bluuebunny/crossref_metadata_2025 huggingface dataset:
Real-time profiling of kidney tubular fluid nitric oxide concentrations in vivo
Authors: Levine David Z. , Iacovitti Michelle , Burns Kevin D. , Zhang Xueji ,   (With the Technical Assistance of Amy Slater)
Published: December 2017

To directly determine intratubular nitric oxide concentrations ([NO]) in vivo, we modified amperometric integrated electrodes (WPI P/N ISO-NOP007), which are highly sensitive to NO and not affected by ascorbic acid, nitrite, l-arginine, or dopamine. Although reactive lengths were as short as 5 μm long, the electrode still responded rapidly. With the use of kidney surface fluid as the “zero point,” the electrode tip was inserted into tubular segments along the track of a perforation made by a beveled glass pipette. The surface fluid zero point was usually stable as distal, late proximal, and early proximal tubule [NO] levels were measured sequentially in the same nephron. In eight normal rats, distal, late proximal, and early proximal [NO] concentrations were each ∼110 nM. In contrast, in nine 5/6 nephrectomized rats 2 wk postsurgery, although [NO] also did not differ among distal, late proximal, and early proximal segments, levels were approximately fourfold higher than those in normal rats and were significantly reduced after NG-monomethyl-l-arginine administration. These are the first quantitative in vivo tubular fluid [NO] measurements and show a significant increase in tubular fluid [NO] after renal ablation.
Renal apoptosis parallels ceramide content after prolonged ureteral obstruction in the neonatal rat
Authors: Malik Rajesh K. , Thornhill Barbara A. , Chang Alice Y. , Kiley Susan C. , Chevalier Robert L.
Published: December 2017

Obstructive nephropathy, the primary cause of renal insufficiency in infants, is characterized by progressive renal apoptosis. Ceramide is a sphingolipid known to stimulate apoptosis in the kidney. We investigated the effects of unilateral ureteral obstruction (UUO) on endogenous renal ceramide content and apoptosis in neonatal and adult rats. Animals were subjected to UUO or sham operation on the first day of life and were studied 3–28 days later. Adult rats were similarly treated and then studied 3 or 14 days later. In additional neonatal rats, the obstruction was removed after 5 days, with study at 14 or 28 days. Renal ceramide content was measured by diacylglycerol kinase assay, and apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end-labeling technique. Renal ceramide content was 50-fold higher in the 3-day neonatal compared with the adult kidney and 10-fold higher in the 7-day neonatal compared with the adult kidney, but there was no additional effect of UUO on ceramide content at these ages. However, after 14 or 28 days UUO in the neonate, renal ceramide was elevated compared with sham or intact opposite kidneys, and renal apoptosis was directly related to ceramide content ( r = 0.99, P < 0.001). Moreover, renal ceramide was reduced by relief of obstruction ( P < 0.05). There was less apoptosis in the obstructed kidney of the adult than the neonate, and UUO had no effect on ceramide content at 14 days in the adult. We conclude that prolonged UUO (at least 14 days duration) increases endogenous renal ceramide in the neonatal but not the adult rat. It is likely that this contributes to the prolonged renal apoptotic response of the neonatal obstructed kidney.
Atrial natriuretic peptide attenuates ANG II-induced hypertrophy of renal tubular cells
Authors: Hannken Tete , Schroeder Regine , Stahl Rolf A. K. , Wolf Gunter
Published: December 2017

ANG II arrests LLC-PK1cells in the G1phase of the cell cycle and induces hypertrophy, an effect mediated by induction of p27Kip1. We studied whether atrial natriuretic peptide (ANP) may modulate ANG II-induced hypertrophy and p27Kip1expression in tubular LLC-PK1cells. ANP, through its fragments 3—28and 4—27, prevented ANG II-induced cell cycle arrest. ANP inhibited >80% of ANG II-induced p27Kip1protein expression (Western blots). ANP stimulated expression of MKP-1, a phosphatase involved in dephosphorylation of p44/42 mitogen-activated protein (MAP) kinase, up to 12 h. ANP prevented the ANG II-mediated phosphorylation peak of MAP kinase after 12 h of stimulation. 8-Bromo-cGMP mimicked all the effects of ANP. Transfection with MKP-1 antisense, but not sense, oligonucleotides abolished the modifying role of ANP on ANG II-mediated cell cycle arrest.. The effect of ANP on ANG II-mediated hypertrophy of LLC-PK1cells is regulated on the level of MAP kinase phosphorylation, a key step in the induction of p27Kip1. Although ANP and ANG II both stimulate generation of reactive oxygen species, ANP additionally induces expression of MKP-1, leading to interference with ANG II-mediated MAP kinase phosphorylation.
The organic anion transporter family: from physiology to ontogeny and the clinic
Authors: Sweet Douglas H. , Bush Kevin T. , Nigam Sanjay K.
Published: December 2017

