INVITED REVIEW
Chloride channels in the kidney: lessons learned from knockout animals

Olivier Devuyst1 and William B. Guggino2

1 Division of Nephrology, Université Catholique de Louvain Medical School, B-1200 Brussels, Belgium; and 2 Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
THE CLC FAMILY OF...
CFTR
CONCLUSIONS AND PERSPECTIVES
REFERENCES

Cl- channels are involved in a range of functions, including regulation of cell volume and/or intracellular pH, acidification of intracellular vesicles, and vectorial transport of NaCl across many epithelia. Numerous Cl- channels have been identified in the kidney, based on single-channel properties such as conductance, anion selectivity, gating, and response to inhibitors. The molecular counterpart of many of these Cl- channels is still not known. This review will focus on gene-targeted mouse models disrupting two structural classes of Cl- channels that are relevant for the kidney: the CLC family of voltage-gated Cl- channels and the CFTR. Disruption of several members of the CLC family in the mouse provided useful models for various inherited diseases of the kidney, including Dent's disease and diabetes insipidus. Mice with disrupted CFTR are valuable models for cystic fibrosis (CF), the most common autosomal recessive, lethal disease in Caucasians. Although CFTR is expressed in various nephron segments, there is no overt renal phenotype in CF. Analysis of CF mice has been useful to identify the role and potential interactions of CFTR in the kidney. Furthermore, observations made in CF mice are potentially relevant to all other models of Cl- channel knockouts because they emphasize the importance of alternative Cl- pathways in such models.

Bartter's syndrome; bicarbonate transport; chloride channel; cystic fibrosis transmembrane conductance regulator; Dent's disease; endocytosis; nephrogenic diabetes insipidus; vesicular acidification


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
THE CLC FAMILY OF...
CFTR
CONCLUSIONS AND PERSPECTIVES
REFERENCES

IN PLANTS AND ANIMAL TISSUES, Cl- is the most abundant anion and Cl- transporters are involved in a range of physiological processes, including regulation of cell volume, regulation of intracellular pH, acidification of intracellular vesicles, and transepithelial transport (79). Cl- channels are involved in the vectorial transport of NaCl and fluid across many epithelia. In the kidney, most of the Cl- filtered is reabsorbed, and this process involves different mechanisms operating in the apical and basolateral membranes of tubular epithelial cells. Several Cl- transporters use the energy stored in transmembrane gradients of other ions to move Cl- across the apical plasma membrane often against its electrochemical gradient. Cl- channels located in the basolateral membrane mediate net Cl- efflux from the cell and participate in NaCl reabsorption by the nephron (19). In addition to the specialized, NaCl-reabsorbing function, passive Cl- diffusion through Cl- channels is involved in cell volume regulation, for which the parallel movement of Cl- and K+ through swelling-activated channels results in a reduction of intracellular osmolytes when cells are exposed to external hypotonicity (96). Transport of Cl- through Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers operating in intercalated cells (5), or through the Na+-linked Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger operating in proximal tubule (PT) cells (140), is involved in intracellular pH regulation. Chloride ions are also important for acidification of intracellular vesicles, because the influx of negative charges partially neutralizes the transmembrane potential generated by the electrogenic proton ATPase (H+-ATPase). The existence of a Cl- conductance in endosomal vesicles is indeed necessary both for endosomal acidification (113) and normal progression and recycling along the endocytic apparatus (182).

Numerous Cl- channels have been identified in all segments of the nephron, based on single-channel properties such as conductance, anion selectivity, gating, and response to inhibitors (19). The molecular counterpart of many Cl- channels in the kidney is still not known, and this review will focus on two structural classes of Cl- channels that have been cloned and sequenced: the CLC channels family and the CFTR. Mutations of two members of the CLC Cl- channels family (ClC-5 and ClC-Kb) in humans lead to two distinct inherited diseases of the kidney: Dent's disease (102), and Bartter's syndrome (155), respectively. These two human diseases are completed by the diabetes insipidus symptoms in the ClC-K1 knockout mouse (111). Loss-of-function mutations affecting CFTR are responsible for cystic fibrosis (CF), the most common autosomal recessive, lethal disease in Caucasians (186). We will detail how phenotypical analyses in mouse models and human diseases have provided new insights into the diverse roles played by Cl- channels in the kidney (Table 1).

                              
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Table 1.   Gene-targeted mouse models of chloride channel disruption investigated at the renal level


    THE CLC FAMILY OF VOLTAGE-GATED CLminus CHANNELS
TOP
ABSTRACT
INTRODUCTION
THE CLC FAMILY OF...
CFTR
CONCLUSIONS AND PERSPECTIVES
REFERENCES

Several members of the CLC family of voltage-gated Cl- channels have been identified in the mammalian kidney (for review, see Refs. 79 and 183). The first member of this family, ClC-0, was isolated by Jentsch and colleagues (80) from the electric organ of the Torpedo. To date, at least nine different CLC genes, highly conserved during evolution, have been identified in mammals (79). Biochemical studies performed chiefly on ClC-0 suggested that CLC channels have a double-barelled, homodimeric configuration, with each CLC subunit containing its own pore (105). An alternative model for ClC-1 was proposed, based on a single pore composed of two subunits (49). Different numbers of transmembrane domains of CLC channels have also been suggested (50, 144). The two issues have been clarified by the recent crystallization of two bacterial CLC proteins at 3.0-Å resolution (46). The CLC channels are homodimers with a double-barreled configuration. Each subunit has its own pore and contains 18 alpha -helices, 17 of which are inserted into the membrane. Each subunit is composed of two halves that are structurally related but have opposite orientations within the membrane. This antiparallel orientation brings together residues that will form the Cl- ion selectivity filter (46).

With the exception of ClC-1, which is predominantly expressed in skeletal muscle, all other CLC channels have been detected in the kidney. Three members of the CLC family (ClC-K1, ClC-Kb, and ClC-5) were found to be involved in kidney disease in either humans or mice. The broadly expressed ClC-2 channel was postulated to have a role in early nephrogenesis (73), but its disruption in mice did not confirm this hypothesis (21).

