bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of metabolic acidosis on the PTH receptor-adenylate cyclase system of canine kidney
Authors: Bellorin-Font E. , Humpierres J. , Weisinger J. R. , Milanes C. L. , Sylva V. , Paz-Martinez V.
Published: December 2017

The phosphaturic action of parathyroid hormone (PTH) is blunted during metabolic acidosis. Previous studies suggest that the activation of renal cortical adenylate cyclase by PTH is decreased under this condition. However, the mechanisms underlying the defect are not completely defined. The present studies were designed to examine the interaction of PTH with its receptor-adenylate cyclase system in basolateral cortical membranes from dogs with metabolic acidosis. Chronic metabolic acidosis was induced in seven normal dogs. Venous blood pH decreased to 7.21 +/- 0.01 and serum bicarbonate to 12.58 +/- 0.32 meq/liter. In seven control dogs blood pH was 7.38 +/- 0.002 and serum bicarbonate was 20.14 +/- 0.26 meq/liter. The kidneys were surgically removed and basolateral membranes were prepared by differential centrifugation and ultracentrifugation in discontinuous sucrose density gradients for studies of adenylate cyclase activity and hormone-receptor binding. Metabolic acidosis resulted in a significant decrease in PTH-dependent adenylate cyclase activity (Vmax 2,119 +/- 150 pmol cAMP X mg prot-1 .30 min-1 vs. 3,548 +/- 116 in the controls). The PTH concentration giving half-maximal activation of adenylate cyclase was unchanged. However, PTH-receptor binding showed similar affinity and binding capacity in both groups of membranes. Basal enzyme activity was also similar. In the presence of the GTP analogue 5'-guanylylimidodiphosphate, PTH-dependent adenylate cyclase activity remained markedly decreased in the acidotic dog membranes compared with the controls. The ability of NaF to stimulate enzyme activity was also depressed in the membrane of acidotic dogs. Enzyme activity in the presence of Mn2+ was similar in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Characteristics of water diffusion in the rabbit proximal convoluted tubule
Authors: Berry C. A.
Published: December 2017

Water diffusion in the in vitro microperfused rabbit proximal convoluted tubule was measured to assess the transepithelial pathway for water movement and the physical characteristics of the water permeation pathway. The measured diffusive water permeability (PDW(MEASURED] was high, 5 X 10(-3) cm/s, suggesting a transcellular transport route. Simultaneously measured n-butanol permeability (PDNB(MEASURED], an index of the cytoplasmic resistance to water diffusion, was 3.6 X 10(-3) cm/s. PDW(MEASURED], PDNB(MEASURED) and the free solution diffusion coefficients for water and n-butanol were used to derive a minimum membrane PDW (PDW(MEMBRANE] of 12.5 X 10(-3) cm/s. These data suggest that at least 60% of the transepithelial resistance to water diffusion resides in the cell cytoplasm. Measurement of the temperature dependence of PDW(MEASURED) and PDNB(MEASURED) gave an apparent activation energy of PDW(MEMBRANE) that was constant between 20 and 40 degrees C at about 4.3 kcal/mol. The organic mercurial sulfhydryl reagent p-chloromercuribenzene sulfonate, which has been shown to reduce diffusive water permeability in the red cell membrane by 50%, reduced PDW(MEMBRANE) by 54% without affecting PDNB(MEASURED). These last two independent lines of evidence are consistent with water diffusion through small, aqueous, protein-bounded channels.
Renal vasodilation with ureteral occlusion and prostaglandins: attenuation by histamine H1 antagonists
Authors: Banks R. O. , Jacobson E. D.
Published: December 2017

We evaluated the effects of histamine receptor antagonists on the renal vasodilatory responses to ureteral occlusion (UO), to the intrarenal infusion of prostaglandins E2, I2, A2, D2 and E1, and to bradykinin. We also determined the effects of meclofenamic acid, a cyclooxygenase inhibitor, on histamine-induced renal vasodilation and the effects of 2-methylhistamine (2-MeH), and H1 agonist, on glomerular filtration rate (GFR) and renal blood flow (RBF). Experiments were performed on adult mongrel dogs anesthetized with pentobarbital sodium. RBF was measured with an electromagnetic flow probe. Neither UO-induced nor prostaglandin- (PG) induced renal vasodilation was affected by infusion of the histamine H2 receptor antagonist cimetidine into the renal artery at 10(-5) M/min. On the other hand, renal artery infusion of the H1 receptor antagonist chlorpheniramine (CP) at 10(-5) M/min blocked UO-induced renal vasodilation [RBF increased 34 +/- 4% (SE) prior to but only 2 +/- 2% during infusion of CP) and markedly attenuated PGI2-, PGA2-, and PGE2-induced increases in RBF (CP inhibited 64 +/- 9% of the PGE2-induced renal vasodilation). CP had less effect on the renal vasodilation associated with infusion of PGD2 or PGE1 and had no effect on the vasodilation induced by bradykinin. Infusion of exogenous histamine (1 micrograms X kg-1 X min-1) into the renal artery prior to ureteral occlusion resulted in a typical H1 + H2-mediated vasodilatory response (RBF increased 53 +/- 7%).(ABSTRACT TRUNCATED AT 250 WORDS)
PGE2, forskolin, and cholera toxin interactions in rabbit cortical collecting tubule
Authors: Nadler S. P. , Hebert S. C. , Brenner B. M.
Published: December 2017