The organic anion transporter (OAT) family handles a wide variety of clinically important compounds (antibiotics, nonsteriodal anti-inflammatory drugs, etc.) and toxins. However, little is known about their appearance during development despite documented differences in the handling of anionic drugs among neonates, children, and adults. A similar spatiotemporal pattern of mRNA expression of the OATs (OAT1–4) during kidney development suggests that OAT genes may be useful in understanding the mechanisms of proximal tubule maturation. Moreover, OAT expression in unexpected extrarenal sites (e.g., spinal cord, bone, skin) has also been detected during development, possibly indicating a role for these transporters in the formation or preservation of extrarenal tissues. The cloning of these transporters also paves the way for computer-based modeling of drug-transporter interactions at the molecular level, potentially aiding in the design and assessment of new drugs. Additionally, increased understanding of single nucleotide polymorphisms in OATs and other transporters may eventually allow the use of a patient's expression profile and polymorphisms to individualize drug therapy.
A numerical model of acid-base transport in rat distal tubule
Authors: Chang Hangil , Fujita Toshiro
Published: December 2017

The purpose of this study is to develop a numerical model that simulates acid-base transport in rat distal tubule. We have previously reported a model that deals with transport of Na+, K+, Cl−, and water in this nephron segment (Chang H and Fujita T. Am J Physiol Renal Physiol 276: F931–F951, 1999). In this study, we extend our previous model by incorporating buffer systems, new cell types, and new transport mechanisms. Specifically, the model incorporates bicarbonate, ammonium, and phosphate buffer systems; has cell types corresponding to intercalated cells; and includes the Na/H exchanger, H-ATPase, and anion exchanger. Incorporation of buffer systems has required the following modifications of model equations: new model equations are introduced to represent chemical equilibria of buffer partners [e.g., pH = p Ka+ log10(NH3/NH4)], and the formulation of mass conservation is extended to take into account interconversion of buffer partners. Furthermore, finite rates of H2CO3-CO2interconversion (i.e., H2CO3⇌ CO2+ H2O) are taken into account in modeling the bicarbonate buffer system. Owing to this treatment, the model can simulate the development of disequilibrium pH in the distal tubular fluid. For each new transporter, a state diagram has been constructed to simulate its transport kinetics. With appropriate assignment of maximal transport rates for individual transporters, the model predictions are in agreement with free-flow micropuncture experiments in terms of HCO[Formula: see text] reabsorption rate in the normal state as well as under the high bicarbonate load. Although the model cannot simulate all of the microperfusion experiments, especially those that showed a flow-dependent increase in HCO[Formula: see text] reabsorption, the model is consistent with those microperfusion experiments that showed HCO[Formula: see text] reabsorption rates similar to those in the free-flow micropuncture experiments. We conclude that it is possible to develop a numerical model of the rat distal tubule that simulates acid-base transport, as well as basic solute and water transport, on the basis of tubular geometry, physical principles, and transporter kinetics. Such a model would provide a useful means of integrating detailed kinetic properties of transporters and predicting macroscopic transport characteristics of this nephron segment under physiological and pathophysiological settings.
Angiotensin II AT2receptors inhibit growth responses in proximal tubule cells
Authors: Zimpelmann Joseph , Burns Kevin D.
Published: December 2017

Angiotensin II (ANG II) subtype 2 (AT2) receptors are expressed in the adult kidney, but the effects of AT2receptor activation are unclear. The proximal tubule cell line LLC-PK1was transfected with a plasmid containing cDNA for the rat AT2receptor. In transfected cells, specific binding of125I-labeled ANG II was detected (dissociation constant = 0.81 nM), with inhibition by the AT2antagonist PD-123319, and no effect of the AT1antagonist losartan. ANG II (10−7M) significantly inhibited mitogen-activated protein kinase (MAPK) activity in transfected cells, associated with decreased phosphorylation of the extracellular signal-related kinases ERK1 and ERK2. ANG II stimulated phosphotyrosine phosphatase activity within 5 min, an effect blocked by PD-123319 and the phosphatase inhibitor vanadate. In transfected cells, ANG II inhibited epidermal growth factor-stimulated [3H]thymidine incorporation, an effect reversed by vanadate. In contrast, vanadate did not block ANG II-stimulated apoptosis of transfected cells. In summary, AT2receptors in proximal tubule cells inhibit MAPK activity and stimulate phosphotyrosine phosphatase. AT2receptor-induced inhibition of mitogenesis is mediated by phosphatase activation, whereas effects on apoptosis are insensitive to phosphatase inhibition. The data suggest that AT2receptors inhibit cell growth via distinct signaling pathways in the proximal tubule.
Embryonic origin and lineage of juxtaglomerular cells
Authors: Sequeira Lopez Maria Luisa S. , Pentz Ellen S. , Robert Barry , Abrahamson Dale R. , Gomez R. Ariel
Published: December 2017

To define the embryonic origin and lineage of the juxtaglomerular (JG) cell, transplantation of embryonic kidneys between genetically marked and wild-type mice; labeling studies for renin, smooth muscle, and endothelial cells at different developmental stages; and single cell RT-PCR for renin and other cell identity markers in prevascular kidneys were performed. From embryonic kidney day 12 to day 15 ( E12 to E15), renin cells did not yet express smooth muscle or endothelial markers. At E16 renin cells acquired smooth muscle but not endothelial markers, indicating that these cells are not related to the endothelial lineage, and that the smooth muscle phenotype is a later event in the differentiation of the JG cell. Prevascular genetically labeled E12 mouse kidneys transplanted into the anterior chamber of the eye or under the kidney capsule of adult mice demonstrated that renin cell progenitors originating within the metanephric blastema differentiated in situ to JG cells. We conclude that JG cells originate from the metanephric mesenchyme rather than from an extrarenal source. We propose that renin cells are less differentiated than (and have the capability to give rise to) smooth muscle cells of the renal arterioles.
Puromycin aminonucleoside damages the glomerular size barrier with minimal effects on charge density
Authors: Hjalmarsson Clara , Ohlson Maria , Haraldsson Börje
Published: December 2017

