bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence against oxidant injury as a critical mediator of postischemic acute renal failure
Authors: Gamelin L. M. , Zager R. A.
Published: December 2017

The purpose of this study is to confirm previous evidence for reactive oxygen species (ROS) as critical mediators of postischemic renal injury by documenting lipid peroxidation after ischemic-hypoxic insults and by demonstrating that antioxidants confer protection. Renal malondialdehyde (MDA) concentrations, an index of lipid peroxidation, were measured using uncorrected and tissue-chromagen-corrected methods in 1) cortical (C), outer medullary stripe (OMS), inner medullary (IM) whole renal tissues, and C and OMS mitochondria obtained 15 min after in vivo renal artery occlusion (RAO; x 45 min); 2) C, OMS, and IM whole tissues obtained 15 min after completing 45 min of ischemia in an isolated perfused kidney; and 3) isolated proximal tubular cell (PTC) suspensions after 45 min of hypoxia with 15 min of reoxygenation. Despite significant oxygen deprivation-induced injury in each of these systems, no significant rise in MDA concentrations could be documented, with the sole exception of the in vivo IM region (by uncorrected MDA assay only). The latter rise could be attributed to medullary vascular congestion causing a hemoglobin-induced artifact in the MDA assay. Sixty-minute in vivo RAO plus reflow also did not raise MDA. To validate the MDA assay 4.2 mM H2O2 was added to PTC. An abrupt fourfold rise in MDA resulted. Pretreatment of 30- and 45-min RAO rats with two antioxidants (allopurinol or superoxide dismutase) failed to confer functional or morphological protection. We conclude that ROS may not be critical consistent mediators of in vivo postischemic acute renal failure.
Effect of systemic pH on pHi and lactic acid generation in exhaustive forearm exercise
Authors: Hood V. L. , Schubert C. , Keller U. , Muller S.
Published: December 2017

To investigate whether changes in systemic pH affect intracellular pH (pHi), energy-rich phosphates, and lactic acid generation in muscle, eight normal volunteers performed exhaustive forearm exercise with arterial blood flow occluded for 2 min on three occasions. Subjects ingested 4 mmol/kg NH4Cl (acidosis; A) or NaHCO3 (alkalosis; B) or nothing (control; C) 3 h before the exercise. Muscle pHi and phosphocreatine (PCr) content were measured with 31P-nuclear magnetic resonance (31P-NMR) spectroscopy during exercise and recovery. Lactate output during 0.5–7 min of recovery was calculated as deep venous-arterial concentration differences times forearm blood flow. Before exercise, blood pH and bicarbonate were lower in acidosis (7.303 +/- 0.009, 18.6 +/- 0.5 meq/l) than alkalosis (7.457 +/- 0.010, 32.2 +/- 0.7 meq/l) and intermediate in control (7.389 +/- 0.007, 25.3 +/- 0.6 meq/l). Lactic acid output during recovery was less with A (245 +/- 39 mumol/100 ml) than B (340 +/- 55 mumol/100 ml) (P less than 0.05) and intermediate in C (293 +/- 31 mumol/100 ml). PCr utilization and resynthesis were not affected by extracellular pH changes. pHi did not differ before exercise (A, 7.04 +/- 0.01; B, 7.09 +/- 0.01; C, 7.06 +/- 0.01) or at its end (A, 6.28 +/- 0.07; B, 6.28 +/- 0.11; C, 6.31 +/- 0.09). Hence systemic acidosis inhibited and alkalosis stimulated lactic acid output. These findings suggest that systemic pH regulates cellular acid production, protecting muscle pH, at the expense of energy availability.
Angiotensin II modulates the intrarenal effects of atrial natriuretic peptide
Authors: Siragy H. M. , Lamb N. E. , Rose C. E. , Peach M. J. , Carey R. M.
Published: December 2017

The mechanism by which atrial natriuretic peptide (ANP) increases renal water and solute excretion is not fully understood. We studied the renal effects of ANP and angiotensin II (ANG II) separately and together in uninephrectomized conscious dogs (n = 7) in sodium metabolic balance (80 meq/day). Exogenous ANG II and ANP were without measurable systemic effects as demonstrated by absence of changes in blood pressure, plasma aldosterone concentration, and plasma renin activity. The quantity of ANG II that had significant renal effects that were without measurable systemic effects was 0.2 pmol.kg-1.min-1. Three infusion rates of ANP had significant renal effects (1, 10, and 20 pmol.kg-1.min-1). These quantities of ANP caused significant diuresis, natriuresis, kaliuresis, and increased glomerular filtration rate without significant changes in renal plasma flow. ANG II alone caused significant antidiuresis, antinatriuresis, and decreased glomerular filtration rate and renal plasma flow. When ANG II and ANP were given together, no change in urinary flow rate, urinary sodium or potassium excretion, or renal plasma flow was observed, whereas glomerular filtration rate increased. Filtration fraction increased significantly with ANG II and ANP separately and together. Intrarenal ANP prevents the ANG II-induced decrement in urinary sodium excretion and urine flow rate. ANP may play an important role in escape from the sodium-retaining action of intrarenal ANG II.
Na+ pump in renal tubular cells is regulated by endogenous Na+-K+-ATPase inhibitor from hypothalamus
Authors: Cantiello H. F. , Chen E. , Ray S. , Haupert G. T.
Published: December 2017

