Sequence-Based Screening of Patients With Idiopathic Polyarteritis Nodosa, Granulomatosis With Polyangiitis, and Microscopic Polyangiitis for Deleterious Genetic Variants in ADA2.

OBJECTIVE: Deficiency of adenosine deaminase 2 (DADA2) is a monogenic form of vasculitis that can resemble polyarteritis nodosa (PAN). This study was undertaken to identify potential disease-causing sequence variants in ADA2 in patients with idiopathic PAN, granulomatosis with polyangiitis (GPA), or microscopic polyangiitis (MPA). METHODS: Patients with idiopathic PAN (n = 118) and patients with GPA or MPA (n = 1,107) were screened for rare nonsynonymous variants in ADA2 using DNA sequencing methods. ADA-2 enzyme activity was assessed in selected serum samples. RESULTS: Nine of 118 patients with PAN (7.6%) were identified as having rare nonsynonymous variants in ADA2. Four patients (3.4%) were biallelic for pathogenic or likely pathogenic variants, and 5 patients (4.2%) were monoallelic carriers for 3 variants of uncertain significance and 2 likely pathogenic variants. Serum samples from 2 patients with PAN with biallelic variants were available and showed markedly reduced ADA-2 enzyme activity. ADA-2 enzyme testing of 86 additional patients revealed 1 individual with strongly reduced ADA-2 activity without detectable pathogenic variants. Patients with PAN and biallelic variants in ADA2 were younger at diagnosis than patients with 1 or no variant in ADA2, with no other clinical differences noted. None of the patients with GPA or MPA carried biallelic variants in ADA2. CONCLUSION: A subset of patients with idiopathic PAN meet genetic criteria for DADA2. Given that tumor necrosis factor inhibition is efficacious in DADA2 but is not conventional therapy for PAN, these findings suggest that ADA-2 testing should strongly be considered in patients with hepatitis B virus-negative idiopathic PAN.

Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

Old Dogs, New Tricks: Monogenic Autoinflammatory Disease Unleashed.

Autoinflammatory diseases are inborn disorders of the innate immune system characterized by episodes of systemic inflammation that are mediated largely by myeloid cells. The field of autoinflammatory diseases has been established since 1999, following the identification of the first genes underlying periodic fever syndromes. This review focuses on developments that have transformed the field in the last two years. We discuss three newly described monogenic autoinflammatory diseases [deficiency of adenosine deaminase 2 (DADA2), a subtype of macrophage activation syndrome (MAS), and stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI)], discuss the possibilities of somatic mosaicism and digenic inheritance, and give an update on new concepts in pathways involved in familial Mediterranean fever (FMF). Finally, the new monogenic autoinflammatory disease haploinsufficiency of A20 (HA20) underscores the placement of monogenic diseases in the firmament of common autoinflammatory phenotypes. The advances in the last two years have shed light on the pathophysiology of several autoinflammatory diseases and have elucidated new pathways that play a role in innate immunity.

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease.

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-κB signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB-mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB-dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

The role of interleukin-1 beta in the pathophysiology of Schnitzler's syndrome.

INTRODUCTION: Schnitzler's syndrome (SchS) is a disabling autoinflammatory disorder, characterized by a chronic urticarial rash, an M-protein, arthralgia, and other signs of systemic inflammation. Anti-interleukin-1 (IL-1) beta antibodies are highly effective, but the pathophysiology is still largely unknown. Here we studied the effect of in-vivo IL-1 inhibition on serum markers of inflammation and cellular immune responses. METHODS: Eight patients with SchS received monthly subcutaneous (s.c.) injections with 150 mg canakinumab for six months. Blood was drawn for measurement of serum markers of inflammation (12 times per patient) and for functional and phenotypic analysis of both freshly isolated and toll-like receptor (TLR)-ligand-stimulated peripheral blood mononuclear cells (PBMCs) (five times per patient). All data were compared to results of healthy controls. RESULTS: IL-6 levels in serum and in lysates of freshly isolated PBMCs and serum myeloid-related protein (MRP8)/14 and S100A12 levels correlated with disease activity. In vitro, LPS stimulation resulted in higher IL-6 and IL-1 beta production in PBMCs from symptomatic SchS patients compared to healthy controls, whereas patient cells were relatively hyporesponsive to poly:IC and Pam3Cys. The mRNA microarray of PBMCs showed distinct transcriptomes for controls, symptomatic patients and anti-IL-1-treated patients. Numbers of T- and B-cell subsets as well as M-protein concentrations were not affected by IL-1 inhibition. Free light chain levels were elevated in 4 out of 8 patients. CONCLUSIONS: In conclusion, patient PBMCs are hyperresponsive to LPS, and clinical efficacy of IL-1 beta inhibition in patients with SchS is associated with in-vivo and ex-vivo suppression of inflammation. Interestingly, patient PBMCs showed divergent responses to TLR2/6, TLR3 and TLR4 ligands. Our data underscore that IL-1 beta plays a pivotal role in SchS.

