The kidney contains the body’s second largest number ofdifferent cell types, modulating the great diversity of differentfunctions that allow complex homeostasis to occur. A featurethat unites all these cell types in the postglomerular nephronis that they are highly polarized; one surface being is in contactwith the urine and the other with interstitial fluid. As such,there is a strict requirement for vectorial transport of ions,proteins, and other molecules across both apical and basolateralplasma membranes. The cell surface resident proteins by whichthis transport occurs are tremendously varied, and their repertoiresvary at different points along the nephron. Moreover, highlyspecialized machinery is required to bring these transport proteinsto their appropriate site of action. This is a dynamic process,with regulated recycling between the cell surface and intracellularvesicular structures. The purpose of this review is to highlightvarious renal diseases where the pathogenetic mechanism involvesdisruption of one or other of these processes.
Resident proteins are synthesized and assembled in the roughendoplasmic reticulum (ER), where there is an in-built qualitycontrol and degradation mechanism that is designed to ensurethat only properly folded and assembled proteins pass onwardsthrough the Golgi apparatus before being directed to their finaldestination (1). Quality control is achieved by the associationof ER chaperone molecules with unfolded or misfolded polypeptidechains. If these proteins fail to assume their correct structure,they are retained in the ER and targeted for ubiquitinationand degradation by the 26S proteasome. While disease-causingmutations may result in complete failure of transcription ortranslation of the encoded protein, many others will resultin structurally abnormal proteins. Although a full discussionof ER function is beyond the scope of this review, it is clearthat in many diseases the ER is a common site for retentionof these mutant proteins, and loss-of-function is often manifestedby intracellular accumulation of abnormal protein that shouldhave been delivered to the cell surface (2). Some publishedexamples of renal diseases where this has been documented includethe following: the familial juvenile hyperuricemic nephropathy/medullarycystic disease constellation (caused by mutations in the uromodulingene UMOD [3,4]), where uromodulin is seen within loop of Henleepithelial cells rather than at the apical surface (5,6); thevarious forms of Bartter syndrome, including the recently describedtype 4, where the problem is that Barttin (an essential co-factorfor CLC-Kb’s basolateral chloride transport function inthe loop) becomes intracellularly retained when mutated andretards CLC-Kb with it, presumably because of failure of correctassembly of the two subunits (7); Gitelman syndrome, which ofteninvolves retention of the mutant thiazide-sensitive NaCl co-transporterin the distal convoluted tubule (8,9); and X-linked or autosomalrecessive nephrogenic diabetes insipidus, where missense vasopressinreceptor or aquaporin-2 (AQP2) mutants respectively are retainedwithin collecting duct principal cells (10,11).
Normal membrane proteins progress from the ER to the Golgi (Figure 1),where they may be modified (for example by the additionof carbohydrate moieties), and on to the trans-Golgi network(TGN) by means of a complex process that allows for both anterogradeand retrograde trafficking. Thereafter, different regulatedprocesses direct proteins from the TGN to the apical, basolateral,or endosomal compartments, depending on their functions. Many,but by no means all of the molecular players in this orchestratedsystem have been elucidated, and some human diseases have providednatural knockout models, contributing to this knowledge. Anexample that includes a renal phenotype is Lowe syndrome, arare X-linked disorder characterized by severe mental retardation,congenital cataracts, and renal Fanconi syndrome. The defectiveprotein is OCRL, which appears to have two functions. First,it is a phosphatase that resides in the TGN, removing the 5'phosphate group from another TGN resident, phosphatidylinositol-4,5-bisphosphate(PI-4,5-P2) (12), which is known to play a role in the regulationof vesicular trafficking (13). Phosphorylation and dephosphorylationof PI molecules signals either the recruitment or the activationof proteins essential for vesicular transport. Second, Ocrl1associates with Rab GTPases (14), and crosstalk between PI metabolitesand guanosine triphosphatases is an important feature of theseregulatory mechanisms. Thus, the association of proximal tubulardysfunction with absence of an important modifier of PI-4,5-P2reveals its importance in coordinated intracellular traffickingin the nephron.
