear to be multiple potential pathways for NKCC2 activation—a WNK4-dependent SPAK/OSR1 pathway, a WNK4-independent SPAK/OSR1 pathway, a SPAK/OSR1-independent WNK4 pathway, and a WNK4-SPAK/OSR1 independent pathway. It should be noted that NKCC2 phosphorylation has a smaller effect on activity than it does for NKCC1 and NCC. Thus changes in NKCC2 phosphorylation may not always reflect large changes in NKCC2 activity.

Though the relevance to humans is unclear, studies in mouse medullary TAL cells have shown that vasopressin switches furosemide-sensitive apical Cl ⁻ transport in the TAL from a K ⁺ -independent NaCl mode to the classic Na ⁺ -K ⁺ -2Cl ⁻ mode. Underscoring the metabolic advantages of paracellular Na ⁺ transport, which is critically dependent on the apical entry of K ⁺ via Na ⁺ -K ⁺ -2Cl ⁻ cotransport (see earlier), vasopressin accomplishes a doubling of transepithelial NaCl transport without affecting cellular ²² Na ⁺ uptake (an indicator of the transcellular component of NaCl transport). This doubling in transepithelial absorption occurs without an increase in O ₂ consumption, highlighting the energy efficiency of ion transport by the TAL. The mechanism of this shift in transport mode involves splice variants of mouse NKCC2 with a novel, shorter C-terminus that confer sensitivity to cAMP when coexpressed with full-length NKCC2. These shorter splice variants appear to encode furosemide-sensitive, K ⁺ -independent NaCl cotransporters when expressed alone in Xenopus oocytes. However, the in vivo relevance of these phenomena is not clear, and it is unknown whether similar splice variants exist in species other than mouse.

In addition to its acute effects on NKCC2, vasopressin increases transepithelial NaCl transport by activating apical K ⁺ channels and basolateral Cl ⁻ channels in the TAL. ^, Details have yet to emerge of the regulation of the basolateral CLC-K2/barttin Cl ⁻ channel complex by vasopressin, cAMP, and related pathways. However, the apical K ⁺ channel ROMK is directly phosphorylated by protein kinase A on three serine residues (S25, S200, and S294 in the ROMK2 isoform). Phosphorylation of at least two of these three serines is required for detectable K ⁺ channel activity in Xenopus oocytes. Mutation of all three serines to alanine abolishes phosphorylation and transport activity, and all three serines are required for full channel activity. Phosphorylation of S25 by the kinase SGK1 stimulates trafficking of the channel to the cell membrane, without affecting channel gating In contrast, phosphorylation of S200 and S294 modulates P _O via effects on pH-dependent gating and activation by binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the C-terminal domain of the channel. ^,

Vasopressin also has considerable long-term effects on transepithelial NaCl transport by the TAL. Sustained increases in circulating vasopressin result in hypertrophy of medullary TAL cells, accompanied by a doubling in baseline active NaCl transport. Water restriction or treatment with DDAVP also results in an increased NKCC2 protein abundance in rat TAL cells. Consistent with a direct effect of vasopressin-dependent signaling, expression of NKCC2 is reduced in mice with a heterozygous deletion of the G _s stimulatory G protein, through which the V ₂ R activates cAMP generation. Increases in cAMP are thought to induce transcription of the SLC12A1 gene that encodes NKCC2 directly, given the presence of a cAMP-response element in the 5′ promoter. ^, Abrogation of the tonic negative effect of PGE ₂ on cAMP generation with indomethacin also results in a considerable increase in abundance of the NKCC2 protein. Finally, in addition to these effects on NKCC2 expression, water restriction or DDAVP treatment increases abundance of the ROMK protein at the apical membrane of TAL cells.

A role for β3-adrenergic receptors in activation of NKCC2 has also been proposed, with administration of the selective agonist BRL37344 increasing NKCC2 phosphorylation ex vivo in wild-type but not β3-adrenergic receptor knockout mice, which also displayed a mild Na ⁺ – and K ⁺ -wasting phenotype.

Stimulation of transepithelial NaCl transport by cAMP-generating hormones (e.g., vasopressin and PTH) is modulated by several negative neurohumoral influences (see Fig. 6.10 ). In particular, extracellular Ca ²⁺ and PGE ₂ exert dramatic inhibitory effects on ion transport by TAL and distal nephron segments through a plethora of synergistic mechanisms. Extracellular Ca ²⁺ and PGE ₂ both activate the G _i inhibitory G protein in TAL cells, opposing the stimulatory, G _s -dependent effects of vasopressin on intracellular levels of cAMP. ^, Extracellular Ca ²⁺ exerts its effect through the calcium-sensing receptor (CaSR), which is expressed at the basolateral membrane of TAL cells; PGE ₂ primarily signals through EP ₃ prostaglandin receptors. ^{, ,} Increases in intracellular Ca ²⁺ due to the activation of the CaSR and other receptors directly inhibit cAMP generation by a Ca ²⁺ -inhibitable adenylate cyclase that is expressed in the TAL, accompanied by an increase in phosphodiesterase-dependent degradation of cAMP ( Fig. 6.15 ). ^, These negative stimuli likely inhibit baseline transport in the TAL; abrogation of the negative effect of PGE ₂ with indomethacin increases NKCC2 protein abundance, whereas targeted deletion of the CaSR in mouse kidney activates NKCC2 via increased N-terminal phosphorylation.

Inhibitory effects of the calcium-sensing receptor (CaSR) on transepithelial NaCl transport in the thick ascending limb.

(A) Activation of the basolateral CaSR inhibits the generation of cAMP in response to vasopressin and other hormones (see text for details). (B) Stimulation of phospholipase A ₂ by the CaSR leads to liberation of arachidonic acid, which is in turn metabolized to 20-HETE (20-hydroxyeicosatetraenoic acid) or prostaglandin E ₂ (PGE ₂ ). 20-HETE iinhibits apical Na ⁺ -K ⁺ -2Cl ⁻ cotransport, apical K ⁺ channels, and the basolateral Na ⁺ -K ⁺ -ATPase.

From Hebert SC. Calcium and salinity sensing by the thick ascending limb: a journey from mammals to fish and back again. Kidney Int Suppl. 2004;91:S28–S33.

Activation of the CaSR and other receptors in the TAL also results in the downstream generation of arachidonic acid metabolites, with potent negative effects on NaCl transport (see Fig. 6.15 ). Extracellular Ca ²⁺ thus activates phospholipase A ₂ (PLA ₂ ) in TAL cells, generating arachidonic acid, which, in response to activation of the CaSR in TAL, is metabolized by cytochrome P450 ω-hydroxylase to 20-HETE. 20-HETE has potent negative effects on apical Na ⁺ -K ⁺ -2Cl ⁻ cotransport, apical K ⁺ channels, and the basolateral Na ⁺ -K ⁺ -ATPase. ^, PLA ₂ -dependent generation of 20-HETE also underlies in part the negative effect of bradykinin and AngII on NaCl transport. ^, Activation of the CaSR also induces tumor necrosis factor–α (TNF-α) expression in the TAL, which activates COX-2 and thus generation of PGE ₂ (see Fig. 6.15 ); this PGE ₂ in turn results in additional inhibition of NaCl transport. Reduction of TNF and NKCC2 isoform expression in vivo via shRNA silencing revealed that TNF promotes natriuresis in response to salt-loading through NKCC2A.

The relative importance of the CaSR in the regulation of NaCl transport by the TAL is strikingly illustrated by the phenotype of a subset of patients with CaSR gain-of-function mutations. In addition to the typical suppressed PTH and hypocalcemia caused by gain-of-function mutations in the CaSR (autosomal dominant hypoparathyroidism), these patients manifest a hypokalemic alkalosis, polyuria, and increases in circulating renin and aldosterone. ^, Therefore the persistent inhibition of NaCl transport in the TAL by these overactive mutants of the CaSR causes a Bartter-like syndrome.

