bluuebunny/crossref_metadata_2025 huggingface dataset:
Double-barreled chloride channels of collecting duct basolateral membrane
Authors: Sansom S. C. , La B. Q. , Carosi S. L.
Published: December 2017

Microelectrode studies have shown that the basolateral membrane of the principal cells (PC) of the rabbit cortical collecting duct (CCD) contains Cl(-)-conductive pathways. To determine the properties of single Cl- channels we prepared the basolateral membrane for patch clamping by incubating the CCD in collagenase and/or tearing the basement membrane with a fine needle. When high concentrations of collagenase were used, only a small nonselective channel was observed. In low concentrations or the absence of collagenase, however, we identified a Cl- channel (g46) in both cell-attached and excised patches. The Cl- channel gated rapidly between three equally spaced substates of 0 (S0), 23 (S1), and 46 pS (S2) and slowly between states C (inactive) and S0. The conductance of each substate was not voltage dependent between pipette potentials from -60 to +60 mV (cell attached). The probability that the channel gated from C to S0 increased with hyperpolarizing potentials, but the probability that g46 was in substate S0 increased with depolarizing patch potentials. This channel was similar to that described by Miller for the nonexcitable membrane of the electric organ of Torpedo californica. Because g46 was the most frequently observed basolateral membrane channel and was voltage dependent at physiological potentials, it is probably the channel responsible for the dominant Cl- conductance of PC.
Glutamate dehydrogenase activities in microdissected rat nephron segments: effects of acid-base loading
Authors: Wright P. A. , Knepper M. A.
Published: December 2017

A method for measurement of glutamate dehydrogenase (GDH) activity in single renal tubules was employed to determine the distribution and regulation of GDH in tubule segments. Fresh microdissected tubules from collagenase-treated kidneys were permeabilized by hyposmotic shock and freezing. The rate of conversion of alpha-ketoglutarate, NH4+, and NADH to glutamate and NAD was measured at 37 degrees C fluorometrically. Very high activities were found in proximal tubule segments (150-210 pmol.min-1.mm tubule length-1), intermediate values (40-90 pmol.min-1.mm-1) in distal convoluted tubules, cortical thick ascending limbs, connecting tubules, medullary thick ascending limbs, and lower values (5-30 pmol.min-1.mm-1) in cortical collecting ducts, inner medullary collecting ducts, outer medullary collecting ducts, outer medullary thin limbs, and inner medullary thin limbs. To determine the effects of acid-base loading on GDH activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to the animals' drinking water for 7 days. Acid intake by the rats increased GDH activity in S1 and S2 proximal tubules by threefold, with no effect in other segments, including S3 proximal tubules. Alkali intake decreased GDH activity in the S3 proximal tubule by 40%, with no effect in other segments. We conclude that GDH activities are highest in proximal tubule segments and are regulated only in proximal tubule segments. Thus the results are consistent with the view that the proximal tubule is the chief site of the regulated production of ammonium in the kidney.
Effects of mitogens and other agents on rat mesangial cell proliferation, pH, and Ca2+
Authors: Ganz M. B. , Perfetto M. C. , Boron W. F.
Published: December 2017

We investigated effects of various agents on proliferation, intracellular pH (pHi), and intracellular calcium [( Ca2+]i) of rat mesangial cells (MCs) in early passages (2-5). Serum-starved MCs incubated in HCO3- were exposed to one of the following: fetal calf serum (FCS), serotonin, angiotensin II (ANG II), arginine vasopressin (AVP), bombesin (Bom), bradykinin (BK), epidermal growth factor (EGF), epinephrine (Epi), interleukin 1 (IL-1), norepinephrine (NE), neuropeptide Y, oxytocin, substance P (SP), platelet-derived growth factor, or 12-O-tetradecanoylphorbol-13-acetate (TPA). We assessed DNA synthesis from [3H]thymidine uptake during exposure to test agent. All agents except ANG II, NE, Bom, and SP were mitogenic. When MCs were incubated in a HCO3(-) -free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium, maximal mitogenic responses to FCS, AVP, and EGF were 41, 44, and 55% (P less than 0.01) lower, respectively, than those in presence of HCO3-. In absence of HCO3-, agents other than BK and IL-1 produced a biphasic pHi response characterized by a transient acidification followed by a prolonged alkalinization that was both Na(+)-dependent and amiloride-sensitive. In presence of HCO3-, agents produced only a small and gradual acidification, except for IL-1 and Epi. Addition of all agonists except IL-1, EGF, and TPA produced significant transient increases in [Ca2+]i, the magnitudes of which were similar in HCO3- and non-HCO3- buffers. These results demonstrate that, in presence of HCO3-, agents (i.e., NE and ANG II) can produce typical [Ca2+]i transients and still not cause MC proliferation. Conversely, an agent may cause proliferation without eliciting a short-term change in either [Ca2+]i or pHi (i.e., IL-1), a change in [Ca2+]i but not pHi (i.e., Epi), or a change in pHi but not [Ca2+]i (i.e., TPA). Thus, at least for MCs, proliferation in HCO3- can be dissociated from early agonist-induced changes in pHi and [Ca2+]i.
Pressor effect of endothelium-derived relaxing factor inhibition in conscious virgin and gravid rats
Authors: Umans J. G. , Lindheimer M. D. , Barron W. M.
Published: December 2017

