bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstitution and partial purification of calcium transport activity from rat kidney cortex
Authors: Sugimura K. , Abramowitz J. , Tsukamoto Y. , Suki W. N.
Published: December 2017

An ATP-dependent Ca2+ uptake system from rat renal cortical basolateral membranes was solubilized with Triton X-100 and reconstituted into liposomes with lecithin. In the presence of Mg2+, Ca2+ uptake in the reconstituted vesicles was time and ATP dependent and was inhibited by vanadate. Ca2+ uptake in basolateral membrane vesicles depleted of endogenous calmodulin was enhanced by exogenous calmodulin and depressed by R-24571. This sensitivity to calmodulin and R-24571 was lost upon reconstitution in the presence and absence of leupeptin. Vesicles containing Ca2+ uptake activity were separated by gradient centrifugation after Ca2+ was taken up and accumulated as calcium phosphate in the vesicles. This resulted in Ca2+ uptake activity that was enriched 25 times. However, Ca(2+)-dependent adenosinetriphosphatase (ATPase) activity was not enriched significantly. This Ca(2+)-ATPase had two kinetic forms for Ca2+: one was a high-affinity low-capacity form; the other had a low affinity and high capacity. The Ca(2+)-ATPase activity also had two kinetic forms for ATP. All kinetic forms were inhibited by Mg2+. Vanadate, calmodulin, and R-24571 had no effects on Ca(2+)-ATPase activity. A protein doublet of Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates was demonstrated at 125 and 136 kDa in the purified vesicles. This doublet was not altered by addition of leupeptin throughout the purification.
Role of endothelium-derived relaxing factor in renal autoregulation in conscious dogs
Authors: Baumann J. E. , Persson P. B. , Ehmke H. , Nafz B. , Kirchheim H. R.
Published: December 2017

In six chronically instrumented, conscious dogs the hypothesis was tested that the release of endothelium-derived relaxing factor (EDRF) is important for autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR). RBF was measured by a Transonic flowmeter. Renal perfusion pressure was servo-controlled by an aortic cuff. EDRF synthesis was inhibited by NG-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg iv). L-NAME increased mean systemic blood pressure (30 mmHg) and decreased heart rate (-40 beats/min), but it left autoregulation of RBF and GFR intact. However, basal RBF decreased markedly (2.24 +/- 0.32 ml.min-1.g-1 with L-NAME vs. 3.91 +/- 0.64 ml.min-1.g-1 for control, P less than 0.01), whereas basal GFR was not significantly influenced (0.37 +/- 0.05 ml.min-1.g-1 with L-NAME vs. 0.42 +/- 0.06 ml.min-1.g-1 for control). Hence filtration fraction increased with L-NAME [27.6 +/- 1.7% vs. 19.3 +/- 1.3% (P less than 0.01)]. The lower limit of autoregulation remained unchanged for RBF (64 +/- 5 mmHg with L-NAME vs. 63 +/- 3 mmHg for control) and increased slightly for GFR (74 +/- 2 mmHg with L-NAME vs. 67 +/- 1 mmHg for control, P less than 0.01). In conclusion, basal EDRF activity tonically influences renal resistance vessels; however, EDRF release is not primarily involved in the process of renal autoregulation. The maintenance of GFR suggests that this effect is localized in preglomerular as well as in postglomerular arterioles.
Chloride channels in apical and basolateral membranes of CCD cells (RCCT-28A) in culture
Authors: Dietl P. , Stanton B. A.
Published: December 2017

Previously, we found that isoproterenol activates whole cell Cl- conductance by a pathway involving adenosine 3',5'-cyclic monophosphate and protein kinase A (PKA) in a renal cell line (RCCT-28A) derived from the cortical collecting duct. The goal of the present study was to determine whether PKA activates Cl- channels in the apical and/or basolateral membrane. Using the patch clamp technique we found a 305-pS Cl- channel, described previously (22), located exclusively in the apical membrane and an outwardly rectifying Cl- channel (13/96 pS) located exclusively in the basolateral membrane. The outward rectifier was highly selective to Cl- versus cations, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, but was not regulated by cytoplasmic pH or Ca2+. Neither isoproterenol nor PKA activated the 305-pS Cl- channel. In contrast, PKA activated a subset of outwardly rectifying channels in inside-out patches. In another subset of outwardly rectifying channels, formation of the inside-out configuration increased channel activity. These channels, however, were not sensitive to PKA. In conclusion, these experiments show that isoproterenol increases the Cl- conductance of RCCT-28A cells by activating a subset of outwardly rectifying Cl- channels located in the basolateral membrane.
Expression of vacuolar H(+)-ATPase in mouse osteoclasts during in vitro differentiation
Authors: Wang Z. Q. , Hemken P. , Menton D. , Gluck S.
Published: December 2017

Osteoclasts express high levels of a vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) on the ruffled membrane which they employ to dissolve bone mineral by acidifying their site of attachment on bone. The factors that control amplification of H(+)-ATPase during osteoclast differentiation are poorly understood. We examined the expression of vacuolar H(+)-ATPase in a cell culture system in which mouse spleen cells can be induced to differentiate into osteoclasts by coculture with a mouse bone marrow stromal cell line. We found that the coculture system produced active osteoclasts, identified as multinucleated cells with staining for tartrate-resistant acid phosphatase activity that formed genuine resorption pits in bone. These cells developed high levels of H(+)-ATPase expression in culture, and omission of dexamethasone or 1 alpha,25-dihydroxyvitamin D3 from the coculture system each partially suppressed the expression of H(+)-ATPase. The results demonstrate that the spleen and PA6 cell coculture system may be useful for investigating the factors that control the induction of H(+)-ATPase amplification that occurs during osteoclast differentiation.
Cellular variability in the development of tight junctions after activation of protein kinase C
Authors: Ellis B. , Schneeberger E. E. , Rabito C. A.
Published: December 2017