TAL cells express high levels of the membrane-bound, glycosylphosphatidylinositol (GPI)-anchored protein, uromodulin (Tamm-Horsfall glycoprotein), which is not expressed by macula densa cells but has been shown to be expressed in the early part of the DCT. Uromodulin is released by proteolytic cleavage at the apical membrane and is secreted as the most abundant protein in normal human urine (20 to 100 mg/day). Uromodulin has a host of emerging roles in the physiology and biology of the TAL. A high-salt diet increases uromodulin expression, suggesting a role in ion transport. In this regard, uromodulin facilitates membrane trafficking and function of the NKCC2 protein, with similar effects on apical ROMK protein. ^,

Mutations in the UMOD gene encoding uromodulin are associated with autosomal dominant tubulointerstitial kidney disease, type 1. This syndrome includes progressive tubulointerstitial damage and chronic kidney disease (CKD), variably penetrant hyperuricemia, gout, and renal cysts. Common (≈0.8) genetic variants in the UMOD promoter are associated with a 20% higher risk of CKD and a 15% higher risk for hypertension. ^, These polymorphisms are associated with higher renal uromodulin expression and higher urinary uromodulin excretion. ^,

Uromodulin-transgenic mice also manifest salt-sensitive hypertension due to activation of the SPAK kinase and activating N-terminal phosphorylation of NKCC2. Human hypertensive subjects homozygous for susceptibility variants in UMOD appear to have an analogous phenotype, with exaggerated natriuresis in response to furosemide compared with those who are homozygous for protective variants. These findings are compatible with the stimulatory effects of uromodulin on NKCC2 and ROMK—that is, a net gain of function in TAL transport. ^,

The DCT reabsorbs about 10% of filtered NaCl. ^, The apical absorption of Na ⁺ and Cl ⁻ by the DCT is electroneutral and inhibited by thiazide diuretics. Apical absorption is mediated by NCC, which has high affinities for both Na ⁺ and Cl ^−283,285 and measured Hill coefficients of about 1 for each ion, consistent with electroneutral cotransport.

NCC expression is the defining characteristic of the DCT ( Fig. 6.17 ). The human SLC12A3 gene encodes three isoforms (NCC1, NCC2, and NCC3), but only NCC3 has been studied extensively because NCC1 and NCC2 are not expressed in rats or mice. Human isoforms may undergo differential regulation because NCC1 and NCC2 contain a region in their carboxyl-termini that is absent from NCC3 and contains a serine (S811), which undergoes phosphorylation and contributes to cotransporter activity. Loss-of-function mutations in the SLC12A3 gene cause Gitelman syndrome, familial hypokalemic alkalosis with hypomagnesemia, and hypocalciuria. Mice with homozygous deletion of the Slc12a3 gene encoding NCC exhibit marked morphologic defects in the early DCT, with a reduction in the absolute number of DCT cells and changes in ultrastructural appearance. Similarly, thiazide treatment promotes marked apoptosis of the proximal part of DCT, suggesting that thiazide-sensitive NaCl cotransport plays an important role in modulating growth and regression of this nephron segment.

Transport pathways for NaCl and K ⁺ in distal convoluted tubule (DCT) cells (A) and principal cells of the connecting tubule (CNT) and cortical collecting duct (CCD) (B).

Aqp-2, 3/4, Aquaporin-2, aquaporin-3/4; ENaC, epithelial Na ⁺ channel; KCC4, K ⁺ -Cl ⁻ cotransporter-4; NCC, thiazide-sensitive NaCl cotransporter; NHE-2, Na ⁺ -H ⁺ exchanger-2; ROMK, renal outer medullary K ⁺ channel.

Coexpression of NCC and ENaC occurs in the “late DCT” (DCT2) of many species, either in the same cells or in adjacent cells in the same tubule. While ENaC is primarily the Na ⁺ transport pathway of CNT and CCD cells, there is evidence for other Na ⁺ and Cl ⁻ entry pathways in DCT cells. The Na ⁺ -H ⁺ exchanger NHE2 (SLC9A2) is coexpressed with NCC at the apical membrane of rat DCT cells. As in the PT, perfusion of the DCT with formate and oxalate stimulates DIDS-sensitive NaCl transport that is distinct from the thiazide-sensitive transport mediated by NCC. Therefore a parallel arrangement of Na ⁺ -H ⁺ exchange and Cl ⁻ anion exchangers may contribute to electroneutral NaCl absorption by the DCT (see Fig. 6.17 ). The Na ⁺ -H ⁺ antiporter NHE2 (SLC9A2) and the anion exchanger SLC26A6 may also be expressed in DCT cells. ^,

At the basolateral membrane, as in other nephron segments, Na ⁺ exits via Na ⁺ -K ⁺ -ATPase and the DCT appears to have the highest Na ⁺ -K ⁺ -ATPase activity of the entire nephron (see Fig. 6.3 ). ^, Although CLC-K1 mRNA can also be detected in microdissected DCT segments, ^, several lines of evidence indicate that Cl ⁻ primarily exits the basolateral membrane via the CLC-K2 channel. First, the basolateral membrane of rabbit DCT contains Cl ⁻ channel activity with functional characteristics similar to those of CLC-K2. ^, Second, CLC-K2 protein is expressed at the basolateral membrane of DCT and CNT cells. Third, CLC-K2 knockout mice display a loss of response to furosemide and a markedly blunted response to thiazide, implicating this channel in chloride reabsorption along both the TAL and DCT. Finally, loss-of-function mutations in CLC-Kb, the human ortholog of CLC-K2, cause classic Bartter syndrome (Bartter syndrome type 3). This Bartter subtype has a phenotype that is typically intermediate between Bartter syndrome type I and Gitelman syndrome, consistent with loss of function in DCT. ^{, ,}

Basolateral membrane K ⁺ channels play a critical role in DCT function. Cell-attached patches in basolateral membranes of microdissected DCTs detect an inward-rectifying K ⁺ channel, with characteristics similar to those of heteromeric KIR4.1/KIR5.1. ^{, ,} Basolateral membranes of the DCT express immunoreactive KIR4.1 and KIR5.1 protein. ^{, ,} While KIR4.2 mRNA has been detected in DCT cells, one study detected no KIR4.2 mRNA or protein in DCT, validated by loss of detection along the PT in KIR4.2 knockout mice. Patients with loss-of-function mutations in the KCNJ10 gene that encodes KIR4.1 develop a syndrome including epilepsy, ataxia, sensorineural deafness, and tubulopathy (EAST or SeSAME syndrome). ^, The associated tubulopathy includes hypokalemia, metabolic alkalosis, hypocalciuria, and hypomagnesemia. ^, Mice in which Kcnj10 has been deleted following development demonstrate hypokalemic alkalosis with hypocalciuria and suppression of NCC abundance, indicating a key role of this channel in supporting transepithelial NaCl transport. ^{, ,} While KIR4.1 activity is detected in the TAL, KIR4.1 disruption in mice has no significant effect on TAL membrane potential or NKCC2 expression, so its physiologic relevance is unclear. In contrast, Kcnj10 disruption markedly depolarizes the basolateral cell membrane of DCT cells, indicating KIR4.1/KIR5.1 channels play a key role in setting the membrane potential in the DCT. ^, Humans with mutations in KCNJ16 (encoding KIR5.1) present with hypokalemia, but in contrast to EAST syndrome, they display metabolic acidosis rather than metabolic alkalosis, possibly due to effects on proximal tubule ammoniagenesis. The roles of KIR5.1 in renal ion transport are likely to be complex and species dependent because Kcnj16 disruption in mice increases NCC activity but causes a Gitelman-like phenotype in Dahl salt-sensitive rats. In addition to sensing the membrane potential, the KIR4.1/KIR5.1 channels at the basolateral membrane of DCT cells are hypothesized to function in basolateral K ⁺ recycling, maintaining adequate Na ⁺ -K ⁺ -ATPase activity for NaCl absorption and other aspects of DCT function.

Early studies showed that dietary NaCl deprivation activates thiazide-sensitive NaCl transport along the DCT. When distal salt delivery was increased further, by administering loop diuretics continuously and administering saline, increases in transport capacity were observed, together with considerable hypertrophy of DCT cells. These effects involve components of the renin-angiotensin-aldosterone system, as discussed later.

Aldosterone has also been suggested as a factor that modulates NCC, which would make the DCT part of the aldosterone-sensitive distal nephron. Administration of aldosterone to adrenalectomized rats activated NCC. Dietary salt restriction and exogenous mineralocorticoids were also shown to increase the abundance of NCC and phosphorylated, active NCC. Rat DCT cells were shown to express 11-beta hydroxysteroid dehydrogenase type 2 (11βHSD2) at low levels, and its deletion from mice resulted in hypertrophy of DCT cells and higher abundance of phosphorylated NCC.

