bluuebunny/crossref_metadata_2025 huggingface dataset:
Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity
Authors: Bennett W. M. , Mela-Riker L. M. , Houghton D. C. , Gilbert D. N. , Buss W. C.
Published: December 2017

Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. [3H]leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis.
Amiloride-sensitive cation channel in apical membrane of inner medullary collecting duct
Authors: Light D. B. , McCann F. V. , Keller T. M. , Stanton B. A.
Published: December 2017

We determined the role of ornithine decarboxylase (ODC) in compensatory renal hypertrophy (CRH) by relating renal ODC activity and polyamine content to kidney size, expressed as a percent of body weight, 1 wk after unilateral nephrectomy (UN). In normal rats, renal ODC activity increased from 11.0 +/- 7.4 (SD) to 36.7 +/- 15.4 pmol 14CO2.min-1.g wet wt-1 3 h after UN (P less than 0.002); 1 wk later the remaining kidney weight had increased from 0.38 to 0.46% body weight (P less than 0.001). Renal concentration of putrescine, the product of ODC's decarboxylation of ornithine, was increased 3, 8, and 48 h after UN, but concentrations of polyamines synthesized later in the pathway, spermidine and spermine, were not appreciably affected. Pretreatment with difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, as a 1% drinking water solution inhibited both base-line renal ODC activity and putrescine concentration as well as increases stimulated by UN, although concentrations of spermidine and spermine were not decreased. In these rats, CRH still occurred, since kidney weight increased from 0.36 to 0.46% (P less than 0.001). A 2% DFMO solution caused depletion of all three polyamines, but CRH took place nevertheless. In hypophysectomized rats, both increased renal ODC activity and CRH occurred as well, indicating that these two consequences of UN do not require intact pituitary function. Although increased renal ODC activity and CRH after UN are correlated in normal and hypophysectomized rats, CRH takes place in rats treated with DFMO despite inhibition of ODC activity and depletion of polyamines.(ABSTRACT TRUNCATED AT 250 WORDS)
Suppression of acidification along inner medullary collecting duct
Authors: Bengele H. H. , McNamara E. R. , Schwartz J. H. , Alexander E. A.
Published: December 2017

The purpose of these experiments was to evaluate the effect of acute respiratory alkalosis (ARA) and chronic bicarbonate drinking (CBD) on inner medullary collecting duct (IMCD) acidification. Microcatheterization was used to measure pH and PCO2, and samples were simultaneously obtained for measurement of bicarbonate, titratable acid (TA), and ammonium. In ten ARA rats (arterial pH was 7.54 +/- 0.02; PCO2 was 20 +/- 1 mmHg), IMCD equilibrium pH was not different (deep pH was 5.65 +/- 0.06 and PCO2 was 20 +/- 1 mmHg; tip pH was 5.54 +/- 0.07 and PCO2 was 22 +/- 1 mmHg). Delivery of bicarbonate, TA, and ammonium also did not differ between collection sites. Thus net acidification along the IMCD was negligible. Nine rats drank NaHCO3 for 5-8 days (pH = 7.48 +/- 0.02) but did not receive NaHCO3 during the experiment so that arterial pH fell to 7.40 +/- 0.01. IMCD equilibrium pH was different at deep (pH was 5.68 +/- 0.06; PCO2 was 32 +/- 1 mmHg) and tip (pH was 5.57 +/- 0.04; PCO2 was 27 +/- 1 mmHg; P less than or equal to 0.05) collection sites. However, only minimal changes in the delivery of bicarbonate, TA, and ammonium were noted, and net acidification along the IMCD was negligible. In ten control rats, net acidification was 219 nmol/min between collection sites (P less than 0.001). We conclude that ARA and CBD abolish acidification along the IMCD. In addition, CBD produces an intrinsic modification along the IMCD, which suppresses acid secretion and persists after acute recovery from alkalemia.
Modulation of Na+-H+ exchange activity by intracellular Na+, H+, and Li+ in IMCD cells
Authors: Wall S. M. , Kraut J. A. , Muallem S.
Published: December 2017

In the present study, cultured inner medullary collecting duct (IMCD) cells loaded with the pH-sensitive dye 2',7' bis(carboxyethyl)-carboxyfluorescein were used to study the interaction of H+, Na+, and Li+ with the intracellular face of the Na+-H+ exchanger. The interaction of Na+ with the external face of the exchanger followed simple saturation kinetics with an apparent affinity for Na+ of 19.5 +/- 2 mM (n = 3) at an extracellular pH of 7.4. Extracellular H+ and Li+ inhibit Na+o-H+i exchange, mainly due to interference with Na+ binding to an external site or the same enzyme intermediate species in the turnover cycle of the Na+-H+ exchanger. The interaction of H+ with the internal face of the exchanger does not follow simple saturation kinetics. The calculated Hill coefficient for H+ interaction with intracellular sites was 1.92. Na+ and Li+ also interact with the intracellular face of the Na+-H+ exchanger. This interaction of Li+ and Na+ with internal sites results in inhibition of Na+-H+ exchange activity in the forward direction (Na+ influx/H+ efflux). The inhibitory effect of intracellular Na+ and Li+ is pHi dependent and most apparent under physiological conditions, i.e., at an intracellular pH of 7.1, an extracellular pH of 7.4, and an extracellular Na+ of 140 mM. This study shows the importance of intracellular Na+ concentration in determining exchanger activity.
Time course and vectorial nature of albumin metabolism in isolated perfused rabbit PCT
Authors: Park C. H.
Published: December 2017

