bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium homeostasis in conscious dogs after chronic cardiac denervation
Authors: Kaczmarczyk G. , Schmidt E.
Published: December 2017

The ability to regulate renal sodium excretion after an acute reduction of total body sodium by peritoneal dialysis (PD) and subsequent dietary sodium repletion was investigated in 12 [6 intact, 6 chronically cardiac denervated (CD)] conscious, chronically instrumented dogs. For 10 days, balance experiments were performed with daily measurements of mean arterial blood pressure (MABP), right atrial pressure (RAP), and heart rate (HR). The prepared diet contained 0.5 (days 1-3 after PD) or 2.5 mmol Na.kg body wt-1.day-1 (control day and days 4-9 after PD). Control values were all similar in both groups except higher fasting plasma renin activities (PRA) were observed in the CD dogs [2.6 +/- 0.4 vs. 1.0 +/- 0.2 ng angiotensin I (ANG I).ml-1.h-1; P less than 0.05]. Days 1-4 after PD, RAP fell in both groups by 2-3 cmH2O, and renal sodium excretion decreased abruptly. PRA increased to 22.8 +/- 4.1 (intact) and 29.9 +/- 4.9 ng ANG I.ml-1.h-1 (CD dogs) (day 3 after PD). Both groups continued to retain sodium, and when it was available again, PRA decreased. After the amount of sodium lost by PD was regained, the intact dogs remained in a balanced equilibrium. In the CD dogs, PRA was still above control, and they retained sodium in excess (+ 1.9 +/- 0.1 mmol/kg body wt). We conclude that the cardiac nerves are not essential for stimulating PRA and sodium retention after an acute sodium deficit. However, the inhibition of PRA and the rapid adjustment of sodium balance during sodium repletion is impaired after cardiac denervation.
Na(+)-dependent biotin transport into brush-border membrane vesicles from rat kidney
Authors: Baur B. , Wick H. , Baumgartner E. R.
Published: December 2017

The mechanisms of biotin reabsorption in rat kidney cortex were investigated using isolated brush-border membrane vesicles. An inwardly directed Na+ gradient specifically stimulated a transient biotin overshoot. Biotin transport was not affected by a valinomycin-induced K(+)-diffusion potential, and biotin(-)-Na+ stoichiometry was found to be 1:1. As a function of concentration, the uptake showed saturation in the presence of a Na+ gradient with an apparent Michaelis constant (Km) of 55 microM and Vmax of 217 pmol.mg protein-1.25 s-1. Desthiobiotin, 250 microM, norbiotin, bisnorbiotin, thioctic acid, valeric acid, probenecid, and nonanoic acid inhibited the transport of 30 microM biotin, whereas other biotin derivatives, as well as biocytin and organic acids found in the urine of biotinidase-deficient patients, did not. Preloading of the vesicles with biotin, desthiobiotin, norbiotin, and thioctic acid in the presence of Na+ increased initial uptake of biotin from the incubation medium (trans-stimulation). Our results indicate that biotin absorption in rat kidney fulfills the criteria for a specific carrier-mediated and electroneutral Na(+)-biotin- cotransport in a 1:1 ratio. The results are discussed in context with congenital biotinidase deficiency in humans.
Cell swelling activates basolateral membrane Cl and K conductances in rabbit proximal tubule
Authors: Welling P. A. , O'Neil R. G.
Published: December 2017

The ionic basis of volume regulation was assessed in the nonperfused rabbit proximal tubule (S2 segment) by use of simultaneous measurements of tubule volume via video-optical imaging techniques and basolateral membrane voltage (Vbl) and relative ionic conductance via conventional microelectrodes. Both cell volume (9.9 +/- 0.70 nl/cm tubule length) and Vbl (-42.8 +/- 3.6 mV) remained stable in the control isotonic Ringer solution (290 mosmol/kg). When the osmolality of the bathing medium was reduced to 150 mosmol/kg, tubules swelled 72% above base line within 1 min and subsequently regulated over the course of the next 4-6 min to a steady state 20 +/- 3% above the initial base-line volume. Cell swelling was accompanied by a transient hyperpolarization of Vbl of -14.3 +/- 2.0 mV (HCO3-containing Ringer) and -10.0 +/- 0.7 mV (HCO3-free Ringer). Although the hyperpolarization was not inhibited by barium in the presence of bicarbonate buffer, addition of 2 mM Ba to a bicarbonate-free Ringer depolarized Vbl by +22 mV and abolished both the relative potassium conductance and the hyperpolarization accompanying cell swelling (delta Vbl = -4.6 +/- 0.6 mV). Furthermore, the relative conductance of K at the basolateral membrane increased from 0.16 in the isotonic control medium to 0.34 at the peak of cell swelling. Because the hyperpolarization of Vbl ensued after cells had swollen approximately 10% above base line, a modest threshold volume and time delay may be involved in triggering the volume-dependent activation of the K conductance. In parallel studies, the change in Vbl on a rapid step-change in bath Cl (49 to 4.9 mM) averaged 5.3 +/- 1.0 mV in the isotonic solution and increased to +11.3 +/- 2.1 (P less than or equal to 0.05) at the peak of cell swelling. This represented an increase in the relative Cl conductance of 0.08 to 0.20, which could only be attributed to an absolute increase in the basolateral membrane Cl conductance and not to a reduction in the other major basolateral membrane conductances. It is concluded that cell swelling results in an increase in both Cl and K conductance, which may underlie subsequent cell volume regulation.
Angiotensin II stimulation of Na(+)-H+ exchange in proximal tubule cells
Authors: Saccomani G. , Mitchell K. D. , Navar L. G.
Published: December 2017