Puromycin aminonucleoside (PAN) has been suggested to reduce glomerular charge density, to create large glomerular “leaks,” or not to affect the glomerular barrier. Therefore, we analyzed glomerular charge and size selectivity in vivo and in isolated kidneys perfused at 8°C (cIPK) in control and PAN-treated rats. The fractional clearances (θ) for albumin and Ficoll of similar hydrodynamic size were 0.0017 ± 0.0004 and 0.15 ± 0.02, respectively, in control cIPKs. Two-pore analysis gave similar results in vivo and in vitro, with small- and large-pore radii of 47–52 and 85–105 Å, respectively, in controls. Puromycin increased the number of large pores 40–50 times, the total pore area over diffusion distance decreased by a factor of 25–30, and the small-pore radius increased by 33% ( P < 0.001 for all comparisons of size selectivity and θ). The effect of PAN was less dramatic on the estimated wall charge density, which was 73% of that of controls. We conclude that puromycin effectively destroys the glomerular size barrier with minimal effects on charge density.
p53 Protects renal inner medullary cells from hypertonic stress by restricting DNA replication
Authors: Dmitrieva Natalia , Michea Luis , Burg Maurice
Published: December 2017

We previously found that p53 upregulation by hypertonicity protected renal inner medullary collecting duct (mIMCD3) cells from apoptosis. The purpose of the present study was to investigate the mechanism by which p53 protects the cells. We now find that hypertonicity (NaCl added to a total of 500 mosmol) inhibits DNA replication and delays G1-S transition as concluded from analysis of cell cycle distributions and bromodeoxyuridine (BrDU) incorporation rates. Lowering of p53 with p53 antisense oligonucleotide attenuated such effects of hypertonicity, resulting in an increased number of apoptotic cells in S phase and cells with >4 N DNA. Results with synchronized cells are similar, showing that cells in the early S phase are more sensitive to hypertonicity. Immunocytochemistry revealed that p53 becomes phosphorylated on Ser15and translocates to the nucleus in S both in isotonic and hypertonic conditions. Caffeine (2 mM) greatly reduces the p53 level and Ser15phosphorylation, followed by a remarkable increase of DNA replication rate, by failure of hypertonicity to inhibit it, and by reduction of cell number during hypertonicity. Finally, inhibition of DNA replication by the DNA polymerase inhibitor aphidicolin significantly improves cell survival, confirming that keeping cells in G1and decreasing the rate of DNA replication is protective and that these actions of p53 most likely are what normally help protect cells against hypertonicity.
Neurotransmitter-stimulated ion transport by cultured porcine vas deferens epithelium
Authors: Sedlacek Roger L. , Carlin Ryan W. , Singh Ashvani K. , Schultz Bruce D.
Published: December 2017

A collagenase-based dissociation technique has been developed to routinely establish monolayer cultures of freshly isolated porcine vas deferens epithelium. Cells isolated from each tissue are transferred to 25-cm2tissue culture flasks and grown in a standard cell culture medium. Flasks reach confluency in 3–4 days, and cells are subsequently seeded onto permeable supports. Cultured cells display a monolayer cobblestone appearance and are immunoreactive to anti-ZO-1 and anti-cytokeratin antibodies. Electron microscopy is employed to demonstrate the presence of junctional complexes and microvilli. When evaluated in modified Ussing chambers, cultured monolayers exhibit a basal lumen negative potential difference, high electrical resistance (>1,000 Ω · cm2), and respond to norepinephrine, vasopressin, ATP, adenosine, and histamine, with changes in short-circuit current indicative of anion secretion. Responses are significantly attenuated in Cl−- and/or HCO[Formula: see text]-free solutions. Attempts to further optimize culture conditions have shown that chronic exposure to insulin increases proliferation rates. Thus the culture method described will reliably produce viable neurotransmitter-responsive cell monolayers that will allow for the characterization of vas deferens epithelial function and associated control mechanisms.
In vivo visualization of characteristics of renal microcirculation in hypertensive and diabetic rats
Authors: Yamamoto Tokunori , Tomura Yuichi , Tanaka Hiroyoshi , Kajiya Fumihiko
Published: December 2017

