bluuebunny/crossref_metadata_2025 huggingface dataset:
Oncotic effects across isolated perfused renal tubular basement membrane
Authors: Williams J. C.
Published: December 2017

Peritubular protein concentration affects the rate of volume absorption by the proximal tubule, and this effect may be mediated across the tubular basement membrane. To examine the permeability properties of this membrane, segments of mouse proximal straight tubule were isolated and perfused, and cells were removed using deoxycholate. Volume loss from the isolated perfused basement membrane was measured using blue dextran as a volume marker. Each segment was exposed alternately to solutions containing either no colloid or 6% bovine serum albumin. With rates of perfusion averaging 15.9 +/- 1.3 nl/min, rates of volume loss averaged 0.7 +/- 1.7 with no protein in the bath but were significantly higher with albumin, 16.5 +/- 2.1 nl.min-1.mm-1 (n = 14; mean length = 0.4 +/- 0.04 mm). Significant volume flux was also seen when the bath was changed to contain dextran [weight-averaged mol wt (Mw) approximately 70,000 or approximately 500,000]. In separate experiments, the net flux of albumin with albumin in the bath averaged 4.2 +/- 1.2 pmol.min-1.mm-1 (n = 5). The results show that this basement membrane has a high water permeability and significantly restricts the movement of albumin. Permeabilities for osmotic water flow and albumin were estimated from modeling fluxes in individual segments and found to average 70.6 +/- 9.8 cm/s (sigma = 0.1; 11.4 +/- 1.6 for sigma = 1) and 1.6 +/- 0.3 micron/s (assuming sigma = 0.1; 0.8 +/- 0.2 for sigma = 1), respectively. Using these permeabilities, it is demonstrated that interstitial protein may significantly reduce hydrostatic pressure in the intercellular space by oncotic action across the basement membrane.
Exogenous cGMP prevents decrease in diuresis and natriuresis induced by inhibition of NO synthesis
Authors: Lahera V. , Navarro J. , Biondi M. L. , Ruilope L. M. , Romero J. C.
Published: December 2017

We previously demonstrated that the intravenous infusion of the specific inhibitor of nitric oxide (NO) synthesis, NG-nitro-L-arginine methyl ester (L-NAME), over a period of 60 min elevates mean arterial pressure (MAP) and reduces renal hemodynamics and excretory function. The objective of the present study was to determine the ability of a guanosine 3',5'-cyclic monophosphate (cGMP) analogue, 8-bromo-cGMP (8-BrcGMP), in preventing the increase in MAP and the reductions in renal plasma flow (RPF), glomerular filtration rate (GFR), urine flow (UV), and sodium excretion rate (UNaV) induced by intravenous infusion of L-NAME in rats. As expected, the infusion of L-NAME (50 micrograms.kg-1.min-1) increased (P < 0.05) MAP and reduced (P < 0.05) RPF, GFR, UV, and UNaV. The administration of 8-BrcGMP (100 micrograms.kg-1.min-1) and L-NAME resulted in no change in MAP, RPF, and GFR. However, decreased (P < 0.05) UV and UNaV were still observed. When 8-BrcGMP (200 micrograms.kg-1.min-1) and L-NAME were infused together, no significant changes in MAP or in renal function were observed. To prove the specificity of the 8-BrcGMP preventive effects, dibutyryl cAMP (200 micrograms.kg-1.min-1) and L-NAME (50 micrograms.kg-1.min-1) were infused together. Under these conditions, MAP, RPF, GFR, UV, and UNaV were modified in a manner similar to that observed during the infusion of L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)
Metabolic clearance of vasopressin and an analogue resistant to vasopressinase in human pregnancy
Authors: Davison J. M. , Sheills E. A. , Philips P. R. , Barron W. M. , Lindheimer M. D.
Published: December 2017