We used pressor responses to N-methylarginine (NMA), a specific inhibitor of endothelium-derived relaxing factor (EDRF) biosynthesis, to assess the contribution of EDRF to control of basal blood pressure in conscious, chronically instrumented virgin and gravid rats. Hypotheses were that NMA would raise blood pressure in conscious animals in spite of intact cardiovascular reflexes and that differing effects in virgin and pregnant animals would reveal contributions of EDRF to the physiological vasodilation of pregnancy. Basal mean arterial pressure (MAP) and heart rate (HR) were 106 +/- 12 mmHg and 394 +/- 27 beats/min (n = 31) in virgin and 102 +/- 8 mmHg and 378 +/- 18 beats/min (n = 14) in gravid rats. After NMA (150 mg/kg iv), increments in MAP and decreases in HR were similar in each group (virgin, delta MAP = 38 +/- 12 mmHg, delta HR = -65 +/- 27 beats/min, n = 14; gravid, delta MAP = 33 +/- 8 mmHg, delta HR = -63 +/- 20 beats/min, n = 6). Pretreatment with excess L-arginine reduced pressor responses to NMA by 80% in both pregnant and virgin animals. In contrast, L-arginine had no significant effect on the pressor response to phenylephrine (6.4 mg/kg iv). EDRF contributes importantly to regulation of basal blood pressure in conscious animals, and pregnancy does not alter the pressor effect of EDRF inhibition in the rat.
Efficacy of nifedipine to prevent systemic and renal vasoconstrictor effects of endothelin
Authors: Madeddu P. , Yang X. P. , Anania V. , Troffa C. , Pazzola A. , Soro A. , Manunta P. , Tonolo G. , Demontis M. P. , Varoni M. V. , et al.
Published: December 2017

We investigated whether systemic and renal vasoconstriction induced by porcine endothelin (endothelin 1) is prevented by nifedipine in awake normotensive rats. Endothelin (0.07-1.4 nmol/kg iv) induced a long-lasting increase in mean blood pressure (MBP) and a decrease in renal blood flow (RBF). Maximal decrease in RBF was 25 +/- 7% (0.07 nmol/kg), 40 +/- 2 (0.35), 67 +/- 5 (0.70), and 74 +/- 8 (1.4). Hemodynamic parameters were back to base line within 35 +/- 5 min (0.07 nmol/kg), 43 +/- 6 (0.35), 60 +/- 4 (0.70), and 81 +/- 7 (1.4). Intravenous bolus injection of either angiotensin II (ANG II, 0.006-0.024 nmol/kg) or norepinephrine (0.40-1.60 nmol/kg) caused a dose-related short-lasting increase in MBP and a decrease in RBF. Endothelin was less potent than ANG II (1:3.42) and more potent than norepinephrine (1:0.015) as a renal vasoconstrictor. Nifedipine (1 mg/kg ip) was equally effective in preventing the increase in MBP caused by endothelin, norepinephrine, or ANG II. It exerted a weaker protection on the renal hemodynamic response to endothelin compared with the inhibition of the other two vasoconstrictors. Thus the regression line representing the relationship between endothelin-induced changes in MBP and RBF was steeper in rats given nifedipine (slope: vehicle, -1.33; nifedipine, -5.50; P less than 0.05). These studies suggest that nifedipine can partially prevent systemic and renal vasoconstriction caused by exogenously administered endothelin in awake normotensive rats.
Oxidative stress in isolated rat renal proximal and distal tubular cells
Authors: Lash L. H. , Tokarz J. J.
Published: December 2017

Suspensions of purified proximal tubular (PT) and distal tubular (DT) cells were isolated from rat kidney cortical cells by Percoll density-gradient centrifugation and were used to investigate susceptibility of these regions of the nephron to oxidative injury. Exposure to tert-butyl hydroperoxide (tBH), menadione (MD), or H2O2 produced significantly greater cytotoxicity, as assessed by leakage of lactate dehydrogenase, in DT cells than in PT cells. The order of cytotoxic potency in both cell types was MD greater than tBH greater than H2O2. Preincubation of PT and DT cells with 5 mM glutathione (GSH) or 5 mM dithiothreitol delayed tBH-induced cytotoxicity, indicating a protective role of GSH. Addition of buthionine sulfoximine and acivicin with GSH, to inhibit GSH synthesis and degradation, eliminated the protective effect of GSH, indicating that protection by GSH in DT cells is not dependent on uptake of the intact tripeptide. Incubation of both PT and DT cells with tBH resulted in oxidation of GSH to glutathione disulfide. Activities of five detoxication enzymes were significantly higher in PT cells, indicating that a diminished ability to detoxify reactive metabolites may contribute to the higher intrinsic susceptibility of DT cells to oxidative injury.
Exaggerated renal vascular reactivity to angiotensin and thromboxane in young genetically hypertensive rats
Authors: Chatziantoniou C. , Daniels F. H. , Arendshorst W. J.
Published: December 2017

The objective of this study was to test the hypothesis that angiotensin II and thromboxane A2 (TxA2) contribute to the elevated renal vascular resistance observed during the development of genetic hypertension. In 6-wk-old anesthetized spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats, renal blood flow (electromagnetic flowmetry) and carotid arterial pressure were measured during bolus injections of different doses of angiotensin II and U46619 (stable receptor agonist of TxA2) into the renal artery before and during inhibition of prostaglandin synthesis by indomethacin. In all cases, arterial pressure remained unchanged at the pre-injection levels. Under control conditions, angiotensin II reduced renal blood flow in SHR almost twice as much as in WKY. This strain difference was abolished by inhibition of prostaglandin synthesis, suggesting that a deficiency in the action of endogenous vasodilator prostaglandins is responsible for the enhanced response to angiotensin II in SHR. Under control conditions, the TxA2-receptor agonist produced similar reductions of renal blood flow in SHR and WKY. However, after indomethacin, the agonist-induced vasoconstriction was twice as large in SHR as in WKY, suggesting that SHR kidneys have an increased vascular reactivity to TxA2, which is unmasked when indomethacin reduces elevated levels of endogenous TxA2. These findings indicate important strain differences between young SHR and WKY in the renal vascular response to angiotensin II and TxA2 that may contribute to the renal vasoconstriction observed during the development of genetic hypertension.
Inhibition by (D-Trp12,Tyr34)bPTH(7-34)amide of PTH and PTHrP effects on Pi transport in renal cells
Authors: Pizurki L. , Rizzoli R. , Bonjour J. P.
Published: December 2017