ATP-Induced IL-1ß Specific Secretion: True Under Stringent Conditions.

Interleukin-1ß is a potent proinflammatory cytokine, of which processing and secretion are tightly regulated. After exposure to various stimuli, mononuclear phagocytes synthesize the inactive precursor (pro-IL-1ß), which is then cleaved intracellularly by caspase-1 and secreted. A widely used method for in vitro secretion of IL-1ß employs LPS-primed human peripheral blood monocytes. Subsequently, adenosine triphosphate (ATP) is added to the cells in order to trigger the P2X7 receptor resulting in processing and secretion of mature IL-1ß. However, it is often reported that secretion is due to cytotoxic effects of ATP with P2X7 receptor-activation-related cell death. We have challenged this concept and demonstrate IL-1ß specific secretion, since there is no increase in cell death and IL-1α and IL-18 are not released in the same cultures. More importantly we show that these conclusions can only be drawn under stringent experimental conditions.

TLR2/TLR4-dependent exaggerated cytokine production in hyperimmunoglobulinaemia D and periodic fever syndrome.

OBJECTIVE: The autoinflammatory hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is characterized by recurrent episodes of fever and inflammation. As part of the mevalonate kinase deficiency spectrum, it is caused by MVK mutations, resulting in decreased mevalonate kinase activity in the isoprenoid pathway. Although IL-1ß is considered a major cytokine in its pathogenesis, IL-1 blockade is not successful in a proportion of patients. We aimed to further characterize the pro-inflammatory cytokine profile of HIDS. METHODS: Peripheral blood mononuclear cells from HIDS patients and healthy donors were incubated with several stimuli. Cytokine concentrations were detected by ELISA. To analyse mRNA and protein expression, we performed quantitative RT-PCR and western blot, respectively. RESULTS: We observed significant differences in cytokine production when cells were incubated with ligands for Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerization domain-containing 2 (NOD2). The increased ratio between active and inactive caspase-1 protein in HIDS patients could explain why these cells are more easily triggered to secrete IL-1ß. This is apparently not regulated at the transcriptional level, since expression levels of caspase-1 and IL-1ß mRNA were similar in patients and controls. Both anakinra and tocilizumab treatment resulted in decreased inflammation, both ex vivo as well as in vivo. CONCLUSION: The increased cytokine secretion in HIDS is specific for TLR2, TLR4 and NOD2 ligation. Although IL-1ß is important in the HIDS pathology, our data suggest it is a multicytokine disease. A more rigorous clinical trial is required to determine whether IL-6 receptor blockade may be considered in patients not responding to anakinra treatment.

The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases.

BACKGROUND: The extracellular domains of cytokine receptors are released during inflammation, but little is known about the shedding of Toll-like receptors (TLR) and whether they can be used as diagnostic biomarkers. METHODS: The release of sTLR2 and sTLR4 was studied in in-vitro stimulations, as well as in-vivo during experimental human endotoxemia (n = 11, 2 ng/kg LPS), and in plasma of 394 patients with infections (infectious mononucleosis, measles, respiratory tract infections, bacterial sepsis and candidemia) or non-infectious inflammation (Crohn's disease, gout, rheumatoid arthritis, autoinflammatory syndromes and pancreatitis). Using C-statistics, the value of sTLR2 and sTLR4 levels for discrimination between infections and non-infectious inflammatory diseases, as well as between viral and bacterial infections was analyzed. RESULTS: In-vitro, peripheral blood mononuclear cells released sTLR2 and sTLR4 by exposure to microbial ligands. During experimental human endotoxemia, plasma concentrations peaked after 2 hours (sTLR4) and 4 hours (sTLR2). sTLR4 did not correlate with cytokines, but sTLR2 correlated positively with TNFα (rs = 0.80, P < 0.05), IL-6 (rs = 0.65, P < 0.05), and IL-1Ra (rs = 0.57, P = 0.06), and negatively with IL-10 (rs = -0.58, P = 0.06), respectively. sTLR4 had a similar area under the ROC curve [AUC] for differentiating infectious and non-infectious inflammation compared to CRP: 0.72 (95% CI 0.66-0.79) versus 0.74 (95% CI 0.69-0.80) [P = 0.80], while sTLR2 had a lower AUC: 0.60 (95% CI 0.54-0.66) [P = 0.0004]. CRP differentiated bacterial infections better from viral infections than sTLR2 and sTLR4: AUC 0.94 (95% CI 0.90-0.96) versus 0.58 (95% CI 0.51-0.64) and 0.75 (95% CI 0.70-0.80), respectively [P < 0.0001 for both]. CONCLUSIONS: sTLRs are released into the circulation, and suggest the possibility to use sTLRs as diagnostic tool in inflammatory conditions.