Bovine hypothalamus contains a high affinity, specific, reversible inhibitor of mammalian Na+-K+-ATPase. Kinetic analysis using isolated membrane fractions showed binding and dissociation rates of the hypothalamic factor (HF) to be (like ouabain) relatively long (off rate = 60 min). To determine whether the kinetics of inhibition in intact cells might be more consistent with regulation of physiological processes in vivo, binding and dissociation reactions of HF in intact renal epithelial cells (LLC-PK1) were studied using 86Rb+ uptake and [3H]ouabain binding. As with membranes, a 60-min incubation with HF inhibited Na+-K+-ATPase in LLC-PK1 cells. In contrast to membrane studies, no prolonged incubation with LLC-PK1 was needed to observe inhibition of Na+-K+-ATPase. HF caused a 33% inhibition of ouabain-sensitive 86Rb+ influx within 10 min. Incubation of cells with HF followed by washout showed rapid reversal of pump inhibition and a doubling of pump activity. The dose-response curve for HF inhibition of LLC-PK1 86Rb+ uptake showed a sigmoidal shape consistent with an allosteric binding reaction. Thus HF is a potent regulator of Na+-K+-ATPase activity in intact renal cells, with binding and dissociation reactions consistent with relevant physiological processes.
Protease effects on adenylate cyclase in potassium-depleted rabbit kidney
Authors: Raymond K. H. , Holland S. D. , Hymer T. K. , McKinney T. D. , Katz M. S.
Published: December 2017

Potassium depletion in rabbits induces a renal concentrating defect in vivo and decreased hydrosmotic response to arginine vasopressin (AVP) in isolated cortical collecting tubules (CCT) perfused in vitro. The molecular basis of the AVP resistance in potassium depletion was investigated by comparing AVP-responsive adenylate cyclase activities in CCT from potassium-depleted and control rabbits. Vasopressin-responsive enzyme activity was impaired in CCT dissected from kidneys of potassium-depleted rabbits but not when kidneys were treated with collagenase to improve microdissection conditions. Potassium depletion also depressed parathyroid hormone (PTH)-stimulated adenylate cyclase activity in proximal straight tubules (PST) dissected from untreated but not collagenase-treated kidneys. Commercially available collagenase, which also contains other proteolytic enzymes, increased AVP-sensitive adenylate cyclase activity in control CCT, and trypsin treatment of CCT dissected without collagenase abolished the decrease in AVP-sensitive activity induced by potassium depletion. Inclusion of trypsin inhibitor during collagenase treatment of kidneys lowered AVP response in CCT from potassium-depleted rabbits. These results demonstrate that potassium depletion impairs hormone-sensitive adenylate cyclase of CCT (and PST) by a protease-sensitive mechanism.
Parathyroid hormone stimulates ammoniagenesis in canine renal proximal tubular segments
Authors: Chobanian M. C. , Hammerman M. R.
Published: December 2017

To determine whether parathyroid hormone (PTH) affects ammoniagenesis in renal proximal tubule, we measured ammonia production in suspensions of canine renal proximal tubular segments incubated with 10 mM L-glutamine in the absence or presence of 10(-7) M PTH. Productions of ammonia were linear functions of time for 120 min and averaged 231 +/- 55 and 311 +/- 67 mumol ammonia/g protein in the absence and presence of PTH, respectively. When measured over the range of 10(-11)-10(-7) M PTH, half-maximal stimulation of ammonia production occurred between 10(-10) and 10(-9) M PTH. Maximal production of ammonia was observed at 10(-8) M PTH. Incubation of proximal tubular segments with PTH increased levels of adenosine 3',5'-cyclic monophosphate (cAMP) in vitro. Ammonia production was significantly enhanced by incubation of segments with the cAMP analogue, 8-bromoadenosine 3',5'-cyclic monophosphate. PTH also increased ammonia production in segments incubated with 1 mM L-glutamine. We conclude that PTH stimulates ammonia production in canine renal proximal tubular segments. This effect appears to be mediated, at least in part, through cAMP in vitro. Such stimulation could reflect a direct action of PTH on the proximal tubule to enhance ammoniagenesis in vivo.
Glutathione protects against exogenous oxidant injury to rabbit renal proximal tubules
Authors: Messana J. M. , Cieslinski D. A. , O'Connor R. P. , Humes H. D.
Published: December 2017

Glutathione, comprising a major portion of cellular nonprotein thiols, plays a central role in a diverse group of cell metabolic functions. Glutathione and related enzyme systems have been shown to protect against both toxin and oxidant-induced injury in several organ systems. The role of glutathione in protecting renal epithelia against oxidant stress has not been investigated previously. We report here the response of enriched, isolated rabbit renal proximal tubule segments to oxidant stress induced by tert-butyl hydroperoxide. In addition, the effects of glutathione depletion by various biochemical means and of exogenous glutathione supplementation on the response of tubule segments to tert-butyl hydroperoxide exposure are described. Depletion of cell glutathione by several distinct methods potentiates oxidant-induced injury. Augmentation of cellular glutathione affords significant protection against exogenous oxidant stress. The protective effect of glutathione may reside in its ability, in conjunction with glutathione peroxidase, to arrest the propagation of lipid peroxidation and, therefore, to minimize alterations in plasma membrane permeability. The results of this study do not exclude the possibility that glutathione prevents tert-butyl hydroperoxide induced oxidation of critical sulfhydryl groups of catalytic or structural proteins associated with control of cell cation homeostasis. These results confirm the important role of glutathione in protecting renal tubular epithelia against oxidant stress.
Effect of an altered glutathione content on renal ischemic injury
Authors: Scaduto R. C. , Gattone V. H. , Grotyohann L. W. , Wertz J. , Martin L. F.
Published: December 2017