We developed a videomicroscope system with a charge-coupled device camera and evaluated it in the investigation of the glomerular microcirculation in normal [Wistar-Kyoto (WKY)], spontaneously hypertensive (SHR), and streptoyotocin-induced diabetic rats (STZ). In WKY, the diameter of the afferent arterioles (Af) was 11.9 ± 0.7 μm and that of the efferent arterioles (Ef) was 8.9 ± 0.7 μm. Af and Ef in each glomerulus could be visualized simultaneously with continuous recording of blood pressure and renal blood flow. In SHR, Af diameter was constricted to ∼60% of that in WKY. A dose-dependent dilation of Af and Ef was observed after administration of barnidipine (1–10 μg/kg iv), a calcium channel antagonist, in all three groups. No change was seen in the Af-to-Ef diameter ratio (Af/Ef ratio) in WKY. In SHR, the Af/Ef ratio increased significantly because of the marked dilation of Af after barnidipine administration. In contrast, barnidipine dilated Ef in STZ, causing a significant reduction in the Af/Ef ratio. This system can analyze in vivo glomerular microcirculation and systemic macrocirculation simultaneously, allowing more direct investigation of the characteristics of and acute changes in glomerular microcirculation in pathological animals.
Structural determinants of glomerular permeability
Authors: Deen William M. , Lazzara Matthew J. , Myers Bryan D.
Published: December 2017

Recent progress in relating the functional properties of the glomerular capillary wall to its unique structure is reviewed. The fenestrated endothelium, glomerular basement membrane (GBM), and epithelial filtration slits form a series arrangement in which the flow diverges as it enters the GBM from the fenestrae and converges again at the filtration slits. A hydrodynamic model that combines morphometric findings with water flow data in isolated GBM has predicted overall hydraulic permeabilities that are consistent with measurements in vivo. The resistance of the GBM to water flow, which accounts for roughly half that of the capillary wall, is strongly dependent on the extent to which the GBM surfaces are blocked by cells. The spatial frequency of filtration slits is predicted to be a very important determinant of the overall hydraulic permeability, in keeping with observations in several glomerular diseases in humans. Whereas the hydraulic resistances of the cell layers and GBM are additive, the overall sieving coefficient for a macromolecule (its concentration in Bowman's space divided by that in plasma) is the product of the sieving coefficients for the individual layers. Models for macromolecule filtration reveal that the individual sieving coefficients are influenced by one another and by the filtrate velocity, requiring great care in extrapolating in vitro observations to the living animal. The size selectivity of the glomerular capillary has been shown to be determined largely by the cellular layers, rather than the GBM. Controversial findings concerning glomerular charge selectivity are reviewed, and it is concluded that there is good evidence for a role of charge in restricting the transmural movement of albumin. Also discussed is an effect of albumin that has received little attention, namely, its tendency to increase the sieving coefficients of test macromolecules via steric interactions. Among the unresolved issues are the specific contributions of the endothelial glycocalyx and epithelial slit diaphragm to the overall hydraulic resistance and macromolecule selectivity and the nanostructural basis for the observed permeability properties of the GBM.
CaR-mediated COX-2 expression in primary cultured mTAL cells
Authors: Wang Dairong , An Shao-Jian , Wang Wen-Hui , McGiff John C. , Ferreri Nicholas R.
Published: December 2017

Primary cultures of medullary thick ascending limb (mTAL) cells retain the capacity to express calcium-sensing receptor (CaR) mRNA and protein. Increases in cyclooxygenase-2 (COX-2) mRNA accumulation, protein expression, and PGE2synthesis were observed in a dose- and time-dependent manner after exposure of these cells to extracellular calcium (Ca[Formula: see text]). Moreover, transfection of mTAL cells with a CaR overexpression vector significantly enhanced COX-2 expression and PGE2production in response to calcium compared with cells transfected with an empty vector. Challenge with the CaR-selective agonist poly-l-arginine (PLA) also increased COX-2 mRNA accumulation, protein expression, and PGE2synthesis. Furthermore, Ca[Formula: see text]- and PLA-mediated PGE2production was abolished in the presence of NS-398 or nimesulide, two different COX-2-selective inhibitors. These data suggest that intracellular signaling mechanisms initiated via activation of CaR contribute to COX-2-dependent PGE2synthesis in the mTAL. Because Ca[Formula: see text] concentration varies along Henle's loop, calcium may contribute to salt and water balance via a COX-2- and CaR-dependent mechanism. Thus novel calcimimetics might be useful in conditions such as hypertension in which manipulation of extracellular fluid volume provides beneficial effects.
Na-K-ATPase activity decreases with aging in female rat brain synaptosomes
Authors: Fraser Cosmo L. , Arieff Allen I.
Published: December 2017

To understand why elderly females are better able to tolerate hyponatremia, we measured brain Na-K-ATPase activity to determine whether this adaptive mechanism was affected by age. Using synaptosomes from 2-, 12-, and 19-mo-old female rats, we show in our results that Na-K-ATPase activity changes with age in female rats. Enzyme activity was significantly ( P = 0.0026) reduced (17%) from 0.416 ± 0.01 at 2 mo to 0.345 ± 0.01 at 12 mo and reduced ( P = 0.0001) (34%) to 0.274 ± 0.02 μmol · min−1· mg protein−1at 19 mo. To determine whether this decrease was due to reduced transport function of the Na-K-ATPase pump, we performed potassium transport using rubidium (86Rb+) as tracer. Ouabain-sensitive potassium uptake at 2 mo was 16.18 ± 1.31 nmol/mg protein, was significantly ( P = 0.0063) reduced (39%) to 9.79 ± 1.44 nmol/mg at 12 mo, and was significantly ( P = 0.0003) reduced (62%) to 6.12 ± 1.05 nmol/mg protein at 19 mo. On the contrary, Na-K-ATPase activity remained elevated in males during aging. These data suggest that the Na-K-ATPase pump in female rat brain synaptosomes is decreased with increased age, and that this decrease is probably due in part to decreased potassium transport by the Na-K-ATPase pump.
α2-Adrenergic-mediated tubular NO production inhibits thick ascending limb chloride absorption
Authors: Plato Craig F. , Garvin Jeffrey L.
Published: December 2017

