bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipase A2 is involved in mediating the effect of extracellular Ca2+ on apical K+ channels in rat TAL
Authors: Wang W. , Lu M. , Balazy M. , Hebert S. C.
Published: December 2017

Raising extracellular Ca2+ (Ca2+o) stimulating the Ca(2+)-sensing receptor (CaR) decreased the activity of the apical 70-pS K+ channel via a cytochrome P-450-dependent mechanism in the thick ascending limb (TAL) of the rat kidney [W. H. Wang, M. Lu, and S. C. Hebert. Am. J. Physiol. 270 (Cell Physiol. 39): C103-C111, 1996]. We have now used the patch-clamp technique and fluorescent dyes to investigate the signaling mechanism by which this effect is produced. Addition of 500 microM gadolinium (Gd3+), an agent which has been shown to activate the CaR (E. M. Brown, G. Gamba, D. Riccardi, M. Lombardi, R. Butters, O. Kifor, A. Sun, M. A. Hediger, J. Lytton, and S. C. Hebert. Nature 366: 575-580, 1993), mimics the inhibitory effect of raising Ca2+o from 1.1 to 5 mM on channel activity. Effects of the high Ca2+o and Gd3+ were abolished by blockade of phospholipase A2 (PLA2) but not by inhibition of phospholipase C (PLC). Raising Ca2+o also increased 20-hydroxyeicosatetraenoic acid production significantly. To investigate the effect of stimulation of the CaR on intracellular Ca2+ (Ca2+i), we used the acetoxymethyl ester of fura 2 to monitor the Ca2+i. Raising Ca2+o from 1.1 to 5 mM increased the Ca2+i significantly from 50 to 150 nM. However, addition of thapsigargin failed to abolish the effect of 5 mM Ca2+o on Ca2+i. Also, application of Gd3+ only slightly increased the Ca2+i, suggesting that elevation of the Ca2+i by high Ca2+o was the result of an influx of Ca2+ rather than enhanced Ca2+ release from Ca2+ stores. That the increase in Ca2+ influx is not mainly responsible for the effect of stimulating the CaR on channel activity is further supported by experiments in which 500 microM Gd3+ inhibited the K+ channel in cell-attached patches in a Ca(2+)-free bath. Furthermore, addition of 500 microM Gd3+ or 5 mM Ca2+o decreased intracellular Na+ measured with fluorescent sodium indicator, suggesting inhibition of Na+ transport. We conclude that PLA2 is involved in the stimulation of the CaR-induced inhibition of apical K+ channels in the TAL.
Pleiotropic upregulation of Na(+)-dependent cotransporters by retinoic acid in opossum kidney cells
Authors: de Toledo F. G. , Beers K. W. , Dousa T. P.
Published: December 2017

All-trans-retinoic acid (atRA) is a regulator of cellular growth and differentiation. We investigated whether atRA can upregulate Na(+)-dependent cotransporters in opossum kidney (OK) cells and thus increase uptake from tubular fluid of several solutes needed for growth during early stages of ontogenesis. In OK cells, incubation with atRA for 24 h increased the Na+ gradient-dependent cotransports of phosphate, L-proline, L-glutamic acid, and SO(4)2- by a similar degree (approximately 40%) that was prevented by pretreatment with actinomycin D. In contrast, activities of other Na(+)-dependent transporters, Na(+)-K(+)-adenosinetriphosphatase, gamma-glutamyltranspeptidase, and leucine aminopeptidase, were unchanged by atRA. Cell proliferation determined by [3H]thymidine incorporation was not increased by atRA. The stimulatory effects of atRA and phosphate deprivation on Na(+)-Pi cotransport demonstrated additivity, whereas the combination of atRA and 3,5,3'-triiodothyronine did not. atRA stimulated Na(+)-Pi cotransport in LLC-PK1 cells with an analogous time course and to a similar extent as observed in OK cells. We conclude that atRA stimulates several Na(+)-dependent cotransporters via a genomic mechanism and may represent a synchronous adaptation to nutritional requirements of early phases of ontogenesis.
Differential activities of H+extrusion systems in MDCK cells due to extracellular osmolality and pH
Authors: Feifel Elisabeth , Krall Markus , Geibel John P. , Pfaller Walter
Published: December 2017

The aim of the present study was to obtain detailed information on MDCK cell proton secretion characteristics under various growth conditions. Confluent monolayers cultured on glass coverslips were adapted over 48 h to media with different osmolality and pH (200 mosmol/kgH2O, pH 7.4; 300 mosmol/kgH2O, pH 7.4; and 600 mosmol/kgH2O, pH 6.8) corresponding to the luminal fluid composition of the collecting duct segments found in the in renal cortex, the outer stripe of outer medulla and inner medulla. Proton fluxes were determined from the recovery of intracellular pH following an acid load induced by an NH4Cl pulse times the corresponding intrinsic buffering power (βi). The intracellular buffering power was found to change only with culture medium osmolality but not with culture medium pH. In addition to an amiloride and Hoe-694-sensitive Na+/H+exchange, Madin-Darby canine kidney (MDCK) cells possess a Sch-28080-sensitive, K+-dependent H+extrusion mechanism that is increased upon adaptation of monolayers to hyperosmotic-acidic culture conditions. A significant contribution of the bafilomycin A1-sensitive vacuolar H+-ATPase could be found only in cells adapted to hyposmotic culture conditions. Exposure of MDCK cells to 10−5or 10−7M aldosterone for either 1 or 18 h did not alter the H+extrusion characteristics significantly. The results obtained show that different extracellular osmolality and pH induce different MDCK phenotypes with respect to their H+-secreting systems.
A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating
Authors: Choe Han , Zhou Hao , Palmer Lawrence G. , Sackin Henry
Published: December 2017