Renal ischemia and reperfusion have been shown to be associated with an enhanced renal lipid peroxidation. Because glutathione (GSH) serves to protect cells from oxidative stress, the role of GSH in renal ischemia was investigated. The content of renal GSH in the rat declined to 40% of control values during 35 min of renal artery occlusion. Renal GSH levels only partially recovered after 120 min of blood reflow. To assess the significance of this effect, renal GSH levels were altered before occlusion of the renal artery. Rats were treated with either buthionine sulfoximine (BSO) or glutathione monoethylester (GSH-ester) to lower or elevate, respectively, renal GSH levels. The ischemia-induced changes in renal ATP, ADP, and AMP after 35 min of ischemia and 90 min of blood reflow were not affected by prior alteration of renal GSH levels. The ischemia-induced decrease in the respiratory control of isolated cortex mitochondria was also unaffected. In control animals, ischemia of 35 min increased urine flow rate 3.2-fold and decreased GFR to 29% of normal values during the reflow period. Similar changes occurred in kidneys with a depleted GSH level. In kidneys with an elevated GSH, however, both urine flow rate and GFR were decreased to values 50 and 3% of normal, respectively. Morphological analysis demonstrated that ischemia produced an enhanced degree of damage with an increase in cast formation in kidneys pretreated with GSH-ester; however, the ester also produced morphological changes in nonischemic kidneys. The severity of ischemic damage was similar in kidneys with a lower GSH content when compared with controls. We conclude that renal GSH is depleted by ischemia but depletion of renal GSH with BSO before ischemia has no effect on ischemic-induced damage to the kidney. However, ischemic-induced renal dysfunction is enhanced when GSH is elevated with glutathione monoethylester before ischemia.
pH in principal cells of frog skin (Rana pipiens): effects of amiloride and potential
Authors: Drewnowska K. , Biber T. U.
Published: December 2017

Intracellular pH (pHi) and apical cell membrane potential (Va) were determined in principal cells of frog skin (Rana pipiens) with double-barrel micro-electrodes. In the Northern and Southern varieties, respectively, pHi is 0.38 and 0.26 pH units below bath pH. Amiloride, applied apically, causes reversible intracellular acidification at concentrations of 10(-5) M or higher. Voltage clamp-induced hyperpolarization and depolarization of Va result in intracellular acidification and alkalinization, respectively. This response of pHi is inhibited or abolished when the apical side is treated with 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Amiloride-induced intracellular acidification is not exclusively due to the hyperpolarization of Va that accompanies amiloride treatment since 1) amiloride causes greater acidification than equivalent voltage clamp-induced hyperpolarization of Va, 2) amiloride-induced acidification persists in DIDS-treated tissues, and 3) there is no correlation between hyperpolarization of Va and intracellular acidification occurring after amiloride. We conclude that pHi is below the extracellular pH. Amiloride causes intracellular acidification that may be in part connected with hyperpolarization of Va. However, a major component of amiloride-induced acidification is due to other factors, possibly inhibition of apical Na+-H+ exchange. The inhibitory effect of apically applied DIDS suggests that the voltage dependent changes in pHi are related to movement of HCO3 (or OH) ions across the apical cell membrane.
Renal phosphate transport in humoral hypercalcemia of malignancy
Authors: Sartori L. , Insogna K. L. , Barrett P. Q.
Published: December 2017

Fisher rats bearing the H-500 Leydig cell tumor (LCT) develop humoral hypercalcemia of malignancy (HHM), which is accompanied by hypophosphatemia and hyperphosphaturia. To better define the mechanisms underlying the changes in phosphate metabolism, the activity of sodium-dependent phosphate uptake (Na+-Pi) in microvillus membrane vesicles (MMV) isolated from the renal cortex of LCT-bearing rats was studied. Ten days after tumor transplantation the animals became hypercalcemic, hypophosphatemic, and hyperphosphaturic, and LCT-MMV showed a specific decrease in Na+-Pi. A kinetic analysis revealed evidence for both a high- and a low-affinity system of Na+-Pi. The Vmax of both the low-affinity system and the high-affinity system were significantly reduced in LCT-MMV. These changes in Na+-Pi transport were similar to those induced by parathyroid hormone. In day-10 tumor-bearing animals, daily injections of dichloromethylene diphosphonate (2.5 mg.kg-1.day-1) prevented the onset of hypercalcemia but not the reduction in Na+-Pi in LCT-MMV. Our data suggest that in this animal model of HHM there is a specific and persistent impairment of Na+-Pi uptake at the level of the renal cortical brush-border membrane, which contributes to the derangement in phosphate metabolism.
Characteristics of papillary collecting duct cells in primary culture
Authors: Husted R. F. , Hayashi M. , Stokes J. B.
Published: December 2017

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.
Transport and histochemical studies of bicarbonate handling by the alligator kidney
Authors: Ventura S. C. , Northrup T. E. , Schneider G. , Cohen J. J. , Garella S.
Published: December 2017

The alligator excretes a persistently alkaline urine despite consuming an acid-residue diet. The amount of bicarbonate excreted is greater than the amount filtered, evidencing tubular secretion of bicarbonate. The parallel urinary excretion of ammonium maintains external acid balance. To investigate putative renal mechanisms responsible for the concurrent excretion of large quantities of ammonium bicarbonate, we used acridine orange fluorescence methodology in microvesicles prepared from the proximal tubule brush border to assess the activity of the Na+-H+ antiporter, and histochemical methods (cobalt sulfide precipitation) to assess carbonic anhydrase localization. We found no evidence for the presence of a functioning Na+-H+ antiporter, the protein known to be responsible for the majority of bicarbonate reabsorption in mammals; Na+-H+ exchange in vesicles from the alligator kidney failed to exhibit saturation kinetics, showed no affinity for lithium, and was not inhibited by amiloride. Sensitive histochemical techniques failed to reveal carbonic anhydrase activity anywhere in the proximal tubule but detected an abundance of enzyme activity in the basolateral membranes and nuclei of distal tubular cells. In the connecting segment and collecting duct, cells without carbonic anhydrase alternated with cells containing carbonic anhydrase; in the latter, the enzyme was localized to the basolateral and luminal membranes, the nucleus and, to a lesser extent, throughout the cytoplasm. We conclude that the proximal tubule of the alligator kidney is devoid of the machinery necessary for the transport of large amounts of bicarbonate. The principal site at which bicarbonate is added to the final urine appears to be the distal tubule, at which site carbonic anhydrase is widespread.
K+ channel currents in basolateral membrane of distal convoluted tubule of rabbit kidney
Authors: Taniguchi J. , Yoshitomi K. , Imai M.
Published: December 2017