To define further the mechanism whereby prostaglandin (PG) E2 inhibits the hydroosmotic response to ADH, we studied the interactions of PGE2 with ADH and two nonhormonal activators of adenylate cyclase, forskolin and cholera toxin, in the isolated perfused rabbit cortical collecting tubule. Forskolin increased hydraulic conductivity (LP) in a dose-dependent fashion and to a degree comparable with ADH-stimulated LP. Forskolin also augmented maximal ADH-stimulated LP, from 135 +/- 15 (SE) to 174 +/- 7 . 10(-7) cm . s-1 . atm-1. Following a 45-min lag phase, 10(-9) M cholera toxin at 37 degrees C increased LP to 107 +/- 12 . 10(-7) cm . s-1 . atm-1, a response that was stable with time. In paired studies at both 25 and 37 degrees C, PGE2 reversibly inhibited ADH-stimulated LP by 45 and 47%, respectively. However, the same protocols with PGE2 and forskolin failed to reveal any inhibitory effect of PGE2 on forskolin-stimulated LP. PGE2 reversibly inhibited cholera toxin-stimulated LP, from 124 +/- 15 to 100 +/- 15 . 10(-7) cm . s-1 . atm-1. These results support the view that PGE2 inhibits ADH-stimulated LP by inhibiting the synthesis of cAMP and suggest that this inhibition occurs at a functional site at or distal to the nucleotide regulatory protein of adenylate cyclase.
Sodium absorption and potassium secretion in rabbit colon during sodium deficiency
Authors: Turnheim K. , Plass H. , Grasl M. , Krivanek P. , Wiener H.
Published: December 2017

Reducing the daily Na intake of rabbits from approximately 4.4 to 0.1 meq/kg body wt increases plasma aldosterone levels and the rate of amiloride-sensitive Na transport in the descending colon two- to threefold. The stimulation of Na transport is a result of an increase in the maximum transport capacity of the epithelium, whereas the affinity of Na to its transport system is not altered. Simultaneous with enhanced Na absorption, there is statistically significant K secretion of 0.25 mu eq . cm-2 . h-1 under short-circuit conditions. Transepithelial current-voltage relations in the absence and presence of amiloride were used to determine the Na permeability of the apical membrane and the intracellular Na activity of the Na-transporting cells. The Na content of the amiloride-sensitive cells was estimated from the kinetics of absorptive Na tracer fluxes. The stimulation of active Na transport under conditions of dietary Na restriction is associated with parallel increases in apical membrane Na permeability and the Na content of the amiloride-sensitive cells, but the intracellular Na activity and the activity of the epithelial Na-K-ATPase are not significantly altered. Taken together, these results suggest that endogenous aldosterone increases the number of conducting Na entry sites in the apical membrane of colonic epithelium and that there is activation of additional Na pump units in the basolateral membrane, brought about by cell swelling and possibly by an increase in the fraction of epithelial cells that participate in active Na transport.
Stimulation of apical Na permeability and basolateral Na pump of toad urinary bladder by aldosterone
Authors: Palmer L. G. , Speez N.
Published: December 2017

The effect of aldosterone on Na entry and Na exit from the toad urinary bladder epithelium was studied using current-voltage analysis of the apical cell membrane to measure apical Na permeability (PNa) and the intracellular Na activity (Nac). Varying the activity of Na in the mucosal solution elicited parallel changes in active Na transport (INa) and Nac, allowing the activation of the basolateral Na pump by Nac to be evaluated. Five hours after addition of aldosterone, INa increased 130% and PNa increased 140% relative to controls. The pump rate at an arbitrarily chosen value of Nac = 4 mM [Ip(4)] increased 53%. Eighteen hours after addition of the hormone, INa increased 500%, PNa increased 680%, and Ip(4) increased 110%. ADH increased INa and PNa without changing Ip(4), whereas KCN decreased all three parameters. A change in the activation curve for the pump induced by aldosterone was also observed in the presence of mucosal nystatin, which permeabilized the apical membrane, allowing Nac to be controlled. Attempts to distinguish an effect on the maximal pump rate from one on the affinity for Na were equivocal. The results imply that aldosterone has independent stimulatory effects on Na entry across the apical membrane and Na exit across the basolateral membrane but that the stimulation of the entry process is considerably stronger.
Thromboxane synthetase inhibition improves function of hydronephrotic rat kidneys
Authors: Klotman P. E. , Smith S. R. , Volpp B. D. , Coffman T. M. , Yarger W. E.
Published: December 2017

Twenty-four hours of complete unilateral ureteral obstruction (UUO) produces intense renal vasconstriction in the rat even after release of obstruction. In the ex vivo perfused hydronephrotic rabbit kidney, bradykinin stimulates increased production of the vasoconstrictor autocoid thromboxane. In the present study, we measured basal and bradykinin-stimulated thromboxane and prostaglandin E2 production by UUO and contralateral rat kidneys perfused ex vivo. Furthermore, we evaluated thromboxane synthetase inhibition by imidazole and by two of its substituted derivatives, UK 37248 and UK 38485, in vitro. We compared these in vitro findings with in vivo measurements of renal hemodynamics and excretory function before and after the intrarenal artery administration of thromboxane synthetase inhibitors. Both basal and bradykinin-stimulated thromboxane and prostaglandin E2 production were significantly increased in hydronephrotic kidneys. Imidazole and its substituted congeners were effective inhibitors of bradykinin-stimulated thromboxane B2 production in vitro. However, the substituted imidazoles were more potent, more efficacious, and more selective for thromboxane synthetase inhibition than the parent compound. In vivo, administration of imidazole into the renal artery of the UUO kidney improved function slightly, whereas administration of UK 37248 or UK 38485 doubled renal blood flow and excretory function but did not restore them to normal. We conclude that the hydronephrotic rat kidney produces increased amounts of the vasoconstrictor eicosanoid thromboxane and that thromboxane is an important mediator of vasoconstriction in this model of disease.
Intrarenal localization of angiotensinogen mRNA by RNA-DNA dot-blot hybridization
Authors: Fried T. A. , Simpson E. A.
Published: December 2017

