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Circadian rhythmicity in renal function suggests rhythmic adaptations in renal metabolism. To decipher the role of the circadian clock in renal metabolism, we studied diurnal changes in renal metabolic pathways using integrated transcriptomic, proteomic, and metabolomic analysis performed on control mice and mice with an inducible deletion of the circadian clock regulator Bmall in the renal tubule (cKOt). With this unique resource, we demonstrated that approximately 30% of RNAs, approximately 20% of proteins, and approximately 20% of metabolites are rhythmic in the kidneys of control mice. Several key metabolic pathways, including [NAD.sup.+] biosynthesis, fatty acid transport, carnitine shuttle, and [beta]-oxidation, displayed impairments in kidneys of cKOt mice, resulting in perturbed mitochondrial activity. Carnitine reabsorption from primary urine was one of the most affected processes with an approximately 50% reduction in plasma carnitine levels and a parallel systemic decrease in tissue carnitine content. This suggests that the circadian clock in the renal tubule controls both kidney and systemic physiology., Introduction Most basic physiological functions, including the process of urine formation in kidneys, exhibit substantial daily oscillations orchestrated by the circadian system. This circadian system allows organisms to anticipate and [...]

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can [...]

GLUT9 (SLC2A9) is a newly described urate transporter whose function, characteristics, and localization have just started to be elucidated. Some transport properties of human GLUT9 have been studied in the Xenopus laevis oocyte expression system, but the type of transport (uniport, coupled transport system, stoichiometry....) is still largely unknown. We used the same experimental system to characterize in more detail the transport properties of mouse GLUT9, its sensitivity to several uricosuric drugs, and the specificities of two splice variants, mGLUT9a and mGLUT9b. [[sup.14]C]urate uptake measurements show that both splice variants are high-capacity urate transporters and have a gm of ~650 [micro]M. The well-known uricosuric agents benzbromarone (500 [micro]M) and losartan (1 mM) inhibit GLUT9-mediated urate uptake by 90 and 50%, respectively. Surprisingly, phloretin, a glucose-transporter blocker, inhibits [[sup.14]C]urate uptake by ~50% at 1 mM. Electrophysiological measurements suggest that urate transport by mouse GLUT9 is electrogenie and voltage dependent, but independent of the [Na.sup.+] and [Cl.sup.-] transmembrane gradients. Taken together, our results suggest that GLUT9 works as a urate (anion) uniporter. Finally, we show by RT-PCR performed on RNA from mouse kidney microdissected tubules that GLUT9a is expressed at low levels in proximal tubules, while GLUT9b is specifically expressed in distal convoluted and connecting tubules. Expression of mouse GLUT9 in the kidney differs from that of human GLUT9, which could account for species differences in urate handling. uric acid; SLC2A9; splice variants

Adjustment of [Na.sup.+] balance in extracellular fluids is achieved by regulated [Na.sup.+] transport involving the amiloride-sensitive epithelial [Na.sup.+] channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and [Na.sup.+] channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Uspl0), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Uspl0 in ENaC-transfected HEK293 cells causes a more than fivefold increase in amiloride-sensitive [Na.sup.+] currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of [alpha]- and [gamma]-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Uspl0 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Uspl0 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Uspl0. In [mCCD.sub.c11] cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC. sorting nexin 3; epithelial sodium channel

The consequences of extracellular proteases on the amiloride-sensitive Na2+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta and gamma of the rat or Xenopus epithelial Na2+ channel (ENaC) were studied. Results indicated that extracellular proteases can increase the open probability of the epithelial sodium channel through an effect which is not related to the activation of a G protein-coupled receptor, but instead is induced by proteolysis of a protein that neither belongs nor is it related to the sodium channel.

Molecular characterization of two isoforms of new G-protein coupled receptor kinases (GRK) has been performed in rat kidney cDNAs. The two rat GRKs showed similar genomic sequence with previously identified human GRKs. They also both have the ability to phosphorylate a synthetic peptide which can be attributed to the presence of similar NH2-terminal domain of the GRKs. However, it was observed that the GRKs differ in the COOH-terminal sequences which would have an effect on their membrane targeting mechanisms.

Gly and Ser, the amino acid residues present in the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC), reduce the channel's affinity for amiloride block and prevent ion permeation, on mutation. The alpha subunit determines the pharmacological properties of the channel. The mutations in beta and gamma unit alter amiloride affinity and single channel conductances and activate binding sites for channel block by divalent cations. This indicates that these subunits participate in ion permeation through the channel.

Reverse transcription and polymerase chain reaction techniques were used to show that two isoforms of calcitonin receptors can be found in rat kidney. However, the CT1a isoform was the only type found in the kidney cortex, outer medulla and papilla, and predominantly in the cortical thick ascending limb of loop of Henle. Lesser concentrations were found in the medullary thick ascending limb, cortical collecting duct and the glomerulus.

A study was conducted to examine if insulin can directly influence electrolyte movement in isolated cortical (CTAL) and medullary thick ascending limbs (MTAL) microperfused in vitro, and to examine the various concentrations which are affected by insulin. Continuous and reversible enhancement of transepithelial potential difference (Vt) which is lumen-positive in the mouse CTAL and MTAL, is caused by insulin. NaCl reabsorbtion in the TAL and divalent cations transfer in the CTAL are directly incited by insulin.

Results indicate that the receptor activity modifying protein-3 (RAMP-3) is assembled with the calcitoni receptor-like receptor (CRLR) to yield a multiple N-glycosylated species using 2 to 4 consensus sites. Data show that inefficient use of Asn28 and Asn57 consensus sites result in the expression of multiple N-glycosylated RAMP3 species and that N-glycans participate in CRLR/RAMP3 receptor's function.