Stimulation of α2-adrenergic receptors inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (THAL) expresses α2-receptors. We hypothesized that selective α2-receptor activation decreases NaCl absorption by cortical THALs through activation of NOS and increased production of NO. We found that the α2-receptor agonist clonidine (10 nM) decreased chloride flux ( JCl) from 119.5 ± 15.9 to 67.4 ± 13.8 pmol · mm−1· min−1(43% reduction; P < 0.02), whereas removal of clonidine from the bath increased JClby 20%. When NOS activity was inhibited by pretreatment with 5 mM NG-nitro-l-arginine methyl ester, the inhibitory effects of clonidine on THAL JClwere prevented (81.7 ± 10.8 vs. 71.6 ± 6.9 pmol · mm−1· min−1). Similarly, when the NOS substrate l-arginine was deleted from the bath, addition of clonidine did not decrease THAL JClfrom control (106.9 ± 11.6 vs. 132.2 ± 21.3 pmol · mm−1· min−1). When we blocked the α2-receptors with rauwolscine (1 μM), we found that the inhibitory effect of 10 nM clonidine on THAL JClwas abolished, verifying that α2, rather than I1, receptors mediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did not increase in response to clonidine, whereas pretreatment with 150 nM wortmannin abolished the clonidine-mediated inhibition of THAL JCl, indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 μM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL JCl. In conclusion, α2-receptor stimulation decreases THAL JClby increasing NO release and stimulating guanylate cyclase. These data suggest that α2-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diuretic effects of clonidine in vivo.
Modulation of aldosterone-induced stimulation of ENaC synthesis by changing the rate of apical Na+entry
Authors: Dijkink Lisette , Hartog Anita , Van Os Carel H. , Bindels René J. M.
Published: December 2017

Primary cultures of immunodissected rabbit connecting tubule and cortical collecting duct cells were used to investigate the effect of apical Na+entry rate on aldosterone-induced transepithelial Na+transport, which was measured as benzamil-sensitive short-circuit current ( Isc). Stimulation of the apical Na+entry, by long-term short-circuiting of the monolayers, suppressed the aldosterone-stimulated benzamil-sensitive Iscfrom 320 ± 49 to 117 ± 14%, whereas in the presence of benzamil this inhibitory effect was not observed (335 ± 74%). Immunoprecipitation of [35S]methionine-labeled β-rabbit epithelial Na+channel (rbENaC) revealed that the effects of modulation of apical Na+entry on transepithelial Na+transport are exactly mirrored by β-rbENaC protein levels, because short-circuiting the monolayers decreased aldosterone-induced β-rbENaC protein synthesis from 310 ± 51 to 56 ± 17%. Exposure to benzamil doubled the β-rbENaC protein level to 281 ± 68% in control cells but had no significant effect on aldosterone-stimulated β-rbENaC levels (282 ± 68%). In conclusion, stimulation of apical Na+entry suppresses the aldosterone-induced increase in transepithelial Na+transport. This negative-feedback inhibition is reflected in a decrease in β-rbENaC synthesis or in an increase in β-rbENaC degradation.
Localization and functional characterization of Na+/H+exchanger isoform NHE4 in rat thick ascending limbs
Authors: Chambrey Régine , St. John Patricia L. , Eladari Dominique , Quentin Fabienne , Warnock David G. , Abrahamson Dale R. , Podevin René-Alexandre , Paillard Michel
Published: December 2017

The Na+/H+exchanger NHE4 was cloned from a rat stomach cDNA library and shown to be expressed predominantly in the stomach and less dramatically in the kidney. The role and precise localization of NHE4 in the kidney are still unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification of thick ascending limbs and collecting ducts, respectively. The results indicate that NHE4 is highly expressed in basolateral membranes of thick ascending limb and distal convoluted tubule, whereas collecting ducts from cortex to inner medulla and proximal tubules showed weaker basolateral NHE4 expression. Western blot analysis of NHE4 in membrane fractions prepared from the inner stripe of the outer medulla revealed the presence of a 95-kDa protein that was enriched in basolateral membrane vesicles isolated from medullary thick ascending limbs. The inhibition curve of H+-activated22Na uptake by 5-( N-ethyl- N-isopropyl)amiloride (EIPA) was consistent with the presence, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 μM. Kinetic analyses showed that the extracellular Na+dependence of NHE4 activity followed a simple hyperbolic relationship, with an apparent affinity constant of 12 mM. Intravesicular H+activated NHE4 by a positive cooperative mechanism. NHE4 had an unusual low affinity for intravesicular H+with a half-maximal activation value of p K6.21. We conclude that NHE4, like NHE1, is expressed on the basolateral membrane of multiple nephron segments. Nevertheless, these two proteins exhibited dramatically different affinities for intracellular H+, suggesting that they may play distinct physiological roles in the kidney.
Mechanical strains induced by tubular flow affect the phenotype of proximal tubular cells
Authors: Essig Marie , Terzi Fabiola , Burtin Martine , Friedlander Gérard
Published: December 2017