MEFV mutations affecting pyrin amino acid 577 cause autosomal dominant autoinflammatory disease.

OBJECTIVES: Autoinflammatory disorders are disorders of the innate immune system. Standard genetic testing provided no correct diagnosis in a female patient from a non-consanguineous family of British descent with a colchicine-responsive autosomal dominant periodic fever syndrome. We aimed to unravel the genetic cause of the symptoms. METHODS: Whole exome sequencing was used to screen for novel sequence variants, which were validated by direct Sanger sequencing. Ex vivo stimulation with peripheral blood mononuclear cells was performed to study the functional consequences of the mutation. mRNA and cytokine levels were measured by quantitative PCR and ELISA, respectively. RESULTS: Whole exome sequencing revealed a novel missense sequence variant, not seen in around 6800 controls, mapping to exon 8 of the MEFV gene (c.1730C>A; p.T577N), co-segregating perfectly with disease in this family. Other mutations at the same amino acid (c.1730C>G; p.T577S and c.1729A>T; p.T577S) were found in a family of Turkish descent, with autosomal dominant inheritance of familial Mediterranean fever (FMF)-like phenotype, and a Dutch patient, respectively. Moreover, a mutation (c.1729A>G; p.T577A) was detected in two Dutch siblings, who had episodes of inflammation of varying severity not resembling FMF. Peripheral blood mononuclear cells from one patient of the index family showed increased basal interleukin 1ß mRNA levels and cytokine responses after lipopolysaccharide stimulation. Responses normalised with colchicine treatment. CONCLUSIONS: Heterozygous mutations at amino acid position 577 of pyrin can induce an autosomal dominant autoinflammatory syndrome. This suggests that T577, located in front of the C-terminal B30.2/SPRY domain, is crucial for pyrin function.

Mutations in the mevalonate kinase (MVK) gene cause nonsyndromic retinitis pigmentosa.

OBJECTIVE: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disorder characterized by night blindness and peripheral vision loss, and in many cases leads to blindness. Despite extensive knowledge about genes involved in the pathogenesis of RP, the genetic cause remains elusive in many patients. In this study, we aimed to identify novel genes that are involved in the cause of RP. DESIGN: We present a case series with mutations in the mevalonate kinase (MVK) gene. PARTICIPANTS: A total of 769 patients with nonsyndromic RP and 174 Dutch control individuals participated in this study. METHODS: Exome sequencing analysis was performed in a proband of Dutch origin who was initially diagnosed with nonsyndromic autosomal recessive RP. Mutations in MVK were identified and subsequently tested for segregation within the patient's family and screened in a large cohort of patients with genetically unsolved RP. Patients with mutations underwent extensive clinical reexamination. MAIN OUTCOME MEASURES: Digital fundus photography, spectral-domain optical coherence tomography (OCT), and fundus autofluorescence analysis were performed in patients with MVK mutations. Mevalonate kinase (MK) enzyme activity was analyzed in cultured lymphoblastoid cells, and mevalonic acid levels were measured in urine samples. RESULTS: Exome variant filtering and prioritization led to the identification of compound heterozygous mutations in MVK (p.I268T and p.A334T) in the proband and her affected brother. Screening of our nonsyndromic RP patient cohort revealed an additional individual who was homozygous for the p.A334T alteration. Clinical reevaluation of all 3 patients showed a classic form of RP with variable extraocular symptoms, such as history of recurrent childhood febrile crises in 2 patients, mild ataxia in 1, and renal failure in 1. All 3 affected individuals showed a significantly decreased MK activity and highly elevated levels of urinary mevalonic acid. CONCLUSIONS: Although the MK activity in cells and mevalonic acid concentrations in urine are strongly aberrant and comparable to that in patients with systemic mevalonate kinase deficiency (MKD), only mild clinical symptoms related to this syndrome were observed in our patients. In the current article, we add another phenotype to the spectrum of diverging disorders associated with mutations in MVK.

