Further evidence for functional recovery of AQP2 mutations associated with nephrogenic diabetes insipidus.

Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Disease-causing AQP2 mutations induce nephrogenic diabetes insipidus (NDI), a condition that challenges the bodily water balance by producing large urinary volumes. In this study, we characterize three new AQP2 mutations identified in our lab from NDI patients (A120D, A130V, T179N) along the previously reported A47V variant. Using Xenopus oocytes, we compared the key functional and biochemical features of these mutations against classical recessive (R187C) and dominant (R254Q) forms, and once again found clear functional recovery features (increased protein stability and function) for all mutations under study. This behaviour, attributed to heteromerization to wt-AQP2, challenge the classical model to NDI which often depicts recessive mutations as ill-structured proteins unable to oligomerize. Consequently, we propose a revised model to the cell pathophysiology of AQP2-related NDI which accounts for the functional recovery of recessive AQP2 mutations.

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.

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.

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.

Stimulating effect of external Myo-inositol on the expression of mutant forms of aquaporin 2.

Myo-inositol (MI; hexahydroxycyclohexane, C(6)H(6)O(12)) is a small neutral molecule used as a compatible osmolyte in the kidney medulla. At high concentrations, MI appears to act as a chemical chaperone and was shown to promote plasma membrane expression of the impaired cystic fibrosis chloride channel (Delta508-CFTR). In the present study, we measured whether MI could increase expression of two human aquaporin 2 (AQP2) mutants which were recently identified as causing nephrogenic diabetes insipidus (NDI). Both proteins (D150E and G196D) were expressed in Xenopus laevis oocytes, but only D150E displayed an increase in oocyte water permeability (P (f)). Adding 5 mM MI to the bathing solution for 24 h produced a 50% increase in the D150E-associated P (f), while it had no effect on noninjected oocytes or on oocytes expressing wt-AQP2 or G196D. Western blots performed on purified plasma membrane preparations confirmed that MI increased the amount of D150E present at the plasma membrane, while G196D was always undetectable. X. laevis oocytes are remarkably impermeable to MI, and the effect of MI on D150E expression does not require the presence of intracellular MI. The effect of external MI was dose-dependent (K (0.5) was 130 microM) and specific with respect to other forms of inositols. Further studies on a second group of AQP2 mutants causing NDI showed that K228E activity was similarly stimulated by MI, while V71M, A70D and S256L were not. It is concluded that physiological concentrations of extracellular MI can stimulate the expression of a specific subgroup of AQP2 mutants.

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 (approximately 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.

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.

Effects of hyperosmolarity on the Na+ -myo-inositol cotransporter SMIT2 stably transfected in the Madin-Darby canine kidney cell line.

Myo-inositol (MI) is a compatible osmolyte used by cells to compensate for changes in the osmolarity of their surrounding milieu. In kidney, the basolateral Na(+)-MI cotransporter (SMIT1) and apical SMIT2 proteins are homologous cotransporters responsible for cellular uptake of MI. It has been shown in the Madin-Darby canine kidney (MDCK) cell line that SMIT1 expression was under the control of the tonicity-sensitive transcription factor, tonicity-responsive enhancer binding protein (TonEBP). We used an MDCK cell line stably transfected with SMIT2 to determine whether variations in external osmolarity could also affect SMIT2 function. Hyperosmotic conditions (+200 mosM raffinose or NaCl but not urea) generated an increase in SMIT2-specific MI uptake by three- to ninefold in a process that required protein synthesis. Using quantitative RT-PCR, we have determined that hyperosmotic conditions augment both the endogenous SMIT1 and the transfected SMIT2 mRNAs. Transport activities for both SMIT1 and SMIT2 exhibited differences in their respective induction profiles for both their sensitivities to raffinose, as well as in their time course of induction. Application of MG-132, which inhibits nuclear translocation of TonEBP, showed that the effect of osmolarity on transfected SMIT2 was unrelated to TonEBP, unlike the effect observed with SMIT1. Inhibition studies involving the hyperosmolarity-related MAPK suggested that p38 and JNK play a role in the induction of SMIT2. Further studies have shown that hyperosmolarity also upregulates another transfected transporter (Na(+)-glucose), as well as several endogenously expressed transport systems. This study shows that hyperosmolarity can stimulate transport in a TonEBP-independent manner by increasing the amount of mRNA derived from an exogenous DNA segment.

SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine.

This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na(+) coupled (SMIT1 and SMIT2) and one is H(+) coupled (HMIT). Through inhibition studies using selective substrates such as d-chiro-inositol (DCI, specific for SMIT2) and l-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 +/- 0.040 mM), DCI (0.31 +/- 0.06 mM), and phlorizin (Pz; 0.016 +/- 0.007 mM); low affinity for glucose (36 +/- 7 mM); and no affinity for l-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was approximately 40-fold lower in vesicles (K(i) = 0.94 +/- 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.

Expression and functionality of the Na+/myo-inositol cotransporter SMIT2 in rabbit kidney.