To examine the nature of ion-conductive pathways in the basolateral membrane of rabbit distal convoluted tubule (DCT), we recorded single-channel currents from the tubule segment isolated from collagenase-treated kidney. Using cell-attached patch pipettes filled with 130 mM KCl, 5.4 mM CaCl2, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), we observed K+ channels in the basolateral membrane of DCT, having two different single-channel conductances of 48.7 +/- 1.4 (n = 9) and 60.6 +/- 1.4 pS (n = 7). Both types of channels were completely blocked by 0.1 mM BaCl2. Both channels have same open probability of approximately 0.5 at the intrinsic basolateral membrane voltage and were recorded with similar incidence. Mean open and closed times were 31.5 +/- 5.2 and 41.3 +/- 16.0 ms for the smaller channel, and 31.5 +/- 5.1 and 36.7 +/- 8.7 ms for the larger channel, respectively. These kinetic properties did not show any clear voltage dependence in both channels. Because of apparent similarity of channel kinetics, it is possible that both activities might represent different states of the same channel. For definite conclusion, however, further investigations are necessary. In three recordings from 54 successful patches, we observed a flickering channel with rapid kinetics, which was insensitive to 1 meq/l Ba2+. The conductance of this channel was 76.6 pS (n = 2). The extrapolated zero current voltage was 76.0 mV (n = 2), indicating that this channel is permeable to K+. From these results, we suggest that K+ channels constitute conductive pathways for K+ in the basolateral membrane of rabbit DCT.
Elevated Ca2+ does not inhibit the 1,25(OH)2D3 response to phosphorus restriction
Authors: Bushinsky D. A. , Nalbantian-Brandt C. , Favus M. J.
Published: December 2017

Phosphorus restriction and parathyroid hormone (PTH) are the most potent stimuli for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] biosynthesis. Elevated arterial blood ionized calcium (Ca2+) inhibits the PTH-stimulated pathway for 1,25(OH)2D3 production. To determine whether the increase in Ca2+ due to chronic metabolic acidosis (CMA) would prevent stimulation of 1,25(OH)2D3 by dietary phosphorus restriction, rats were fed either a normal (NPD, 0.65%)- or low (LPD, 0.1%-phosphorus diet for 10 days. Ammonium chloride (NH4Cl) was added (1.5%) to the drinking water of some rats (CMA) while others served as nonacidemic controls. LPD increased serum 1,25(OH)2D3 levels in the absence of CMA and CMA did not affect the increase of 1,25(OH)2D3. LPD decreased serum phosphorus and increased Ca2+. Serum 1,25(OH)2D3 levels were correlated inversely with serum phosphorus and directly with Ca2+. Using stepwise linear regression the correlation between phosphorus and 1,25(OH)2D3 accounted for the majority of the variance contributed by both phosphorus and Ca2+ (F = 12.30, P less than 0.001). Increased Ca2+ induced by CMA does not inhibit the rise in serum 1,25(OH)2D3 during LPD, indicating that during phosphorus restriction Ca2+ does not inhibit production of 1,25(OH)2D3. Thus the mechanism whereby PTH and phosphorus restriction stimulate 1,25(OH)2D3 production differ; increased Ca2+ blocks the PTH-mediated rise in 1,25(OH)2D3 but does not alter the 1,25(OH)2D3 response to phosphorus restriction.
Amiloride transport in rabbit renal brush-border membrane vesicles
Authors: Wright S. H. , Wunz T. M.
Published: December 2017

Rabbit renal brush-border membrane vesicles (BBMV) were used to study amiloride transport across the luminal membrane of proximal tubular cells. An outwardly directed H+ gradient (pHi 6.0; pHo 7.5) stimulated 8 microM [14C]-amiloride uptake into BBMV and supported a transient "active" accumulation of substrate consistent with the presence of an amiloride-H+ exchange process. Uptake was inhibited, in the presence or absence of a pH gradient, by 1 mM unlabeled amiloride or 20 mM tetraethylammonium (TEA). Amiloride transport was not directly affected by the presence of 100 mM Na+ in the extravesicular medium, suggesting that Na-H exchange did not mediate amiloride flux. Amiloride transport was a saturable process with a maximal flux (under pH gradient conditions) of 3 nmol.mg-1.min-1 and an apparent Kt of 8 microM. TEA acted as a competitive inhibitor of this process with an apparent Ki of approximately 80 microM, similar to the Kt of TEA transport via the TEA-H+ exchanger. Likewise, amiloride acted as a competitive inhibitor of TEA uptake with an apparent Ki of approximately 11 microM. Preloading BBMV with 1-2 mM TEA stimulated the rate of amiloride uptake and supported a transient active accumulation of amiloride. We conclude that amiloride and TEA are transported by a common pathway in BBMV, which involves a carrier-mediated exchange with H+ and which may play a role in the tubular secretion of these compounds.
Renal actions of neuropeptide Y in the primate
Authors: Echtenkamp S. F. , Dandridge P. F.
Published: December 2017