ClC-K1 and Nephrogenic Diabetes Insipidus

ClC-K1 and its human homologue ClC-Ka. The ability to concentrate urine is an essential function of the mammalian kidney, which is achieved by water reabsorption in the collecting duct (CD) according to the osmolar gradient across the tubule. In response to increased plasma osmolality, the neurohypophysal antidiuretic hormone arginine vasopressin (AVP) is released and binds to its specific V2 receptors (V2R) located in the principal cells of the CD. The binding of AVP to V2R activates the stimulatory GTP-binding protein Gs, which in turn stimulates adenylate cyclase. The subsequent increase in the cytosolic levels of cAMP leads to protein kinase A activation, phosphorylation of the water channel aquaporin-2 (AQP2), and, eventually, its insertion into the apical membrane (90). On the other hand, urine concentration necessitates the generation of an osmotic gradient extending from the corticomedullary junction to the inner medulla. In the outer medulla, the gradient is generated by the countercurrent multiplication of the transepithelial reabsorption of NaCl in the thick ascending limb (TAL) of the loop of Henle (56). Both NaCl reabsorption and urea recycling play a crucial role in maintaining high interstitial osmolality in the inner medulla (110). Any disruption of the complex mechanism of urinary concentrating ability will potentially lead to diabetes insipidus, a clinical condition qualified as "central," if the urinary concentrating ability is corrected by the exogenous administration of AVP or an analog, or "nephrogenic," if the kidney remains unresponsive to AVP (118).

The inner medulla is characterized by a variable density of long-looped nephrons containing the thin ascending limb (tAL) of Henle's loop. The tAL has long been considered to play an important role by diluting urine and maintaining the hypertonicity of the interstitium through reabsorption of NaCl. In vitro perfusion studies showed that the tAL has the highest Cl- permeability of the nephron segments (77). Following the descending thin limb, where urine has been concentrated through aquaporin-1 (AQP1)-mediated water permeability, the tAL constitutes the first diluting segment of the Henle's loop, and this is achieved through the passive reabsorption of NaCl (78). Insights into the molecular basis of NaCl reabsorption in the tAL were provided by Kondo et al. (93) and Yoshitomi et al. (193), whose work showed evidence for the presence of stilbene-sensitive Cl- channels in both luminal and basolateral membranes of the tAL. Using a PCR cloning strategy, Uchida and colleagues (173) isolated ClC-K1, a member of the CLC family that is almost exclusively expressed in the kidney. The homologue in humans was named ClC-Ka (88). The rat ClC-K1 mRNA is mainly expressed in the tAL, and its expression is significantly increased by dehydration (173). These data were confirmed at the protein level, with the ultrastructural localization of ClC-K1 in both the apical and basolateral membranes of tAL epithelial cells (175, 176). When ClC-K1 is expressed in Xenopus laevis oocytes, Cl- currents are induced and regulated by extracellular pH and Ca2+ concentration. In contrast to other functionally analyzed CLC channels, the gating of ClC-K1 is not voltage dependent. These currents are inhibited by furosemide [inhibition constant (Ki) 100 µM] and stimulated by N-ethyl-maleimide (175). Because rapid NaCl exit from the tAL was thought to be a component of the countercurrent multiplication system in the inner medulla (78, 92), it was suggested that ClC-K1 plays a role in urinary concentration (175).

Nephrogenic diabetes insipidus in the Clcnk1 knockout mouse. The hypothesis that ClC-K1 is a major Cl- channel mediating transepithelial Cl- transport in tAL and thereby participating in urinary concentration was confirmed by a mouse model in which ClC-K1 had been deleted by homologous recombination (111) (Fig. 1). There were no apparent differences among Clcnk1-/-, Clcnk1+/-, and Clcnk1+/+ mice in survival, gross physical appearance, and organ morphology. The plasma values of creatinine, Na+, K+, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were similar among the three groups. However, compared with Clcnk1+/+ and Clcnk1+/- mice, Clcnk1-/- mice showed a fivefold increase in urine volume, coupled to a significant decrease in urine osmolality. Water deprivation induced a 27% weight loss in Clcnk1-/- mice compared with 12% weight loss in Clcnk1+/+ and Clcnk1+/- mice. These manifestations were associated with a minimal increase in urine osmolality after administration of the V2R agonist dDAVP, confirming the clinical picture of nephrogenic diabetes insipidus. In vitro microperfusion studies showed that establishment of a lumen-to-bath Cl- gradient in the tAL did not yield a Cl- conductance in Clcnk1-/- mice, in contrast to that observed in Clcnk1+/+ and Clcnk1+/- mice (111).


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Fig. 1.   Intrarenal distribution and physiological roles of the chloride channels ClC-5, ClC-K1, and ClC-Kb/K2. In proximal tubule (PT) cells, ClC-5 colocalizes with H+-ATPase in subapical endosomes, where it may provide the Cl- conductance necessary for endosomal acidification, a prerequisite for endocytosis. In cells lining the thin ascending limb (tAL) of Henle's loop, apical and basolateral ClC-K1 channels mediate transcellular Cl- permeability. In the cells of the thick ascending limb (TAL), the apical NKCC2 cotransporter, which drives Na+, K+, and Cl- uptake, requires the K+ channel ROMK to recycle K+ across the apical membrane, whereas Cl- exits the cell through channels formed by ClC-Kb (alpha -subunit) and barttin (beta -subunit). ClC-5 also colocalizes with H+-ATPase in the alpha -type, acid-secreting intercalated cells (alpha IC) of the collecting duct (CD), in which its role remains to be defined. G, glomerulus; the filled oval symbols (junctional complexes) delineate apical and basolateral plasma membrane domains.

A series of elegant studies in the Clcnk1 mouse gave further insights into the countercurrent multiplication process of Henle's loop. Akizuki et al. (3) showed that the creatinine clearance, the ratio of osmolar clearance to creatinine clearance, as well as the fractional excretion of Na+, Cl-, and urea were not significantly different between Clcnk1-/- and Clcnk1+/+ mice, clearly demonstrating that polyuria in Clcnk1-/- mice is not osmotic diuresis but water diuresis. The Clcnk1-/- mice were characterized by increased urinary AVP excretion, in both control and dehydrated conditions, thus demonstrating the reactivity of the hypothalamus to high plasma osmolality. In these conditions, the water diuresis observed in Clcnk1-/- mice could be due to either a loss of water permeability in the CD or impairment in the generation of hypertonicity in the inner medulla. The first possibility was ruled out by showing increased expression of AQP2 by Northern analysis in Clcnk1-/- mice, with a clear immunolocalization of AQP2 in the apical area of CD principal cells. Because the transcription of AQP2 is regulated by the V2R and a cAMP-responsive element in its promoter (43, 174), increased expression of AQP2 was interpreted as the result of increased secretion of AVP in Clcnk1-/- mice. The second hypothesis was substantiated by demonstrating that papillary osmolality was significantly lower in Clcnk1-/- mice in both control and dehydrated conditions, an effect that was attributed to a significant decrease in the accumulation of Na+, Cl-, and urea in the inner medulla (3). Furthermore, Liu et al. (100) characterized the nature of NaCl transport in the tAL using in vitro microperfusion. Neither Cl- transport inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) nor acidification of the bathing fluid, which typically inhibit the major Cl- transport pathway in tAL of Clcnk1+/+ mice, changed the transepithelial diffusion voltage (Vd) values in Clcnk1-/- mice. In contrast, both Clcnk1+/+ and Clcnk1-/- mice showed the same protamine-sensitive Na+ conductance of the paracellular shunt pathway.