We studied the effect of (D-Trp12,Tyr34)bovine parathyroid hormone-(7-34)amide [(D-Trp12,Tyr34)bPTH(7-34)NH2], a recently described highly potent antagonist of parathyroid hormone (PTH), on the adenosine 3',5'-cyclic monophosphate (cAMP) and the sodium-dependent phosphate transport (NaPiT) responses to bPTH-(1-34) or PTH-related protein [PTHrP(1-34)] in renal epithelial cells. In this system, bPTH and PTHrP increased cAMP production and inhibited NaPiT in a similar manner. (D-Trp12,Tyr34)bPTH(7-34)NH2 did not influence either cAMP or NaPiT, but markedly attenuated the responses to PTH or PTHrP. The effect was concentration dependent, and a maximal inhibition of PTH or PTHrP effects was obtained with a 10(3)- to 10(4)-fold greater concentration of the antagonist. In contrast, the same concentration of the unsubstituted counterpart, (Tyr34)bPTH(7-34)NH2, which abolished the PTH- or PTHrP-induced stimulation of cAMP production, did not affect the inhibition of NaPiT caused by either peptide. Thus (D-Trp12,Tyr34)bPTH(7-34)NH2 inhibited the effects of PTH and PTHrP on both cAMP synthesis and Pi transport in renal cells. Because of the effects of this analogue on a transport function affected by these two peptides under physiological and pathophysiological conditions, it appears to be a promising antagonist.
Neurogenic control of pressure natriuresis in conscious dogs
Authors: Ehmke H. , Persson P. B. , Seyfarth M. , Kirchheim H. R.
Published: December 2017

In this study we investigated the interaction of the sympathetic nervous system with renal perfusion pressure (RPP) in the short-term control of sodium excretion (UNa V). Pressure natriuresis curves (PNCs) were determined in 13 conscious dogs on a normal-salt diet during control conditions, bilateral common carotid occlusion (CCO), CCO combined with an intrarenal prazosin infusion, and during an intrarenal methoxamine infusion. RPP was reduced in controlled steps by inflation of a cuff placed around the renal artery. For controls, a reduction in RPP resulted in a strong decrease in urine output and UNaV. In all dogs, the PNC was closely related to individual resting blood pressure; UNaV fell to less than 50% of control (10-20 mmHg below resting blood pressure). A baroreflex activation of the sympathetic nervous system by CCO shifted PNC to the right by 10-15 mmHg (n = 8). Sensitivity of pressure natriuresis was not affected by CCO. The shift was blocked when the selective alpha 1-adrenoceptor antagonist prazosin was infused intrarenally during CCO (n = 9). Without CCO, prazosin had no effects on urine flow rate or UNaV at the control RPP. Similar to CCO, intrarenal infusion of the selective alpha 1-adrenoceptor agonist methoxamine shifted PNC to the right by 15-20 mmHg (n = 4). Neither renal blood flow nor glomerular filtration rate was significantly different between control and any experimental condition. These results indicate that the sympathetic nervous system regulates UNaV by shifting the PNC through intrarenal alpha 1-adrenoceptors without altering the sensitivity of pressure natriuresis.
Renal actions of endothelin: interaction with prostacyclin
Authors: Chou S. Y. , Dahhan A. , Porush J. G.
Published: December 2017

The renal actions of endothelin were examined by infusing it intrarenally in anesthetized dogs at 4 ng.min-1.kg-1 without affecting arterial blood pressure or cardiac output. Endothelin infusion caused a transient and significant increase in renal blood flow (RBF) by 13 +/- 2%, followed by large decreases in RBF and glomerular filtration rate (GFR; by 26 +/- 2 and 23 +/- 7%, respectively) but did not alter urine flow rate or absolute sodium excretion. After endothelin infusion, renal venous and arterial plasma 6-ketoprostaglandin F1 alpha increased from 250 +/- 58 and 117 +/- 31 to 1,044 +/- 249 and 617 +/- 211 pg/ml, respectively, and its renal output increased from 339 +/- 99 to 963 +/- 202 pg.min-1.g-1 (P less than 0.01 for all). The renal prostacyclin synthesis was augmented by endothelin without stimulating the renal renin release or norepinephrine output. Inhibition of prostaglandin synthesis with indomethacin partially prevented the early renal vasodilation induced by endothelin, which then caused a more pronounced decline in RBF and GFR (by 65 +/- 7 and 54 +/- 8%, respectively). With suppression of prostacyclin synthesis, inhibition of renin release by endothelin was observed. Thus the vasoconstrictive effects of endothelin on renal hemodynamics are significantly modified by its ability to enhance production of vasodilators, including prostacyclin.
Model of TGF-proximal tubule interactions in renal autoregulation
Authors: Cupples W. A. , Wexler A. S. , Marsh D. J.
Published: December 2017

