Download pdf - Nephrogenic Diabetes Insipidus in Children - Springer · Nephrogenic Diabetes Insipidus in Children Nine V. A. M. Knoersa* and Elena N. Levtchenkob aDepartments of Medical Genetics,

Nephrogenic Diabetes Insipidus in Children

Nine V. A. M. Knoersa* and Elena N. LevtchenkobaDepartments of Medical Genetics, University Medical Centre Utrecht, Utrecht, The NetherlandsbDepartment of Pediatric Nephrology, Department of Growth and Regeneration, University Hospitals Leuven,Katholieke Universiteit Leuven, Leuven, Belgium

History

First familial cases with diabetes insipidus were described byMcIlraith in 1892; however, he did notdistinguish between renal and neurohormonal forms of the disorder [1]. The renal type of diabetesinsipidus was appreciated as a separate entity more than 50 years ago, when it was describedindependently by two investigators: Forssman [2] in Sweden and Waring et al. [3] in the UnitedStates. In 1947, Williams and Henry [4] noticed that injection of antidiuretic hormone (ADH) indoses sufficient to induce systemic side effects could not correct the renal concentrating defect. Theycoined the term nephrogenic diabetes insipidus. Subsequent studies revealed active hormone in theserum and urine of affected persons and lent further support to the theory of renal unresponsivenessto vasopressin. Nephrogenic diabetes insipidus is synonymous with the terms vasopressin- or ADH-resistant diabetes insipidus and diabetes insipidus renalis.

Definition and Clinical Manifestations

Congenital nephrogenic diabetes insipidus (NDI) is a rare inherited disorder, characterized byinsensitivity of the distal nephron to the antidiuretic effects of the neurohypophyseal hormonearginine vasopressin (AVP). As a consequence, the kidney loses its ability to concentrate urine,which may lead to severe dehydration and electrolyte imbalance (hypernatremia andhyperchloremia). Patients with NDI have normal birth weight and pregnancies are not complicatedby polyhydramnios. The urine-concentrating defect in NDI is present from birth, and manifestationsof the disorder generally emerge within the first weeks of life. With breast milk feedings, infantsusually thrive and do not develop signs of dehydration. This is because human milk has a low saltand protein content and therefore a low renal osmolar load. With cows’ milk formula feedings, theosmolar load to the kidney increases, resulting in an increased demand for free water. This is usuallynot provided by oral feeding, and therefore hypernatremic dehydration appears. Irritability, poorfeeding, and poor weight gain are usually the initial symptoms [5]. Patients are eager to suck but mayvomit during or shortly after the feeding. Dehydration is evidenced by dryness of the skin, loss ofnormal skin turgor, recessed eyeballs, increased periorbital folding, depression of the anteriorfontanel, and a scaphoid abdomen. Intermittent high fever is a common complication of thedehydrated state, predominantly in very young children. Body temperature can be normalized byrehydration. Seizures can occur but are rare and most often seen during therapy, particularly ifrehydration proceeds too rapidly. Constipation is a common symptom in childrenwith NDI. Nocturiaand nocturnal enuresis are common complaints later in childhood.

*Email: [email protected]

Pediatric NephrologyDOI 10.1007/978-3-642-27843-3_36-1# Springer-Verlag Berlin Heidelberg 2014

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Untreated, most patients fail to grow normally. In a retrospective study of 30 male NDI patients,most children grew below the 50th percentile, most of them having standard deviation (SD) scoreslower than�1 [6]. Some well-treated patients, however, may achieve normal adult height. Catch-upgrowth occurs at least in some patients after normalization of water and electrolyte balance,especially in those with adherence to treatment. Bone maturation is generally not delayed [7].Weight-for-height SD scores are initially low, followed by global normalization at schoolage [6]. Initial feeding problems and the ingestion of large amounts of low-caloric fluid resultingin a decreased appetite may play roles in failure to thrive seen in NDI [8, 9]. Furthermore, it ispossible that repeated episodes of dehydration have some as yet undetermined negative effects ongrowth.

Mental retardation has long been considered an important complication of untreated NDI andassumed to be a sequel of recurrent episodes of severe brain dehydration and cerebral edema causedby overzealous attempts at rehydration [10–12]. Additional evidence underscoring the assumptionthat NDI has adverse effects on the cerebrum is provided by several reports describing intracranialcalcifications in NDI patients [13, 14]. Such lesions are generally considered to be the result ofhemorrhage or necrosis. Most of the reported patients with cerebral calcifications were mentallyretarded. Nowadays mental retardation is rare due to earlier recognition and treatment of NDI. Exactestimates of the current frequency of mental retardation under modern treatment are unknown, but inthe largest psychometric study ever reported, only 2 of the 17 male NDI patients (aged 3–30 years)had a total intelligence quotient more than 2 SD below the norm. Fourteen patients had anintelligence score within or above the normal range and one patient had a general index scorebetween �1 and �2 SD [15].

The psychological development of NDI patients is influenced by a persistent desire for drinkingand the need for frequent voiding, which compete with playing and learning. Therefore, many NDIpatients are characterized by hyperactivity, distractibility, short attention span, and restlessness. Inthe psychometric study mentioned earlier, the criteria for attention-deficit/hyperactivity disorderwere met in 8 of 17 tested NDI patients [15].

Persistent polyuria can result in the development of megacystis, trabeculated bladder wall,hydroureter, and hydronephrosis [6, 16, 17]. Urinary tract distension may be seen on ultrasoundexamination even in infants [18] and young children [19]. Potential complications of urinary tractdilatation are rupture of the urinary tract, infection, intractable pain, improper bladder function,and/or kidney failure. These complications may occur as early as the second decade of life. Large-capacity hypotonic bladder dysfunction might require clean intermittent catheterization[17]. Patients should be trained to void regularly in order to assure that maximal urinary bladdercapacity remains within normal range. Both patient groups with AVPR2 and AQP2 mutations candevelop urinary tract dilatation and bladder dysfunction [16, 20].

