General Features

Rocha and Kokko and Burg and Green first demonstrated the salient characteristics of salt absorption in rabbit medullary and cortical TAL segments, respectively. First, net salt absorption resulted in a lumen-positive transepithelial voltage (Ve, mV), which could be abolished by furosemide, and in dilution of the luminal fluid. Second, the transport of Cl ⁻ under these circumstances occurred against both electrical and chemical gradients, and hence involved an active transport process. Third, both net chloride absorption and the transepithelial voltage depended on (Na ⁺ , K ⁺ )-ATPase activity, present in large amounts along the basolateral membrane of this segment. A final curious feature of the TAL is that this segment is a “hybrid epithelium” possessing a very low permeability to water, yet a high ionic conductance ( Table 34.2 ). The ionic conductance determined from the fluxes of ²² Na ⁺ and ³⁶ Cl ⁻ is approximately 20–50 mS/cm ² , and is cation selective (P _Na /P _Cl =2–6). This high electrical conductance is unusual among epithelia with low water permeabilities.

Table 34.2 presents a summary of the important transport properties of the rabbit, mouse, and rat TAL which are relevant to the concentrating and diluting functions of this nephron segment. A low permeability to water is required for the TAL to function as a diluting segment. Hebert et al., and subsequently Hebert demonstrated that the apical cell membrane constitutes the major barrier to transcellular water flow in this segment.

Studies of the electrophysiologic ( Table 34.3 ) and biochemical properties of intact, isolated, perfused thick limb segments, and of apical and basolateral membranes of thick limb cells have provided insights into the specific transport mechanisms involved in salt absorption, and the origin of the lumen-positive transepithelial voltage in this nephron segment. A model for salt absorption by the TAL, which integrates the results of these studies, is shown in Figure 34.1 . Net Cl ⁻ absorption by the TAL is a secondary active transport process. Luminal Cl ⁻ entry into the cell is mediated by an electroneutral Na ⁺ -K ⁺ -2Cl ⁻ co-transport process driven by the favorable electrochemical gradient for sodium entry. More specifically, the net driving force for the entry of Cl ⁻ into the cell is determined by the sum of the chemical gradients for Na ⁺ , K ⁺ , and Cl ⁻ . Since the Na ⁺ gradient is maintained by the continuous operation of the basolateral membrane (Na ⁺ ,K ⁺ )-ATPase pump, apical entry of Cl ⁻ via the co-transporter ultimately depends on the operation of the basolateral (Na ⁺ ,K ⁺ )-ATPase. Accordingly, maneuvers that inhibit ATPase activity, such as removal of K ⁺ from or addition of ouabain to, peritubular solutions leads to dissipation of the Na ⁺ gradient and subsequent inhibition of apical membrane Cl ⁻ entry.

A model depicting the major pathways involve in NaCl absorption mechanism by the thick ascending limb.

Dashed lines indicate passive movements down electrochemical gradient.

From Reeves, W. B., and Andreoli, T. E. (2008). Sodium chloride transport in the loop of Henle, distal convoluted tubule, and collecting duct. In “The Kidney: Physiology and Pathophysiology,” 4th edn, 849–888, Alpern, R. J., and Hebert, S. C. (eds.). Elsevier.

In contrast to the electroneutral entry of Cl ⁻ across the apical membrane, the majority of Cl ⁻ efflux across the basolateral membrane proceeds through conductive pathways. A favorable electrochemical gradient for Cl ⁻ efflux through dissipative pathways has been demonstrated by Greger et al. in the rabbit cTAL. In this study, an intracellular Cl ⁻ activity of 22 mM, measured using single Cl ⁻ -selective microelectrodes, was substantially above the equilibrium value (5 mM) predicted from the intracellular voltage. Intracellular Cl ⁻ is maintained at concentrations above electrochemical equilibrium by the continued entry of Cl ⁻ via the apical Na ⁺ -K ⁺ -2Cl ⁻ co-transporter. Blocking Cl ⁻ entry through this pathway with furosemide or substitution of extracellular Cl ⁻ by gluconate, caused the intracellular Cl ⁻ activity to fall to a value close to its equilibrium. In addition to electrodiffusive efflux of Cl ⁻ across the basolateral membrane, a component of electroneutral KCl co-transport has been proposed in some species, and the the K ⁺ -Cl ⁻ co-transporter, KCC4, has been shown to be present at the TAL basolateral membrane.

In addition, according to the model in Figure 34.1 , the potassium that enters TAL cells via the Na ⁺ -K ⁺ -Cl ⁻ co-transporter recycles, to a large extent, across the apical membrane by way of a K ⁺ conductive pathway. This apical K ⁺ recycling serves several purposes. First, it ensures a continued supply of luminal potassium in order to sustain Na ⁺ -K ⁺ -Cl ⁻ co-transport. Without recycling, the luminal K ⁺ concentration would fall rapidly as a consequence of K ⁺ entry via Na ⁺ -K ⁺ -Cl ⁻ co-transport, and would limit net NaCl absorption. Second, the apical membrane potassium current provides a pathway for net potassium secretion by the TAL, which is an active process, ultimately driven by the (Na ⁺ ,K ⁺ )-ATPase, proceeding in the face of a lumen-positive transepithelial potential. Third, under open circuit conditions, the transcellular and paracellular pathways form a current loop in which the currents traversing the two pathways are of equal size but opposite direction. The potassium current from cell to lumen polarizes the lumen and causes an equivalent current to flow from lumen to bath through the paracellular pathway. Since the paracellular pathway is cation-selective (P _Na /P _Cl =2–6), the majority of the current through the paracellular pathway is carried by sodium moving from the lumen to the interstitium. This paracellular absorption of sodium increases the efficiency of sodium transport by the TAL. With reference to Figure 34.1 , for each Na ⁺ transported through the cell – and requiring utilization of ATP – one Na ⁺ is transported through the paracellular pathway without any additional expenditure of energy. Finally, the apical K ⁺ current satisfies the continuity requirement imposed by a high degree of conductive Cl ⁻ efflux across basolateral membranes. A small component of sodium transport by the TAL (5–10%) is accounted for by sodium bicarbonate absorption. Sodium bicarbonate absorption is thought to be mediated by an apical membrane amiloride-sensitive Na ⁺ /H ⁺ exchanger and a basolateral membrane electrogenic 3Na ⁺ /(HCO ₃ ⁻ ) co-transporter. The following sections will describe the individual components of the mechanism for TAL salt transport ( Figure 34.1 ) in greater detail.

