Transgenic mice and their impact on kidney research.

The kidney is a key organ in the maintenance of ion and fluid homeostasis and specific transport systems localized along the nephron guarantee this function. Due to its large functional heterogeneity, experiments on the whole organ level cannot be easily performed, and thus more refined tools are needed, like for example the development of specific recombination systems to gain knowledge on the physiological role of single proteins implicated in ion transport. This review introduces the transgenic technology developed over the past decades, and then focuses on recent strategies for generating kidney-specific gene targeting, over-expression, and gene ablation in mice, that will help to understand the physiological role of proteins implicated in salt and water balance in the kidney.

Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.

The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.

Liddle's syndrome caused by a novel missense mutation (P617L) of the epithelial sodium channel beta subunit.

OBJECTIVE: The aim of the study was to search for mutations of SCNN1B and SCNN1G in an Italian family with apparently dominant autosomal transmission of a clinical phenotype consistent with Liddle's syndrome. METHODS: Genetic analysis was performed in the proband, his relatives, and 100 control subjects. To determine the functional role of the mutation identified in the proband, we expressed the mutant or wild-type epithelial sodium channel in Xenopus laevis oocytes. RESULTS: A novel point mutation, causing an expected substitution of a leucine residue for the second proline residue of the conserved PY motif (PPP x Y) of the beta subunit was identified in the proband. The functional expression of the mutant epithelial sodium channel in X. laevis oocytes showed a three-fold increase in the amiloride-sensitive current as compared with that of the wild-type channel. CONCLUSION: This newly identified mutation adds to other missense mutations of the PY motif of the beta subunit of the epithelial sodium channel, thus confirming its crucial role in the regulation of the epithelial sodium channel. To our knowledge, this is the first report of Liddle's syndrome in the Italian population, confirmed by genetic and functional analysis, with the identification of a gain-of-function mutation not previously reported.

Hyperpolarized lithium-6 as a sensor of nanomolar contrast agents.

Lithium is widely used in psychotherapy. The (6)Li isotope has a long intrinsic longitudinal relaxation time T(1) on the order of minutes, making it an ideal candidate for hyperpolarization experiments. In the present study we demonstrated that lithium-6 can be readily hyperpolarized within 30 min, while retaining a long polarization decay time on the order of a minute. We used the intrinsically long relaxation time for the detection of 500 nM contrast agent in vitro. Hyperpolarized lithium-6 was administered to the rat and its signal retained a decay time on the order of 70 sec in vivo. Localization experiments imply that the lithium signal originated from within the brain and that it was detectable up to 5 min after administration. We conclude that the detection of submicromolar contrast agents using hyperpolarized NMR nuclei such as (6)Li may provide a novel avenue for molecular imaging.

Mice carrying ubiquitin-specific protease 2 (Usp2) gene inactivation maintain normal sodium balance and blood pressure.

Ubiquitylation plays an important role in the control of Na⁺ homeostasis by the kidney. It is well established that the epithelial Na⁺ channel ENaC is regulated by the ubiquitin-protein ligase NEDD4-2, limiting ENaC cell surface expression and activity. Ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs). One such DUB, USP2-45, was identified previously as an aldosterone-induced protein in the kidney and is also a circadian output gene. In heterologous expression systems, USP2-45 binds to ENaC, deubiquitylates it, and enhances channel density and activity at the cell surface. Because the role of USP2-45 in renal Na⁺ transport had not been studied in vivo, we investigated here the effect of Usp2 gene inactivation in this process. We demonstrate first that USP2-45 protein has a rhythmic expression with a peak at ZT12. Usp2-KO mice did not show any differences from wild-type littermates with respect to the diurnal control of Na⁺ or K⁺ urinary excretion and plasma levels either on a standard diet or after acute and chronic changes to low- and high-Na⁺ diets, respectively. Moreover, they had similar aldosterone levels on either a low- or high-Na⁺ diet. Blood pressure measurements using telemetry did not reveal variations compared with control mice. Usp2-KO mice did not display alterations in expression of genes involved in sodium homeostasis or the ubiquitin system, as evidenced by transcriptome analysis in the kidney. Our data suggest that USP2 does not play a primary role in the control of Na⁺ balance or blood pressure.