The metabolic clearance rate (MCR) of arginine vasopressin (AVP) increases fourfold during human pregnancy. To explore whether circulating vasopressinase may play a role in this change, six women underwent a three-tier infusion clearance study, twice, in random order, to determine the MCRs of either AVP or 1-deamino-8-D-AVP (dDAVP, an analogue resistant to degradation by vasopressinase). Volunteers were tested in late pregnancy (LP), 24-48 h postdelivery (PD), and 5-6 (PP1) and 10-12 (PP2) wk postpartum, thus examining MCRs when vasopressinase levels were high, before and after removal of the placenta (LP and PD), and when plasma enzyme activity was becoming (PP1) and became (PP2) undetectable. Manipulation of infusate permitted comparison of MCRs at three plasma levels whose range was similar at each test period. PAVP and PdDAVP (2.2 and 10 pg/ml, respectively, during the initial infusion) increased to 8 and 31 pg/ml, stepwise increments that had no influence on respective MCRs (AVP: 3.4, 2.2, 0.77, and 0.67 l/min during LP, PD, PP1, and PP2 compared with 0.18, 0.21, 0.17, and 0.15 l/min for dDAVP). Comparison of similar and submaximal urinary osmolality revealed PdDAVP values three- to fourfold greater than PAVP. Von Willebrand factor (VWF) and factor VIIIc levels increased almost fourfold in response to dDAVP during pregnancy, but only doubled in the nonpregnant state; these differences did not reach significance. We conclude that although AVP disposal rates increase fourfold in pregnancy, those of dDAVP change little, suggesting a role of vasopressinase in the increased MCR of AVP in gestation (as well as in the genesis of certain polyuric disorders of pregnancy).(ABSTRACT TRUNCATED AT 250 WORDS)
Model of ammonium and bicarbonate transport along LDL: implications for alkalinization of luminal fluid
Authors: Mejia R. , Flessner M. F. , Knepper M. A.
Published: December 2017

Luminal fluid exiting the proximal convoluted tubule of a juxtamedullary nephron is alkalinized as it passes through the long-loop thin descending limb of Henle (LDL). Three potential mechanisms of alkalinization are: 1) concentration of bicarbonate by water abstraction, 2) direct bicarbonate entry, and 3) NH3 entry. We have used a mathematical model of the LDL to investigate these mechanisms. With permeabilities of HCO3-, NH3, and NH4+ measured for subsegments of the chinchilla LDL [M. F. Flessner, R. Mejia, and M. A. Knepper. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33):F388-F396, 1993], the osmotic water permeability of each segment [C.-L. Chou and M. A. Knepper. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32):F417-F426, 1992], and appropriate parameters from the literature, we have used the model to calculate hypothetical pH, HCO3- concentration, and NH3 concentration of the luminal fluid as it descends the LDL within an assumed interstitium. After eliminating each mechanism in turn by setting the appropriate permeability to zero, we recalculated the axial profiles. Our results suggest that, although all three mechanisms individually contribute to LDL alkalinization, NH3 entry likely plays the dominant role.
HCO3- transport in rat CCD: rapid adaptation by in vivo but not in vitro alkalosis
Authors: Gifford J. D. , Ware M. W. , Luke R. G. , Galla J. H.
Published: December 2017

Acute chloride-depletion alkalosis (CDA) in vivo results in sustained net total carbon dioxide (tCO2) secretion in vitro in the rat cortical collecting duct (CCD) for several hours. To determine whether altering in vitro pH and electrolytes similarly result in tCO2 secretion, CCD were incubated for 1 h at 37 degrees C in an alkalotic environment similar to in vivo arterial pH, PCO2, and electrolytes (pH 7.6, 40 mM HCO3). The in vitro alkalosis incubation had no effect on tCO2 transport. Second, alteration of the magnitude of vivo alkalosis was correlated with in vitro tCO2 transport. After generation of CDA by intraperitoneal dialysis against 154 mM HCO3-, rats received an infusion for 2.5 h of either 5% dextrose to maintain alkalosis or 154 mM NaCl at differing rates to partially correct or fully correct the systemic alkalosis. After in vitro isolation and perfusion, in vitro tCO2 flux correlated with in vivo Cl- balance (r2 = 0.82), serum HCO3- (r2 = 0.84), and arterial H+ concentration (r2 = 0.78), but not with K+ balance (r2 = 0.33). These findings suggest that: 1) the regulation of tCO2 transport in vitro correlates with the degree of systemic alkalosis and Cl- balance in vivo, and 2) simulating alkalotic pH and electrolytes in vitro does not rapidly alter transport as does in vivo CDA within a similar time. Taken together, pH and electrolyte changes alone cannot account for the rapid adaptation of tCO2 transport in the CCD, but an in vivo factor(s) contributes importantly to alter tCO2 transport in magnitude and direction that would tend to restore normal acid-base balance.
High-salt diet upregulates activity and mRNA of renal Na(+)-K(+)-ATPase in Dahl salt-sensitive rats
Authors: Nishi A. , Celsi G. , Aperia A.
Published: December 2017