Diagnostic Procedures

The observation of polyuria in a dehydrated infant, together with the finding of a high serum sodiumconcentration and inappropriately diluted urine (Uosm < Posm), provides presumptive evidencefor a renal concentrating defect. To confirm the concentrating defect and to distinguish the renal formof diabetes insipidus from the central form, a vasopressin test is performed with 1-desamino-8-D-arginine vasopressin (DDAVP), a synthetic analogue of the natural arginine vasopressin that


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produces a high and prolonged antidiuretic effect. In the test, DDAVP (10 mg for infants<1 year old,20 mg for children>1 year old) is administered intranasally. Urine is collected during the subsequent5.5 h. The first collected portion of the urine should be discarded. The maximal urine osmolality inany collected aliquot is chosen as a measure of the concentrating capacity [21]. After DDAVPadministration, NDI patients are (1) unable to increase urinary osmolality, which remains below200 mOsm/kg H2O (normal values: <1 year old>600, between 1 and 2 years old between 600 and800, >2 years old >800 mOsm/kg H2O), and (2) cannot reduce urine volume or free-waterclearance.

Plasma vasopressin levels are normal or only slightly increased in affected children. Otherlaboratory findings have been described, which mainly result from chronic dehydration. Serumsodium concentration is generally elevated and may be above 170 mmol/L. There is also anincrease in serum chloride concentration and retention of urea and creatinine. All values arenormalized by adequate rehydration. In addition, reduced glomerular filtration rate (GFR) andrenal blood flow can return to normal when a normal hydration state has been achieved.

The primary congenital form of NDI has to be differentiated from central diabetes insipidus (dueto lack of AVP) and from the secondary or acquired forms, which are much more common [22].In our experience, the urinary osmolality obtained after DDAVP administration in secondarydisorders is always higher than in NDI. Several secondary causes, some of which will be discussedlater, are listed in Table 1.

Table 1 Causes of secondary nephrogenic diabetes insipidus

Monogenetic diseases associated with secondary NDI

Renal Fanconi syndromes

Bartter syndrome (type 1 or type 2)

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis

Distal renal tubular acidosis (dRTA)

Apparent mineralocorticoid excess (AME)

Ciliopathies (nephronophthisis, Bardet–Biedl syndrome, etc.)

Other renal diseases

Obstructive uropathy

Renal dysplasia

Postischemic damage

Amyloidosis

Sarcoidosis

Chronic renal failure

Renal impairment in sickle-cell disease or trait

Drug induced

Lithium

Ifosfamide

Amphotericin B

Tetracyclines

Biochemical abnormalities

Hypercalcemia, hypercalciuria, and nephrocalcinosis

Hypokalemia


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Cellular Physiology of Arginine Vasopressin’s Antidiuretic Action in theDistal Nephron

The physiologic action of vasopressin on the renal collecting duct has been one of the mostintensively studied processes in the kidney. Arginine vasopressin (AVP, ADH) is synthesized onthe ribosomes of the magnocellular neurons of the supraoptic and paraventricular nuclei of thehypothalamus as a large biologically inactive bound form. Within storage granules, the hormone iscleaved into the biologically active form and transported down the neuronal axons to the posteriorpituitary and stored there. Following appropriate stimuli, AVP is secreted from the posterior pituitaryinto the circulation as biologically active hormone. AVP release is regulated by changes in plasmaosmolality (by >2 %) but can also occur in response to nonosmotic stimuli. These nonosmoticstimuli are generally related to changes in either total blood volume or the distribution of extracel-lular fluid. Patients with depleted effective circulating volume may secrete ADH even in thepresence of low plasma osmolality. In addition, physical pain, emotional stress, and certain drugs(e.g., nicotine) influence the release of AVP. In its effector organ, the kidney, AVP binds tovasopressin type-2 (V2) receptors on the basolateral membrane of the principal inner medullarycollecting duct cells and of the arcade cells (Fig. 1, review in Ref. [23]). The arcades are long, highlybranched renal tubule segments that connect distal convoluted tubules of several deep andmidcortical nephrons to the origin of cortical collecting ducts. Upon binding of AVP, the V2 receptoris activated and then stimulates GTP loading of the small GTPase-aGS – subunit of its coupledtrimeric G-protein – eventually leading to dissociation of the G-protein from the receptor. GTP-aGS

can then bind to the membrane-associated adenylate cyclase (AC), activating it, which results in anincrease in intracellular cyclic adenosine monophosphate (cAMP) from adenosine triphosphate(ATP). The elevated cAMP levels stimulate protein kinase A (PKA), leading to phosphorylationof AQP2 which in turn initiates a redistribution of aquaporin-2 (AQP2) water channels fromintracellular vesicles to the apical plasma membrane, rendering this membrane water permeable.The increase in apical membrane permeability allows water to flow from the tubule lumen to thehypertonic medullary interstitium, via AQP2 in the apical membrane and via AQP3 and AQP4,constitutive water channels in the basolateral membrane. This then leads to the formation ofconcentrated urine. Upon fluid intake, AVP release into the blood decreases, AQP2 is redistributedinto intracellular vesicles, and water reabsorption is reduced. Katsura et al. have shown that theAVP-regulated recycling of AQP2 can occur at least six times with the same molecules [24].

In recent years, our knowledge of the AQP2 dynamics in the cell has increased significantly(Fig. 1). For further details the reader is also referred to several excellent reviews on this subject(review in Refs. [23, 25–28]).

AQP2 is 1 of the 13members of the aquaporin family of water channels. After transcription AQP2is folded into its native monomeric conformation in the endoplasmic reticulum, and homotetra-merization takes place [29]. The tetramers are forwarded to the Golgi apparatus, where two out offour monomers are complex N-glycosylated. These functional water channels are then stored inendosomal vesicles to be transported to the apical membrane [30].

Phosphorylation of a PKA-consensus site in AQP2, the serine at position 256 in the cytoplasmiccarboxy-terminus, is absolutely essential for AQP2 delivery to the apical membrane [31, 32]. Inaddition, it has been shown that anchoring of PKA to PKA-anchoring proteins (AKAPs), whichensures targeting of PKA to AQP2-bearing vesicles, is another prerequisite for AVP-mediated AQP2translocation [33]. Studies using oocytes as a model system indicated that for plasma membranelocalization three out of four monomers in an AQP2 tetramer need to be phosphorylated [34].