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a "compatible osmolyte". Currently, three MI cotransporters have been identified: two are Na(+)-dependent (SMIT1 and SMIT2) and one is H(+)-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence of d-chiro-inositol and l-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes.

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na(+)-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.

The role of the GX9GX3G motif in the gating of high voltage-activated Ca2+ channels.

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A >> G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating.

Identification of a disulfide bridge linking the fourth and the seventh extracellular loops of the Na+/glucose cotransporter.

The Na+/glucose cotransporter (SGLT1) is an archetype for the SLC5 family, which is comprised of Na+-coupled transporters for sugars, myo-inositol, choline, and organic anions. Application of the reducing agent dithriothreitol (DTT, 10 mM) to oocytes expressing human SGLT1 affects the protein's presteady-state currents. Integration of these currents at different membrane potentials (Vm) produces a Q-V curve, whose form was shifted by +25 mV due to DTT. The role of the 15 endogenous cysteine residues was investigated by expressing SGLT1 constructs, each bearing a single mutation for an individual cysteine, in Xenopus oocytes, using two-microelectrode voltage-clamp electrophysiology and fluorescent labeling. 12 of the 15 mutants were functional and could be separated into three distinct groups based on the effect of the mutation on the Q-V curve: four mutants did not perturb the transferred charge, six mutants shifted the Q-V curve towards negative potentials, and two mutants (C255A and C511A) produced a shift in the positive direction that was identical to the shift produced by DTT on the wild-type (wt) SGLT1. The double mutant C(255,511)A confirms that the effects of each single mutant on the Q-V curve were not additive. With respect to wt SGLT1, the apparent affinities for alpha-methylglucose (alphaMG) were increased in a similar manner for the single mutants C255A and C511A, the double mutant C(255,511)A as well as for wt SGLT1 treated with DTT. When exposed to a maleimide-based fluorescent probe, wt SGLT1 was not significantly labeled but mutants C255A and C511A could be clearly labeled, indicating an accessible cysteine residue. These residues are presumed to be C511 and C255, respectively, as the double mutant C(255,511)A could not be labeled. These results strongly support the hypothesis that C255 and C511 form a disulfide bridge in human SGLT1 and that this disulfide bridge is involved in the conformational change of the free carrier.

Expression of the sodium-myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells.

Myo-inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na(+)-dependent transporters such as SMIT1. We have recently identified a second Na(+)-myo-inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin-Darby canine kidney (MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo-inositol by this cell line is 20-fold larger than that seen in native MDCK cells. The affinity for myo-inositol of MDCK cells transfected with SMIT2 is slightly lower (K(m)= 334 microm) than that found in voltage-clamped Xenopus laevis oocytes expressing SMIT2 (K(m)= 120 microm). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2-transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5-fold when submitted to 24 h hypertonic treatment (+200 mosmol l(-1)). The SMIT2-MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.

Glucose accumulation can account for the initial water flux triggered by Na+/glucose cotransport.

Over the last decade, several cotransport studies have led to the proposal of secondary active transport of water, challenging the dogma that all water transport is passive. The major observation leading to this interpretation was that a Na+ influx failed to reproduce the large and rapid cell swelling induced by Na+/solute cotransport. We have investigated this phenomenon by comparing a Na+/glucose (hSGLT1) induced water flux to water fluxes triggered either by a cationic inward current (using ROMK2 K+ channels) or by a glucose influx (using GLUT2, a passive glucose transporter). These proteins were overexpressed in Xenopus oocytes and assayed through volumetric measurements combined with double-electrode electrophysiology or radioactive uptake measurements. The osmotic gradients driving the observed water fluxes were estimated by comparison with the swelling induced by osmotic shocks of known amplitude. We found that, for equivalent cation or glucose uptakes, the combination of substrate accumulations observed with ROMK2 and GLUT2 are sufficient to provide the osmotic gradient necessary to account for a passive water flux through SGLT1. Despite the fact that the Na+/glucose stoichiometry of SGLT1 is 2:1, glucose accumulation accounts for two-thirds of the osmotic gradient responsible for the water flux observed at t = 30 s. It is concluded that the different accumulation processes for neutral versus charged solutes can quantitatively account for the fast water flux associated with Na+/glucose cotransport activation without having to propose the presence of secondary active water transport.

Controversy regarding the secondary active water transport hypothesis.

Historically, water transport across biological membranes has always been considered a passive process, i.e., the net water transport is proportional to the gradients of hydrostatic and osmotic pressure. More recently, this dogma was challenged by the suggestion that secondary active transporters such as the Na/glucose cotransporter (SGLT1) could perform secondary active water transport with a fixed stoichiometry. In the case of SGLT1, the stoichiometry would consist of one glucose molecule to two Na+ ions to 220-400 water molecules. In the present minireview, we summarize and criticize the evidence supporting and opposing this water cotransport hypothesis. Published and unpublished observations from our own laboratory are also presented in support of the idea that transport-dependent osmotic gradients begin to build up immediately after cotransport commences and are fully responsible for the cell swelling observed.