The more recent recognition that dietary potassium intake is a powerful NCC and DCT regulatory factor has modified the view of aldosterone’s role. Vallon and colleagues documented that dietary potassium deprivation increased the abundance of phosphorylated NCC in mice. Two groups subsequently showed that low potassium intake can increase NCC abundance and activity even in the setting of high-salt intake, ^, and another showed that high potassium intake can suppress NCC, even when dietary salt intake is low. Because high potassium diets are associated with high aldosterone secretion and low NCC activity, the effects of potassium thus predominate over those of sodium and aldosterone. It is now apparent that DCT cells, and NCC, are exquisitely sensitive to plasma [K ⁺ ]. Effects of K ⁺ occur rapidly, as a short-term gavage with high potassium solution leads to rapid NCC dephosphorylation. Similarly, raising the plasma potassium concentration, either by potassium infusion, by administering the ENaC blocker amiloride, or by deleting Na ⁺ channels strikingly reduces the abundance of phosphorylated NCC. These effects on the abundance of phosphorylated NCC are functionally relevant, as directional changes in phosphorylated NCC abundance, in the setting of potassium challenge, are associated with concordant changes in thiazide-sensitive natriuresis. The mechanisms involving WNK kinases in the potassium effect are discussed later.

The recognition that potassium plays a dominant role in regulating NCC and the DCT suggested that some effects observed during aldosterone infusion might be secondary to induced potassium imbalance. While mice with nephron-specific disruption of the mineralocorticoid receptor (MR) exhibit low NCC activity, as would be expected if aldosterone stimulates NCC directly, NCC could be stimulated to normal levels by dietary K ⁺ restriction, showing effects of MR deletion on potassium balance were responsible for changes in NCC. ^, Similarly, mosaic deletion of MR in renal epithelial cells revealed no differences in NCC abundance and phosphorylation between DCT cells that did and did not express MRs, and dietary Na ⁺ restriction upregulated NCC to a similar extent in all cells. Finally, in mice with deletion of the MR target α-ENaC, switching mice from normal diet to a high-Na ⁺ /low-K ⁺ diet normalized plasma [K ⁺ ] and the reduced abundances of total and phosphorylated NCC. In summary, the preponderance of evidence suggests that the effects of plasma [K ⁺ ] are dominant and that aldosterone plays only a modifying role in regulating NCC.

A central role in the regulation of DCT Na ⁺ and Cl ^– transport is played by WNK1 and WNK4, key regulatory kinases in the distal nephron that were initially identified as two of the causative genes for familial hyperkalemic hypertension (FHHt; also known as pseudohypoaldosteronism type II or Gordon syndrome). FHHt is in every respect the mirror image of Gitelman syndrome, encompassing hypertension, hyperkalemia, hyperchloremic metabolic acidosis, suppressed plasma renin activity and aldosterone, and hypercalciuria. Furthermore, FHHt behaves like a gain of function in NCC and/or the DCT in that treatment with thiazides typically results in resolution of the entire syndrome. However, simple transgenic overexpression of NCC in DCT does not replicate the phenotype in mice, indicating specific effects of the mutant WNK1 and WNK4 alleles. ^,

According to a highly simplified scheme ( Fig. 6.18 ), WNK kinases activate the downstream kinases SPAK and/or OSR1 by binding to them along their conserved carboxyl terminal domains and directly phosphorylating them. Activated SPAK and OSR1, enhanced by interactions with MO25 (Cab39), then bind to and activate NCC by phosphorylation key residues in the NCC amino terminal cytoplasmic domain.

Role of WNK kinases in the differential effects of extracellular fluid (ECF) volume depletion and hyperkalemia ([K ⁺ ]) on transport along the distal nephron.

In the lumen, Na ⁺ delivery to the aldosterone-sensitive distal nephron (ASDN, depicted as lower part of tubule) is determined by the extent of Na ⁺ entry into the DCT (depicted as upper part of tubule) and reabsorption by the thiazide-sensitive NaCl cotransporter (NCC). WNK4, likely together with kidney-specific (KS) WNK1, activates NCC. This process is stimulated by angiotensin II (Ang II), which also enhances aldosterone secretion by the adrenal gland to stimulate the epithelial Na ⁺ channel, ENaC. Sodium reabsorption along both the DCT and ASDN corrects ECF volume depletion. During hyperkalemia, NCC is turned off via increased intracellular chloride (modulated by K ⁺ flux through KIR4.1/KIR5.1 channels), increasing Na ⁺ delivery to the ASDN, where it is reabsorbed primarily by ENaC, promoting K secretion.

Mutations in WNK1 and WNK4 cause FHHt by increasing WNK protein abundance along the DCT. Intronic mutations in WNK1 enhance the expression of a full-length kinase-active form of WNK1 along the DCT, where it is normally expressed only at low levels. Mutations in the WNK4 coding region cluster around an acid-rich conserved region of the protein or near the carboxyl terminal domain. These mutations disrupt WNK4 binding to proteins that are essential for WNK4 degradation (see later). The WNK1 gene has at least three separate promoters and a number of alternative splice forms. The predominant intrarenal WNK1 isoform is generated via a promoter that bypasses the N-terminal exons that encode the kinase domain, yielding a kinase-deficient short form of the protein (kidney-specific WNK1, “KS-WNK1”), while the full-length WNK1 is referred to as long-WNK1 (L-WNK1).

Several lines of evidence suggest WNK4 rather than L-WNK1 is the major NCC-activating WNK kinase in vivo. First, disruption of WNK4 in mice dramatically reduces NCC activity. ^, Second, when mice with an FHHt-like phenotype induced by mutation of the protein KLHL3 (see later) are interbred with WNK4 knockout mice, increased WNK1 abundance does not compensate for the loss of WNK4, suggesting a key role for WNK4. Third, transgenic mice expressing the FHHt-causing WNK4 mutant Q562E display DCT hyperplasia; when bred with NCC knockout mice, this hyperplasia was completely suppressed. In addition, these data support other studies suggesting that changes in NaCl entry via NCC can modulate hyperplasia or regression of the DCT. ^{, , , ,} Generation of mice with L-WNK1 deletion specifically in the kidney may shed more light on its contribution to NCC regulation in vivo, but evidence suggests that KS-WNK1 plays a major role in NCC activation. While early in vitro studies suggested KS-WNK1 inhibits WNK4 and thus NCC, more recent studies suggest KS-WNK1 may play a critical role in assembly of cytoplasmic protein complexes called “WNK bodies” that facilitate activation of the WNK4-SPAK/OSR1 pathway. ^,

WNKs, especially WNK4, appear to be sensitive to inhibition by chloride ( Fig. 6.19 ), which directly binds to the catalytic site and inhibits autophosphorylation and activation. ^, Thus when intracellular chloride concentration is low, WNK kinases are maximally activated, and when it is higher, WNK kinases are less active. This chloride binding is critical for the potassium-sensing function of DCT cells. Reduction in potassium intake and/or hypokalemia leads to reduced basolateral K ⁺ concentration in the DCT and subsequent KIR4.1/KIR5.1-dependent hyperpolarization. ^{, ,} Hyperpolarization has been proposed to lead to chloride exit via basolateral CLC-K2 channels; the resulting reduction in intracellular chloride activates the WNK-SPAK/OSR1 cascade, resulting in NCC phosphorylation and activation. ^, This model helps explain the activating effect of potassium depletion on NCC, the inhibitory effect of potassium loading on NCC, and critical role of the DCT and NCC in potassium homeostasis.

Detailed scheme of WNK kinase regulation.

NCC is phosphorylated and activated by SPAK, which is phosphorylated and activated by WNK4, which is activated by low intracellular chloride concentration. WNKs can interact with a cullin-ring ligase, via the adaptor KLHL3, leading to WNK ubiquitination and subsequent degradation by the proteasome. When dephosphorylated, NCC can be removed from the apical plasma membrane.

