bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of an acute saline infusion on fluid and electrolyte metabolism in humans
Authors: Drummer C. , Gerzer R. , Heer M. , Molz B. , Bie P. , Schlossberger M. , Stadaeger C. , Rocker L. , Strollo F. , Heyduck B. , Bauer K. , Warberg J. , Baisch F. , Christensen J. , Konig A. , Norsk P.
Published: December 2017

Several hormonal systems participating in body fluid and electrolyte homeostasis were investigated in six healthy volunteers in a supine body position during a period of 9 days and nights. Under strictly controlled conditions, striking circadian rhythms were observed for plasma levels of vasopressin, renin, aldosterone, guanosine 3',5'-cyclic monophosphate, cortisol, and epinephrine. Nocturnal decreases and diurnal increases in urine flow rate and urinary excretion of electrolytes were observed and closely paralleled the urinary excretion of urodilatin. During 48 h after an acute isotonic saline infusion (2 liters within 25 min) and after a 48-h control experiment the urinary excretion of H2O and electrolytes, and simultaneously the alterations in endocrine systems participating in body fluid homeostasis, were determined. Urine flow and urinary electrolyte excretion rates were significantly increased during 2 days after the saline infusion. The largest increase in urinary fluid and electrolyte excretion was observed between 3 and 22 h postinfusion. These long-term changes were paralleled by altered H2O and Na balances and also by elevated body weights that returned to baseline values with an approximate half-life of 7 h. These data suggest that vasopressin, atrial natriuretic peptide, and catecholamines are unlikely to be of major importance for the renal response to this hypervolemic stimulus. The renin-aldosterone system was suppressed during 2 days postinfusion. This suppression correlated with the effects of saline load on Na excretion. However, the closest relation with Na excretion was observed for the kidney-derived member of the atrial natriuretic peptide family, urodilatin, which was considerably increased during the long-term period up to 22 h postinfusion. Thus these data show that the human body in supine position requires approximately 2 days to regulate the amount of Na and H2O provided by an acute saline infusion. The data also suggest that urodilatin and the renin-aldosterone system might participate in the long-term renal response to an acute saline infusion and also in the mediation of circadian urinary excretion rhythms.
Transport of beta-hydroxybutyrate and acetoacetate along rat nephrons: a micropuncture study
Authors: Ferrier B. , Martin M. , Janbon B. , Baverel G.
Published: December 2017

The transport of ketone bodies across the luminal membrane of the nephron was studied by means of micropuncture techniques in rats in normal acid-base state. The concentration of beta-hydroxybutyrate (beta-HB) and acetoacetate (AcAc) in plasma, tubular fluid and urine was measured by an ultramicromethod using enzymatic cycling. At endogenous plasma ketone body concentration, approximately 80% of the filtered load of beta-HB and AcAc was reabsorbed in the proximal convoluted tubule, the remaining fraction being almost completely reabsorbed between the late proximal convoluted and the distal tubule; under these conditions, the urinary excretion of ketone bodies was less than 1% of the filtered load. A progressive elevation to steady-state levels of plasma beta-HB resulted in a progressive reduction of the fractional reabsorption of beta-HB and AcAc in the proximal convoluted tubule, which means that reabsorption of ketone bodies in this nephron segment is saturable. No net secretion of ketone bodies could be demonstrated along the nephron even at the highest plasma ketone body concentrations reached. In clearance experiments, the capacity of the rat kidney for reabsorbing both beta-HB and AcAc was found to be limited by a maximal tubular capacity (Tm). The data suggest that, in the young Wistar rat nephron, most of the reabsorption of ketone bodies is carrier mediated.
pH dependence of the action of arginine vasopressin in renal collecting tubule
Authors: Ishikawa S. , Okada K. , Saito T.
Published: December 2017

We determined whether extracellular pH (pHe) and intracellular pH (pHi) modulate the cellular actions of arginine vasopressin (AVP) in rat renal papillary collecting tubule cells in culture. AVP significantly increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) production and cellular free calcium concentration ([Ca2+]i). pHe ranging from 6.8 to 8.0 distributed the pHi between 6.94 and 7.27. The acidified pHe reduced the AVP- and forskolin-induced cAMP production, the AVP-mobilized [Ca2+]i, and [3H]AVP receptor binding, and the alkalinized pHe enhanced the AVP- and forskolin-produced cAMP. Intracellular acidification occurred under three different conditions as follows: using carbonyl cyanide-m-chlorophenylhydrazone (CCCP), acetate buffer, and bicarbonate buffer with a reduced concentration of bicarbonate. Intracellular acidification significantly diminished both the AVP- and forskolin-induced increases in cAMP production and the AVP-mobilized [Ca2+]i but did not alter [3H]AVP receptor binding. Intracellular alkalinization by NH4Cl or chloride-free bicarbonate buffer, in contrast, augmented them. These results indicate that alterations in pHi modulate the cellular action of AVP to produce cAMP and mobilize [Ca2+]i in renal papillary collecting tubule cells. Also, reduced receptor binding of AVP is involved in the mechanism of the effects of low pHe.
Calcium-dependent inhibitory step in control of renin secretion
Authors: Park C. S. , Hong C. D. , Honeyman T. W.
Published: December 2017