Phorbol 12-myristate 13-acetate (PMA) decreases the tight junction conductance (TJC) during the reorganization of LLC-PK1A monolayers, but has the opposite effect in LLC-PK1B4, MDCK, and MDCK4 cells. Because no protein synthesis was required for the effects of PMA on the TJC of LLC-PK1A monolayers, we conclude that the regulation of the tight junction by protein kinase C (PKC) is a posttranslational event. In LLC-PK1A monolayers with existing tight junctions, PMA produced an initial increase in the TJC that reverted later to control values despite the continuous presence of PMA and cycloheximide. The inhibitory effect of PMA on the other cell lines was not revertible. A downregulation of total PKC activity and phorbol ester receptors was only observed during the reorganization of LLC-PK1A monolayers. PMA further increases this downregulation. This indicates that the peculiar response to PMA observed in LLC-PK1A monolayers is the result of two concurrent events: 1) the early activation of the enzyme just before the reorganization of the tight junctions begin, and 2) its late downregulation induced after prolonged exposure to phorbol esters. We conclude that PKC regulates the development of the occluding junctions, but through different mechanisms dependent on the characteristics of the cells.
Hormone and autacoid regulation of cAMP production in rat IMCD subsegments
Authors: Maeda Y. , Terada Y. , Nonoguchi H. , Knepper M. A.
Published: December 2017

The inner medullary collecting duct (IMCD) of the rat consists of two structurally and functionally distinct segments, i.e., the initial and the terminal IMCD. To identify factors that may regulate the transport function in the IMCD segments, we assessed whether catecholamines, carbachol, prostaglandin E2 (PGE2), bradykinin, glucagon, calcitonin, parathyroid hormone, or epidermal growth factor affects adenosine 3',5'-cyclic monophosphate (cAMP) production in microdissected tubules in the presence and absence of arginine vasopressin (AVP, 0.1 nM). All experiments were performed in the presence of 3-isobutyl-1-methylxanthine, and cAMP was measured by radioimmunoassay. Epinephrine (greater than or equal to 50 nM) and clonidine (greater than or equal to 1 microM) markedly decreased AVP-induced cAMP levels in both IMCD segments. However, phenylephrine did not show an effect. The inhibitory effect of epinephrine was blocked by yohimbine (50 nM) but not by prazosin (50 nM). In isolated perfused terminal IMCDs, epinephrine inhibited AVP-stimulated urea permeability. Isoproterenol (1 microM), in the absence of AVP, caused a significant increase in cAMP level only in the initial IMCD. Propranolol (1 microM) inhibited this isoproterenol effect, but atenolol did not. Dopamine (less than or equal to 1 microM) had no effect on cAMP levels in either IMCD segment. Carbachol, PGE2, and the various peptide hormones had no effect on cAMP levels (+/- AVP) in either IMCD segment. We conclude that an adrenergic beta 2-receptor is present only in the initial IMCD, where its occupation increases cAMP production. We conclude also that an adrenergic alpha 2-receptor is present in both IMCD segments, where its occupation inhibits AVP-induced cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)
Denervation inhibits early increase in Na(+)-H+ exchange after uninephrectomy but does not suppress hypertrophy
Authors: Mackovic-Basic M. , Fan R. , Kurtz I.
Published: December 2017

Na(+)-H+ exchange in the rat proximal tubule luminal membrane increases approximately 30% within 15 min after the contralateral uninephrectomy. The present study was designed to test whether altered renal sympathetic nerve outflow to the remaining kidney is the underlying mechanism of increased antiport activity and whether suppression of Na(+)-H+ antiport activity by renal denervation inhibits renal hypertrophy in the remaining kidney after uninephrectomy. Sprague-Dawley rats were divided into four groups: 1) sham operated, 2) uninephrectomized, 3) uninephrectomized with prior denervation of the remaining kidney, and 4) contralateral renal denervation. Na(+)-H+ antiport activity (brush-border vesicles), Na(+)-K(+)-ATPase activity (basolateral vesicles), and kidney weight were measured days 1-7. On days 1 and 7, Na(+)-H+ antiport activity and Na(+)-K(+)-ATPase activities were significantly greater in uninephrectomized rats. Denervation of the remaining kidney before contralateral uninephrectomy prevented the stimulation of the antiporter and Na(+)-K(+)-ATPase activity, but failed to inhibit renal hypertrophy by day 7. In separate experiments, contralateral renal denervation alone without removal of the kidney stimulated the Na(+)-H+ antiporter and Na(+)-K(+)-ATPase activity. Kidney weight, however, remained unchanged. The results demonstrate a dissociation between the activation of the Na(+)-H+ antiporter and induction of renal hypertrophy in vivo.
Basolateral Na(+)-independent Cl(-)-HCO3- exchange in primary cultures of rat IMCD cells
Authors: Kraut J. A. , Hart D. , Nord E. P.
Published: December 2017