Experiments were performed to evaluate the effect of angiotensin II (ANG II) on the sodium transport activity of isolated intact rabbit proximal tubule cells. Initial rates of 22Na(+) uptake were measured in Na+-depleted and ouabain-treated cells in the presence of an opposing H+ gradient (pHin less than pHout). ANG II (10(-12)-10(-9) M) stimulated the initial rate of 22Na+ uptake by 33 +/- 2%, whereas amiloride (0.5 mM) inhibited both basal and ANG II-stimulated 22Na+ uptake. ANG II-stimulated rate of 22Na+ uptake was inhibited by the receptor antagonist saralasin. Additional experiments were performed to evaluate the effect of ANG II on the rate of recovery of pHin in acid-loaded proximal tubule cells. Cells were acid loaded by an NH4Cl pulse in the presence of the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. ANG II increased the initial rate of intracellular alkalinization, and this effect was inhibited by amiloride (1.0 mM). ANG II stimulation increased the Vmax of H+ efflux (from 0.53 +/- 0.02 to 0.64 +/- 0.04 pH units/min) without changing the Km for extracellular Na+. The present findings indicate that physiological concentrations of ANG II stimulate an amiloride-sensitive Na+-H+ antiport in proximal tubule cells.
Effects of prostacyclin on the cAMP system in cultured rat inner medullary collecting duct cells
Authors: Veis J. H. , Dillingham M. A. , Berl T.
Published: December 2017

Rat inner medullary collecting tubule (RIMCT) cells produce arachidonate derivatives including prostacyclin (PGI2). In RIMCT cells, PGI2 causes a dose-dependent increase in adenosine 3',5'-cyclic monophosphate (cAMP; fmol/micrograms protein) from a basal level of 15.6 +/- 1.7 to 32.4 +/- 5.7 at 0.3 microM, 63.3 +/- 8.3 at 3 microM, and 103.5 +/- 9.4 at 30 microM PGI2. At concentrations of arginine vasopressin (AVP) from 10(-7) to 10(-9) M, cAMP was greater in the presence than absence of 3 microM PGI2, suggesting independent sites of action. To assess whether the PGI2 effect is mediated by the prostaglandin E2 (PGE2) receptor, desensitization studies were performed. A 6-h preincubation with 10 microM PGE2 blunted the response to 3 microM PGE2 by 90 +/- 2% but the PGI2 response was decreased by only 31 +/- 5%, P less than 0.001. Carbaprostacyclin (carba-PGI2), a stable analogue of PGI2, blunted the cAMP response to PGI2 by 94 +/- 3% but to PGE2 by only 46 +/- 7%, P less than 0.005. The postreceptor effect of PGI2 on components of the adenylate cyclase was examined. The response to forskolin was markedly potentiated by PGI2. PGI2 (3 microM) caused an increase in cAMP of 67 fmol/micrograms over basal in the absence of forskolin, of 164 fmol/micrograms at 10(-7) M forskolin, of 386 fmol/micrograms at 10(-6) M forskolin, and of 563 fmol/micrograms at 10(-5) M forskolin. The response of PGI2 was likewise potentiated by forskolin. Water permeability alone or in response to AVP in isolated perfused inner medullary collecting tubules was not affected by carba-PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)
Myo-inositol does not modulate PI turnover in MDCK cells under hyperosmolar conditions
Authors: Shayman J. A. , Wu D.
Published: December 2017

The relationship between free cellular myo-inositol concentration and phosphatidylinositol turnover was evaluated in Madin-Darby canine kidney (MDCK) cells under isosmolar and hyperosmolar conditions. MDCK cells exposed to high extracellular sodium chloride were documented to increase their free myo-inositol content as measured by gas-liquid chromatography in both a time- and concentration-dependent manner. Measurement of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate mass failed to reveal changes under conditions where the myo-inositol concentration was more than threefold higher compared with control conditions. CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase activity, measured in plasma membranes from MDCK cells grown under control and hyperosmolar conditions, was kinetically similar with comparable observed Michaelis constant (Km) and maximal rate of enzyme reaction. Moreover, the apparent Km was significantly below the estimated intracellular myo-inositol concentration consistent with exposure of the transferase to saturating concentrations of myo-inositol under both conditions. Finally, bradykinin-stimulated myo-inositol trisphosphate mass was measured by use of a competitive binding assay. Both basal and hormone-stimulated myo-inositol 1,4,5-trisphosphate levels were not significantly different under control and hyperosmolar conditions. These data indicate that bulk free myo-inositol content is unlikely to regulate phosphatidylinositol turnover and myo-inositol trisphosphate formation under hyperosmolar conditions.
Kinetics and Na independence of amino acid uptake by blood side of perfused sheep choroid plexus
Authors: Segal M. B. , Preston J. E. , Collis C. S. , Zlokovic B. V.
Published: December 2017

The kinetic constants and sodium independence of amino acid uptake by the basolateral face (blood side) of the isolated perfused choroid plexus of the sheep were investigated. Uptake of 3H-labeled L-alanine, glycine, L-glutamine, L-leucine, L-glutamate, and L-lysine was not significantly inhibited when the sodium level of the perfusate was lowered to less than 6 mM. The cerebrospinal fluid (CSF) secretion rate was, however, reduced by 65.6%. The Michaelis constants for L-serine, glycine, L-phenylalanine, L-glutamate, and L-arginine were measured and varied between 25.4 microM for L-arginine and 2.6 microM for L-glutamate. The Vmax for the amino acids varied by about the same range as Km, with L-serine having the highest Vmax of 28.8 nmol.min-1.g-1, and L-phenylalanine having the lowest at 4.6 nmol.min-1.g-1. At normal plasma concentrations of 30-155 microM for individual amino acids, the carriers for glycine, L-phenylalanine, and L-glutamate would be almost fully saturated (greater than 90%), while those for L-serine and L-arginine would be greater than 60% saturated. This restricted uptake of amino acids by the basolateral face of the choroid plexus, coupled to clearance of these substances out of the CSF (5), could account for the low levels in the CSF compared with that in the plasma.
Kinetics of system A amino acid uptake by muscle: effects of insulin and acute uremia
Authors: Maroni B. J. , Haesemeyer R. W. , Kutner M. H. , Mitch W. E.
Published: December 2017