Previous models, assuming constant reabsorption in the proximal tubule, have shown that tubuloglomerular feedback (TGF) can explain only a fraction of glomerular filtration rate (GFR) and renal blood flow autoregulation. Increased arterial pressure inhibits proximal tubule fluid reabsorption, an effect that should increase the efficacy of TGF because of the resulting increased flow rate in the loop of Henle. Models describing pressure and flow in a glomerulus and a nephron were derived to test this prediction. The models were coupled by a TGF function with tubular flow rate at the end of the proximal tubule (superficial nephron) or at the macula densa (juxtamedullary nephron) as input and with afferent arteriolar resistance as output. In agreement withd others, the model predicted that TGF alone could account for about one-half of autoregulation. Pressure-dependent inhibition of proximal reabsorption increased the ability of TGF to account for autoregulation, providing compensation for increases in arterial pressure comparable to published whole kidney values. The inclusion of an approximation of an effect of arterial pressure on TGF marginally improved predicted autoregulation. Although the results suggest that the proximal tubule-TGF interaction can provide a quantitatively adequate explanation for autoregulation, they also indicate that the effect of the interaction is spent at arterial pressures greater than 130 mmHg. Additional mechanisms are required to extend this range.
Electrical properties of smooth muscle cell membrane in renal pelvis of rabbits
Authors: Seki N. , Suzuki H.
Published: December 2017

Intracellular recordings were made to study the electrical properties of smooth muscle cells in the rabbit renal pelvis. The muscle cells exhibited spontaneous oscillation in the membrane potential (slow wave). The slow waves were regular and were resistant to tetrodotoxin and sympathomimetic or parasympathomimetic antagonists, findings indicative of myogenic activity. The membrane was depolarized by an increase in extracellular concentration of K+ ([K+]o), decrease in [Na+]o, inhibition of the electrogenic Na(+)-K+ pump by ouabain or K(+)-free solution, and the application of norepinephrine (NE, greater than 10(-6) M). The maximum slope of the membrane depolarization produced by a 10-fold increase in [K+]o was approximately 48 mV. Reductions in [Ca2+]o inhibited the generation of slow waves with no marked change in the membrane potential. Depolarizations produced by any given method increased the frequency and decreased the amplitude of the slow wave, and NE had the most potent accelerating action on the frequency. Hyperpolarization of the membrane by 1-5 mV with extracellularly applied current stimuli reduced the frequency, and a strong hyperpolarization (greater than 5 mV) blocked the generation of slow waves. Electrophysiological properties of the slow waves obtained with tissues of the renal pelvis and intestinal smooth muscles were compared.
Postnatal development of oxidative enzymes in various rat nephron segments: effect of weaning on different diets
Authors: Bastin J. , Djouadi F. , Geloso J. P. , Merlet-Benichou C.
Published: December 2017

Activities of fumarase and 3-hydroxyacylCoA dehydrogenase (3-OHDH) were determined in homogenates of rat kidneys between day 21 of gestation and postnatal day 10 and in single isolated nephron segments at postnatal days 16, 21, and 30, and in adult segments. For 3-OHDH activity, main developmental changes were found in proximal convoluted (PCT) and straight tubules (PST) and were characterized by an overshoot of adult level from postnatal days 21 to 30 (59.7 +/- 3.0 and 37.5 +/- 3.4 at day 21 vs. adult values 27.1 +/- 1.5 and 22.7 +/- 1.5 mol.kg dry wt-1.h-1). When rats were precociously weaned on day 16 and fed a diet containing lipid to equal 13% of total caloric intake, a significant decrease in 3-OHDH activity was observed in some parts of the nephron. These changes could be prevented by maintaining early weaned animals on high-fat diet providing 70% of total calories as lipid. Results suggest that changes in fat content of diet during kidney maturation can in part regulate 3-OHDH activity in some nephron segments. Fumarase activity increased 2.6-fold in the medullary thick ascending limb between days 16 and 30; pattern of development was similar to the one reported for Na(+)-K(+)-ATPase activity in this segment. High levels of both enzymes were reached noticeably earlier during development in PCT and PST than in medullary thick ascending limb, which emphasizes metabolic heterogeneity of developing rat kidney nephron.
Glomerular endothelin receptors during initiation and maintenance of ischemic acute renal failure in rats
Authors: Wilkes B. M. , Pearl A. R. , Mento P. F. , Maita M. E. , Macica C. M. , Girardi E. P.
Published: December 2017

Glomerular endothelin (ET) receptors were studied in normal Sprague-Dawley rats and in rats with ischemic acute renal failure (ARF) induced by a 60-min occlusion of the left renal artery (right kidney intact). In normal rats ET bound to specific glomerular receptor sites [equilibrium affinity constant (Kd), 46.6 +/- 5.8 pM; receptor number (Ro), 1,167 +/- 160 fmol/mg (n = 7)]. ET infusion (90 ng.kg-1.min-1, intra-arterially) raised mean arterial pressure by 32 +/- 4 mmHg, lowered renal blood flow (RBF) by 62% and glomerular filtration rate (GFR) by 49%, and reduced the number of glomerular ET receptor sites by 62%. Reduced ET binding could not be explained by prior occupancy, because acid treatment (which dissociates bound ET from its receptors) did not increase receptor number. If elevated ET levels contributed to decreased RBF and GFR in ARF, glomerular ET receptors would be expected to down-regulate. In rats with ischemic ARF there were no differences in the number or affinity of glomerular ET receptors in the clamped or contralateral kidneys. Additional studies demonstrated that the downregulation response to ET infusion was intact in ARF. The data demonstrate that glomerular ET receptors are unaltered in ischemic ARF and do not support a role for increased glomerular ET in the alterations of renal hemodynamics in this model.
A calcium-permeable stretch-activated cation channel in renal proximal tubule
Authors: Filipovic D. , Sackin H.
Published: December 2017