Apical Na ⁺ -K ⁺ -Cl ⁻ Co-Transport

The predominant mode for Cl ⁻ entry into the TAL cell is via a Na ⁺ -K ⁺ -2Cl ⁻ co-transporter. A characteristic feature of this transporter is its sensitivity to inhibition by furosemide, bumetanide, and other 5-sulfamoylbenzoic acid derivatives. The first demonstration of dependence on luminal sodium for Cl ⁻ absorption was reported by Greger in isolated perfused rabbit cortical TAL segments. Moreover, a requirement for luminal potassium has been demonstrated for sodium and chloride absorption in both mouse medullary and rabbit cortical thick ascending segments perfused in vitro . As a result of these studies, it is now recognized that Cl ⁻ absorption in the TAL generally depends on the simultaneous presence of Na ⁺ and K ⁺ (or NH ₄ ) in the lumen.

Measurement of isotope flux into TAL cells or membrane vesicles prepared from the inner stripe of outer medulla have delineated further the ionic requirements and stoichiometry of 1Na ⁺ -1K ⁺ -2Cl ⁻ co-transport. There is a general agreement that the co-transporter in the thick ascending limb conforms to this stoichiometry under most experimental conditions. However, under certain circumstances, which will be discussed below, apical membrane NaCl entry may be independent of luminal potassium. Early work suggested differences in apparent affinity constant for chloride along TAL which suggested axial heterogeneity in the properties of the Na ⁺ -K ⁺ -2Cl ⁻ co-transporter, because Greger, and Hus-Citharel and Morel examined the cortical TAL, while Koenig et al. and Burnham et al. prepared membranes from the medulla. An axial heterogeneity in the regulation of co-transporter activity as the thick limb ascends from the medulla into the cortex might be anticipated. This is explained by differences in affinity constant for ions of axially distributed along TAL of alternatively spliced variants of the Na ⁺ -K ⁺ -2Cl ⁻ co-transporter.

SLC12A1 gene encodes for the absorptive form of the co-transporter, referred to as BSC1 or NKCC2, located exclusively at the apical membrane of TAL, simultaneously cloned by Gamba et al. and Payne et al. Molecular biology of this gene is extensively discussed in Chapter 32 . Inactivating mutations in the SLC12A1 gene is the cause of Bartter syndrome type I. This syndrome, characterized by hypokalemia, metabolic alkalosis, hyperaldosteronism, and low blood pressure, results from a defect in salt absorption by the TAL, providing strong support for the conclusion that NKCC2 is responsible for apical Na ⁺ -K ⁺ -2Cl ⁻ co-transport in the thick ascending limb. The NKCC2 cDNA encodes a protein containing ~1100 amino acids and having a predicted molecular weight of 115–120 kDa. The observed molecular weight, however, is approximately 150 kDa due to extensive glycosylation. Hydropathy analysis of the amino acid sequence predicts a protein having 12 putative transmembrane spanning domains (TM). Six isoforms of NKCC2 have been identified that are results of alternative splicing in the 5′ and 3′ regions of the NKCC2 gene. Three 5′ splice products, termed A, B, and F, are expressed in all mammalian species. These variants differ only in a 96 bp region, which encodes the amino acids forming half of the second TM and interconnecting segment between TM2 and 3. The isoforms show differential expression within the thick ascending limb. Using RT-PCR, Yang et al. examined the distribution of NKCC2 isoforms in single nephron segments. The A isoform was found in both the cortical and medullary TAL, the B isoform was restricted to the cortical TAL, while the F isoform was present in the medullary, but not cortical, TAL and, to a lesser extent, in the outer medullary collecting duct. Altough the three variants mediate Na ⁺ -K ⁺ -2Cl ⁻ co-transport, they have different transport properties. The A and B isoforms have higher affinities for Na ⁺ , K ⁺ , and Cl ⁻ than the F isoform. The A isoform appears to have a greater transport capacity than the other isoforms (see Figure 32.3 of Chapter 32 ). Thus, the presence of the A and F isoform in the medullary thick ascending limb could account for the observed high rates of NaCl transport relative to the cortical segment ( Table 34.2 ), while the expression of the A and B isoforms in the cortical thick ascending limb may subserve the ability to achieve lower luminal NaCl concentrations in that segment. As discussed with detail in Chapter 32 , difference in affinity for ions among the NKCC2 variants B and F has been attributed to only six residues within these exons.