The time course and vectorial nature of renal metabolism of albumin (Alb) were studied. The tubular absorption, accumulation, and hydrolysis of Alb and the release of the hydrolysis products were determined in the isolated rabbit proximal convoluted tubule (PCT) perfused with tritiated Alb ([3H3C]Alb) at 36.4 micrograms/ml. The Alb absorption across the apical membrane was constant (99.9 +/- 4.9 x 10(-3) ng.min-1.mm-1). In contrast, the accumulation and hydrolysis of Alb in the cells increased nonlinearly with time. The bulk of the tritium that accumulated in the cells was associated with intact [3H3C]Alb. Only the final hydrolysis products were released from the cells and these first appeared in the peritubular bath 6–7 min after the start of perfusion of the tubule with [3H3C]Alb. The hydrolysis product was not detectable in the tubule lumen. The proteolytic activity correlated linearly with the protein load to the cells, characteristic of first-order kinetics and a high-capacity system. The results suggest that the renal tubular handling of proteins proceeds from the apical to the basolateral aspect of the cell. The transcellular processing of Alb is rapid and can occur in 6–7 min. The accumulation of intact protein in the cell and the first-order kinetics of hydrolysis of the absorbed protein suggest that the rate-limiting step in proximal tubular handling of proteins may include the initial hydrolysis of protein or reside in steps that precede the hydrolysis.
NMR studies of renal phosphate metabolites in vivo: effects of hydration and dehydration
Authors: Wolff S. D. , Eng C. , Balaban R. S.
Published: December 2017

The present study characterizes the 31P-nuclear magnetic resonance (NMR) spectrum of rabbit kidneys in vivo and evaluates the effect of hydration on phosphorous metabolites including the organic solute glycerophosphorylcholine (GPC). Cortical phosphorylethanolamine is the predominant component of the phosphomonoester region of the 31P spectrum. The contribution of blood to the spectrum is mainly from 2,3 diphosphoglycerate, which comprises approximately 30% of the inorganic phosphate region. Acute infusion of 0.9% saline decreases the sodium content of the inner medulla by greater than 50% in 15 min as shown by 23Na imaging. Despite this medullary Na dilution, no change in renal GPC content was observed for greater than 1 h even with the addition of furosemide (2.5 mg/kg) or furosemide and antidiuretic hormone (0.125 U/kg). However, 20 h of chronic hydration with 0.45% saline did result in a 30% decrease in renal GPC content when compared with dehydrated animals. These findings are consistent with GPC not playing a role in the short-term regulation of the medullary intracellular milieu in response to acute reductions in medullary Na content.
Coupled transport of p-aminohippurate by rat kidney basolateral membrane vesicles
Authors: Pritchard J. B.
Published: December 2017

p-Aminohippuric acid (PAH) transport by basolateral membrane (BLM) vesicles isolated from rat renal cortex was stimulated very little by a Na+ gradient (out greater than in). However, when micromolar concentrations of glutaric acid or alpha-ketoglutaric acid were added in the presence of a out greater than in Na+ gradient, PAH uptake was accelerated greater than 20-fold and an overshoot of greater than fivefold was produced. Other anions, e.g., fumarate, stimulated PAH uptake very modestly under these conditions (approximately 2-fold), and that stimulation was totally prevented by short circuiting, i.e., with K+ (in = out) and valinomycin. Glutarate-stimulated uptake was inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and probenecid and was slightly stimulated by the imposition of an inside-negative membrane potential. Furthermore, even in the absence of a Na+ gradient, glutarate-loaded vesicles exhibited a marked acceleration of PAH uptake (5-fold) and a modest overshoot (2.5-fold). These results suggest an indirect coupling of BLM PAH uptake to the Na+ gradient by a cyclic accumulation (Na+-dependent) of glutarate followed by its efflux from the vesicle in exchange for PAH. This coupled system was absent in apical membranes. Thus net secretory transport of PAH may entail Na+-dependent, glutarate-driven PAH uptake at the BLM, followed by the exit of PAH into the lumen down its electrochemical gradient, probably in exchange for other anions, e.g., Cl-, HCO3-, or OH-.
Mesangial cells express PDGF mRNAs and proliferate in response to PDGF
Authors: Shultz P. J. , DiCorleto P. E. , Silver B. J. , Abboud H. E.
Published: December 2017

Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin and is released and/or synthesized by platelets, macrophages, endothelial cells, and rat mesangial cells. In the present investigation, we found that human glomerular mesangial cells in culture release a PDGF-like protein which competes for 125I-PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. The competing and to a lesser extent the mitogenic activities present in the conditioned medium are partially recognized by an anti-PDGF antibody. Northern blot analysis of poly(A)+ RNA from human mesangial cells demonstrates the expression of both PDGF A- and B-chain mRNAs. PDGF also binds to mesangial cells in a specific manner and stimulates DNA synthesis and cell proliferation. These data suggest that a PDGF-like protein secreted by mesangial cells or released from platelets, monocytes, or endothelial cells during glomerular inflammation may function as an autocrine or a paracrine growth factor for these cells. The biological role of PDGF in mediating proliferative and other inflammatory events in the glomerulus remains to be identified.
Secretion of endolymph by semicircular canals of the shark
Authors: Garvin J. L. , Spring K. R. , Santi P. A.
Published: December 2017