WNK kinases are regulated by Cullin 3 (CUL3) and Kelch-like 3 (KLHL3), components of an E3 ubiquitin ligase complex that targets the WNKs for degradation (see Fig. 6.19 ). ^{, ,} Mutations in the CUL3 and KLHL3 genes also cause FHHt and account for the majority of cases. Disease-associated mutations in KLHL3 abrogate binding to WNK4 and vice versa. In turn, disease-associated mutations in CUL3 may deplete levels of both CUL3 and KLHL3, preventing WNK degradation. ^, Physiologically, phosphorylation of KLHL3 by protein kinase C, downstream of AngII, also prevents the interaction between KLHL3 and WNK4, leading to NCC activation. More information about the mechanisms of FHHt and the roles of regulatory proteins can be found in Chapters 16 and 44 . More recently, similar mutations in WNK1 have been identified, but these mutations cause a renal tubular acidosis type IV phenotype rather than FHHt and appear to preferentially affect degradation of KS-WNK1 rather than L-WNK1.

Contributions and interactions with other kinases including WNK3, SGK1, and a truncated form of WNK1 (see later) also contribute to the complexity of NCC activation by WNK4 and SPAK/OSR1 kinases, as do CUL3 and KLHL3. ^{, , ,} Furthermore, signals that activate or inhibit NCC must be integrated with the WNK4-SPAK/OSR1 pathway. For example, the activation of NCC by the AngII receptor type 1 (AT ₁ R) appears to require the downstream activation of SPAK by WNK4. ^, Increased delivery of glucose or fructose to the DCT was shown to activate NCC through WNK4 and SPAK via activation of the CaSR. Furthermore, local synthesis of uromodulin by the DCT or increased delivery of shed uromodulin from the TAL appears to activate NCC in the DCT1. Changes in circulating and local levels of AngII, aldosterone, vasopressin, K ⁺ , uromodulin, and delivery of solutes from upstream are thus expected to have different and often opposing effects on the activity of NCC in the DCT (see also Fig. 6.18 and “Integrated NaCl and K ⁺ Transport in the Distal Nephron”). ^{, , ,}

The apical membrane of CNT cells and principal cells contain prominent Na ⁺ and K ⁺ conductances, without a measurable apical conductance for Cl ⁻ ( Fig. 6.17 ). ^{, , ,} Entry of Na ⁺ occurs via the highly selective epithelial Na ⁺ channel (ENaC), which is sensitive to micromolar concentrations of amiloride ( Fig. 6.20 ). This selective absorption of positive charge generates a lumen-negative PD, the magnitude of which varies considerably as a function of mineralocorticoid status and other factors. This lumen-negative PD serves to drive the following critical processes: 1. K ⁺ secretion via apical K ⁺ channels; 2. paracellular Cl ⁻ transport through the adjacent tight junctions; and/or 3. electrogenic H ⁺ secretion via adjacent type A intercalated cells.

Maximal expression of the amiloride-sensitive epithelial Na ⁺ channel (ENaC) at the plasma membrane requires the coexpression of all three subunits (α-, β-, and γ-ENaC).

(A) Amiloride-sensitive current in Xenopus oocytes expressing the individual subunits and various combinations thereof. (B) Surface expression is markedly enhanced in Xenopus oocytes that coexpress all three subunits. The individual cDNAs were engineered with an external epitope tag; expression of the channel proteins at the cell surface is measured by binding of a monoclonal antibody (M ₂ Ab∗) to the tag. Poly A ⁺ , Polyadenylated mRNA.

A from Canessa CM, Schild L, Buell G, et al. Amiloride-sensitive epithelial Na ⁺ channel is made of three homologous subunits. Nature. 1994;367:463–467. B from Firsov, D, Schild L, Gautschi I, et al. Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci U S A. 1996;93:15370–15375.

ENaC is a heteromeric channel complex formed by the assembly of separate, homologous subunits, denoted α-, β-, and γ-ENaC. These channel subunits share a common structure, with intracellular N- and C-terminal domains, two transmembrane segments, and a large glycosylated extracellular loop. Xenopus oocytes expressing α-ENaC alone have detectable Na ⁺ channel activity (see Fig. 6.20 ), which facilitated the initial identification of this subunit by expression cloning; functional complementation of this modest activity was then used to clone the other two subunits by expression cloning. Full channel activity requires the coexpression of all three subunits, which causes a dramatic increase in expression of the channel complex at the plasma membrane (see Fig. 6.20 ). The single-channel characteristics of heterologously expressed ENaC are essentially identical to those of the amiloride-sensitive channel detectable at the apical membrane of CCD cells. ^, Determination of the structure of the assembled channel by cryo-electron microscopy revealed that ENaC assembles with a stoichiometry of 1α:1β:1γ subunits.

Recessive loss-of-function mutations in the three subunits of ENaC are a cause of pseudohypoaldosteronism type I. ^, Patients with this syndrome typically present with severe neonatal salt-wasting, hypotension, acidosis, and hyperkalemia. This dramatic phenotype underscores the critical roles of ENaC activity in renal NaCl reabsorption, maintenance of the extracellular fluid volume, K ⁺ secretion, and H ⁺ secretion. In contrast, gain-of-function mutations in all three ENaC subunits cause Liddle syndrome, an autosomal dominant hypertensive syndrome accompanied by suppressed aldosterone and variable hypokalemia. Most mutations associated with Liddle syndrome disrupt interactions between a PPxY motif in the C-terminus of channel subunits with the NEDD4-2 ubiquitin ligase leading to increased apical membrane expression of the channel. Mutations in the extracellular loops of α-ENaC and γ-ENaC have also been identified, ^, with the α-ENaC mutation increasing intrinsic activity of the channel.

ENaC protein is detectable at the apical membrane of CNT cells and principal cells in the CCD, OMCD, and IMCD ( Fig. 6.17 ). ^, However, several lines of evidence support the hypothesis that the CNT makes the dominant contribution to amiloride-sensitive Na ⁺ reabsorption by the distal nephron:

• 1.

  Amiloride-sensitive Na ⁺ currents in the CNT are twofold to fourfold higher than in the CCD; the maximal capacity of the CNT for Na ⁺ reabsorption is estimated to be about 10 times higher than that of the CCD.

• 2.

  Targeted deletion of α-ENaC in the collecting duct abolishes amiloride-sensitive currents in CCD principal cells but does not affect Na ⁺ or K ⁺ homeostasis; the residual ENaC expression in the late DCT and CNT of these knockout mice easily compensates for the loss of the channel in CCD cells.

• 3.

  Na ⁺ -K ⁺ -ATPase activity in the CCD is considerably less than that of the DCT (see also Fig. 6.3 ), suggesting a greater capability for transepithelial NaCl absorption by the DCT and CNT.

• 4.

  Apical recruitment of ENaC subunits in response to dietary Na ⁺ restriction begins in the CNT, with progressive recruitment of subunits in the downstream CCD at lower levels of dietary Na ⁺ ; although the CNT plays a dominant role in ENaC-mediated sodium transport, it does so primarily in an aldosterone-independent mechanism, with aldosterone-mediated sodium transport in the CCD involved in the finely tuned regulation of sodium transport. ^,

• 5.

  Patch-clamp analysis of knock-in mice homozygous for a Liddle syndrome ENaC mutant showed that the primary site of increased Na ⁺ reabsorption is the DCT2/CNT rather than the CNT/CCD.

There are two major pathways for Cl ⁻ absorption in the CNT and CCD—paracellular transport across the tight junction and transcellular transport across type B intercalated cells ( Fig. 6.21 ). ^, The CNT and CCD are “tight” epithelia, with comparatively low paracellular permeability that is not selective for Cl ⁻ over Na ⁺ . However, voltage-driven paracellular Cl ⁻ transport in the CCD may play a considerable role in transepithelial NaCl absorption. The CNT, DCT, and collecting duct express claudin-3, claudin-4, and claudin-8; claudin-8 in particular may function as a paracellular cation barrier that prevents backleak of Na ⁺ , K ⁺ , and H ⁺ in the distal nephron. ^, Claudin-4 and claudin-8 have been proposed to interact to form a paracellular pathway for Cl ⁻ in the collecting duct, thus mediating transepithelial Cl ⁻ absorption via the paracellular pathway. CCD-specific knockout of claudin-4 in mice leads to NaCl wasting and hypotension, while CCD-specific disruption of claudin-8 causes hypotension, hypokalemia, and metabolic alkalosis. This model proposes that claudin-4 and claudin-8 are functionally interdependent, but one study showed that claudin-4 levels did not change under conditions where claudin-8 expression changed. Rather, claudin-8 appears to functionally interact with ENaC, possibly to prevent solute and water backflow and couple paracellular and transcellular permeability. Regulated changes in paracellular permeability may also contribute to Cl ⁻ absorption by the CNT and CCD. In particular, WNK4 increases paracellular Cl ⁻ permeability in transfected MDCK II cell lines. FHHt-associated WNK4 also increases paracellular permeability, due perhaps to an associated hyperphosphorylation of claudin proteins. ^, Claudin-4–mediated chloride conductance is also negatively regulated by cleavage in its second extracellular loop by channel-activating protease 1 (cap1). Claudin-8 was shown to be a target of KLHL3-mediated proteasomal degradation, with FHHt-associated KLHL3 (see “ Regulation of NaCl Transport in the Distal Convoluted Tubule”) displaying impaired interaction.