We found that acute renal failure (ARF) depresses maximal responsiveness to insulin of system A amino acid transport in muscle [Maroni, Karapanos, and Mitch. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F74-F80, 1986]. To examine mechanisms for this change, we measured the kinetics of system A in response to insulin and ARF in epitrochlearis muscle. Insulin stimulation increased the diffusion constant (KD) approximately twofold in muscles from both ARF and control rats, suggesting that insulin can modulate nonsaturable alpha-(methylamino)isobutyrate uptake. Compared with basal values, insulin did not significantly change maximal transport velocity (Vmax) in ARF or control rats; however, insulin decreased Michaelis constant (Km) by 79 and 63%, respectively. Inhibition of protein synthesis with cycloheximide did not prevent stimulation of system A by insulin. Acute uremia had no effect on nonsaturable uptake or basal system A kinetics. However, ARF produced opposing effects on insulin-stimulated system A kinetics. There was a approximately 47% decrease in Vmax, which was partially offset by a 66% reduction in Km. In summary, both insulin and ARF modulate system A kinetics by mechanisms that are independent of protein synthesis.
AVP reduces transepithelial resistance across IMCD cell monolayers
Authors: Mishler D. R. , Kraut J. A. , Nagami G. T.
Published: December 2017

The inner medullary collecting duct (IMCD) is an important site of action for arginine vasopressin (AVP). To examine the mode of action of AVP in this segment, we measured the change in transepithelial resistance of cultured rat IMCD cells grown to confluence on collagen-coated Millicell culture plate inserts in response to AVP. Resistance was measured by use of an EVOM voltage-ohm meter. AVP at 10(-11)-10(-8) M caused a fall in resistance of 6.9 +/- 1.3 to 14.0 +/- 1.4 omega.cm2 (P less than 0.05 to less than 0.01 vs. no AVP), which was reversed by removal of AVP or addition of 10(-6) M amiloride. Pretreating the apical surface of IMCD cells with trypsin had no effect on resistance but totally prevented the antidiuretic hormone-induced fall in resistance. Pretreating the apical surface with trypsin and amiloride did not prevent the fall in resistance to AVP. Addition of 10(-9) M AVP or 10(-6) M forskolin increased 2-min adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by 55 or 96%, respectively. Stimulation of endogenous cAMP accumulation by forskolin or the addition of exogenous 8-bromo-cAMP caused no change in resistance. To examine the relationship between intracellular calcium [( Ca2+]i) and AVP action, the response of [Ca2+]i to AVP was measured by use of fura-2. AVP induced no change in [Ca2+]i in IMCD cells in suspension, on glass cover slips, or on permeable supports. Ionomycin (25 nM) increased [Ca2+]i in IMCD cells and lowered resistance across monolayers, but the fall in resistance was not blocked by amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)
Heterogeneity of Pi transport by BBM from superficial and juxtamedullary cortex of rat
Authors: Levi M.
Published: December 2017

There is an axial heterogeneity for proximal tubular phosphate (Pi) transport. Sodium gradient-dependent Pi transport (Na-Pi cotransport) is greater in the proximal convoluted tubule (PCT) than in the proximal straight tubule (PST). In brush-border membrane vesicles (BBMV) isolated from the superficial cortex (SC-BBM) and the juxtamedullary cortex (JMC-BBM), we have also found a heterogeneity in BBM Na-Pi cotransport activity. The greater Na-Pi cotransport activity in SC-BBM was not caused by an alteration in the stoichiometry (2 Na+:1 HPO42-) or the activation of the Na-Pi cotransporter by Na+ (KNa = 37 in SC-BBM vs. 44 mM Na in JMC-BBM, P = NS, not significant). Despite a higher affinity for Pi in JMC-BBM (KPi = 156 in SC-BBM vs. 105 microM Pi in JMC-BBM, P less than 0.001), the Vmax for Na-Pi cotransport was higher in the SC-BBM (Vmax = 2,939 in SC-BBM vs. 1,421 pmol Pi.5 s-1.mg BBM protein-1 in JMC-BBM, P less than 0.001). Na gradient-dependent Pi-protectable phosphonoformic acid (Na-PFA) equilibrium binding studies revealed that the higher Vmax for Na-Pi cotransport in SC-BBM was in part due to a higher number of Na-Pi cotransport units (Bmax = 4.24 in SC-BBM vs. 2.67 nmol PFA.30 min-1.mg BBM protein-1 in JMC-BBM, P less than 0.001). In addition, the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (rDPH), which is inversely related to fluidity, was lower in SC-BBM (rDPH = 0.247 in SC-BBM vs. 0.254 in JMC-BBM, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron
Authors: Cheval L. , Doucet A.
Published: December 2017

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5–;30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20–30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.
Dopamine1-receptor blockade inhibits ANP-induced phosphaturia and calciuria in rats
Authors: Ortola F. V. , Seri I. , Downes S. , Brenner B. M. , Ballermann B. J.
Published: December 2017