Sustained efficacy of the monoclonal anti-interleukin-1 beta antibody canakinumab in a 9-month trial in Schnitzler's syndrome.

OBJECTIVES: Schnitzler's syndrome is a chronic disabling autoinflammatory disorder, characterised by chronic urticaria, paraproteinemia and systemic inflammation. The interleukin (IL) 1 receptor antagonist anakinra is a very effective treatment, but requires daily injection and blocks both IL-1α and IL-1ß. Canakinumab is a selective human monoclonal anti-IL-1ß antibody with a long half-life. We investigated the long-term efficacy and safety of canakinumab in Schnitzler's syndrome. METHODS: In an open-label, single-treatment arm trial, eight patients with Schnitzler's syndrome received monthly injections with 150 mg canakinumab subcutaneously for 6 months, followed by a 3-month observation period. Primary outcome was complete or clinical remission at day 14. Secondary outcome measures included inflammatory markers, quality of life, time to relapse, safety and tolerability. RESULTS: After stopping anakinra, patients developed moderate to severe clinical symptoms. Canakinumab induced complete or clinical remission at day 14 in all eight patients. Median C-reactive protein concentrations decreased from 169 mg/l at baseline to less than 10 mg/l on day 14 and remained low or undetectable. One patient discontinued participation on day 39 because of return of symptoms while all others remained in complete or clinical remission during the 6-month treatment period. Relapse after last canakinumab dose occurred within 3 months in four patients. For two patients, remission continued several months post-study. Five patients reported at least one adverse event, predominantly mild upper respiratory tract infections. One patient died in a traffic accident. CONCLUSIONS: In this 9-month study, monthly 150 mg canakinumab injection was an effective and well-tolerated treatment for Schnitzler's syndrome. Our data demonstrate that IL-1ß plays a pivotal role in this disease. CLINICALTRIALS.GOV: NCT01276522.

The influence of concentration/meditation on autonomic nervous system activity and the innate immune response: a case study.

OBJECTIVE: In this case study, we describe the effects of a particular individual's concentration/meditation technique on autonomic nervous system activity and the innate immune response. The study participant holds several world records with regard to tolerating extreme cold and claims that he can influence his autonomic nervous system and thereby his innate immune response. METHODS: The individual's ex vivo cytokine response (stimulation of peripheral blood mononuclear cells with lipopolysaccharide [LPS]) was determined before and after an 80-minute full-body ice immersion during which the individual practiced his concentration/meditation technique. Furthermore, the individual's in vivo innate immune response was studied while practicing his concentration/mediation technique during human endotoxemia (intravenous administration of 2 ng/kg LPS). The results from the endotoxemia experiment were compared with a historical cohort of 112 individuals who participated in endotoxemia experiments in our institution. RESULTS: The ex vivo proinflammatory and anti-inflammatory cytokine response was greatly attenuated by concentration/meditation during ice immersion, accompanied by high levels of cortisol. In the endotoxemia experiment, concentration/meditation resulted in increased circulating concentrations of catecholamines, and plasma cortisol concentrations were higher than in any of the previously studied participants. The individual's in vivo cytokine response and clinical symptoms after LPS administration were remarkably low compared with previously studied participants. CONCLUSIONS: The concentration/meditation technique used by this particular individual seems to evoke a controlled stress response. This response is characterized by sympathetic nervous system activation and subsequent catecholamine/cortisol release, which seems to attenuate the innate immune response.

Hyper-IgD syndrome or mevalonate kinase deficiency.

PURPOSE OF REVIEW: The hyper-IgD and periodic fever syndrome (HIDS) is one of the classical monogenetic hereditary autoinflammatory disorders, and together with the more severe mevalonic aciduria it is also known as 'mevalonate kinase deficiency' (MKD). In this study, we will give an overview of the primary research on mevalonate kinase deficiency published in the past 2 years. RECENT FINDINGS: Besides an inventory of a number of recent case reports, literature review shows there are several interesting developments in the basic field of research. First, a group of articles was recently published on chemically instead of genetically induced MKD mouse and cell models, investigating the effects of several isoprenoid pathway intermediates. Second, another study confirms a role for small GTPases and their isoprenylation in the inflammatory response in mevalonate kinase deficiency. Lastly, there are now, finally, modest new indications about the role of IgD. SUMMARY: Both pathophysiological studies and clinical observations in the last 2 years have supported the central role of IL-1 in HIDS. There are some intriguing results and hypotheses about the link between isoprenoid metabolism and the IL-1 pathway through geranylgeranylation that deserve to be further examined.

The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans.

