Ionic Permeability and Electrical Potential Differences in Necturus Kidney Cells

The cellular concentrations of Na, K, and Cl have been measured in kidney slices of the amphibian, Necturus maculosus. Permeability coefficients have been determined for Na, K, Cl, Rb, Cs, and choline, from studies both of the uptake of radioactive isotopes and the rate of cell swelling in anisotonic solutions. The results of both methods were found to agree well. Measurements were also made of electrical potential differences across the peritubular face of the kidney cells using bathing solutions in which the electrolyte composition and concentrations could be varied. The data obtained are consistent with a model cell in which the potential difference arises as a result of differences in Na permeability relative to K on the two faces of the cell. The intracellular Na concentration is considered to be regulated by a Na-K coupled pump located at the peritubular face of the cell.     

In Vivo Study of Transepithelial Potential Difference (TEPD) in Proximal Convoluted Tubules of Rat Kidney by Synchronization Modulation Electric Field

Synchronization modulation (SM) electric field has been shown to effectively activate function of Na⁺/K⁺ pumps in various cells and tissues, including skeletal muscle cells, cardiomyocyte, monolayer of cultured cell line, and peripheral blood vessels. We are now reporting the in vivo studies in application of the SM electric field to kidney of living rats. The field-induced changes in the transepithelial potential difference (TEPD) or the lumen potential from the proximal convoluted tubules were monitored. The results showed that a short time (20 s) application of the SM electric field can significantly increase the magnitude of TEPD from 1–2 mV to about 20 mV. The TEPD is an active potential representing the transport current of the Na/K pumps in epithelial wall of renal tubules. This study showed that SM electric field can increase TEPD by activation of the pump molecules. Considering renal tubules, many active transporters are driven by the Na⁺ concentration gradient built by the Na⁺/K⁺ pumps, activation of the pump functions and increase in the magnitude of TEPD imply that the SM electric field may improve reabsorption functions of the renal tubules.     

大鼠肾近曲小管、远曲小管和集合管壁厚的性别差异

运用光学显微镜对雌、雄大鼠的近曲小管、远曲小管和集合管的壁厚分别进行了测量,结果发现:雌、雄大鼠的近曲小管和集合管存在明显的性别差异(P<0.05);而远曲小管差异不明显(P>0.05).研究结果提示:雌、雄大鼠近曲小管和集合管的功能可能存在显著的性别差异.     

Mis-regulation of Mammalian Target of Rapamycin (mTOR) Complexes Induced by Albuminuria in Proximal Tubules

High albumin concentrations in the proximal tubule of the kidney causes tubulointerstitial injury, but how this process occurs is not completely known. To address the signal transduction pathways mis-regulated in renal injury, we studied the modulation of mammalian target of rapamycin (mTOR) complexes by physiologic and pathophysiologic albumin concentrations in proximal tubule cells. Physiologic albumin concentrations activated the PI3K/mTORC2/PKB/mTORC1/S6 kinase (S6K) pathway, but pathophysiologically high albumin concentrations overactivated mTORC1 and inhibited mTORC2 activity. This control process involved the activation of ERK1/2, which promoted the inhibition of TSC2 and activation of S6K. Furthermore, S6K was crucial to promoting the over activation of mTORC1 and inhibition of mTORC2. Megalin expression at the luminal membrane is reduced by high concentrations of albumin. In addition, knockdown of megalin mimicked all the effects of pathophysiologic albumin concentrations, which disrupt normal signal transduction pathways and lead to an overactivation of mTORC1 and inhibition of mTORC2. These data provide new perspectives for understanding the molecular mechanisms behind the effects of albumin on the progression of renal disease.     

A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.     

Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na⁺ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na⁺ + K⁺-ATPase and the ouabain-insensitive, K⁺-insensitive, and furosemide-sensitive Na⁺-ATPase, which participate in the active Na⁺ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na⁺-ATPase activity (C1P₅₀ ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na⁺-ATPase. Conversely, we did not observe any effect on the Na⁺ + K⁺-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na⁺-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.     