The role of anion exchange in the regulation of intracellular pH (pHi) under base load and steady-state conditions was investigated in confluent monolayers of rat inner medullary collecting duct (IMCD) cells in primary culture using the pH-sensitive fluoroprobe 2,7-bis(carboxyethyl)-5(6')-carboxyfluorescein (BCECF). Recovery of pHi after imposition of a base load induced either by replacement of HCO3-/CO2 by N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at the same extracellular pH (pHo) or deletion of Cl- from a HCO3-/CO2-buffered solution had an absolute requirement for Cl-, was Na+ independent, and was inhibited approximately 90% by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). When pHo was decreased by lowering HCO3- concentration in the constant presence of 5% CO2, the rate of decrement in pHi was significantly blunted in the absence of Cl-. Imposition of a positive or negative diffusion potential of equal but opposite magnitude did not modify the anion exchange rate, confirming the electroneutrality of the process. Under steady-state conditions, pHi of cells bathed in a HCO3-/CO2-buffered solution was 7.33 +/- 0.06, significantly lower than that of cells bathed in a nominally HCO3-/CO2-free buffer (7.50 +/- 0.04), indicating that under physiological conditions the pathway functions as a base extruder. In studies performed on cells grown on permeable supports, the anion exchange pathway was found to be confined exclusively to the basolateral-equivalent cell surface. In summary, confluent monolayers of rat IMCD cells in primary culture possess a Na(+)-independent, DIDS-inhibitable electroneutral Cl(-)-HCO3- exchange pathway that is confined to the basolateral cell surface. The transporter is an important determinant of steady-state pHi and is the predominant mechanism whereby the cell recovers from imposed elevations in pHi.
Angiotensin II receptor subtypes in cultured rat renal mesangial cells
Authors: Ernsberger P. , Zhou J. , Damon T. H. , Douglas J. G.
Published: December 2017

The selective angiotensin (ANG II) antagonists losartan (DuP 753) and PD 123319 have been shown to bind selectively to AT1 and AT2 subtypes, respectively. To characterize ANG II receptor subtypes in mesangial cells, washed membranes were incubated with 0.1 to 0.5 nM 125I-ANG II and increasing concentrations of competitors. The inhibition of 125I-ANG II binding by losartan and PD 123319 was biphasic, and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation comprised 86% of the total and showed high affinity for ANG II and losartan, but low affinity for the AT2 antagonists PD 123319 and CGP42112A, and thus appear identical to the recently cloned AT1 subtype. The remaining 14% of the sites showed nearly 100-fold lower affinity for losartan and 10,000-fold higher affinity for PD 123319 relative to AT1 sites. However, another AT2-selective antagonist, CGP42112A, showed little affinity for these sites. Both classes of binding sites were inhibited by guanosine 5'-O-(3-thiophosphate) and pertussis toxin treatment. We propose that there are two distinct G protein-coupled ANG II receptor subtypes (AT1A and AT1B) present in renal mesangial cells.
In vitro perfusion of chinchilla thin limb segments: segmentation and osmotic water permeability
Authors: Chou C. L. , Knepper M. A.
Published: December 2017

The thin limb segments of the long loop of Henle are thought to play important roles in the urinary concentrating mechanism. In this study, we present new approaches to the identification, dissection, and in vitro perfusion of individual thin limb segments from all levels of the chinchilla renal medulla, including the deepest portions of the papilla. We have applied these techniques to the investigation of the osmotic water permeability along the chinchilla long loop of Henle. The results demonstrate that the osmotic water permeability of the thin descending limb is not uniformly high along its length, as previously thought, but that the distal 20% of the long-loop descending limb has a very low water permeability (approximately 50 microns/s). The transition to the low water permeability region of the thin descending limb is accompanied by a relatively abrupt change in morphology (increased cellularity and decreased diameter) that is readily perceptible in the perfused segments and even in the dissection dish. In contrast, the upper part of the chinchilla long-loop thin descending limb had an extremely high osmotic water permeability (greater than 2,000 microns/s) as observed in other species. Thin ascending limbs from deep in the inner medulla had water permeabilities that were indistinguishable from zero, as previously found in thin ascending limbs from near the inner-outer medullary junction. The presence of a low-water-permeability portion of the long-loop thin descending limb in chinchilla may have important implications with regard to the inner medullary concentrating process. A relatively low osmotic water permeability (397 microns/s) was also found in the deep inner medullary portion of the thin descending limb from the rat.
Renal hemodynamic actions of lipoxins in rats: a comparative physiological study
Authors: Katoh T. , Takahashi K. , DeBoer D. K. , Serhan C. N. , Badr K. F.
Published: December 2017

Interactions between either the arachidonate 5-lipoxygenase (5-LO) and 15-LO or 5-LO and 12-LO can lead to the formation of lipoxins. Although the functional significance of lipoxygenase products of arachidonic acid has been recognized increasingly in glomerular inflammation, less is known regarding the specific effects of lipoxins on renal hemodynamics. Here, we examine the renal actions of lipoxin (LX) A4, LXB4, and, the recently identified 7-cis-11-trans-LXA4, which were administered into the renal artery of the euvolemic male Munich-Wistar rat. LXA4 caused increases in renal plasma flow (RPF) and glomerular filtration rate (GFR) in a dose-dependent manner. These effects were reversed during cyclooxygenase inhibition. LXB4 decreased both RPF and GFR, and its mode of action was independent of cyclooxygenase activity. 7-cis-11-trans-LXA4 decreased RPF and GFR, which effects were abolished during systemic infusion of the leukotriene D4 receptor antagonist SKF 104353. Results indicate that each of these lipoxins displays distinct hemodynamic effects via a specific mode of action on the renal vasculature of rats. Moreover, they suggest mechanisms whereby lipoxins may participate in the changes of renal hemodynamics that occur during glomerular inflammatory processes.
Glucocorticoids inhibit colonic aldosterone-induced conductive Na+ absorption in adrenalectomized rat
Authors: Bastl C. P. , Schulman G. , Cragoe E. J.
Published: December 2017