Isolated Necturus proximal tubules were split to expose the apical membrane surface for patch clamping. When both pipette and bath solutions contained only Ca, N-methyl-D-glucamine, and methanesulfonate, inwardly directed Ca currents were observed through a stretch-activated (SA) cation channel with conductance of 18 +/- 1 pS (n = 19). The SA cation channel exhibited little discrimination among Na, K, and Ca but was at least nine times more selective for cations than anions. The channel was not significantly gated by either membrane potential or cytosolic Ca. However, application of 15 cmH2O suction to patch pipette significantly increased the mean number of open channels by a factor of 6.5, from 0.04 +/- 0.02 to 0.26 +/- 0.08 (n = 11). Ca currents through the SA cation channel were reversibly blocked by 10 microM gadolinium, which was applied to outside surface of excised patches. This is similar to gadolinium block of stretch-activated channels in Xenopus oocytes (X.-C. Yang and F. Sachs. Science Wash. DC 243: 1068-1071, 1989). A Ca-dependent, maxi-K channel (92 +/- 9 pS, n = 5) was also found at the apical membrane of the same proximal tubules. In some cases this maxi-K channel appeared to be indirectly activated by pipette suction, raising the possibility that Ca influx through the SA cation channel may regulate K efflux via the maxi-K channel. Such a process could mediate cell volume regulation and maintain electrolyte homeostasis during normal variations in Na-substrate cotransport.
Release of acetylcholine from isolated canine renal tissue
Authors: Pirola C. J. , Alvarez A. L. , Finkielman S. , Nahmod V. E.
Published: December 2017

Slices of canine kidney were incubated with [3H]choline and Ringer-Krebs solution for 30 min. Secretion of [3H]acetylcholine ([3H]ACh) was evoked by either 1) an electrical field stimulation (0.5-1 Hz, 2 ms, 20 V) or 2) high-potassium (57 mM) superfusion for 3 min to depolarize nerve terminals. With these stimulatory schedules, the 3H release mainly consists of [3H]-ACh. The ACh release is Ca2+ dependent and blocked in presence of a Mg2(+)-rich medium. Thus suppression of ongoing neuronal activity by tetrodotoxin and blockade of nicotinic receptors by hexamethonium reduced the electrically evoked ACh release. With both stimulatory procedures, the secretory response was modified by pilocarpine and atropine. In addition, the release of [3H]ACh produced by electrical field stimulation was inhibited by neostigmine, indicating the presence of functional presynaptic receptors in cholinergic putative terminals. Activation of these receptors depresses ACh secretion. The disruption of extrinsic renal nerves 7 days before did not diminish [3H]ACh release caused by K+ stimulation and, conversely, increased [3H]ACh released by electrical field stimulation. These data support the presumed existence of cholinergic nerve terminals in the canine kidney that can release ACh under suitable conditions of stimulation.
Rat kidney band 3 mRNA modulation in chronic respiratory acidosis
Authors: Da Silva Junior J. C. , Perrone R. D. , Johns C. A. , Madias N. E.
Published: December 2017

Recent evidence indicates the existence of a protein related to the erythroid chloride-bicarbonate exchanger (band 3 protein) in the basolateral aspect of type A intercalated cells of the distal nephron. To probe the possible participation of this transporter in the renal adaptation to chronic hypercapnia, we examined the steady-state abundance of band 3 mRNA in the kidney during respiratory acidosis of variable duration. Total RNA was isolated from renal cortex and medulla of rats maintained in a 10% CO2 atmosphere for 2 or 5 days and from contemporaneous controls. The RNA was analyzed by Northern blot assay using cDNA probes for band 3 and beta-actin genes. Using a 3' cDNA probe encoding the membrane-associated domain of band 3 protein that is involved in anion exchange, we found a two- to threefold increase in steady-state mRNA levels (whether or not correction for the beta-actin signals was applied) in renal cortex and medulla at 5 days of hypercapnia. Similar, but less definitive, increases were observed at the 2-day time point. Using a 5' cDNA probe encoding an erythroid-protein segment absent from the kidney band 3 major transcript, we detected meager hybridization in renal tissue and no measurable variation during hypercapnia. Use of splenic RNA as a positive control for the 5' probe disclosed marked reduction of band 3 mRNA levels in hypercapnia, indicating organ specificity of band 3 gene expression. We conclude that steady-state levels of kidney band 3 mRNA increase in chronic respiratory acidosis as a result of transcriptional or posttranscriptional regulatory mechanisms. This adaptation might be involved in the augmentation of renal acidification characteristic of chronic hypercapnia.
Abnormal proximal tubule apical membrane protein composition in X-linked hypophosphatemic mice
Authors: Ford D. M. , Molitoris B. A.
Published: December 2017

The hypophosphatemic (Hyp) mouse is characterized by an isolated X-linked defect in proximal tubular phosphate (Pi) reabsorption associated with a decreased maximum velocity (Vmax) and a normal affinity (Km). To directly investigate the underlying cellular defect proximal tubular brush-border membranes (BBM) from normal control (Con) and Hyp male littermates were examined for differences in cholesterol content, total and individual phospholipid composition, phospholipid incorporation rates, membrane fluidity, and by two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide protein electrophoresis. The cholesterol content, total and individual phospholipid profiles, phospholipid incorporation rates, and membrane fluidity of Con and Hyp BBM samples were comparable. However, the two-dimensional gel electrophoreses of Con and Hyp BBM proteins, run simultaneously under identical conditions, revealed a protein with an apparent abnormal isoelectric migration pattern in Hyp BBM samples. This protein had an apparent molecular weight 56,000 and an apparent pI of 7.2 and was consistently evident on Hyp gels (n = 3) but not on Con gels (n = 3). The appearance of this protein band was associated with a diminution in staining of a control protein of comparable apparent molecular weight but markedly lower apparent pI.
Carbonic anhydrase and proton secretion in turtle bladder mitochondrial-rich cells
Authors: Fritsche C. , Kleinman J. G. , Bain J. L. , Heinen R. R. , Riley D. A.
Published: December 2017