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 ± 0.02 to near zero ( n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093–4099, 1996). However, the apparent p K a of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent p K a for inward conductance from 6.5 ± 0.01 ( n = 8, wild type) to 7.0 ± 0.02 ( n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent p K a in the opposite direction, from 6.5 ± 0.01 ( n = 8, wild type) to 6.0 ± 0.02 ( n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent p K a via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS ( n = 8, wild type) to 17.4 ± 1 pS ( n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.
Cyclooxygenase-2 is expressed in bladder during fetal development and stimulated by outlet obstruction
Authors: Park John M. , Yang Tianxin , Arend Lois J. , Smart Ann M. , Schnermann Jurgen B. , Briggs Josephine P.
Published: December 2017

Studies were undertaken to assess expression of inducible cyclooxygenase (COX)-2 in bladder during fetal development and COX-1 and COX-2 expression after outlet obstruction. Bladder tissue or bladder progenitor tissue was harvested from CD-1 murine embryos at embryonic days 11.5( E11.5), E14.5, E17.5, E20.5 (newborn), and from adult. Bladder obstruction was created in adult female mice by ligating the urethra, and bladders were harvested after 3–24 h of obstruction. Gene expression was assessed by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. COX-2 was highly expressed at the early stages of bladder development and declined progressively throughout gestation. In adult bladder, both COX-1 and COX-2 were detectable at low levels under basal conditions. An ∼30-fold increase in COX-2 mRNA was seen after 24 h of obstruction. In contrast, COX-1 did not change with obstruction. COX-2 mRNA levels peaked at 6 h of obstruction. In regional bladder-distention models, COX-2 induction was confined to the area of distention. Bladder outlet obstruction stimulates COX-2 expression dramatically, reactivating a gene that is highly expressed during fetal development.
Peptide YY receptor distribution and subtype in the kidney: effect on renal hemodynamics and function in rats
Authors: Blaze C. A. , Mannon P. J. , Vigna S. R. , Kherani A. R. , Benjamin B. A.
Published: December 2017

This study characterizes the location and subtype of peptide YY (PYY) receptors in rat and rabbit kidney and the effect of PYY on renal function and renal hemodynamics in rats. Receptor autoradiography performed on kidney sections revealed a dense concentration of specific high-affinity binding sites [dissociation constant ( Kd) = 0.7 ± 0.1 nM] in the papilla of the rat, as well as cortical and papillary binding in the rabbit (papilla, Kd = 1.6 ± 0.6 nM) and some medullary binding in both species. In the rat papilla, neuropeptide Y (NPY) and the Y1agonist [Leu31, Pro34]NPY competed with PYY for binding ( Kd = 1.1 ± 0.4 nM and 1.6 ± 0.5 nM, respectively), but NPY-(13—36) (Y2agonist) and pancreatic polypeptide (PP, Y4agonist) were without effect, demonstrating that the PYY receptor in the rat papilla is of the Y1subtype. In the rabbit papilla, NPY and NPY-(13—36) competed with PYY ( Kd = 0.5 ± 0.1 and 3.1 ± 0.6 nM, respectively), but [Leu31, Pro34]NPY and PP were without effect, evidence that the PYY receptor in the rabbit papilla is of the Y2subtype. Infusion of PYY into rats (47 pmol ⋅ kg−1⋅ min−1) increased mean arterial pressure (103 ± 6 to 123 ± 8 mmHg) and decreased renal plasma flow (13 ± 1.8 to 8.4 ± 2.1 ml/min) but produced no significant change in glomerular filtration rate or sodium excretion. Injection of PYY or angiotensin II directly into the renal artery caused a dose-related vasoconstriction, which was less intense but of longer duration for PYY than for angiotensin II. These results show that receptors for PYY are widely distributed in the kidney and that exogenously administered PYY causes renal vasoconstriction and may influence renal sodium excretion.
Apical ANG II-stimulated PLA2activity and Na+flux: a potential role for Ca2+-independent PLA2
Authors: Becker B. N. , Cheng H.-F. , Harris R. C.
Published: December 2017