There is experimental evidence that the apical co-transporter in TAL might operate as a simple NaCl symporter under certain conditions. Eveloff and co-workers have reported potassium-independent, furosemide-sensitive NaCl transport in isolated rabbit TAL cells and membrane vesicles prepared from TAL cells. Potassium dependence of the co-transporter might be subject to physiologic regulation. Eveloff and Calamia have shown that under isotonic conditions the chloride-dependent, furosemide-sensitive component of sodium uptake is independent of potassium, but that under hypertonic conditions the sodium uptake becomes K ⁺ -dependent. Sun et al. have also reported that, in perfused mouse medullary TAL segments, basal sodium chloride transport was K ⁺ -independent, but that upon stimulation of salt transport by ADH NaCl transport became dependent on luminal K ⁺ . The NKCC2 variants at the carboxyl terminal domain help to explain this discrepancy. Alternative splicing at the 3′ end of NKCC2 produces additional isoforms with either long (C9) or short (C4) carboxy-termini. Under isotonic conditions, the truncated (C4) isoforms do not mediate Na ⁺ -K ⁺ -2Cl ⁻ co-transport. Under hypotonic conditions, however, the C4 isoforms mediate K ⁺ -independent NaCl co-transport, which is inhibited by cAMP and may account for the K ⁺ -independent NaCl transport noted in earlier studies. Of note, the C4 isoform inhibit the transport activity of the full-length (C9) isoforms when the two are co-expressed, and this inhibition is relieved by cAMP. The inhibition of C9 NKCC2 isoforms by C4 isoforms suggests a physical interaction between these proteins. Biochemical studies have established that NKCC2 exists as a dimer. Thus, it is possible that different combinations of NKCC2 isoforms within the dimer could produce transporters with a wide variety of functional properties. Moreover, the subunit composition of the dimers may be a point of physiologic regulation. Alternatively, the C4 isoform could alter membrane trafficking or stability of the C9 isoform. The C4 isoform resides predominantly in subapical vesicles rather than the cell membrane. Meade et al. found that co-expression of the C4 isoform reduced the abundance of C9 isoform in Xenopus oocyte membranes. The reduction in cell surface C9 isoform was reflected by a commensurate reduction in bumetanide-sensitive Rb uptake. The inhibitory effect of C4 on C9 cell surface localization could be prevented by cAMP or by disruption of microtubules, suggesting that C4 alters the trafficking of C9 in or out of the apical membrane.

Evidence for a model of two distinct binding sites for Cl ⁻ with differing affinities follows from studies of Forbush and Palfrey on ³ H-bumetanide binding to renal medullary membranes. In these studies, Na ⁺ , K ⁺ , and Cl ⁻ were all required for bumetanide to bind to canine renal medullary membranes. The K _a values for sodium and potassium were 2 mM and 1 mM, respectively. The effect of Cl ⁻ concentration on bumetanide binding was biphasic. At low concentrations (<5 mM), Cl ⁻ enhanced ³ H-bumetanide-binding. These data are consistent with a model in which the binding of Cl ⁻ to a high-affinity site exposes a second lower-affinity site that may be occupied either by bumetanide or by the second Cl ⁻ . However, functional studies using chimeras constructed between the ubiquitously expressed Na ⁺ -K ⁺ -2Cl ⁻ co-transporter NKCC1 encoded by SLC12A2 gene and NKCC2, as well as in wild-type and mutant variants A, B, and F of NKCC2, revealed that changes in chloride affinity are not paralleled by changes in bumetanide affinity, and vice versa , questioning the competition of chloride and bumetanide for a single-binding site (see Chapter 32 on NaCl co-transporters).

The binding of ions to the co-transporter is thought to be ordered and cooperative. Thus, sodium binds to the co-transporter first, and promotes binding of a Cl ⁻ and then K ⁺ . Binding of K ⁺ to its site, in turn, promotes binding of the second Cl ⁻ to the co-transporter. Once fully occupied, the co-transporter–ion complex translocates to the internal surface of the cell membrane, where debinding occurs in the same order. The full reaction sequence of binding and debinding results in inward Na ⁺ /K ⁺ /2Cl ⁻ transport. Partial reactions permit K ⁺ /K ⁺ and Na ⁺ /Na ⁺ exchange.

Apical Potassium (K) Channels

Studies of isolated, perfused, thick limb segments have established that the predominant, and perhaps only, conductance across the apical membrane is to potassium. Blockade of the apical K conductance by luminal barium in mouse medullary thick ascending limb (mTAL) decreases the transepithelial electrical conductance (G _e ) by roughly 50%, while increasing the apical-to-basolateral membrane resistance ratio (Ra/Rb) from 1.9 to 12.9. Moreover, changes in the luminal K ⁺ concentration produce essentially Nernstian changes in the apical membrane electrical potential. Electrophysiological studies demonstrated that the apical membrane K ⁺ conductance is sufficient to recycle the potassium uptake via the Na ⁺ -K ⁺ -2Cl ⁻ co-transporter.

The properties of the apical membrane K ⁺ conductance have been studied in plasma membrane vesicles prepared from outer renal medulla. Conductive K ⁺ fluxes in these vesicles can be measured by loading the vesicles with a high concentration of potassium, and then removing the potassium from the external solutions. Tracer amounts of ⁸⁶ Rb ⁺ are then added to the extravesicular solutions to begin uptake. Under these conditions, the outwardly directed K ⁺ gradient creates an inside-negative diffusion potential that drives ⁸⁶ Rb ⁺ uptake into the vesicles. Burnham et al. and Reeves et al. were thus able to demonstrate barium-sensitive ⁸⁶ Rb ⁺ flux in membranes from rabbit outer medulla. The Rb ⁺ flux was conductive as judged by its inhibition via collapse of the intravesicular voltage or by measurement of intravesicular voltage using voltage-sensitive dyes. The K _0.5 value for barium inhibition of Rb ⁺ flux was 50–100 µM. The barium-sensitive Rb ⁺ flux was also dependent on the calcium activity within the vesicles. Moreover, in reconstituted proteoliposomes prepared from porcine outer medulla, the calcium dependence of the K ⁺ conductance was modulated by a high-affinity (K _0.5 =0.1 nM) calmodulin-binding site.

The patch-clamp technique has identified two types of apical K ⁺ channels in the TAL, a low-conductance (35 pS) K ⁺ channel and an intermediate-conductance (70 pS) K ⁺ channel in the apical membrane of the TAL. The 35 pS K ⁺ channel is observed in the apical membrane of rabbit, rat, and mouse. Moreover, exposure of the cytoplasmic surface of the patch to ATP inhibited channel activity. In addition to the 35 pS K ⁺ channel, Bleich et al. and Wang described an intermediate conductance (60–70 pS) K ⁺ channel in rat TAL segments. Like the low conductance K ⁺ channel, the 70 pS K ⁺ channel was inhibited by ATP and barium. In addition, the channel activity was voltage-dependent with depolarization increasing its activity; the channel could be blocked by quinine, verapamil, and diltiazem, and was inhibited at low cytosolic pH. The activities of both the intermediate and low conductance K ⁺ channels are increased by high dietary potassium. An overview regarding regulation of apical K ⁺ channels in the TAL is summarized in Figure 34.2 , and detailed information can also be found in the review articles.