Licorice-induced hypertension and common variants of genes regulating renal sodium reabsorption.

AIM: To study if gene alterations affecting renal sodium reabsorption associate with susceptibility to licorice-induced hypertension.METHODS: Finnish subjects (n = 30) with a previously documented incident of licorice-induced hypertension were recruited for the study using a newspaper announcement. Their previous clinical and family histories as well as serum electrolyte levels were examined. DNA samples from all individuals were screened for variants of the genes encoding 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) and alpha-, beta-, and gamma-subunits of the epithelial sodium channel (ENaC).RESULTS: Upon licorice predisposition, the patients had a mean blood pressure of 201/118 mmHg. Circulating potassium, renin, and aldosterone levels were low. No significant DNA variations were identified in the 11betaHSD2 gene. Four subjects were heterozygous for beta- and gammaENaC variants previously shown to be associated with hypertension. Furthermore, a novel G insertion (2004-2005insG) in the SCNN1A gene encoding the alphaENaC was identified in two subjects. The frequency of these ENaC variants was significantly higher in subjects with licorice-induced hypertension (6/30 i.e. 20%) than in blood donors (11/301 i.e. 3.7%, P = 0.002).CONCLUSIONS: Defects of the 11betaHSD2 gene do not constitute a likely cause for licorice-induced hypertension. Variants of the ENaC subunits may render some individuals sensitive to licorice-induced metabolic alterations and hypertension.

Molecular determinants of desensitization in an ENaC/degenerin channel.

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Å above the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

The 19th Ion Channel Meeting. September 2008, France.

The annual meeting of the French Ion Channels Society, held on the Mediterranean coast of France, is aimed at gathering the international scientific community working on various aspects of ion channels. In this report of the 19th edition of the meeting, held in September 2008, we summarize selected symposia on aspects of the ion channel field from fundamental to clinical research. The meeting is an opportunity for leading investigators as well as young researchers to present and discuss their recent advances and future challenges in the ion channel field.

Focused ion beam scanning electron microscopy in biology.

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB-SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three-dimensional data, FIB-SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block-face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo-) transmission electron microscopy. Here, we will present an overview of the development of FIB-SEM and discuss a few points about sample preparation and imaging.

Association with {beta}-COP regulates the trafficking of the newly synthesized Na,K-ATPase.

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and β-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, β-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase β-subunit, the Na,K-ATPase α-subunit interacts with β-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase β-subunit, the α-subunit does not interact with β-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and β-subunits, newly synthesized α-subunit associates with β-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and β-subunits to form the pump holoenzyme. The interaction with β-COP was reduced by mutating a dibasic motif at Lys(54) in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase β-subunit expression. Although the Lys(54) α-subunit reaches the cell surface without need for β-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the β-subunit.

Architectural and Functional Similarities between Trimeric ATP-Gated P2X Receptors and Acid-Sensing Ion Channels

ATP-gated P2X receptors and acid-sensing ion channels are two distinct ligand-gated ion channels that assemble into trimers. They are involved in many important physiological functions such as pain sensation and are recognized as important therapeutic targets. They have unrelated primary structures and respond to different ligands (ATP and protons) and are thus considered as two different ion channels. As a consequence, comparisons of the biophysical properties and underlying mechanisms have only been rarely made between these two channels. However, the recent determination of their molecular structures by X-ray crystallography has revealed unexpected parallels in the architecture of the two pores, providing a basis for possible functional analogies. In this review, we analyze the structural and functional similarities that are shared by these trimeric ion channels, and we outline key unanswered questions that, if addressed experimentally, may help us to elucidate how two unrelated ion channels have adopted a similar fold of the pore.

A novel dominant mutation of the Nav1.4 alpha-subunit domain I leading to sodium channel myotonia.