These data obtained in Clcnk1 mice provided the first demonstration that a molecular deletion in the tAL affects the countercurrent mechanism in vivo. They also showed that generation of hypertonicity in the inner medulla probably results from multiple interactions between NaCl and urea permeability in the tAL (3). Finally, these studies indicated that ClC-K1 mediates Cl- transport sensitive to NPPB or pH in the tAL and that the paracellular shunt pathway of this segment has a selective Na+ permeability (100).

ClC-K2/b and Bartter's Syndrome

ClC-K2 and its human homologue ClC-Kb. ClC-K2 was isolated from rat kidney by using a PCR-based cloning strategy (2). This Cl- channel shares an ~80% amino acid identity with ClC-K1 and is also exclusively expressed in the kidney (2). The human isoform ClC-Kb (which is 95% identical to ClC-Ka) has also been identified in the kidney (88). One homologue has been identified in the rabbit (rbClC-Ka) (196). The intrarenal distribution of ClC-K2 is clearly distinct from that of ClC-K1. In situ hybridization with specific riboprobes directed against the nonhomologous 3'-untranslated regions of ClC-K1 and ClC-K2 demonstrated the absence of colocalization between the two transcripts. It also showed abundant expression of ClC-K2 in the distal convoluted tubule (DCT), connecting tubule (CT), and cortical CD and moderate expression in the medullary TAL (192). The homology between ClC-K1 and ClC-K2 hampered the generation of specific antibodies against ClC-K2 (172, 183), but the difficulty has been overcome by staining Clcnk1-/- kidneys with an anti-ClC-K antibody that recognizes both ClC-K1 and ClC-K2 (91). This study confirmed that ClC-K2 is broadly expressed in the distal nephron, including TAL, DCT, CT, and intercalated cells of the CD. In all these locations, ClC-K2 is located in the basolateral membrane (91).

Bartter's syndrome and mutations in ClC-Kb. Although it was initially not possible to functionally express ClC-Kb/K2 in X. laevis oocytes (88), its high degree of sequence homology with other members of the CLC family predicted that it likely plays a role in transepithelial transport of Cl- in the distal nephron. Even before the generation of a ClC-Kb/K2 knockout mouse, Simon et al. (155) verified that hypothesis by reporting mutations of the CLCNKB gene in patients with Bartter's syndrome. Bartter's syndrome is an autosomal recessive renal tubular disorder characterized by salt wasting, hypokalemic alkalosis, and hypercalciuria (142). Because the syndrome is similar to the action of the loop diuretic furosemide, it was suggested that Bartter's syndrome results from impaired NaCl reabsorption in the distal nephron/diluting segment of the loop of Henle. Simon et al. (155-157) actually proved that assertion by documenting three types of Bartter's syndromes: Bartter type I (due to mutations in the apical Na+-K+-2Cl- cotransporter NKCC2); Bartter type II (due to mutations in the apical K+ channel ROMK); or Bartter type III (due to mutations in the basolateral Cl- channel ClC-Kb). A fourth variant, that associates Bartter's syndrome with sensorineural deafness, has recently been linked to mutations in BSND, a gene that encodes barttin, an integral membrane protein that acts as an essential beta -subunit for ClC-Ka and ClC-Kb Cl- channels (18, 48). This remarkable collection of genetic and functional evidence puts ClC-Kb and its crucial beta -subunit barttin into a functional relationship with the apical Na+-K+-2Cl- cotransporter and the ROMK K+ channel, suggesting a mechanism byn which Cl- is taken up apically by the cotransporter and exits basolaterally via ClC-Kb (Fig. 1).

There is no knockout mouse model for Bartter's syndrome type III, corresponding to a disruption of ClC-K2. A mouse model of Bartter's syndrome type I was provided by inactivating the Slc12a1 gene that codes for the apical NKCC2 of the TAL in the mouse (166). The Slc12a1-/- pups died within 2 wk, characterized by low weight, hydronephrosis, dehydration, renal failure, and impaired ability to concentrate urine. Treatment of Slc12a1-/- pups with indomethacin enabled some of them to survive into adulthood. The Slc12a1-/- adult mice presented nephrogenic diabetes insipidus, characterized by polyuria and failure to concentrate urine after administration of a vasopressin analog. In contrast to Clcnk1 knockout mice, in which the primary deficit is located in the tAL, the nephrogenic diabetes insipidus in Slc12a1-/- mice is explained by the lack of apical NKCC2 in the TAL, resulting in the abolition of transcellular and paracellular NaCl reabsorption, which eventually alters the generation of the osmotic gradient necessary for urine concentration (166).

ClC-5 and Dent's Disease

Identification and characterization of ClC-5. The CLCN5 gene that codes for the ClC-5 Cl- channel was identified by positional cloning in families with Dent's disease, an X-linked renal tubular disorder that includes low-molecular-weight proteinuria, generalized PT dysfunction (renal Fanconi syndrome), hypercalciuria, nephrocalcinosis, kidney stones, and renal failure (51). ClC-5 encodes a 746-amino acid protein and shares ~80% homology with ClC-3 (83) and ClC-4 (181). These three channels form a branch of the CLC family with only ~30% amino acid identity with other branches of the family (79). When ClC-5 is expressed in X. laevis oocytes (52, 102, 160) or in Chinese hamster ovary cells (52), it induces strong outwardly rectifying Cl- currents that are inhibited by acidic extracellular pH. The Cl- currents elicited by injection of ClC-5 in X. laevis oocytes require voltages of +20 mV, a condition rarely reached in most cells, except for the urinary bladder of the amphibian Necturus (67). Because some CLC channels require beta -subunits to form heteromers (48), it is possible that acting in combination with beta -subunits could modify this voltage dependence.