The regulatory step for Ca(2+)- and calmodulin-dependent control of renin secretion was studied in rabbit renal cortical slices. Renin secretion was stimulated by adding the K(+)-H+ exchange ionophore nigericin or a weak base, benzylamine, to Ca(2+)-free incubation medium. Both nigericin and benzylamine can produce chemiosmotic swelling of acidic intracellular organelles in a pH gradient-dependent manner. Swelling of renin secretory granules may account for the stimulatory effects of these two agents on renin secretion. The stimulation of renin secretion by each of these agents was prevented by including Ca2+ in the incubation medium. The inhibitory effects of Ca2+ were reversed by the calmodulin antagonist calmidazolium, suggesting that the Ca2+ inhibition may be mediated by calmodulin. However, when renin secretion was first stimulated by nigericin or benzylamine in Ca(2+)-free medium, subsequent addition of Ca2+ to the incubation medium failed to reverse the stimulation. These results therefore suggest that Ca2+ acting through calmodulin may exert its inhibitory effects on renin secretion by blocking an early step in the cellular events that lead to renin secretion such as pH gradient-dependent swelling of renin secretory granule.
Attenuation of endothelin effects by a chloride channel inhibitor, indanyloxyacetic acid
Authors: Takenaka T. , Epstein M. , Forster H. , Landry D. W. , Iijima K. , Goligorsky M. S.
Published: December 2017

We have recently proposed that the actions of endothelin (ET) are in part mediated by opening of chloride channels (K. Iijima, L. Lin, A. Nasjletti, and M. S. Goligorsky. Am. J. Physiol. 260 (Cell Physiol. 29: C982-C992, 1991). In the present study the ability of a chloride channel inhibitor, an indanyloxyacetic acid (IAA-94), to block ET-induced effects was examined in cultured vascular smooth muscle cells (VSMC) by spectrofluorometry and direct videomicroscopic visualization of the renal microcirculation in isolated perfused hydronephrotic kidneys (IPHK). A fluorescein isothiocyanate (FITC)-labeled IAA-94 analogue showed specific binding to VSMC. IAA-94 (30 microM) neither affected basal cytosolic calcium concentration ([Ca2+]i) in VSMC nor peak response to ET, but it significantly curtailed sustained elevation of [Ca2+]i (half-time recovery was 147 +/- 23 vs. 248 +/- 33 s in control, P less than 0.05). IAA-94 blunted ET-induced membrane depolarization from 24.5 +/- 3.3 to 8.0 +/- 1.8 mV. In IPHK, ET constricted afferent arterioles (AA) by 29 +/- 2% (18.7 +/- 0.8 to 13.2 +/- 0.6 microns, P less than 0.001). Isradipine reversed this ET-induced vasoconstriction. Pretreatment with IAA-94 did not alter AA diameter, but markedly attenuated ET-induced AA constriction (reduction of AA diameters by only 9 +/- 2%, P less than 0.001). The subsequent addition of isradipine (0.1-1 microM) did not further dilate AA. Our data indicate that IAA-94 markedly attenuates AA vasoconstriction elicited by ET and suggest that ET-induced opening of chloride channels, membrane depolarization, and subsequent activation of voltage-dependent calcium channels contribute to the vasoconstrictor mechanisms of this peptide.
Mercury inhibits Na-K-ATPase primarily at the cytoplasmic side
Authors: Anner B. M. , Moosmayer M.
Published: December 2017

The investigation of active Na-K transport inhibition by mercury is difficult to perform in a cell because of the presence of numerous other membrane and intracellular proteins modifiable by mercury. Thus purified Na-K-adenosinetriphosphatase (ATPase) molecules performing active transport in an artificial membrane are required to demonstrate unequivocally the inhibition of active transport by mercury. We made use of a single population of Na-K-ATPase liposomes filled with ATP and Na to show mercury inhibition of active 86Rb transport mediated by both the inside-out and right-side-out pumps in the same liposome. The effect of HgCl2 on the Na-K-ATPase in cell-like and reversed orientation was measured in comparison with convallatoxin. A dilution series showed that 10 microM externally added HgCl2 inhibited the active 86Rb transport at the cytoplasmic side first; at 50 microM both pump populations were blocked, indicating either membrane permeation by HgCl2 and inhibition at the internal intracellular domains or onset of extracellular action at higher HgCl2 concentration. The results show that the metal-binding interface of Na-K-ATPase molecule is profoundly implicated in active ion transport and that the intracellular part of the Na-K-ATPase molecule presents the primary target for mercury action.
Colocalization of GAPDH and band 3 (AE1) proteins in rat erythrocytes and kidney intercalated cell membranes
Authors: Ercolani L. , Brown D. , Stuart-Tilley A. , Alper S. L.
Published: December 2017

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.2.12) (GAPDH) is a multifunctional protein that associates with the cytoplasmic face of intact human erythrocyte membranes. This association has been postulated to be critically dependent on the interaction of GAPDH with the highly acidic NH2-terminal domain of the principal integral membrane protein of the erythrocyte plasma membrane, the band 3 anion exchanger (AE1). This domain is not conserved in murine erythrocyte AE1 and is fully deleted in the alternatively spliced AE1 isoform that is expressed in the kidney. The lack of conservation of this domain has been proposed to explain the reported absence of GAPDH association with rodent erythrocyte membranes. To determine whether GAPDH could be associated with AE1 proteins in rodent cell membranes, specific rabbit antibodies to peptide sequences of rat GAPDH and mouse AE1 were used to immunolocalize these proteins in sequential semithin sections of rat erythrocytes and kidney medulla. In rat erythrocytes, GAPDH immunoreactivity was predominantly membrane associated and colocalized with AE1. In the kidney medulla, GAPDH was concentrated in the basolateral membrane of type A intercalated cells, where it colocalized with the alternatively spliced kidney form of AE1. GAPDH immunoreactivity was not detected in the plasma membrane of any other cell type in the kidney, indicating its predominant association with AE1-rich membranes. If this membrane interaction occurs via AE1 binding, then GAPDH must have binding sites in addition to those previously described for such binding in human AE1.
Extracellular pH modifies adaptive response to high K+ in cultured canine kidney cells
Authors: Manuli M. A. , Lorenz J. M.
Published: December 2017