Neuropeptide Y (NPY) is a potent vasoconstrictor peptide contained in sympathetic nerve terminals and is co-released with norepinephrine. Previous studies in the rat have suggested that NPY influences renal sodium reabsorption and renin release. However, little is known about the physiological effects of NPY on the kidney in the human. In the present study NPY was infused intravenously and directly into the renal artery of the primate Macaca fascicularis, an experimental model of the human. Intravenous NPY infusion at doses of 20-1,000 ng.kg-1.min-1 produced dose-dependent rises in renal vascular resistance with minimal changes in arterial pressure. Urine flow and sodium excretion were changed significantly only at doses of NPY that significantly reduced renal blood flow and filtration rate. Arterial plasma renin activity and renin secretion rate were not significantly altered at any dose of NPY. Intrarenal infusion of NPY at doses of 20-400 ng.kg-1.min-1 produced potent dose-dependent renal vasoconstriction with minimal changes in arterial pressure. Under these conditions sodium excretion was significantly reduced concurrent with decreases in renal blood flow and glomerular filtration rate. However, no significant changes in arterial plasma renin activity or renin secretion rate were found at any dose of NPY. These data indicate that in the nonhuman primate NPY is a potent renal vasoconstrictor agent that has variable effects on renal excretory and secretory function, which may be secondary to its vasoconstrictor actions.
Chronic indomethacin increases rabbit cortical collecting tubule Na+-K+-ATPase activity
Authors: Cordova H. R. , Kokko J. P. , Marver D.
Published: December 2017

To help determine whether prostaglandin-mediated inhibition of transport in the isolated rabbit cortical collecting tubule (CCT) is related to a suppression of sodium pump activity, adrenalectomized rabbits on replacement dexamethasone were treated for 3 days with vehicle or the prostaglandin inhibitor, indomethacin. On the day of death, rabbits treated with indomethacin had a significantly reduced urinary prostaglandin E2 (PGE2) excretion rate compared with vehicle-treated animals (107 +/- 7 vs. 415 +/- 129 ng.kg body wt-1.day-1, indomethacin vs. vehicle, P less than 0.05). In addition, CCTs obtained from indomethacin-treated rabbits had a significantly higher Na+-K+-ATPase activity than controls (2.66 +/- 0.33 vs. 1.17 +/- 0.33 mol Pi.kg dry wt-1.h-1, 37 degrees C, P less than 0.005); Mg-ATPase activity was invariant. Despite the elevated CCT Na+-K+-ATPase activity, there was no evidence of a sustained increase in Na reabsorption and/or K excretion by the kidney. Thus at death, the fractional excretions of Na and K, urinary Na/K ratios and plasma K values in rabbits given indomethacin were not significantly different from control values. There was also no evidence for a direct effect of either indomethacin or PGE2 on Na+-K+-ATPase activity when added directly to CCT broken-cell assays. Therefore in vivo chronic indomethacin administration leads to a rise in CCT Na+-K+-ATPase activity, which is coincident with a fall in urinary PGE2, suggesting that endogenous PGE2 may suppress the activity of this enzyme by a cell-mediated process.
Volume regulation determinants in isolated proximal tubules in hypertonic medium
Authors: Lohr J. W. , Sullivan L. P. , Cragoe E. J. , Grantham J. J.
Published: December 2017

Isolated nonperfused proximal tubules maintained their cell volume at a constant level [isovolumetric regulation (IVR)] when extracellular osmolality was gradually increased above 295 mosmol at 1.5 mosmol/min. Cell volume remained constant between 295 and 361 +/- 7 mosmol in medium containing HCO3- -CO2 as the major buffer. In N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid-buffered medium, IVR maintained cell size constant between 295 and 370 +/- 11 mosmol; thus, the volume regulatory process did not require the presence of external HCO3- -CO2. Omission of medium phosphate and sulfate from defined bathing medium had no effect on the volume regulatory process, whereas acetate (5 mM) was required to maintain cell volume constant. Hypertonic IVR was inhibited by acetazolamide (10(-6) M), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (2.5 x 10(-5) M), and, in medium in which choline replaced all but 25 mM of Na+, amiloride (1 mM). These results suggest the involvement of carbonic anhydrase, Cl-, and HCO3- transport, and Na-H exchange in IVR; and provide indirect support for the view that IVR in hyperosmotic medium depends on the intracellular accumulation of electrolytes resulting from altered rates of membrane ionic exchange mechanisms.
Independent desensitization of rat renal thick ascending limbs and collecting ducts to ADH
Authors: Dublineau I. , Elalouf J. M. , Pradelles P. , de Rouffignac C.
Published: December 2017

Recent micropuncture studies have demonstrated that administration of high doses of 1-deamino-8-D-arginine vasopressin (dDAVP), a synthetic analogue of vasopressin (AVP), causes desensitization of the thick ascending limb to AVP but may leave unaltered the effect of this hormone on the permeability to water of the collecting duct. In the present experiments, desensitization to AVP was studied by measuring adenosine 3',5'-cyclic monophosphate (cAMP) synthesis in microdissected cortical thick ascending limbs (CTAL) and cortical collecting ducts (CCD) incubated in vitro. Desensitization was induced by intramuscular injections of dDAVP (2 micrograms/day for 3 days). In a first series of experiments, performed on Brattleboro rats lacking circulating AVP, the effects of AVP on cAMP accumulation were reduced by 30% in CTAL of the rats given dDAVP, whereas in CCD no reduction was noted. Desensitization of CTAL was selective for AVP (i.e., homologous), the effects of glucagon being unaltered. In a second series of experiments, performed on Sprague-Dawley rats, a marked (up to 75% 2 h after dDAVP injection), homologous and reversible desensitization of CTAL to AVP was observed. However, here again no desensitization was obtained in CCD, indicating that in the normal rat, administration of 2 micrograms dDAVP also elicited preferential desensitization of CTAL.
Postischemic renal injury is mediated by neutrophils and leukotrienes
Authors: Klausner J. M. , Paterson I. S. , Goldman G. , Kobzik L. , Rodzen C. , Lawrence R. , Valeri C. R. , Shepro D. , Hechtman H. B.
Published: December 2017