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PKA is the main kinase for AQP2 phosphorylation, but other kinases may potentially participatein the regulation of AQP2 trafficking. Besides PKA sites, putative phosphorylation sites for PKG,PKC, and casein kinase II are also present in the AQP2 sequence.

The molecular machinery for the docking and fusion of AQP2-containing vesicles with the apicalmembrane is similar to the process of synaptic vesicle fusion with the presynaptic membrane andinvolves vesicle (v) SNAREs (soluble NSF attachment protein receptors) and target membrane(t) SNAREs. The apical membrane-specific t-SNARE is syntaxin 4, which interacts specifically with

Fig. 1 Intracellular signal transduction pathway initiated by AVP binding to V2R. Via activation of adenylate cyclaseand cAMP-production stimulation, PKA is activated and phosphorylates its target proteins AQP2, Rho-GDI, andCREB-1. The transcription factor CREB-1-p stimulates AQP2 transcription, Rho-GDI-p initiates actin reorganizationrequired for AQP2 transport, and AQP2-p homotetramers are transported to the apical membrane. There they render themembrane permeable for water, which is reabsorbed from the passing pro-urine and transported back into thebloodstream by AQP3 and AQP4. Rab5-mediated AQP2 endocytosis by clathrin-coated vesicles is triggered byshort-chain ubiquitination and leads to termination of the response. Internalized AQP2 vesicles are transported toearly and late endosomes as well as multivesicular bodies (MVBs) for storage. From MVBs they can then either belysosomally degraded or recycled via the Rab11-dependent slow-recycling pathway (From Ref. [23], with kindpermission from Springer Science and Business Media)


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the v-SNARE protein VAMP2 located on the cytoplasmic side of AQP2-containing endosomalvesicles [35–38]. V- and t-SNAREs are recycled by the AAA-type ATPase NSF. Reorganization ofthe actin cytoskeleton is another important mechanism required for AQP2 transport and accumula-tion at the apical membrane [39, 40]. The actin cytoskeleton most likely provides a network thatanchors the AQP2-bearing vesicles in the unstimulated cell. Vasopressin has been shown todepolymerize apical F-actin in rat inner medullary collecting duct, resulting in the fusion ofAQP2-carrying vesicles with the apical membrane [41], indicating that reorganization of the apicalactin network may be critical in promoting the trafficking of AQP2-bearing vesicles. Rho inhibitionthrough PKA-mediated phosphorylation of Rho-GDP dissociation inhibitor (Rho-GDI) is shown tobe a key event for actin reorganization inducing AQP2 translocation [39, 40].

Counterbalancing increased expression on the plasma membrane, AQP2 is internalized. Duringthis endocytotic process, AQP2 accumulates in clathrin-coated pits and is internalized via a clathrin-mediated process [42]. Endocytosis is regulated by short-chain ubiquitylation at lysine 270 (K270)in the AQP2 terminal tail [43, 44].

To be available for recycling, AQP2-containing endosomes need to be redistributed to theperinuclear region. This process is mediated by dynein-dependent transport along microtubules[45, 46]. Specificity of the endocytotic AQP2 internalization is mediated by Rab5 protein, aneffector-binding factor involved in plasma membrane-to-early-endosome transport [47]. From theendosomal system – early/late endosomes and/or multivesicular bodies (MVBs) – AQP2 is eitherrecycled by the Rab11-dependent slow-recycling pathway or marked for lysosomal degradation[48]. Prolonged K270 ubiquitylation induces MVB trafficking and localization to internal vesiclesof MVBs followed by lysosomal degradation, while deubiquitylation increases localization to earlyendosomes and the limiting membrane of MVBs and enables AQP2 recycling [44].

Long-term adaptation to circulating AVP levels, for instance, in a dehydrated state, is accom-plished by increasing the expression of AQP2 mRNA and protein. PKA-mediated phosphorylationof a cAMP-responsive element-binding protein 1(CREB-1) stimulates synthesis of AQP2 bybinding to the AQP2 gene promotor and activating its transcription, which increases intracellularAQP2 levels [49].

Genetics

Three different inheritance patterns of NDI have been recognized. In most cases (about 90 %), NDIis transmitted as an X-linked recessive trait (MIM304800). In these families, female carriers who areusually unaffected transmit the disease to sons, who display the complete clinical picture [2, 4,50]. In 1988, the major NDI locus was mapped to the distal region of the long arm of theX chromosome (Xq28) [51], and in 1992 mutations in the AVPR2 gene were shown to underlieX-linked NDI [52–54]. In a minority of families (about 10 %), the transmission and phenotypiccharacteristics of NDI are not compatible with an X-linked trait. In these families, females displaythe complete clinical picture of NDI and are clinically undistinguishable from affected male familymembers [55–57]. Family pedigrees suggested the existence of both an autosomal recessive (MIM222000) and an autosomal dominant form (MIM 125800) of NDI. It was subsequently demonstratedthat both autosomal forms of NDI are caused by mutations in the AVP-sensitive aquaporin-2 waterchannel [58, 59]. The prevalence of NDI is not exactly known, but the disease is assumed to be rare.The estimate of the prevalence of NDI in Quebec, Canada, is 8.8:1,000,000 males [60]. In the Dutchpopulation of about 16 million, at least 50 different families are known.