A cell scheme illustrating the inhibitory and stimulatory factors for regulation of apical and basolateral ion channels.

An ATP-dependent K ⁺ channel was cloned from rat kidney by Ho et al. This channel, termed ROMK (Renal Outer Medullary K ⁺ channel), is the prototype for a large family of inward rectifying K ⁺ channels (K _IR channels). K _IR channels have two transmembrane spanning domains, intracellular N- and C-termini, and an extracellular helical domain between the two transmembrane domains. Structural studies of other K _IR channels have established that the channels exist as heteromers of four K _IR subunits. The extracellular helical domain appears to form the outer vestibule and selectivity filter of the channel, while the second transmembrane domain lines the pore cavity.

ROMK ⁺ channels have three alternatively spliced forms and they are ROMK2 (Kir1.1b), ROMK3 (Kir1.1c), and ROMK6 (Kir1.1d). The encoded ROMK proteins differ at the beginning of the N-terminus; ROMK2 has the shortest N-terminus and splicing adds either 19 or 26 amino acids for ROMK1 or ROMK3, respectively. Relative ROMK mRNA abundance measured by competitive PCR has shown that ROMK2 and ROMK3 are much more abundant than ROMK1 or ROMK6 in rat kidney. Moreover, single-nephron PCR analysis indicated that ROMK1 is expressed in the collecting duct, while both ROMK2 and ROMK3 are expressed in the medullary and cortical thick ascending limb. Immunolocalization of ROMK proteins, using antibodies that recognized all three isoforms, revealed apical membrane localization in the medullary and cortical thick ascending limb, the late distal tubule, the connecting tubule, and the cortical collecting duct. Oddly, the expression of ROMK in the thick limb was heterogeneous, with some cells lacking demonstrable staining. In contrast, the expression of the Na ⁺ -K ⁺ -2Cl ⁻ co-transporter was uniform within the TAL. This implies that apical K ⁺ recycling by the ROMK-positive cells may be sufficient to supply K ⁺ for Na ⁺ -K ⁺ -2Cl ⁻ transport in the ROMK-negative cells.

The 35 pS channel conductance of ROMK corresponds to the low conductance K ⁺ channel described by Wang et al. The current–voltage relations exhibit weak inward rectification, which is due to blocking of the channel by intracellular Mg ²⁺ or polyamines. The activity of this channel is modulated by intracellular pH and by intracellular ATP, through both phosphorylation-dependent and phosphorylation-independent pathways. The maintenance of ROMK ⁺ channel activity requires protein kinase A (PKA)-mediated phosphorylation and deletion of any two of three PKA-phosphorylation sites inactivates ROMK. Phosphorylation of ROMK affects both channel activity (P _o ) and the number of functional channels in the plasma membrane. In addition, PKA-induced phosphorylation of ROMK enhances the sensitivity of the ROMK ⁺ channel to phosphatidylinositol phosphates (PIP2).

Nucleotides have both inhibitory and stimulatory effects on ROMK activity. At micromolar concentrations, ATP stimulates ROMK activity via PKA-mediated phosphorylation and production of PIP ₂ . Millimolar concentrations of ATP inhibit ROMK activity. The inhibition by ATP can be relieved by increasing concentrations of ADP or by PIP ₂ . Certain other members of the K _IR family are also inhibited by ATP. The ATP sensitivity of these channels–for example, K _IR 6.2–is endowed by the association of regulatory subunits. These regulatory subunits, such as the sulfonylurea receptor (SUR) and CFTR, belong to the ABC gene family which is characterized by nucleotide-binding domains. The sulfonylurea receptor, together with K _IR 6.2, forms the glibenclamide-sensitive, ATP-sensitive K ⁺ channel that controls insulin secretion by pancreatic β-cells. The K _IR /SUR channels are more sensitive to inhibition by ATP than those of heterologously expressed ROMK ⁺ channels. In addition, the native 35 pS K ⁺ channel, but not the heterologously expressed ROMK ⁺ channel, is inhibited by glibenclamide. These observations have prompted the search for regulatory subunits for the ROMK ⁺ channel. Both SUR2B and CFTR are expressed in the thick ascending limb and collecting duct, making them candidates for ROMK regulation. In transfection studies, co-expression of ROMK with either SUR2B or CFTR dramatically increases both the nucleotide dependence and glibenclamide sensitivity of ROMK. Immunoprecipitation studies confirmed that one of the ROMK isoforms, ROMK2, physically interacts with SUR2B. The role of CFTR in regulating the native ROMK ⁺ channels has been further suggested by the finding that PKA-induced regulation of ATP sensitivity of the native 35 pS K ⁺ channel is compromised in CFTR knockout mice. Figure 34.3 is a cartoon illustrating the current view regarding the composition of the native ATP-sensitive K ⁺ channel in the apical membrane of the TAL.

Assembly of the native ATP-sensitive K channels in the TAL with ATP-binding cassette [ABC] proteins.

(a) The proposed hetero-octameric complex forming the native ATP-sensitive K channels in the TAL with four K _IR subunits and four sulfonylurea receptor [SUR] subunits. (b) The topology of CFTR and ROMK channels proposed to form kidney K _ATP channels. A single nucleotide-binding domain [NBD, 1] is present on the C-terminus of ROMK while two NBDs [numbered 2 and 3] are found on CFTR. (c) The topology of the sulfonylurea receptor, SUR2B, and ROMK proposed to form kidney K _ATP channels.