The renal renin-angiotensin system plays an important role in the control of renal hemodynamics and in the etiology of some types of hypertension. Angiotensinogen is the prohormone for angiotensins I and II. In the present communication, we report for the first time the presence of mRNA coding for angiotensinogen in the kidney. Indeed, the intrarenal location appears to be predominantly in the renal medulla. Additionally, an investigation of the effect of uninephrectomy on the intrarenal angiotensinogen mRNA suggests that regulation of mRNA levels in the kidney does occur.
Effects of hypercalcemia and parathyroid hormone on blood pressure in normal and renal-failure rats
Authors: Iseki K. , Massry S. G. , Campese V. M.
Published: December 2017

Hypertension is common in primary hyperparathyroidism, but the mechanisms are not clear. Significant hypercalcemia induces elevation in blood pressure (BP), whereas excessive parathyroid hormone (PTH) lowers BP. However, in chronic renal failure (CRF) and secondary hyperparathyroidism, the hypercalcemia-induced hypertension is more severe. We examined the interaction between PTH and calcium on BP in normal rats and in those with CRF. Calcium caused a dose-related rise in serum calcium and a rise in mean arterial pressure (MAP). For a comparable rise in serum calcium, the increment in MAP in parathyroidectomized (PTX) rats (7 +/- 3 mmHg) was significantly lower (P less than 0.05) than in sham PTX rats (19 +/- 7.3 mmHg). In PTX rats receiving PTH, the MAP response to calcium infusion (17 +/- 2.4 mmHg) was similar to that in the sham PTX rats. The infusion of similar amounts of calcium in CRF rats caused a greater rise in serum calcium. In CRF-PTX rats, the changes in MAP during calcium infusion were significantly lower (P less than 0.05) than in CRF-sham PTX animals, despite similar rise in serum calcium. For a comparable rise in serum calcium, the rise in MAP in CRF rats was greater than in normal rats. These data suggest that the presence of PTH plays an important permissive role for the hypertensive action of the hypercalcemia.
Effects of diuretic drugs on Na, Cl, and K transport by rat renal distal tubule
Authors: Velazquez H. , Wright F. S.
Published: December 2017

Diuretic drugs were used to characterize mechanisms involved in transporting sodium, chloride, and potassium across the wall of surface distal tubules of the rat kidney using in vivo microperfusion techniques. Both furosemide and chlorothiazide inhibited sodium and chloride absorption but did not affect the rate of potassium secretion or the transepithelial voltage. However, chlorothiazide inhibited sodium and chloride absorption more completely than furosemide and was additive to the effect of furosemide; furosemide was ineffective if chlorothiazide was already present. In contrast to the effect of furosemide, bumetanide did not affect sodium and chloride absorption but did increase potassium secretion. Amiloride reduced sodium absorption and potassium secretion without affecting net chloride absorption. These effects were additive to those of chlorothiazide. In the loop of Henle bumetanide was more effective than furosemide in inhibiting net sodium potassium and chloride absorption. It appears that cells of the distal tubule in the rat possess an Na-Cl cotransport mechanism that differs from the Na-K-2Cl cotransport mechanism found in the thick ascending limb. Sodium transport also proceeds via a conductive pathway that is inhibited by amiloride. The two modes of sodium transport, conductive and coupled to chloride, may occur in different cell types along the distal tubule.
Effect of amino acid infusion on renal hemodynamics in humans
Authors: Castellino P. , Coda B. , DeFronzo R. A.
Published: December 2017

The effect of amino acid (AA) infusion on renal hemodynamics was examined in 19 healthy subjects. Thirteen subjects participated in the first protocol (normal protein intake) and six in the second protocol (low protein intake). The first protocol consisted of three studies: study 1 (n = 13), AA were infused over 3 h to increase plasma amino acid levels two- to threefold; study 2 (n = 7), AA were infused with somatostatin and peripheral replacement of insulin, glucagon, and growth hormone; study 3 (n = 6), somatostatin was infused with basal hormonal replacement as in study 2. During study 1, glomerular filtration rate (GFR) rose by 20% (from 107 +/- 5 to 128 +/- 4 ml . 1.73 m-2 . min-1, P less than 0.001). Renal plasma flow (RPF) increased by a similar percentage (599 +/- 35 to 704 +/- 33 ml . 1.73 m-2 . min-1, P less than 0.001). When somatostatin was infused with AA (study 2), neither GFR nor RPF changed from base line. Somatostatin infusion alone (study 3) had no effect on GFR or RPF. During protocol 2, six subjects received a low-protein diet (40 g/day) for 7 days and AA were infused as per study 1. Base-line GFR (104 +/- 5 to 96 +/- 4 ml . 1.73 m-2 . min-1 and RPF (593 +/- 32 to 507 +/- 23 ml . 1.73 m-2 . min-1) both decreased (P less than 0.02) after the low-protein diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Factors affecting renal handling of sodium, hydrogen ions, and bicarbonate by the fetus
Authors: Kesby G. J. , Lumbers E. R.
Published: December 2017

Renal excretion of acid and reabsorption of bicarbonate was studied in 17 chronically catheterized fetal sheep aged 121-143 days. The rates of excretion of titratable acid (0.16-6.2 mumol/min) and ammonium (1.2-9.7 mumol/min) were variable. Urinary phosphate excretion was significantly greater (P less than 0.001) than the excretion of titratable acid. Of the filtered bicarbonate load 80-100% was reabsorbed. In 9 of the 17 fetuses net acid excretion was positive. Bicarbonate, sodium, and chloride reabsorption were related to glomerular filtration rate (GFR) (P less than 0.0005). The increase of GFR results in an increase in the excretion of titratable acid (P less than 0.001), phosphate (P less than 0.0005), and ammonium (P less than 0.001). These relationships could account for the age-dependent increase in renal excretion of acid (P less than 0.0005), ammonium (P less than 0.025), and bicarbonate reabsorption (P less than 0.0005). Arterial pH affected the rates of excretion of titratable acid (P less than 0.005), ammonium ions (P less than 0.05), and net acid (P less than 0.025). It is concluded that the fetal kidneys can excrete protons and generate bicarbonate. This ability increases with age due mainly to the concomitant increase in GFR.
Regulation of renal Na-K-ATPase in the rat: effect of uninephrectomy
Authors: Mujais S. K. , Kurtzman N. A.
Published: December 2017