Low-dose glucocorticoids induce only electroneutral Na absorption in rat colon. High-dose dexamethasone induces both electroneutral and electrogenic Na absorption. Aldosterone induces only electrogenic Na absorption and inhibits basal electroneutral transport in distal colon. To define the interrelationship between glucocorticoid-mediated and aldosterone-mediated Na absorption, adrenalectomized rats were treated with aldosterone and either glucocorticoid-receptor-specific doses of dexamethasone or the specific glucocorticoid RU26988. In combination the steroids did not increase Na and Cl absorption in proximal and distal colon and transmural potential difference (PD) and K secretion in distal colon as much as aldosterone alone. Na absorption was not inhibited by spironolactone or amiloride (10(-4) M). Transport in both segments was by Na-H exchange as demonstrated by marked inhibition by amiloride (10(-3) M) and the Na-H antiport inhibitor 5-(N-ethyl-N-isopropyl)amiloride. Thus glucocorticoids not only decreased Na absorption but also produced a marked qualitative change in the mode of Na absorption in aldosterone-treated rats. Acute dexamethasone infusion in rats pretreated with aldosterone decreased Na absorption and transmural PD within 30 min, suggesting inhibition of electrogenic Na absorption at a step distal to synthesis of aldosterone-induced proteins. These findings suggest that upregulation of one Na absorptive mechanism downregulates the other. This may explain why in intact unstressed rats there is little or no conductive Na absorption, despite sufficient endogenous steroid to occupy the aldosterone receptor. It may also explain why in proximal colon of intact rats, despite the presence of aldosterone receptors, even prolonged aldosterone exposure does not induce significant conductive Na absorption.
Effects of adenosine on ion transport in rat medullary thick ascending limb
Authors: Beach R. E. , Good D. W.
Published: December 2017

Previously, we demonstrated that adenosine (Ado) was released by the medullary thick ascending limb (MTAL) during hypoxia. The present experiments were designed to examine the effects of Ado and adenosine analogues on net chloride (JCl) and bicarbonate (JHCO3) absorption by the isolated, perfused MTAL of the rat. Ado, 10 nM, in the presence or absence of arginine vasopressin (AVP, 10(-10) M) reduced JCl by 50%. The inhibition of Ado was reproduced with the selective A1 agonist, N-6-phenylisopropyladenine (2 nM), and was reversed by 8-cyclopentyl-1,3-dipropylxanthine, an A1-receptor antagonist. Thus the inhibition of JCl is likely mediated through A1 receptors. In contrast, Ado had no effect on (JHCO3) either in the presence or absence of AVP. Ado also had no influence on the effect of AVP to inhibit JHCO3. The lack of effect on JHCO3 suggests that the inhibition of JCl by Ado is unlikely to be mediated through changes in cellular adenosine 3',5'-cyclic monophosphate. These results support the hypothesis that Ado released into the renal medulla during hypoxia may protect the MTAL from ischemic injury by directly inhibiting NaCl absorption and reducing transport-related oxygen consumption.
Cytoskeleton disruption and apical redistribution of proximal tubule Na(+)-K(+)-ATPase during ischemia
Authors: Molitoris B. A. , Dahl R. , Geerdes A.
Published: December 2017

The polar distribution of Na(+)-K(+)-ATPase to the basolateral membrane of proximal tubule cells is essential for the efficient and vectorial reabsorption of Na+ and may be dependent on the formation of a metabolically stable, detergent-insoluble complex of Na(+)-K(+)-ATPase with the actin membrane cytoskeleton. The present studies utilized immunocytochemical techniques to demonstrate and quantify the apical redistribution of Na(+)-K(+)-ATPase during mild ischemia (15 min) that occurred in proximal (1.3 +/- 0.9 vs. 4.5 +/- 1.1 particles/100 microns surface membrane, P less than 0.01) but not distal tubule cells. Treatment of control apical membranes with 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C), a fluidizing agent, markedly increased membrane fluidity without any effect on Na(+)-K(+)-ATPase activity. In brush-border membrane vesicles isolated after ischemia, however, A2C further increased an already elevated Na(+)-K(+)-ATPase activity. During ischemia, total cellular Na(+)-K(+)-ATPase activity remained unaltered, but the Triton X-100-soluble (noncytoskeleton associated) fraction of Na(+)-K(+)-ATPase increased significantly following 15 and 30 min. There was a corresponding decrease in the Triton X-100-insoluble fraction of Na(+)-K(+)-ATPase, with the ratio of detergent-soluble to -insoluble Na(+)-K(+)-ATPase increasing from 13 +/- 2 to 32 +/- 5% (P less than 0.01) during 30 min of ischemia. Western blot analysis of the Triton X-100-soluble fraction, following 30 min of ischemic injury, revealed the presence of Na(+)-K(+)-ATPase, actin, fodrin, and uvomorulin. However, in a fraction highly enriched for Na(+)-K(+)-ATPase, neither actin, fodrin, nor uvomorulin was detected.(ABSTRACT TRUNCATED AT 250 WORDS)
Localization, synthetic regulation, and biology of renal atriopeptin-like prohormone
Authors: Ritter D. , Chao J. , Needleman P. , Tetens E. , Greenwald J. E.
Published: December 2017

We recently demonstrated the synthesis and secretion of an atriopeptin (AP)-like prohormone in rat neonatal and adult cortical kidney cell cultures. However, these cultures contained proximal as well as distal tubular epithelial cells; thus characterization of the peptide synthetic cell was not possible. Also, by immunohistochemical techniques, we localized this AP-like prohormone to the distal cortical nephron in adult rat kidney. In this study, we examined further details of the kidney cortical cell type that expresses and secretes this AP-like peptide in adult renal cortical cell cultures, its regulation by adenylate cyclase via adenosine 3',5'-cyclic monophosphate (cAMP) generation, and its ability to stimulate guanylate cyclase. Tubular fragments were derived from cortical tissue of adult Sprague-Dawley rats and separated into four fractions on Percoll density gradient. Cell cultures generated from fraction 3 secreted 5- to 10-fold the amount of this renal peptide compared with fractions 2 and 4. Further cell culture characterization was performed by agonist-stimulated cAMP formation, kallikrein localization, and prostaglandin E2 formation. From these analyses, it was determined that tissue band 3 was enriched for distal cortical connecting tubules. To further evaluate whether mammalian distal nephron synthesizes an AP-like protein, we determined that two immortalized mouse cell lines, derived from either the distal convoluted tubule or cortical collecting tubule, synthesized a radiolabeled AP after being pulsed with [35S]-methionine.(ABSTRACT TRUNCATED AT 250 WORDS)
Critical role of bicarbonate in calcium release from bone
Authors: Bushinsky D. A. , Sessler N. E.
Published: December 2017