The electrophysiologic properties of the ROMK channels have strongly suggested that ROMK is the native 35 pS K ⁺ channel expressed in the thick ascending limb. In support of this view, no low-conductance K ⁺ channels were detected by patch-clamp analysis in ROMK knockout mice. Moreover, deleting the gene product encoding ROMK also abolished the expression of intermediate conductance K ⁺ channels in the thick ascending limb. It has been proposed that the intermediate conductance channel may be a heteromeric protein containing ROMK and other, as yet unidentified, subunits. The notion that one of the ROMK channels is the predominant channel responsible for apical K ⁺ recycling in the thick ascending limb is strongly supported by the finding of mutations in the ROMK gene in some families with Bartter syndrome. Thus, the presence of ROMK mutations as a cause of Bartter syndrome indicates the important role of ROMK in net salt absorption by the thick ascending limb.

Basolateral Potassium (K) Channels

Basolateral K ⁺ channels in the TAL play an important role in the regulation of transepithelial transport. They are responsible for generating basolateral membrane potential which is essential for Cl diffusion across the basolateral membrane in the TAL. Activation of basolateral K ⁺ channels in the TAL is expected to hyperpolarize the basolateral cell membrane, thereby augmenting the driving force for Cl exit across the basolateral membrane. In contrast, decreasing basolateral K ⁺ channel activity depolarizes the cell membrane potential, thereby diminishing the driving force for Cl ⁻ exit across the basolateral membrane. Consequently, inhibition of Cl ⁻ exit leads to an increase in intracellular Cl ⁻ concentration which decreases the activity of NKCC2, probably via inhibiting WNK3-SPAK.

The physiological importance of the basolateral K ⁺ channels in maintaining transepithelial membrane transport in the TAL and distal nephron is best demonstrated in SeSAME disease ( Se izures, S ensorineural deafness, A taxia, M ental retardation, and E lectrolyte imbalance). This disease is the result of defective gene product encoding KCNJ10, an inwardly-rectifying K ⁺ channel, which is also expressed in the basolateral membrane of TAL and distal nephron. The renal phenotypes of SeSAME disease are hypokalemia, metabolic alkalosis, and hypomagnesemia. Presumably, defective basolateral K ⁺ channels cause membrane potential depolarization, thereby decreasing Cl ⁻ diffusion across the basolateral membrane. Consequently, an increase in intracellular Cl ⁻ concentrations results in the inhibition of the apical Na entry through NKCC2, thereby suppressing Na absorption and diminishing the lumen-positive potential in the TAL. A decrease in the lumen-positive potential results in inhibition of magnesium absorption through the paracellular pathway in the TAL. Also, the inhibition of Na absorption in the TAL increases Na delivery to the distal nephron, accordingly stimulating Na absorption in the expanse of K ⁺ in the connecting tubule and causing K ⁺ -wasting. Therefore, an alteration in the basolateral K ⁺ channel activity in the TAL has a significant effect on transepithelial transport not only in the TAL, but also in the distal nephron segment.

Patch-clamp experiments demonstrated that an inwardly-rectifying 50 pS K ⁺ channel and a Na ⁺ and Cl ⁻ activated 140–180 pS K ⁺ channel are present in the basolateral membrane of the TAL. The 50 pS K ⁺ channel is highly-expressed, and may be the main K ⁺ channel in the basolateral membrane of the TAL. The regulation of the 50 pS K ⁺ channels has been intensively studied and several factors, including protein kinase A (PKA), 20-hydroxyeicosatetraenoic acid (20-HETE), and external Ca ²⁺ , have been identified to modulate the 50 pS K ⁺ channels ( Figure 34.2 ). Arachidonic acid inhibits the basolateral 50 pS K ⁺ channels in the TAL through the CYP-omega-hydroxylase-dependent metabolism, and 20-HETE mediates the effect of arachidonic acid on the K ⁺ channel. Also, raising the external Ca ²⁺ inhibited the basolateral 50 pS K ⁺ channels in the TAL by a PKC-dependent mechanism. Two lines of evidence suggest that the effect of the external Ca ²⁺ on the basolateral 50 pS K ⁺ channels was the result of stimulating the Ca ²⁺ -sensing receptor (CaSR) which is expressed in the TAL : (1) the inhibitory effect of raising the external Ca ²⁺ on the 50 pS K ⁺ channels was observed only in cell-attached patches, but not in excised patches; (2) the effect of raising external Ca ²⁺ on the 50 pS K ⁺ channel was absent in the presence of the CaSR antagonist. It is possible that the CaSR might directly interact with the basolateral 50 pS K ⁺ channels in the TAL. This speculation is supported by reports from several studies. First, the basolateral 50 pS K ⁺ channels in the TAL might represent heterotetramers made of Kir4.1/Kir5.1, because the basolateral K ⁺ channels in the distal tubules with similar biophysical properties to those of the 50 pS K ⁺ channel in the TAL are composed of Kir4.1 and Kir5.1. Second, immunostaining experiments demonstrated that Kir4.1 and Kir5.1 were expressed in the basolateral membrane of the TAL. Finally, the study using immunoprecipitation performed in the cells transiently transfected with Kir4.1 and the CaSR has demonstrated that Kir4.1 was associated with the CaSR. The regulation of the basolateral 50 pS K ⁺ channel by the external Ca ²⁺ should play a role in the modulation of transepithelial Na transport and concentrating ability in the TAL. Since the active reabsorption of NaCl ⁻ in the water-impermeable TAL is essential for urinary concentrating mechanism, inhibition of NaCl ⁻ reabsorption in the TAL should result in a decrease in concentrating ability. Indeed, it has been reported that hypercalcemia impairs urinary concentrating ability.