The semicircular canals of the vestibular labyrinth of the dogfish shark, Squalus acanthias, may serve as a simple in vitro system for the study of the ionic transport mechanisms involved in endolymph formation. Electron microscopy showed that the epithelium was made up of at least three distinct cell types divided into separate regions running the length of the canals. Secretion of endolymph was studied in isolated canals by the split droplet method; when the lumens of the canals were filled with shark Ringer a potassium-rich fluid was secreted into the lumen at a rate of 0.34 microliter.cm-2.min-1. The K concentration of the secreted fluid averaged 112 mM; the calculated rate of K secretion was 2.3 mumol.cm-2.h-1, comparable to recent measurements in mammalian utricle. Fluid secretion was dependent on active transport and was inhibited by ouabain, bumetanide, or methazolamide in the external bathing solution. Fluid secretion was unaffected by the K channel blocker, Ba, in the luminal droplet or by the adenylate cyclase stimulator, forskolin, in the external bathing solution. For electrophysiological analysis, isolated canals were perfused in a chamber designed for voltage/current clamping; an axial wire was inserted into the canal lumen and constant-current pulses were passed to determine tissue resistance. When the luminal fluid was high-K Ringer, transepithelial potential difference was -1.1 mV (lumen negative) and resistance 37 omega.cm2. Dilution and bi-ionic potential measurements showed that the epithelium of the canals exhibited only slight cation selectivity. These results are consistent with a model for endolymph secretion involving cotransport secondary to the ion gradients created by the Na+-K+-ATPase.
Corticosterone's metabolite is an agonist for Na+ transport stimulation in A6 cells
Authors: Duncan R. L. , Grogan W. M. , Kramer L. B. , Watlington C. O.
Published: December 2017

This study tests the hypothesis, in A6 epithelia, that 1) corticosterone stimulates active Na+ transport (short-circuit current, Isc) by an additional receptor mechanism to the type I (mineralocorticoid) and type II (glucocorticoid) mechanisms shared with aldosterone (Aldo) and 2) that the agonist may be 6 beta-OH-corticosterone made in the effector cell. The dose-response relationship of corticosterone at 24 h resolves into two components, by curve fitting, with a 50% effective concentration (EC50) for 10% of maximum Isc stimulation of 2 X 10(-9) M and an EC50 for the other 90% of 3 X 10(-7) M. The EC50 of the smaller component correlates with the apparent dissociation constant (K'd) of corticosterone for high affinity (type II) nuclear binding sites shared with Aldo. In unlabeled analogue competition studies Aldo and corticosterone displaced nuclear binding equally below 10(-8) M [3H]corticosterone, indicating only shared sites. However, nonshared saturable sites (displaced by corticosterone but not by Aldo) were found at [3H]-corticosterone concentrations above 10(-8) M. Concentration-binding curves performed with [3H]corticosterone, in presence of 1,000 X Aldo to displace shared sites, revealed a single class of binding sites with a half-maximal saturation of 2 X 10(-7) M, which is quite similar to the EC50 of the lower affinity component of Isc stimulation by corticosterone at 24 h. Reversed phase high-pressure liquid chromatography of nuclear extracts indicates that the saturable component of bound [3H] was 6 beta-OH-[3H]corticosterone derived from [3H]corticosterone. Thus, A6 cells metabolize corticosterone to 6 beta-OH-corticosterone, which in turn occupies lower-affinity receptors not shared with Aldo or corticosterone, to mediate most of the active Na+ transport stimulation by corticosterone.
Intrarenal renin inhibition increases renal function by an angiotensin II-dependent mechanism
Authors: Siragy H. M. , Lamb N. E. , Rose C. E. , Peach M. J. , Carey R. M.
Published: December 2017

ACRIP is a competitive inhibitor of renin in which an analogue of statine, (3R,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, is incorporated into analogues of porcine renin substrate. ACRIP inhibits the enzymatic activity of renin, thus blocking the initiation of the angiotensin cascade. We studied the intrarenal action of ACRIP in small quantities without measurable systemic effects on renal function. In the first experiment, ACRIP was administered intrarenally at 0.02, 0.2, and 2 micrograms.kg-1.min-1 to uninephrectomized conscious dogs (n = 6) in metabolic balance at sodium intake of 10 meq/day. ACRIP, in doses of 0.02 and 0.2 micrograms.kg-1.min-1, markedly increased urine sodium excretion (UNaV) from 5.8 +/- 1.4 to 15.1 +/- 5.1 and 19.9 +/- 3.2 mu eq/min, respectively. Urinary flow rate (UV) underwent a similar increase and glomerular filtration rate (GFR) increased from 25.7 +/- 2.5 to 35.6 +/- 2.5 at 0.02 micrograms.kg-1.min-1 of ACRIP. Renal plasma flow (RPF), plasma renin activity (PRA), and plasma aldosterone concentration (PAC) were not affected. At 2 micrograms.kg-1.min-1, ACRIP traversed the kidney in quantities large enough to produce a reduction in systemic PRA and mean arterial pressure and caused natriuresis, diuresis, and increased GFR. In a second experiment, ACRIP was administered intrarenally at 0.2 micrograms.kg-1.min-1 in a separate group (n = 4) under identical conditions. ACRIP-induced increases in UV and UNaV were completely blocked by concurrent intrarenal administration of angiotensin II. The results indicate that intrarenal angiotensin II acts as a physiological regulator of renal sodium and fluid homeostasis.
ANF inhibits vasopressin-induced Ca2+ mobilization and contraction in glomerular mesangial cells
Authors: Meyer-Lehnert H. , Tsai P. , Caramelo C. , Schrier R. W.
Published: December 2017

