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Physiology and Pathophysiology of the NaCl Co-Transporters in the Kidney
This chapter reviews the molecular physiology of the NaCl co-transporters, with emphasis on the thick ascending limb of Henle’s loop Na + -Cl − co-transporter and the distal convoluted tubule Na + -Cl − co-transporter that serve as the receptors for the loop diuretics and thiazide-type diuretics, respectively. Inactivating mutations of these co-transporters produces the hypokalemic metabolic alkalosis syndromes known as Bartter’s and Gitelman’s disease, respectively, and their altered regulation by mutant kinases is associated with a salt-sensitive form of human hypertension. Thus, these proteins are potentially involved in complex disease such as essential hypertension.
Keywords
sodium; potassium; chloride; pore; ion channel; carrier; pump; diffusion; electrodiffusion
Introduction
There are four types of electroneutral co-transporter systems that have been identified: (1) the sulfamoylbenzoic (or bumetanide)-sensitive Na + -K + -2Cl − co-transporter; (2) the sulfamoylbenzoic (or bumetanide)-sensitive Na + -Cl − co-transporter; (3) the benzothiadiazine (or thiazide)-sensitive Na + -Cl − co-transporter; and (4) the dihydroindenyloxy-alkanoic acid (DIOA)-sensitive K + -Cl − co-transporter. All these possibilities are encoded by members of the family of solute carriers SLC12, according to the classification of the Human Genome Organization. Two genes of this family, SLC12A1 encoding the apical Na + :K + :2Cl − co-transporter, NKCC2 (also known as BSC1), and SLC12A3, encoding the Na + :Cl − co-transporter, NCC (also known as TSC1), are particularly relevant to kidney physiology, pharmacology, and pathophysiology. NKCC2 and NCC play a key role in salt reabsorption of the thick ascending limb of Henle’s loop and distal convoluted tubule, respectively, with consequent effects in potassium, calcium, and acid–base homeostasis. They also serve as receptors for the loop diuretics (furosemide, bumetanide, ethacrinic acid) and thiazide-type diuretics (chlortalidone, hydrochlorothiazide, metolazone), respectively, that are heavily used in the management of patients with arterial hypertension or edematous states, such a cardiac failure, chronic liver disease, chronic renal disease or nephrotic syndrome. Inactivating mutations of NKCC2 cause Bartter syndrome type I, and of NCC cause Gitelman’s syndrome. Additionally, dysregulation of NCC is implicated in the genesis of pseudohypoaldosteronism type II, and it is proposed that both co-transporters belong to the hypertension susceptibility genes. In this chapter we will discuss the cation-coupled chloride co-transporters, with particular emphasis on NKCC2 and NCC.
The SLC12A family was identified in the early 1990s with the cloning of the thiazide-sensitive Na + -Cl − co-transporter, followed by two genes encoding the Na + -K + -2Cl − co-transporters, and is composed of nine related genes. Later, the four genes encoding K + :Cl − co-tranporters that were named KCC1, KCC2, KCC3, and KCC4 were identified.
Figure 32.1 depicts a phylogenetic tree of all members of the SLC12 family for which functional properties are known, as well as the chromosome to which each gene has been mapped in humans, the inherited disease that results from inactivating mutations of the gene, and the consequence of knocking-out each gene in the mouse. Two branches within the family are clearly identified. The Na + -driven branch encompasses three genes: SLC12A1 and SLC12A2 encode the Na + :K + :2Cl − co-transporters, NKCC2 and NKCC1, respectively. NKCC2 is a kidney-specific gene, with expression restricted to the apical membrane of the thick ascending limb of Henle’s loop (TAL). In contrast, NKCC1 is a ubiquitously expressed protein that is located at the basolateral membrane of secretory epithelial cells, as well as in many non-epithelial cells (neurons, fibroblasts, erythrocytes, etc.). SCL12A3 is the third gene of the Na + -driven branch, and encodes the thiazide-sensitive Na + :Cl − co-transporter, NCC. It is mainly expressed in the apical membrane of the distal convoluted tubule (DCT). Initially it was thought that NCC is a gene with restrictive expression in the kidney; however, later it was demonstrated to be expressed at the protein level along the gut, in bone, and in the lens. Although a thiazide-sensitive Na + -Cl − co-transporter has been postulated to exist in other tissues, blood vessels, pancreas, peripheral blood mononuclear cells, gallbladder and heart, its presence at the molecular level has not been demonstrated. In bone, NCC activity is associated with the rate of bone formation. Many clinical studies have shown that thiazide diuretics in elderly subjects promote an increase in bone mineral density and help to prevent pathological fractures. Consistent with this beneficial effect of thiazides, NCC is expressed in osteoblasts of rat and human bones, and addition of thiazides to osteoblasts in culture increases the formation of mineralized nodules. This effect of thiazides was not present after NCC expression was reduced by transfecting cells with an NCC antisense plasmid.