Calcium release from cultured bone is pH dependent; net calcium flux (JCa) from bone increases with decreasing pH. At a similar decrement in pH there is greater JCa when acidosis is produced by a low medium bicarbonate concentration ([HCO3-]), a model of metabolic acidosis (Met), compared with an increased medium PCO2, a model of respiratory acidosis (Resp). To separate the role of [HCO3-] from that of pH in inducing JCa we cultured calvariae for 3 h under three different neutral (pH approximately 7.4) isohydric environments [control (Ctl), fully compensated Met (C-Met), or fully compensated Resp (C-Resp)] and two different acid (pH approximately 7.1) isohydric environments (Met or Resp). During neutral pH (Ctl, C-Met, and C-Resp) there was JCa from bone during C-Met (decreased [HCO3-]), no net flux during Ctl (normal [HCO3-]), and JCa into bone during C-Resp (increased [HCO3-]); and JCa was correlated inversely with [HCO3-] (r = -0.824, n = 36, P less than 0.001). During acid pH there was greater JCa from bone during Met (decreased [HCO3-]) than during Resp (normal [HCO3-]); and JCa was again correlated inversely with [HCO3-] (r = -0.848, n = 22, P less than 0.001). JCa from bone during Met and Resp was greater than C-Met and C-Resp, respectively. The addition of the osteoclastic inhibitor salmon calcitonin did not alter the relative JCa results. Thus at a constant pH the magnitude of JCa from cultured neonatal mouse calvariae appears dependent on the [HCO3-]; the lower the [HCO3-], the greater the calcium efflux.(ABSTRACT TRUNCATED AT 250 WORDS)
Calorie restriction decreases microalbuminuria associated with aging in barrier-raised Fischer 344 rats
Authors: Van Liew J. B. , Davis F. B. , Davis P. J. , Noble B. , Bernardis L. L.
Published: December 2017

Renal function as a sensitive biomarker of aging has been studied in specific pathogen-free (SPF) Fischer 344 rats (n = 211), and results are presented according to animal age (5, 8, 12, 18, 24 mo), sex, and diet (ad libitum vs. 40% calorie restriction). Plasma creatinine concentration, endogenous creatinine clearance, total protein excretion, and albumin excretion were measured. Kidney histology was evaluated by light microscopy. In both calorie-restricted and ad libitum-fed animals, kidney weight (KW) and body weight (BW) showed parallel changes with age. The KW-to-BW ratio was unaffected by age in all groups. There was no alteration in plasma creatinine concentration as a function of age or diet. In these SPF animals there was also no change in glomerular filtration rate with age. In animals fed ad libitum, albumin and protein excretion increased with age (females: 0.39 +/- 0.05 at 5 mo vs. 7.4 +/- 2.6 mg protein.24 h-1.g KW-1 at 24 mo; males: 4.1 +/- 0.6 at 5 mo vs. 15 +/- 3 mg protein.24 h-1.g KW-1 at 24 mo). The higher protein excretion rate in all males at 5 mo reflected the excretion of sex-dependent low-molecular-weight proteins that commenced with sexual maturation. Calorie restriction prevented the age-dependent increase in total protein excretion. Kidney histopathology was positively correlated with total protein and albumin excretion. Microalbuminuria preceded the development of lesions detectable by light microscopy. These observations support the concept that microalbuminuria in this model is a sensitive and early biomarker of nephropathy that can be monitored easily and noninvasively.
ANF inhibits norepinephrine-stimulated fluid absorption in rat proximal straight tubules
Authors: Garvin J. L.
Published: December 2017

We have previously shown that atrial natriuretic factor (ANF) only inhibits fluid absorption in the proximal straight tubule after the tissue has been exposed to angiotensin (ANG) II. In this paper, the interaction between ANF and norepinephrine (NE) in the proximal straight tubule was investigated. ANF alone (10(-9) M) added to the bath had no significant effect on fluid absorption. In contrast, when tubules were first treated with 10(-7) M NE, 10(-9) M ANF reduced fluid absorption from 0.63 +/- 0.09 to 0.41 +/- 0.04 nl.mm-1.min-1. NE alone stimulated fluid absorption by 33%. Dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP; 50 microM) mimicked the effects of ANF, reducing fluid absorption from 0.73 +/- 0.06 to 0.35 +/- 0.07 nl.mm-1.min-1 in NE-treated tubules. However, it had no effect on fluid absorption in the absence of NE. These studies suggest that 1) ANF can regulate fluid absorption in the proximal nephron; 2) this inhibition occurs only after transport has been stimulated by agents such as NE (or ANG II); 3) cGMP is part of the second messenger system of ANF in the rat proximal straight tubule; and 4) the primary interaction between ANF and NE (or ANG II) occurs in the second messenger cascades subsequent to steps that affect levels of cGMP.
Effect of cold exposure and nutrient intake on sympathetic nervous system activity in rat kidney
Authors: Daly P. A. , Young J. B. , Landsberg L.
Published: December 2017