Type 1 angiotensin II (ANG II) receptors (AT1R), which mediate proximal tubule (PT) salt and water reabsorption, undergo endocytosis and recycling. Prior studies in a PT-like model (LLC-PKCl4cells expressing rabbit AT1R) (LLC-PK-AT1R cells) determined that quinacrine, a nonspecific phospholipase A2(PLA2) inhibitor, and the haloenol lactone suicide substrate (HELSS), a Ca2+-independent PLA2inhibitor, attenuated apical (AP) AT1R recycling. Further studies were undertaken to examine the association between AT1R endocytotic movement and PLA2activity in this model. AP ANG II (100 nM) increased[Formula: see text]arachidonic acid ([Formula: see text]AA) release 4.4 ± 0.38-fold in LLC-PK-AT1R cells cultured on permeable supports. Basolateral (BL) ANG II had no significant effect. Reversed-phase high-performance liquid chromatography confirmed that AP ANG II stimulated free [Formula: see text]AA release. Quinacrine, HELSS, and palmitoyl trifluoromethyl ketone, another Ca2+-independent PLA2inhibitor, inhibited AP ANG II-stimulated [Formula: see text]AA release, as did inhibiting AP AT1R internalization with phenylarsine oxide. The role of HELSS-inhibitable AA release in ANG II-mediated22Na flux was examined, given the effects of AT1R-mediated PLA2activity on salt and water reabsorption. AP ANG II (100 nM) stimulated22Na flux (AP → BL), a response inhibited by HELSS. Thus, in this model, AP AT1R activated PLA2with concomitant22Na flux (AP → BL), suggesting a link between AP AT1R endocytotic movement, AT1R-stimulated PLA2activity, and22Na flux in this model. The effects of HELSS suggest that Ca2+-independent PLA2activity may be involved in this AP ANG II response.
Endothelin-1 increases rat distal tubule acidification in vivo
Authors: Wesson Donald E. , Dolson George M.
Published: December 2017

Because endothelin receptor inhibition blunts increased distal tubule acidification induced by dietary acid, we examined whether endothelin-1 (ET-1) increases acidification of in vivo perfused distal tubules of anesthetized rats. ET-1 was infused intra-aortically (1.4 pmol ⋅ kg−1⋅ min−1) into control animals and into those with increased distal tubule HCO3secretion induced by drinking 80 mM NaHCO3solution for 7–10 days. ET-1 increased distal tubule acidification in both control and NaHCO3animals. Increased acidification in control animals was mediated by increased distal tubule H+secretion (23.7 ± 2.2 vs. 18.7 ± 1.7 pmol ⋅ mm−1⋅ min−1, P < 0.05) with no changes in HCO3secretion. By contrast, ET-1 increased distal tubule acidification in NaHCO3animals predominantly by decreasing HCO3secretion (−9.5 ± 1.0 vs. −18.7 ± 1.8 pmol ⋅ mm−1⋅ min−1, P < 0.001) with less influence on H+secretion. When indomethacin was infused (83 μg ⋅ kg−1⋅ min−1) to inhibit synthesis of prostacyclin, an agent previously shown to increase HCO3secretion in the distal tubule, ET-1 increased distal tubule H+secretion in both control (24.3 ± 2.2 vs. 15.7 ± 1.6 pmol ⋅ mm−1⋅ min−1, P < 0.02) and NaHCO3(20.0 ± 2.0 vs. 13.6 ± 1.4 pmol ⋅ mm−1⋅ min−1, P < 0.05) without affecting HCO3secretion. The data show that ET-1 increases distal tubule acidification in vivo and can do so by increasing H+secretion and by decreasing HCO3secretion when the latter is augmented by dietary NaHCO3.
Immunolocalization of AE2 anion exchanger in rat kidney
Authors: Alper Seth L. , Stuart-Tilley Alan K. , Biemesderfer Daniel , Shmukler Boris E. , Brown Dennis
Published: December 2017

The cellular and subcellular localizations of the AE2 anion exchanger in rat kidney have remained elusive despite detection of moderately abundant AE2 mRNA and AE2 polypeptide in all kidney regions. In this report a simple epitope unmasking technique has allowed the immunolocalization of AE2 antigenic sites in basolateral membranes of several rat kidney tubular epithelial cells. AE2 immunostaining was faint or absent in the glomerulus and proximal tubule, present in descending and ascending thin limbs, and stronger in the medullary thick ascending limb (MTAL). A lower staining intensity was found in cortical thick ascending limbs and even less in the distal convoluted tubule. In contrast, there was an enhanced staining in the macula densa. In principal cells (PC) of the connecting segment, AE2 was undetectable but gradually increased in intensity along the collecting duct, with strongest staining in inner medullary collecting duct (IMCD) PC. A sodium dodecyl sulfate-sensitive AE2-related Golgi epitope was also detected in some interstitial and endothelial cells of the inner medulla and in epithelial cells of IMCD and MTAL. Colchicine treatment of the intact animal altered the distribution of this Golgi-associated epitope but left plasmalemmal AE2 undisturbed. Reverse transcription-polymerase chain reaction detected AE2a, AE2b, and AE2c2 but not AE2c1 transcripts in rat kidney mRNA. The results suggest a widespread occurrence of the AE2 protein in several renal epithelial cell types.
Cloning and characterization of maxi K+channel α-subunit in rabbit kidney
Authors: Morita Takashi , Hanaoka Kazushige , Morales Marcelo M. , Montrose-Rafizadeh Chahrzad , Guggino William B.
Published: December 2017