In rats, ClC-5 is abundantly expressed in the kidney, and much lower expression is observed in brain, liver, lung, and testis (160). In the kidney, ClC-5 is located in the PT, where it colocalizes with the vacuolar H+-ATPase in subapical endosomes, and in the alpha -type intercalated cells of the cortical CD, where it again colocalizes with apical H+-ATPase (42, 62, 107, 138) (Fig. 1). A diffuse, intracellular pattern of ClC-5 expression has also been reported in the medullary TAL of Henle's loop, at least in rat and human kidney (42, 107). Subcellular fractionation analyses have shown that ClC-5 colocalizes with H+-ATPase and the small GTPases rab4 and rab5a in endosomes (42, 177). The intracellular distribution of ClC-5 and its colocalization with endocytosed proteins have been confirmed in opposum kidney cells that endogenously express ClC-5 (42), as well as in ClC-5-transfected MDCK and COS-7 cells (62). Schwake et al. (145) have identified a proline-rich motif in the COOH terminus of ClC-5 resembling the PY motif, which is critical for the internalization of the endothelial sodium channel ENaC by clathrin-mediated endocytosis (153), and shown that this motif is important for the endocytosis of ClC-5 itself.

Mutations of CLCN5 and Dent's disease. Mutations of CLCN5 were found in Dent's disease and three other phenotypic variants referred to as X-linked recessive nephrolithasis, X-linked recessive hypophosphataemic rickets, and the idiopathic low-molecular-weight proteinuria of Japanese children (70, 102, 103). Mutations of ClC-5 that are associated with Dent's disease abolish or markedly reduce the Cl- currents elicited in heterologous expression systems (75, 101-103). Low-molecular-weight proteinuria represents the most consistent manifestation of Dent's disease and is almost always detected in female carriers (103, 135). In contrast, there is considerable interfamilial and intrafamilial variability in other manifestations of the disease, including hypercalciuria, PT solute wasting, hypercalciuria, distal tubule disorders, and rickets (141, 188, 194). There are no clear correlations between the clinical phenotype of Dent's disease and the >35 mutations of CLCN5 identified thus far (141, 194).

ClC-5 and endocytosis in the PT. The expression of ClC-5 in PT cells and its colocalization with H+- ATPase to subapical endosomes suggested that ClC-5 may have a role in the counterion transport mechanism that facilitates endosomal acidification (42, 62, 107), despite the fact that its biophysical properties (outward rectification and inhibition by acidic pH) are apparently not ideal for such a role (52, 102). The endosomes form part of the megalin receptor-mediated endocytic pathway that reabsorbs proteins such as albumin and low-molecular-mass (<70 kDa) proteins [e.g., alpha 1-microglobulin, beta 2-microglobulin, vitamin D binding protein (DBP), Clara cell protein (CC16)] that are freely filtered and almost totally reabsorbed by PT cells (29). It is well established that a Cl- conductance is necessary for acidification of PT endosomes (9, 134) (Fig. 2). Luminal acidification of the endosomes is required for the distribution and degradation of internalized ligands in the endocytic pathway (182) and for the progression of early endosomes to late endosomes/lysosomes (7). Endosomal acidification is generated primarily by the vacuolar H+-ATPase, with acidification increasing progressively from endocytic vesicles and early endosomes to late endosomes and, ultimately, lysosomes. The varying degree of endosomal acidification depends on whether the electrogenic H+-ATPase is antagonized by other transporters (e.g., by the electrogenic Na+ pump) or facilitated by a coupled Cl- conductance (113, 152). Although the exact nature of the Cl- channels operating in endosomes remains undefined (22, 143), it has been hypothesized that an impaired acidification of the endosomal apparatus due to loss of function of ClC-5 could affect endocytosis, thus causing the systematic occurrence of low-molecular-weight proteinuria in Dent's disease (42, 62, 102). However, it is still unknown whether luminal acidification is required for the formation of primary endocytic vesicles or whether poor entry in the endocytic apparatus is a secondary defect, e.g., due to inefficient recycling of a rate-limiting partner of the endocytic machinery.


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Fig. 2.   Acidification of the endocytic pathway: role of chloride conductance. The endocytic pathway in proximal tubule cells involves coated pits (CP) and coated vesicles (CV), followed by early endosomes (E1) that form recycling vesicles (RV) or mature to late endosomes (E2) and lysosomes (L). There is a progressive, ATP-dependent acidification of the endosomes (pH 5.0-6.0) and lysosomes (pH 4.6-5.0). This acidification is mediated by the vacuolar H+-ATPase, which generates both a pH gradient and a membrane potential (Delta Psi ). The presence of a Cl- conductance in the endosomes, such as mediated by ClC-5, dissipates the positive charge gradient and allows organelle acidification. In contrast, the absence of Cl- conductance, such as probably encountered in Dent's disease, is reflected by an inside positive Delta Psi , which inhibits H+ entry and impairs endosomal acidification (inset). Modified from Refs. 22 and 113.

ClC-5 in distal nephron segments. In addition to PT cells, ClC-5 is also expressed in the intercalated cells of the CD (42, 62, 122, 138) and, at least in human and rat kidney, in the TAL (42, 107). In the acid-secreting alpha -type intercalated cells, ClC-5 colocalizes with H+-ATPase in intracellular vesicles and apical plasma membrane (42, 62, 138), and it could play a role in distal urinary acidification (Fig. 1). However, this question remains debatable, because urinary acidification is normal in the majority of patients with Dent's disease (141, 188). The TAL is an important site for regulated Ca2+ reabsorption (142), and Silva et al. (154) recently showed that ClC-5 mRNA expression in the kidney is regulated by parathyroid hormone (PTH). Thus ClC-5 may play a role in Ca2+ homeostasis, although it remains unclear how an apparently intracellular Cl- channel may participate in the positive luminal potential that drives paracellular Ca2+ reabsorption in that nephron segment (142). Furthermore, at variance with Bartter's syndrome (cf. Bartter's syndrome and mutations in ClC-Kb), Dent's disease is not consistently associated with hypercalciuria and is usually not associated with clinical dehydration (141, 188).

Mouse models of ClC-5 disruption. The potential roles of ClC-5 in endocytosis and Ca2+ homeostasis have been substantiated by three distinct mouse models of disrupted ClC-5 expression (Table 1). Luyckx et al. (108) reported the first mouse model of reduced ClC-5 expression by a ribozyme approach, resulting in ~80% reduction in renal ClC-5 protein expression. The transgenic ribozyme-expressing mice showed no obvious phenotype except for a borderline hypercalciuria (that was found only in males), without PT dysfunction and low-molecular-weight proteinuria (108, 194). Of note, the hypercalciuria was prevented by a low-Ca2+ diet, suggesting a role for increased intestinal Ca2+ absorption (108).