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X-Linked Nephrogenic Diabetes Insipidus: Mutations in the AVPR2 Gene

The X-linked form of NDI is caused by inactivating mutations in the AVPR2 gene (MIM 300538)([52–54], reviews in Refs. [23, 28, 61]). AVPR2 is a relatively small gene, consisting of three exonsseparated by two short intervening sequences (introns); two isoforms are known that are generatedby alternative splicing [62]. AVPR2 is localized on the X chromosome on locus Xq28. The cDNAencodes a receptor protein of 371 amino acids, has a predicted molecular mass of approximately40 kDa, and shares the general structure of a G-protein-coupled receptor consisting of sevenhydrophobic transmembrane helices, connected by extracellular and intracellular loops. The recep-tor contains one unique consensus sequence site for N-linked glycosylation in the extracellularamino-terminus [63] and phosphorylation sites for G-protein-coupled receptor kinases (GRK)represented by a serine cluster in the carboxy-terminus [64, 65]. The N-terminal part of the proteinincluding the first transmembrane domain and the positively charged first intracellular loop areimportant for proper insertion and orientation in the membrane [66]. A conserved glutamate-dileucine motif in the intracellular carboxy-terminal part of the receptor is essential for receptortransport from the endoplasmic reticulum (ER) to the Golgi apparatus [67]. Two conserved adjacentcysteines in the C-terminus are palmitoylated, thereby anchoring the carboxy tail to the plasmamembrane and controlling the tertiary structure of this region of the receptor [68].

At this writing, more than 240 distinct disease-causing mutations in AVPR2 have been identified,and the number is constantly increasing ([69, 70] and review in Refs. [23, 28, 61] and www.hgmd.org). The mutations are not clustered in one domain of the V2 receptor but are scattered throughoutthe protein, except for the part coding for the N- and C-terminal tails of the receptor. More than 50 %of the mutations are missense mutations. Nucleotide deletions and insertions causing frameshifts(26 %), nonsense mutations (13 %), large deletions (7 %), large in-frame insertions/duplications(1 %), splice-site mutations (1 %), and complex rearrangements (2 %) account for the remainder ofmutations ([12, 69], and review in Refs. [23, 28, 61, 71]). In addition to these disease-causingmutations, at least 21 AVPR2 variations that do not lead to disease are known. These non-disease-causing variations are most likely polymorphisms that can be found in more than 1 % of thepopulation. The G12E mutation, for example, has also been found in non-affected individuals,suggesting that it belongs to this class of polymorphisms that do not exert a significant effect onproper functioning of the V2 receptor [72]. Several mutations are recurrent as evidenced by the factthat these mutations were found on different haplotypes in ancestrally independent families. Themost frequent of these recurrent mutations (D85N, R106C, R113W, R137H, S167L, and R337X)occur at potential mutational hot spots.

AVPR2 mutations seem to be present in all ethnical groups tested with no preference of onemutation for any ethnic group over others.

The molecular mechanism underlying the renal insensitivity for AVP differs between mutants. Asupcoming pharmacological treatments for NDI likely depend on the underlying mechanism, GPCRmutations in general and V2 mutations in particular have been divided in different classes accordingto their cellular fate [73, 74].

Class I comprises all mutations that lead to improperly processed or unstable mRNA, likepromoter alterations, exon skipping, or aberrant splicing. This class also holds frameshift andnonsense mutations, which result in truncated proteins like W71X, 458delG (frameshift, 161X),and R337X.

Class II mutations are missense or insertions/deletions of one or more nucleotide triplets, resultingin fully translated proteins. Due to the mutation, however, mutant receptors are misfolded andretained in the endoplasmic reticulum (ER), as the ER is the organelle that has the cellular quality


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http://www.hgmd.org

http://www.hgmd.org

control over proper folding and maturation of synthesized proteins. Misfolded proteins are subse-quently mostly targeted for proteasomal degradation [75]. Intracellular entrapment of missense V2Rmutants and their rapid degradation likely represents the most important cause of NDI, as more than50 % of the mutations in V2R are missense mutations and cellular expression revealed that most ofthese result in ER-retained proteins. The amount of retention and degradation varies within thisclass, since different mutations affect protein folding to a different extent, sometimes allowingpartial transport of at least partially active receptors to the plasma membrane [76].

Class III mutations result in full-length receptors expressed at the cell surface, but interfere withproper interaction with their natural ligands, thereby causing reduced/abolished signaling [77]. ClassIII mutations can be subdivided into two minor groups. IIIa mutations interfere with binding of orsignal transduction to the coupled trimeric G-protein, leading to a reduced activation of adenylatecyclase and thus formation of cAMP. Mutations in this group are missense mutations and in-framedeletions, mostly located in transmembrane and intracellular domains. Examples are the D85N andP322S mutations [78]. IIIb mutations interfere with, or reduce, AVP binding. These mutations,which are also mostly missense and small in-frame deletions or insertions, especially involveresidues thought to be in or close to the AVP-binding pocket, of which delR202 is a clearexample [79].

Finally, class IV is assigned to all mutations that neither interfere with protein synthesis ofmaturation, not with ligand binding, but affect other aspects of protein function. The NDI R137Hmutation, located in the well-conserved DRY/H motif of GPCRs, is the best-characterized exampleof this class. The effect of this mutation is constitutive internalization of V2, leading to reducedexpression of the receptor in the plasma membrane and thereby reduced adenylate cyclase-dependent cAMP signaling upon AVP binding [80, 81].

Sometimes, mutants do not exert a full phenotype of a particular class and then often also showfeatures of another class. For example, some V2R missense mutants are partially ER retained(class II), but are also partially expressed in the plasma membrane, where they might show a reducedG-protein coupling (class IIIa) or AVP binding (class IIIb). As such, it provides an explanation forthe observed small antidiuretic response to high doses of DDAVP in NDI patients harboring suchmutations [82].

Genotype-Phenotype Correlations in X-Linked NDIAlmost all mutations in the V2 receptor gene result in a uniform clinical NDI phenotype withpolyuric manifestations in the first weeks of life and poor growth. There are, however, a fewexceptions to this rule. Several mutations appear to be associated with a milder form of NDI,characterized by a later manifestation, not at birth but later in childhood, and without growthretardation. Examples of mutations causing partial NDI are D85N, V88M, G201D, M311V,N317S, P322S, and S329R [83–86]. Functional studies of some of these mutations by in vitroexpression systems have confirmed the partial phenotype of the NDI. P322S is the most remarkableof these three mutations, since another mutation substituting proline 322, namely, P322H, isassociated with a severe phenotype. By in vitro expression of both P322H and P322S in COS-7cells, Ala et al. [79] have shown that the P322H mutant had totally lost the ability to stimulate theGs/adenylate cyclase system, whereas the P322S mutant was able to stimulate adenylate cyclase,albeit less than the wild-type receptor. Thus, the in vitro experiments closely correspond to theclinical phenotype. On the basis of three-dimensional modeling of the P322H and P322S mutantreceptors, a plausible hypothesis to explain the molecular basis for the mild phenotype of the P322Shas been proposed. Based on this modeling, it is suggested that complete loss of function of the


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P322H receptor could be due, in part, to hydrogen bond formation between the His322 side chainand the carboxyl group of Asp85, which does not occur in the P322S receptor [79].