We have identified in rabbit renal cells two alternatively spliced transcripts of the α-subunit rbslo1 and rbslo2. Rbslo1 has a novel “in-frame” 174-bp insertion immediately after the predicted S8 transmembrane segment, whereas rbslo2 has a 104-bp deletion between S9 and S10, creating a frameshift and a premature termination codon. Amino acid identity between mouse maxi K+channel α-subunit ( mslo) and rbslo1 was 99%. Two transcript sizes of 4.2 and 7.5 kb were detected in brain, kidney, stomach, testis, and lung. Rbslo is expressed in glomeruli, thin limbs of Henle’s loop, medullary and cortical thick ascending limbs of Henle’s loop, and cortical, outer, and inner medullary collecting ducts; however, it was rarely detected in proximal convoluted tubules. Rbslo1 is most abundant in inner medulla. Expressed in Xenopus oocytes, rbslo1 generates depolarization-activated, outwardly rectifying K+currents. Rbslo1 expressed in Chinese hamster ovary cells could be activated by depolarization and Ca2+. These data suggest that rbslo transcripts are expressed in multiple nephron segments and that the magnitude of mRNA expression varies among different nephron segments.
Nonlinear filter properties of the thick ascending limb
Authors: Layton H. E. , Pitman E. Bruce , Moore Leon C.
Published: December 2017

A mathematical model was used to investigate the filter properties of the thick ascending limb (TAL), that is, the response of TAL luminal NaCl concentration to oscillations in tubular fluid flow. For the special case of no transtubular NaCl backleak and for spatially homogeneous transport parameters, the model predicts that NaCl concentration in intratubular fluid at each location along the TAL depends only on the fluid transit time up the TAL to that location. This exact mathematical result has four important consequences: 1) when a sinusoidal component is added to steady-state TAL flow, the NaCl concentration at the macula densa (MD) undergoes oscillations that are bounded by a range interval envelope with magnitude that decreases as a function of oscillatory frequency; 2) the frequency response within the range envelope exhibits nodes at those frequencies where the oscillatory flow has a transit time to the MD that equals the steady-state fluid transit time (this nodal structure arises from the establishment of standing waves in luminal concentration, relative to the steady-state concentration profile, along the length of the TAL); 3) for any dynamically changing but positive TAL flow rate, the luminal TAL NaCl concentration profile along the TAL decreases monotonically as a function of TAL length; and 4) sinusoidal oscillations in TAL flow, except at nodal frequencies, result in nonsinusoidal oscillations in NaCl concentration at the MD. Numerical calculations that include NaCl backleak exhibit solutions with these same four properties. For parameters in the physiological range, the first few nodes in the frequency response curve are separated by antinodes of significant amplitude, and the nodes arise at frequencies well below the frequency of respiration in rat. Therefore, the nodal structure and nonsinusoidal oscillations should be detectable in experiments, and they may influence the dynamic behavior of the tubuloglomerular feedback system.
A simplified method for isolation of large numbers of defined nephron segments
Authors: Schafer J. A. , Watkins M. L. , Li L. , Herter P. , Haxelmans S. , Schlatter E.
Published: December 2017

We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37°C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger. Pflügers Arch. 421: 381–387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.
Cl−and K+conductances activated by cell swelling in primary cultures of rabbit distal bright convoluted tubules
Authors: Rubera I. , Tauc M. , Poujeol C. , Bohn M. T. , Bidet M. , De Renzis G. , Poujeol P.
Published: December 2017

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl−currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl−currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I−= Br−> Cl−≫ glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid and 1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 μM LaCl3, 10 μM nifedipine, 100 μM verapamil, 5 μM tamoxifen, and 50 μM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+was without effect on the fully developed Cl−currents. Osmotic shock also activated K+currents in Cl−-free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl−and K+currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.
Zis: a developmentally regulated gene expressed in juxtaglomerular cells
Authors: Karginova Elena A. , Pentz Ellen Steward , Kazakova Irina G. , Norwood Victoria F. , Carey Robert M. , Gomez R. Ariel
Published: December 2017

Renal juxtaglomerular (JG) cells are specialized myoepithelioid cells located in the afferent arteriole at the entrance to the glomerulus. Their main function and distinctive feature is the synthesis and release of renin, the key hormone-enzyme of the renin-angiotensin system that regulates arterial blood pressure. Despite their relevance to health and disease, not much is known about factors that confer and/or maintain JG cell identity. To identify genes uniquely expressed in JG cells, we used a cell culture model and RNA differential display. JG cells cultured for 2 days express renin and renin mRNA, but after 10 days in culture they no longer contain or release renin and renin mRNA is reduced 700-fold. We report one cDNA differentially expressed in the 2-day JG cell culture that detects a 2.6-kb mRNA expressed at higher levels in newborn than adult kidney. Screening a 2-day culture JG cell cDNA library yielded clones representing differentially spliced transcripts. These cDNAs encode one unique protein (Zis) containing zinc fingers and domains characteristic of splicing factors and RNA binding proteins. Northern blot analysis confirmed Zis mRNA expression in differentiated JG cells, and identified an additional unique 1.5-kb transcript. The Zis transcripts are developmentally regulated in kidney and a number of other organs. The features of the Zis protein and its organ distribution suggest a possible role in regulation of transcription and/or splicing, both important steps for controlling developmentally expressed genes.
Localization of the ROMK protein on apical membranes of rat kidney nephron segments
Authors: Xu Jason Z. , Hall Amy E. , Peterson Linda N. , Bienkowski Michael J. , Eessalu Thomas E. , Hebert Steven C.
Published: December 2017