Atrial natriuretic peptide (ANP) is known to enhance the excretion of Pi and Ca, solutes reabsorbed primarily by the proximal tubule. Previous studies have shown that proximal tubule Na transport is inhibited by dopamine (DA), and that the natriuretic action of ANP is blunted by DA-receptor blockade. However, alterations in Na reabsorption cannot localize ANP or DA action to a specific nephron site. Therefore, the possibility that DA mediates the apparent proximal tubule effects of ANP was investigated with the use of Pi and Ca as proximal tubule markers. ANP was infused into normal rats in the presence and absence of specific DA-receptor antagonists, and Na, Pi, and Ca excretion rates were determined. ANP enhanced Na, Pi, and Ca excretion at doses that failed to alter glomerular filtration rate and mean arterial pressure (MAP). DA1-receptor blockade significantly blunted the influence of ANP on urinary Na, Pi, and Ca excretion, whereas DA2-receptor blockade was without effect. MAP and inulin and p-aminohippurate (PAH) clearances remained stable during DA-receptor blockade. Because endogenous ANP levels are elevated in rats with remnant kidneys, and because blockade of endogenous ANP reduces Pi and Ca as well as Na excretion in this model, the effect of DA1-receptor blockade on solute excretion was also examined in rats with 5/6 nephrectomy. DA1-receptor blockade significantly reduced absolute and fractional Na, Pi, and Ca excretion in rats with 5/6 nephrectomy, in the absence of measurable changes in MAP, inulin, or PAH clearance.(ABSTRACT TRUNCATED AT 250 WORDS)
Prostaglandin influences on afferent arteriolar responses to vasoconstrictor agonists
Authors: Inscho E. W. , Carmines P. K. , Navar L. G.
Published: December 2017

The present study was designed to evaluate, at the microvascular level, the ability of prostaglandins E2 (PGE2) and I2 (PGI2) to counteract the afferent vasoconstrictor effects of angiotensin II (ANG II) and norepinephrine (NE). The renal microvasculature of rats pretreated with captopril and indomethacin was studied directly by use of the in vitro blood-perfused juxtamedullary nephron technique combined with videomicroscopy. Afferent arterioles averaged 22.7 +/- 0.6 microns ID (n = 59) under control conditions. Topical administration of PGE2 revealed a concentration-dependent afferent vasoconstriction, whereas PGI2 (10(-7) to 10(-5) M) failed to significantly alter afferent arteriolar diameter. Afferent arterioles constricted during exposure to either 10(-9) M ANG II (-15 +/- 3%, n = 13) or 10(-7) M NE (-19 +/- 3%, n = 13). Addition of PGE2 (10(-6) M) to the bathing solution enhanced the vasoconstrictor influences of ANG II and NE by an additional 18 +/- 6 and 13 +/- 4%, respectively. In contrast, while 10(-6) M PGI2 had no effect on ANG II-induced afferent vasoconstriction, it did produce a 30% attenuation of NE-induced constriction. Furthermore, pretreatment of the tissue with 10(-6) M PGI2 prevented development of NE-induced afferent vasoconstriction. Thus, although local tissue prostanoid concentrations are unknown, it appears that low micromolar concentrations of PGE2 elicit an afferent arteriolar constriction that can accentuate the vascular actions of ANG II and NE on rat juxtamedullary afferent arterioles. In contrast, PGI2 can counteract the vasoconstrictor response to NE, but not ANG II, in this experimental setting.
Early effects of aldosterone on Na-K pump in rat cortical collecting tubules
Authors: Fujii Y. , Takemoto F. , Katz A. I.
Published: December 2017

Sustained exposure to aldosterone (Aldo) increases the abundance and activity of the Na-K pump in cortical collecting tubules (CCT). However, the onset and mechanism of the early interaction of Aldo with the CCT pump, especially in adrenal-intact animals, are unclear. We evaluated the short-term effects of the hormone on Na-K-adenosinetriphosphatase (ATPase) activity and on ouabain-sensitive 86Rb uptake, a measure of the transporting rate of the pump, in microdissected CCT from adrenal-intact rats. Incubation with Aldo (10(-8) M, 2 h) had no effect on Na-K-ATPase activity (Vmax), whereas it produced at least a twofold increase in 86Rb uptake. This effect was generated by physiological concentrations of the hormone (threshold 10(-10) M; apparent K1/2 approximately 10(-9) M), after a short lag of less than or equal to 30 min. Incubation with Aldo in the presence of amiloride or nystatin or in a Na-free medium (choline chloride) did not prevent the enhanced 86Rb uptake seen after Aldo alone; possible interpretations of these observations are discussed. We conclude that Aldo produces a rapid stimulation of pump function in CCT that precedes its induction of new pump synthesis; the physiological significance of this effect is suggested by its occurrence in tubules from adrenal-intact animals within the time frame and concentration range of the hormone's effects on electrolyte transport.
Extracellular matrix regulates proliferation and phospholipid turnover in glomerular epithelial cells
Authors: Cybulsky A. V. , Bonventre J. V. , Quigg R. J. , Wolfe L. S. , Salant D. J.
Published: December 2017

To understand how glomerular epithelial cell (GEC) growth might be regulated in health and disease, we studied the effects of growth factors and extracellular matrix on proliferation and membrane phospholipid turnover in cultured rat GECs. In GECs adherent to type I collagen matrix, epidermal growth factor (EGF), insulin, and serum stimulated DNA synthesis and increased cell number. In addition, GECs proliferated when adherent to type IV collagen, but not to laminin or plastic substrata. Attachment of GECs to the substrata that facilitated proliferation (types I or IV collagen) produced increases in 1,2-diacylglycerol (DAG), an activator of protein kinase C (PKC). Increased DAG was associated with hydrolysis of inositol phospholipids and an increase in inositol trisphosphate and was not dependent on the presence of growth factors. After PKC downregulation (by preincubation with a high dose of phorbol myristate acetate), DNA synthesis was enhanced in GECs adherent to collagen. Thus contact of GECs with collagen matrices is required for serum, EGF, or insulin to induce proliferation. Collagen matrix also activates phospholipase C. As a result, the DAG-PKC signaling pathway desensitizes GECs to the mitogenic effects of growth factors and might promote cell differentiation. Understanding the interaction between GECs, growth factors, and extracellular matrix may elucidate the mechanisms of proliferation during glomerular injury.
Expression of the Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes
Authors: Burnham C. E. , Kidd J. , Palfrey H. C.
Published: December 2017