Transepithelial Cl ⁻ transport by principal (PC) and α- (α-IC) and β- (β-IC) intercalated cells.

The lumen-negative potential difference generated by principal cells drives paracellular Cl ⁻ absorption. Alternatively, transepithelial transport occurs in β-intercalated cells via apical Cl ⁻ -HCO ₃ ⁻ exchange (SLC26A4/pendrin) and basolateral Cl ⁻ exit via CLC-K2.

Modified from Moe OW, Baum M, Berry CA, Rector FC Jr. Renal transport of glucose, amino acids, sodium, chloride, and water. In Brenner BM, ed. Brenner and Rector’s the Kidney. Philadelphia: WB Saunders; 2004. p. 413–452.

Transcellular Cl ⁻ absorption across intercalated cells is thought to play a quantitatively greater role in the CNT and CCD than paracellular transport. In the simplest scheme, this process requires the concerted function of type A and type B intercalated cells, achieving net electrogenic Cl ⁻ absorption without affecting HCO ₃ ⁻ or H ⁺ excretion (see also Fig. 6.21 ). Chloride enters type B intercalated cells via apical Cl ⁻ -HCO ₃ ⁻ exchange, followed by exit from the cell via basolateral Cl ⁻ channels. Recycling of Cl ⁻ at the basolateral membrane of adjacent type A intercalated cells also results in HCO ₃ ⁻ absorption and extrusion of H ⁺ at the apical membrane. The net effect is electrogenic Cl ⁻ absorption.

At the basolateral membrane, intercalated cells display robust Cl ⁻ conductance, with transport characteristics similar to those of CLC-K2/barttin. ^, CLC-K2 protein is also detected at the basolateral membrane of type A intercalated cells, and CLC-K2 activity has been observed in type B cells. ^, The basolateral Na ⁺ -K ⁺ -2Cl ⁻ cotransporter NKCC1 in adjacent type A intercalated cells also plays an evident role in transepithelial Cl ⁻ absorption by the CCD. At the apical membrane, the SLC26A4 exchanger (also known as pendrin) has been conclusively identified as the elusive Cl ⁻ -HCO ₃ ⁻ exchanger of type B and non-A, non-B intercalated cells; this exchanger functions as the apical entry site during transepithelial Cl ⁻ transport by the distal nephron. SLC26A4 mutations cause Pendred syndrome, which manifests as sensorineural hearing loss and goiter; these patients do not have an appreciable renal phenotype. However, pendrin knockout mice develop hypotension during severe dietary NaCl restriction and are resistant to mineralocorticoid-induced hypertension. Pendrin has indirect effects on ENaC abundance and activity by modulating luminal ATP and HCO ₃ ⁻ concentrations; pendrin and ENaC are also both coactivated by AngII, and pendrin expression is also induced by aldosterone. Overexpression of pendrin in intercalated cells in transgenic mice thus causes hypertension, with an increase in ENaC activity and electroneutral NaCl absorption (see later). Conversely, disruption of pendrin in mice decreased ENaC-mediated Na ⁺ absorption by reducing channel P _O and channel density at the apical membrane. Finally, replacement of dietary NaCl with Na ⁺ -HCO ₃ ⁻ results in Cl ⁻ wasting in pendrin knockout mice and increased apical pendrin expression in type B intercalated cells of normal littermate controls. Several groups have reported that pendrin expression is exquisitely responsive to changes in distal chloride delivery. Importantly, mice with deletion of the MR specifically in type B intercalated cells infused with aldosterone display lower Cl ⁻ reabsorption. Therefore pendrin plays a critical role in distal nephron Cl ⁻ absorption, underlining the particular importance of transcellular Cl ⁻ transport in this process. Of broader relevance, these studies underline the important role of Cl ⁻ homeostasis in the maintenance of extracellular volume and pathogenesis of hypertension.

Although thiazide-sensitive NaCl cotransport is considered the exclusive provenance of the DCT, which expresses the canonical thiazide-sensitive transporter NCC (see earlier, “Mechanisms of NaCl Transport in the Distal Convoluted Tubule”), approximately 50% of NaCl transport in rat CCD is electroneutral, amiloride resistant, and thiazide sensitive, ^, and this has also been demonstrated in mouse CCD. This transport activity remains in CCDs from mice with genetic disruption of NCC and ENaC. This thiazide-sensitive, electroneutral NaCl transport has been proposed to be mediated by the parallel activity of the Na ⁺ -driven SLC4A8 Cl ⁻ -HCO ₃ ⁻ exchanger and SLC26A4 Cl ⁻ -HCO ₃ ⁻ exchanger (pendrin; see earlier). SLC4A8 knockout mice display only a mild perturbation of NaCl and water balance due to compensation by NCC; combined Slc4A8 and NCC disruption caused intravascular volume contraction and hypokalemia. However, a newer study demonstrated complete absence of SLC4A8 mRNA from the cortex including the CCD; furthermore, the activity of heterologously expressed SLC4A8 is completely resistant to HCTZ inhibiton. Rather, electroneutral NaCl transport along CCD may involve coupling of pendrin with NHE2.

The apical entry of Na ⁺ requires a basolateral exit of Na ⁺ in type B intercalated cells, evidently mediated by the basolateral Na ⁺ -HCO ₃ ⁻ transporter SLC4A9. Finally, a series of elegant experiments has revealed that electroneutral NaCl transport in type B intercalated cells is uniquely among mammalian renal epithelial cells energized by and thus dependent on the activity of the basolateral H ⁺ -ATPase rather than Na ⁺ -K ⁺ -ATPase activity.

The DCT, CNT, and collecting ducts have generally been considered to constitute the aldosterone-sensitive distal nephron, expressing the MR and 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) enzyme that protects against illicit activation by glucocorticoids. However, data suggest that aldosterone does not directly act on the DCT and CNT, with MR-dependent effects likely to be stimulated by glucocorticoids. ^, Aldosterone is a dominant activator of distal nephron NaCl transport, with a plethora of mechanisms and transcriptional targets. For example, aldosterone increases the expression of the Na ⁺ -K ⁺ -ATPase α ₁ – and β ₁ -subunits in the CCD, in addition to inducing SLC26A4, the apical Cl ⁻ -HCO ₃ ⁻ exchanger of intercalated cells. ^, Aldosterone may also affect paracellular permeability of the distal nephron via posttranscriptional modification of claudins and other components of the tight junction. Impressive progress has been made in understanding the downstream effects of aldosterone on synthesis, trafficking, and membrane-associated activity of ENaC subunits. A detailed discussion of aldosterone’s actions may be found in Chapter 12; here we summarize the major findings of relevance to NaCl transport.

Aldosterone increases abundance of α-ENaC via a glucocorticoid-response element in the promoter of the SCNN1A gene. Aldosterone also relieves tonic inhibition of the SCNN1A gene by a complex that includes the DOT1A (disruptor of telomere silencing splicing variant a ) and AF9 and AF17 transcription factors. An aldosterone-dependent reduction in promoter methylation is also involved. This transcriptional activation results in an increased abundance of α-ENaC protein in response to exogenous aldosterone or dietary NaCl restriction ( Fig. 6.22 ); this response is blunted by spironolactone, indicating involvement of the MR. At baseline, α-ENaC transcripts in the kidney are less abundant than those encoding β- and γ-ENaC (see Fig. 6.22 ), so induction of α-ENaC is thought to relieve a major bottleneck in the processing and trafficking of active ENaC complexes.

Immunofluorescence images of connecting tubule (CNT) profiles in kidneys from adrenalectomized rats (ADX) and from ADX rats 2 and 4 h after aldosterone (aldo) injection.