Figure 1. Simplified schema for trafficking of plasma membrane proteins in a polarized epithelial cell. Proteins synthesized in the endoplasmic reticulum (ER) are trafficked sequentially through the Golgi (G) and trans-Golgi network (TGN), by which point they should be normally folded and subunits assembled. Different endosomal structures (E) then carry cargo to apical or basolateral compartments (lower half of Figure, solid arrows) from which recycling may occur. Degradation (dotted arrows, upper half of Figure) requires delivery to lysosomes (L). Abnormal proteins are targeted from the Golgi to the 26S proteasome for disposal (not shown). * Some basolateral proteins transit the apical compartment before final delivery. Apical and basolateral compartments are separated by intercellular tight junctions. N, nucleus.
The final residency of membrane proteins is governed by sortingmotifs contained within their sequences, which interact specificallywith the cellular sorting machinery. These complexes are incorporatedinto distinct vesicular structures whose surface componentsfurther direct sorting by virtue of interactions between attachmentproteins known as SNARES (15). The mechanisms and machinerythat control this trafficking are currently under extensiveinvestigation, and it is becoming clear that there are multiplepathways for vectorial transport. Moreover, these are dynamicprocesses, with turnover of cargo between compartments in bothforward and reverse directions (Figure 1) becoming increasinglyrecognized (16).
Several different basolateral plasma membrane targeting motifshave been described, generally residing in the cytosolic C-terminaldomains of transmembrane proteins. These include the YXXØmotif (where X is any amino acid, and Ø is one possessinga hydrophobic side chain), the NPXY motif, and the di-leucinemotif. The first of these is the best characterized. YXXØ-containingproteins associate with one of the adaptor protein (AP) complexes,which are heterotetrameric structures. AP-1 is found in theTGN and endosomes (17). In polarized epithelial cells, the AP-1Btype and AP-4 have been implicated in sorting from the TGN tothe basolateral cell surface (18–20), while AP-2 functionsin the opposite direction to internalize proteins from the surface(21). In contrast, AP-3 appears to bind cargo destined for thelysosome (22). Lysosomes are a major site of intracellular degradation,and classical lysosomal targeting motifs also involve tyrosineor leucine residues (reviewed in reference 16).
The mechanism of apical membrane targeting is at present lesswell understood, particularly because there is emerging evidencefor crosstalk and transient movement between apical and basolateralcompartments (23). Like basolateral sorting, apical sortinginformation is thought to be localized in the cytosolic domainof transmembrane proteins; in the case of proteins anchoredto the membrane via a GPI anchor, sorting information may beprovided by lipid moieties.
A number of renal diseases are now recognized to be associatedwith aberrant trafficking. This may be because of failure ofnormal recycling from the membrane, or mis-targeting away fromthe correct cell surface compartment, or misdirection into thewrong intracellular organelle. Examples of each of these arediscussed below.
In 1963, Liddle described a three-generation kindred with autosomaldominant inheritance of early-onset hypertension and hypokalemicalkalosis (24). The clinical picture in Liddle syndrome is consistentwith hyperaldosteronism, but aldosterone levels are suppressed—often,as in the original report, to undetectable levels. The abnormalitiesin Liddle syndrome can be ameliorated by a low-salt diet plusantagonists of the epithelial sodium channel of the collectingduct, amiloride or triamterene, but they are not improved bymineralocorticoid receptor antagonists. This suggested excessivesodium reabsorption in the kidney as the primary defect ratherthan activity of an unknown mineralocorticoid. The index patientreceived a cadaveric renal transplant in 1989, after which herdisorder resolved, with normalization of the aldosterone andrenin responses to salt restriction. This confirmed a primaryrenal tubular origin for the salt retention and hypertension(25).
Using a positional cloning approach, Shimkets et al. (26) foundthat Liddle syndrome is associated with mutation in the -subunitof the epithelial sodium channel (ENaC) gene, after which similaralterations in the -subunit were identified in other kindreds(27). By and large, these mutations are premature stop codons,resulting in truncations of the cytoplasmic C-terminal tailof the relevant subunit. Consistent with the dominant inheritancepattern, these are associated with a gain-of-function in ENaC.