The ATP-sensitive, inwardly rectifying K+channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR1.2) have been identified in kindreds with neonatal Bartter’s syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252–391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34–49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluorescence studies using anti-ROMK COOH-terminal antibody showed intense apical staining along the loop of Henle and distal nephron; staining with preimmune and immunoabsorbed serum was negative. When colocalized with distal nephron markers [the thiazide-sensitive cotransporter (rTSC1), the bumetanide-sensitive cotransporter (rBSC1), the vacuolar type H+-ATPase, and neuronal nitric oxide synthase (NOS I)], the ROMK protein was found primarily at the apical border of cells in the TAL, macula densa, distal convoluted tubule, and connecting tubule. Within the CCD, the ROMK protein was expressed in principal cells and was absent from intercalated cells. The tubule localization and polarity of ROMK staining are consistent with the distribution of ROMK mRNA and provide more support for ROMK being the low-conductance K+secretory channel in the rat distal nephron.
Systemic and renal hemodynamic changes in the luteal phase of the menstrual cycle mimic early pregnancy
Authors: Chapman Arlene B. , Zamudio Stacy , Woodmansee Whitney , Merouani Aicha , Osorio Fritz , Johnson Ann , Moore Lorna G. , Dahms Thomas , Coffin Carolyn , Abraham William T. , Schrier Robert W.
Published: December 2017

Blood pressure decreases during early pregnancy in association with a decrease in peripheral vascular resistance and increases in renal plasma flow and glomerular filtration rate. These early changes suggest a potential association with corpora lutea function. To determine whether peripheral vasodilation occurs following ovulation, we studied 16 healthy women in the midfollicular and midluteal phases of the menstrual cycle. A significant decrease in mean arterial pressure in the midluteal phase of the cycle (midfollicular of 81.7 ± 2.0 vs. midluteal of 75.4 ± 2.3 mmHg, P< 0.005) was found in association with a decrease in systemic vascular resistance and an increase in cardiac output. Renal plasma flow and glomerular filtration rate increased. Plasma renin activity and aldosterone concentration increased significantly in the luteal phase accompanied by a decrease in atrial natriuretic peptide concentration. Serum sodium, chloride, and bicarbonate concentrations and osmolarity also declined significantly in the midluteal phase of the menstrual cycle. Urinary adenosine 3′,5′-cyclic monophosphate (cAMP) excretion increased in the luteal compared with the follicular phase, whereas no changes in urinary cGMP or NO2/NO3excretion were found. Thus peripheral vasodilation occurs in the luteal phase of the normal menstrual cycle in association with an increase in renal plasma flow and filtration. Activation of the renin-angiotensin-aldosterone axis is found in the luteal phase of the menstrual cycle. These changes are accompanied by an increase in urinary cAMP excretion indicating potential vasodilating mediators responsible for the observed hemodynamic changes.
Effect of cyclosporin A on the expression of tissue kallikrein, kininogen, and bradykinin receptor in rat
Authors: Wang Cindy , Ford Philbert , Chao Caroline , Chao Lee , Chao Julie
Published: December 2017

The tissue kallikrein-kinin system is involved in vasodilation and blood pressure regulation. In the present study, we investigated the effects of chronic cyclosporin A (CsA) administration on blood pressure and the expression of tissue kallikrein, kininogen, and bradykinin receptor in normotensive Wistar rats. Chronic administration of CsA significantly increased systolic blood pressure compared with control rats ( n = 6, P < 0.01), although body weight was significantly lower than control rats ( n = 6, P < 0.01). The development of hypertension was accompanied by the altered expression of kallikrein-kinin system components. Immunoreactive renal kallikrein and urinary excretion of tissue kallikrein levels were increased by chronic administration of CsA ( n = 5 or 6, P < 0.05). Levels of N-tosyl-l-phenylalanine chloromethyl ketone-trypsin and kallikrein-releasable kininogens in sera increased in response to chronic CsA treatment ( n = 5 or 6, P < 0.05). Chronic CsA treatment significantly increased renal kallikrein, bradykinin B2receptor, and hepatic kininogen mRNA levels. The increased levels of tissue kallikrein-kinin system components were accompanied by significant increases in 24-h urine excretion and water intake after chronic CsA treatment ( n = 5, P < 0.05). These results suggest that enhanced activity of the tissue kallikrein-kinin system may compensate for the CsA-induced vasoconstriction and hypertension.
cDNA cloning and localization of OCRL-1 in rabbit kidney
Authors: Erb Brian C. , Velázquez Heino , Gisser Monique , Shugrue Christine A. , Reilly Robert F.
Published: December 2017