BACKGROUND: Mutations in SCN4A may lead to myotonia. METHODS: Presentation of a large family with myotonia, including molecular studies and patch clamp experiments using human embryonic kidney 293 cells expressing wild-type and mutated channels. RESULTS: In a large family with historic data on seven generations and a clear phenotype, including myotonia at movement onset, with worsening by cold temperature, pregnancy, mental stress, and especially after rest after intense physical activity, but without weakness, the phenotype was linked with the muscle sodium channel gene (SCN4A) locus, in which a novel p.I141V mutation was found. This modification is located within the first transmembrane segment of domain I of the Na(v)1.4 alpha subunit, a region where no mutation has been reported so far. Patch clamp experiments revealed a mutation-induced hyperpolarizing shift (-12.9 mV) of the voltage dependence of activation, leading to a significant increase (approximately twofold) of the window current amplitude. In addition, the mutation shifted the voltage dependence of slow inactivation by -8.7 mV and accelerated the entry to this state. CONCLUSIONS: We propose that the gain-of-function alteration in activation leads to the observed myotonic phenotype, whereas the enhanced slow inactivation may prevent depolarization-induced paralysis.

Glomerular hyperfiltration and increased proximal sodium reabsorption in subjects with type 2 diabetes or impaired fasting glucose in a population of the African region.

BACKGROUND. Glomerular hyperfiltration (GHF) is a well-recognized early renal alteration in diabetic patients. As the prevalence of GHF is largely unknown in populations in the African region with respect to normal fasting glucose (NFG), impaired fasting glucose (IFG) and type 2 diabetes [diabetes mellitus (DM)], we conducted a cross-sectional study in the Seychelles islands among families including at least one member with hypertension. METHODS. The glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and proximal tubular sodium reabsorption were measured using inulin, p-aminohippurate (PAH) and endogenous lithium clearance, respectively. Twenty-four-hour urine was collected on the preceding day. RESULTS. Of the 363 participants (mean age 44.7 years), 6.6% had IFG, 9.9% had DM and 63.3% had hypertension. The prevalence of GHF, defined as a GFR >140 ml/min, was 17.2%, 29.2% and 52.8% in NFG, IFG and DM, respectively (P trend <0.001). Compared to NFG, the adjusted odds ratio for GHF was 1.99 [95% confidence interval (CI) 0.73-5.44] for IFG and 5.88 (2.39-14.45) for DM. Lithium clearance and fractional excretion of lithium were lower in DM and IFG than NFG (P < 0.001). CONCLUSION. In this population of African descent, subjects with impaired fasting glucose or type 2 diabetes had a high prevalence of GHF and enhanced proximal sodium reabsorption. These findings provide further insight on the elevated incidence of nephropathy reported among African diabetic individuals.

Acid-sensing channels in human bladder: expression, function and alterations during bladder pain syndrome.

Purpose: To examine the possible role of H+-activated acid-sensing ion channels (ASICs) in pain perception we characterized their expression in bladder dome biopsies of Bladder Pain Syndrome (BPS) patients and controls, in cultured human urothelium and in urothelial TEU-2 cells.Materials and Methods: Cold cut biopsies from the bladder dome were obtained in 8 asymptomatic controls and 28 patients with symptoms of BPS. ASIC expression was analyzed by QPCR and immunofluorescence. The channel function was measured by electrophysiology.Results: ASIC1a, ASIC2a and ASIC3 mRNAs were detected in human bladder. Similar amounts of ASIC1a and -3 were detected in detrusor smooth muscle, whereas in urothelium ASIC3 levels were higher than -1a. ASIC2a mRNA levels were lower than either -1a or -3 in both layers. ASIC currents were measured in TEU-2 cells and in primary cultures of human urothelium, and ASIC expression was confirmed by QPCR. Differentiation of TEU-2 cells caused an up-regulation of ASIC2a and ASIC3, and a down-regulation of ASIC1a mRNAs. BPS patients showed an up-regulation of ASIC2a and -3 mRNA, whereas ASIC1a remained unchanged. In contrast, the mRNA levels of TRPV1 were down-regulated during BPS. All differences were statistically significant (p<0.05)Conclusions: Several different ASIC subunits are expressed in human bladder and TEU-2 cells, where their levels are regulated during urothelial differentiation. An up-regulation of ASIC2a and -3 in BPS suggests their involvement in increased pain and hyperalgesia. A down-regulation of TRPV1 mRNA levels might indicate a different regulatory mechanism, controlling its expression in human bladder.

An Adeno-Associated Virus-Based Intracellular Sensor of Pathological Nuclear Factor-κB Activation for Disease-Inducible Gene Transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.