It has been suggested that atrial natriuretic factor (ANF) may increase glomerular filtration rate (GFR) via alteration of the glomerular ultrafiltration area, which is at least in part regulated by the contractile state of mesangial cells. The present study therefore investigated the effect of ANF (atriopeptin III) on arginine vasopressin (AVP)-induced Ca2+ mobilization and contraction in cultured rat mesangial cells. AVP (10(-8) M) increased intracellular Ca2+ ([ Ca2+]i) as measured by the quin2 method (142.1 +/- 3.7 vs. 297.9 +/- 8.9 nM, P less than 0.001). This effect, however, was diminished by 10(-8) M ANF (297.9 +/- 8.9 vs. 233.1 +/- 9.3 nM, P less than 0.0025). ANF alone did not affect basal [Ca2+]i. AVP-stimulated 45Ca2+ efflux (cpm.mg prot-1.30 s-1) from mesangial cells within 30 s (1,432 +/- 70 basal vs. 2,751 +/- 287 AVP at 30 s, P less than 0.001). This effect of AVP on 45Ca2+ efflux was also inhibited by ANF (1,403 +/- 62 basal vs. 1,584 +/- 87 AVP and ANF, NS); ANF alone had no effect on spontaneous 45Ca2+ efflux. Furthermore, ANF inhibited AVP-induced 45Ca2+ uptake, which is purportedly one of the major determinants of the sustained phase of cell contraction. Cell contraction was assessed by digital imaging analysis. AVP (10(-7) M) caused contraction of 33.8 +/- 2.0% of the cells, but the contractile response decreased to 20.7 +/- 2.3% in the presence of 10(-9) M ANF and was virtually abolished in the presence of 10(-8) M ANF. Guanosine 3',5'-cyclic monophosphate (cGMP) has been proposed as the second messenger for ANF. ANF (10(-7) M) stimulated cGMP production significantly (0.54 +/- 0.12 vs. 22.30 +/- 5.91 pmol.mg prot-1.10 min-1, P less than 0.01), but it had no effect on cGMP at concentrations of 10(-8) M and below. 8-Bromo-cGMP (8-BrcGMP) attenuated the stimulatory effect of AVP on [Ca2+]i, 45Ca2+ efflux, and 45Ca2+ uptake. AVP-induced cell contraction was also decreased in the presence of 2 mM 8-BrcGMP (33.8 +/- 2.0% vs. 18.8 +/- 2.6%, P less than 0.005). The present results suggest that ANF may increase GFR in part by blocking the cellular action of AVP and possibly other vasoconstrictors on glomerular mesangial cells, thus affecting glomerular filtration area. A role of cGMP as second messenger for ANF is possible, but it appears unlikely to be the only mediator for the effects of ANF.
Inhibition of rat glomerular visceral epithelial cell growth by heparin
Authors: Adler S.
Published: December 2017

The effect of several glycosaminoglycans and sulfated polysaccharides on the growth of cultured rat glomerular visceral epithelial cells (GEC) was studied in vitro. Heparin, one preparation of heparan sulfate proteoglycan, dextran sulfate, and pentosan polysulfate significantly inhibited the growth of several GEC clones studied (36.0-77.1% inhibition at 100 micrograms/ml). Other glycosaminoglycans studied did not affect GEC growth. Growth inhibition by heparin was dose related and did not appear to reflect cytotoxicity. Heparins with high or low affinity for antithrombin inhibited growth to similar degrees. When heparin was fractionated into high- and low-anticoagulant activity fractions by physicochemical means the high activity fraction displayed significantly greater growth inhibition. The degree of growth inhibition significantly correlated with serum concentration in the media (r = 0.64; P less than 0.001). Removal of heparin binding factors from serum resulted in a loss of this correlation as well as less overall growth inhibition. These experiments suggest that interactions of GEC with heparan sulfates and other heparin-like molecules in the extracellular matrix may be important in the control of GEC growth.
Effect of vasopressin on intracellular [Ca] and Na transport in cultured toad bladder cells
Authors: Wong S. M. , Chase H. S.
Published: December 2017

Intracellular free [Ca] [( Ca]i) and transepithelial sodium transport were measured simultaneously in cultured toad bladder cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy while sodium transport was measured as the short-circuit current (Isc) with a voltage clamp. Following stimulation with vasopressin [Ca]i and Isc rose in parallel to maximal values within 10 min. [Ca]i increased from 65 +/- 5 to 123 +/- 12 nM and Isc, from 11 +/- 3 to 25 +/- 6 microA (n = 4). The vasopressin-induced rise in [Ca]i correlated significantly with the increase in Isc, suggesting that the rise in [Ca]i might be necessary for the increase in Isc. If so, then adenosine 3',5'-cyclic monophosphate (cAMP), which mimics the natriferic action of vasopressin, should also increase [Ca]i. Although cAMP increased [Ca]i to a peak value of 32 +/- 13% (P less than 0.05) above control at 10 min, the rise in Isc did not parallel the increase in [Ca]i. Isc peaked instead at 20 min, rising to 114 +/- 25% (P less than 0.05) over control, during which time [Ca]i returned to base line. This result suggested that a steady state increase in [Ca]i was not necessary for the natriferic action of cAMP. This notion was confirmed in experiments in which the vasopressin-induced increase in [Ca]i was prevented by bathing the tissue in a low-[Ca] buffer. Under these conditions, Isc increased 37 +/- 9% above control (P less than 0.05, n = 4) even though [Ca]i remained largely unchanged. Our results suggest that although vasopressin increases [Ca]i in toad bladder cells, the rise in [Ca]i does not seem to play a role in the natriferic response. These experiments also demonstrate the utility of making simultaneous measurements of ion transport and [Ca]i, which allow direct examination of calcium's role in mediating ion transport.
Effects of renal artery pressure on interstitial pressure and Na excretion during renal vasodilation
Authors: Granger J. P. , Scott J. W.
Published: December 2017