Renal sympathetic nervous system (SNS) responses to environmental temperature and diet were evaluated using [3H]norepinephrine ([3H]NE) turnover as the index of sympathetic activity. Pharmacological studies first demonstrated that renal NE was localized principally within storage granules of renal sympathetic nerves and regulated by central sympathetic outflow. Acute exposure to cold (4 degrees C), which increased cardiac SNS activity (P < 0.00005), had no effect on renal SNS. A 48-h fast suppressed renal [3H]NE turnover by 37% (P = 0.00024) and cardiac [3H]NE turnover by 48% (P = 0.00608). Dietary supplementation with sucrose did not affect [3H]NE turnover in kidney in either of two separate experiments, although it increased cardiac NE turnover in both. On the other hand, lard feeding significantly increased [3H]NE turnover in both kidney and heart, whereas dietary protein supplementation exerted no effect on either renal or cardiac [3H]NE turnover. These studies demonstrate a unique pattern of sympathetic regulation in kidney, one which is highly responsive to fasting and dietary fat, but not to cold exposure or dietary sucrose.
Effects of dietary protein and salt on rat renal osmolytes: covariation in urea and GPC contents
Authors: Peterson D. P. , Murphy K. M. , Ursino R. , Streeter K. , Yancey P. H.
Published: December 2017

Renal medullary cells contain high levels of (glycine) betaine, glycerophosphorylcholine (GPC), myo-inositol, and sorbitol. Two functions of these have been proposed: 1) that they are compatible osmolytes which regulate cell volume (against high external NaCl) without inhibiting proteins and 2) that methylamines (GPC and betaine) are counteracting osmolytes which stabilize proteins against perturbation from high renal urea. As a test of the latter, osmolyte contents in kidney medullas were measured in rats subjected to three types of dietary manipulation: 1) diets with protein and NaCl contents varied oppositely, 2) diets with a constant low NaCl and varied protein content, and 3) a low-calorie diet. With low-protein and low-calorie diets, only renal contents of urea, GPC, and inositol decreased; betaine and sorbitol contents increased such that contents of total nonurea organic osmolytes remained constant. With high-protein diets, only renal contents of sodium, urea, and GPC increased, with the latter giving total organic osmolytes a consistent correlation to sodium. Across all diets, the only consistent (linear) correlations were 1) between urea and GPC contents, supporting previous suggestions that GPC is the major counteractant to urea, and 2) between total organic osmolytes and sodium (but not urea) contents, as predicted by the compatible osmolytes hypothesis.
Endothelin-1 is an autocrine factor in rat inner medullary collecting ducts
Authors: Kohan D. E. , Padilla E.
Published: December 2017

Endothelin-1 (ET-1) may be an important factor in the regulation of inner medullary collecting duct (IMCD) physiology. This segment of the nephron synthesizes ET-1, expresses endothelin receptors, and responds to exogenous ET-1 by reducing Na(+)-K(+)-ATPase activity and water transport. Taken together, these findings suggest an autocrine role for ET-1 in the regulation of IMCD function; however, because of the polarized nature of the IMCD, it is not known if ET-1 secretion, receptors, and receptor activation occur on the same side of the cell. To examine this question, rat IMCD cells were grown to confluence on semipermeable membranes. These cells exhibited polar morphology with high transepithelial electrical resistances. Immunoreactive ET-1 was secreted primarily into the basolateral side. Furthermore, 125I-ET-1 bound predominantly to the basolateral surface. Finally, ET-1 (10(-8) M) stimulated prostaglandin E2 production only when added to the basolateral side. These data indicate, therefore, that ET-1 is capable of autocrine regulation of IMCD cells and that this effect occurs predominantly on the basolateral side.
Effect of vasoactive agents on induction of Egr-1 in rat mesangial cells: correlation with mitogenicity
Authors: Rupprecht H. D. , Dann P. , Sukhatme V. P. , Sterzel R. B. , Coleman D. L.
Published: December 2017

The early growth response gene 1 (Egr-1) is a member of the family of immediate early response genes. Egr-1 encodes a nuclear phosphoprotein that binds a specific nonameric DNA sequence through three zinc-finger domains and functions as a transcriptional activator. We tested whether the vasoactive agents platelet-derived growth factor (PDGF), arginine vasopressin (AVP), serotonin (5-HT), and angiotensin II (ANG II) induced Egr-1 mRNA in cultured rat mesangial cells (MCs) and investigated the role of protein kinase C (PKC) in mediating the induction process. PDGF, AVP, and 5-HT induced Egr-1 mRNA within 15 min, reaching peak levels at 45-60 min. After PDGF and 5-HT stimulation, Egr-1 mRNA levels returned to baseline within 4 h, whereas AVP induced a sustained increase for up to 8 h. There was a very close correlation between doses required for Egr-1 induction and induction of MC proliferation. ANG II was a very weak MC mitogen and induced only a small increase in Egr-1 mRNA. Comparison of control cells with cells depleted of PKC by 48 h of PMA treatment revealed that induction of Egr-1 by PDGF and 5-HT is independent of PKC. In contrast, however, the Egr-1 response to AVP was diminished in PKC-depleted cells. AVP induced Egr-1 mRNA 10.9-fold in control cells, compared with 7.8-fold in PKC-depleted cells. Egr-1 mRNA after AVP stimulation remained elevated in control cells for up to 8 h but returned to baseline after 120 min in PKC-depleted cells. Similar results were obtained using the PKC-inhibitor H-7. Using immunocytochemistry, PDGF and AVP were found to induce Egr-1 protein within 30 min localized to the nucleus. We conclude that there is a strong correlation between induction of Egr-1 after stimulation with PDGF, AVP, 5-HT, and ANG II and the proliferative response elicited by these agents in MCs. AVP induces Egr-1 by both PKC-dependent and PKC-independent pathways, whereas the effects of PDGF and 5-HT are independent of PKC.
Diabetic rat glomerular mesangial cells display normal inositol trisphosphate and calcium release
Authors: Hurst R. D. , Whiteside C. I. , Thompson J. C.
Published: December 2017