In the kidney, functional ENaC channels are expressed at theapical surface of collecting duct principal cells and are composedof at least three subunits: , , and (28). The stoichiometrymay be 2:1:1 (29) or 1:1:1 (30,31). ENaC activity is mainlyregulated by variation in the number of channels present atthe cell surface. When necessary, ENaC are removed from theapical surface by ubiquitination and internalization, followedby proteasome-mediated degradation (Figure 2A). Ubiquitinationcan occur at the N-terminal lysine residues of - and -subunits(32), and the E3 ligases responsible are members of the Nedd4family (33). Under normal circumstances, ENaC activity is regulatedby aldosterone and by a variety of hormones, including insulin,ANP, and ADH (29,34), acting via a range of intracellular kinases.Both Nedd4 and the C-terminal cytoplasmic tail of ENaC havebeen shown to be phosphorylated (35). Nedd4 is phosphorylatedvia the action of SGK-1 (serum and glucocorticoid kinase 1),the function of which is upregulated by aldosterone (36,37).After Nedd4 is phosphorylated, it loses the ability to interactwith ENaC, leading to the observed increase in activity (Figure 2B).
Figure 2. ENaC recycling. ENaC at the apical (urinary) surface of collecting duct principal cells are trimers; for clarity, one subunit only is shown, (A) Normally, apically inserted ENaC is prevented from transporting Na+ by the action of Nedd4–2 (and possibly other E3 ligases), which binds to ENaC’s C-terminal-domain PY motif and ubiquitinates the molecule, signaling internalization and degradation. (B) ENaC is upregulated by aldosterone, which causes SGK-1 (serum and glucocorticoid kinase 1) to phosphorylate Nedd4-2, which prevents its interaction with ENaC, such that more channels remain at the surface. (C) In Liddle syndrome, C-terminal truncations in either the or subunits (or missense mutations involving their PY motifs) similarly disrupt Nedd4-2 binding.
The first clue as to the mechanism of disease in Liddle syndromecame from the finding of a de novo missense mutation in a highlyconserved -subunit proline residue within the C-terminus ofthe -subunit, identifying this as the critical region of themolecule (38). Snyder (39) reported that this conserved proline-richmotif, present in the C-terminus of all three subunits, reproducedthe affect of Liddle truncations when mutated and expressedin Fischer rat thyroid epithelia. The significance of thesefindings was clarified with the discovery that it is this proline-richregion, since termed the PY motif, that mediates removal ofENaC from the cell surface via direct interaction with Nedd4(Figure 2C). Nedd4 is an E3 ligase that was demonstrated tobind ENaC by association of its WW domains with the XPPXY motifpresent in each ENaC subunit (40–42). Recently, it hasbeen demonstrated that both Nedd4–2, a splice variantof Nedd4, and WWP2, a related family member, can regulate ENaC(43,44). Other ubiquitin machinery proteins such as TSG101 arealso potentially implicated (45). It is concluded that ENaClacking the PY motif are inefficiently removed from the cellsurface, leading to increased channel concentration and thusincreased sodium reabsorption (46). Although these findingswere not initially devoid of controversy, evidence for the alternativehypothesis, that mutations affect open probability (the activitylevel of individual channels) of ENaC, has not been substantiated.
In vitro evidence of internalization failure has also been substantiatedby animal studies. Mice transgenic for the R566X -subunit mutationthat was present in the original kindred show hypervolemia,hypertension, and metabolic alkalosis (47). In mutant comparedwith wild-type mice, open probability of ENaC was similar; transepithelialpotential differences were higher; urinary sodium excretionwas lower on recovery from sodium depletion; and ENaC were retainedat the apical membrane (48,49).
Nephrogenic diabetes insipidus (NDI) is a condition that resultsfrom failure of the kidney to concentrate urine in responseto circulating antidiuretic hormone (vasopressin, AVP), whichis released from the hypothalamus in response to hypernatremiaor hypovolemia. Most cases of NDI are acquired; for example,secondary to hypokalemia, hypercalcemia, or lithium toxicity.Of the primary forms of NDI, three patterns of inheritance aredescribed: X-linked recessive, autosomal recessive, and autosomaldominant. Clinical presentation of these different forms issimilar, usually occurring within the first 2.5 yr of life andoften in the neonatal period. Polyuria is the primary clinicalsequela, with secondary polydipsia, but common presenting symptomsinclude anorexia, vomiting, constipation, fever, and failureto thrive. Growth retardation occurs commonly, and mental retardationhas been described (50). If the diagnosis is made promptly,all of these complications can be prevented by adequate waterintake (51).