The chronic interactive and independent effects of extracellular pH and K+ on renal Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity and active K+ transport were studied in the Madin-Darby canine kidney (MDCK) cell line. Confluent cell monolayers were incubated for 24 h in control (4 mM) or high (7.5 mM) K+ medium at acid (6.8) or neutral (7.4) pH. Under acid pH conditions, exposure to high K+ elicited a rise of 133% in maximum Na(+)-K(+)-ATPase activity and 66% in active K+ uptake. In contrast, high K+ had no effect on enzyme activity or K+ uptake at neutral pH. Detergent-activated Na(+)-K(+)-ATPase assay demonstrated a latent pool of enzyme at acid pH-control K+, which seemed to account entirely for the increase in Na(+)-K(+)-ATPase activity after exposure to high K+. The effects of pH appeared unrelated to HCO3- and Cl- concentration in the extracellular environment. We conclude that the upregulatory effect of high K+ on renal Na(+)-K(+)-ATPase is pH dependent. The data suggest that a pool of catalytically inactive enzyme exists only at acid extracellular pH at K+ concentrations in the normal physiological range and that K+ adaptation, at least initially, is the result of recruitment of this latent intracellular pool. In the intact cell extracellular K+ and luminal pH may interact to modify catalytic turnover rate as well as bioavailability of Na(+)-K(+)-ATPase.
Dietary Na and ACE inhibition effects on renal tissue angiotensin I and II and ACE activity in rats
Authors: Fox J. , Guan S. , Hymel A. A. , Navar L. G.
Published: December 2017

This study was designed to improve and validate methods for the accurate and consistent quantitation of angiotensin (ANG) I and II levels in rat kidney and to determine the effects on renal ANG I and II of changes in dietary sodium intake and ANG-converting enzyme (ACE) inhibition. Kidneys from pentobarbital-anesthetized rats were rapidly removed and homogenized in methanol before extraction and purification of ANG peptides by solid-phase extraction and high-performance liquid chromatography (HPLC). Recoveries of 125I-ANG I and II were greater than 80%. Reversed-phase HPLC of the partially purified methanol extract showed that greater than 75% of the ANG I- and greater than 82% of the ANG II-like immunoreactivity coeluted with ANG I and II, respectively. Dietary sodium deprivation (0.003 meq/g) and excess (1.34 meq/g) for 7 days significantly (P less than 0.01) increased and decreased renal ANG I (296 +/- 30 and 82.6 +/- 15.8 vs. 161 +/- 18 fmol/g) and ANG II (216 +/- 16 and 45.6 +/- 11.8 vs. 98 +/- 16 fmol/g) contents, respectively. Plasma ANG I and II levels showed similar changes. ACE activity was significantly upregulated by sodium deprivation in both kidney (44% increase) and plasma (30% increase). In rats fed normal chow, infusion of enalaprilat for 1 h abolished plasma ACE activity but decreased renal ACE activity by only 58%. ACE inhibition increased renal and plasma ANG I levels 2.8- and 12-fold, respectively, and decreased renal and plasma ANG II levels 75-78%.(ABSTRACT TRUNCATED AT 250 WORDS)
Substrate responses to acid-base alterations in dog renal proximal tubules
Authors: Simpson D. P. , McSherry N. , Scarbrough E. , Zweifel K.
Published: December 2017

Proximal tubules from dog kidney were incubated for 2-6 min with low concentrations of pyruvate, glutamine, and malate. When initial medium citrate was between 0 and 0.5 mM and alpha-ketoglutarate was between 0 and 0.1 mM, concentrations of these two substrates in tubules and media after incubation were lower with 10 than with 40 mM HCO3-. Malate levels in tubules and media changed in the opposite direction. CO2 formation from labeled citrate or alpha-ketoglutarate was greater at low than at high HCO3-. In tubules treated with digitonin to disrupt the cell membrane, differences in citrate and alpha-ketoglutarate concentrations in tubules at high and low HCO3- were eliminated. The effects of acid-base changes seen in intact tubules on malate levels and on the rate of oxidation of labeled citrate persisted after digitonin pretreatment. These results show that acute effects of acid-base changes on citrate, alpha-ketoglutarate, and malate levels observed in intact renal cortex can be reproduced in isolated tubules. They suggest that these changes are related to changes in levels of citrate and alpha-ketoglutarate in cytoplasm without corresponding changes within mitochondria.
Dopamine DA1 receptor agonist, fenoldopam, reverses glycine-induced hyperfiltration in rats
Authors: Wang Y. X. , Gellai M. , Brooks D. P.
Published: December 2017

Stimulation of dopamine DA1 receptors can prevent glomerular hyperfiltration in streptozotocin-induced diabetic rats. In the present study we have therefore investigated whether the DA1 agonist, fenoldopam, can prevent glycine-induced hyperfiltration. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured by inulin and p-aminohippurate clearances in conscious chronically instrumented rats. Glycine (3.7 mg/min iv; n = 8) significantly increased GFR by 37% (from 1.09 +/- 0.53 to 1.49 +/- 0.11 ml.100 g-1.min-1, P less than 0.01), ERPF by 23% (from 2.96 +/- 0.30 to 3.64 +/- 0.43 ml.100 g-1.min-1, P less than 0.05), and filtration fraction (FF) by 13% (from 0.39 +/- 0.04 to 0.44 +/- 0.05, P less than 0.05). Fenoldopam, at a dose (1 microgram.kg-1.min-1 iv; n = 8) that increased ERPF by 26%, decreased FF by 13%, but did not change GFR, significantly attenuated the glycine-induced hyperfiltration. In the presence of fenoldopam, glycine resulted in only an 8% increase in GFR (from 1.08 +/- 0.07 to 1.17 +/- 0.09 ml.100 g-1.min-1; n = 8). ERPF increased by 20% (from 3.34 +/- 0.24 to 4.00 +/- 0.21 ml.100 g-1.min-1, P less than 0.05), and FF decreased by 13% (from 0.34 +/- 0.03 to 0.29 +/- 0.02, P less than 0.05). Infusion of the DA1-selective antagonist, Sch 23390, abolished the effects of fenoldopam. Thus DA1 receptor activation can prevent glomerular hyperfiltration induced by glycine.
Role of nucleoside uptake in renal postischemic ATP synthesis
Authors: Van Waarde A. , Avison M. J. , Thulin G. , Gaudio K. M. , Shulman R. G. , Siegel N. J.
Published: December 2017