Antibodies against the α-, β-, and γ-subunits of ENaC reveal absent expression of the former in ADX rats, with progressive induction by aldosterone. All three subunits traffic to the apical membrane in response to aldosterone. This coincides with rapid aldosterone induction of the SGK kinase in the same cells; SGK is known to increase the expression of ENaC at the plasma membrane (see text for details). Bar ≅ 15 μm.

From Loffing J, Zecevic M, Féraille E, et al. Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK. Am J Physiol Renal Physiol. 2001;280:F675–F682.

Aldosterone also plays a role in trafficking of ENaC subunits to the plasma membrane by regulating accessory proteins that interact with preexisting ENaC subunits. Aldosterone rapidly induces expression of serum and glucocorticoid-induced kinase-1 (SGK1); coexpression of SGK1 with ENaC subunits in Xenopus oocytes potently activates ENaC activity due to increased plasma membrane expression. ^{, ,} Importantly, an analogous redistribution of ENaC subunits from the cytoplasm to the apical membrane occurs in the CNT and early CCD, in response to aldosterone infusion or NaCl restriction (see Fig. 6.22 ). ^{, ,} This redistribution of ENaC in the CNT correlates with induction of SGK1 protein.

SGK1 modulates membrane expression of ENaC by interfering with regulated endocytosis of its channel subunits. This is mediated by the ubiquitin ligase NEDD4-2, which is thought to ubiquitinate ENaC subunits, resulting in their removal from the cell membrane and degradation in lysosomes and the proteosome. PPxY domains in the C-termini of all three ENaC subunits bind to WW domains of NEDD4-2. These PPxY domains are deleted, truncated, or mutated in patients with Liddle syndrome, leading to a gain of function in channel activity. ^, Coexpression of NEDD4-2 with wild-type ENaC markedly inhibits channel activity due to removal from the cell membrane, whereas channels bearing Liddle syndrome mutations are resistant. Phosphorylation of NEDD4-2 by SGK1, which also possesses a PPxY motif, abrogates the ability of NEDD4-2 to inhibit ENaC. ^, Aldosterone also stimulates NEDD4-2 phosphorylation in vivo. NEDD4-2 phosphorylation in turn results in ubiquitin-mediated degradation of SGK1, indicating feedback regulation in this system. Aldosterone also reduces NEDD4-2 protein expression in cultured CCD cells, and acetylation of ENaC antagonizes ENaC ubiquitination, suggesting additional levels of regulation. ^,

Induction of SGK1 by aldosterone thus appears to stimulate redistribution of ENaC subunits from the cytoplasm to the apical membrane of CNT and CCD cells. There is considerable axial heterogeneity in the recruitment of ENaC to the plasma membrane, which begins in the CNT and only extends into the CCD and OMCD in NaCl–restricted or aldosterone-treated animals. ^, The underlying causes of this progressive axial recruitment are not yet clear, but NEDD4-2 expression is inversely related to the apical distribution of ENaC, with low expression in the CNT and increased expression levels in the CCD. Therefore the relative balance among SGK1, ENaC, and NEDD4-2 likely figures prominently in membrane recruitment of ENaC subunits.

NEDD4-2 and ENaC are part of a larger regulatory complex that includes the signaling protein RAF-1, stimulatory aldosterone-induced chaperone GILZ1 (glucocorticoid-induced leucine zipper-1), and scaffolding protein CNK3. ^, The mTORC2 (mammalian target of rapamycin complex 2) kinase complex is another component, catalyzing upstream activation of SGK1 and thus inducing activation of ENaC. ^,

Despite altered apical trafficking of ENaC in SGK1 knockout mice, ENaC activity is normal even after aldosterone administration, suggesting that other aldosterone-induced proteins contribute to ENaC activation via MRs. ^, For example, another aldosterone-induced protein, ankyrin G, a cytoskeletal protein involved in vesicular trafficking, increases ENaC activity in cultured CCD cells by promoting its plasma membrane insertion from recycling endosomes.

Finally, aldosterone indirectly activates ENaC channels through the induction of channel-activating proteases, which increase open channel probability by cleavage of the extracellular domains of α- and γ-ENaC. Western blotting of kidney lysates from rats subjected to NaCl restriction or aldosterone treatment has revealed α- and γ-ENaC subunits of lower molecular mass than those detected in control animals, consistent with proteolytic cleavage. ^, Proteases that have been implicated in the processing of ENaC include furin, elastase, and three membrane-associated proteases denoted CAP1-3 (channel-activating protease-1, protease-2, and protease-3). Filtered proteases such as plasmin may also contribute to ENaC activation in nephrotic syndrome. CAP1 was initially identified from Xenopus A6 cells as an ENaC-activating protease; the mammalian ortholog is an aldosterone-induced protein in principal cells. ^, Urinary excretion of CAP1, also known as prostasin, is increased in hyperaldosteronism, with a reduction after adrenalectomy. CAP1 is tethered to the plasma membrane by a GPI linkage, whereas CAP2 and CAP3 are transmembrane proteases. ^, All three proteases activate ENaC by increasing the P _o of the channel, without increasing membrane expression. However, kidney-specific CAP1 and global CAP2 knockout mice display normal sodium balance in vivo, which may reflect compensation by other CAPs. Proteolytic cleavage of ENaC appears to activate the channel by removing the self-inhibitory effect of external Na ⁺ . In the case of furin-mediated proteolysis of α-ENaC, this appears to involve the removal of an inhibitory domain from within the extracellular loop. ^, Extracellular Na ⁺ appears to interact with a specific acidic cleft in the extracellular loop of α-ENaC, causing inhibition of the channel. Unprocessed channels at the plasma membrane are thought to function as a reserve pool, capable of rapid protease-mediated activation.

Although not typically considered an antinatriuretic hormone, vasopressin has well-characterized stimulatory effects on NaCl transport by the CCD. ^, Vasopressin directly activates ENaC in murine CCD, increasing channel P _o . In perfused rat CCD, vasopressin and aldosterone can have synergistic effects on Na ⁺ transport. Furthermore, water and Na ⁺ -restriction synergistically increase ENaC P _o in murine CCDs. Prostaglandins inhibit this effect of vasopressin, particularly in the rabbit CCD, in part through reductions in vasopressin-generated cAMP. ^, There are, however, considerable species-dependent differences in the interactions between vasopressin and negative modulators of NaCl transport in the CCD including prostaglandins, bradykinin, endothelin, and α ₂ -adrenergic tone. ^, Regardless, cAMP rapidly increases the Na ⁺ conductance of apical membranes in the CCD by increasing ENaC localization to the plasma membrane and open channel probability. ^, Notably, cAMP inhibits retrieval of ENaC subunits from the plasma membrane via PKA-dependent phosphorylation of the phosphoacceptor sites in NEDD4-2 that are targeted by SGK1. Therefore both aldosterone and vasopressin converge on NEDD4-2 in the regulation of ENaC activity in the distal nephron. Analogous to the effect on trafficking of aquaporin-2 in principal cells, cAMP also seems to stimulate exocytosis of ENaC subunits to the plasma membrane. Finally, similar to the long-term effects of vasopressin on aquaporin-2 and NKCC2 expression, chronic treatment with DDAVP increases abundances of the β- and γ-ENaC subunits. ^,

Activation of ENaC by vasopressin directly affects water homeostasis. Hypernatremic mice treated with the ENaC inhibitor benzamil thus exhibit further increases in serum tonicity due to a reduction in urinary osmolality. In adrenalectomized mice, which lack circulating aldosterone, vasopressin maintains ENaC activity in the distal nephron. In addition to inducing secretion of aldosterone, which activates ENaC, AngII itself directly activates amiloride-sensitive Na ⁺ transport in perfused CCDs; blockade by losartan or candesartan suggests that this activation is mediated by AT ₁ Rs. Perfusion of tubules with AngI stimulates ENaC, an effect blocked by angiotensin-converting enzyme (ACE) inhibition with captopril, suggesting that intraluminal conversion of AngI to AngII can occur in the CCD. Importantly, luminal AngII has a greater effect on ENaC activity than bath AngII does. In contrast to PT cells that express extremely low levels of renin, CNT cells express high levels of renin. Angiotensinogen secreted by the PT may thus be converted to AngII in the CNT via locally generated renin and ACE and/or related proteases. AngII also activates chloride absorption across intercalated cells via a pendrin (SLC26A4) and H ⁺ -ATPase-dependent mechanism, as well as via activation of CLC-K2.