The oculocerebrorenal syndrome of Lowe (OCRL) is a hereditary multisystem disorder characterized by congenital cataracts, mental retardation, renal tubular dysfunction, and progressive renal insufficiency. Tubular abnormalities include proximal tubular dysfunction, a distal acidification defect, and a possible impairment of urinary concentrating ability. The most important renal manifestation of Lowe’s syndrome is a progressive loss of kidney function associated with a glomerular lesion that progresses to end-stage renal disease in either the third or fourth decade. The gene responsible for Lowe’s syndrome, OCRL-1, was recently identified by positional cloning, and mutations were demonstrated in many affected patients. In the present study reverse transcription-polymerase chain reaction (RT-PCR) was used to clone a partial-length cDNA encoding rabbit renal OCRL-1. There is a high degree of similarity between rabbit and human sequences, with nucleotide and amino acid identities of 92% and 97%, respectively. Northern analysis identified a 5.4-kb transcript that is expressed in both rabbit kidney cortex and medulla. Isolated nephron-segment RT-PCR showed that OCRL-1 is expressed in all segments studied: the glomerulus, proximal tubule, medullary and cortical thick ascending limb, distal convoluted tubule, connecting tubule, cortical collecting duct, and outer medullary collecting duct. Defective OCRL-1 expression in these regions may play a pathogenetic role in the renal manifestations of this syndrome.
Control of excretion of potassium: lessons from studies during prolonged total fasting in human subjects
Authors: Lin Shih-Hua , Cheema-Dhadli Surinder , Gowrishankar Manjula , Marliss Errol B. , Kamel Kamel S. , Halperin Mitchell L.
Published: December 2017

A deficit of K+of close to 300 mmol develops in the first 2 wk of fasting, but little further excretion of K+occurs, despite high levels of aldosterone and the delivery of ketoacid anions that are not reabsorbed in the distal nephron. Our purpose was to evaluate how aldosterone could have primarily NaCl-retaining, rather than kaliuretic, properties in this setting. To evaluate the role of distal delivery of Na+, four fasted subjects recieved an acute infusion of NaCl to induce a natriuresis. To assess the role of distal delivery of [Formula: see text], five fasted subjects were given an infusion containing NaHCO3. The natriuresis induced by an infusion of NaCl caused only a small rise in the rate of excretion of K+(0.8 ± 0.1 to 1.9 ± 0.3 mmol/h); in contrast, when [Formula: see text]replaced Cl−in the infusate, K+excretion rose to 8.3 ± 2.2 mmol/h, despite little excretion of[Formula: see text] (urine, pH 5.8) and similar rates of excretion of Na+. The transtubular K+concentration gradient was 19 ± 3 with [Formula: see text] and 6 ± 2 with NaCl. We conclude that the infusion of NaHCO3led to an increase in K+excretion, likely reflecting an increased rate of distal K+secretion. With a low distal delivery of[Formula: see text], aldosterone acts as a NaCl-retaining, rather than a kaliuretic, hormone.
Effect of glandular kallikrein on distal nephron HCO 3 − secretion in rats and on HCO 3 − secretion in MDCK cells
Authors: Vallés P. , Ebner S. , Manucha W. , Gutierrez L. , Marin-Grez M.
Published: December 2017

Renal kallikrein is localized in the connecting tubule cells and secreted into the tubular fluid at late distal nephron segments. The present experiments were performed to further test the hypothesis that renal kallikrein reduces bicarbonate secretion of cortical collecting duct (CCD). The effect of orthograde injections of pig pancreatic kallikrein (1 or 3 μg/ml) into the renal tubular system was investigated. Urine fractions (Fr) were collected after a 2-min stop flow. Changes in the urine fraction with respect to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first two urine fractions collected after glandular kallikrein administration (kallikrein, 1 μg/ml, P < 0.05; kallikrein, 3 μg/ml, P < 0.01).[Formula: see text] secretion of collecting ducts was significantly reduced dose dependently by orthograde and also reduced by retrograde pig pancreatic kallikrein administration. Release of kinins into the fractions was not affected by the retrograde kallikrein injection, even though the kallikrein activity increased considerably (2.26 ± 0.2 vs. 1.55 ± 0.2, P < 0.05). Adequacy of retrograde injections for delivering substances to the CCD was demonstrated by injecting colloidal mercury and detecting the appearance of this mercury in the renal cortex by transmission electron microscopy. The integrity of the renal tissue after a retrograde ureteral injection was confirmed by scanning electron microscopy. These results confirm and extend previous data (M. Marin-Grez and P. Vallés. Renal Physiol. Biochem. 17: 301–306, 1994; and M. Marin-Grez, P. Vallés, and P. Odigie. J. Physiol. 488: 163–170, 1995) showing that renal kallikrein reduces bicarbonate secretion at the CCD, probably by inhibiting [Formula: see text]transported by a mechanism unrelated to its kininogenase activity. Support for this assessment was obtained in experiments testing the effect of kallikrein on the luminal bicarbonate secretion of a subpopulation of Madin-Darby canine kidney cells capable of extruding the anion. Kallikrein inhibited[Formula: see text]/Cl−exchange, and the degree of inhibition was dose dependent. This inhibition occurred in the absence of kininogen in the bathing solution.
Primary structure and functional expression of a cortical collecting duct Kirchannel
Authors: Welling Paul A.
Published: December 2017