Neutrophils have been implicated as central mediators in myocardial and skeletal muscle ischemia-reperfusion injury. This study tests whether these cellular elements and the chemoattractant leukotriene (LTB4) play a role in postischemic renal failure. Anesthetized rats underwent 45 min of left renal pedicle clamping. Five minutes after reperfusion, LTB4 levels were elevated to 1.42 ng/ml (P less than 0.05); thromboxane (Tx)B2 was 2,840 pg/ml, higher than 503 pg/ml in sham controls (P less than 0.05); renal artery blood flow was 67% of preclamping values at 1 min of reperfusion compared with 111% in sham (P less than 0.05). At 24 h, creatinine levels were 4.6 mg/dl (P less than 0.05). At 24 h, creatinine levels were 4.6 mg/dl (P less than 0.05); histology showed acute tubular necrosis (ATN). Neutrophil depletion by rabbit antiserum (n = 8) led during reperfusion to reduced LTB4 and TxB2 levels, 1.04 ng/ml and 1.043 pg/ml (P less than 0.05); increased renal blood flow of 174% (P less than 0.05); reduced creatinine levels of 1.8 mg/dl (P less than 0.05); and limited ATN. Pretreatment with diethycarbamazine prevented the increases in LTB4 and TxB2 (P less than 0.05), increased renal blood flow (P less than 0.05), minimized creatinine increase to 1.7 mg/dl (P less than 0.05), and reduced ATN. These data indicate that neutrophils and LTB4 play a role in ischemia-induced Tx synthesis and mediate postischemic renal injury.
Fish oil diet decreases prostacyclin and increases resistance in isolated rat kidneys
Authors: Kasiske B. L. , O'Donnell M. P. , Keane W. F.
Published: December 2017

The mechanisms whereby diet-induced alterations in fatty acids may affect renal structure and function are unknown. Kidneys from rats fed chow supplemented with 18% (wt/wt) coconut oil (CO, n = 8), sunflower seed oil (SO, n = 7), or fish oil (FO, n = 8) were isolated from systemic influences of the diets and perfused with a cell-free medium. The FO diet caused a twofold reduction in prostaglandin (PG) I2 (urine excretion of 6-keto-PGF1 alpha) compared with SO and CO. Urine PGE2 and thromboxane (Tx) B2 were similar in the three diet groups. There was a 22% reduction in perfusate flow associated with the decrease in PGI2 in the FO group (42 +/- 6 ml.min-1.g-1) compared with the CO (54 +/- 7 ml.min-1.g-1) and SO (54 +/- 8 ml.min-1.g-1) groups (mean +/- SD, P less than 0.05). The glomerular filtration rate (GFR) was similar in the three groups during the initial base-line determination but subsequently declined to a greater degree in the FO group. Morphologically, tubular injury was most extensive in the FO group, and the distribution of the injury indicated that it was caused, at least in part, by ischemia. The decline in GFR and the degree of histological injury were more closely linked to the diminished PGI2 production. Thus dietary FO supplementation caused decreased renal PGI2, increased renal vascular resistance, and an increased susceptibility to ischemic tubular cell injury in the isolated kidney.
Different mechanisms of adaptive increase in Na+-Pi cotransport across renal brush-border membrane
Authors: Yusufi A. N. , Szczepanska-Konkel M. , Hoppe A. , Dousa T. P.
Published: December 2017

We explored the biochemical mechanism by which thyroid hormone (T3) and low-phosphate diet (LPD) cause an adaptive increase in Na+-Pi cotransport across renal brush-border membrane (BBM). The rate of Na+-Pi cotransport was determined by 32Pi uptake by BBM vesicles (BBMV), and the number of Na+-Pi symporters was assessed by binding of [14C]phosphonoformic acid (PFA) on BBMV. In BBMV of both T3-treated rats and LPD-fed rats, the Na+ gradient-dependent 32Pi uptake increased (Vmax increased; Km Pi was not changed). The Na+-dependent [14C]PFA binding on BBMV increased (higher Vmax, no change in Km PFA) in response to T3, but it remained unchanged in rats fed LPD. Both the increase of Na+-Pi cotransport and of Na+-dependent [14C]PFA binding in response to T3 were blocked by actinomycin D or cycloheximide. Addition of benzyl alcohol to BBMV in vitro increased Na+-Pi cotransport, but [14C]PFA binding did not change; the [3H]phlorizin binding and cotransports of other solutes decreased or did not change. The exposure of BBMV to cholesterol decreased Na+-Pi cotransport without changing [14C]PFA binding. We suggest that the adaptive increase of Na+-Pi cotransport elicited by T3 is due to an increase in number of Na+-Pi cotransporters in BBM. In contrast, in response to LPD the number of Na+-Pi cotransporters is unchanged, and the increased Na+-Pi cotransport is due to faster translocation of Na+ with Pi due to enhanced fluidity of BBM.
Flow dependence of K+ secretion in cortical distal tubules of the rat
Authors: Malnic G. , Berliner R. W. , Giebisch G.
Published: December 2017

Superficial distal tubules were pump perfused (range 0-35 nl/min), generally with solutions similar in composition to early distal tubule fluid, in control, K+–depleted, acutely K+–loaded, and K+–adapted rats with the use of double-barreled resin-reference microelectrodes to measure K+ and Na+ activities and transepithelial potential differences (PD). When perfusion rate decreased from 35 to 5 nl/min in control animals, [K+] increased from 2 mM to between 10 and 20 mM, remaining at these levels as perfusion rate was decreased further. In low-K+ rats, the change in K+ activity with flow was greatly attenuated. In K+–loaded and K+–adapted rats, [K+] was higher than in controls at all flow rates. Na+ concentrations and lumen-negative PD increased with high flow rates in control rats. Addition of 10(-3) M amiloride blocked the increase in luminal K+ with low flow rates. In the physiological range of late distal flow rates, luminal [K+] remains constant and similar to the concentration attained in the steady state. At higher flow rates, [K+] declines, and K+ balance, PD, and Na+ reabsorption modulate the relationship between K+ secretion and flow rate.
Guanosine nucleotide-dependent activation of the amiloride-blockable Na+ channel
Authors: Garty H. , Yeger O. , Yanovsky A. , Asher C.
Published: December 2017