The effects of flow-induced mechanical strains on the phenotype of proximal tubular cells were addressed in vivo and in vitro by subjecting LLC-PK1and mouse proximal tubular cells to different levels of flow. Laminar flow (1 ml/min) induced a reorganization of the actin cytoskeleton and significantly inhibited the expression of plasminogen activators [tissue-type (tPA) activity: 25% of control cells; tPA mRNA: 70% of control cells; urokinase (uPA) mRNA: 56% of control LLC-PK1cells]. In vivo, subtotal nephrectomy (Nx) decreased renal fibrinolytic activity and uPA mRNA content detectable in proximal tubules. Nx also induced a reinforcement of the apical domain of the actin cytoskeleton analyzed by immunofluorescence. These effects of flow on tPA and uPA mRNA were prevented in vitro when reorganization of the actin cytoskeleton was blocked by cytochalasin D and were associated, in vitro and in vivo, with an increase in shear stress-responsive element binding activity detected by an electrophoretic mobility shift assay in proximal cell nuclear extracts. These results demonstrate that tubular flow affects the phenotype of renal epithelial cells and suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases.
Alternative splicing and diversity of renal transporters
Authors: Gamba Gerardo
Published: December 2017

The growing molecular identification of renal transporter genes is revealing that alternative splicing is common among transporters. In this paper, I review the physiological consequences of alternative splicing in some genes encoding renal transporters in which spliced isoforms have recently been identified. In some cases, the spliced isoforms resulted in nonfunctional proteins, which, however, possess a dominant negative effect on the cotransporter function, suggesting that the presence of such isoforms can be important in the functional regulation of the transporter. In most transporter genes, however, the spliced isoforms have been shown to be functional, resulting in a variety of physiological consequences, including, for example, changes in the polarization of isoforms to the apical or basolateral membrane, changes in pharmacological or kinetic properties, and changes in tissue distribution or intrarenal localization. In some cases, although the spliced isoform is functional, the consequence of splicing is still unknown. Different regulation among isoforms is an interesting possibility. Thus the diversity of several renal transporters is enhanced by alternative splicing mechanisms.
AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex
Authors: Jo Inho , Ward Donald T. , Baum Michelle A. , Scott John D. , Coghlan Vincent M. , Hammond Timothy G. , Harris H. William
Published: December 2017

We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCζ, and an RII-binding protein (relative molecular mass ∼90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCζ. With the use of small-particle flow cytometry, RII and PKCζ were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex.32P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.
Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron
Authors: Wang Tong , Hropot Max , Aronson Peter S. , Giebisch Gerhard
Published: December 2017

This study assessed the functional role of Na+/H+exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid ( Jv) and HCO[Formula: see text]( JHCO3) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced Jvand JHCO3, but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, JHCO3was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, JHCO3was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na+/H+exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.
Prostaglandins potentiate U-46619-induced pulmonary microvascular dysfunction
Authors: Wright Joseph K. , Kim Lawrence T. , Rogers Thomas E. , Turnage Richard H.
Published: December 2017

The induction of cyclooxygenase is an important event in the pathophysiology of acute lung injury. The purpose of this study was to examine the synergistic effects of various cyclooxygenase products (PGE2, PGI2, PGF2α) on thromboxane A2(TxA2)-mediated pulmonary microvascular dysfunction. The lungs of Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer containing indomethacin and PGE2 (5 × 10− 8 to 1 × 10− 7 M), PGF2α (7 × 10− 9 to 5 × 10− 6 M), or PGI2 (5 × 10− 8 to 2 × 10− 5 M). The TxA2-receptor agonist U-46619 (7 × 10− 8 M) was then added to the perfusate, and then the capillary filtration coefficient ( K f), pulmonary arterial pressure (Ppa), and total pulmonary vascular resistance (Rt) were determined. The K f of lungs perfused with U-46619 was twice that of lungs perfused with buffer alone ( P = 0.05). The presence of PGE2, PGF2α, and PGI2 within the perfusate of lungs exposed to U-46619 caused 118, 65, and 68% increases in K f, respectively, over that of lungs perfused with U-46619 alone ( P < 0.03). The Rt of lungs perfused with PGE2 + U-46619 was ∼30% greater than that of lungs exposed to either U-46619 ( P < 0.02) or PGE2 ( P < 0.01) alone. When paired measurements of Rt taken before and then 15 min after the addition of U-46619 were compared, PGI2 was found to attenuate U-46619-induced increases in Rt ( P < 0.01). These data suggest that PGE2, PGI2, and PGF2α potentiate the effects of TxA2-receptor activation on pulmonary microvascular permeability.
Attenuation of force deficit after lengthening contractions in soleus muscle from trained rats
Authors: Gosselin Luc E.
Published: December 2017