Maintenance of a negative membrane potential in the cortical collecting duct (CCD) principal cell depends on a small-conductance, inward-rectifying basolateral membrane K+(Kir) channel. In the present study, a candidate cDNA encoding this K+channel, CCD-IRK3, was isolated from a mouse collecting duct cell line, M1. CCD-IRK3shares a high degree of homology with a human brain inward-rectifier K+channel (Kir2.3). By Northern analysis, CCD-IRK3transcript (2.9 kb) was readily detected in M1 CCD cells but not in Madin-Darby canine kidney, LLC-PK1, Chinese hamster ovary, or monkey kidney fibroblast cell lines. CCD-IRK3-specific reverse transcription-polymerase chain reaction confirmed bonafide expression in the kidney. Functional expression studies in Xenopus oocytes revealed that CCD-IRK3operates as strongly inward-rectifying K+channel. The cation selectivity profile of CCD-IRK3[ionic permeability values ( PK/ Pi), Tl ≥ Rb ≥ K+≫ NH4> Na; inward-slope conductance ( GK/ Gi), Tl ≥ K+≫ NH4> Na > Rb] is similiar to the macroscopic CCD basolateral membrane K+conductance ( GK/ Gi, K+≫ NH4> Rb; PK/ Pi, Rb ≈ K+≫ NH4). CCD-IRK3also exhibits the pharmacological features of the native channel. Patch-clamp analysis reveals that CCD-IRK3functions as a high open probability, voltage-independent, small-conductance channel (14.5 pS), consistent with the native channel. Based on these independent lines of evidence, CCD-IRK3is a possible candidate for the small-conductance basolateral Kirchannel in the CCD.
Hypotonicity increases transcription, expression, and action ofEgr-1in murine renal medullary mIMCD3 cells
Authors: Zhang Zheng , Cohen David M.
Published: December 2017

In cells of the murine renal inner medullary collecting duct (mIMCD3) cell line, acute hypotonic shock (50% dilution of medium with sterile water but not with sterile 150 mM NaCl) increased Egr-1 mRNA abundance 2.5-fold at 6 h, as determined by Northern analysis. This increase was accompanied by increased Egr-1 transcription, as quantitated by luciferase reporter gene assay. Increased transcription was dose dependent, additive with other Egr-1 transcriptional activators, and occurred in the absence of overt cytotoxicy, as quantitated via a fluorometric viability assay. In addition, hypotonic stress increased Egr-1 protein abundance, which was accompanied by augmented Egr-1-specific DNA binding ability, as measured via electrophoretic mobility shift assay. Increased DNA binding was further associated with increased transactivation by Egr-1, demonstrated through transient transfection of mIMCD3 cells with a luciferase reporter gene driven by tandem repeats of the Egr-1 DNA consensus sequence. Taken together, these data indicate that hypotonic stress activates Egr-1 transcription, translation, DNA binding, and transactivation in renal medullary cells. This phenomenon might play a role in the acquisition of the adaptive phenotype in response to hypotonic stress in cells of the renal medulla in vivo.
A functional CFTR-NBF1 is required for ROMK2-CFTR interaction
Authors: McNicholas Carmel M. , Nason Malcolm W. , Guggino William B. , Schwiebert Erik M. , Hebert Steven C. , Giebisch Gerhard , Egan Marie E.
Published: December 2017

In a previous study on inside-out patches of Xenopus oocytes, we demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) enhances the glibenclamide sensitivity of a coexpressed inwardly rectifying K+ channel, ROMK2 (C. M. McNicholas, W. B. Guggino, E. M. Schwiebert, S. C. Hebert, G. Giebisch, and M. E. Egan. Proc. Natl. Acad. Sci. USA 93: 8083–8088, 1996). In the present study, we used the two-microelectrode voltage-clamp technique to measure whole cell K+ currents in Xenopus oocytes, and we further characterized the enhanced sensitivity of ROMK2 to glibenclamide by CFTR. Glibenclamide inhibited K+currents by 56% in oocytes expressing both ROMK2 and CFTR but only 11% in oocytes expressing ROMK2 alone. To examine the role of the first nucleotide binding fold (NBF1) of CFTR in the ROMK2-CFTR interaction, we studied the glibenclamide sensitivity of ROMK2 when coexpressed with CFTR constructs containing mutations in or around the NBF1 domain. In oocytes coinjected with ROMK2 and a truncated construct of CFTR with an intact NBF1 (CFTR-K593X), glibenclamide inhibited K+ currents by 46%. However, in oocytes coinjected with ROMK2 and a CFTR mutant truncated immediately before NBF1 (CFTR-K370X), glibenclamide inhibited K+ currents by 12%. Also, oocytes expressing both ROMK2 and CFTR mutants with naturally occurring NBF1 point mutations, CFTR-G551D or CFTR-A455E, display glibenclamide-inhibitable K+currents of only 14 and 25%, respectively. Because CFTR mutations that alter the NBF1 domain reduce the glibenclamide sensitivity of the coexpressed ROMK2 channel, we conclude that the NBF1 motif is necessary for the CFTR-ROMK2 interaction that confers sulfonylurea sensitivity.
Redistribution of villin to proximal tubule basolateral membranes after ischemia and reperfusion
Authors: Brown Dennis , Lee Richard , Bonventre Joseph V.
Published: December 2017