The degree of identity at the protein level between NKCC1 and NKCC2 is ~60%, and between NCC and the NKCCs is ~50%. The degree varies, however, within specific domains of the co-transporters. It is >80% in some of the central hydrophobic membrane spanning domains, ~50% in the carboxyl terminal domain, and <10% in the amino terminal domain and most of the interconnecting loops that are oriented toward the extracellular side.
The K + -driven branch is composed of four genes, SLC12A4 to SLC12A7, encoding the K + :Cl − co-transporters KCC1 to KCC4, respectively. KCC1 is ubiquitously expressed, while KCC2 is only present in neurons. KCC3 and KCC4 are expressed in several tissues, including the kidney. Along the nephron KCC3 has been shown to be present only in the proximal tubule, while KCC4 is also expressed in the basolateral membrane of TAL and the intercalated cells of collecting duct (CD). The degree of identity is about 60% between KCCs and about 25% with the members of the Na + -driven branch.
Two additional genes are classified within the SLC12A family (not shown in Figure 32.1 ) due to a degree of identity of ~20% with either the Na + -driven or the K + -driven branches. SLC12A8 gene (human chromosome 3) encodes a protein of 714 amino acid residues that has been identified as a psoriasis susceptibility gene by two independent groups. One report suggests that this protein may translocate polyamines and amino acids across the plasma membrane. SLC12A9 (human chromosome 7) encodes a 918 residue protein originally named as co-transporter interacting protein (CIP) for its ability to modulate the activity of NKCC1. Its topological similarity and the 25% identity with other members of the family suggest that it is likely that CIP transports substrates that have not been identified.
The Physiology of NaCl Co-Transporters in the Kidney
The electroneutral cation–chloride co-transporters translocate Cl − ions together with a cation, which can be Na + , K + or both, maintaining the requirement of electroneutrality: 1Na + -1Cl − , or 1K + -1Cl − or 1Na + -1K + -2Cl − stoichiometry. The direction of the transport process is defined by the cation gradient. Therefore, NKCC1, NKCC2, and NCC move ions inward across the plasma membrane, while KCCs move ions outward. Because Na + and K + are returned to the steady-state by the Na + :K + -ATPase, the activity of the SLC12A co-transporters is considered to be primarily regulation of the intracellular chloride concentration [Cl − ] i , a role that is critical to some physiological processes. One of these is cell volume regulation. During cell shrinking, due to increased osmolarity of the extracellular medium, the regulatory volume increase response stimulates transport mechanisms to enhance the intracellular osmolarity, including activation of NKCC1 > NKCC2 > NCC. In contrast, during cell swelling, the regulatory volume decrease promotes the activation of KCCs to reduce intracellular osmolarity. Because basically every cell expresses NKCC1 and KCC1, this cell volume regulatory mechanism is universal.
Another major function of electroneutral cation–chloride co-transporters is the setting of the intraneuronal chloride concentration, either above or below its electrochemical equilibrium potential. For this reason, the activity of these co-transporters is critical in determining the polarity and magnitude of the effect of neurotransmitters that gate Cl − channels in postsynaptic membranes, such as GABA. It is known that before birth GABA behaves mostly as an excitatory neurotransmitter, in neurons in which intracellular chloride is above the electrochemical equilibrium due to more prominent expression of NKCC1 than KCC2. After birth, however, GABA becomes predominantly an inhibitor transmitter, due to the inversion of NKCC1/KCC2 ratio of expression that lowers intracellular chloride below equilibrium.
A third major role of the SLC12A co-transporters that will occupy our interest for the rest of this chapter is the transepithelial movement of ions. NCC and NKCC2 are polarized to the apical membrane of DCT and TAL, respectively, where they participate in renal salt reabsorption. NKCC1 is expressed in the basolateral membrane of several chloride-secreting epithelia, where its activity is critical to provide the cell with chloride ions to be secreted in the apical membrane. The K + :Cl − co-transporters are also involved in epithelial movement of ions. One example is KCC4 that is expressed in the intercalated cells of the CD, where it plays a role in the chloride efflux required to maintain hydrogen secretion and thus, acid–base homeostasis.