Effects of guanosine nucleotides on the epithelial Na+ channel were studied in apical membrane vesicles derived from the toad bladder epithelium. Trapping 10 microM guanosine-5'-O-(thiotriphosphate) (GTP gamma S) in vesicles evoked two- to fourfold increase in the amiloride-sensitive (Na+ channel-mediated) 22Na+ uptake. The nucleotide had no significant effect on the amiloride-insensitive 22Na+ uptake or the valinomycin-mediated 86Rb+ uptake in the same membranes. The stimulatory action of GTP gamma S was mimicked by 5'-guanylylimidiodiphosphate (GppNHp) and could at least partly be reversed by guanosine-5'-O-(thiodiphosphate) (GDP beta S) (10-fold excess). GTP itself and adenosine-5'-O-(thiotriphosphate) (ATP gamma S) had no sustained effect on Na+ transport in vesicles. Thus it appears that the epithelial Na+ channel is directly or indirectly regulated by the occupancy of a guanosine-specific site, probably the alpha subunit of a G protein. The possibility that GTP gamma S acts indirectly by activating a membrane-bound, GTP-dependent enzyme the product of which modulates the channel conductance was assessed by measuring 22Na+ fluxes in membrane vesicles prepared to contain products of such enzymes. None of the reagents tested [adenosine 3',5' cyclic monophosphate (cAMP), guanosine 3',5y cyclic monophosphate (cGMP), inositol 1,4,5-trisphosphate (IP3), and diacylglycerol (DAG)] increased the tracer flux in vesicles or altered its response to GTP gamma S.
Effect of lumen pH on cell pH and cell potential in rabbit proximal tubules
Authors: Kuwahara M. , Ishibashi K. , Krapf R. , Rector F. C. , Berry C. A.
Published: December 2017

To determine the effect of luminal pH on cell pH and basolateral cell membrane potential difference (Vbl) of rabbit proximal convoluted tubules, Vbl was measured by conventional microelectrodes and intracellular pH was measured microfluorometrically. Lowering lumen pH acidified the cell and depolarized Vbl. Three factors contributed to depolarization of Vbl. Lowering lumen pH decreased apical cell membrane potassium permeability (PK) as indicated by the following: 1) at lumen pH 7.4 raising lumen [K] depolarized Vbl; 2) lowering lumen pH eliminated the depolarization of Vbl induced by increasing lumen [K]. An additional effect was suggested by the following: lumen Ba2+ blunted, but did not eliminate, the Vbl response to lowering lumen pH. An effect on basolateral K permeability (PK) via its effect on cell pH was suggested by the fact that lowering lumen pH dramatically reduced the depolarization induced by increasing bath [K]. Lowering lumen pH might influence Vbl by inhibiting H+-HCO3- transport. Addition of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath solution hyperpolarized Vbl and enhanced the depolarization induced by lowering luminal pH. At luminal pH 6.0 SITS had no effect, suggesting elimination of H+ secretion. Addition of 1 mM luminal amiloride had no effect on Vbl or the response of Vbl to lowering luminal pH, but in the presence of amiloride SITS still hyperpolarized Vbl, suggesting amiloride-insensitive electrogenic H+ secretion. These results suggest that lumen pH-dependent depolarization of Vbl is due to 1) a decrease in apical PK; 2) cell acidification with secondary effects on basolateral PK; and 3) a decrease in apical electrogenic H+ transport.
Determinants of ammonia entry along the rat proximal tubule during chronic metabolic acidosis
Authors: Simon E. E. , Merli C. , Herndon J. , Cragoe E. J. , Hamm L. L.
Published: December 2017

The technique of in vivo microperfusion was used to examine the determinants of ammonia entry along the rat proximal tubule under conditions of chronic metabolic acidosis (CMA). When perfused with a 5 mM bicarbonate-containing perfusate, collected fluid ammonia concentrations remained constant with increasing flow rate and thus ammonia entry was highly flow-rate dependent. Ammonia entry was also flow-rate dependent using a 25 mM bicarbonate perfusate but entry reached a plateau as perfusion rate increased. Also, ammonia entry tended to be lower at all perfusion rates with the 25 mM perfusate compared with the 5 mM bicarbonate perfusate, but this was most evident at the highest perfusion rate (45 nl/min). The decline in ammonia entry was associated with increasing collected fluid bicarbonate concentrations, suggesting that there was inhibition of diffusion trapping of ammonia. The effects of Na+-H+ exchange inhibition on ammonia entry were examined using the amiloride analogue, 5-(N-ethyl-N-isopropyl)amiloride. With a 25 mM bicarbonate-containing perfusate, the amiloride analogue caused a significant decrease in bicarbonate reabsorption but a nonsignificant decrease in ammonia entry associated with a significant rise in collected fluid bicarbonate concentration. When the potential effects of decreased diffusion trapping of ammonia were eliminated with 12 and 5 mM bicarbonate-containing perfusates, the analogue had no effect on ammonia entry despite significant inhibition of bicarbonate reabsorption. Thus ammonia entry in CMA is moderately affected by tubule fluid pH but is highly flow-rate dependent. There were no effects of inhibition of Na+-H+ exchange above those expected from inhibition of diffusion trapping of ammonia.
Effects of serosal anion composition on the permeability properties of rabbit urinary bladder
Authors: Donaldson P. J. , Chen L. K. , Lewis S. A.
Published: December 2017