This study has examined the temporal profile and the segmental localization along the rat nephron of the increase in Na-K-ATPase produced by uninephrectomy, and the role of the adrenal gland in the generation of the increase in enzyme activity. In adrenal-intact rats, an increase in Na-K-ATPase activity in the cortical collecting tubule (CCT) was observed at 1 wk (140 +/- 13% of sham, P less than 0.05) and sustained at 2 wk (140 +/- 8% of sham, P less than 0.05). In contrast, the enhancement of enzyme activity in the proximal convoluted tubule (PCT) was transient (at 1 wk: 164 +/- 20% of sham, P less than 0.05; and at 2 wk: 97 +/- 9% of sham, P greater than 0.5). No changes in Na-K-ATPase activity were observed in the other nephron segments studied: pars recta, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and medullary collecting tubule. In adrenalectomized rats, CCT enzyme activity was lower than in adrenal-intact rats (761 +/- 84 vs. 1,984 +/- 276 pmol X mm-1 X h-1, P less than 0.001) and was not altered by uninephrectomy (849 +/- 91 pmol X mm-1 X h-1, NS). We conclude that the increase in Na-K-ATPase activity following uninephrectomy is restricted to two segments of the nephron and follows a distinctive pattern in each. In the PCT a transient enhancement in enzyme activity is observed, whereas in the CCT the increase in Na-K-ATPase is sustained and requires the presence of an intact adrenal gland.
Angiotensin II effects on microvascular diameters of in vitro blood-perfused juxtamedullary nephrons
Authors: Carmines P. K. , Morrison T. K. , Navar L. G.
Published: December 2017

The purpose of this study was to determine the specific renal microvascular segments that are functionally responsive to angiotensin II (ANG II) and other vasoactive hormones. Experiments were performed on juxtamedullary tissue from captopril-treated rats during perfusion with blood at a constant pressure of 110 mmHg. Epifluorescence videomicroscopy was utilized to measure diameters of arcuate and interlobular arteries (ART), mid- (MA) and late- (LA) afferent arterioles, and efferent arterioles (EA). Norepinephrine (700 nM) significantly decreased, and sodium nitroprusside (380 nM) increased, inside diameters of all segments. Topical application of ANG II (0.01 to 1 nM) induced significant reductions in diameters of all vessel segments: ART, 17.5 +/- 2.0%; MA, 19.6 +/- 2.5%; LA, 13.5 +/- 1.5%; and EA, 16.9 +/- 2.7%. The preglomerular response to ANG II was blocked by saralasin (10 microM) and, in most cases, was dose dependent; however, an initial hypersensitivity to low ANG II doses (30% decrease in diameter) was exhibited by 38% of the preglomerular vessels studied. Under these experimental conditions, single-nephron glomerular filtration rate decreased significantly in response to 0.01 nM ANG II exposure. These observations demonstrate that physiological concentrations of ANG II can elicit receptor-dependent and reversible vasoconstriction of the juxtamedullary nephron microvasculature at both pre- and postglomerular sites.
Regulation of cytosolic free calcium concentration in cultured renal epithelial cells
Authors: Cheung J. Y. , Constantine J. M. , Bonventre J. V.
Published: December 2017

The relative contribution by intracellular organelles to the regulation of cytosolic free Ca2+ concentration was investigated in isolated cultured renal LLC-PK1 cells. Exchangeable mitochondrial and nonmitochondrial Ca2+ represented 10 and 50% of total cell Ca2+ content, respectively. In the absence of Mg2+, Ca2+ added to cells made permeable with digitonin was rapidly sequestered by the mitochondria so that basal cytosolic free Ca2+ concentration (approximately 100 nM) was reestablished within minutes. Provision of Mg2+ and ATP resulted in biphasic Ca2+ buffering behavior. A slow, low-capacity system buffered exogenous Ca2+ to a steady-state level of 50-300 nM. Ca2+ buffering at these low Ca2+ concentrations was likely by the endoplasmic reticulum, since vanadate but not ruthenium red blocked the Ca2+ buffering ability. After the endoplasmic reticulum was saturated with Ca2+, exogenous Ca2+ was buffered by a fast, high-capacity organelle to a new steady-state level of 600-1,500 nM. The high-capacity system was identified as the mitochondria, since ruthenium red but not vanadate abolished Ca2+ buffering and resulted in release of previously sequestered Ca2+. Addition of inositol-1,4,5-trisphosphate (IP3) resulted in rapid release from and reuptake of Ca2+ by the endoplasmic reticulum in digitonin-treated cells. We conclude that, under physiological conditions, most of the cell Ca2+ is stored in endoplasmic reticulum which is the functionally important organelle in buffering small changes in Ca2+ at the resting cytosolic free Ca2+ level. The mitochondrial compartment represents a high-capacity Ca2+ buffering system that may serve important physiological functions when large loads of Ca2+ are presented to the cell. In addition, IP3 mobilizes Ca2+ stored in the endoplasmic reticulum and may serve as the intracellular messenger to raise cytosolic free Ca2+ concentration transiently in response to stimuli.
Electrically neutral Na+-H+ exchange in endosomes obtained from rabbit renal cortex
Authors: Gurich R. W. , Warnock D. G.
Published: December 2017

