Yoann Lussier

Characterization of D150E and G196D aquaporin-2 mutations responsible for nephrogenic diabetes insipidus: importance of a mild phenotype.

Aquaporin-2 (AQP2) is a water channel responsible for the final water reabsorption in renal collecting ducts. Alterations in AQP2 function induce nephrogenic diabetes insipidus (NDI), a condition characterized by severe polyuria and polydipsia. Three patients affected with severe NDI, who were compound heterozygous for the AQP2 mutations D150E and G196D, are presented here along with a mildly affected D150E homozygous patient from another family. Using Xenopus oocytes as an expression system, these two mutations (G196D and D150E) were compared with the wild-type protein (AQP2-wt) for functional activity (water flux analysis), protein maturation, and plasma membrane targeting. AQP2-wt induces a major increase in water permeability (P(f) = 47.4 +/- 12.2 x 10(-4) cm/s) whereas D150E displays intermediate P(f) values (P(f) = 12.5 +/- 3.0 x 10(-4) cm/s) and G196D presents no specific water flux, similar to controls (P(f) = 2.1 +/- 0.8 x 10(-4) cm/s and 2.2 +/- 0.7 x 10(-4) cm/s, respectively). Western blot and immunocytochemical evaluations show protein targeting that parallels activity levels with AQP2-wt adequately targeted to the plasma membrane, partial targeting for D150E, and complete sequestration of G196D within intracellular compartments. When coinjecting AQP2-wt with mutants, no (AQP2-wt + D150E) or partial (AQP2-wt + G196D) reduction of water flux were observed compared with AQP2-wt alone, whereas complete loss of function was found when both mutants were coinjected. These results essentially recapitulate the clinical profiles of the family members, showing a typical dominant negative effect when G196D is coinjected with either AQP2-wt or D150E but not between AQP2-wt and D150E mutant.

MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17-SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters.

Aquaporin-2: new mutations responsible for autosomal-recessive nephrogenic diabetes insipidus—update and epidemiology

It is clinically useful to distinguish between two types of hereditary nephrogenic diabetes insipidus (NDI): a ‘pure’ type characterized by loss of water only and a complex type characterized by loss of water and ions. Patients with congenital NDI bearing mutations in the vasopressin 2 receptor gene, AVPR2, or in the aquaporin-2 gene, AQP2, have a pure NDI phenotype with loss of water but normal conservation of sodium, potassium, chloride and calcium. Patients with hereditary hypokalemic salt-losing tubulopathies have a complex phenotype with loss of water and ions. They have polyhydramnios, hypercalciuria and hypo- or isosthenuria and were found to bear KCNJ1 (ROMK) and SLC12A1 (NKCC2) mutations. Patients with polyhydramnios, profound polyuria, hyponatremia, hypochloremia, metabolic alkalosis and sensorineural deafness were found to bear BSND mutations. These clinical phenotypes demonstrate the critical importance of the proteins ROMK, NKCC2 and Barttin to transfer NaCl in the medullary interstitium and thereby to generate, together with urea, a hypertonic milieu. This editorial describes two new developments: (i) the genomic information provided by the sequencing of the AQP2 gene is key to the routine care of these patients, and, as in other genetic diseases, reduces health costs and provides psychological benefits to patients and families and (ii) the expression of AQP2 mutants in Xenopus oocytes and in polarized renal tubular cells recapitulates the clinical phenotypes and reveals a continuum from severe loss of function with urinary osmolalities <150 mOsm/kg H2O to milder defects with urine osmolalities >200 mOsm/kg H2O.

New autosomal recessive mutations in aquaporin-2 causing nephrogenic diabetes insipidus through deficient targeting display normal expression in Xenopus oocytes.

Aquaporin‐2 (AQP2), located at the luminal side of the collecting duct principal cells, is a water channel responsible for the final concentration of urine. Lack of function, often occurring through mistargeting of mutated proteins, induces nephrogenic diabetes insipidus (NDI), a condition characterized by large urinary volumes. In the present study, two new mutations (K228E and V24A) identified in NDI‐affected individuals from distinct families along with the already reported R187C were analysed in comparison to the wild‐type protein (AQP2‐wt) using Xenopus laevis oocytes and a mouse collecting duct cell‐line (mIMCD‐3). Initial data in oocytes showed that all mutations were adequately expressed at reduced levels when compared to AQP2‐wt. K228E and V24A were found to be properly targeted at the plasma membrane and exhibited adequate functionality similar to AQP2‐wt, as opposed to R187C which was retained in internal stores and was thus inactive. In coexpression studies using oocytes, R187C impeded the functionality of all other AQP2 variants while combinations with K228E, V24A and AQP2‐wt only showed additive functionalities. When expressed in mIMCD‐3 cells, forskolin treatment efficiently promoted the targeting of AQP2‐wt at the plasma membrane (>90%) while K228E only weakly responded to the same treatment (∼20%) and both V24A and R187C remained completely insensitive to the treatment. We concluded that both V24A and K228E are intrinsically functional water channels that lack a proper response to vasopressin, which leads to NDI as found in both compound mutations studied (K228E + R187C and V24A + R187C). The discrepancies in plasma membrane targeting response found in both expression systems stress the need to evaluate such data using mammalian cell systems.

Functional Recovery of AQP2 Recessive Mutations Through Hetero-Oligomerization with Wild-Type Counterpart.

Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Mutations in the protein induce nephrogenic diabetes insipidus (NDI), which challenges the water balance by producing large urinary volumes. Although recessive AQP2 mutations are believed to generate non-functional and monomeric proteins, the literature identifies several mild mutations which suggest the existence of mixed wt/mut tetramers likely to carry function in heterozygotes. Using Xenopus oocytes, we tested this hypothesis and found that mild mutants (V24A, D150E) can associate with wt-AQP2 in mixed heteromers, providing clear functional gain in the process (62 ± 17% and 63 ± 17% increases, respectively), conversely to the strong monomeric R187C mutant which fails to associate with wt-AQP2. In kidney cells, both V24A and D150E display restored targeting while R187C remains in intracellular stores. Using a collection of mutations to expand recovery analyses, we demonstrate that inter-unit contacts are central to this recovery process. These results not only present the ground data for the functional recovery of recessive AQP2 mutants through heteromerization, which prompt to revisit the accepted NDI model, but more importantly describe a general recovery process that could impact on all multimeric systems where recessive mutations are found.

Mice deficient for ERAD machinery component Sel1L develop central diabetes insipidus

Deficiency of the antidiuretic hormone arginine vasopressin (AVP) underlies diabetes insipidus, which is characterized by the excretion of abnormally large volumes of dilute urine and persistent thirst. In this issue of the JCI, Shi et al. report that Sel1L-Hrd1 ER-associated degradation (ERAD) is responsible for the clearance of misfolded pro-arginine vasopressin (proAVP) in the ER. Additionally, mice with Sel1L deficiency, either globally or specifically within AVP-expressing neurons, developed central diabetes insipidus. The results of this study demonstrate a role for ERAD in neuroendocrine cells and serve as a clinical example of the effect of misfolded ER proteins retrotranslocated through the membrane into the cytosol, where they are polyubiquitinated, extracted from the ER membrane, and degraded by the proteasome. Moreover, proAVP misfolding in hereditary central diabetes insipidus likely shares common physiopathological mechanisms with proinsulin misfolding in hereditary diabetes mellitus of youth.