The Na(+)-K(+)-2Cl- cotransporter (NKCCT) is important in mediating net salt transport in a variety of cells. As a first step in obtaining structural information on this transport system, we attempted to express it in Xenopus laevis oocytes by injection of poly(A)+ mRNA from rat kidney. Four days after injection, batches of oocytes were tested for 86Rb uptake in presence and absence of 10 microM bumetanide to distinguish that fraction of influx mediated by NKCCT. In the absence of bumetanide, the oocytes formed a bimodal distribution with respect to 86Rb uptake, with some oocytes accumulating significantly more 86Rb than others. In the presence of bumetanide, or when Na was replaced with choline, that group of oocytes taking up more 86Rb did not appear. Sham-injected oocytes did not accumulate sufficient 86Rb to be distinguishable above background. Taken together, these data suggest that some of the mRNA-injected oocytes expressed the rat renal NKCCT. Crude poly(A)+ mRNA was separated by acid-urea agarose gel electrophoresis, and fractions were injected into oocytes. One fraction corresponding to messages of approximately 7 kilobases in length induced a bumetanide-sensitive 86Rb influx resembling that seen with total mRNA. Poly(A)+ mRNA fractionated on sucrose density gradients gave similar results. It is concluded that the rat kidney NKCCT has been expressed in Xenopus oocytes from a high molecular weight fraction of poly(A)+ mRNA.
Segmental distribution of epidermal growth factor binding sites in rabbit nephron
Authors: Breyer M. D. , Redha R. , Breyer J. A.
Published: December 2017

The kidney possesses epidermal growth factor (EGF) receptors and is a major site of synthesis for the EGF precursor, prepro-EGF. To examine the segmental localization of EGF receptors in the rabbit kidney, we characterized 125I-labeled EGF binding to micro-dissected rabbit nephron segments. Specific binding constituted 70-80% of total binding and was saturable with an apparent Kd of 8 nM. Kinetic studies (0 degrees C) revealed an association t1/2 of 20.7 min and a dissociation t1/2 of 27 min. Competition studies revealed that 125I-EGF binding was inhibited by unlabeled EGF or its homologue transforming growth factor-alpha, but not by parathyroid hormone or insulin. Mapping studies showed specific 125I-EGF binding (attomoles per centimeter) was highest in proximal straight tubules, followed by proximal convoluted tubules, cortical collecting ducts, inner medullary collecting ducts, outer medullary collecting ducts, and distal convoluted tubules. Specific binding to glomeruli was also observed. Interestingly, no specific binding of 125I-EGF to thick ascending limbs, a site of EGF precursor synthesis, was observed. These studies suggest potential sites of action for EGF in the rabbit kidney.
Modulation of osmolytes in MDCK cells by solutes, inhibitors, and vasopressin
Authors: Heilig C. W. , Brenner R. M. , Yu A. S. , Kone B. C. , Gullans S. R.
Published: December 2017

MDCK cells accumulate organic osmolytes in response to hyperosmotic NaCl-supplemented medium. We examined time course and inhibitor sensitivity of myo-inositol, sorbitol, and glycerophosphorylcholine (GPC) accumulation in MDCK cells exposed to hyperosmotic NaCl-, D-glucose-, or mannitol-supplemented media. In NaCl medium, cells preferentially accumulated inositol and GPC. In comparison, in glucose medium cells preferentially accumulated sorbitol and GPC. Inositol demonstrated a late (72-96 h) accumulation in glucose medium, although less than in NaCl medium. Mannitol medium did not significantly stimulate accumulation of any of these three osmolytes at 24 h, suggesting that hyperosmolality alone is not sufficient stimulus for their accumulation in this time frame. GPC accumulation was very rapid in glucose medium, and fell to the level induced by NaCl medium at 96 h (approximately 50 nmol/mg protein). Inositol and sorbitol accumulated more gradually, each reaching greater than 400 nmol/mg protein after 96 h. Sorbitol was still accumulating at 96 h, whereas inositol plateaued at 72-96 h. Phlorizin or sorbinil blocked accumulation of inositol or sorbitol, respectively. Sorbitol and GPC accumulation in glucose medium were partially inhibited in absence of serum or in presence of 1 microM vasopressin. Thus NaCl and glucose appear to stimulate specific cellular mechanisms responsible for accumulation of inositol, sorbitol, and GPC in MDCK cells. This accumulation is also modulated by constituents of serum.
Sodium and calcium share the electrogenic 2 Na(+)-1 H+ antiporter in crustacean antennal glands
Authors: Ahearn G. A. , Franco P.
Published: December 2017