Intrafamilial variability of the X-linked NDI phenotype has also been described. A nice exampleis the case described by Kalenga et al., who reported a Belgian family in which the R137H mutationwas associated with severe NDI in the proband but with very mild NDI in his affected brother[87]. Genetic and/or environmental modifying factors are likely to account for this intrafamilialphenotype variability.

The Autosomal Recessive and Autosomal Dominant Forms of NephrogenicDiabetes Insipidus: Mutations in the Aquaporin-2 Water Channel

Both the autosomal recessive and the autosomal dominant types of NDI are caused by mutations inthe AQP2 water channel gene (MIM 107777; GenBank accession number z29491). The humanAQP2 gene is a small gene consisting of 4 exons, comprising 5 kb genomic DNA. The 1,5 kbmRNAencodes a protein of 271 amino acids which has a predicted molecular weight of 29 kDa [88]. AQP2belongs to a family of membrane integral proteins, aquaporins, which function as selective watertransporters throughout the plant and animal kingdom. In mammals, 13 different aquaporins havebeen identified to date, 8 of which (aquaporins 1–4, 6–8, and 11) are highly expressed in the kidney.Like other aquaporins, AQP2 is assembled in the membrane as a homotetramer in which each29 kDa monomer, consisting of six membrane-spanning a-helical domains and intracellular N- andC-termini, is a functional water channel. The six transmembrane domains are connected by fiveloops (A through E). The mechanism of selectivity for water of aquaporins in general has beenrevealed in the homologous AQP1 protein [89, 90] and has further been strengthened by moleculardynamics modeling approaches [91, 92] and was recently also confirmed for AQP2 [93]. The waterpore is formed between the first and sixth transmembrane domains and is lined by the intracellularB-loop and the extracellular E-loop. AQP2 is exclusively localized in the apical membrane and asubapical compartment of collecting duct cells. It is upregulated by dehydration or AVP, indicatingthat it is the AVP-regulated water channel.

To date, 46 putative disease-causing mutations in AQP2 have been identified in families withautosomal recessive NDI ([69, 70, 94–96], reviews in Refs. [23, 61, 71]). These include 38 missensemutations, 2 nonsense mutations, 3 small deletions, and 3 splice-site mutations. Most mutations arefound between the first and last transmembrane domain of AQP2. Expression studies in Xenopuslaevis oocytes have revealed that most AQP2 missense mutations that cause recessive NDI are classII mutations. Thus, these mutations lead to misfolding of the mutant protein, retention in theendoplasmic reticulum (ER), and rapid degradation of AQP2 (review in Refs. [23, 78]). Inagreement with extensive degradation, AQP2 could not be detected in the urine of patients withrecessive NDI [97]. When overexpressed in oocytes and Chinese hamster (CHO) cells, six ofthese AQP2 mutants (A147T, T126M, G64R, L22V, A47V, and T125M) confer water permeability[98, 99]. This indicates that at high expression levels, these AQP2 mutant proteins escape from theER and are routed to the plasma membrane, where they are functional. In terms of possible treatmentstrategies, these results are of high importance, since they suggest that functional channels may bestimulated to reach the plasma membrane by restoring mutant trafficking (discussed later intreatment).

One AQP2 missense mutation, P262L, located in the AQP2 C-terminal tail, a region until thenbelieved to result in dominant NDI, surprisingly was found to be involved in recessive NDI [100].In cell biological experiments, it was shown that the P262L mutant is a functional water channel that


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forms heterooligomers with wt-AQP2. These wt-AQP2/AQP2-P262L heterotetramers are located inthe apical membrane, indicating that the apical sorting of wild-type AQP2 is dominant over themissorting signal of AQP2-P262L. This is different from dominant NDI, because in this formmutants retain wt-AQP2 in intracellular locations (see below). The recessive inheritance in the twopatients encountered (patients were heterozygous for a R187C or A190T mutation on one allele,combined with a P262Lmutation on the other allele) can be explained as follows: AQP2-R187C andAQP2-A190T are retained in the ER and do not interact with AQP2-P262L. AQP2-P262L foldsproperly and assembles in homotetramers, but will be retained mainly in intracellular vesicles. Theconsequent lack of sufficient AQP2 proteins in the apical membrane of the patients’ collecting ductcells explains their NDI phenotype. In the parents coding for wt-AQP2 and AQP2-R187C or AQP2-A190T, wt-AQP2 will not interact with either mutant but will form homotetrameric complexes, ofwhich the insertion into the apical membrane will be regulated properly by vasopressin and will givea healthy phenotype. In the parents coding for wt-AQP2 and AQP2-P262L, both proteins likelyassemble into heterotetramers. The dominancy of wt-AQP2 sorting on the localization of AQP2-P262Lwill result in proper AVP-regulated trafficking of the heterotetrameric complexes to the apicalmembrane and will also give a healthy phenotype [100].