After ischemia and reperfusion, severe alterations in the cytoskeletal organization of renal tubular epithelial cells have been reported. These effects, accompanied by a modification in the polarized distribution of some membrane transport proteins, are especially evident in the proximal tubule. In normal proximal tubule cells, actin is concentrated in apical brush border microvilli, along with the actin-binding protein villin. Because villin plays an important role in actin bundling and in microvillar assembly but can also act as an actin-fragmenting protein at higher calcium concentrations, we examined the effects of ischemic injury and reperfusion on the distribution of villin and actin in proximal tubule cells of rat kidney. Using specific antibodies against villin and actin, we show that these proteins redistribute in parallel from the apical to the basolateral plasma membrane within 1 h of reperfusion after ischemia. Ischemia alone had no effect on the staining pattern. Repolarization of villin to the apical membrane begins within hours after reperfusion with enhanced apical localization over time during the period of regeneration. This apical repolarization of villin is accompanied by the migration of actin back to the apical membrane. These results show not only that villin may be involved in the initial disruption of the actin cytoskeleton during reperfusion injury but also that its migration back to the apical domain of these cells accompanies the reestablishment of a normal actin distribution in the brush border.
H+secretion is inhibited by clostridial toxins in an inner medullary collecting duct cell line
Authors: Alexander Edward A. , Shih Theodora , Schwartz John H.
Published: December 2017

Renal epithelial cell H+secretion is an exocytic-endocytic phenomenon. In the inner medullary collecting duct (IMCD) cell line, which we have utilized as a model of renal epithelial cell acid secretion, we found previously that acidification increased exocytosis and alkalinization increased endocytosis. It is likely, therefore, that the rate of proton secretion is regulated by the membrane insertion and retrieval of proton pumps. There is abundant evidence from studies in the nerve terminal and the chromaffin cell that vesicle docking, membrane fusion, and discharge of vesicular contents (exocytosis) involve a series of interactions among so-called trafficking proteins. The clostridial toxins, botulinum and tetanus, are proteases that specifically inactivate some of these proteins. In these experiments we demonstrated, by immunoblot and immunoprecipitation, the presence in this IMCD cell line of the specific protein targets of these toxins, synaptobrevin/vesicle-associated membrane proteins (VAMP), syntaxin, and synaptosomal-associated protein-25 (SNAP-25). Furthermore, we showed that these toxins markedly inhibit the capacity of these cells to realkalinize after an acid load. Thus these data provide new insight into the mechanism for H+secretion in the IMCD.
Regulation of glomerular and proximal tubule renin mRNA by chronic changes in dietary NaCl
Authors: Tank Julia E. , Henrich William L. , Moe Orson W.
Published: December 2017

Renal adaptations to chronic changes in dietary NaCl and extracellular fluid volume involve both glomerular and tubular mechanisms that result in preservation of glomerular filtration rate and modifications of renal tubular transport to secure external NaCl balance. Although the systemic renin-angiotensin system (RAS) mediates some of these responses, the possible contributions of local glomerular and proximal tubule RASs in these adaptations have not been examined. Thus, in this study, glomeruli and proximal tubules were microdissected from rats adapted to high (4.0%)-, normal (0.5%), or low (0.01%)-NaCl diets, and renin mRNA was measured using quantitative competitive reverse transcription-polymerase chain reaction. After 4 days of the diets, glomerular renin mRNA abundance was increased 100% by the low-NaCl diet ( P < 0.05) and suppressed 50% ( P < 0.01) by the high-NaCl diet compared with controls. Renin mRNA in proximal tubules was stimulated 230% ( P < 0.05) by the low-NaCl diet and tended to be suppressed (68% decrease, not significant) by the high-NaCl diet. When the high-NaCl diet was continued for 2 wk, proximal tubule renin mRNA was suppressed by 89% ( P < 0.05). This study provides evidence that glomerular and proximal tubule renin transcript levels are regulated by chronic changes in dietary NaCl, suggesting that local RASs contribute to the renal adaptations in response to chronic alterations in NaCl.
Bradykinin induces tubulin phosphorylation and nuclear translocation of MAP kinase in mesangial cells
Authors: Jaffa Ayad A. , Miller Bradley S. , Rosenzweig Steven A. , Naidu Padma S. , Velarde Victoria , Mayfield Ronald K.
Published: December 2017

Glomerular hypertension and glomerular hypertrophy act early and synergistically to promote glomerular injury in diabetes. We have previously shown that increased renal kinin production contributes to the glomerular hemodynamic abnormalities associated with diabetes. Glomerulosclerosis, characterized by mesangial cell proliferation and matrix expansion, is the final pathway leading to renal failure. The signal(s) initiating mesangial cell proliferation is ill defined. In the present study, we utilized immunofluorescence, immunoprecipitation, and immunoblotting techniques to identify substrates that are tyrosine phosphorylated in response to bradykinin action in mesangial cells. Immunofluorescence microscopy of mesangial cells stained with anti-phosphotyrosine (anti-PY) antibodies following bradykinin treatment (10−9–10−6M) revealed a dose-dependent increase in the labeling of cytoplasmic and nuclear proteins. Immunoprecipitation with anti-PY, followed by immunoblot revealed bradykinin-induced tyrosyl phosphorylation of tubulin and mitogen-activated protein kinase (MAPK). Confocal microscopy of mesangial cells stained for MAPK indicated that bradykinin stimulation resulted in translocation of MAPK from the cytoplasm to the nucleus by 2 h. These data demonstrate that bradykinin action results in the tyrosine phosphorylation of cellular proteins in mesangial cells and suggest a role for tubulin and MAPK in the signaling cascade of bradykinin leading to altered mesangial function.