Na uptake by short-circuited epithelial brush-border membrane vesicles of Atlantic lobster (Homarus americanus) antennal gland labyrinth was Cl independent, amiloride sensitive, and stimulated by a transmembrane H+ gradient [( H]i greater than [H]o; i is internal, o is external). Na influx (2.5-s uptake) was a sigmoidal function of [Na]o (25-400 mM) when pHi = 5.0 and pHo = 8.0 and followed the Hill equation for binding cooperatively [apparent maximal influx (Jmax) = 271 nmol.mg protein-1.s-1, apparent affinity constant for Na (KNa) = 310 mM Na, and Hill coefficient (n) = 2.41]. Amiloride acted as a competitive inhibitor of Na binding to two external sites with markedly dissimilar apparent amiloride affinities (Ki1 = 14 microM; Ki2 = 1,340 mM). Electrogenic Na-H antiport by these vesicles was demonstrated by equilibrium-shift experiments in which an imposed transmembrane electrical potential difference was the only driving force for exchange. A transport stoichiometry of 2 Na to 1 H was demonstrated with the static-head technique in which a balance of driving forces was attained with 10:1 Na gradient and 100:1 H gradient. External Ca, like amiloride, was a strong competitive inhibitor of Na-H exchange, acting at two sites on the outer vesicular face with markedly different apparent divalent cation affinities (Ki1 = 20 microM; Ki2 = 500 microM). Ca-H exchange by electrogenic Na-H antiporter was demonstrated in complete absence of Na by use of an outward H gradient in presence and absence of amiloride. Both external amiloride (Ki1 = 70 microM; Ki2 = 500 microM) and Na (Ki1 = 12 mM; Ki2 = 380 mM) were competitive inhibitors of Ca-H exchange. These results suggest that the electrogenic 2 Na-1 H exchanger characterized for this crustacean epithelium may also have a role in organismic Ca balance.
Angiotensin II induces cellular hypertrophy in cultured murine proximal tubular cells
Authors: Wolf G. , Neilson E. G.
Published: December 2017

In the present study we demonstrate that a murine proximal tubular cell line (MCT cells), expressing angiotensin II (ANG II) receptors [dissociation constant (Kd) = 0.89 nM; receptor density (R0) = 46,900 receptors/cell] in culture, can be induced to hypertrophy after the daily addition of exogenous ANG II (10(-8) M). This hypertrophic response was characterized by an increase in total cellular protein content, by an enhancement of [3H]leucine incorporation into precipitable proteins, and by an augmentation in cell size by cytofluorography. This ANG II effect producing MCT cell enlargement was demonstrable in the absence of cellular proliferation. Proliferation of MCT cells, however, could be induced by epidermal growth factor (EGF) or platelet-derived growth factor (PDGF), and pretreatment of rested MCT cells with ANG II further enhanced EGF-induced cell division. ANG II-induced hypertrophy in MCT cells was factor specific, in that it could be blocked with saralasin, and not induced by angiotensin I (ANG I). This hypertrophic response was also independent of prostaglandin E2 synthesis but was transducible by pertussis toxin-sensitive G proteins and involved, to some extent, the activation of Na(+)-H+ exchange. ANG II, as well as EGF and/or PDGF, moreover, could induce the cellular oncogenes c-fos, c-myc, c-N-ras, but not c-cis, which suggests that early gene activation is probably not a specific prerequisite for hypertrophy. Our findings demonstrate that ANG II, in culture, can be a single-factor event capable of inducing hypertrophy in proximal tubular cells.
12-HETE modulates Na-coupled uptakes in proximal tubular cells: role of diacylglycerol kinase inhibition
Authors: Friedlander G. , Le Grimellec C. , Sraer J. , Amiel C.
Published: December 2017

Inhibition of diacylglycerol (DAG) kinase, an alternative way to increase the cellular DAG level, was shown to reproduce, in renal proximal tubular cells, the inhibitory effect of protein kinase C (PKC) activators on Na-Pi and Na-alpha-methyl-D-glucopyranoside (MGP) cotransport. To evaluate whether 12S-hydroxyeicosatetraenoic acid (12S-HETE) or 12R-HETE, a DAG kinase inhibitor in endothelial cells, has a similar effect in proximal tubular cells, we studied the influence of this lipoxygenase product on Na-dependent uptake of Pi, MGP, and alanine, as well as on [14C]arachidonate-DAG content and [32P]phosphatidic acid (PA) content in rabbit proximal tubular cells grown as a primary culture. 12-HETE (1-10 microM) decreased [32P]PA content and stimulated [14C]DAG accumulation in a concentration-dependent manner. The labeled phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin contents were not modified. 12-HETE also decreased DAG kinase activity of cell membranes. 12-HETE (10 microM) decreased the maximum velocity of Pi uptake by 36% and that of MGP uptake by 44% but did not affect alanine uptake. The effect of 12-HETE on transport was potentiated by calcium ionophore A23187 and was blunted by PKC downregulation. The effects of 12-HETE on lipid composition and transport were mimicked by R 59022, a pharmacological DAG kinase inhibitor. Neither arachidonic acid nor prostaglandin E2 reproduced the effects of 12-HETE. We conclude that in the proximal tubule, 12-HETE affected Na-dependent Pi and MGP cotransport through stimulation of PKC and that 12-HETE-induced activation of PKC is mediated by the inhibition of DAG kinase.
Urodilatin, a natriuretic factor from kidneys, can modify renal and cardiovascular function in men
Authors: Saxenhofer H. , Raselli A. , Weidmann P. , Forssmann W. G. , Bub A. , Ferrari P. , Shaw S. G.
Published: December 2017