Renal vasodilation has a marked effect on the pressure-natriuresis relationship. The purpose of this study was to determine the role of renal interstitial hydrostatic pressure (RIHP) in mediating the effect of renal perfusion pressure (RPP) on urinary sodium excretion rate (UNaV) in control and vasodilated kidneys. The effects of RPP on UNaV and RIHP were determined in dogs under control conditions and during renal vasodilation with acetylcholine (Ach, 2.0 micrograms.kg-1.min-1) or secretin (SEC, 0.025 micrograms.kg-1.min-1). Decreases in RPP in control kidneys from 130 to 60 mmHg decreased UNaV from 2.9 +/- 0.1 to 0.6 +/- 0.3 microeq/min and fractional excretion of Na (FENa) from 0.15 +/- 0.08 to 0.06 +/- 0.04%. These changes were associated with significant reductions in RIHP (8.9 +/- 0.6 to 5.6 +/- 1.2 mmHg). In Ach-vasodilated kidneys, reductions in RPP from 130 to 60 mmHg decreased UNaV from 149.8 +/- 52.4 to 0.2 +/- 0.1 microeq/min and FENa from 3.42 +/- 1.18 to 0.012 +/- 0.01%. RIHP decreased from 17.8 +/- 3.4 to 8.4 +/- 1.3 mmHg, despite autoregulation of RBF. Renal vasodilation with SEC, which did not affect RIHP, had only a small effect on the relationship between RPP and UNaV. These data suggest that RIHP may be playing an important role in mediating the effect of RPP on UNaV.
Importance of anion in hypotonic volume regulation of rabbit proximal straight tubule
Authors: Welling P. A. , Linshaw M. A.
Published: December 2017

Rabbit proximal straight tubules rapidly swell to a maximum volume when abruptly immersed into hypotonic medium. However, in a second, slower phase, termed volume regulatory decrease (VRD), tubules shrink toward their basal volume due to the efflux of K, accompanying anion and water. In the present study, we investigated the nature of the anion during hypotonic volume regulation. We removed Cl and/or the HCO3 buffer to assess their relative importance in VRD, and we used furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and barium to investigate the nature of the transmembrane anion pathway in VRD. Isosmotic replacement of peritubular Cl with gluconate had no effect on either tubule volume in isotonic medium or the initial osmometric swelling response in hypotonic medium. However, the VRD in such Cl-depleted tubules was significantly inhibited. Control tubules regulated 81% below their maximal volume in dilute medium. By contrast, Cl-depleted tubules regulated only 39%. This inhibitory effect could not be attributed to the absence of peritubular Cl or to the presence of gluconate. The absence of the HCO3 buffer or the presence of SITS (0.5 mM) had no inhibitory effect on the rate or extent of VRD. Furosemide alone (1 mM) also had no inhibitory effect on VRD. However, whereas barium alone delays VRD, addition of furosemide to barium-treated tubules further slowed their maximal rate of fluid efflux and delayed VRD even more.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of synthetic tumoral PTH-related peptide on cAMP production and Na-dependent Pi transport
Authors: Pizurki L. , Rizzoli R. , Moseley J. , Martin T. J. , Caverzasio J. , Bonjour J. P.
Published: December 2017

Malignant hypercalcemia can be associated with a biochemical syndrome very similar to that encountered in primary hyperparathyroidism. The putative tumoral factor responsible for this syndrome has been isolated very recently from conditioned medium of a cultured lung squamous cell carcinoma (BEN), cDNA clones characterized, and an amino-terminal fragment synthesized. We investigated and compared the effect of this synthetic amino-terminal fragment of parathyroid hormone-related peptide [PTHrP-(1-34)], to purified PTHrP-(1-141) isolated from the same lung squamous cell carcinoma, and to bovine parathyroid hormone [bPTH-(1-34)] on adenosine 3',5'-cyclic monophosphate (cAMP) production and sodium-dependent phosphate transport (NaPiT) in opossum kidney (OK) epithelial cells. PTHrP-(1-34) and bPTH-(1-34) were equipotent in eliciting a 30-fold increase of cAMP production. NaPiT, as assessed by measuring the initial rate of Pi uptake, was inhibited in a concentration-dependent manner by either synthetic peptide. Half-maximal inhibition was observed with approximately 0.03-0.1 nmol/l of either bPTH-(1-34) or PTHrP-(1-34). At 10 nmol/l, either peptide produced an inhibition of 55 +/- 4 and 53 +/- 6%, respectively. This effect was specific for Pi, since the Na-dependent transport of glucose or alanine was not altered by either peptide. In OK cells dose-dependent stimulation of cAMP production and inhibition of NaPiT were also observed with purified native PTHrP-(1-141). In LLC-PK1 cells, which are devoid of PTH receptors, none of the peptides affected NaPiT. These results demonstrate a direct and specific effect of tumoral PTHrP on cAMP production and NaPiT in cultured renal epithelial cells in a way similar to bPTH.(ABSTRACT TRUNCATED AT 250 WORDS)
Dopamine1 receptors in rat kidneys identified with 125I-Sch 23982
Authors: Felder R. A. , Jose P. A.
Published: December 2017