The purposes of this study were 1) to determine the extent to which endurance training reduces the functional deficit induced by lengthening contractions in the soleus (Sol) muscle and 2) to determine whether young and old rats training at a comparable relative exercise intensity would demonstrate a similar protective effect from lengthening-contraction-induced injury. Young (3-mo-old) and old (23-mo-old) male Fischer 344 rats were randomly assigned to either a control or exercise training group [young control (YC), old control (OC), young trained (YT), old trained (OT)]. Exercise training consisted of 10 wk of treadmill running (15% grade, 45 min/day, and 5 days/wk) such that by the end of training the young and old rats were exercising at 27 and 15 m/min, respectively. After training, contractile properties of the Sol muscle were measured in vitro at 26°C. The percent decrease in maximal isometric specific force (Po) was determined after a series of 20 lengthening contractions (20% strain from optimal muscle length, 1 contraction every 5 s). After the lengthening-contraction protocol, Sol muscle Po was decreased by ∼26% (19.6 vs. 14.6 N/cm2) and 28% (14.8 vs. 9.6 N/cm2) in the YC and OC rats, respectively. After exercise training, the reduction in Po was significantly ( P < 0.05) attenuated to a similar degree (∼13%) in both YT rats (18.7 vs. 16.2 N/cm2) and OT rats (15.8 vs. 13.7 N/cm2). It is concluded that exercise training attenuates the force deficit after repeated lengthening contractions to a comparable extent in young and old rats training at a similar exercise intensity.
Interaction of the effects between vitamin D receptor polymorphism and exercise training on bone metabolism
Authors: Tajima Orie , Ashizawa Noriko , Ishii Tomoo , Amagai Hitoshi , Mashimo Tomoko , Liu Li Jing , Saitoh Shinichi , Tokuyama Kumpei , Suzuki Masashige
Published: December 2017

Bone metabolism is strongly influenced by heredity and environmental factors. To investigate interaction of the effects between vitamin D receptor polymorphism by Fok I and resistance exercise training on bone metabolism, young male subjects with FF genotype (F, n = 10) and Ff or ff genotypes (f, n = 10) followed 1 mo of weight training, and changes in bone metabolism were compared. An additional 14 subjects served as a sedentary control. Biomarkers of bone formation, bone-specific alkaline phosphatase, and osteocalcin were significantly increased by training in both F and f groups. 1,25-Dihydroxyvitamin D3, known to upregulate bone formation, was also increased by the training in the f but not in the F group. Bone resorption assessed by cross-linked NH2-terminal teropeptide of type I collagen was significantly suppressed by the training, and the decrease in F was greater and longer lasting than that in f group. In conclusion, stimulation of bone formation and suppression of bone resorption occurred within 1 mo in young men. Despite a significant increase in 1,25-dihydroxyvitamin D3 in the f group but not in the F group, the response of bone metabolism to the training in the F was similar to or greater than that in f group, suggesting a functional difference between vitamin D receptor genotypes f and F.
Fiber-type-specific αB-crystallin distribution and its shifts with T3and PTU treatments in rat hindlimb muscles
Authors: Atomi Yoriko , Toro Kyoko , Masuda Tsuyoshi , Hatta Hideo
Published: December 2017

Changes in αB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5,3′-triiodothyronine (T3) and propylthiouracil (PTU) treatments. Cellular distributions of αB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti-αB-crystallin antibody. αB-crystallin content in both soleus and EDL muscles was significantly decreased after T3, and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. αB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx ≥ IIa in soleus muscle. The relation was basically unchanged in both muscles after T3treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean αB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein αB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle.
Developmental myosin heavy chains in the adult human diaphragm: coexpression patterns and effect of COPD
Authors: Nguyen Taitan , Shrager Joseph , Kaiser Larry , Mei Lijuan , Daood Monica , Watchko Jon , Rubinstein Neal , Levine Sanford
Published: December 2017

In preliminary experiments we noted developmental (i.e., embryonic and neonatal) myosin heavy chains (MHCs) in the diaphragms of patients with severe chronic obstructive pulmonary disease (COPD). We hypothesized that this finding represented new fiber formation secondary to injury associated with the mechanical stress of COPD or previously undescribed MHCs in the human diaphragm. To distinguish between these possibilities, we analyzed diaphragmatic biopsies obtained from 9 patients with severe COPD (forced expiratory volume in 1 s = 21 ± 2% predicted, residual volume = 283 ± 22% predicted) and 10 age-matched controls. First, using immunocytochemistry with specific monoclonal antibodies, we noted that control diaphragms had greater proportions of fibers expressing embryonic (50 ± 2 vs. 28 ± 3%, P < 0.0001) and neonatal (52 ± 2 vs. 32 ± 3%, P < 0.001) MHCs than COPD diaphragms. Second, SDS-PAGE demonstrated that these developmental MHCs represented only a very small fraction of the diaphragmatic MHC content. Third, the RT-PCR demonstrated mRNA coding for embryonic and neonatal MHCs in COPD and control diaphragms. Last, COPD and control diaphragms exhibited normal histology on light microscopy. We conclude that the presence of developmental MHC isoforms does not indicate new fiber formation in diaphragms of patients with severe COPD. Although these results represent the first systematic description of embryonic and neonatal MHCs in normal adult human diaphragms, their function remains to be elucidated.
Historical perspectives of cellular oxygen sensing and responses to hypoxia
Authors: Lahiri Sukhamay
Published: December 2017