Urodilatin is a newly identified analogue of human atrial natriuretic factor-(99-126) [ANF-(99-126)], which has recently been isolated from human urine and has 32 amino acid residues [ANF-(95-126)]. To investigate renal and cardiovascular effects in men, eight healthy subjects received injections of 25, 50, and 100 micrograms urodilatin iv compared with 50 micrograms ANF-(99-126) and placebo. Blood pressure decreased (P less than 0.05) after 50 micrograms ANF-(99-126), whereas urodilatin lowered diastolic blood pressure only at the highest dose (P less than 0.01). Heart rate increased (P less than 0.05-0.01) dose dependently after urodilatin injections. Glomerular filtration rate rose after 100 micrograms (from 120 +/- 3 to 156 +/- 7 ml.min-1.1.73 m-2, P less than 0.001) and 50 micrograms urodilatin (from 116 +/- 7 to 149 +/- 13 ml.min-1.1.73 m-2, P less than 0.01) but not after 25 micrograms urodilatin, ANF-(99-126), or placebo. Effective renal plasma flow was not significantly modified. Diuresis and excretion of sodium, chloride, and guanosine 3',5'-cyclic monophosphate increased (P less than 0.001) dose dependently; effects of 25 micrograms urodilatin equaled those of 50 micrograms ANF-(99-126). Plasma renin, aldosterone, and catecholamines were unchanged. We conclude that urodilatin can acutely modify renal and cardiovascular function in men and seems to exert more potent renal effects than ANF-(99-126).
Factors affecting the ratio of different organic osmolytes in renal medullary cells
Authors: Moriyama T. , Garcia-Perez A. , Burg M. B.
Published: December 2017

Renal medullary cells contain high concentrations of sorbitol, inositol, glycerophosphorylcholine (GPC), and betaine, which balance the variably high osmolality of extracellular NaCl. We found that PAP-HT25 (rabbit renal medullary) cells in tissue culture increase their content of all four when medium osmolality is increased by adding NaCl and urea. However, this requires that betaine be added to medium in addition to customary constituents. Some factors affecting the mix of organic osmolytes in these cells during hypertonicity are as follows. 1) Urea in medium increases cell GPC and tends to decrease others, particularly betaine. 2) With small increases in medium NaCl, intracellular inositol is highest, whereas sorbitol predominates with large NaCl increases. 3) When osmolality is suddenly decreased, these four organic osmolytes exit rapidly from cells, but in differing relative amounts (betaine much greater than sorbitol greater than inositol much greater than GPC). 4) Altering cell betaine levels (by varying betaine in medium) causes reciprocal changes in cell sorbitol (by affecting aldose reductase activity) and vice versa, whereas inositol and GPC are less affected. 5) Raising medium glucose concentration (from which sorbitol is synthesized) increases cell sorbitol and decreases cell inositol and betaine. 6) Decreasing the amount of GPC in cells (by removing choline from medium) causes small changes in betaine and sorbitol, but not in inositol. Changing the amount of inositol does not affect the others. Similar interrelations may operate in vivo to vary the mix of organic osmolytes in renal medulla.
Bladder contributes to eicosanoids excreted in urine
Authors: Reyes A. A. , Klahr S.
Published: December 2017

The excretion of eicosanoids was examined in urine collected from the bladder or the ureter in normal rats. We found that the excretion of thromboxane B2 (the stable metabolite of thromboxane A2), prostaglandin E2, and 6-ketoprostaglandin F1 alpha was significantly greater in urine collected from the bladder than in urine collected from the ureter. This suggests that the bladder contributes significantly to the amount of eicosanoids present in the urine.
Effect of acute pH change on mitochondrial glutamine transport
Authors: Sastrasinh M. , Sastrasinh S.
Published: December 2017

The uptake of [3H]glutamine in submitochondrial particles (SMP) was measured at varying medium pH. Glutamine transport, but not glutamine binding, was inversely related to medium pH (range 6.5-8.5). Glutamine uptake was highest at medium pH of 6.5 (4.59 +/- 0.4 pmol.mg-1.30 s-1) and lowest at medium pH of 8.0 (1.99 +/- 0.3 pmol.mg-1.30 s-1). The effect of medium pH on glutamine transport was rapidly reversible. Changes in pH gradient (delta pH) had no influence on the rate of glutamine transport. Kinetics of mitochondrial glutamine transport was studied at pH 6.5 and 8.5 to further elucidate the mechanism by which pH alters glutamine transport. Glutamine concentration at half-maximal velocity ([S]0.5) was 9.07 and 13.32 mM at pH 6.5 and 8.5, respectively (P less than 0.02). The maximal velocity (Vmax) was 1,417.72 +/- 185.69 pmol.mg-1.15 s-1 at pH 6.5 and 910.95 +/- 192.85 pmol.mg-1.15 s-1 at pH 8.5 (P less than 0.01). Thus both the affinity and the Vmax of the transport system were enhanced in acidic pH. These data imply that intracellular pH has influence over glutamine transport into the mitochondrial matrix and, consequently, renal NH3 production. Glutamine delivery to the matrix via changes in cytosolic pH may be one of the regulators for ammoniagenesis during acute acid-base disorders.
Evidence for a preglomerular oxygen diffusion shunt in rat renal cortex
Authors: Schurek H. J. , Jost U. , Baumgartl H. , Bertram H. , Heckmann U.
Published: December 2017

Although blood flow to the renal cortex is high and oxygen extraction is low, the renal cortex is remarkably susceptible to hypoxia. Because erythropoietin production has been localized mainly to the renal cortex, the aim of this study was to find a common denominator for both the high susceptibility to hypoxia and oxygen sensing within the renal cortex. By direct measurement of oxygen pressure with microcoaxial needle sensors at superficial glomeruli of the in situ kidney of anesthetized Munich-Wistar-Fromter rats, we obtained mean partial pressure of O2 (PO2) values of 46 +/- 13 (SD) mmHg (n = 71). The simultaneously measured systemic PO2 in arterial blood was 90 +/- 8 mmHg (n = 54). Changing the respirator gas from air to pure oxygen enhanced systemic arterial PO2 to 593 +/- 27 mmHg, whereas PO2 at the superficial glomeruli increased only to a mean of 80 +/- 28 mmHg (n = 71). These data suggest significant preglomerular shunting of oxygen within the cortical vasculature, most likely between interlobular vessels, which are arranged in a countercurrent fashion and represent quantitatively the largest contact area between arteries and veins within the renal cortex.
Myo-inositol and D-glucose transport in rat glomerular and cultured mesangial cells
Authors: Whiteside C. I. , Thompson J. C. , Ohayon J.
Published: December 2017