The basic paradigm of AVP-mediated reabsorption of water inthe collecting system involves the circulating hormone bindingto a receptor (V2R) on the basolateral side of the principalcell, which leads indirectly to the shuttling of water channels(AQP2) from intracellular vesicles to the apical surface ofthis polarized cell (Figure 3A) (52,53). Water is thereby ableto enter the otherwise impermeable cell from the collectingduct lumen, down an osmotic gradient, and then passes throughother water channels (AQP3 and AQP4) in the basolateral membrane,to be reclaimed into the interstitium.
Figure 3. Autosomal dominant NDI. (A) AVP binds to its receptor (V2R) on the basolateral membrane of the principal cell in the collecting duct. This results in activation of protein kinase A (PKA), via cAMP-mediated signaling, which phosphorylates aquaporin-2 (AQP2) and leads to shuttling of the AQP2 homotetramer from intracellular vesicles to the apical membrane, rendering the cell permeable to water. (B) In the case of AQP2 mutations causing autosomal dominant NDI, mutant subunits in the AQP2 tetramer exert a dominant negative effect, leading to aberrant basolateral surface expression or intracellular retardation. The apical membrane then remains relatively impermeable to water. AC, adenylyl cyclase.
The detail of some of these steps has not yet been fully elucidated,but V2R is known to be a G-protein coupled receptor, which onbinding its agonist results in activation of adenylate cyclase,production of cyclic AMP (cAMP) with consequent activation ofprotein kinase A (PKA), and phosphorylation of a serine residue(S256) in the C-terminus of AQP2 (54). This is followed by translocationof the AQP2-containing vesicles to the apical surface, withincorporation of AQP2 into the plasma membrane in tetramericform. V2R itself, having bound its ligand, is phosphorylatedand internalized by a clathrin-mediated mechanism, resultingin downregulation.
Recent data have added to the complexity of this model, withevidence that cell surface expression of AQP2 may be regulatedby pathways other than the cAMP cascade. Elevated cGMP, stimulatedby hormones such as ANP, may also lead to recruitment of AQP2to the cell surface, as may depolymerization of the actin cytoskeleton(55). In addition, it has been observed that constitutive recyclingof AQP2 occurs between vesicles and the cell surface, independentof changes in the levels of cAMP (56). This alone does not resultin accumulation of AQP2 at the surface because of a dynamicequilibrium between vesicle fusion with the plasma membrane,and endocytosis. The latter is of potential interest therapeutically,because it has been demonstrated in cell culture that blockingthis endocytosis results in cell surface accumulation of AQP2,independent of the function of V2R, thus bypassing any defectin the receptor or in the subsequent signaling cascade (57).
Using cell culture systems, studies of the molecular pathogenesisof the three inherited forms of the disease have revealed adiversity of points at which intracellular processing and traffickingof proteins may be disrupted, some of which may emerge as potentialtherapeutic targets.
X-linked recessive NDI is the most common form, accounting forapproximately 90% of cases. The disease is caused by loss offunction mutations in the AVPR2 gene on the long arm of theX-chromosome, which encodes the seven-transmembrane domain V2Rprotein (58–60). Over 160 mutations have been describedin all regions of the gene. In some of these mutations, theresulting protein is so severely truncated it could not be functional.As noted above, it is likely that such proteins would be retainedby the ER and then degraded.
However, while this is the fate of over 70% of V2R mutants,this form of the disease merits further discussion because thereis evidence that in some cases cell surface expression of functionalV2R can be rescued pharmacologically. Morello et al. (61) investigatedtwo different cell permeable V2R antagonists using nonpolarizedcell systems and observed rescue in 8 of 15 mutants. Tan etal. (62), using polarized cells, observed cell surface rescuein 2 of 3 mutants. This was achieved not only with a permeableV2R antagonist, but also by reducing the temperature of thecell culture. It was suggested that both of these interventionsenabled stabilization or completion of protein folding, allowingsubsequent escape from the ER or Golgi.
Autosomal recessive NDI accounts for approximately 10% of inheritedcases. It is caused by mutations in the AQP2 gene (63), almostall of which appear to be retained in the ER, suggesting thatin each case, incomplete or misfolding of the mutant proteinoccurs. In some of these cases it has been demonstrated thatthe mutant protein was monomeric, rather than the normal homotetramericconfiguration (64).