The responses to acute and chronic hypoxia begin with oxygen sensing, and this historical perspective is written in line with this concept. The earliest pertinent work started with studies on fermentation in yeast in the 17th century, before the discovery of oxygen. It required 200 yr to localize the oxygen sensing within the cells and another 100 yr to discover the cellular oxidation reactions. Today, the consensus is that the mitochondrial respiratory chain is in part the site of oxygen sensing. In addition, membrane-bound NAD(P)H oxidase possibly takes part in oxygen sensing. Oxygen-sensing mechanisms occur in a tissue-specific fashion. For example, the carotid body responds to hypoxia promptly by eliciting a ventilatory response, whereas erythropoietin production in response to hypoxia requires more time, involving new expression of genes. The mechanism has therefore moved from the cells to genes.
Single-unit recordings of arterial chemoreceptors from mouse petrosal ganglia in vitro
Authors: Donnelly David F. , Rigual Ricardo
Published: December 2017

A preparation was developed that allows for the recording of single-unit chemoreceptor activity from mouse carotid body in vitro. An anesthetized mouse was decapitated, and each carotid body was harvested, along with the sinus nerve, glossopharyngeal nerve, and petrosal ganglia. After exposure to collagenase/trypsin, the cleaned complex was transferred to a recording chamber where it was superfused with oxygenated saline. The ganglia was searched for evoked or spontaneous unit activity by using a glass suction electrode. Single-unit action potentials were 57 ± 10 (SE) ( n = 16) standard deviations above the recording noise, and spontaneous spikes were generated as a random process. Decreasing superfusate[Formula: see text] to near 20 Torr caused an increase in spiking activity from 1.3 ± 0.4 to 14.1 ± 1.9 Hz ( n = 16). The use of mice for chemoreceptor studies may be advantageous because targeted gene deletions are well developed in the mouse model and may be useful in addressing unresolved questions regarding the mechanism of chemotransduction.
Effects of the RBC membrane and increased perfusate viscosity on hypoxic pulmonary vasoconstriction
Authors: Deem Steven , Berg John T. , Kerr Mark E. , Swenson Erik R.
Published: December 2017

Red blood cells (RBCs) augment hypoxic pulmonary vasoconstriction (HPV) in part by scavenging of nitric oxide (NO) by Hb (Deem S, Swenson ER, Alberts MK, Hedges RG, and Bishop MJ, Am J Respir Crit Care Med 157: 1181–1186, 1998). We studied the contribution of the RBC compartmentalization of Hb to augmentation of HPV and scavenging of NO in isolated perfused rabbit lungs. Lungs were initially perfused with buffer; HPV was provoked by a 5-min challenge with hypoxic gas (inspired O2 fraction 0.05). Expired NO was measured continuously. Addition of free Hb to the perfusate (0.25 mg/ml) resulted in augmentation of HPV and a fall in expired NO that were similar in magnitude to those associated with a hematocrit of 30% (intracellular Hb of 100 mg/ml). Addition of dextran resulted in a blunting of HPV after free Hb but no change in expired NO. Blunting of HPV by dextran was not prevented by NO synthase inhibition with N ω-nitro-l-arginine and/or cyclooxygenase inhibition. RBC ghosts had a mild inhibitory effect on HPV but caused a small reduction in expired NO. In conclusion, the RBC membrane provides a barrier to NO scavenging and augmentation of HPV by Hb. Increased perfusate viscosity inhibits HPV by an undetermined mechanism.
No association between the angiotensin-converting enzyme ID polymorphism and elite endurance athlete status
Authors: Rankinen Tuomo , Wolfarth Bernd , Simoneau Jean-Aimé , Maier-Lenz Dirk , Rauramaa Rainer , Rivera Miguel A. , Boulay Marcel R. , Chagnon Yvon C. , Pérusse Louis , Keul Joseph , Bouchard Claude
Published: December 2017

Several studies have reported that the insertion (I) allele of the angiotensin-converting enzyme ( ACE) I/deletion (D) polymorphism is associated with enhanced responsiveness to endurance training and is more common in endurance athletes than in sedentary controls. We tested the latter hypothesis in a cohort of 192 male endurance athletes with maximal oxygen uptake ≥75 ml ⋅ kg−1 ⋅ min−1and 189 sedentary male controls. The ACE ID polymorphism in intron 16 was typed with the three-primer polymerase chain reaction method. Both the genotype ( P = 0.214) and allele ( P = 0.095) frequencies were similar in the athletes and the controls. Further analyses in the athletes revealed no excess of the I allele among the athletes within the highest quartile (> 80 ml ⋅ kg−1 ⋅ min−1) or decile (>83 ml ⋅ kg−1 ⋅ min−1) of maximal oxygen uptake. These data from the GENATHLETE cohort do not support the hypothesis that the ACE ID polymorphism is associated with a higher cardiorespiratory endurance performance level.