Myo-inositol (MI) is the precursor of membrane-bound phosphoinositides important for transmembrane signaling. This study examines whether freshly isolated whole glomeruli can be used to characterize intact cell transport kinetics for MI and D-glucose. Transport properties of cultured mesangial cells are compared. Glomeruli greater than 95% tubule free were isolated from Sprague-Dawley rat kidney cortex by means of selective sieving. Scanning electron microscopy revealed substantial damage of glomerular epithelial cells, whereas endothelial and mesangial cells remained intact. Specific [3H]MI uptake (7-180 min, 37 degrees C) was observed in presence of 5.5 mM D-glucose when L-[14C]glucose was used as a marker of nonspecific uptake. With ouabain (3 mM), or when Na was replaced with N-methyl-D-glucamine, choline, or Li, specific MI uptake was reduced by 95%. A single high-affinity, Na-dependent MI transport site on glomerular cells with Km of 16.5 +/- 1.4 (SE) microM and Vmax of 947 +/- 56 (SE) fmol.mg protein-1.min-1 was observed for 0.75-100 microM MI. D-glucose competitively inhibited MI transport. Specific D-[3H]-glucose transport was Na independent. Phlorizin inhibition of D-glucose and MI uptake was in keeping with Na-independent D-glucose transport. Km and Vmax for MI uptake in cultured mesangial cells were 42.7 +/- 7.1 microM and 1,474 +/- 192 fmol.mg protein-1.min-1, respectively. We conclude that freshly isolated glomeruli can be used to study cellular transport function that may be modified in disease states.
Na+ transport in isolated rat CCD: effects of bradykinin, ANP, clonidine, and hydrochlorothiazide
Authors: Rouch A. J. , Chen L. , Troutman S. L. , Schafer J. A.
Published: December 2017

We examined the effects of bradykinin (BK), atrial natriuretic peptide (ANP), hydrochlorothiazide (HCTZ), and clonidine on Na+ transport in isolated perfused cortical collecting ducts from rats treated with deoxycorticosterone. Arginine vasopressin was present in the bathing solution at 220 pM. Clonidine (1 microM, bathing solution) depolarized transepithelial potential difference (PDT) from -11.9 +/- 2.0 (SE) to -7.4 +/- 1.7 mV (P less than 0.001), hyperpolarized basolateral membrane potential difference (PDbl) from -85 +/- 1 to -87 +/- 1 mV (P less than 0.01), and increased the fractional resistance of the apical membrane (FRa) from 0.81 +/- 0.02 to 0.86 +/- 0.02 (P less than 0.03), indicating that it inhibited the Na+ conductance of the luminal membrane. BK (1 or 10 nM) or ANP (10 nM) in the bathing solution had no effect on PDT, PDbl, or FRa. BK, ANP, or 0.1 mM luminal HCTZ also had no effect on lumen-to-bath 22Na+ flux (J1----b), whereas we showed previously that clonidine inhibits J1----b by 30% (L. Chen, M. Paris, S. K. Williams, M. C. Reif, and J. A. Schafer. Kidney Int. 37: 366, 1990). Luminal addition of Na+ channel blockers amiloride (10 microM) or benzamil (1 microM) reduced J1----b to a level not significantly different from bath-to-lumen 22Na+ flux measured previously (M. Reif, S. L. Troutman, and J. A. Schafer. J. Clin. Invest. 77: 1291-1298, 1986), and neither BK nor HCTZ had any further effect. These results show that transcellular Na+ transport occurs exclusively through the apical membrane amiloride-sensitive channel, and this conductance is inhibited by clonidine but not by BK, ANP, or HCTZ.
Vasopressin is involved in renal effects of high-protein diet: study in homozygous Brattleboro rats
Authors: Bouby N. , Trinh-Trang-Tan M. M. , Coutaud C. , Bankir L.
Published: December 2017

The present study was designed to test the possible role of vasopressin in the renal response to dietary protein. This possibility was suggested by the similarity of effects on renal function and morphology of chronic high-protein intake and chronic stimulation of urine concentration. Adult male Brattleboro rats, genetically unable to produce vasopressin, were fed high-protein (32% casein = HP, n = 8) or low-protein (10% casein = LP, n = 9) diet for 7 wk. Renal function was evaluated by clearance techniques based on 24-h urine collections in metabolic cages. The response to a single injection of the vasopressin analogue 1-desamino-8-D-arginine vasopressin (DDAVP) was also tested. Kidney weight and height of the different renal zones were assessed at the end of the study. HP diet increased urea excretion nearly sevenfold. Water intake increased by 57% (P less than 0.001) and urine flow rate by 71% (P less than 0.01). Urine osmolality rose from 104 to 181 mosmol/kgH2O (P less than 0.001). At variance with what occurs in rats with endogenous vasopressin (Sprague-Dawley; Bouby, N., et al. Kidney Int 34: 4-12, 1988), HP diet increased creatinine clearance per unit body weight by only 14% and did not change free water clearance, renal mass, and height of inner stripe of outer medulla. However, the rise in urine osmolality and drop in free water clearance after DDAVP were significantly greater in HP- than in LP-fed Brattleboro rats.(ABSTRACT TRUNCATED AT 250 WORDS)