Since diabetes may cause cellular myo-inositol depletion, we investigated whether the observed in vitro hypocontractile response of streptozotocin (STZ)-treated rat glomeruli to angiotensin II (ANG II) is associated with an alteration in inositol trisphosphate (IP3) mobilization of intracellular Ca2+. Contraction of diabetic isolated glomeruli induced by ANG II (5 microM), measured in vitro by changes in the planar area, was reduced by 60%, compared with normal up to 60 min (P < 0.05). In cells of isolated glomeruli, preloaded with myo-[3H]inositol, production of [3H]inositol phosphates ([3H]IPs) and [3H]inositol trisphosphate ([3H]IP3) was analyzed by Dowex chromatography. ANG II (1 microM) evoked an immediate peak (5-10 s) in total [3H]IPs of 60.5 +/- 18.8% (mean +/- SE) above basal (nonstimulated state) in normal glomeruli, and 88.4 +/- 19.4% in diabetic condition [not significant (NS), n = 8]. At 60 s, the normal and diabetic total [3H]IPs responses were not significantly different from each other. The immediate (10 s) [3H]IP3 response from normal glomeruli, 8.1 +/- 7.9% above basal, was not significantly different from that of diabetic glomeruli, 15.7 +/- 7.4%. ANG II receptor-mediated rise in cytosolic Ca2+ in the cells of normal and diabetic isolated glomeruli was compared by measuring the efflux of 45Ca2+. Isolated glomeruli were preloaded with 45Ca2+. Following ANG II stimulation, peak 45Ca2+ efflux values at 1 min were 141.7 +/- 15.9% (normal) vs. 143.7 +/- 7.8% (diabetic) of baseline (100%), respectively (NS, n = 4). Thapsigargin, 2 microM, specifically prevented ANG II-stimulated and IP3-mediated 45Ca2+ efflux (73% inhibition, P < 0.001) from cells of whole glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)
Involvement and source of calcium in volume regulatory decrease of collapsed proximal convoluted tubule
Authors: Breton S. , Beck J. S. , Cardinal J. , Giebisch G. , Laprade R.
Published: December 2017

We examined the role of Ca2+ in the volume regulatory decrease (VRD) of rabbit collapsed proximal tubules. Reduction of bath osmolality by 125 mosmol/kgH2O led to an initial cell swelling of 62.3 +/- 7.5% followed by a partial regulatory phase bringing cell volume to a value of 13.3 +/- 2.9% above control (n = 5). This swelling was accompanied by a transient intracellular Ca2+ ([Ca2+]i) increase from 174 +/- 33 to 306 +/- 67 nM (P < 0.05, n = 8). In the same condition, but in absence of extracellular Ca2+ ([Ca2+]e) [1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], VRD following hypotonic shock was identical to that observed in presence of [Ca2+]e (n = 5), and [Ca2+]i increased transiently from 136 +/- 29 to 161 +/- 31 nM (P < 0.05, n = 5). Addition of 100 microM 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an agent known to inhibit Ca2+ release from intracellular stores, did not affect the initial cell swelling (63.4 +/- 4.2%), and VRD occurred to the same extent (25.0 +/- 7.1%, n = 4), although at a lower rate. In these conditions, [Ca2+]i, which was 113 +/- 30 nM in the isotonic solution, decreased progressively to 81 +/- 20 nM over the 5-min hypotonic period (n = 5). Mere preincubation with 100 microM TMB-8 before hypotonic shock led to a VRD identical to that observed in presence of Ca2+ and absence of TMB-8 while still blocking the Ca2+ release, with cell Ca2+ decreasing progressively from 179 +/- 32 to 87 +/- 21 nM (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)
Fetal-maternal fluid and electrolyte relations during chronic fetal urine loss in sheep
Authors: Wlodek M. E. , Harding R. , Thorburn G. D.
Published: December 2017

Our aim was to determine the effects of prolonged removal of fetal urine during late gestation on fetal-maternal fluid and electrolyte relationships. We measured the volume and composition of fetal urine and amniotic and allantoic fluids and the composition of fetal and maternal plasma in sheep before and during continuous urine drainage, which began at 130 days of gestation and continued until the onset of labor; a control group was also studied. The response to fetal urine drainage occurred in two phases. In the "acute" phase (1-3 days), amniotic and allantoic fluid volumes decreased significantly, presumably due to their reabsorption into the fetal chorionic circulation or swallowing of amniotic fluid by the fetus. During the "chronic" phase, starting 3-5 days after urine drainage, a significant reversal in the transplacental osmotic gradient occurred due to a decrease in maternal plasma osmolality. During the entire drainage period (14.1 +/- 1.1 days, mean +/- SE, n = 5) at least 542 ml/day of water and 24 mmol/day of electrolytes passed from the mother into the fetal circulation and fetal plasma osmolality was unchanged. We conclude that, despite the loss of substantial water and electrolytes, the fetus is able to maintain its growth and fluid and electrolyte homeostasis by obtaining water and electrolytes initially from the amniotic and allantoic fluids and subsequently from its mother. The movement of water and electrolytes to the fetus would have been facilitated by the reversed transplacental osmotic gradient.
Ouabainlike factor in Milan hypertensive rats
Authors: Ferrandi M. , Minotti E. , Salardi S. , Florio M. , Bianchi G. , Ferrari P.
Published: December 2017