Endocytotic vesicles (i.e., endosomes) were prepared from rabbit renal cortex following the intravenous injection of horseradish peroxidase. The endosomal population was derived from a 100,000-g pellet and was found at equilibrium in the lightest area of a sucrose density gradient. This population was separate from other organelles as determined by enzyme markers and had a 2.5-fold enrichment of horseradish peroxidase specific activity compared with the homogenate specific activity corrected for soluble horseradish peroxidase activity. The endosomes contain an oligomycin-insensitive, electrogenic H+-translocating ATPase. They also contain an electroneutral Na+-H+ exchanger. This exchanger is not inhibited by amiloride, and lithium is not a substrate for the exchanger. Lithium does inhibit the Na+-H+ exchanger when added prior to the addition of sodium. The Michaelis constant for sodium of the endosomal Na+-H+ exchanger was found to be 10.0 mM. These data indicate that a population of endosomes from rabbit renal cortex contain an electrogenic H+-ATPase and an electroneutral Na+-H+ exchanger and that the exchanger is distinct from the brush-border membrane Na+-H+ antiporter.
Regulation of single potassium ion channels from apical membrane of rabbit collecting tubule
Authors: Hunter M. , Lopes A. G. , Boulpaep E. , Giebisch G.
Published: December 2017

The regulation of K+-channel activity from the apical membrane of the rabbit cortical collecting tubule was studied using the patch-clamp technique. Using inside-out patches, channel open probability was determined as a function of calcium and barium concentration and transmembrane potential. Channel open probability was increased by raising bath (cytoplasmic) calcium concentration, with an apparent Ka of 2.4 microM. Mean channel open time also increased during this maneuver. The channel was reversibly inhibited by barium, applied to the cytoplasmic face, with an apparent Ki of 12 microM. Depolarization of the transmembrane potential increased channel open probability. With 1 mM calcium in the bath solution, the open probability was one-half maximal at -55 mV. It is concluded that this channel is the probable route for transcellular K+ secretion by the cortical collecting tubule and that procedures likely to increase intracellular calcium and/or depolarize the apical membrane will cause an increased potassium secretion.
Substrate support of medullary thick ascending limb oxygen consumption
Authors: Chamberlin M. E. , Mandel L. J.
Published: December 2017

The oxygen consumption of medullary thick ascending limb tubule suspensions was measured in the presence and absence of exogenous glucose. In the absence of exogenous glucose, the control oxygen consumption decreased 15%. Under identical conditions, the nystatin-stimulated oxygen consumption was inhibited 36%, indicating that oxidation of endogenous substrates could not meet the ATP demand of the fully stimulated Na+-K+-ATPase. Addition of inhibitors of fatty acid oxidation (bromooctanoate and tetradecylglycidic acid) further inhibited oxygen consumption, revealing that endogenous fats are oxidized in control and nystatin-stimulated states. Inhibition of endogenous carbohydrate (2-deoxy-D-glucose present) or amino acid (aminooxyacetate present) oxidation suppressed the nystatin response. In the presence of 10 mM glucose, only aminooxyacetate inhibited the nystatin-stimulated oxygen consumption, suggesting that the involvement of the malate-aspartate shuttle in redox balance during increased glycolytic flux. Addition of fatty acids or acetoacetate increased the control, nystatin-stimulated and uncoupler-stimulated oxygen consumption of tubules suspended in 10 mM glucose, indicating that these tubules oxidize exogenous fatty acids and ketones and oxidation of exogenous glucose alone may not meet all the energy demands of the tissue. Conversely, the addition of organic acids failed to enhance control oxygen consumption, possibly due to the absence of transport systems for these compounds in the medullary thick ascending limb tubules.(ABSTRACT TRUNCATED AT 250 WORDS)
Renal handling of cadmium in perfused rat kidney and effects on renal function and tissue composition
Authors: Diamond G. L. , Cohen J. J. , Weinstein S. L.
Published: December 2017

Isolated rat kidneys perfused with a Krebs-Ringer bicarbonate (KRB) solution containing 1 microM CdCl2 plus 6% substrate-free albumin (SFA) and a mixture of substrates accumulated substantially less cadmium in tissue than kidneys perfused with 1 microM CdCl2 in a protein-free KRB solution containing the same substrates: 11 vs. 205 nmol Cd/g dry wt. Decreasing the glomerular filtration rate (GFR) by occluding the ureters of kidneys perfused in the absence of albumin did not change the rate of net tissue uptake of cadmium (Cd), suggesting that the kidney can extract Cd from the peritubular capillary fluid and that net uptake of Cd is not dependent on the reabsorption of filtered Cd. The tissue accumulation of large quantities of Cd (1.8 mumol Cd/g dry wt), which established levels of non-metallothionein-bound Cd exceeding 1 mumol Cd/g dry wt, caused no changes in either GFR, perfusion flow rate, fractional reabsorption of Na+, fractional reabsorption of K+, fractional reabsorption of glucose, or free-water clearance. However, discrete changes in renal tissue K+ content were observed. Exposure to 1 microM CdCl2 resulted in a net loss of renal tissue K+ in rat kidneys perfused with substrate-enriched KRB containing 6% albumin. Exposure to 0.8 microM or 7 microM CdCl2 completely prevented K+ loss from kidneys perfused with a substrate-enriched, protein-free KRB solution.
Handling of lysozyme in isolated perfused proximal tubules
Authors: Nielsen J. T. , Nielsen S. , Christensen E. I.
Published: December 2017