This study describes the effects of serosal Cl- and HCO3- substitution on transepithelial Na+ transport and basolateral membrane properties of the rabbit urinary bladder. Replacement of Cl- with NO3-, SCN-, and Br- had no effect on transepithelial Na+ transport or the basolateral membrane potential (Vbl). However, gluconate, isethionate, and cyclamate (anions that were shown previously to be not as permeable as Cl- through the basolateral membrane anion channel), decreased transepithelial Na+ transport and depolarized Vbl. Replacement of HCO3- also produced a decrease in transepithelial Na+ transport and a depolarization of Vbl. Utilizing conventional and K+-specific microelectrodes, we found the depolarization to be due to a reduction in basolateral K+ conductance. This depolarization was reversible only when Cl- was returned to the serosal solution, the normally permeant anion NO3- being unable to affect repolarization, suggesting that both the K+ and Cl- conductance are reduced during depolarization. A lag period of some 4 min preceded the repolarization of Vbl. The Na+-H+ exchange blocker amiloride prolonged the lag phase associated with repolarization, whereas niflumic acid, a Cl-(-)HCO3- exchange blocker (in red blood cells) reduced the magnitude of Vbl repolarization. Because of the possible involvement of the exchangers it is believed that the lag phase represents a volume-dependent and/or pH-dependent reactivation of the basolateral membrane conductances.
Neural control of distal tubular bicarbonate and fluid transport
Authors: Wang T. , Chan Y. L.
Published: December 2017

Although previous studies have shown that inhibition of proximal tubular reabsorption of bicarbonate and fluid partially accounts for bicarbonaturia and diuresis observed in the denervated kidney, the role of the distal convoluted tubule in the neural regulation of electrolyte excretion has not been defined. This study was designed to examine the effects of denervation on bicarbonate and fluid reabsorption in the distal convoluted tubules by using in situ microperfusion methods. Early distal tubules were perfused with a solution containing 15 mM bicarbonate at a rate of 12 nl/min, and fluid samples were collected from the late distal tubule. Bicarbonate concentrations were measured as total CO2 by microcalorimetry. The rate of fluid absorption (Jv) was determined using [methoxy-3H]inulin as a volume marker. Before denervation, the rate of bicarbonate absorption (JHCO3-) was 24.7 +/- 2.6 peq.min-1.mm-1, and Jv was 1.80 +/- 0.14 nl.min-1.mm-1. After denervation, marked reductions in JHCO3- (51%) and Jv (60%) were observed. In contrast, sham denervation did not affect JHCO3- or Jv significantly. Our results indicate that the distal convoluted tubule can reabsorb bicarbonate and fluid under physiological conditions. We conclude that the transport processes in this segment of the nephron are regulated by renal nerve activity.
Renal metabolism and urinary excretion of thromboxane B2 in the rat
Authors: Benigni A. , Chiabrando C. , Perico N. , Fanelli R. , Patrono C. , FitzGerald G. A. , Remuzzi G.
Published: December 2017

We wanted to evaluate whether the kidney tissue can metabolize thromboxane (Tx) B2 and, specifically, whether the 2,3-dinor metabolite might be formed in the kidney and excreted in the urine. For this purpose, we used an isolated perfused kidney preparation exposed to vehicle or TxB2 at different infusion rates. Approximately 96% of the total TxB2 infused was recovered in the venous effluent, whereas approximately 1% was found in urine. Isolated perfused kidneys exposed to [3H]TxB2 eliminated in the urine 1.2% of the [3H]TxB2 infused, measured by thin-layer chromatographic analysis, and actively metabolized [3H]TxB2 to 2,3-dinor-TxB2, 11-dehydro-TxB2, and possibly 2,3,4,5-tetranor-TxB1. No metabolites of TxB2 were recovered in the venous effluent. As a marker of renal TxB2 metabolic activity, urinary 2,3-dinor-TxB2 was quantified by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry. The 2,3-dinor-TxB2 was detected both before and during TxB2 infusion in urinary samples but not in the venous effluent. The ratio of 2,3-dinor-TxB2-TxB2 increased during the infusion reaching a peak value immediately after stopping the TxB2 infusion. These results indicate that, in the rat, the kidney tissue metabolizes TxB2 to 2,3-dinor-TxB2, and both TxB2 and 2,3-dinor-TxB2 are excreted in the urine.
Role of organic anions in renal response to dietary acid and base loads
Authors: Brown J. C. , Packer R. K. , Knepper M. A.
Published: December 2017

Bicarbonate is formed when organic anions are oxidized systemically. Therefore, changes in organic anion excretion can affect systemic acid-base balance. To assess the role of organic anions in urinary acid-base excretion, we measured urinary excretion in control rats, NaHCO3-loaded rats, and NH4Cl-loaded rats. Total organic anions were measured by the titration method of Van Slyke. As expected, NaHCO3 loading increased urine pH and decreased net acid excretion (NH4+ + titratable acid - HCO3-), whereas NH4Cl loading had the opposite effect. Organic anion excretion was increased in response to NaHCO3 loading and decreased in response to NH4Cl loading. We quantified the overall effect of organic ion plus inorganic buffer ion excretion on acid-base balance. The amounts of organic anions excreted by all animals in this study were greater than the amounts of NH4+, HCO3-, or titratable acidity excreted. In addition, in response to acid and alkali loading, changes in urinary organic anion excretion were 40-50% as large as changes in net acid excretion. We conclude that, in rats, regulation of organic anion excretion can contribute importantly to the overall renal response to acid-base disturbances.