At present ten families have been described with autosomal dominant NDI, initially uncovereddue to father-to-son transmission of the disease. In these families subsequent sequencing of theAQP2 gene revealed putative disease-causing mutations of one AQP2 allele. The identified muta-tions in AQP2 comprise small deletions, insertions, and missense mutations ([69], review in Refs.[23, 101]). All mutations causing dominant NDI are located in the coding region of the C-terminaltail of AQP2, which is not part of the pore-forming segment but contains important sorting signalsthat govern intracellular transport of the protein [78, 102]. Indeed, all mutants AQP2 proteins foundin dominant NDI appeared to be folded functional water channels that were sorted to othersubcellular locations in the cell than wt-AQP2, e.g., late endosomes/lysosomes and the basolateralmembrane. Because none of these mutants was misfolded, they were, in contrast to AQP2mutants inrecessive NDI, able to interact and form heterotetramers with wt-AQP2. Due to this wt-mutantinteraction and the dominancy of the missorting signals in the mutant protein, the wt-mutantcomplexes are also missorted. Formation of heterotetramers with wt-AQP2 has been shown formost of the dominant AQP2 mutants. For instance, expression studies in polarized cell lines haverevealed that the dominant AQP2-E258K mutant is routed to the Golgi complex or late endosomes/lysosomes [59]. In co-expression studies with wild-type AQP2, a dominant-negative effectwas observed, caused by impaired routing of wild-type AQP2 to the plasma membrane afterhetero-oligomerization with the E258K mutant [103]. Mistargeting to the basolateral membranehas been reported for the AQP2-721delG, AQP2-763-772del, AQP2-812-818del, and AQP2-779-780insA mutants [102, 104]. The AQP2-727delG mutant was shown to interfere with the routing ofwild-type AQP2 to the apical membrane by its mistargeting to the basolateral membrane andlate endosomes/lysosomes [104]. The loss of appropriate AQP2 heterotetramer trafficking indominant NDI is caused by several mechanisms. The phosphorylation site at Ser256, serving asan introducible apical sorting signal, may be inactivated, overruled by basolateral sorting signals, orreprogrammed to induce basolateral sorting, all causing intracellular misrouting ([105–108] andreview in Ref. [23]).

Dominant NDI patients have a milder phenotype when compared to milder recessive forms ofNDI, suggesting that some wt-homotetramers are formed that are able to reach the apicalmembrane [103].


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Differential Diagnosis Between the X-Linked and the Autosomal Forms of NDIWith a few exceptions, no differences in clinical symptoms between X-linked and autosomalrecessive forms of NDI can be observed, nor in the time of onset of the disease. Only in a minorityof patients with the X-linked form of NDI, namely, those individuals carrying V2R mutations withpartial insensitivity to AVP, the disease onset is not directly after birth but later in childhood. Ingeneral the initial symptoms in most autosomal dominant cases also appear later in childhood.

Male patients with X-linked NDI can be discriminated from patients with autosomal recessiveNDI on the basis of their extrarenal reaction to the intravenous administration of the syntheticV2-vasopressin analogue 1-desamino-8-D-arginine vasopressin (DDAVP). Patients with autosomalrecessive NDI show a decrease in blood pressure, an accelerated heart rate, and an increase in vonWillebrand factor, factor VIII, and tissue-type plasminogen activator levels, whereas in patients withX-linked NDI, these extrarenal responses are absent as a result of an extrarenal mutant V2R [109]. Infemale patients, the interpretation of this intravenous DDAVP test is more complicated. Althoughabsence of the extrarenal responses to intravenous administration of DDAVP in females clearlypoints to the presence of a V2R defect, a normal response cannot be interpreted as indicative of adefect beyond the V2R and thus an AQP2 defect. For instance, a symptomatic female patientdescribed byMoses et al., who was shown to be heterozygous for a V2Rmutation, showed a twofoldincrease in factor VIII activity after administration of DDAVP [110]. The discrepancy between therenal and extrarenal response to DDAVP in these female V2R mutation carriers might be explainedby variability in the pattern of X-inactivation between different tissues.

Nephrogenic Diabetes Insipidus in Females

Several families have been described in which females show classical clinical and laboratoryfeatures of NDI. After the identification of AQP2 mutations as a cause for autosomal recessiveNDI, and in some cases for autosomal dominant NDI, a satisfying explanation for the completemanifestation of the disease in some females had been found. However, several families have beenreported in which symptomatic females do not have an AQP2 defect but are heterozygous for a V2

receptor defect [111–114]. In some of these women, maximal urinary osmolality after DDAVPadministration does not exceed 200 mosmol/L. Of interest, in some of the reported families,asymptomatic female family members shared the same V2 receptor mutation with the manifestingfemales [111, 115]. The most likely explanation for the existence of different phenotypes in carriersof a V2 receptor mutation, varying from no symptoms to complete manifestation of the disorder, isskewed X-inactivation [116]. This hypothesis was underlined by studies investigating theX-inactivation patterns in peripheral blood leukocytes of female carriers via the detection of amethylated trinucleotide repeat in the human androgen receptor gene [117]. In asymptomaticfemales random X-inactivation was found, while in most female carriers who showed clinicalNDI symptoms, skewed X-inactivation patterns occurring preferentially to normal X alleles wererecognized. In a few females with over clinical NDI, however, randomX-inactivation was identified.

In conclusion, clinical NDI phenotypes may correlate with the X-inactivation patterns in femaleswith heterozygote V2R mutations. In some female carriers, however, the clinical phenotype cannotbe predicted by evaluation of X-inactivation patterns in peripheral blood cells, probably due to thefact that X-inactivation ratios within an individual may vary between different tissues.


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Acquired Nephrogenic Diabetes Insipidus

Although the hereditary forms of NDI are relatively rare, a wide range of pathologic conditions anddrug treatments can lead to acquired NDI (Table 1). In our experience, the urine osmolality obtainedafter DDAVP administration in these acquired disorders is always higher than in congenital NDI. Allthese disorders have been shown to coincide with decreased expression of AQP2 or deregulatedAQP2 trafficking to the apical membrane [118–123]. For instance, prolonged treatment withlithium, the drug of choice for treating bipolar disorders and prescribed to 1 in 1,000 of thepopulation, leads to the development of NDI in at least 20% if treated individuals [124]. Thedevelopment of lithium-NDI is believed to occur in two phases. In the first short-time phase, lithiumcauses a decrease in AQP2 expression ([125], review in Ref. [126]). Lithium enters the cells viaepithelial sodium channel (ENaC) and accumulates in principal cells [125, 127]. How lithiumdownregulates AQP2 is not clear but likely involves glycogen synthase kinase type 3b, which isimportant in AVP-regulated antidiuresis and is inhibited by lithium [128–130]. Lithium alsoinfluences AQP2-mediated water reabsorption by increased tubular release of prostaglandin E2[129]. In the second phase, lithium reduces the percentage of principal cells in the collecting duct tothe advantage of intercalated cells, involved in acid-base homeostasis [131]. The exact contributionof this collecting duct remodeling in the lithium-induced resistance to vasopressin remains to beelucidated.