In contrast, the rare dominantly inherited NDI, also causedby mutations in AQP2, appears to result from mistargeting offunctional protein to the wrong cellular compartment (Figure 3B).Six mutations have been studied to date, using a varietyof cell systems. In each case, mutant protein is able to passthrough the ER and to assemble with wildtype protein, encodedby the normal allele, into functional heterotetramers. A dominantnegative mechanism has been proposed whereby the mutant componentsof the heterotetramer influence its targeting (65,66). Therehave been some differences in the observed final destinationof the heterotetramers among these six mutations. For example,the missense mutant AQP2-E258K was reported to target to theGolgi complex of Xenopus oocytes, whereas the deletion mutantAQP2–727delG was reported to accumulate in late endosomes,lysosomes, and the basolateral plasma membrane in MDCK cells(66,67). Clearly, different mutations might cause disease bydifferent mechanisms, but it is also likely that traffickingbehavior of proteins varies between different in vitro cellsystems, and caution is required when attempting to infer invivo pathophysiology. In particular, the commonly used oocytesystem is nonpolarized, whereas in vivo the collecting ductprincipal cell is highly polarized. Indeed, three other AQP2mutants have behaved differently in oocytes and polarized MDCKcells, the mutant tetramer appearing at the (wrong) cell surfacein the polarized cell line in each case, but being retainedintracellularly in the nonpolarized line (68).
When mutant AQP2 is delivered erroneously to the basolateralrather than to the apical surface, thus disrupting the functionof the cell, possible mechanisms include loss of an apical targetingsignal in the protein, or the gain or activation of a basolateralsignal that overrides any inherent apical signal. Recently,Kamsteeg et al. (69) described a case of dominantly inheritedNDI resulting from a frame-shift mutation that caused an extensionof the C-terminus of AQP2 and the introduction of two basolateraltargeting motifs, one tyrosine-based and one di-leucine. Theyproposed that these new motifs present on the mutant componentsof the heterotetramer dominated any apical motifs present inthe mutant or wild-type subunits. This mechanism is likely tobe particularly rare, but it does provide an interesting contrastto the mechanism described below for a form of dominant distalrenal tubular acidosis, where loss of a basolateral sortingmotif results in aberrant targeting.
AQP2 has been dissected further to try to delineate targetingmotifs involved in its shuttling between intracellular vesiclesand the apical cell surface. Van Balkom et al. (70) identifieda region in the C-terminal tail that was required for apicallocalization, but the cytosolic N-terminal domain was also requiredto achieve localization in the intracellular vesicles with subsequentmovement to the apical surface when the action of AVP was simulated.Not surprisingly then, the trafficking of AQP2 appears to becomplex, involving more than one motif.
Distal renal tubular acidosis (dRTA) is a disease of defectiveurinary acidification that is caused by dysfunction of -intercalatedcells (-IC) in the collecting system. DRTA is characterizedby hypokalemic metabolic acidosis, metabolic bone disease, andnephrocalcinosis and/or nephrolithiasis. As with NDI, most casesof dRTA are acquired; for example in association with autoimmunedisease such as Sjögren syndrome (71). Of the inheritedforms, autosomal dominant dRTA (ddRTA) generally presents later,occasionally in adulthood, and with milder phenotype, than doesthe recessive form (72). Treatment for both is with alkali replacement,which corrects most of the biochemical abnormalities.
In the -IC, protons are secreted actively across the apicalsurface of the cell by the H+-ATPase into the collecting ductlumen. This process is coupled to the reclamation of bicarbonateions across the basolateral plasma membrane via the chloride-bicarbonateexchanger, AE1 (Figure 4A) (73). Mutations in SLC4A1, encodingAE1, are to date the only genetic cause of ddRTA (74–78)
Figure 4. Polarized function of -IC and ddRTA. (A) In cortical collecting duct -IC, acid-base balance is fine-tuned by proton pumps on the apical surface of the cell that actively secrete acid into the duct. This process is coupled to the reclamation of bicarbonate ions into the interstitium, via chloride-bicarbonate exchanger AE1, whose expression is confined to the basolateral surface. (B) In at least one form of autosomal dominant distal renal tubular acidosis, loss of a C-terminal basolateral targeting sequence results in a nonpolarized distribution of AE1, such that a proportion of the protein reaches the apical surface, thus disrupting the electrochemical balance of the cell.