The role of nucleoside uptake in the enhanced metabolic recovery seen with postischemic ATP.MgCl2 was assessed by determining the effect of S-(p-nitrobenzyl)-6-thioinosine (NBTI) on postischemic ATP recovery in rats given normal saline (NS), ATP.MgCl2, or adenosine after 45 min of bilateral renal ischemia. In NS-infused animals, postischemic administration of NBTI (250 nmol) had no significant effect on the pattern of ATP recovery. In animals given 50 mumol ATP.MgCl2, coinfusion of NBTI significantly reduced the renal ATP content 2 h after reperfusion but blocked only one-half of the enhancement in renal ATP content compared with animals given ATP.MgCl2 alone. In animals postischemically infused with [2,5,8-3H]ATP.MgCl2 (50 mumol) there was significant labeling of nucleotides, nucleosides, and bases after 2 h of reperfusion. The specific activity of the adenosine pool was consistent with significant label uptake in the form of adenosine. Coinfusion of NBTI led to a significant reduction in label incorporation into renal ATP and total adenine nucleotide pools. These data are consistent with an important role for an NBTI-sensitive nucleoside uptake mechanism in the enhanced metabolic and functional recovery observed in ischemically injured kidney treated by postischemic infusion of ATP.MgCl2.
A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C
Authors: Force T. , Bonventre J. V. , Flannery M. R. , Gorn A. H. , Yamin M. , Goldring S. R.
Published: December 2017

The signal transduction pathways of the recently cloned porcine kidney calcitonin (CT) receptor were evaluated. This receptor, when stably transfected into MC-3T3 cells, avidly bound salmon CT (SCT) [dissociation constant (Kd) = 4 nM]. Incubation with SCT resulted in a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) [50% effective concentration (EC50) = 0.02 nM] in transfected cells (referred to as PC-1 cells). Binding kinetics and cAMP dose response relationships were similar to those of the native receptor in LLC-PK1 cells. PC-1 cells also responded to calcitonin gene-related peptide (CGRP), but the EC50 value for cAMP accumulation was more than three orders of magnitude higher than for SCT. Exposure of PC-1 cells to SCT (5 nM to 1 microM) produced a dose-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]i), whereas CGRP did not. The initial rise in [Ca2+]i was not dependent on extracellular Ca2+, suggesting that SCT induced release of Ca2+ from intracellular stores. SCT also increased inositol trisphosphate production in PC-1 cells. In conclusion, the cloned, transfected porcine CT receptor functionally couples to and activates both adenylyl cyclase and phospholipase C. This dual coupling is also a characteristic of the parathyroid hormone receptor, which has significant homology in amino acid sequence with the CT receptor.
Growth-regulatory properties of atrial natriuretic factor
Authors: Appel R. G.
Published: December 2017

Recent evidence supports the notion that atrial natriuretic factor (ANF) has growth-regulatory properties. In the adrenal gland, ANF inhibits growth specifically in the zona glomerulosa. In the kidney, ANF causes an antimitogenic, antiproliferative effect in cultured glomerular mesangial cells. In vascular smooth muscle, ANF inhibits cell proliferation (hyperplasia) as well as cell growth (hypertrophy). ANF has growth-regulatory properties in a variety of other tissues such as brain, bone, myocytes, red blood cell precursors, and endothelial cells. The cellular mechanisms involved in the growth-regulatory actions of ANF are not totally clear. Evidence supports involvement of both biological and clearance receptors for ANF, guanosine 3',5'-cyclic monophosphate (cGMP) and cGMP-independent mechanisms, and protooncogene expression. For the most part, the actions reviewed in this report represent in vitro phenomena, and it is unclear whether these effects are relevant to normal physiology or pathophysiology. Nevertheless, an emerging hypothesis is developing which states that circulating and autocrine/paracrine factors such as ANF interact to regulate the vasomotor tone and cellular growth of a variety of tissues such as vascular smooth muscle and the glomerulus.
Signal transduction events whereby PGF2 alpha inhibits the ammoniagenic response to acute acidosis
Authors: Sahai A. , Sandler R. S. , Xu G. , Shayman J. A. , Tannen R. L.
Published: December 2017

Subconfluent cultures of LLC-PK1 cells were incubated for 1 h in Krebs-Henseleit buffer (KHB) of pH 7.4 or 6.8 to investigate the signal transduction events associated with prostaglandin F2 alpha (PGF2 alpha) inhibition of ammonia metabolism. Exposure of these cultures to PGF2 alpha (0.1 ng/ml) inhibited the acute low pH stimulation of ammonia production and to a lesser degree alanine formation in a manner analogous to the response exhibited with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment with an inhibitor of protein kinase C [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, i.e., H-7] or utilization of cultures with downregulated protein kinase C activity abolished the inhibitory response to PGF2 alpha. Exposure to PGF2 alpha for 10 min in KHB of pH 6.8 resulted in an activation of protein kinase C, as demonstrated by a significant increase in membrane-bound enzyme activity. Incubation of the cells with PGF2 alpha in KHB of pH 6.8 also resulted in a significant increase in inositol trisphosphate formation. Treatment of the cultures with verapamil in calcium-containing medium or removal of calcium from the incubating medium resulted in a significant loss of the PGF2 alpha inhibitory response on both ammonia and alanine production. Furthermore, under conditions of calcium-free incubation, PGF2 alpha had no significant effect on protein kinase C activity. Because both PGF2 alpha- and TPA-induced inhibition of ammoniagenic response to acute acidosis was prevented by amiloride, the underlying mechanism may involve protein kinase C-mediated changes in intracellular pH. These results indicate that the activation of protein kinase C plays a key role in mediating PGF2 alpha inhibition of ammoniagenesis.
Discordant aspects of aldosterone resistance in potassium depletion
Authors: Mujais S. K. , Chen Y. , Nora N. A.
Published: December 2017