Because the activity of the basolateral K ⁺ channels is regulated by the external Ca ²⁺ /Mg ²⁺ concentrations at a physiologically relevant range (1 to 2 mM), the CaSR-mediated regulation of the membrane transport in the TAL is mainly through controlling the basolateral K ⁺ channel activity. Figure 34.4 is a cell scheme illustrating the role of basolateral K ⁺ channels in mediating the effect of stimulation of the CaSR on Na ⁺ and divalent cation transport in the TAL. Under physiological conditions, the basolateral K ⁺ channels maintain the cell membrane potential such that it could sustain a constant Cl ⁻ diffusion across the basolateral membrane. An increase in the external Ca ²⁺ (hypercalcemia) activates the CaSR, thereby inhibiting basolateral K ⁺ channels and decreasing the driving force for Cl ⁻ diffusion across the basolateral membrane. Inhibition of Cl ⁻ exit is expected to lead to hyperpolarization of transepithelial voltage (V _te ). A less positive V _te would diminish the reabsorption of Na ⁺ and divalent cations such as Ca ²⁺ and Mg ²⁺ . Indeed, it has been reported that an increase in plasma Ca ²⁺ /Mg ²⁺ level enhanced urinal Ca ²⁺ /Mg ²⁺ excretion.

A cell scheme illustrating the role of stimulation of Ca ²⁺ ensing receptor (CaSR) in regulation of apical and basolateral K channels, thereby affecting the transepithelial transport of Na and divalent cations.

(Abbreviations: PLC: phospholipase C; PLA2: phospholipase A2; PKC: protein kinase C; 20-HETE: 20-hydroxyeicosatetraenoic acid.)

Basolateral Membrane Cl ⁻ Channel and Transporter

Synchronous Na ⁺ /H ⁺ : Cl ⁻ /HCO ₃ ⁻ Exchange

An additional form of apical membrane NaCl entry has been observed in the mouse cTAL. Friedman and Andreoli found that net Cl ⁻ absorption and the transepithelial voltage were doubled when CO ₂ and HCO ₃ ⁻ were added to the external solutions bathing cTAL segments. Since the (CO ₂ HCO ₃ ⁻ )-stimulated rate of NaCl absorption did not result in net CO ₂ transport, and could be abolished by the lipophilic carbonic anhydrase inhibitor ethoxyzolamide or by the luminal addition of the anion exchange inhibitor SITS or DIDS, it was proposed that the apical membrane of the mouse cTAL contains parallel, near-synchronous Na ⁺ /H ⁺ :Cl ⁻ /HCO ₃ ⁻ exchangers in addition to a Na ⁺ -K ⁺ -Cl ⁻ co-transporter. Subsequent studies have shown that, like the mouse mTAL, the apical membrane of the mouse cTAL contains a potassium conductance, and that both the (CO ₂ HCO ₃ ⁻ )-dependent and (CO ₂ HCO ₃ ⁻ )-independent components of NaCl absorption require luminal potassium. Thus, the cation exchange process may proceed as (Na ⁺ , K ⁺ )/2 H ⁺ . Both the (CO ₂ HCO ₃ ⁻ )-dependent and -independent components of NaCl absorption in mouse cTAL segments were equally susceptible to inhibition by luminal bumetanide (K _i =5–8×10–7 M). Addition of CO ₂ and HCO ₃ ⁻ to the bathing solutions has no effect on net NaCl transport in either rabbit cTAL or mouse mTAL. Both the rat and mouse mTAL do contain Na ⁺ /H ⁺ exchangers in their apical membranes. However, in these segments, Na ⁺ /H exchange plays a role in net HCO ₃ ⁻ transport and cell pH regulation, rather than transcellular NaCl absorption.

Antidiuretic Hormone

The ADH affects NaCl absorption in the ascending limb of Henle, and thereby regulates the countercurrent multiplication process. Support for this contention was provided by the demonstration, in some species, of an ADH-induced increase in adenylate cyclase activity and protein kinase activity in the medullary, but not cortical, portions of the TAL.

Table 34.4 summarizes the effects of ADH on salt transport in in vitro microperfused medullary TAL segments of mouse, rat, and rabbit. Hall and Varney established that ADH increased the lumen-positive transepithelial voltage and the net rate of tracer Cl ⁻ absorption in the mouse medullary TAL. Several laboratories have confirmed these effects of ADH on the transepithelial voltage and unidirectional Cl ⁻ flux in the mouse, and have demonstrated that the effect of ADH on NaCl transport was restricted to the medullary portion of this segment. Moreover, the maximal stimulation of the transepithelial voltage in the mouse medullary TAL occurred at hormone concentrations of approximately 20 pg/ml or 10 µU/ml, found during states of antidiuresis. A similar increase in transepithelial voltage can be obtained in the mouse medullary TAL, but not the cortical TAL, by the addition of cAMP analogs or forskolin, a nonhormonal activator of adenylate cyclase to the peritubular media. In contrast, ADH, even at peritubular concentrations of 250 µU/ml, has no effect on V _e in the mouse cortical TAL. Taken together, these results indicate that the mouse medullary TAL and cortical TAL are functionally heterogeneous with respect to the effect of ADH on net NaCl absorption, and that stimulation of net NaCl absorption in the mouse medullary TAL by ADH is mediated by cAMP. The effect of ADH in TAL does not seem to be contant in all species. ADH causes a variable increase in cyclic AMP production in the rabbit TAL, but pharmacologic concentrations of ADH had no effects on either the transepithelial voltage or tracer Cl ⁻ efflux in this species. ADH or the V ₂ selective analog dDAVP increased NaCl absorption from the TAL on homozygous Brattleboro (central diabetes insipidus) rats. In humans evidence is limited. Studies in pump-perfused human kidneys rejected for transplantation have shown ADH-sensitive adenylate cyclase activity in collecting ducts, but not in TAL segments. Since the ability of humans to elaborate concentrated urine is limited compared to the mouse, the failure of ADH to increase cAMP in the human TAL is consistent with Morel’s suggestion that the response of medullary TAL segments to ADH correlates directly with urinary concentrating ability.

Mechanisms of ADH Affect on TAL

The mechanism whereby ADH stimulates salt transport in the medullary TAL has been studied most extensively in the mouse. The effects of ADH in this segment are mediated predominantly through interaction of the hormone with the vasopressin V ₂ receptor coupled to adenylate cyclase. Thus, ADH stimulates adenylate cyclase activity in the TAL, and cAMP analogs and forskolin mimic the effects of ADH on transport in this segment. Cholera toxin stimulates salt transport in the TAL, indicating that the V ₂ receptor is coupled to the catalytic subunit of adenylate cyclase by a stimulatory guanine nucleotide-binding protein, G _s . The catalytic subunit of adenylate cyclase may, in turn, be influenced by calmodulin. Takaichi and Kurokawa have shown that calmodulin inhibitors inhibited ADH-sensitive cAMP production in isolated mouse medullary TAL segments. Likewise, Ausiello and Hall showed that the addition of exogenous calmodulin stimulated ADH-sensitive cAMP production by LLC PK ₁ cells.