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL-1ß in macrophages. Inflammasome activation in macrophages results from differences in cell-wall architecture between yeasts and hyphae and is partly mediated by the dectin-1/Syk pathway. These results define the dectin-1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.

Inflammasome-independent modulation of cytokine response by autophagy in human cells.

Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohn's disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient mice have shown that disruption of autophagy increases processing of IL-1ß and IL-18 through an inflammasome-dependent manner. Using complementary approaches either inducing or inhibiting autophagy, we describe modulatory effects of autophagy on proinflammatory cytokine production in human cells. Inhibition of basal autophagy in human peripheral blood mononuclear cells (PBMCs) significantly enhances IL-1ß after stimulation with TLR2 or TLR4 ligands, while at the same time reducing the production of TNFα. In line with this, induction of autophagy by starvation inhibited IL-1ß production. These effects of autophagy were not exerted at the processing step, as inflammasome activation was not influenced. In contrast, the effect of autophagy on cytokine production was on transcription level, and possibly involving the inhibition of p38 mitogen activated protein kinase (MAPK) phosphorylation. In conclusion, autophagy modulates the secretion of proinflammatory cytokines in human cells through an inflammasome-independent pathway, and this is a novel mechanism that may be targeted in inflammatory diseases.

Mouse dendritic cells matured by ingestion of apoptotic blebs induce T cells to produce interleukin-17.

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of antinuclear autoantibodies. Increased apoptosis and reduced clearance of apoptotic material have been assigned a role in the pathogenesis of SLE, but the underlying mechanisms remain elusive. During apoptosis apoptotic blebs are formed in which autoantigens are clustered. The cellular remnants after blebbing are referred to as apoptotic cell bodies. We undertook this study to compare the effects of apoptotic blebs and apoptotic cell bodies on maturation of dendritic cells (DCs) and their T cell stimulatory capacity in a murine setting. METHODS: The uptake by DCs of apoptotic blebs and apoptotic cell bodies was analyzed by flow cytometry and confocal microscopy. DC maturation and DC-induced T cell activation were determined by measuring expression of costimulatory molecules using flow cytometry and by measuring production of cytokines using enzyme-linked immunosorbent assay. RESULTS: DCs internalized apoptotic blebs more efficiently than apoptotic cell bodies. Incubation of DCs with apoptotic blebs resulted in increased CD40 and CD86 expression and increased interleukin-6 (IL-6) and tumor necrosis factor alpha production, while apoptotic cell bodies had no stimulatory effects. Using chloroquine, apoptotic bleb-induced DC maturation was shown to be independent of Toll-like receptors 3, 7, and 9. Interestingly, in cocultures with allogeneic T cells, bleb-matured DCs induced production of IL-2, interferon-gamma, and, in particular, IL-17, suggesting a Th1/Th17 response. CONCLUSION: Apoptotic blebs, in contrast to apoptotic cell bodies, induce DC maturation, thereby providing DCs with increased Th17 cell stimulatory capacity. These data imply that apoptotic bleb-induced DC maturation represents an important driving force in the autoimmune response in SLE.

Repulsion between Lys258 and upstream arginines explains the missorting of the AQP2 mutant p.Glu258Lys in nephrogenic diabetes insipidus.

Regulation of body water homeostasis occurs by the vasopressin-dependent sorting of aquaporin-2 (AQP2) water channels to and from the apical membrane of renal principal cells. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease that renders the kidney unresponsive to vasopressin, resulting in polyuria and polydipsia. The AQP2 mutant c.772G>A; p.Glu258Lys (AQP2-E258K) causes dominant NDI by oligomerizing with wild-type AQP2 and missorting of this AQP2 complex to multivesicular bodies instead of the apical membrane. The motif causing this missorting of AQP2-E258K was identified here. Functional analyses and plasma membrane expression studies of truncation mutants in oocytes revealed that AQP2-E258K shortened to Leu259 is still intracellular retained. Alanine scanning and glutamic acid to arginine exchanges revealed increased function and plasma membrane expression for AQP2-E258K mutants with the following additional changes: Leu259Ala, Arg252Glu, Arg253Glu, or Arg252Ala-Arg254Ala, or for the AQP2 mutant p.Glu258Ala, indicating that the motif RRRxxxK(258)L confers AQP2-E258K retention. Fusion of this motif to aquaporin-1 also resulted in missorting of that water channel, indicating that this retention motif is transferable. In conclusion, our data reveal that the RRRxxxKL motif and repulsion between K258 and the arginine-triplet within this motif are the primary cause of missorting of AQP2-E258K in NDI.