Electron Spin Resonance Studies of Ionic Permeability Properties of Thylakoid Membranes of Beta vulgaris and Avicennia germinans

Measurement of intrathylakoid aqueous volumes by electron spin resonance spectroscopy was used to study ionic permeability properties of thylakoid membranes isolated from Beta vulgaris L. and Avicennia germinans L. The thylakoids behaved as perfect osmometers in the presence of sorbitol and betaine. Thylakoids exposed to hypertonic solutions of NaCl and KCl shrank and subsequently swelled, requiring 10 minutes to regain their original volume. The initial influx rate calculated from the kinetics of changes in intrathylakoid volume in response to 450 millimolar gradients of NaCl and KCl was 2.3 × 10⁻¹³ moles per square centimeter per second. These data show that the passive permeability to NaCl and KCl was low.     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Action Potential in the Transverse Tubules and Its Role in the Activation of Skeletal Muscle

The double sucrose-gap method was applied to single muscle fibers of Xenopus. From the "artificial node" of the fiber, action potentials were recorded under current-clamping condition together with twitches of the node. The action potentials were stored on magnetic tape. The node was then made inexcitable by tetrodotoxin or by a sodium-free solution, and the wave form of the action potential stored on magnetic tape was imposed on the node under voltage-clamp condition (simulated AP). The twitch height caused by the simulated AP's was always smaller than the twitch height produced by the real action potentials, the ratio being about 0.3 at room temperature. The results strongly suggest that the transverse tubular system is excitable and is necessary for the full activation of twitch, and that the action potential of the tubules contributes to about 70 % of the total mechanical output of the normal isotonic twitch at 20°C. Similar results were obtained in the case of tetanic contraction. At a temperature near 10°C, twitches produced by the simulated AP were not very different (85 % of control amplitude) from the twitches caused by real action potentials. This indicates that the excitability of the tubules becomes less necessary for the full activation of twitch as the temperature becomes lower.     

A Novel Transporter of SLC22 Family Specifically Transports Prostaglandins and Co-localizes with 15-Hydroxyprostaglandin Dehydrogenase in Renal Proximal Tubules

We identified a novel prostaglandin (PG)-specific organic anion transporter (OAT) in the OAT group of the SLC22 family. The transporter designated OAT-PG from mouse kidney exhibited Na⁺-independent and saturable transport of PGE₂ when expressed in a proximal tubule cell line (S₂). Unusual for OAT members, OAT-PG showed narrow substrate selectivity and high affinity for a specific subset of PGs, including PGE₂, PGF_2α, and PGD₂. Similar to PGE₂ receptor and PGT, a structurally distinct PG transporter, OAT-PG requires for its substrates an α-carboxyl group, with a double bond between C13 and C14 as well as a (S)-hydroxyl group at C15. Unlike the PGE₂ receptor, however, the hydroxyl group at C11 in a cyclopentane ring is not essential for OAT-PG substrates. Addition of a hydroxyl group at C19 or C20 impairs the interaction with OAT-PG, whereas an ethyl group at C20 enhances the interaction, suggesting the importance of hydrophobicity around the ω-tail tip forming a “hydrophobic core” accompanied by a negative charge, which is essential for substrates of OAT members. OAT-PG-mediated transport is concentrative in nature, although OAT-PG mediates both facilitative and exchange transport. OAT-PG is kidney-specific and localized on the basolateral membrane of proximal tubules where a PG-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase, is expressed. Because of the fact that 15-keto-PGE₂, the metabolite of PGE₂ produced by 15-hydroxyprostaglandin dehydrogenase, is not a substrate of OAT-PG, the transport-metabolism coupling would make unidirectional PGE₂ transport more efficient. By removing extracellular PGE₂, OAT-PG is proposed to be involved in the local PGE₂ clearance and metabolism for the inactivation of PG signals in the kidney cortex.     

Potential, Current, and Ionic Fluxes across the Isolated Retinal Pigment Epithelium and Choroid

A flux chamber was utilized for in vitro studies of a membrane formed by the retinal pigment epithelium and choroid of the eye of the toad (Bufo arenarum and Bufo marinus). A transmembrane potential of 20 to 30 mv was found, the pigment epithelium surface positive with respect to the choroidal surface. Unidirectional fluxes of chloride, sodium, potassium, and calcium were determined in the absence of an electrochemical potential difference. A net transfer of chloride from pigment epithelium to choroid accounted for a major fraction of the mean short-circuit current. A small net flux of sodium from choroid to pigment epithelium was detected in Bufo marinus. In both species of toads, however, about one-third of the mean short-circuit current remained unaccounted for. Manometric determinations of bicarbonate suggested an uptake of this ion at the epithelial surface of the membrane but did not provide evidence of a relationship between this process and the short-circuit current.