Table 34.5 lists the effects of ADH on several electrophysiologic parameters of mouse medullary TAL segments perfused in vitro . The approximate doubling of the equivalent short-circuit current results from an increase in the transepithelial voltage and an increase of roughly 20% in the transepithelial conductance. Moreover, the ADH-induced increase in transepithelial conductance, G _e , is referable to an increase in the barium-sensitive or transcellular conductance, Gc. ADH has no effect on the magnitude or permselectivity of the paracellular conductance, G _s ( Table 34.5 ).

ADH increases both the apical and basolateral membrane conductance. The apical membrane of TAL cells is, as noted previously, predominantly K ⁺ conductive. In the presence of luminal furosemide to block K ⁺ uptake via the Na ⁺ -K ⁺ -2Cl ⁻ co-transporter, mouse TAL segments exhibit net K ⁺ secretion that is increased by ADH. Because the electrochemical driving force for K ⁺ secretion is presumably constant in the presence of furosemide, it was suggested that the increase in K ⁺ secretion was due to an increase in apical membrane K ⁺ conductance. In support of this notion, using membrane vesicles prepared from rabbit outer medulla, Reeves et al. demonstrated that in vitro exposure to cAMP-dependent protein kinase and ATP specifically enhanced barium-sensitive rates of ⁸⁶ Rb ⁺ uptake. This finding has been confirmed in subsequent work that examined the effects of phosphorylation on the activity of ROMK channels. ROMK is phosphorylated by cAMP-dependent protein kinase at a number of sites, and this phosphorylation increases the activity of the channel.

The ADH-induced increase in transepithelial NaCl absorption can be blocked by luminal furosemide, and therefore represents an increase in apical Na ⁺ -K ⁺ -2Cl ⁻ co-transport activity. The mechanism whereby ADH increases apical co-transport activity is unclear. Sun et al. demonstrated that in isolated perfused mouse TAL segments, ADH changed the stoichiometry of NaCl entry from 1Na ⁺ :1Cl ⁻ to 1Na ⁺ :1 K ⁺ :2Cl ⁻ . This change in stoichiometry, accompanied by apical K ⁺ recycling, could create a lumen-positive potential to drive Na ⁺ reabsorption via the paracellular pathway. Phosphorylation of the cloned Na ⁺ -K ⁺ -2Cl ⁻ co-transporter NKCC2 by cAMP-dependent protein kinase does not appear to directly increase co-transporter activity. However, phosphorylation may influence co-transporter activity by modulating the interactions between NKCC2 isoforms. As noted above, an isoform with a truncated C-terminus, denoted C4, can inhibit the activity of the full-length NKCC2. However, the inhibition by the C4 isoform can be relieved by phosphorylation by cAMP-dependent protein kinase. Thus, ADH may increase apical Na ⁺ -K ⁺ -2Cl ⁻ entry by releasing NKCC2 from tonic inhibition by truncated isoforms. Of note, the short C4 variant is expressed in medullary TAL, but not in cortical TAL, providing a potential explanation for the lack of ADH effect on cortical TAL discussed above. Chronic exposure to ADH may increase apical Na ⁺ -K ⁺ -2Cl ⁻ entry by increasing the abundance of NKCC2.

The predominant portion of the ADH-induced increase in cellular conductance is accounted for by an increase in the basolateral membrane Cl ⁻ conductance. Two mechanisms have been suggested for the hormone-dependent increase in basolateral Cl ⁻ conductance. Schlatter and Greger proposed that the ADH-induced increase in intracellular cAMP results in a direct increase in chloride channel activity. Such a mechanism has been amply demonstrated in Cl ⁻ secreting epithelia, such as the trachea and intestine. In support of their proposal, Schlatter and Greger demonstrated that cAMP and ADH elicited a fall in the fractional resistance of the basolateral membrane in mouse medullary TAL segments, even when cell Cl ⁻ activity was kept at low levels by blocking apical Cl ⁻ entry with furosemide. Likewise, using patch-clamp analysis, Paulais and Teulon found that preincubation of mouse cortical TAL segments with forskolin or cAMP analogs increased the number of Cl ⁻ channels observed in basolateral membrane patches. It is now known that NKCC2 is sensitive to intracellular chloride concentration: chloride depletion activates the co-transporter by inducing phosphorylation of key amino terminal domain threonines of the co-transporter by a pathway involving the soluble kinases WNK3 and SPAK. Additionaly, elimination of TAL basolateral chloride channels in Bartter’s type III and IV reduces salt reabsorption by TAL, producing the disease. Thus, one mode of regulation of NKCC2 can be achieved by modulating intracellular chloride concentration in TAL.

Alternatively, ADH might enhance Cl ⁻ conductance indirectly by increasing apical membrane Cl ⁻ entry. According to this proposal, an ADH-dependent activation of apical membrane Na ⁺ -K ⁺ -2Cl ⁻ co-transporter and K ⁺ channels leads to an increase in intracellular Cl ⁻ activity, and a subsequent increase in basolateral Cl ⁻ conductance. In support of this view, Molony et al. found, also in mouse medullary TAL, that the ADH-dependent rise in cellular and basolateral conductance were much lower when Cl ⁻ entry was inhibited by furosemide than in control conditions. Studies of Cl ⁻ channels incorporated from basolaterally enriched renal medullary membrane vesicles into planar lipid bilayers have demonstrated several properties of these channels that may account for an intracellular Cl ⁻ concentration-dependent rise in basolateral Cl ⁻ conductance. First, the activity of the Cl ⁻ channels is increased with membrane depolarization evidently due to the basolateral membrane depolarization that follows ADH stimulation of TAL segments. Second, Cl ⁻ channels in the vesicles behave like Goldman rectifiers, so that increases in intracellular Cl ⁻ cause an increase in the outward single-channel conductance. Third, channel activity is dependent on the intracellular Cl ⁻ concentration, such that increases in Cl ⁻ over the range 2–50 mM result in large increases in the open time probability of these Cl ⁻ channels. In this respect, it should be recognized that the time-averaged conductance of a basolateral Cl ⁻ channel is given by the product g _Cl P _o , where g _Cl is unit channel conductance and P _o is open time probability.