Isolated, perfused proximal tubules from rabbit were used to study the luminal endocytic uptake, digestion, and transcellular transport of 125I-lysozyme. Ten tubules were perfused for 20 min with 125I-lysozyme and [14C]inulin and then with tracer-free perfusate for additional 40 min before fixation. The uptake and digestion of lysozyme was calculated per millimeter tubule length. The transfer of intact lysozyme from perfusate to the bath was measured and compared with the transfer of inulin. Five tubules were processed for electron microscope autoradiography, and the grain distribution was analyzed quantitatively. The results show that 2.7% of the perfused amount of lysozyme was taken up, and 21.3% of the absorbed protein was digested. The present experiments demonstrate that the transfer of intact lysozyme from lumen to bath is not significantly different from the transfer of inulin. The autoradiographic analysis showed that lysozyme was localized mainly in endocytic vacuoles and lysosomes after 60 min of perfusion.
Effects of parathyroid hormone on net proton flux from neonatal mouse calvariae
Authors: Bushinsky D. A.
Published: December 2017

Bone mineral buffers protons during acute metabolic acidosis; whether parathyroid hormone (PTH) augments proton buffering is controversial. To determine whether PTH augments proton buffering by bone, we cultured neonatal mouse calvariae with or without PTH (10(-8) M) for 3 h in medium that was physiologically acid (pH approximately 7.20), neutral (pH approximately 7.40), or alkaline (pH approximately 7.60). Over the entire pH range studied there was less influx of protons into calvariae treated with PTH than into control calvariae, indicating that PTH does not augment but instead inhibits proton buffering by bone. To determine whether chronic exposure to PTH is necessary to augment proton buffering, calvariae were incubated with PTH for 24 h before a 3-h culture. Calcium efflux from calvariae exposed to PTH (10(-8) M) for 24 h exceeded that of controls. When these same calvariae were recultured for 3 h in fresh medium, PTH-treated and control calvariae behaved similarly, with net efflux of protons into acid, neutral, and alkaline media. Regardless of whether PTH is added at the time of exposure to acid medium or 24 h before calvariae cultured with PTH do not buffer protons to a greater extent than controls.
Vasopressin receptors from cultured mesangial cells resemble V1a type
Authors: Jard S. , Lombard C. , Marie J. , Devilliers G.
Published: December 2017

Mesangial cells respond to vasopressin by contraction and increased prostaglandin production. The purpose of the present study is to characterize vasopressin receptors from these cells. Glomeruli were isolated from rat kidneys and plated for explant growth of mesangial cells. Membranes were prepared from cells grown for 6 wk and tested for their ability to bind [3H]vasopressin (lysine vasopressin). These membranes contained a single class of specific vasopressin binding sites [equilibrium dissociation constant (Kd) = 10 +/- 1 nM, maximal binding capacity (Bmax) = 270 +/- 7 fmol/mg protein for 5 determinations]. Vasopressin induced a dose-dependent (apparent Kact value = 2 nM) accumulation of labeled inositol phosphates in myo[3H]inositol-prelabeled mesangial cells incubated in the presence of 10 mM of Li. Conversely, vasopressin failed to alter the adenylate cyclase activity of mesangial cell membranes. Competition experiments with a series of vasopressin structural analogues that have different degrees of affinity for V2-(renal), V1a- (vascular and hepatic), and V1b- (adenohypophyseal) receptors, indicated that vasopressin receptors from rat glomerular mesangial cells resemble the V1a- receptor subtype.
Response of collecting tubule cells to aldosterone and potassium loading
Authors: Kashgarian M. , Ardito T. , Hirsch D. J. , Hayslett J. P.
Published: December 2017

To investigate the sites of action of aldosterone (Aldo) and chronic potassium loading (high K) and to determine whether dietary K can modulate collecting duct structure independent of Aldo, a morphometric study of the structure of the initial collecting tubule (ICT), the cortical collecting tubule (CCT), the medullary collecting duct in the outer stripe (MCDOS) and the inner stripe (MCDIS), and the papillary collecting duct (PCD) was carried out in a rat model in which plasma levels of Aldo were clamped at basal or physiologically elevated levels and high K could be induced without concomitant hyperaldosteronism. Elevated levels of Aldo increased cell area (Ac) and basolateral membrane surface (BLM) from control values in the ICT, CCT, and MCDOS but not in the MCDIS or PCD. High K with normal physiological levels of Aldo increased BLM in the ICT and CCT without a concomitant change in AC. No response was seen in MCDOS, MCDIS, or PCD. It is concluded that Aldo can modulate principal cell structure in the collecting duct system in segments extending from the ICT through the MCDOS in the rat and that high K independent of Aldo can influence collecting duct structure only in the ICT and CCT.
Acidification is inhibited in late proximal convoluted tubule during chronic metabolic alkalosis
Authors: Liu F. Y. , Cogan M. G.
Published: December 2017

In vivo microperfusion was used to assess the changes in the active and passive components of bicarbonate absorption in the rat late proximal tubule during chronic metabolic alkalosis. In tubules perfused with 40 mM bicarbonate, net bicarbonate absorption was inhibited and normal flow dependence was attenuated during alkalosis, compared with values in normal tubules perfused with 40 or even 25 mM bicarbonate concentrations. Under all conditions, bicarbonate back leak was small and contributed little to alterations in net bicarbonate transport, even though bicarbonate permeability was reduced by approximately 75% during chronic metabolic alkalosis and was flow dependent. Suppression of net bicarbonate absorption during chronic metabolic alkalosis was instead attributable to inhibition of proton secretion as a function of both luminal bicarbonate concentration and flow rate. At the highest level of bicarbonate delivery to yield maximal acidification rates, proton secretion during alkalosis was diminished by 38% (from 216 +/- 15 to 133 +/- 10 peq X mm-1 X min-1, P less than 0.001). In conclusion, despite extracellular volume contraction, potassium deficiency, and reduction in bicarbonate permeability during chronic metabolic alkalosis, net bicarbonate absorption in the late proximal convoluted tubule is depressed as a function of luminal bicarbonate concentration and flow rate because acidification is inhibited by hyperbicarbonatemia/alkalemia.
Ultrastructural localization of acidic compartments in cells of isolated rabbit PCT
Authors: Larsson L. , Clapp W. L. , Park C. H. , Cannon J. K. , Tisher C. C.
Published: December 2017