Aldosterone resistance, defined as absent kaliuretic response to exogenous hormone, has been described in K depletion. It is not clear whether the absent kaliuresis is due to activation of K-conserving mechanisms or to failure of activation of the Na-K pump in cortical collecting tubules (CCT) by mineralocorticoids. Adrenalectomized male Sprague-Dawley rats were allocated to either a normal or low-K diet. Na-K pump activity (pmol.mm-1.h-1) in microdissected CCT and medullary collecting tubules (MCT, inner stripe of the outer medulla) was determined at 7 or 21 days after allocation to the dietary groups before and after exogenous aldosterone (50 micrograms twice daily, for 3 days). K depletion led to progressive hypertrophic changes in the CCT and MCT manifest in an increase in basal Na-K pump activity. In both K repletion and short-term K depletion (7 days), aldosterone led to the expected increase in CCT Na-K pump activity. With long-term K depletion, the CCT Na-K pump response to aldosterone was blunted. In the MCT where under normal conditions the Na-K pump is aldosterone unresponsive, an increasing aberrant responsiveness to the mineralocorticoid was observed with progressive K depletion. We conclude that apparent aldosterone resistance in short-term K depletion is likely due to activation of K-conserving mechanisms with early preservation of the CCT biochemical response to the hormone. With long-term K depletion, a blunted biochemical response to aldosterone may contribute to the absent kaliuretic response. In the MCT, K depletion led to the development of aberrant responsiveness to aldosterone.
Time course of renal glutamate dehydrogenase induction during NH4Cl loading in rats
Authors: Wright P. A. , Packer R. K. , Garcia-Perez A. , Knepper M. A.
Published: December 2017

To study mechanisms involved in renal glutamate dehydrogenase (GDH) regulation in response to systemic acid loading, we have measured blood pH, ammonium excretion, renal GDH mRNA levels, and GDH activity in rats. Acid intake (0.28 M NH4Cl in drinking water for 3 days) increased GDH mRNA levels in the renal cortex, but had no effect in the outer stripe of the outer medulla, inner stripe of the outer medulla, or the inner medulla. Rats were subjected to a step change in acid intake by alkali loading for 3 days (7.2 meq NaHCO3 per day in food slurry) and shifting to acid loading for up to 7 days (7.2 meq NH4Cl in food slurry). Ammonium excretion rose rapidly, increasing by 14-fold in the first 24-h period and 38-fold in the second 24-h period. Cortical GDH mRNA levels were increased relative to alkali-loaded values by 3.7-fold in 24 h, 4.3-fold in 4 days, but only 2.2-fold in 7 days. GDH activity was unchanged after 24 h of acid intake, but was significantly increased after 48 h. We concluded the following: 1) GDH mRNA is present in all regions of the kidney, but levels increase in response to acid loading only in the renal cortex; 2) GDH mRNA levels increase within 1 day after the initiation of acid loading, but the associated increase in functional enzyme activity takes 2 or more days; and 3) the large increases in ammonium excretion that occur in the first day after initiation of acid loading are not dependent on increased GDH activity.
ANF and bradykinin synergistically inhibit transport in M-1 cortical collecting duct cell line
Authors: Stoos B. A. , Carretero O. A. , Garvin J. L.
Published: December 2017

Previous data suggest that atrial natriuretic factor (ANF) and bradykinin (BK) interact to increase Na+ and water excretion. We propose that this interaction is due to a synergistic action that inhibits Na+ absorption in the distal nephron. We examined the effects of BK and ANF on transport by monolayers of a cortical collecting duct cell line, M-1. BK (10(-8) M) had no effect on short-circuit current (Isc). Similarly, ANF (10(-8) M) did not inhibit Isc. In contrast, Isc decreased by 18% (from 57 +/- 8 to 46 +/- 6 microA/cm2) when BK and ANF were added simultaneously at this concentration (P less than 0.05). Because guanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase C are implicated in the second messenger cascades of ANF and BK, we investigated their potential roles in mediating this interaction. Dibutyryl-cGMP (10(-4) M) inhibited Isc from 33 +/- 4 to 22 +/- 3 microA/cm2 (P less than 0.05) in the presence of BK but not in its absence. Staurosporine and calphostin C, inhibitors of protein kinase C, completely blocked the decrease in Isccaused by simultaneous addition of ANF and BK. cAMP levels in M-1 cells were not affected by either ANF alone or BK alone; however, when cultures were treated with both hormones, cAMP decreased from 856 +/- 56 to 332 +/- 26 fmol/10(6) cells (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of S6 kinase activity in renal proximal tubule
Authors: Harris R. C.
Published: December 2017