Two groups have independently generated and characterized knockout mouse models for Dent's disease by targeted disruption of part of the exon 5 and/or exon 6 of Clcn5 (129, 184). The loss of ClC-5 mRNA and protein in the kidney of both Clcn5-/Y mice was demonstrated by Northern blotting and immunoblotting with NH2-terminal antibodies (129, 184). The Clcn5-/Y mice reported by Wang et al. (184) were found to have remarkable similarities to Dent's disease, including low-molecular-weight proteinuria, generalized aminoaciduria, glycosuria, phosphaturia, and hypercalciuria. Renal failure, which is generally only observed in adults with Dent's disease (188), was not observed in the Clcn5-/Y mice, and this may partly be due to the young age (5-9 wk) of the mice at the time of the initial assessment. In contrast, some young Clcn5-/Y mice already showed renal calcifications (184) and subsequently developed nephrocalcinosis and parenchymal lesions including mild fibrosis and renal cysts (Devuyst O, Guggino S, and Guggino WB, unpublished observations). The Clcn5-/Y mouse reported by Piwon et al. (129) was also characterized by low-molecular-weight proteinuria and generalized PT dysfunction, but it lacked hypercalciuria. As discussed elsewhere, the heterogeneity in terms of urinary Ca2+ excretion may reflect genetic background, differences in residual ClC-5 expression, dietary phosphorus intake, or other environmental conditions (26, 194).

The two knockout mouse models provided new insights into the role of ClC-5 in the PT and the pathophysiology of PT dysfunction in Dent's disease. Both Clcn5-/Y mice showed low-molecular-weight proteinuria in parallel with defective apical endocytosis, suggesting that ClC-5 is a key mediator of the Cl- conductance that is necessary for early endosomal acidification and receptor-mediated reabsorption in PT cells (129, 184) (Fig. 2). Any failure in this process would not only lead to low-molecular-weight proteinuria but also a loss of amino acids and glucose, presumably by a failure to recycle their specific transporters to the apical cell membrane (89). Recent studies showed that other members of the CLC family of Cl- channels fulfill similar functions in other cells and organelles. Kornak et al. (94) have generated a Clcn7 knockout mouse, demonstrating that ClC-7 provides the Cl- conductance required for acidifying the extracellular resorption lacuna in osteoclasts. Mutations in CLCN7 have been identified in patients with familial (autosomal dominant or recessive) osteopetrosis, a condition characterized by impaired bone and cartilage resorption (33, 94). In another example, the broadly expressed ClC-3 channel is involved in granular acidification in the insulin-secreting pancreatic beta  cell (13), and its disruption in mice impairs acidification of synaptic vesicles and induces severe postnatal degeneration of the retina and hippocampus (162).

The phenotypical analysis of ClC-5 knockout mice also provided interesting hypotheses to explain the phosphaturia and hypercalciuria inconsistently observed in Dent's disease. Piwon et al. (129) showed that mice lacking ClC-5 had reduced renal expression of megalin, together with a slight, 1.7-fold increase in urinary PTH. Because megalin mediates the endocytosis and degradation of PTH in PT cells (68), they hypothesized that the reduced endocytosis of filtered PTH in Clcn5-/Y mice may lead to the subsequent stimulation of functional apical PTH receptors in PT cells (170). The PTH-mediated regulation of renal phosphate reabsorption involves internalization and subsequent lysosomal degradation of the type II Na+-phosphate cotransporter (NaPi-2) (169), which mediates most of the phosphate transport in the PT (14). Accordingly, Piwon et al. (129) showed reduced expression and enhanced internalization of NaPi-2 in the PT of Clcn5-/Y mice, which could explain the increased phosphaturia in these mice. The apical stimulation of PTH receptors may also promote the internalization of the Na+-H+ exchanger NHE3 in PT cells, which may contribute to the reduced NaCl reabsorption in Clcn5-/Y mice (129).

The 1alpha -hydroxylase enzyme expressed in PT cells converts the 25-hydroxyvitamin D substrate in its active metabolite, 1,25-dihydroxyvitamin D. A marked increase in the urinary excretion of the 50-kDa DBP, suggesting a lower delivery of 25-hydroxyvitamin D substrate to the 1alpha -hydroxylase, was observed in the Clcn5-/Y mice (129, 184). It is thus conceivable that endocytic defect observed in Clcn5-/Y mice is reflected in terms of vitamin D and Ca2+ homeostasis. Piwon et al. (129) observed lower levels of 1,25-dihydroxyvitamin D, partially compensated for by slightly increased PTH levels, in their Clcn5-/Y mice. In contrast, increased levels of 1,25-dihydroxyvitamin D were found in most patients with Dent's disease (135, 188) and also in the Clcn5-/Y mouse as reported by Wang et al. (184; Guggino S, unpublished observations). The complexity of hormonal regulation and environmental factors may explain differences in vitamin D metabolism and urinary Ca2+ excretion between the two strains of Clcn5-/Y mice. Further studies will also be necessary to distinguish between the potential contributions of intestinal absorption and bone resorption in maintaining a normal plasma Ca2+ despite hypercalciuria in patients with Dent's disease (188) and Clcn5-/Y mice (184). A certain degree of Ca2+ reabsorption in the PT occurring via the megalin receptor-mediated endocytosis (30) may account for part of the observed hypercalciuria in patients with Dent's disease and Clcn5-/Y mice (184). However, it is important to note that regulated Ca2+ reabsorption occurs in the TAL and DCT, segments in which the role of ClC-5 remains to be substantiated. Alternatively, the expression of ClC-5 in enterocytes, where it colocalizes with H+-ATPase in endosomes (177), may also suggest a primary defect in intestinal regulation by vesicular recycling of the epithelial Ca2+ channel ECaC (69).

ClC-2 and the Kidney

ClC-2 was cloned by homology to ClC-1 (168). The ClC-2 Cl- channel can be activated by hyperpolarization, acidic extracellular pH, and osmotic cell swelling (79). Northern analysis demonstrated that ClC-2 is ubiquitously expressed, with relatively high levels detected in the brain, intestine, and kidney (168). It has been suggested that ClC-2 is involved in lung development (120) and may provide an alternative pathway for Cl- secretion in CF airway epithelial cells (147; see Mouse Models of CF and the Concept of Alternative Pathways for Cl- Conduction). The identification of alternatively spliced ClC-2 isoforms together with Cl- currents slowly activated by hyperpolarization in embryonic epithelia at the tip of the ureteric bud suggested that ClC-2 exerts a role in early nephrogenesis (73). Further studies in rat kidney suggested that ClC-2 expression may be modulated both by AVP (117) and aldosterone (124). The above hypotheses were not supported by the disruption of ClC-2 in mice (21). Indeed, the Clcn2-/- mice did not show abnormalities in lung or kidney development but revealed a severe degeneration of the testes and the photoreceptors of the retina. This unexpected phenotype could be explained by the role played by ClC-2 in the interaction (e.g., pH regulation) between the vulnerable germ cells and photoreceptors and their supporting epithelial cells that form the blood-testis and blood-retina barriers, respectively (21).