Acidic intracellular compartments were identified in cells of isolated perfused rabbit proximal convoluted tubules (PCT) with the weak base, N-(3-[(2,4-dinitrophenyl)amino]propyl)-N-(3-aminopropyl)methylamine, dihydrochloride (DAMP), a congener of dinitrophenol (DNP) and immunogold staining at the ultrastructural level in Lowicryl-embedded tissue. DAMP is protonated upon entrance into an acidic compartment and binds to protein. Glutaraldehyde fixation cross-links the DAMP-protein complex to maintain the complex in its original environment. Monoclonal antibodies directed against DNP cross-react with DAMP to identify its location in tissue sections. Accumulation of colloidal gold-conjugated antibodies to the monoclonal anti-DNP antibodies, indicating the presence of an acidic environment, was found in large endocytic vacuoles (diam greater than 0.5 micron) and lysosomes, and to a lesser extent in endocytic vesicles. Qualitatively, activity greater than background was not found in other organelles, including the Golgi apparatus and the cell nuclei. The specificity of the technique was tested by treating tubules with the ionophore, monensin, to collapse the intracellular pH gradient. This treatment resulted in the complete disappearance of the specific localization of colloidal gold particles. Quantitative analysis of the number of colloidal gold particles per area of cross-sectioned cell organelle demonstrated that monensin especially affected those counts obtained over large endocytic vacuoles and lysosomes. The present ultrastructural observations, therefore, identify endocytic vesicles, endocytic vacuoles, and lysosomes as acidic intracellular compartments in isolated rabbit PCT. This technique affords the opportunity to identify the location of acidic compartments along the entire nephron at the ultrastructural level.
Vasopressin increases cytosolic free calcium in LLC-PK1 cells through a V1-receptor
Authors: Burnatowska-Hledin M. A. , Spielman W. S.
Published: December 2017

We examined the effects of arginine vasopressin (AVP), parathyroid hormone (PTH), and bradykinin (BK) on the cytosolic free calcium concentration ([Ca]i) in cultured LLC-PK1 and MDCK kidney cell lines by use of the fluorescent Ca chelator fura-2. In LLC-PK1 cells, the addition of AVP but not [1-desamino-8-D-arginine]vasopressin (dDAVP, V2 agonist), PTH, or BK (10(-6) M) caused a significant increase in [Ca]i. The AVP-induced increase in [Ca]i from 61 +/- 6 to 225 +/- 44 nM (n = 7, P less than 0.01) was rapid and transient, returning to base line in 2 to 3 min. The effect of AVP was dose dependent and was present at 1 (61% increase) but not 5 min after extracellular Ca was removed. The effect of 10(-6) M AVP could be blocked with the pressor (V1) antagonist, d(CH2)5Tyr(Me)AVP, but not dDAVP. In MDCK cells, BK, but not AVP and PTH, increased [Ca]i from 146 +/- 11 to 281 +/- 31 nM (n = 9, P less than 0.001). The removal of extracellular Ca (5 min), reduced but did not abolish this effect. These results indicate that [Ca]i mobilized by activation of V1-receptors may mediate AVP-regulated function in some transporting epithelia.
Regulation of brain water and electrolytes during acute hyperosmolality in rats
Authors: Cserr H. F. , DePasquale M. , Patlak C. S.
Published: December 2017

Regulation of brain water and electrolytes during acute hyperosmolal states has been studied in anesthetized rats. Rats were injected intravenously or intraperitoneally with hypertonic NaCl, mannitol, or sucrose (hyperosmolal series) or with isotonic NaCl (isosmolal controls). Terminal plasma osmolality varied from 290 to 385 mosmol/kg and the experimental duration from 15 to 120 min. Osmotically induced water loss from brain tissue for the different protocols was only 26-78% of that predicted for ideal osmotic behavior, revealing a degree of tissue volume regulation, and the brain gained Na, Cl, and K. This gain was sufficient to account quantitatively for tissue volume regulation at 120 min of hypernatremia but not at shorter times or during mannitol- or sucrose-induced hyperosmolality. Water loss and electrolyte uptake occur simultaneously, over 30 min, which limits the degree of brain shrinkage. Results of this analysis of the time course and magnitude of tissue electrolyte gain during acute hyperosmolality form the basis for the following two studies of the volume regulatory influx of electrolyte from plasma and CSF, respectively.
Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments
Authors: Sands J. M. , Nonoguchi H. , Knepper M. A.
Published: December 2017

The inner medullary collecting duct (IMCD) is widely viewed as a single renal tubule segment with homogeneous properties. However, recent morphological and functional studies have raised the possibility that the initial and terminal parts of the IMCD may differ. To test this possibility further and to localize sites of action of arginine vasopressin (AVP) along the IMCD, we measured osmotic water permeability (Pf) and urea permeability (Purea) in isolated perfused rat IMCDs. In the initial third of the IMCD, 10 nM AVP increased Pf from 16 +/- 8 to 148 +/- 50 micron/s. The terminal two-thirds of the IMCD had a significantly higher basal Pf (70 +/- 12 micron/s), which increased to 186 +/- 25 micron/s with AVP. The initial IMCD had a relatively low basal Purea (3 +/- 1 X 10(-5) cm/s), which did not change with AVP. The terminal IMCD had a significantly higher basal Purea (17 +/- 4 X 10(-5) cm/s), which increased to a very high value (69 +/- 15 X 10(-5) cm/s) with AVP. The results support the premise that (from the point of view of vasopressin effects on water and urea transport) there are two functionally distinct parts of the inner medullary collecting duct: an initial part that resembles the cortical or outer medullary portions of the mammalian collecting duct and a terminal part that resembles the toad urinary bladder. The significance of these findings for the urinary concentrating mechanism is discussed.
Alpha- and beta-adrenergic receptors in proximal tubules of rat kidney
Authors: Sundaresan P. R. , Fortin T. L. , Kelvie S. L.
Published: December 2017