Luminal perfusion with ATP or uridine triphosphate (UTP) inhibits amiloride-sensitive Na ⁺ transport and reduces ENaC P _o in the CCD via activation of luminal P2Y ₂ purinergic receptors. ^, P2Y ₂ receptor knockout mice display salt-resistant hypertension due in part to upregulation of NKCC2 activity in the TAL. Resting ENaC activity is also increased, but suppressed aldosterone and downregulation of the α-subunit of ENaC blunt the role of amiloride-sensitive transport. ^, Administration of exogenous mineralocorticoid to clamp mineralocorticoid activity at higher levels reveals that P2Y ₂ receptor activation may be a major mechanism for the modulation of ENaC P _o in response to changes in dietary NaCl. Increased dietary NaCl thus leads to increased urinary ATP and UTP excretion in mice. Endogenous ATP from principal cells inhibits ENaC, and ENaC activity is not responsive to increased dietary NaCl in P2Y ₂ receptor knockout mice. ^, In addition, the activation of apical ionotropic purinergic receptors, likely P2X ₄ and/or P2X ₄ /P2X ₆ , can inhibit or activate ENaC, depending on luminal Na ⁺ concentration; these receptors may also participate in fine-tuning ENaC activity in response to dietary NaCl. ATP-derived adenosine may also contribute to lower NaCl reabsorption along the CCD by selectively inhibiting ClC-K2 activity in intercalated cells via the adenosine 1 receptor (A ₁ R).

As in other nephron segments, NaCl transport by the CNT and CCD is modulated by metabolites of arachidonic acid generated by cytochrome P450 monooxygenases. In particular, arachidonic acid inhibits ENaC channel activity in the rat CCD via generation of the epoxygenase product 11,12-EET (epoxyeicosatrienoic acid) by the CYP2C23 enzyme. Disruption of Cyp4a10, another P450 monooxygenase, in mice, results in salt-sensitive hypertension. Urinary excretion of 11,12-EET is reduced in these knockout mice, with a blunted effect of arachidonic acid on ENaC channel activity in the CCD. These mice also became normotensive after treatment with amiloride, indicative of in vivo activation of ENaC. It appears that deletion of Cyp4a10 reduces activity of the murine ortholog of rat CYPC23 (CYP2C44 in mouse) and/or related expoxygenases via reduced generation of a ligand for PPARα (peroxisome proliferator–activated receptor α) that induces epoxygenase activity. The mechanism(s) whereby 11,12-EET inhibits ENaC are unknown as yet. However, renal 11,12-EET production is known to be salt sensitive, suggesting that generation of this mediator may serve to reduce ENaC activity during high dietary NaCl intake.

Finally, activation of PPARγ by thiazolidinediones results in amiloride-sensitive hypertension, suggesting in vivo activation of ENaC. ^, Thiazolidinediones (TZDs; e.g., rosiglitazone, pioglitazone, and troglitazone) are insulin-sensitizing drugs used for the treatment of type II diabetes. Treatment with these agents is frequently associated with fluid retention, suggesting an effect on renal NaCl transport. Indeed, selective deletion of the murine PPARγ gene in principal cells abrogates the increase in amiloride-sensitive transport seen in response to TZDs. ^, This effect by itself does not explain edema since primary ENaC activation leads to hypertension and hypokalemia, not to edema; therefore the mechanisms of TZD-induced edema are complex. Conversely, mice with disruption of α-ENaC specifically along CNT/CCD display blunted increases in total body water and extracellular fluid volume in response to rosiglitazone administration, suggesting the effects of PPARγ are mediated through ENaC. TZDs appear to induce transcription of the genes encoding γ-ENaC and SGK1; SGK1 knockout mice display attenuated TZD-associated edema. ^{, ,} Notably, however, other studies have failed to detect an effect of TZDs on ENaC activity, which may instead activate a nonspecific cation channel within the IMCD. ^, Regardless, the beneficial effect of spironolactone in patients with type 2 diabetes with TZD-associated volume expansion is consistent with in vivo activation of NaCl absorption in the distal nephron. In addition, the risk of peripheral edema is increased considerably in patients treated with both TZDs and insulin therapy. Insulin appears to activate ENaC via SGK1-dependent mechanisms; PPARγ is required for the full activating effect of insulin on ENaC, such that this clinical observation may reflect synergistic activation of ENaC by insulin and TZDs. ^{, ,}

Approximately 90% of filtered K ⁺ is reabsorbed by the PT and loop of Henle (see Fig. 6.23 ); the fine-tuning of renal K ⁺ excretion occurs in the remaining distal nephron. The bulk of regulated secretion occurs in the DCT2 and principal cells within the CNT and CCD, whereas K ⁺ reabsorption primarily occurs in the OMCD (see later). Generally, the CCD is considered the primary site for distal K ⁺ secretion, partially due to the greater ease with which this segment is perfused and studied. However, as is the case for NaCl absorption (see “Connecting Tubules and the Cortical Collecting Duct: Apical Na ⁺ Transport” ) , the bulk of distal K ⁺ secretion appears to occur before the CCD, within the DCT2 and CNT under basal conditions. ^,

In principal cells, apical Na ⁺ entry via ENaC generates a lumen-negative PD, which drives passive K ⁺ exit through apical K ⁺ channels. Distal K ⁺ secretion is therefore dependent on delivery of adequate luminal Na ⁺ to the CNT and CCD, essentially ceasing when luminal Na ⁺ drops below 8 mmol/L. At steady state, urinary K ⁺ excretion equals intake, but Na ⁺ intake can affect K ⁺ excretion over the short term, such that excretion is enhanced by excess Na ⁺ intake and reduced by Na ⁺ restriction. ^, Secreted K ⁺ enters principal cells via the basolateral Na ⁺ -K ⁺ -ATPase, which also generates the gradient that drives apical Na ⁺ entry via ENaC (see Fig. 6.23 ).

Two major subtypes of apical K ⁺ channels function in K ⁺ secretion by the CNT and CCD; a small-conductance (SK), 30 pS channel and a large-conductance, Ca ²⁺ -activated, 150 pS (maxi-K or BK) channel ( Fig. 6.17 ). ^{, ,} The density and high P _o of the SK channel are sufficient to mediate the bulk of K ⁺ secretion in the CCD under baseline conditions. Notably, SK channel density is considerably higher in the CNT than CCD, consistent with the greater capacity for Na ⁺ absorption and K ⁺ secretion in the CNT. The SK has been identified as ROMK. ROMK protein has been localized at the apical membrane of principal cells, ^, and SK channel activity is absent from apical membranes of the CCD in ROMK (encoded by Kcnj1 ) knockout mice. ROMK knockout mice are normokalemic with increased excretion of K ⁺ , illustrating the considerable redundancy in distal K ⁺ secretory pathways; distal K ⁺ secretion in these mice is mediated by apical BK channels (see later). ^, However, dietary K ⁺ loading induced hyperkalemia in ROMK1 knockout mice, consistent with a role in K ⁺ secretion along the CCD. Of interest, loss-of-function mutations in human KCNJ1 genes are associated with Bartter syndrome; ROMK expression is critical for the 30 pS and 70 pS channels that generate the lumen-positive PD in the TAL (see Fig. 6.14 ). ^, These patients typically have slightly higher serum K ⁺ levels than those with other forms of Bartter syndrome. Patients with severe neonatal hyperkalemia have also been described, presumably the result of a transient developmental deficit in apical BK channel activity. ^, Note that Bartter syndrome due to KCNJ1 disruption may specifically reflect a defect in ROMK2 and/or ROMK3 function since, consistent with absence of ROMK1 along TAL, ROMK1 knockout mice do not display a Bartter syndrome phenotype.