    CFTR
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ABSTRACT
INTRODUCTION
THE CLC FAMILY OF...
CFTR
CONCLUSIONS AND PERSPECTIVES
REFERENCES

CFTR and CF

The CFTR protein contains 1,480 amino acids and is a member of the ATP-binding cassette (ABC) transporters family. CFTR is composed of two transmembrane domains (TMD1 and TMD2) and two nucleotide-binding domains (NBD1 and NBD2), separated by a large, polar, regulatory (R) domain containing multiple phosphorylation sites (for a review, see Ref. 151). Mutations in the CF gene that encodes CFTR are responsible for CF, the most common autosomal recessive disease in Caucasians (37, 136, 186). CFTR is located primarily in the apical membrane of epithelial cells, where it provides a pathway for transepithelial Cl- movement and regulates other types of channels, such as the outwardly rectifying Cl- channels (ORCC) (148), the Na+ channel ENaC (164), and the inwardly rectifying, ATP-sensitive renal K+ channel ROMK2 (112). Thus CFTR is central in determining transepithelial ion transport and fluid movement across epithelia. In the intestine, pancreas, and sweat gland secretory coil, CFTR plays a key role in fluid and electrolyte secretion, and in sweat gland duct and airway epithelia it participates in fluid and electrolyte absorption (37, 186). Failure of protein kinase A-mediated phosphorylation and activation of CFTR Cl- channels accounts for most of the clinical manifestations of CF, which can include airway disease, pancreatic failure, meconium ileus, male infertility, and elevated levels of salt in sweat (20, 136).

In addition to impaired Cl- channel activity, a defect in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion may participate in the pathophysiology of CF in the pancreas (131). Among the >= 900 putative disease-causing mutations in the CF gene, there are a few that are characterized by normal Cl- channel activity (37). A recent study of 16 such mutations expressed in HEK 293 cells has shown that they are associated with a significant alteration of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport activity of control cells transfected with wild-type CFTR (28). Of note, mutants associated with <15% of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport activity were associated with pancreatic insufficieny, whereas >30% activity predicted pancreatic sufficiency (28). It is postulated that the loss of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport associated with these mutations results in acidic fluid secretion that may affect mucin viscosity, bacterial binding, or activation of digestive enzymes (28). Future studies will be necessary to state precisely whether the defect in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion reflects an impaired ability of the mutated CFTR to conduct HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (76) or, alternatively, to regulate Cl--coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport (97).

CFTR in the Kidney

Numerous studies have identified CFTR in the mammalian kidney (159). CFTR mRNA is abundantly expressed in the cortex and outer medulla but much less in the inner medulla (116). Segmental analysis by RT-PCR showed that CFTR is expressed in all nephron segments, including PT, thin limbs of Henle, TAL, distal tubules, and CD (116). Immunostaining for CFTR has been detected in the apical domain of both proximal and distal tubules of the mature human kidney (36). CFTR is also expressed early in the embryonic human and rat kidney, with a transient upregulation in the ureteric bud during the late stage of branching morphogenesis (41, 72). This pattern of expression in the mammalian kidney is similar to that observed during branching morphogenesis in pancreas and lung (71). Cl- channels with properties similar to CFTR have been identified in several nephron segments. A PKA-activated Cl--conductive pathway attributed to CFTR has been demonstrated in rat kidney cortical brush-border membrane vesicles (15). In addition, CFTR-like Cl- currents have been detected in cultured rabbit DCT cells (167), rat and mouse inner medullary CD cells (74, 179), and mouse cortical CD cells (98). The exact role of CFTR in the normal renal epithelium, which primarily reabsorbs most of the filtered NaCl, is not yet established.

A splice variant of CFTR is expressed in rat and human kidney and shows distinct regulation during nephrogenesis (41, 72). This truncated CFTR variant lacks 145 bp of exons 13 and 14, resulting in a frameshift and a premature stop codon. The truncated, 75-kDa isoform (TNR-CFTR) contains the TMD1, NBD1, and R domains. It is functional when expressed in X. laevis oocytes, with single-channel properties very similar to full-length CFTR but less efficient processing and plasma membrane expression (116).

CFTR and Intracellular Organelles

In parallel with its role in apical membrane Cl- permeability and salt transport, it has been suggested that CFTR may also function within intracellular organelles of the endosomal and biosynthetic pathways (for a review, see Ref. 22). Studies in cultured cells have shown that forskolin-stimulated increases in cAMP result in a significant inhibition of endocytic activity in the human colonic epithelial cell line T84 (24) and the CF pancreatic cell line expressing wild-type CFTR (23), suggesting that CFTR may regulate plasma membrane recycling. Furthermore, CFTR has been identified and partially purified in endosomes from rabbit kidney (35), in agreement with the presence of protein kinase A-mediated Cl- permeability in renal endosomes (9). Thus CFTR-mediated Cl- conductance may participate in regulating and maintaining an acidic pH of intracellular organelles. Consistent with this hypothesis, Barash et al. (12) demonstrated a significant acidification defect in trans-Golgi and prelysosomal compartments in both immortalized respiratory epithelial cells and nasal polyps from CF patients. The restricted acidification appears to be due to a high membrane potential (Delta Psi ) secondary to the lack of counterion conductance (12, 22). It may also explain the decreased sialylation of mucins and other glycoproteins secreted by CF cells (12) and the chronic colonization of CF airway by pathogens such as Pseudomonas, which bind avidly to asialated glycosphingolipids (95). However, the role of CFTR in modulating organelle pH has remained controversial, which probably reflects the use of different cell lines and the assessment of pH in different organelles (55, 106, 150). Furthermore, recent studies in CF lung epithelial cells showed an hyperacidification of cellubrevin-labeled endosomes that was attributed to a loss of CFTR inhibitory effect on Na+ transport in this subset of endosomes (130).