The proximal tubule undergoes hypertrophy in response to loss of functioning renal mass and hyperplasia following injury by ischemia or nephrotoxins. Both hypertrophic growth and cell proliferation are characterized by increases in the rate of protein synthesis. To investigate regulation of protein synthesis in mammalian proximal tubule cells, potential peptide mediators of proximal tubule growth, epidermal growth factor (EGF) and angiotensin II, were studied in cultured rabbit proximal tubule cells. Although only EGF stimulated DNA synthesis, both agonists stimulated protein synthesis. One potential regulatory mechanism of eukaryotic protein synthesis involves phosphorylation of ribosomal protein S6 by activation of a specific serine/threonine kinase (S6 kinase). Both EGF and angiotensin II stimulated S6 kinase activity and S6 phosphorylation. Phorbol 12-myristate 13-acetate was also found to activate S6 kinase, and 24 h of pretreatment to deplete protein kinase C inhibited subsequent S6 kinase activation by a high concentration (10(-6) M) of angiotensin II. To determine whether S6 kinase was also activated in the kidney in vivo, S6 kinase activity was examined after ablation of renal mass. Within 1 h after contralateral nephrectomy, S6 kinase activity increased in rat renal cortex. In summary, both EGF and angiotensin II stimulated protein synthesis and S6 kinase activity in cultured proximal tubule cells, and S6 kinase activity also increased in renal cortex after contralateral nephrectomy.
Increased functional differentiation of rabbit proximal tubule cells cultured in glucose-free media
Authors: Blais A. , Jalal F. , Crine P. , Paiement J. , Berteloot A.
Published: December 2017

We have determined the influence of glucose (Glc)-free medium on the growth and differentiation of rabbit kidney proximal tubule cells (PTC) in primary cultures. The specific growth rates and the protein-to-volume ratios are shown to be independent of the culture conditions. In contrast, the functional expression of four brush-border membrane enzyme markers was found to decline steadily and in the same way from day 3 in culture up to late confluence in Glc-containing medium, and different evolution patterns and high expression levels were observed up to confluence in a Glc-free glutamine (Gln)-supplemented medium. Electron microscopy clearly showed, however, that the functional and morphological differentiation of the brush-border membrane is not correlated. Finally, by use of an indirect immunofluorescent technique in combination with flow cytometry, it is demonstrated that confluent cells grown in Glc and Gln media form homogeneous cell populations of PTC. It is thus concluded that the functional differentiation of rabbit kidney PTC in primary cultures is strongly dependent upon the energy source present in the culture medium.
Effects of formate and oxalate on volume absorption in rat proximal tubule
Authors: Wang T. , Giebisch G. , Aronson P. S.
Published: December 2017

We examined the effects of formate and oxalate on the rate of fluid absorption (Jv) in the rat proximal convoluted tubule in situ. Proximal tubules were microperfused with a high-Cl-, low-HCO3- Ringer solution (pH 6.7), and the peritubular capillaries were perfused with a standard Ringer solution (pH 7.4), simulating conditions in the late proximal tubule. Jv, a measure of transtubular NaCl absorption under these conditions, was calculated from the change in luminal [3H]inulin. Addition of formate in the physiological range (500 microM) to the luminal perfusate increased Jv by 45%; addition of 500 microM formate to both luminal and capillary perfusates increased Jv by 57%. Similarly, addition of oxalate in the physiological range (5 microM) to the luminal perfusate increased Jv by 37%; addition of 5 microM oxalate to both luminal and capillary perfusates increased Jv by 57%. The stimulatory effects of formate and oxalate perfused in the lumen and capillaries were not additive. Addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS, 0.1 mM) to the luminal perfusate had no effect on baseline Jv measured in the absence of added formate and oxalate but completely abolished the increment in Jv induced by formate and oxalate. Addition of the Cl(-)-channel blocker diphenylamine-2-carboxylate (DPC, 0.2 mM) to the capillary perfusate had no effect on baseline Jv but completely abolished the increment in Jv induced by formate and oxalate.(ABSTRACT TRUNCATED AT 250 WORDS)
Renal acid-base physiology in marine teleost, the long-horned sculpin (Myoxocephalus octodecimspinosus)
Authors: Maren T. H. , Fine A. , Swenson E. R. , Rothman D.
Published: December 2017

We have sought to define urinary acid-base excretion in the marine teleost using the long-horned sculpin, Myoxocephalus octodecimspinosus. Urine flow (1.7 ml.h-1.kg-1) is relatively high, and glomerular filtration rate is very low (2.9 ml.h-1.kg-1). The urine-to-plasma ratio of inulin is 2. Renal clearance of p-aminohippurate is very high (108 ml.h-1.kg-1); phosphate and divalent cations are also secreted. In this framework we found urinary pH to average 6.6, but infusion of acid or alkali elicited a pH range of 6.1-7.8. Untreated fish may also have alkaline urine; so it is not surprising that precipitates of calcium or magnesium phosphate are sometimes found in bladder. These are of fine sandy quality and never cause blockage. Infusion of buffer (imidazole) increased the concentration of titratable acid 11-fold and output 2.5-fold. Carbonic anhydrase inhibitors had no effect on any urinary component, and histochemical studies revealed that cytoplasm and membranes did not yield the specific cobalt stain for the enzyme. An alkaline load (NaHCO3) is rapidly dissipated by gill excretion, mediated in part by carbonic anhydrase. An acid load (HCl) is rapidly dissipated by gill excretion, not dependent on carbonic anhydrase, and some renal excretion. Comparison and contrast of the low rates of HCO3- reabsorption in the marine teleost (and elasmobranch) with those of mammals suggest strongly that renal carbonic anhydrase evolved in connection with these high reabsorptive rates beginning in freshwater fish and continuing through amphibia and birds.
Localization of mRNAs coding for isozymes of plasma membrane Ca(2+)-ATPase pump in rat kidney
Authors: Magosci M. , Yamaki M. , Penniston J. T. , Dousa T. P.
Published: December 2017