Treatment

Conventional TreatmentSymptomatic treatment of NDI is focused on establishing and maintaining normovolemia byreplacing urinary water losses and reducing urinary volume. Adequate supply of fluid to preventdehydration is the most important component of the therapy. For reducing urine output, a low-solutediet is applied to diminish the renal osmolar load and decrease obligatory water excretion [132]. Ini-tially, a diet low in sodium (1 mmol/kg per day) as well as protein (2 g/kg per day) wasrecommended. However, severe limitations of dietary protein may introduce serious nutritionaldeficiencies. Therefore, it is preferable to prescribe dietary restriction of sodium only.

Diuretics such as hydrochlorothiazide (2–4 mg/kg per 24 h) were the first class of drugs shown tobe effective in lowering the urine volume in NDI [133]. When combined with a reduction of saltintake, hydrochlorothiazide reduces urine volume by 20–50 % of baseline values. However,thiazide-induced hypokalemia may cause further impairment or urine-concentrating ability inpatients with NDI. Another possible risk associated with hypokalemia is cardiac arrhythmia.Simultaneous administration of potassium salt is therefore advised in most cases. Very low dailysodium intake in combination with thiazide diuretics should be avoided to prevent the developmentof hyponatremia.

There is ample evidence that the combined administration of hydrochlorothiazide with either aprostaglandin-synthesis inhibitor, such as indomethacin (2 mg/kg per 24 h), or the potassium-sparing diuretic amiloride, is much more effective in reducing urine volume than the thiazidediuretic alone [134–138]. Prolonged use of prostaglandin-synthesis inhibitors, however, is oftencomplicated by gastrointestinal and hematopoietic side effects. Gastrointestinal complaints andcomplications include anorexia, nausea, vomiting, abdominal pain, ulceration, perforation, andhemorrhage. Hematopoietic reactions include neutropenia, thrombocytopenia, and, rarely, aplastic


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anemia. In addition, renal dysfunction has been described during indomethacin therapy, most oftenconsisting of a slight reduction in GFR.

In patients, who are not tolerating indomethacin, selective inhibitors of cyclooxygenase-2(COX-2) might be helpful [139]. Caution in using indomethacin and selective COX-2 inhibitorsin NDI is warranted as their administration can potentially lead to the acute deterioration of renalfunction in dehydrated patients.

Amiloride counterbalances the potassium loss from prolonged use of thiazides and thus preventshypokalemia. Since amiloride appears to have only minor long-term side effects, the combination ofhydrochlorothiazide (2–4 mg/kg/24 h) with amiloride (0.3 mg/kg/24 h) is the first choice oftreatment. Our personal experience of more than 20 years with the amiloride-hydrochlorothiazidecombination, however, indicates that amiloride is less well tolerated in young children below the ageof 4–6 years because of persistent nausea. Therefore we advise the temporary use of the combinationof indomethacin-hydrochlorothiazide in these young children.

For a long time the following mechanism for the antidiuretic effect of thiazides in NDI has beenproposed: thiazides reduce sodium reabsorption in the distal tubule by inhibition of the NaClcotransporter (NCC). This subsequently results in increased sodium excretion, extracellular volumecontraction, decreased glomerular filtration rate, and increased proximal sodium and waterreabsorption. Consequently, less water and sodium reach the collecting tubules and less water isexcreted [140, 141]. This long-standing hypothesis has been challenged by Magaldi, who reportednew insights into the possible mechanism of action, based on microperfusion studies in rat innermedullary collecting duct (IMCD) [142, 143]. In these studies it was shown that in the absence ofvasopressin, hydrochlorothiazide, when added to the luminal side, increased osmotic and diffusionalwater permeabilities, thus, decreasing water excretion. When prostaglandins were added, the effectof thiazides decreased. This finding may offer one explanation why indomethacin potentiates theeffect of thiazides in NDI [143]. Antidiuretic effect of thiazides is associated with an increase inAQP2 expression in collecting duct cells [144]. Long-term side effects of chronic thiazide admin-istration such as hyperuricemia, alterations in serum lipid spectrum, and glucose intolerance shouldbe monitored.

Although the drugs mentioned above reduce urine excretion, they are unable to achieve urinevolumes produced in healthy individuals. Therefore the general problem remains, although thesymptoms are relieved. Consequently, current research focuses on methods to treat NDI on a morecausative level than solely try to fight the symptoms (Fig. 2; for detailed reviews [23, 28]).

Therapeutic Strategies for Treatment of X-Linked NDIBecause in vitro expression studies reveal that the majority of AVPR2 mutations in X-linked NDIresult in normal protein that is retained within the endoplasmic reticulum (ER), agents that restoreplasma routing are under investigation as potential treatments. Promising agents are cell-permeableV2R antagonists and agonists that in vitro rescue the intracellular retention of several V2R mutants[145–148]. An important problem with the antagonists is that once the mutant V2R is rescued to thebasolateral membrane, the antagonist needs to be displaced by high concentrations of AVP/DDAVPto induce cAMP signaling. Therefore, low-affinity antagonists are believed to have the highestclinical value. However, their efficiency in rescuing is lower than that of high-affinity ligands, andthe high concentrations required to be administered for sufficient activity by low-affinity antagonistsmight lead to severe complications in patients. The use of non-peptide agonists has somewhatcircumvented this problem since they do not need displacement to activate V2R. All high-affinity


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agonists have been shown to induce receptor maturation as well as translocation to the plasmamembrane and to elicit a cAMP response [148].