The Thiazide-Sensitive Na − -Cl − Co-Transporter
NCC is the major salt transport pathway in the apical membrane of mammalian DCT cells which mediates reabsorption of 5–10% of glomerular filtrate. In the early DCT (DCT1) NCC is fully in charge of salt reabsorption, while in the late DCT (DCT2) it shares the responsibility with the sodium channel ENaC. This is an important difference, since DCT1 is not considered to be part of the aldosterone-sensitive distal nephron, due to the lack of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which prevents illicit occupation of the mineralocorticoid receptor by cortisol. Thus, NCC is susceptible to aldosterone regulation only in DCT2 cells. The molecular mechanism of salt reabsorption in DCT is illustrated in Figure 32.2 . The Na + gradient that drives transport from the lumen to the interstitium is generated and maintained by the intense activity of Na + /K + -ATPase that is polarized to the basolateral membrane. Potassium entering the cell through the Na + /K + pump is secreted by the luminal membrane via K + channels and by an apical K + -Cl − co-transporter. Thus, potassium secretion rate is determined, at least in part, by the rate of Na + -Cl − reabsorption. In addition, NCC modulates magnesium and calcium reabsorption, the latter in an inverse relationship with sodium reabsorption. The lower the sodium reabsorption, the higher the calcium reabsorption, and vice versa . As shown in Figure 32.2 , NCC is the target for thiazide-type diuretics (metolazone, hydrochlorothiazide, chlortalidone, bendroflumethiazide). Because thiazides are recommended for the treatment of arterial hypertension, some edematous states, and renal stone disease, this group of drugs are among the most commonly prescribed medicines in the world.
The Loop-Diuretic-Sensitive Na + -K + -2Cl − Co-Transporter 2 (NKCC2)
NKCC2 is the major salt transport pathway in the apical membrane of the mammalian TAL, a nephron segment where 15–20% of glomerular filtrate is reabsorbed. In addition to its role in salt reabsorption, NKCC2 activity also serves to keep the countercurrent multiplication mechanism by promoting salt concentration in the renal medulla and thus, the renal ability to concentrate urine. Divalent cation (Ca 2+ and Mg 2+ ) and ammonium (NH 4 + ) reabsorption in the TAL also requires the activity of NKCC2 (for reviews see ). As shown in Figure 32.2 , in the case of Ca 2+ and Mg 2+ this is due to the fact that simultaneous operation of NKCC2 with the basolateral chloride channels CLC-KB, the Na +/ K + -ATPase, and the apical inwardly rectifying K + channel known as ROMK, promotes the generation of positive voltage within the TAL lumen, which in turn drives the reabsorption of a second cation via a paracellular pathway, which could be Na + , Ca 2+ or Mg 2+ . Thus, in contrast to what occurs in DCT, in the TAL reducing salt reabsorption is associated with reducing calcium reabsorption. Because of this, loop diuretics are often used in the clinical setting for the management of life-threatening hypercalcemia. In the case of ammonium, this is due to the fact that NH 4 can use potassium transport pathways for its translocation through the plasma membrane. Thus, NKCC2 can also operate as the Na + :NH 4 + :2Cl − co-transporter.
NKCC2 is an important target in cardiovascular and renal pharmacology, because inhibition of this co-transporter is the base of the loop diuretic actions that are the most potent natriuretic agents available for clinical use (furosemide, ethacrinic acid, bumetanide). Loop diuretics decrease the salt reabsorption in TAL, producing significant natriuresis and diuresis. Because loop diuretics reduce interstitial osmolarity of the renal medulla, another mechanism of action is to reduce the concentration capacity of the kidney. Any increase in salt delivery to the macula densa is expected to be compensated by reducing the glomerular filtration rate due to the tubuloglomerular balance. This compensation is absent, however, in the presence of loop diuretics, because the salt-sensing protein in macula densa is also NKCC2. Since the B variant of NKCC2 is the most abundant variant in macula densa cells, and it is not expressed in shark kidney, which also lacks a macula densa, it has been proposed that NKCC2B is the Cl − sensor in TAL cells. It has been difficult to prove this hypothesis because the variant-specific knockout mice lacking NKCC2B developed a compensatory increase in NKCC2A expression in the TAL cells, and only exhibited a very slight shift to the right of the tubuloglomerular feedback curve. Similarly, the isoform-specific null mice for NKCC2A variant also showed slight changes in tubuloglomerular feedback. These observations suggest that both isoforms working together compose the Cl − sensor in the macula densa.
Increasing net NaCl reabsorption in TAL by hormones generating cAMP via Gs-coupled receptors such as vasopressin, glucagon, parathyroid hormone, β-adrenergic, and calcitonin is a fundamental mechanism for regulating salt transport in this nephron segment. Of these hormones, the most important is the antidiuretic hormone vasopressin, which increases NaCl absorption by TAL through a mechanism that appears to involve trafficking of NKCC2 to the apical plasma membrane. Supporting this proposal it has been observed in medullary TAL that most NKCC2 protein is located in intracellular vesicles, and that addition of cAMP increases the expression of NKCC2 in the apical membrane by activating the exocytosis, rather than by inhibiting the endocytosis of NKCC2-containing vesicles. Additionally, long-term increases in vasopressin are associated with increased expression of NKCC2 and maximal urinary concentration ability.