We have studied localization of mRNAs coding isozymes of rat plasma membrane Ca(2+)-adenosinetriphosphatase pump (rPMCA) in the rat kidney, with use of reverse transcription (RT) with subsequent amplification by polymerase chain reaction (PCR). When zones of the kidney were separated by macrodissection, a large amount of mRNA coding isozyme rPMCA1 was found in all zones; mRNA for isozyme rPMCA2 was abundant in cortex and in outer medulla, and mRNA for isozyme rPMCA3 was prominent in outer medulla. The mRNAs were analyzed in microdissected cortical nephron segments by use of RT-PCR approach described previously [T. Moriyama, H. R. Murphy, B. M. Martin, and A. Garcia-Perez. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1470-F1474, 1990]. We detected mRNA for isozyme rPMCA2 in microdissected distal convoluted tubules (DCT) and in cortical thick ascending limbs (CTAL) and, less consistently, also in proximal convoluted tubule and in glomeruli. The mRNA for isozyme rPMCA1 was abundant in glomeruli but was absent in all examined cortical tubular segments. Our results document that mRNAs for all three major isozymes of rPMCA are present and show a unique distribution in the three major zones of rat renal parenchyma. Specific mRNA coding for rPMCA2 was detected in cortical tubules, namely in CTAL and DCT, whereas mRNA coding isozyme rPMCA1 was found in glomeruli. We suggest that isozyme rPMCA2 might be specifically related to epithelial cells and their function, whereas rPMCA1 is probably a component of nonepithelial cells including these in glomeruli.
Renal receptors for atrial and C-type natriuretic peptides in the rat
Authors: Brown J. , Zuo Z.
Published: December 2017

Receptors for alpha-atrial natriuretic peptide (alpha-ANP) and C-type natriuretic peptide [CNP-(1-22)] were quantified in kidneys from adult Wistar rats by in vitro autoradiography. 125I-labeled alpha-ANP (100 pM) bound reversibly to glomeruli, outer medullary vasa recta, and inner medulla with an apparent dissociation constant (Kd) of 3–6 nM. The presence of 10 microM des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4– 23) (C-ANP), a specific ligand of the ANPR-C subtype of alpha-ANP receptor, inhibited approximately 50% of the glomerular binding of 125I-alpha-ANP, and this moiety of glomerular binding was also inhibited by CNP-(1–22) with an apparent inhibitory constant (Ki) of 10.47 +/- 7.59 nM. C-ANP and CNP-(1–22) showed little affinity for the medullary binding sites of alpha-ANP. 125I-[Tyr0]CNP-(1–22) (110 pM) bound solely to glomeruli and was competitively displaced by increasing concentrations of [Tyr0]CNP-(1–22) with an apparent Kd of 1.42 +/- 0.48 nM. Binding of increasing concentrations (25 pM to 1 nM) of 125I-[Tyr0]CNP-(1–22) in the presence or absence of 1 microM [Tyr0]CNP-(1–22) also demonstrated a high affinity (Kd of 0.41 +/- 0.07 nM) for the glomerular binding of 125I-[Tyr0]CNP-(1–22). Bound 125I-[Tyr0]CNP-(1–22) could be displaced by excess alpha-ANP and excess CNP-(1–22), both with high affinities. The glomerular binding of 125I-[Tyr0]CNP-(1–22) was also prevented by 10 microM C-ANP. Guanosine 3',5'-cyclic monophosphate produced by isolated glomeruli was measured by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of intracellular calcium in hydrogen peroxide-induced renal tubular cell injury
Authors: Ueda N. , Shah S. V.
Published: December 2017

Both reactive oxygen metabolites and calcium have been implicated in ischemic and toxic renal tubular cell injury. However, the role of calcium in oxidant injury to renal tubular cells has not been previously examined. In the present study we examined the role of intracellular free Ca2+ ([Ca2+]i) in H2O2-mediated injury to LLC-PK1 cells, a renal tubular epithelial cell line. H2O2 induced a significant rise in [Ca2+]i within 1 min after exposure of cells to 5 mM H2O2, with a sustained rise in [Ca2+]i during the course of experiments, reaching a value of 1.3 microM at 60 min (n = 10). The rise in [Ca2+]i preceded sublethal cell injury as measured by [3H]adenine release or irreversible cell injury as determined by trypan blue exclusion. Buffering [Ca2+]i with quin-2 (50 microM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) was highly protective against the H2O2-induced cell injury. For example, at 120 min after exposure of cells to 5 mM H2O2, irreversible cell injury was reduced from 45 +/- 8 to 9 +/- 1% (n = 3) by quin-2. The acetoxymethyl ester of quin-2 (quin-2/AM) and BAPTA/AM did not interfere with the trypan blue exclusion assay or scavenge H2O2. Preventing mobilization of Ca2+ from intracellular storage sites using 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 10(-4) M) significantly reduced the rise in [Ca2+]i and thus prevented H2O2-mediated cytotoxicity to LLC-PK1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Comparative effects of arginine vasopressin and oxytocin in cell culture systems
Authors: Briner V. A. , Tsai P. , Choong H. L. , Schrier R. W.
Published: December 2017

Arginine vasopressin (AVP) and oxytocin (OXT) induced contraction in cultured vascular smooth muscle cells (VSMC) and glomerular mesangial cells (GMC). The contractile response of AVP and OXT was paralleled by Ca2+ mobilization as assessed by 45Ca2+ efflux in a dose-dependent manner. The effects of AVP were blocked by pretreating VSMC and GMC with a V1 antagonist. OXT-stimulated effects, however, were not affected by preexposure of VSMC and GMC to an OXT antagonist but were inhibited by the V1 antagonist. Competition studies demonstrated displacement of [3H]AVP from its receptors by unlabeled AVP, the V1 antagonist, and high doses of OXT. The OXT antagonist was the least effective in displacing [3H]AVP. Thus occupancy of the V1 receptor by OXT may initiate signal transduction and contraction in VSMC and GMC in a manner qualitatively similar to that of the AVP agonist. Cultured myometrium cells (MMC) also contracted in response to AVP and OXT. Moreover, 45Ca2+ efflux increased in response to both hormones in a dose-dependent manner. AVP-stimulated contraction and 45Ca2+ efflux were blocked in MMC by pretreatment with V1 antagonist. OXT-induced effects were inhibited by the OXT antagonist but not by the V1 antagonist. Binding experiments showed that [3H]AVP was displaced equally by unlabeled AVP and V1 antagonist. Very high concentrations of OXT antagonist also demonstrated displacement.(ABSTRACT TRUNCATED AT 250 WORDS)
Xanthine oxidase produces O2-. in posthypoxic injury of renal epithelial cells
Authors: Greene E. L. , Paller M. S.
Published: December 2017