With respect to mechanism of disease, it had been realized forseveral years that ddRTA is unlikely to result from simple haploinsufficiency.First, the evidence for this was that numerous heterozygousAE1 mutations have been described that cause the autosomal dominanterythrocyte diseases hereditary spherocytosis and ovalocytosis(the red cell being the other site of expression of AE1), butthese are not associated with a urine acidification defect (79).Second, all of the dRTA-associated AE1 mutations thus far studiedhave demonstrated near-normal anion exchange function when expressedin Xenopus oocytes (74,75,80). The majority of these are missensemutations of R589 (R589H, R589S, and R589C) as well as S613F,and a complex mutation resulting in an 11–amino acid truncationat the C-terminus, R901X.
The hypothesis of AE1 mistargeting as a mechanism of diseasetherefore arose. Possible explanations included the proteinbeing retained intracellularly or reaching the surface but losingits specific basolateral distribution such that the electrochemicalbalance of the cell might be disturbed (Figure 4B). Given that50% of functional AE1 appears to be sufficient for normal urinaryacidification, intracellular retention of the mutant AE1 wouldalso have to be associated with retention of a significant proportionof the wildtype AE1 encoded by the normal allele. Indeed expressionof AE1 mutants in (nonpolarized) HEK293 cell line has suggestedthat they are retained intracellularly and that they exert adominant negative influence by preventing co-expressed wildtypeAE1 from reaching the cell surface (81,82).
Similarly, Toye et al. (80) suggested intracellular retentionof the R901X mutant, this time in MDCK cells. However, the conditionsused did not lead to polarization of the cells, which may havehad an effect on trafficking behavior. We subsequently demonstratedthat in adequately polarized MDCK cells, the R901X mutant proteinappears at both the basolateral and the apical surfaces, witha proportion being retained intracellularly. We went on to confirmthese findings in another mammalian renal epithelial cell line,IMCD (83).
The R901X mutant had offered more of a clue to a possible targetingdefect, as the missing 11–amino acid tail contains thesequence YDEV, which could represent a basolateral targetingmotif of the YXXØ type. We demonstrated that the 26–aminoacid C-terminal cytosolic domain of AE1 containing this putativemotif, when transplanted onto an apical reporter protein inplace of its own C-terminus, caused basolateral redistributionof a proportion of the protein. In addition, we found that thetyrosine component of the motif, Y904, was essential for basolateraltargeting of wildtype AE1. When changed to alanine, a nonpolarizeddistribution was observed.
As for missense mutations involving R589, there are as yet nopublished data from adequately polarized renal epithelial celllines, so it is not clear whether mutation of this residue disruptsa different basolateral targeting motif or whether it causesa conformational change leading to intracellular retention ofthe protein.
Nephropathic cystinosis is characterized by poor growth, renaltubular Fanconi syndrome, glomerular failure, and involvementof other tissues and organ systems (reviewed in reference 84).In untreated children, poor growth is generally evident by 9mo of age. Signs of renal tubular Fanconi syndrome, includingpolyuria, polydipsia, dehydration, and acidosis, appear as earlyas 6 mo of age and are irreversible if tubular damage has alreadysupervened. In untreated patients, glomerular function graduallydeteriorates, resulting in renal failure at approximately 10years of age. Intermediate cystinosis is characterized by allthe typical early manifestations of nephropathic cystinosis,but at a later age. Glomerular failure occurs in all patients,usually between 15 and 25 yr of age. Adolescent nephropathiccystinosis manifests itself first at age 10 to 12 yr with proteinuriadue to glomerular damage rather than with the manifestationsof tubular damage that occur first in infantile cystinosis.There is no excess aminoaciduria and stature is normal. Photophobia,late development of pigmentary retinopathy, and chronic headachesare features. Non-nephropathic cystinosis is characterized byphotophobia only.
The underlying metabolic defect in all forms of cystinosis isdefective transport of cystine, released from the hydrolyticcleavage of peptides, across the lysosomal membrane and intothe cytosol (85,86). The lysosomal build-up of cystine can bereduced by frequent oral administration of cysteamine. Thismay slow or stop the progression of glomerular damage, may attenuatethe Fanconi syndrome, and can delay or prevent the need forrenal transplantation. Growth hormone may also be required.