Renal Manifestations of CF

There is a striking discrepancy between the level of CFTR expression in the human kidney and the lack of clearly documented renal phenotype in CF patients (40, 136, 159, 186). Despite structural and obstructive changes in the vas deferens, epididymis, and seminal vesicles, which suggest that the CF disease process operates during the development of the mesonephric duct (171), CF patients do not show overt developmental abnormalities in the urinary excretory system. Children with CF may present dehydration episodes attributed to excessive NaCl loss in sweat, particularly during warm weather and in case of digestive losses (20), but it is unknown whether a reduced ability of the CF kidney to retain NaCl contributes to these episodes. In contrast, clearance studies have suggested that CF patients have a decreased natriuretic response to Na+ loading and a reduction in free-water clearance, suggestive of impaired renal diluting capacity (17, 44). Increased PT Na+ reabsorption has been documented in some studies (8, 161) but not in others (4). Patients with CF show an increased renal clearance of aminoglycosides (99), which may be due to impaired megalin uptake (114) and/or altered endocytosis in PT cells (139). The issue remains to be clarified, because renal handling of beta 2-microglobulin and other low-molecular-weight proteins is not apparently altered in CF patients (84; Devuyst O, unpublished observations). Microscopic nephrocalcinosis has been described in a series of neonates with CF (82), and CF patients are at increased risk of developing kidney stones (63). At variance with Dent's disease (141, 188), the risk of nephrolithiasis in CF patients is not attributed to hypercalciuria (16) but rather to hyperoxaluria and decreased urinary citrate (126).

Taken together, these data suggest that the putative roles of CFTR during nephrogenesis, and perhaps in the mature kidney, are probably complemented by alternative pathways for Cl- conduction (cf. Mouse Models of CF and the Concept of Alternative Pathways for Cl- Conduction). It must be stressed that subtle abnormalities in development, function, and/or morphology of the kidney may remain clinically silent or, alternatively, be associated with specific, rare mutations of CFTR.

Mouse Models of CF and the Concept of Alternative Pathways for Cl- Conduction

Six knockout mouse models of CF have been generated in which the CF gene has been disrupted by insertion, duplication, or an in-frame stop codon (45, 66, 123, 133, 137, 158). The deletion of phenylalanine at position 508 (Delta F508) accounts for >70% of the CFTR mutations in the human population and induces a characteristic CFTR-processing defect (37). Three Delta F508 mouse models with a similar CFTR-processing defect have been produced (34, 178, 195). In one additional model, the glycine at position 551 has been changed to an aspartate (G551D) (39). The G551D mutation is relatively common in patients with CF and results in a mutated CFTR protein that is processed normally but shows a significant reduction in cAMP-regulated Cl- channel activity (187).

As recently reviewed (59), these mouse models gave previous information on the pathophysiology of CF in different organs. Except for two strains with a milder disease (45, 178), the most striking phenotype in the CF mice has been demonstrated in the intestine, with intestinal blockage and perforation, characteristic pathological changes, and ion transport abnormalities, including a lack of cAMP-mediated Cl- secretion (59). In addition, CFTR plays an important role in the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion that protects the duodenum and jejunum epithelium from acidic gastric effluent (32). Evidence suggests that the cftrtm1Unc mouse has a defect in the ability to secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in both epithelia (57, 59). Whereas lung disease is by far the most common cause of morbidity and mortality in CF patients (20), a surprising observation made in the CF knockout mice is the lack of lung disease and the virtual absence of histological abnormalities, including mucus plugging, that are consistently observed in CF patients (59). Additional differences between the CF mice and humans include the relatively low degree of pancreatic involvement and the lack of pathology in the male reproductive tract (59).

An attractive explanation for the discrepancy between human and mouse CF lung phenotypes is that Cl- channel(s) active in mouse airways may constitute an alternative pathway for Cl- in the absence of CFTR (31). For instance, the upregulation of a Ca2+-mediated Cl- secretory pathway has been evidenced in the nasal mucosa of cftrtm1Unc mice (60) and in the tracheal epithelium of two other CF mouse models (34, 39). In contrast, intestinal epithelia lack such apical Ca2+-activated Cl- secretion (31). It has been suggested that the new family of Ca2+-activated Cl- channels (CaCC or alternatively named CLCA) may provide the molecular counterpart for this Ca2+-activated Cl- conductance (53, 79). Other putative candidates to provide apical membrane Cl- transport in the absence of a functional CFTR include the voltage-activated ClC-2 (119, 147), ORCC (54), and a volume-activated Cl- channel (6, 163). Recent studies have also suggested that ClC-5, which is expressed during lung ontogeny and localized along the luminal surface of the airway epithelium, may also participate in lung Cl- secretion (47). The data summarized above, which suggest the increased expression of alternative pathways in the CF mouse, are potentially relevant to all other models of Cl- channel knockouts because they indicate that the pathophysiological consequences of the loss of a given Cl- channel pathway can be partially compensated for by the induction of another. This possibility, which has been documented in the lung of the CF mice, may apply to other epithelia expessing Cl- channels, including the kidney.

Renal Function in CF Mouse Models

Given the lack of renal manifestations in CF, it is not surprising that there is only limited information about kidney development and function in the CF mouse models (Table 1). The cftrtm1Unc mouse (158) was used to investigate whether CFTR is the molecular counterpart of the 9-pS Cl- channel identified in the basolateral membrane of the TAL of mouse kidney (61). Studies in isolated, microperfused TAL have shown that the basal NaCl reabsorption occurring in that nephron segment mainly by the operation of the apical NKCC2 cotransporter and the basolateral Na+-K+-ATPase and ClC-Kb is increased by cAMP (11). However, the study in cftrtm1Unc mice showed that the 9-pS TAL Cl- channel is active at the same frequency and with the same channel properties in cell-attached patches from cortical TAL tubules of CFTR+/+, CFTR+/-, and CFTR-/- mice. In addition, both the Cl- absorption across isolated TAL epithelium and the overall Na+-K+-Cl- cotransport activity are identical in all mice (109). These results indicate that the 9-pS Cl- channel in the mouse TAL is distinct from CFTR and that CFTR has no influence on the cAMP-stimulated Cl- absorption in this nephron segment (109). The molecular counterpart of this Cl- channel could involve the epithelial basolateral Cl- conductance regulator (EBCR) belonging to the ABC family (180). However, it remains unclear whether EBCR is a channel by itself or modulates other endogenous basolateral Cl- channels located in the TAL.