Proximal tubules were isolated from the rat kidney by collagenase digestion of the cortical tissue followed by Percoll gradient centrifugation. Microscopic and hormone-stimulated adenylate cyclase activity studies proved the purity of the preparation. [3H]Prazosin, [3H]rauwolscine, and [125I]iodocyanopindolol were used to identify and quantitate respectively the alpha 1-, alpha 2- and beta-adrenergic receptors. Proximal tubular (F4) particulate fraction was compared against other cortical nephron segment (F1, F2) fractions and the total collagenase-digested cortex particulate suspension (Ft). Proximal tubules were enriched in alpha 1- and alpha 2-adrenergic receptors compared with Ft (alpha 1-receptor, 100.4 +/- 4.5 vs. 87.4 +/- 4.9; alpha 2-receptor, 250 +/- 16.2 vs. 185.1 +/- 12 fmol/mg protein). The fractions enriched in glomeruli and distal tubular segments (F1, F2) had relatively low concentrations of alpha 1- and alpha 2-adrenergic receptors. In contrast, beta-adrenergic receptor concentration in the proximal tubules was approximately 25% of that in the Ft fraction and approximately 10% of that in the F1 fraction. Isoproterenol-stimulated adenylate cyclase activities in the different fractions corroborated well with the pattern suggested by the [125I]iodocyanopindolol binding studies. Our results suggest that whole-cortex preparation radioligand binding studies may reflect proximal tubular alpha 1- and alpha 2-adrenergic receptor changes quite well. They may, however, miss or give erroneous impressions about beta-adrenergic receptor changes occurring in different cortical nephron segments.
Mechanism of proton-induced bone calcium release: calcium carbonate-dissolution
Authors: Bushinsky D. A. , Lechleider R. J.
Published: December 2017

Protons are buffered and calcium is released by bone during metabolic acidosis. Incubation of neonatal mouse calvariae in acid medium causes net calcium efflux from bone and net proton influx into bone, just as metabolic acidosis does in vivo. To determine whether the calcium carbonate phase of bone mineral is solubilized with increasing proton concentrations, we cultured calvariae for 3 h in medium in which the saturation was varied by changing pH or calcium and phosphate concentrations. We determined the driving force for crystallization by calculating the Gibbs free energy of formation (DG). With alteration of the medium pH, calcium carbonate entry or loss from bone varied linearly with the initial DG for medium calcium carbonate (r = -0.745, n = 41, P less than 0.001) as it did with alteration of the medium calcium and phosphate (r = -0.665, n = 118, P less than 0.001). There was dissolution of calcium carbonate into medium that was unsaturated with respect to calcium carbonate, net flux ceased at saturation, and calcium carbonate entered bone from supersaturated medium, indicating that the medium is in equilibrium with the calcium carbonate phase of bone mineral. Neither the mineral phase brushite nor apatite was in equilibrium with the medium. These observations indicate that in vitro, acute proton-induced calcium efflux is due to dissolution of bone calcium carbonate.
Effects of anion transport inhibitors and ion substitution on Cl- transport in TAL of Henle's loop
Authors: Kondo Y. , Yoshitomi K. , Imai M.
Published: December 2017

To identify the mechanism of Cl- transport across the thin ascending limb of Henle's loop (TAL), we examined effects of anion transport inhibitors and ionic substitution in the isolated segments of hamsters using the in vitro microperfusion technique. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at 10(-3) M changed the NaCl diffusion voltage (Vt) to the orientation that corresponds to the decrease in the Cl(-)-Na+ permeability ratio when it was added either to the bath or to the lumen. DIDS, added to the bath or to the lumen decreased the lumen-to-bath flux coefficient for 36Cl, KCl-(1----b) (X10(-7) cm2/s), from 74.5 +/- 2.1 to 17.9 +/- 3.6 and from 77.7 +/- 3.6 to 51.1 +/- 5.4, respectively, whereas it had little effect on the flux coefficient for 22Na, KNa+(1----b). Elimination of HCO3- slightly increased rather than decreased both NaCl diffusion potential and KCl-(1----b), indicating that Cl- transport is independent of HCO3- transport. Phloretin at 10(-3) M inhibited both diffusion potential and KCl-(1----b) to a similar extent as DIDS did. KNa+(1----b) was also changed little. The inhibitory effect of phloretin was rapid and reversible. Phloridzin was ineffective. Furosemide added to the bath at 10(-3) M decreased Vt and KCl-(1----b) from 29 +/- 0.9 to 5.3 +/- 0.7 mV and from 75.7 +/- 3.9 to 62.8 +/- 4.1 X 10(-7) cm2/s, respectively; but it was ineffective when added to the lumen. Elimination of Na+ caused only a small decrease in KCl-(1----b) from 119.2 +/- 6.9 to 107.3 +/- 8.5 X 10(-7) cm2/s. Elimination of K+ and addition of Ba2+ did not change KCl-(1----b). From these observations, we conclude that Cl- transport across the TAL is distinct from Na+ and is not coupled with Na+, K+, or HCO3-.