All forms of cystinosis have been shown to be allelic (87) andare due to mutations in CTNS (84,88). The CTNS transcript encodesa 367–amino acid protein named cystinosin, which is alysosomal membrane cystine transporter (88–90). Cystinosinappears to function as a proton/cystine symporter (91), protonsbeing pumped into the lysosome via vacuolar ATPases (Figure 5A).It is highly specific for cystine. Cystinosin is predictedto be a seven-transmembrane domain protein, with seven potentialN-glycosylation sites in the N-terminal region and a classictyrosine-based lysosomal targeting signal (GYDQL) in the C-terminaltail (88). Mutation analysis revealed that the correct sortingof cystinosin to the lysosomal membrane requires not only theGYDQL motif but also a second signal, because missense alterationsin this motif caused only partial redirection to the plasmamembrane (89). Site-directed mutagenesis then uncovered a novelconformational motif, the core of which is YFPQA, situated inthe fifth loop of the protein (89). When both motifs were altered,the protein completely relocated to the plasma membrane (Figure 5B).This second motif is the first example of a non-C-terminallysosomal sorting signal.
Figure 5. Cystinosin mistargeting. (A) Wild-type cystinosin resides in the lysosomal membrane of proximal tubular cells, where it transports cystine in association with protons. (B) In some forms of cystinosis, mutations involve one of two lysosomal targeting signals (GYDQL or YFPQA), resulting in mistargeting away from the lysosome, sometimes as far as the cell surface. Thus cystine builds up in the lysosomes, causing cell malfunction.
Thus the trafficking abnormalities that cause cystinosis maybe either because cystinosin is absent or nonfunctional (forexample, where there are truncation mutations) and thereforethe cargo cannot move appropriately, or because this transportercannot itself traffic normally, as would be predicted from reportedmissense mutations in the fifth loop or C-terminal truncations(92,93), both of which affect the targeting signals.
Dent disease is an X-linked recessive renal tubular disordercharacterized by low–molecular weight proteinuria, hypercalciuria,nephrolithiasis, nephrocalcinosis, and progressive impairmentof renal function. Other features may include rickets, hypophosphatemiawith renal phosphate wasting, aminoaciduria, glycosuria, anduricosuria. Consequently the disease may be classified as aform of Fanconi syndrome.
Dent disease is caused by mutations in CLCN5, which encodesCLC-5, a member of a family of mammalian voltage-gated chloridechannels (94). Since the identification of this as the responsiblegene, three similar conditions have been found to be allelicand are often referred to under the collective term of Dentdisease: X-linked recessive nephrolithiasis (95); X-linked recessivehypophosphatemic rickets (96); and idiopathic low–molecularweight proteinuria of Japanese children (97).
CLC-5 is a 746–amino acid protein with multiple membrane-spanningdomains and intracellular N- and C-terminal domains (98). Inthe mammalian nephron, it is expressed in the proximal tubule,in the thick ascending limb, and in IC (99). Electrophysiologicand immunofluorescence studies of transfected cells have demonstratedthat CLC-5 is located predominantly in subapical vesicles, withsome reported cell surface expression. It colocalizes with thevacuolar H+-ATPase in the PT and IC, suggesting that it functionsto permit proton pump–mediated acidification of endosomesby providing the anion influx required to maintain electroneutrality(100). Endosomes recycle between the surface and subapical compartments;in fact, a putative PY internalization signal, similar to thatseen in ENaC, has been identified in CLC-5’s C-terminaldomain (101). Over 60 mutations have now been described in CLCN5,located throughout the gene. Many of these are predicted toresult in loss of function or absence of CLC-5 (102). A resultingreduction in endosome acidification may be associated with disruptionto their cycling and endocytic function (Figure 6) (103,104).
Figure 6. Dent disease. Low–molecular weight proteins (LMWPs undergo receptor-mediated endocytosis in the proximal tubule, possibly involving the receptors megalin and cubilin. Receptors and ligands are internalized by the formation of endosomes, which are progressively acidified by vacuolar proton pumps. Chloride influx via CLC-5 facilitates acidification by maintaining electroneutrality of ion transport. Endosomal recycling brings LMWP receptors back to the apical surface. CLC-5 absence or dysfunction might interrupt endosome cycling at three points (shown by red block arrows): (1) reduced rate of internalization of receptors; (2) disruption of progression of endosomes to lysosomes; (3) recycling of endosomes to the cell surface.