Bladders from actively feeding turtles were processed for carbonic anhydrase (CA) cytochemically. CA-positive cells were identified as microplicated (MP) cells, microvillated (MV) cells, and subluminal (SL) cells. After acute enhancement of H+ secretion with 5% CO2, MP cells displayed extensive microplicae and a reduced density of apical subplasmalemmal vesicles, and they were CA reactive throughout a large part of the cytoplasm including the microplicae. After acute inhibition of H+ secretion with a pH 4.5 mucosal bath, CA staining was excluded from the microplicae and apical subplasmalemmal region of most MP cells, whereas microplicae varied from extensive to reduced, and subapical vesicle density remained elevated. MV cells were characterized by basolateral staining with sparing of the MV and apical subplasmalemmal region in all settings except 1) after 5% CO2 and 2) when MV cells were found in areas in which MP cells were stained to the lumen. These results indicate that CA is active at the site of H+ secretion in MP cells and is correlated with the acute acid-base status of the bladder.
Effects of chronic hyperkalemia on renal production and proximal tubule transport of ammonium in rats
Authors: DuBose T. D. , Good D. W.
Published: December 2017

Free-flow micropuncture experiments were performed to examine directly the effects of chronic hyperkalemia on renal ammonium production, urinary ammonium excretion, and proximal convoluted tubule ammonium transport in the rat in vivo. Munich-Wistar rats were pair-fed either a control or a high-K+ diet for 6-11 days. Chronic K+ loading was associated with an increase in plasma K+ concentration and significant systemic metabolic acidosis. Renal blood flow did not differ in control and high-K+ rats. In the hyperkalemic rats, urinary ammonium excretion was reduced by 40% and whole kidney ammonium production was reduced by 50% compared with controls. In contrast, chronic hyperkalemia had no significant effect on net ammonium transport by either the early or late segment of the proximal convoluted tubule. Chronic hyperkalemia also had no effect on the absolute rate of ammonium delivery to early or late proximal convoluted tubule sites. These results indicate that a change in renal ammonium production does not necessarily correlate with a change in proximal tubule ammonium transport and that reduced urinary ammonium excretion in chronic hyperkalemia is not due to impaired secretion of ammonium by the proximal convoluted tubule. Chronic hyperkalemia may reduce ammonium excretion by decreasing transfer of ammonium from proximal tubules to collecting ducts in the renal medulla.
Mechanisms of inhibition of proximal acidification by PTH
Authors: Sasaki S. , Marumo F.
Published: December 2017

The parathyroid hormone (PTH) effects on luminal Na-H exchange and basolateral Na-HCO3 cotransport were examined in isolated perfused rabbit proximal convoluted tubules. Lumen pH (pHi) and cell pH (pHi) were measured by a fluorescent technique using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The time course of the PTH effect on proximal acidification was first determined by continuous monitoring of pHi in a stop-flow condition. PTH 10(-8) M added to the bath promptly inhibited the acid pHl generation. Adenosine 3',5'-cyclic monophosphate (cAMP) (10(-4) M) added to the bath mimicked the PTH effect. Luminal Na-H exchange was assayed by monitoring changes in pHi (dpHi/dt) in response to luminal Na+ removal in HCO3- free condition. The addition of PTH or cAMP for 5 min decreased dpHi/dt by 30 and 37%, respectively. Basolateral Na-HCO3 cotransport was assayed by measuring dpHi/dt caused by bath Na+ removal. The addition of PTH or cAMP for 5 min did not change the dpHi/dt. PTH also did not alter the dpHi/dt induced by reducing the bath HCO3- from 25 to 5 mM. The addition of PTH or cAMP to the bath slightly reduced pHi by 0.05. These results suggest that 1) PTH inhibits proximal acidification very rapidly and the effect reached a maximum within 10 min, 2) PTH rapidly inhibits luminal Na-H exchange but not basolateral Na-HCO3 cotransport in a short period, and 3) this effect is at least partly mediated by cAMP-dependent mechanisms.
High-NaCl diets increase natriuretic and diuretic responses in salt-resistant but not salt-sensitive SHR
Authors: Mozaffari M. S. , Jirakulsomchok S. , Shao Z. H. , Wyss J. M.
Published: December 2017

This study tested the hypothesis that NaCl-sensitive spontaneously hypertensive rats (SHR-S) display a defect in natriuretic and diuretic responses to acute volume loading that contributes to the rise in arterial pressure observed when the rats are fed a high-NaCl diet. Seven-week-old SHR-S and NaCl-resistant SHR rats (SHR-R) and normotensive (Wistar-Kyoto and Sprague-Dawley rats) were fed high- or basal NaCl diets. After 2.5 wk on the diets, preinstrumented conscious rats received an intravenous infusion (5% body wt; 0.5 ml/min) of isotonic saline, and urine was collected through a bladder catheter for 90 min. Control rats on the high-NaCl diet (compared with basal) excreted a significantly greater percentage of Na+ and volume load. In contrast, SHR-S on high-NaCl diet (compared with basal) had a very small increase in natriuretic response and no increase in diuretic response to volume expansion. The effect of renal denervation on natriuretic and diuretic responses to volume load was tested. In SHR-R on 1 and 8% NaCl diets, renal denervation had little or no effect on these responses, suggesting that renal nerves do not play a prominent role in the dietary NaCl-induced increases in the natriuretic and diuretic responses to volume load. These results demonstrate that NaCl-resistant rats rapidly adapt to diets high in NaCl content with increased natriuretic and diuretic responses to acute volume loading. The failure of SHR-S to adapt to the dietary challenge may result in volume loading and a secondary increase in arterial pressure after feeding.
Localization of endothelin-like immunoreactivity in rat kidneys
Authors: Wilkes B. M. , Susin M. , Mento P. F. , Macica C. M. , Girardi E. P. , Boss E. , Nord E. P.
Published: December 2017