The hypothesis that posthypoxic renal injury is mediated by xanthine oxidase-derived oxygen free radical production was tested in an in vitro model of rat proximal tubule epithelial cells in primary culture subjected to 60 min of hypoxia and 30 min of reoxygenation. Hypoxia-reoxygenation-induced injury, measured as lactate dehydrogenase (LDH) release, was 54.0 +/- 7.1%. Inhibition of xanthine oxidase by 10(-4) M allopurinol attenuated injury (LDH release = 35.5 +/- 3.7%; P less than 0.01). Oxypurinol was similarly effective. Alternatively, cells were treated with 50 or 100 microM tungsten to inactivate xanthine oxidase. Tungsten prevented hypoxia-reoxygenation-induced superoxide radical production (basal = 97 +/- 8, hypoxia-reoxygenation = 172 +/- 12, and plus tungsten = 73 +/- 8 nmol/micrograms protein) and attenuated hypoxia-reoxygenation-induced injury (LDH release: basal = 18.8 +/- 3.0%, hypoxia-reoxygenation = 62.0 +/- 4.8%, plus 50 microM tungsten = 24.8 +/- 5.0%, and plus 100 microM tungsten = 6.0 +/- 0.7%). In addition, hypoxia and reoxygenation increased the ratio of xanthine oxidase to total activity (xanthine oxidase + xanthine dehydrogenase) from 73 to 100%. Therefore xanthine oxidase was responsible for hypoxia-reoxygenation-induced superoxide radical formation and hypoxia-reoxygenation-induced injury. Xanthine oxidase is likely to be the major source of oxygen free radicals during renal ischemia and reperfusion.
Endothelium-derived relaxing factor regulates renin release in vivo
Authors: Sigmon D. H. , Carretero O. A. , Beierwaltes W. H.
Published: December 2017

Endothelium-derived relaxing factor (EDRF), through its inhibitory second messenger guanosine 3',5'-cyclic monophosphate (cGMP), inhibits renin release in vitro. To determine whether EDRF affects renin in vivo, we tested whether EDRF synthesis inhibition could stimulate renin secretion in intact rats. Because EDRF synthesis inhibition increases blood pressure and consequently withdraws sympathetic activity (both renin inhibitory signals), we also studied the effect of L-N omega-nitroarginine methyl ester (L-NAME) when renal perfusion pressure was controlled and during beta-adrenergic blockade. Mean blood pressure (BP), heart rate (HR), and plasma renin activity (PRA) were measured in anesthetized rats before and after EDRF synthesis inhibition by a 10 mg/kg body wt bolus of L-NAME. L-NAME decreased PRA by 67% [from 11.0 +/- 2.7 to 3.7 +/- 0.8 ng angiotensin I (ANG I).ml-1.h-1, n = 12; P less than 0.001], increased BP by 20 +/- 2 mmHg (P less than 0.001), and decreased HR from 332 +/- 8 to 312 +/- 9 beats/min (P less than 0.005). We repeated our experiment in rats instrumented with an intra-aortic balloon catheter to control renal perfusion pressure and pretreated with propranolol to eliminate the beta-adrenergic effect. Under these conditions, L-NAME now increased PRA by 55% (from 6.9 +/- 1.9 to 10.8 +/- 2.6 ng ANG I.ml-1.h-1, n = 12; P less than 0.02), whereas renal perfusion pressure was unchanged (91 +/- 4 vs. 90 +/- 4 mmHg). HR increased slightly from 308 +/- 5 to 315 +/- 3 beats/min (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)
Colocalization and release of angiotensin and renin in renal cortical cells
Authors: Hunt M. K. , Ramos S. P. , Geary K. M. , Norling L. L. , Peach M. J. , Gomez R. A. , Carey R. M.
Published: December 2017

Angiotensin is generated within the kidney, but the precise loci for the formation of angiotensin I (ANG I) and angiotensin II (ANG II) have not been demonstrated. We performed electron microscopy immunocytochemistry in kidney sections of 10-day-old (newborn) and adult Wistar-Kyoto (WKY) rats using specific antibodies to renin, ANG I, ANG II, and angiotensinogen (AO). Renin, ANG I, ANG II, and AO were present in juxtaglomerular (JG) cells. Renin was largely confined to cytoplasmic granules; ANG I and ANG II were colocalized to these granules but also were present in the cytoplasm; AO was distributed throughout the cytoplasm. AO also was present in a renal cortical distribution in proximal tubular cells. Northern blot analysis demonstrated AO mRNA in total kidney and liver but not in renal microvessels. Using the reverse hemolytic plaque assay, we demonstrated release of ANG I and renin from individual renocortical cells of adult WKY rats. Under control conditions, the number of releasing cells was 11 +/- 1 for ANG I and 10 +/- 1 for renin. Addition of rat renin inhibitor (RI) (1 x 10(-5) M), which inhibited renin activity in the medium from 37 to 9 pg ANG I.ml-1.h-1, did not alter ANG I plaque number. Addition of rat AO increased ANG I plaque number to 17 +/- 2 (P less than 0.05). Incubation with both RI and AO prevented the increase in ANG I plaque number obtained with AO alone. Enalapril treatment (7 days; n = 5) increased the number of plaque-forming cells to 22 +/- 2 for ANG I (P less than 0.0005) and to 39 +/- 7 for renin (P less than 0.001). The results suggest an intracellular location for AO and angiotensin and release of renin and ANG I by renal cortical cells and suggest that released angiotensin is produced intracellularly and that secretion of ANG I is augmented by converting enzyme inhibition.