It is not known precisely how absence or reduced activity ofCLC-5 in the endosome and impaired endosomal acidification resultin impaired endocytosis. Normally endocytosed LMWP have beendemonstrated to colocalize with CLC-5 (100). Christensen etal. (105) suggested that reduced apical surface expression ofthe multiligand receptors, megalin and cubilin, involved inLMWP endocytosis could in part be responsible. They demonstratedthat, in a CLC-5 knockout mouse, there was a redistributionof these receptors from the apical surface of proximal tubulecells to endosomes and suggested that this arises from retardationof receptor recycling. They proposed that this, in additionto reduced apical internalization of these receptors and slowerprogression of endosomes to lysosomes, could account for reducedLMWP endocytosis.
This model provides a possible explanation for the proteinuriaassociated with Dent disease, but how CLC-5 mutation might causethe other clinical features of Dent disease is less clear. Usinga CLC-5 knockout mouse, Günther et al. (106) have suggestedthat hyperphosphaturia and hypercalciuria are both indirectconsequences of impaired endocytosis of PTH.
Defects in the intracellular trafficking of CLC-5 itself maybe involved in the mechanism of disease in some cases. Carret al. (107) conducted renal epithelial cell expression studiesof 3 CLC-5 truncated mutants to investigate the possible significanceof the second of two CBS domain motifs in the C-terminal domain.These motifs, approximately 50 residues in length and namedafter their description in cystathione -synthase, are of unknownfunction, but they are present in all members of the CLC family.The intracellular distribution of these three mutant constructswas mainly perinuclear. The authors concluded that this CBSdomain is required for correct intracellular trafficking ofCLC-5.
CLCN5 mutations might therefore result in disruption of traffickingof CLC-5 itself, or by loss of function they might disrupt endosomecycling, which in turn influences the cell surface expressionof receptors that mediate endocytosis. The detail of these complexinteractions remains to be elucidated (Figure 6).
Type I primary hyperoxaluria is a rare autosomal recessive disordercharacterized by high urinary oxalate excretion, progressivebilateral calcium oxalate stone formation, and nephrocalcinosis.Extrarenal deposits of oxalate occur in later stages, includingin the heart, vessels, bones, and retina. Death from renal failureoccurs in childhood or early adult life. Therapy with pyridoxine,phosphates, magnesium, and citrate have proven helpful (108),but combined liver and kidney transplants are required to effecta cure because the metabolic abnormality originates in the liver(109).
The disease is caused by functional defects of the liver enzymealanine-glyoxylate aminotransferase (AGT). This results in failureof catabolism of glyoxalate to glycine, leading to overuse ofthe alternate pathway whereby oxalate is generated. The kidneyrepresents the only excretory pathway for oxalate, which cannotbe further metabolized. AGT normally resides in peroxisomes.In some patients, mutations in the AGT gene on chromosome 2do not lead to loss of the protein, but instead promote mistargetingto the mitochondrion (110,111). AGT mistargeting results fromthe combination of a common Pro11Leu polymorphism, which generatesa cryptic but functionally weak mitochondrial targeting sequence(MTS) and a rare Gly170Arg mutation. The combination of theseincreases the efficiency of this MTS by slowing the rate atwhich AGT dimerizes. This type of mistargeting represents ahighly unusual mechanism of disease.
Over the past few years, an increasing number of human diseaseshave become attributable to defects in the normal dynamic traffickingprocesses in all cells, and investigating the molecular basesfor these will continue to be a fertile field for new insightsinto cell biology. Indeed, such mechanistic defects are notjust the preserve of rare disorders. For example, there is evidencethat a problem in autosomal dominant polycystic kidney diseasemight be aberrant targeting of mutant polycystins, thereby disruptingnormal polarity and cell-cell adhesion (112). However, thesedata are still to some extent preliminary and are the subjectof intensive study. The next decade will undoubtedly increaseour understanding of the complex ways in which the kidney carriesout its many interrelated functions.
We are grateful to the UK National Kidney Research Fund (MAJD)and the Wellcome Trust (FEK) for support. We thank KatherineBorthwick for assistance with manuscript preparation.
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Introduction
Basic Trafficking
-subunit of the epithelial sodium channel (ENaC) gene, after which similar alterations in the 
-subunit were identified in other kindreds (27). By and large, these mutations are premature stop codons, resulting in truncations of the cytoplasmic C-terminal tail of the relevant subunit. Consistent with the dominant inheritance pattern, these are associated with a gain-of-function in ENaC. 
, 