The distribution of endothelin-like immunoreactivity was examined in normal rat kidneys using the immunoperoxidase technique. A specific polyclonal antibody to endothelin 1, which recognized endothelin 1 and its precursor molecule, big endothelin, was raised in rabbits. Immunoperoxidase staining revealed specific endothelin-like immunoreactivity in the renal cortex, medulla, and papilla. Immunostaining density was greatest in the renal papilla where staining was predominantly localized to the vasa rectae of the distal nephron segments. Cytoplasmic immunostaining was noted focally in collecting duct cells in the renal papilla. In the renal medulla, intense immunostaining was identified in the vasa rectae. Cortical immunostaining was localized to the endothelial surfaces of arcuate arteries, veins, arterioles and peritubular capillaries. Glomerular immunostaining followed a capillary loop distribution and appeared to be predominantly localized to endothelial cells with smaller amounts of reaction product overlying the mesangium. The most proximal portion of the proximal tubule brush border and papillary collecting duct epithelium demonstrated focal endothelin-like immunostaining. We conclude that endothelin-like immunoreactivity is widely distributed in renal tissue compatible with important autacrine and paracrine actions in the kidneys.
Vasopressin receptor-mediated endocytosis: current view
Authors: Lutz W. , Salisbury J. L. , Kumar R.
Published: December 2017

A number of peptide hormones have been shown to undergo receptor-mediated endocytosis (RME). RME involves the internalization of receptor-ligand complexes followed by delivery to an intracellular compartment, the endosome, from which ligands or receptors can be delivered to lysosomes or other cellular destinations. Vasopressin, a peptide hormone that plays a role in kidney and vascular physiology, has recently been demonstrated to undergo RME in LLC-PK1 and A10 cells, which express V2- and V1-type vasopressin receptors, respectively. Fluorescent vasopressin analogues are internalized by RME from the basolateral surface of polarized LLC-PK1 cells. The precise role of RME in vasopressin action is uncertain, but it is likely that it is involved in the desensitization of target cells by altering the number of cell surface vasopressin receptors. Alterations in the rate of RME may alter the response of the cell to vasopressin. Fluorescent and biotinylated vasopressins are useful tools for the study of this process.
Dynamics of intrarenal pressures and glomerular filtration rate after acetazolamide
Authors: Leyssac P. P. , Karlsen F. M. , Skott O.
Published: December 2017

The dynamics of intrarenal pressures, early distal tubular fluid conductivity (EDC), and renal flood flow (RBF) were studied in rats given acetazolamide (ACZ), an inhibitor of proximal reabsorption. Glomerular filtration rate (GFR) and end-proximal flow were estimated by clearances of 51Cr-EDTA and lithium. Proximal tubular pressure (Pprox) increased initially by 1.7 +/- 0.1 mmHg after ACZ, causing a decrease in the hydrostatic pressure difference across the glomerular membrane (delta P). EDC increased, and then RBF, glomerular capillary pressure (Pgc), Pprox, and star vessel pressures (Psv) dropped as a result of afferent vasoconstriction. Pprox decreased less than Pgc, resulting in a further decrease in delta P, which after 25–30 s reached a constant level 3-4 mmHg below control. After a transient increase the pressures declined to a new steady state, in which Pprox was equal to control, Pgc was decreased, and distal tubular pressure, end-proximal flow, and EDC were increased. GFR was depressed by 29%. The results indicate that the tubuloglomerular feedback mechanism controls Pgc and Pprox by afferent vasoconstriction, as well as efferent vasodilation. The data also indicate that proximal reabsorption rate is important in determining the changes in delta P by its effect on Pprox at least in the early transient phase.
Alteration in surface ion composition of cultured bone during metabolic, but not respiratory, acidosis
Authors: Chabala J. M. , Levi-Setti R. , Bushinsky D. A.
Published: December 2017

Acidosis produced by a fall in [HCO3-] (metabolic acidosis, Met) produces greater Ca efflux from cultured bone than that produced by a rise in PCO2 (respiratory acidosis, Resp). To compare the effects of Met and Resp on bone surface ion composition we measured the surface abundance of 40Ca, 23Na, and 39K in cultured bone with a scanning ion microprobe utilizing secondary-ion mass spectrometry. Neonatal mouse calvariae were incubated for 24 h in medium simulating either Met (pH = 7.193 +/- 0.034, [HCO3-] = 15.1 +/- 1.4 meq/l), Resp (pH = 7.153 +/- 0.014, PCO2 = 85.4 +/- 1.2 mmHg) or normal physiological (Ctl; pH = 7.484 +/- 0.009, [HCO3-] = 29.7 +/- 0.7, PCO2 = 39.6 +/- 0.3) conditions. The surface of Ctl at 2-nm depth is rich in Na and K relative to Ca (Na/Ca = 25.6, K/Ca = 12.0, ratios of counts/s of secondary ions). Compared with Ctl, Met produced a sharp fall in both Na/Ca (6.5, P less than 0.01) and K/Ca (4.6, P less than 0.01), whereas Resp did not alter Na/Ca (23.8) or K/Ca (15.0). Ca efflux was greater in Met (873 +/- 54 nmol.bone-1.24 h-1) than in Resp (546 +/- 71 nmol.bone-1.24 h-1, P less than 0.01), which was greater than that in Ctl (315 +/- 49 nmol.bone-1.24 h-1, P less than 0.01 vs. Met and vs. Resp).(ABSTRACT TRUNCATED AT 250 WORDS)
Expression of renal Na(+)-Ca2+ exchange activity in Xenopus laevis oocytes
Authors: Milovanovic S. , Frindt G. , Tate S. S. , Windhager E. E.
Published: December 2017