Dopamine1 receptors were studied in rat kidney using the selective dopamine1 antagonist 125I-labeled Sch 23982. The specific binding of 125I-Sch 23982 (defined by 5 microM Sch 23390) to renal cortical homogenates incubated at room temperature was rapid, saturable with time and ligand concentration, and reversible. Analysis of Rosenthal plots revealed a single class of receptors with an apparent dissociation constant of 12.2 +/- 1.9 nM and maximum receptor density of 1.03 +/- 0.15 pmol/mg protein (n = 6). However, competition experiments with the dopamine1 antagonist Sch 23390 revealed a low- and high-affinity binding site with inhibition constants of 1 x 10(-6) and 1 x 10(-8) M, respectively. The competition experiments were also indicative of dopamine1 receptors with stereoselectivity noted for dopamine1 but not for dopamine2 antagonists. The inhibition constants for dopamine1 antagonists and agonists were two orders of magnitude greater in renal cortical than striatal homogenates. Different buffers affected striatal but not renal cortical binding. Autoradiographic studies revealed 125I-Sch 23982 binding in renal cortical but not medullary tissue. These studies confirm the presence of dopamine1 receptors in the cortex of the rat kidney.
Diluting power of thick limbs of Henle. I. Peritubular hypertonicity blocks basolateral Cl- channels
Authors: Molony D. A. , Andreoli T. E.
Published: December 2017

This paper provides the results of experiments intended to assess the mechanism responsible for the suppression of net salt absorption and the attendant spontaneous voltage (Ve, mV) that occurs when isolated thick ascending limbs of Henle (TAL) are exposed to a hypertonic environment. In isolated mouse medullary (MTAL) and cortical (CTAL) segments, as well as in rabbit MTAL segments, increases in peritubular osmolality with urea produced a graded suppression of Ve. This effect was evaluated in further detail in isolated mouse MTAL segments, where 600 mM peritubular urea produced a reversible reduction in Ve and a reversible reduction in the transcellular electrical conductance (Gc; mS.cm-2). There was no detectable effect on the paracellular conductance (Gs; mS.cm-2). Simultaneously, 600 mM peritubular urea also produced hyperpolarization of the voltage across basolateral membranes (mV). Moreover, 600 mM peritubular urea produced virtually the same magnitude reduction in Gc either in the absence or presence of 10(-4) M luminal furosemide. Thus we conclude that peritubular urea hypertonicity directly suppresses the Cl- conductance of basolateral membranes (mS.cm-2).
Renal adaptation to metabolic acidosis in senescent rats
Authors: Prasad R. , Kinsella J. L. , Sacktor B.
Published: December 2017

In this study, we compared results obtained in senescent rats with young rats given an equivalent acid load. We examined the renal changes by giving equivalent acid loads for 48 h to both 6- and 24-mo-old rats. The basal excretion of ammonium was the same in both groups, whereas titratable acids, phosphate, and Ca2+ excretions were increased in the senescent animal. After administration of the acid load, ammonium, phosphate, Ca2+, and titratable acid excretions increased in both age groups, but there were greater absolute increases in ammonium and titratable acid excretions in the young rats. The total acid excreted by the 24-mo rats was reduced 50 (day 1) and 25% (day 2) compared with the young rats, which was reflected by the more severe acidosis in those animals. The portion of total acid excreted as titratable acids in senescent animals was also increased during acidosis when compared with the young animals. In isolated proximal tubule brush-border membrane vesicles, acidosis increased Na+-H+ exchange and decreased Na+-dependent phosphate transport in both age groups. We also found that the basal activity of the Na+-H+ exchanger was not changed with age but the Na+-dependent phosphate transporter was less in the 24-mo rat. The results suggest that physiological regulation of these renal processes remains intact in the aged rat but the responses may be reduced or delayed in the senescent animal.
Synthesis and release of insulinlike growth factor I by mesangial cells in culture
Authors: Conti F. G. , Striker L. J. , Elliot S. J. , Andreani D. , Striker G. E.
Published: December 2017

Mesangial cell proliferation is a common hallmark of many glomerular diseases. The exact mechanisms inducing cell proliferation in glomerulosclerosis are not completely understood, and it remains to be determined whether growth factors play a role in this process. Insulinlike growth factor I (IGF I) has been shown to be synthesized in the kidney, and glomerular mesangial cells have receptors for and exhibit mitogenic response to IGF I. We found that mouse glomerular mesangial cells in culture synthesized and released into the culture medium a molecule with immunological and biological features of IGF I. This molecule specifically bound to mesangial cell IGF I receptors; high-pressure liquid chromatographic analysis provided further evidence of its similarity to human recombinant IGF I. Mesangial cells released into the culture medium 6 ng/10(6) cells of IGF I-like material per 24 h in a time-dependent and actinomycin-D inhibitable fashion. These data suggest that IGF I might be locally released by mesangial cells in the glomerulus and act in an autocrine and paracrine fashion.
The everted renal tubule: a methodology for direct assessment of apical membrane function
Authors: Engbretson B. G. , Beyenbach K. W. , Stoner L. C.
Published: December 2017