The Loop-Diuretic Sensitive Na + -K + -2Cl − Co-Transporter 1 (NKCC1)
NKCC1 is the Na + -K + -2Cl − co-transporter that is present in secretory epithelium, as well as in many non-epithelial cells. At the cellular physiology level, NKCC1 is very important for cell volume and [Cl − ] i regulation. The diverse phenotypes of NKCC1 knockout mice illustrate the role of this transporter in numerous physiological processes. NKCC1 knockout mice feature deafness due to both disrupted epithelial secretion in the labyrinth and a sensorineural defect, infertility due to a deficiency in spermatocyte production, cecum bleeding and blockade of the colon due to impaired intestinal secretion, salivation impairment, and low blood pressure due to vascular and renal effects. Blood pressure in NKCC1 null mice is decreased due to reduced vascular tone. In this regard, the loop diuretic bumetanide decreased blood pressure in normal mice by inhibiting the activity of NKCC1 in vascular beds, and reduced the vascular smooth muscle cells’ myogenic tone. Both effects are not present in the NKCC1 null mice, strongly suggesting that are the results of inhibiting NKCC1 in blood vessels. Of note, however, one study using telemetry to monitor blood pressure day and night for several days failed to confirm hypotension in the NKCC1 knockout mice, and suggested a salt-sensitive component because a significant increase in blood pressure was produced by a high-salt diet. Finally, NKCC1 is expressed in the basolateral membrane of the macula densa cells, where it has been suggested that it modulates renin secretion.
Molecular Biology of the Sodium-Dependent Chloride Co-Transporters
The Thiazide-Sensitive Na + -Cl − Co-Transporter
Following an expression cloning strategy using the functional expression system of Xenopus laevis oocytes, NCC cDNA was first identified at the molecular level from the winter flounder ( Pseudopleuronectes americanus ) urinary bladder. This clone was named as TSC (for thiazide-sensitive co-transporter), and later changed to NCC. The flounder’s transcript produced a 3.7 kb cDNA clone containing a 3609 bp open reading frame that predicted a 1023 amino acid residues protein with a core molecular mass of 112 kDa. The computer-based analysis hydrophobicity/hydrophylicity suggested the putative basic topology of the Na + -coupled-Cl − co-transporters shown in Figure 32.3a . The short amino terminal domain is followed by a central hydrophobic domain containing what appear to be 12 α-helices compatible with transmembrane-spanning segments. A long carboxyl terminal domain follows this. The amino and carboxyl terminal domains are predicted to be located within the cell. The long loop between transmembrane segments 7 and 8 is glycosylated in NCC and NKCC2, and thus presumably in all members of the SLC12A family. In flounder, a shorter 3.0 kb transcript due to alternative splicing is expressed in several tissues including the brain, eye, heart, intestine, gonads, and skeletal muscle. The functional consequence of this variant remains elusive. After the cloning of NCC from the flounder urinary bladder, cDNAs encoding NCC from a variety of mammalian sources were isolated, including rat ( Rattus norvegicus ), mouse ( Mus musculus ), rabbit ( Oryctolagus cuniculus ), and human ( Homo sapiens ). Additionally, the NCC cDNA sequence has been deposited in gene databases for at least another 10 species, mostly mammalians and one birth. The degree of identity between mammalian NCCs is ~90%, and of any mammalian with flounder is ~60%. Molecular identification of putative NCC sequences in eel suggests the existence of two different NCC genes. NCCα is expressed only in eel kidney, while NCCβ was observed in many tissues, but more abundantly in intestine. The amino acid residues of NCCα and NCCβ are 1027 and 1043, respectively. No functional expression was analyzed, but the extend of identity of NCCα or NCCβ with any NCC, NKCC1 or NKCC2 supports the proposition that these eel sequences probably correspond to NCC, since degree of identity of NCCα or NCCβ with any NCC sequence is higher than with any NKCC1 or NKCC2. In mammals, rabbit and human NCC is longer than other species due to the presence of 17–26 amino acid residues in the carboxyl terminal domain. These extra residues were shown to be encoded in humans by a separate exon (exon 20) which is not present in rodents. It is noteworthy that in humans, there is a putative protein kinase A (PKA) site (RPS) within the extra fragment. No functional significance for this extra exon in humans has been reported, but an extensive proteomic analysis of human urinary exosomes revealed, among may proteins found, the presence of several fragments of NCC, some of which have the serine 811 phosphorylated, that correspond to the putative protein kinase A site RPS contained by the human exon 20. The expression of NCC protein has been confirmed using specific antibodies in intestine, bone cells, and lens. Additionally, in silico analysis of NCC expression revealed that NCC transcripts are abundantly present in sensory ganglia, such as trigeminal and dorsal root ganglion. No report has confirmed the presence of NCC protein in this tissue, and its functional significance is unknown.
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