The expression of a renal Na(+)-Ca2+ exchanger by Xenopus oocytes has been investigated. Each oocyte was injected with 50 ng of poly(A)+ RNA from either rat or rabbit kidney or with an equivalent volume of water. Na(+)-Ca2+ exchange was determined 3 days after injection, by measuring Ca2+ uptake by oocytes in the presence or absence of an outwardly directed Na+ concentration gradient. To manipulate Na+ concentration gradients, oocytes were first loaded with Na+ in Ca(2+)-free medium containing 90 mM Na+ and nystatin. They were then exposed to medium containing 45Ca and either 90 mM or 0 Na+. Na(+)-free media contained (in mM) either 90 K+, 90 choline or 85 choline plus 5 K+. Oocytes injected with rat kidney poly(A)+ RNA showed a Na+ gradient-dependent Ca2+ uptake of 6.6 +/- 0.8 (SE, n = 5) pmol.oocyte-1.30 min-1. This is significantly higher than the value of 3.4 +/- 0.5 (SE, n = 5) pmol.oocyte-1.30 min-1 obtained in water-injected oocytes (P less than 0.001). Similar results were obtained using poly(A)+ RNA from rabbit kidney cortex. Neither 10 microM nifedipine nor 0.5 mM D 600 significantly affected this Ca2+ uptake. However, 90% of the Ca2+ uptake was inhibited in the presence of 0.1 mM La3+. The poly(A)+ RNA-induced Na(+)-Ca2+ exchange activity was stimulated by the presence of 5 mM K+ in the extracellular choline solution compared with choline alone. Fractionation experiments indicate that the rat kidney Na(+)-Ca2+ exchanger was encoded by poly(A)+ RNA of 3-4 kb.
Regulation of mesangial cell adhesion and shape by thrombin
Authors: Glass W. F. , Rampt E. , Garoni J. A. , Fenton J. W. , Kreisberg J. I.
Published: December 2017

Adenosine 3',5'-cyclic monophosphate (cAMP) elevation in cultured rat mesangial cells causes urokinase-dependent adhesion loss, stress-fiber fragmentation, and shape change. Thrombin cleaves single-chain urokinase (scu-PA), causing its inactivation, but not two-chain u-PA [tcu-plasminogen activator (PA)] or tissue-type PA. We tested the ability of thrombin to inhibit the effects of cAMP elevation in mesangial cells and inactivate cell-associated scu-PA. In an assay of trypsin-sensitive adhesion, 65.9% of control cells and 5.5% of cells treated with isoproterenol + methylisobutylxanthine (IM) remained adherent. In the presence of 0.01, 0.1, 1.0, and 10.0 unit/ml thrombin, 20.9, 46.6, 50.4, and 53.3%, respectively, of IM-treated cells remained attached. Thrombin also inhibited stress-fiber fragmentation and shape change. The effects of thrombin were blocked by hirudin or antithrombin III plus heparin. Direct zymography in gels containing gelatin and plasminogen revealed loss of a closely spaced pair of PA bands with thrombin treatment (1.0 unit/ml). Hirudin blocked the loss. alpha-Thrombin inactivated by diisopropyl fluorophosphate neither inhibited shape change nor caused loss of the PA bands; however, gamma-thrombin was nearly as active as native alpha-thrombin in both regards. Pretreatment of the cells with as little as 1.0 unit/ml thrombin for 1.0 min caused marked inhibition of shape change and near total loss of the slower migrating u-PA band (of the doublet). The faster migrating band was inhibited less. The results indicate that the slower migrating band represents scu-PA; the nature of the faster migrating band is less certain. Thrombin reversed the adhesion loss and shape change caused by 8-(4-chlorophenylthio)-cAMP and MIX. Thus physiological concentrations of thrombin rapidly inactivate mesangial cell scu-PA and inhibit and reverse cAMP-stimulated adhesion loss and shape change.(ABSTRACT TRUNCATED AT 250 WORDS)
Cellular mechanism of lithium-induced nephrogenic diabetes insipidus in rats
Authors: Yamaki M. , Kusano E. , Tetsuka T. , Takeda S. , Homma S. , Murayama N. , Asano Y.
Published: December 2017

One of the mechanisms by which Li evokes polyuria is thought to be impairment of arginine vasopressin (AVP)-sensitive adenylate cyclase (AdC) in cells of the renal collecting duct. To investigate how AdC is influenced by chronic administration of Li, we created nephrogenic diabetes insipidus (NDI) in rats and microdissected the medullary collecting tubule from both control and NDI rats. In the NDI group, the 10(-6) M AVP-stimulated cAMP contents failed to increase completely, and the levels were significantly lower than that of the control group (10.4 +/- 1.4 vs. 48.4 +/- 4.7 fmol/mm, P less than 0.001). Pretreatment with pertussis toxin (PT), an inhibitor of inhibitory G protein (Gi), did not affect the basal cAMP levels in both groups, although it increased AVP-stimulated cAMP production in the NDI group in a dose- and time-dependent manner. AVP-stimulated cAMP production with over 100 ng/ml PT in the NDI group reached the levels observed in the control group. Incubation with cholera toxin, an agonist of stimulatory G protein (Gs), increased the cAMP content in the two groups to almost equal levels. To exclude the possibility that prostaglandin E2 (PGE2) is involved in the cellular mechanism of Li-induced NDI, the effect of indomethacin (Indo) on PT action was examined. However, Indo (10(-5) M) did not influence either the basal or AVP-dependent cAMP contents. From these results it is suggested that Li impairs AVP-sensitive AdC not through inhibition of Gs but through activation of Gi and that PGE2 may not be involved in the cellular pathogenesis of NDI at least in the rat at the step of cAMP formation.