Ouabainlike factor (OLF) has been extracted from the hypothalamus and adrenals of the ox and rats of the Milan hypertensive strain (MHS) and their normotensive controls (MNS). OLF was identified by its ability to 1) inhibit ouabain-sensitive 86Rb uptake into human erythrocytes, 2) displace [3H]ouabain binding, and 3) inhibit purified dog kidney Na-K-adenosinetriphosphatase (ATPase). Rat and bovine OLF have similar characteristics. Those that are close to ouabain are 1) ligand conditions for maximal inhibitory activity, 2) high-performance liquid chromatography retention time, 3) reversibility of inhibitory activity on Na-K-ATPase, 4) reduced Na-K pump inhibitory activity by K, 5) high affinity for Na-K-ATPase, and 6) no activity on calcium ATPase. OLF does not resemble ouabain in the following characteristics: 1) the capacity of OLF to inhibit ouabain low-affinity Na-K-ATPase isoform is greater than that of ouabain and 2) the capacity of OLF to inhibit renal Na-K-ATPase isoforms is greater when the enzyme is obtained from adult rather than young rats. The yield of OLF is greater from MHS than MNS. These findings represent the first direct evidence that a higher amount of OLF is present in tissues from genetically hypertensive rats than from their inbred normotensive controls, maintained under the same dietary and environmental conditions. This further supports previous observations on the role of OLF in the pathogenesis of MHS hypertension.
Macrophages mediate adverse effects of cholesterol feeding in experimental nephrosis
Authors: Pesek-Diamond I. , Ding G. , Frye J. , Diamond J. R.
Published: December 2017

We tested whether the deleterious effects of hypercholesterolemia, in a progressive glomerular disease model, may be mediated by infiltrating renal macrophages. A single sublethal dose of whole body X-irradiation (XI) delivered to rats with acute puromycin aminonucleoside (PA) nephrosis fed a high-cholesterol (HC) diet resulted in significantly greater inulin and p-aminohippurate (PAH) clearances at 11 days after PA without any alterations in circulating lipid levels, in contrast to nonirradiated HC-fed nephrotic controls. This functional protection was associated with significant declines in both glomerular and cortical interstitial macrophage number. Over the course of this 16-wk model, HC-fed PA rats had significantly less albuminuria as well as significantly fewer glomerulosclerosis (GS) lesions and less mesangial matrix expansion at the end of the study despite an equivalent degree of sustained hypercholesterolemia. This data suggests that reducing the infiltrating glomerular and cortical interstitial macrophage burden with XI during acute PA nephrosis, unaccompanied by any hypolipidemic effect, produces not only early salutary effects on renal function but also a significant amelioration of the progressive glomerulopathic features of this model. This is consistent with the hypothesis that the infiltrating renal macrophage, in large part, directly mediates the adverse effects of hypercholesterolemia in this model.
Chloride transport in a mathematical model of the rat proximal tubule
Authors: Weinstein A. M.
Published: December 2017

The proximal tubule model of this laboratory [Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F860-F873, 1986] has been updated to examine proposed pathways for Cl- transport. Two additional buffer pairs have been added, i.e., HCO2-/H2CO2 and NH3/NH4+. At the luminal cell membrane Cl-/HCO2- and Cl-/HCO3- exchange are considered as pathways for Cl- entry, whereas at the peritubular membrane, Cl- exit occurs by either Na(+)-2HCO3-/Cl- exchange or K(+)-Cl- cotransport. Calculations with this model indicate that absolute proximal reabsorption of both Na+ and Cl- are critically dependent on the rate of luminal Na+/H+ exchange. In contrast, increases in the coefficient for Cl-/HCO2- exchange have little impact on overall Cl- flux, but, by enhancing base secretion, limit the depression of end-proximal HCO3-. Model calculations confirm those of Preisig and Alpern (J. Clin. Invest. 83: 1859–1867, 1989) showing that their measured value of luminal membrane H2CO2 permeability is inadequate to sustain the transcellular Cl- flux as Cl-/HCO2- exchange. Conversely, with sufficiently high H2CO2 permeability, luminal Cl- uptake is enhanced along the tubule, as HCO2- secretion and luminal acidification increase luminal H2CO2 to values severalfold greater than in glomerular filtrate. At the basolateral membrane, the thermodynamic driving force across the Na(+)-2HCO3-/Cl- exchanger is small. Although its contribution to steady-state Cl- exit may be less than the K(+)-Cl- cotransporter, the Na(+)-2HCO3-/Cl- exchanger can be a mechanism by which cytosolic acidification enhances peritubular Cl- transport, when luminal acidification enhances luminal Cl- uptake. A simulation is presented in which impermeant replacement of luminal Na+ leads to enhanced convective Cl- flux across the tight junction and alkalinization of the lateral interspace. In this setting, cytosolic Cl- depletion via the Na(+)-2HCO3-/Cl- exchanger may mimic luminal membrane Na(+)-Cl- cotransport.
Folate transport and binding by cultured human proximal tubule cells
Authors: McMartin K. E. , Morshed K. M. , Hazen-Martin D. J. , Sens D. A.
Published: December 2017

Because mechanisms for the renal regulation of folate excretion are poorly understood, a cell culture system representative of the human proximal tubule (HPT) was used for studies of renal folate transport. After confluent cultures of HPT cells were incubated with 3H-labeled folic acid (PteGlu), binding to the apical membrane was determined by an acid removal procedure, and transport was subsequently measured in solubilized cells. Although PteGlu binding was almost all specific (suppressed by excess unlabeled PteGlu), HPT cells transported PteGlu by specific and nonspecific processes. Specific PteGlu binding and transport were both saturable processes, reaching maxima of 0.5 and 0.1 pmol/mg protein, respectively, with half-maximal constants of 12 and 50 nM, respectively. The PteGlu analogues methotrexate and 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) inhibited both the binding and transport of PteGlu, with 5-CH3-H4PteGlu being more potent (lower half-maximal inhibitory concentration). In contrast, 5-formyltetrahydrofolic acid significantly reduced PteGlu transport at concentrations (100–250 nM) that had no effect on binding. These data suggest that the HPT cells will serve as a good model for studies of renal folate reabsorption. Initial characterization of the transport of folate by HPT cells suggests two distinct processes, binding to a high-affinity membrane folate-binding protein followed by a structurally specific transfer into the cell.