Micropuncture experiments have shown that cAMP stimulates Cl- transport and fluid absorption in rat PT (185), an effect attributed to the presence of cAMP-activated Cl- channels in that nephron segment (1, 149). Kibble et al. (85) investigated PT function in the Delta F508 cftrtm2Cam mouse (34) and showed that renal Na+ clearances under basal conditions or after acute extracellular volume expansion are similar in wild-type and Delta F508 mice. The latter are also characterized by a lack of increase in glomerular filtration rate secondary to volume expansion and a relatively less important increase in proximal Na+ reabsorption than wild-type mice (85). Despite identification of CFTR expression in the outer cortex of wild-type mice by RT-PCR, in situ microperfusion showed that the basal fluid absorptive rate is similar in wild-type and Delta F508 mice and is not significantly influenced by addition of forskolin-dibutyryl cAMP to the perfusate. These data may be explained by a lack of cAMP-dependent whole cell Cl- conductance evidenced in isolated mouse PT cells (85). In addition, Kibble et al. (86) demonstrated that, under chronic dietary salt restriction, Delta F508 mice are equally able to reduce renal Na+ excretion but display a significantly higher amiloride-sensitive Na+-reabsorption than wild-type mice. Thus Delta F508 mice may handle volume expansion and chronic NaCl restriction similarly to wild-type mice, but that could involve more distal parts of the nephron (86). The increased amiloride-sensitive Na+ reabsorption observed in salt-restricted Delta F508 mice is consistent with the proposed interaction between CFTR and ENaC in principal cells of the CD (98). Accordingly, a reduced inhibition of ENaC in the CD of Delta F508 mice could explain the enhanced Na+ absorption in that nephron segment, which may provide an advantage during states of salt deprivation. It must be noted, however, that other mechanisms, such as enhanced secretion of aldosterone, may participate in distal Na+ reabsorption in salt-restricted mice (58).

The cftrtm2Cam Delta F508 CF mouse (34) has also been used to investigate the K+-sparing diuretic effect of glibenclamide. Glibenclamide blocks native K+ channels in renal cells (10), but in vitro studies have demonstrated that coexpression of CFTR with Kir1.1 (ROMK2) is necessary to confer glibenclamide sensitivity on Kir1.1 channels (112). The fact that glibenclamide induced an equivalent diuresis and a similar K+-sparing effect in wild-type and Delta F508 mice indicates that the formation of Kir1.1/CFTR complexes is not required to mediate its diuretic effect in mice (87).


    CONCLUSIONS AND PERSPECTIVES
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ABSTRACT
INTRODUCTION
THE CLC FAMILY OF...
CFTR
CONCLUSIONS AND PERSPECTIVES
REFERENCES

Since the first mouse models of CF described in 1992, the generation of transgenic mice has become an essential tool for understanding the pathophysiology of Cl- channels in the kidney. This is particularly true for Cl- channels belonging to the CLC family, which are implicated in selective nephron segments, where they participate in essential kidney functions such as endocytosis, NaCl reabsorption, or urinary concentration. There is a considerable interplay between the characterization of these mouse models and the discovery of channel diseases in humans. For instance, the phenotype of patients with Dent's disease due to mutations in CLCN5 was consistent with a role played by the Cl- channel ClC-5 in proximal tubule endocytosis (102), a hypothesis that was indeed verified in two independent knockout mice lacking ClC-5 (129, 184). The discovery of mutations in genes coding for ClC-Kb and the beta -subunit barttin not only completed the understanding of genetic heterogeneity in Bartter's syndrome but also provided key insights into the regulation of this class of channels (18, 48). Furthermore, the nephrogenic diabetes insipidus observed in Clcnk1-/- mice suggests that mutations in CLCNKA may be responsible for nephrogenic diabetes insipidus in humans lacking mutations in V2R and AQP2 (172).

There are numerous perspectives for existing and future mouse models targeting Cl- channels. Studies of mice lacking CLC channels will be important to further define the physiological role of some of these channels in the kidney and the phenotypical variability observed in some models. Knockout mice for ClC-k2 or barttin may provide useful models for Bartter's syndrome and give new information about the regulation of CLC channels. Gene targeting will also expand our knowledge of other types of channels that exhibit a Cl- conductance. An interesting example is aquaporin-6 (AQP6), a member of the aquaporin family of water channels that has been identified in rat kidney (191). AQP6 is located in podocytes, PT cells, and alpha -type intercalated cells of the CD, and it appears to be mostly associated with intracellular vesicles (191). The low basal water permeability of AQP6 can be increased by exposure to mercurials or low pH. Interestingly, the latter conditions are also reflected by a significant anion conductance (190). The colocalization of AQP6 with the vacuolar H+-ATPase in intracellular vesicles, as well as the rapid and reversible activation of the anion conductance of AQP6 by acidic pH, led to the suggestion that AQP6 may play a role along other Cl- channels in vesicle acidification (190).

Further characterization of CF mouse models will certainly be useful to identify the role of CFTR in specialized kidney functions such as endocytosis or its interactions with other types of channels and transporters in the nephron (115, 146). These studies may also be useful in investigating the apparent tissue specificity of Delta F508 CFTR trafficking (81, 128) and other mechanisms that could explain the absence of overt renal phenotype in CF. Mating CF mice with other mouse models will also create opportunities to characterize the role of CFTR in the pathophysiology of renal cyst formation (127). The issue is particularly relevant in autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. ADPKD is characterized by the development of multiple cysts in both kidneys, resulting in end-stage renal disease in 50% of patients by age 60 yr (65). Genetic studies have shown that ADPKD is due to mutations of two major genes, PKD1, responsible for ~85% of cases, and PKD2, which accounts for the vast majority of other cases (65). Although the complex mechanisms involved in cystogenesis are not yet understood (27), abnormal fluid secretion is a critical pathogenic mechanism associated with cyst expansion in ADPKD (165). Several lines of evidence have demonstrated that cAMP-dependent Cl- secretion provides the driving force for fluid accumulation into ADPKD cysts and that CFTR is implicated in this process (25, 38, 64). The availability of mice with targeted disruptions of Pkd1 (104) and Pkd2 (189) may give insights into this intriguing question.

Finally, one should remain careful about the general limitations of mouse models, including the occurrence of phenotypical variability despite similar genetic backgrounds, the necessity for specific environmental conditions to reveal a phenotype, and the frequent species differences in the structure and function of a given organ (125, 132). However, even these limitations could prove useful because the phenotypical variability of mutant mice according to genetic background underlies the potential importance of genetic modifiers (121). Thus mouse models will also be valuable to investigate disease-modifying genes, an approach that may give insights into complex pathways and provide new therapeutic strategies in renal diseases.


    ACKNOWLEDGEMENTS

We thank Prof. R. Beauwens and Prof. P. Courtoy for fruitful discussions.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32753, the Fondation Alphonse and Jean Forton, the Fondation pour la Recherche Scientifique Medicale, and Actions de Recherche Concertées 00/05-260.

Address for reprint requests and other correspondence: W. B. Guggino, Johns Hopkins Univ. School of Medicine, Depts. of Physiology and Medicine, 725 North Wolfe St., Baltimore, MD 21205 (E-mail: wguggino{at}bs.jhmi.edu).

10.1152/ajprenal.00184.2002


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ABSTRACT
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CONCLUSIONS AND PERSPECTIVES
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