The apical Ca ²⁺ -activated BK channel plays a critical role in flow-dependent K ⁺ secretion by the CNT and CCD. BK channels have a heteromeric structure, with α-subunits that form the channel pore and modulatory β-subunits that affect the biophysical, regulatory, and pharmacologic characteristics of the channel complex. BK α-subunit transcripts are expressed in multiple segments, and channel protein is detectable at the apical membrane of principal and intercalated cells in the CCD and CNT. The β-subunits are differentially expressed within the distal nephron. Thus β ₁ -subunits are restricted to the CNT, with no expression in intercalated cells, whereas β ₄ -subunits are detectable at the apical membranes of the TAL, DCT, and intercalated cells. ^, Increased distal flow has a well-established stimulatory effect on K ⁺ secretion, due in part to enhanced delivery and absorption of Na ⁺ and to increased removal of secreted K ⁺ . ^, The pharmacology of flow-dependent K ⁺ secretion in the CCD is consistent with dominant involvement of BK channels, and flow-dependent K ⁺ secretion is reduced in mice with targeted deletion of the α ₁ – and β ₁ -subunits. ^, Both mice strains develop hyperaldosteronism that is exacerbated by a high-K ⁺ diet, leading to hypertension in the α ₁ -subunit knockout. Disruption of the β2 subunit also leads to hyperaldosteronism, but flow-induced K ⁺ secretion is normal, suggesting compensation by other isoforms. Ca ²⁺ -dependence of BK activation involves TRPV4. A high-K ⁺ diet increases TRPV4 expression and leads to its redistribution to the apical membrane in CCD, and TRPV4 knockout mice display decreased BK activity in CCD and hyperkalemia after dietary K ⁺ loading.

The greater density of BK channels in intercalated cells in both the CCD and CNT ^, suggested a major role for intercalated cells in K ⁺ secretion. However, the much lower density of Na ⁺ -K ⁺ -ATPase activity in intercalated cells is considered inadequate to support K ⁺ secretion across the apical membrane. The evidence supports a role for the basolateral Na ⁺ -K ⁺ -2Cl ⁻ cotransporter NKCC1 in K ⁺ secretion mediated by apical BK channels. NKCC1 expression at the basolateral membrane of intercalated cells provides an alternative entry pathway for basolateral K ⁺ secreted at the apical membrane. ^, A basolateral Na ⁺ pump, the ouabain-insensitive furosemide-sensitive Na ⁺ -ATPase, whose activity has been detected in cell culture models of intercalated cells, may recycle Na ⁺ across the basolateral membrane in the absence of significant Na ⁺ -K ⁺ -ATPase activity. At the apical membrane, BK-mediated K ⁺ secretion is only partially dependent on luminal Na ⁺ ; K ⁺ secretion would eventually hyperpolarize the membrane in the absence of apical Na ⁺ entry. An intriguing possibility is that apical Cl ⁻ channels allow for the parallel secretion of K ⁺ and Cl ⁻ in intercalated cells.

Mice with a targeted deletion of the β ₄ -subunit exhibit normal K ⁺ excretion on a normal diet but develop hyperkalemia with a blunted increase in K ⁺ excretion and urinary flow rates when fed a high-K ⁺ diet, which increases urinary and tubular flow rates and tubular shear stress. Intercalated cells protrude into the lumen of distal tubules; flow-activated BK channels reduce the cell volume of intercalated cells after K ⁺ loading, reducing tubular resistance, increasing tubular flow rates, and increasing distal K ⁺ secretion. Intercalated cells from β ₄ -knockouts fail to significantly decrease cell volume in response to high-K ⁺ diet.

The physiologic rationale for the presence of two apical secretory K ⁺ channels, ROMK and BK channels, is not completely clear. However, the high density and higher P _o of ROMK channels are perhaps better suited for a role in basal K ⁺ secretion, with additional recruitment of the higher capacity, flow-activated BK channels when additional K ⁺ secretion is required. BK channels may function in partially Na ⁺ -independent K ⁺ secretion by intercalated cells, with ROMK functioning in ENaC- and Na ⁺ -dependent K ⁺ excretion by DCT, CNT, and CCD cells. Regardless, at the whole-organ level, the two K ⁺ channels can substitute for one another, with BK-dependent K ⁺ secretion in ROMK knockout mice and an upregulation of ROMK in the distal nephron of α ₁ -subunit BK knockouts. ^,

Other K ⁺ channels reportedly expressed at the luminal membranes of the CNT and CCD include voltage-sensitive channels such as KV1.3, the calcium-activated, small-conductance SK3 channel; and double-pore K ⁺ channels, such as TWIK-1 and KCNQ1. KCNQ1 is expressed at the apical membrane of principal cells in the CCD, whereas TWIK-1 is expressed at the apical membrane of intercalated cells. ^, The roles of these channels in renal K ⁺ handling are unknown. However, KV1.3 may play a role in distal K ⁺ secretion since specific blockade with margatoxin reduces K ⁺ secretion in CCDs of rat kidneys from animals on a high-K ⁺ diet. Other apical K ⁺ channels in the distal nephron may subserve other physiologic functions. The apical KV1.1 channel may contribute to Mg ²⁺ transport by the DCT by hyperpolarizing the apical membrane, thus increasing the driving force for Mg ²⁺ influx via TRPM6 (transient receptor potential cation channel 6); missense mutations in KV1.1 are a cause of genetic hypomagnesemia.

Basolateral membrane K ⁺ channels appear to set the resting potential of the basolateral membrane of principal cells and function in K ⁺ secretion and Na ⁺ absorption (by recycling K ⁺ to maintain Na ⁺ -K ⁺ -ATPase activity) at the apical membrane. A single predominant basolateral activity has been identified in principal cells from the rat CCD using whole-cell recording techniques under conditions in which ROMK is inhibited (low intracellular pH or presence of the ROMK inhibitor tertiapin-Q). This basolateral current is tetraethylammonium (TEA) insensitive, barium sensitive, and acid sensitive (pK _a ≅ 6.5), with a conductance of about 17 pS and weak inward rectification. Candidate inward-rectifying K ⁺ channel subunits localized at the basolateral membrane of the CCD include KIR4.1, KIR5.1, KIR7.1, and KIR2.3. KIR4.1 and KIR5.1 channels generate a predominant 40 pS basolateral K ⁺ channel in murine principal cells, with both KIR4.1 and KIR5.1 participating in generating the membrane potential that permits Na ⁺ entry through ENaC. ^, Notably, basolateral K ⁺ channel activity increases on a high-K ⁺ diet, suggesting a role in transepithelial K ⁺ secretion. Disruption of KIR5.1 in rats reveals it plays a critical role in collecting duct function, particularly with respect to maintenance of K ⁺ homeostasis. Activation of KIR4.1/KIR5.1 by insulin and IGF-1 may also facilitate Na ⁺ reabsorption along the CCD by hyperpolarizing the basolateral membrane.

Apical K ⁺ -Cl ⁻ cotransport (or functionally equivalent pathways) is also implicated in distal K ⁺ secretion. ^{, , ,} In rat distal tubules, anion-dependent K ⁺ secretion that is not influenced by luminal Ba ²⁺ has been recorded, suggesting that it does not involve apical K ⁺ channel activity. Cl ⁻ -coupled K ⁺ secretion is detectable in the DCT and early CNT. In addition, similar pathways are detectable in rabbit CCD, where a decrease in luminal Cl ⁻ concentration from 112 to 5 mmol/L increases K ⁺ secretion by 48%. A reduction in basolateral Cl ⁻ also decreases K ⁺ secretion without an effect on transepithelial voltage or Na ⁺ transport, and a lumen to bath Cl ⁻ gradient reverses K ⁺ flux to cause K ⁺ absorption. In perfused CCDs from rats treated with mineralocorticoid, vasopressin increases K ⁺ secretion; because this increase in K ⁺ secretion is resistant to luminal Ba ²⁺ (2 mmol/L), vasopressin may stimulate Cl ⁻ -dependent K ⁺ secretion. ^, Pharmacologic study results of perfused tubules are consistent with K ⁺ -Cl ⁻ cotransport mediated by the KCCs. A study using an antibody validated in knockout mice suggests that KCC3a, encoded by the Slc12a9 gene, may mediate electroneutral secretion of KCl in type B or type-non-A/non-B intercalated cells along the CNT. Other functional possibilities for Cl ⁻ -dependent K ⁺ secretion include parallel operation of apical H ⁺ -K ⁺ -exchange and Cl ⁻ -HCO ₃ ⁻ exchange in type B intercalated cells.

A provocative study by Frindt and Palmer serves to underline the importance of ENaC-independent K ⁺ excretion (see also “Integrated NaCl and K ⁺ Transport in the Distal Nephron”). Rats were infused with sufficient amiloride expected to inhibit more than 98% of ENaC activity. Whereas amiloride almost abolished K ⁺ excretion in rats on a normal K ⁺ intake, acute and long-term high-K ⁺ diets led to an increasing fraction of K ⁺ excretion that was independent of ENaC activity (≈50% after 7–9 days on a high-K ⁺ diet).