A technique is described whereby it is possible to evert a 0.5- to 1.0-mm segment of an amphibian renal tubule and perfuse it in vitro. Consequently, the apical membranes of an intact nephron fragment are directly accessible for electrophysiological study. Viability of the cells of everted diluting segments taken from Ambystoma kidney was indicated by 1) failure of the cells to take up trypan blue and 2) the existence of an apical membrane voltage (average 66 mV, cell negative), which decreased predictably in the presence of either 5 mM barium or elevated potassium in the luminal bathing solution. The utility of the everted tubule to patch clamp studies was tested. A large conductance channel that appeared to be selective for potassium could be demonstrated in a cell-attached patch of the apical membrane of an everted initial collecting tubule. The everted tubule preparation not only provides large quantities of apical membrane for patch clamp studies but, more importantly, allows the investigator to control the solutions bathing each membrane surface independently. The application of patch clamp techniques to perfused, everted tubules may then serve to more completely describe the role of the apical membrane in transcellular ion transport.
Ammoniagenesis by cultured human renal cortical epithelial cells: study with 15N
Authors: Nissim I. , States B.
Published: December 2017

The metabolic fate of 15N-labeled glutamine and glutamate in cultured human renal cortical epithelial cells was investigated. The main goal was to elucidate the major pathways of ammoniagenesis depending on varying H+ concentration. Incubations at pH 7.4 or 6.8 were conducted with either 1 mM [5-15N]glutamine, [2-15N]glutamine, [15N]glutamate, or L-[2-15N]-gamma-glutamylmethylamide. The results demonstrate that acute acidosis had little effect on total ammonia generation from glutamine. However, 15NH3 formation from [5-15N]glutamine was significantly higher at pH 7.4 compared with pH 6.8. Conversely, at pH 6.8, 15NH3 production from either [2-15N]-glutamine or [15N]glutamate was twofold higher than at pH 7.4. Thus the observations indicate that acute acidosis had little effect on net ammonia production from glutamine due to decreased flux through glutaminase and concomitant increased flux through glutamate dehydrogenase. When L-[2-15N]-gamma-glutamylmethylamide was utilized as the sole substrate, significantly higher amounts of 15NH3 and 15N-labeled amino acids were formed at pH 6.8 compared with pH 7.4. Addition of either 1 mM pyruvate or alpha-ketoglutarate significantly decreased 15NH3 and increased 15N-amino acid formation from either [2-15N]glutamine or [2-15N]-gamma-glutamylmethylamide. The metabolism of either substrate via transamination reaction was significantly stimulated at acidic pH, presumably due to a depleted pool of alpha-ketoglutarate during the course of the incubations. The data indicate that in addition to glutaminase I and glutamate dehydrogenase, the glutamine aminotransferase (glutaminase II) pathway exists in cultured human renal cells. The data suggest that glutamate dehydrogenase flux and/or the alpha-ketoglutarate dehydrogenase reaction may have an important regulatory role in ammoniagenesis from glutamine and/or glutamate in human kidney during acute acidosis.
Regulation of Na+-K+-ATPase activity in kidney proximal tubules: involvement of GTP binding proteins
Authors: Bertorello A. , Aperia A.
Published: December 2017

This study evaluates the involvement of GTP-dependent regulatory proteins (G-proteins) in the regulation of Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments. Single PCT segments were dissected from rat kidney and permeabilized to allow nucleotides and medium free access to the interior of the cell. A GDP analogue that blocks GTP-dependent activation of the G-protein, GDP beta S (400 microM) significantly inhibited PCT Na+-K+-ATPase activity when Na in the medium (Nam) was greater than or equal to 70 mM. The inhibition was attenuated when Nam was 55 and 35 mM and was no longer significant when Nam was 25 mM. GDP beta S had no inhibitory effect on the activity of purified Na+-K+-ATPase. A nonhydrolyzable GTP analogue, GppNHp (50 microM) significantly increased Na+-K+-ATPase activity when Nam was 25 and 35 mM, but not when Nam was 55-140 mM. Dopamine (DA) and DA1 plus DA2 agonists significantly inhibit Na+-K+-ATPase activity. DA inhibition was competitively abolished by GppNHp. In PCT segments from rats pretreated with pertussis toxin, DA and DA1 plus DA2 agonist inhibition of Na+-K+-ATPase activity was abolished. In PCT segments from rats pretreated with cholera toxin, basal Na+-K+-ATPase activity was increased, but DA significantly inhibited Na+-K+-ATPase activity. Na+-K+-ATPase activity in PCT segments is regulated via a G-protein that stimulates Na+-K+-ATPase activity and a DA-activated pertussis toxin-sensitive G-protein that inhibits Na+-K+-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Electrophysiology of collecting duct H+ secretion: effect of inhibitors
Authors: Koeppen B. M.
Published: December 2017

Segments of the outer medullary collecting duct were isolated from the inner stripe of the rabbit kidney (OMCDi), perfused in vitro, and impaled across their basolateral membranes with voltage-recording microelectrodes. The disulfonic stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-4) M) and the carbonic anhydrase inhibitor acetazolamide (10(-4) M) depolarized the lumen-positive transepithelial voltage (VT) toward 0 mV when added to the bath solution. Concurrently, the basolateral membrane voltage (Vbl) hyperpolarized. The hyperpolarization of Vbl, which averaged 19.3 +/- 2.9 mV (n = 11) for SITS and 22.7 +/- 3.5 mV (n = 11) for acetazolamide, was not due to an alteration in the ionic selectivity of the basolateral membrane, which was highly Cl- selective. The hyperpolarization of Vbl could best be explained by a decrease in the intracellular [Cl-], and the associated shift in the emf for Cl- (ECl) across the basolateral membrane. The decrease in intracellular [Cl-] could be attributed to inhibition of a Cl-HCO3 antiporter in the basolateral membrane. SITS appeared to inhibit this antiporter directly, whereas the effect of acetazolamide was indirect, probably secondary to a decrease in the intracellular [HCO3-]. Finally, both SITS and acetazolamide induced or unmasked an electroneutral K+-coupled transport system in the basolateral membrane.
In vivo indicator dilution kinetics of PAH transport in dog kidney
Authors: Silverman M. , Whiteside C. , Lumsden C. J. , Steinhart H.
Published: December 2017