Fig. 2 Therapeutic approaches to treat NDI. Approaches 1–4 focus on X-linked NDI, 5–6 are suited for autosomalrecessive NDI, and 7 applies to autosomal dominant NDI. (1) Cell-penetrating V2R antagonists induce native foldingand rescue V2R to the basolateral membrane. Displacement of the antagonist by high AVP/DDAVP concentrations isrequired to induce cAMP signaling. (2) Agonists function similarly, but do not need displacement to activate V2R. (3)ER-penetrating agonists can stimulate misfolded V2R without inducing maturation and induce prolonged signaling. (4)Stimulation of EP2 by butaprost activates cAMP production as well as AQP2 phosphorylation and targets AQP2 to theapical membrane without involvement of V2R. (5) Glycerol acts as pharmacological chaperone in high concentrations,rescuing AQP2 mutants from the ER. (6) Hsp90-inhibitor 17-AAG enables AQP2 mutant escape from the ER. (7)Rolipram inhibits PDE-4 and increases AQP2 concentration in the apical membrane by slowing down dephosphory-lation and downregulating re-internalization (From Ref. [23], with kind permission from Springer Science and BusinessMedia)


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The feasibility of treatment with these so-called pharmacologic “chaperones” has been testedin vivo. In individuals with NDI who have missense AVPR2 mutations, Bernier et al. showed thattreatment with a non-peptide V1a receptor antagonist had beneficial effects on urine volume andosmolality starting a few hours after administration. However, the long-term effect of this drug couldnot be tested because the clinical development of this V1a receptor antagonist was interrupted duringthe course of this study as a result of possible interference with the cytochrome P450 metabolicpathway [147].

Remarkably, certain non-peptide V2R agonists, such as OPC51, VA88, and VA89, were shown tobe able to intracellularly stimulate the V2R and increase cAMP production and AQP2 translocationto the apical membrane [149]. In contrast to pharmacochaperone-assisted folding and rescue of thereceptors, the localization andmaturation state of the V2R did not change upon activation, indicatingthat these compounds do not act as molecular chaperones. The mode of action by which receptorstrapped intracellularly can still activate their coupled G-protein and how this stimulates adenylatecyclase is not yet understood. Future in vivo and clinical testing has to confirm whether thepharmacological chaperones and the intracellularly acting non-peptide agonists have the desiredpositive effects in patients and meet the safety requirements.

In patients with X-linked NDI, bypassing the V2R could be an alternative way to treat the disease.By stimulation of the E-prostanoid receptor EP4, NDI symptoms were greatly reduced in a

conditional AVPR2-deletion mouse model [150]. This was due to raised AQP2 levels, most probablyas a consequence of cAMP production caused by EP4 stimulation. Recently, a similar effect wasseen after stimulation of the EP2 receptor by the agonist butaprost [151]. The EP2 receptor is a moreinteresting candidate for treatment of NDI than the EP4 receptor since EP2 agonists have alreadybeen tested in clinical studies for other diseases and have shown promising results concerning safetyissues. However, clinical trials in NDI are necessary to evaluate the effects and safety of EP2agonists for this disorder.

Another potential therapeutic strategy bypassing the V2R could be an activation of the cGMP-signaling pathway. Several groups have shown that nitric oxide donors and atrial natriuretic factorstimulate the insertion of AQP2 in renal epithelial cells in vitro and in vivo via a cGMP-dependentpathway without increasing the expression of AQP2 [152, 153], and the selective cGMP phospho-diesterase inhibitor sildenafil citrate (Viagra) prevents degradation of cGMP resulting in increasedmembrane expression in AQP2 in vitro and in vivo [154]. In a small number of NDI patientssubjected to clinical trials with sildenafil citrate, no decreases in urine volume or increases in urineosmolality were observed (personal communication in Ref. [28]). Alternative AVP-independentstrategies are the use of calcitonin, which has a vasopressin-like effect on AQP2 trafficking andurine-concentrating ability via cAMP-mediated mechanism [155] and of various statins(simvastatin, fluvastatin) that were reported to increase AQP2 expression and water reabsorptionin the kidney via an as yet unknown mechanism [156, 157]. Very recently, using a systemic high-throughput chemical screening procedure, Nomura et al. identified AG-490 (an EGF receptor andJAK-2 kinase inhibitor) as a compound that stimulates AQP2 exocytosis, induces AQP2 membraneaccumulation, and stimulates urine concentration in an AVP-independent manner [158]. Despitethese promising results in in vitro studies and in animal models, none of these compounds has yetbeen translated into therapy of NDI.

Therapeutic Strategies for Treatment of Autosomal NDISimilarly to V2R mutants, the majority of AQP2 mutants causing autosomal recessive NDI aremissense mutations that lead to aberrant folding of AQP2 in the ER. Hence, finding substances thatare able to reestablish natural AQP2 folding holds comparable promises for treatment of recessive


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NDI as it has been shown for the X-linked form. In CHO and MDCK cells, glycerol has proven theapplicability of chemical chaperones to AQP2 by restoring ER export in high concentrations [99].

Yang et al. described partial restoration of cellular AQP2 processing upon treatment of conditionalAQP2-T126M knock-in mice with an Hsp90 inhibitor, 17-allylamino-demethoxygeldanamycin(17-AAG), eventually resulting in improved urinary concentrating ability [159]. The preciseexplanation underlying the beneficial effect of this Hsp inhibitor remains to be elucidated. Further-more, it is not unlikely that Hsp90 inhibition may have severe side effects that outweigh theadvantages [160]. Therefore, lengthened studies addressing safety issues of Hsp90 or other chap-erone inhibitors have to be conducted in order to elucidate the applicability of these compounds inNDI therapy.

Based on the improvement of AVP-dependent cAMP signaling of collecting duct cells in ahypercalcemia-induced NDI mouse model, Sohara et al. also tested the phosphodiesterase-4 inhib-itor Rolipram in the knock-in dominant NDI mice [161]. Their data indicated that Rolipram is able toincrease cAMP levels leading to increased AQP2 phosphorylation and translocation to the apicalmembrane. Phosphodiesterase-4 is a common protein that also is involved in immunosuppressiveand anti-inflammatory pathways, and therefore its inhibition may have severe side effects. Rolipramhas been tested in two male patients with X-linked NDI and did not cause any relief of symptoms[162], but the potential for other PDE inhibitors in the treatment of NDI needs to be examinedfurther.

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