Thus, an ADH-dependent increase in apical Cl ⁻ entry could stimulate basolateral Cl ⁻ conductance to a far greater extent than expected from simple Goldman rectification. In that connection, at least two laboratories have found, in mTAL segments, that reducing intracellular Cl ⁻ with luminal furosemide produced a three-fold reduction in basolateral Cl ⁻ conductance.

Modulation of ADH Effect on TAL Segments

Several factors, such as prostaglandins, peritubular calcium concentration, and peritubular osmolality, have been demonstrated to modulate the actions of ADH on NaCl absorption in the TAL (see the review articles by Hebert and Molony). In isolated mouse and rabbit TAL segments, increases in peritubular osmolality, induced by adding urea or impermeant solutes such as mannitol, rapidly and reversibly inhibit the ADH-stimulated rate of net Cl ⁻ absorption. Peritubular hypertonicity results in a prompt reduction in the transepithelial voltage and in the cellular conductance. Molony and Andreoli determined that hypertonicity inhibits the basolateral membrane chloride conductance. The antagonizing effect on ADH-induced stimulation of net Cl transport is not due to decreasing cAMP, because application of either ADH or cAMP is unable to reverse the hypertonicity-mediated effect. Thus, increasing the absolute magnitude of interstitial osmolality provides a negative feedback signal that can reduce ADH-dependent salt absorption by the mTAL.

PGE ₂ also plays a role in modulating the effect of ADH on NaCl absorption in the mTAL (see Molony’s review article ). In the mouse mTAL, PGE ₂ abolished the effect of ADH on transepithelial voltage and on net NaCl absorption, while it had no effect on NaCl salt absorption in the absence of ADH. Likewise, reported biochemical studies in the mTAL indicate that PGE ₂ has no effect on cellular cAMP concentrations in the absence of ADH, and that PGE ₂ markedly inhibits the ADH-dependent stimulation of cytosolic cAMP concentrations. The TAL, particularly in the macula densa, expresses COX-2. COX-2 expression in these cells may be coupled to renin secretion. Thus, COX-2 expression is increased by salt restriction, diuretics, and in Bartter syndrome all conditions characterized by hyperreninemia. In addition to PGE ₂ , 20-HETE may also modulate the effect of cAMP on Na ⁺ transport in the TAL. McGiff and co-workers demonstrated that 20-HETE inhibits NaCl transport in the TAL by a mechanism involving inhibition of the apical K ⁺ channel and the Na ⁺ -K ⁺ -2Cl ⁻ co-transporter. Moreover, high 20-HETE concentration inhibits the effect of cAMP on the apical K ⁺ channels in the rat TAL.

Hypercalcemia often results in an ADH-resistant urinary concentrating defect, that is, nephrogenic diabetes insipidus. At least part of this concentrating defect results from the inhibition of ADH-stimulated cAMP production in the TAL by calcium. Takaichi and Kurokawa demonstrated that high ambient calcium inhibited cAMP production stimulated by forskolin, indicating that the inhibition probably involved the catalytic subunit of adenylate cyclase. Preincubation of tubule segments with pertussis toxin abolishes the effect of hypercalcemia on cAMP generation, indicating that the inhibition of cAMP generation is mediated through activation of G _i . The effects of hypercalcemia on cAMP production are mediated by a G-protein-coupled CaSR present on the basolateral membrane of TAL cells. As discussed above, stimulation of CaSR also inhibits the apical 70 pS and basolateral 50 pS K channels. Consequently, activation of CaSR is expected to alter NaCl absorption, thereby affecting the concentrating mechanism.

Intracellular Mechanisms for NKCC2 modulation in TAL

The physiological pathways for NKCC2 regulation have begun to be uncovered. The rate of salt reabsorption by TAL depends on the amount of NKCC2 protein expressed in the apical membrane at any given time; that is, the protein half-life in the plasma membrane. As shown in Figure 34.5 , the amount of NKCC2 is the result of a dynamic process involving exocytosis and endocytosis mechanisms. Stimulators of NKCC2, such as cAMP (vasopressin, PTH, glucagon or β-adrenergic stimulation) promote exocytosis over endocytosis. Electron microscopy analysis of mouse TAL and biotinylation of rat TAL apical membrane demonstrated that only between 3–5% of total NKCC2 is present in the apical membrane in basal conditions. Vasopressin stimulates trafficking of NKCC2 towards the plasma membrane in Xenopus oocytes, in mouse TAL, and in rat TAL, by a process that is modulated by the vesicle-associated membrane proteins VAMP2 and VAMP3. Exocytosis of NKCC2 is a continuous dynamic process that is further activated by cAMP, via protein kinase A (PKA). Because PKA inhibition does not completely prevent cAMP-induced increase of NKCC2 presence in the apical membrane, it is possible that cAMP also inhibits endocytosis. Two amino acid residues of NKCC2 become phosphorylated by PKA, Ser126 and Ser874, but the exact consequence of this is not known. Endocytosis of NKCC2 occurs also constitutively in TAL. A fraction recycles toward the apical membrane and another fraction undergoes degradation. Inhibition of endocytosis by cholesterol depletion completely block NKCC2 retrieval from the membrane, increasing NKCC2 presence and chloride transport by TAL. Supporting this view, it has been shown that part of NKCC2 is associated with lipid rafts.

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