In vivo multiple indicator-dilution (MID) data were analyzed using a computer-assisted mathematical model of transepithelial cell transport to determine p-aminohippuric acid (PAH) transport kinetics across the proximal tubular antiluminal (ALM) and luminal (LM) membranes. A bolus of 125I-labeled albumin (plasma reference), [14C]creatinine (interstitial reference), and tracer [3H]PAH was injected into the left renal artery of anesthetized mongrel dogs (n = 21), and immediate serial sampling of the left renal venous and left and right urine outputs was performed (control). MID runs were then repeated in the same dog following intravenous infusion of unlabeled PAH. For all plasma PAH concentrations ([PAH]P), the steady-state unidirectional flux coefficients were calculated at the ALM and LM. The computer-derived unidirectional flux coefficients were in keeping with active ALM transport and passive, carrier-mediated LM transport. The Km calculated for ALM uptake (interstitium to cell) was 0.51 mM. PAH transport was completely inhibited by probenecid. As [PAH]P increased, the renal vein mean transit time ratio t[3H]PAH/t[14C]creatinine was greater than 1.0, indicating backflux from cells into the interstitium, then declined toward unity, as ALM and LM transport became saturated. This study, which used PAH as a model substrate demonstrated the feasibility of utilizing computer-assisted mathematical models to quantitate the kinetics of transepithelial transport from in vivo experimentation.
Reversible downregulation of thiazide diuretic receptors by acute renal ischemia
Authors: Beaumont K. , Vaughn D. A. , Maciejewski A. R. , Fanestil D. D.
Published: December 2017

Receptors for thiazide diuretic drugs in the rat renal cortex have recently been identified through the binding of [3H]metolazone, a potent diuretic with a thiazide-like mechanism of action. The present studies describe the rapid and reversible alterations that occur in thiazide receptors following acute renal ischemia in the rat. The apparent density of thiazide receptors in kidney membranes as measured by the binding of [3H]metolazone was reduced by 90% following 10 min of renal ischemia produced by clamping the renal pedicle. With release of the clamp and subsequent reperfusion for 10 min, thiazide receptor density returned to within 40% of control levels. Ischemia did not alter apparent affinity of receptors for [3H]-metolazone. Sections prepared from renal cortex and incubated in oxygenated media in vitro displayed similar rapid changes in thiazide receptors. Hypoxia of 10- to 30-min duration produced by incubating sections in vitro in nitrogen-saturated media caused a significant decrease in [3H]metolazone binding that was reversible with return to oxygenated media. Similar decreases were obtained in oxygenated sections that were incubated with mitochondrial inhibitors, dinitrophenol and rotenone, but not in sections incubated with ouabain. These results indicate that renal thiazide receptors undergo a rapid and reversible form of regulation and that controlling mechanisms are dependent on metabolic energy.
Depolarization-induced alkalinization in proximal tubules. II. Effects of lactate and SITS
Authors: Siebens A. W. , Boron W. F.
Published: December 2017

Intracellular pH and voltage microelectrodes were used to further characterize the depolarization-induced alkalinization (DIA) observed in isolated perfused proximal tubules of the tiger salamander Ambystoma tigrinum. Tubules were depolarized by raising basolateral [K+] from 2.5 to 50 mM. The solutions were air equilibrated and nominally HCO3- free (estimated [HCO3-] = 0.2 mM). In the preceding study we showed that the DIA is partially blocked by removal of Na+ from only the lumen or only the bath, but completely blocked by bilateral Na+ removal. In the present study we found that bilateral amiloride (1 mM) had no effect on the DIA, suggesting that Na-H exchange is not involved. In contrast, basolateral 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (0.5 mM) partially blocked the DIA, presumably due to inhibition of one or more of three SITS-sensitive basolateral transporters present in amphibian proximal tubules: electrogenic Na-HCO3 cotransport, Na-dependent Cl-HCO3 exchange, and H-lactate cotransport. Bilateral removal of all organic substrates, or of only lactate (Lac-) also blocked the DIA partially. As shown elsewhere (A. W. Siebens, and W. F. Boron, J. Gen. Physiol. 90: 799-831, 1987), in the absence of depolarization, luminal Lac- causes a pHi increase due to luminal Lac- entry via a Na-Lac cotransporter, followed by basolateral Lac- exit via an H-Lac cotransporter sensitive to alpha-cyano-4-hydroxycinnamate (CHC). Three lines of evidence indicate that this Na-Lac/H-Lac mechanism is involved in the DIA. 1) As noted previously, the DIA is partially blocked by luminal Na+ removal. 2) With the DIA partially blocked by basolateral SITS, removal of Lac- from only the lumen blocks the remainder of the DIA. 3) Basolateral CHC partially blocks the DIA. Our data suggest that the DIA is mediated by at least two additive mechanisms, a basolateral transporter that is SITS sensitive and Na+ dependent, and the Na-Lac/H-Lac transport system.