We examined the effect of a high-salt (HS) diet on the regulation of renal cortical Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in young Dahl salt-sensitive (DS) and salt-resistant (DR) rats. The activity of Na(+)-K(+)-ATPase, determined in permeabilized proximal tubule segments, was similar in DS and DR rats on normal salt (NS) diet. HS diet resulted in a twofold increase in proximal tubule Na(+)-K(+)-ATPase activity in DS rats but not in DR rats. The mRNA abundance, which was also similar in DS and DR rats on NS diet, increased after 2 days on HS diet in both innervated and denervated kidneys from DS rats but had no effect in DR rats. The activity of Na(+)-K(+)-ATPase and the content of alpha 1- and beta-protein in cortical homogenate were similar in DS and DR rats on both NS and HS diets. Treatment with benserazide, an inhibitor of dopa decarboxylase, upregulated proximal tubule Na(+)-K(+)-ATPase activity and increased Na(+)-K(+)-ATPase mRNA in DR rats on HS diet. Taken together, these data indicate that there is a primary defect in the dynamic hormonal regulation of Na(+)-K(+)-ATPase activity in intact tubular cells, which might stimulate Na(+)-K(+)-ATPase transcription.
Parathyroid hormone inhibition of vasopressin-induced vascular smooth muscle contraction
Authors: Wang X. , Briner V. A. , Schrier R. W.
Published: December 2017

In various cells, parathyroid hormone (PTH) has been shown to initiate both polyphosphoinositide (PI) breakdown and activation of adenylate cyclase (AC). In vascular smooth muscle cells (VSMC), PI hydrolysis is known to induce contraction, whereas a rise in adenosine 3',5'-cyclic monophosphate (cAMP) causes relaxation. In the present study, the effect of PTH on arginine vasopressin (AVP)-induced VSMC contraction and signal transduction was studied. PTH (10(-7) M) attenuated the percentage of VSMC contracting in response to AVP (10(-7) M; 40 to 26.5%, P < 0.05). This loss of VSMC contractility was not the result of PTH-induced changes in AVP receptor binding. PTH did, however, stimulate VSMC cAMP production in a dose-dependent manner. The effect of PTH on AVP-induced contraction could be mimicked by treating VSMC with the cell-permeant cAMP analogue, 8-(4-chlorophenylthio)-cAMP (ClPheScAMP). The effect of PTH on AVP-induced VSMC contraction was blocked by H-8, an inhibitor of protein kinase A and thus cAMP production. In parallel to the inhibitory effects of ClPheScAMP on VSMC contraction, AVP-stimulated inositol trisphosphate production was also reduced by this permeant cAMP (4,415 to 2,592 cpm/mg protein, P < 0.01). PTH-induced production of cAMP was not blocked by an inhibition of prostaglandin synthesis [PTH 203 vs. PTH + ibuprofen 161 fmol/micrograms protein, not significant (NS)]. In contrast, AVP also stimulated cAMP, but this increase was blocked by ibuprofen.(ABSTRACT TRUNCATED AT 250 WORDS)
Determinants of relative amounts of medullary organic osmolytes: effects of NaCl and urea differ
Authors: Nakanishi T. , Uyama O. , Nakahama H. , Takamitsu Y. , Sugita M.
Published: December 2017

Sorbitol, inositol, betaine, taurine, and glycerophosphorylcholine (GPC) are organic osmolytes that accumulate in the renal inner medulla during antidiuresis. In the cultured cell model, high medium sodium increases all the cell osmolytes and high medium urea increases cell GPC and inositol. It has been difficult, however, to discriminate between the effects of sodium and urea on organic osmolytes in water-deprived animals. To determine the nature of the in vivo responses of osmolyte accumulation induced by extracellular sodium or urea, we measured the medullary organic osmolytes and tested the degree of their linear correlation with urine and tissue parameters in control, dehydrated, salt-loaded, and urea-loaded rats. All of the osmolytes except myo-inositol increased in salt-loaded rats. Betaine and sorbitol contents in dehydrated rats were less than in salt-loaded rats, but other osmolytes increased significantly. Conversely, in urea-loaded rats, only GPC increased significantly, whereas either no change occurred for other osmolytes or sometimes betaine and sorbitol levels decreased. These data suggest that high sodium increases all of the osmolytes except myoinositol, whereas high urea increases only GPC and may decrease the renal medullary contents of betaine and sorbitol. We also demonstrated, using linear regression analysis, that urea and electrolyte in urine as well as tissue correlate well with each osmolyte measured.
Rat kidney band 3 Cl-/HCO3- exchanger mRNA is transcribed from an alternative promoter
Authors: Kudrycki K. E. , Shull G. E.
Published: December 2017

We have previously shown that the rat kidney band 3 Cl-/HCO3- exchanger mRNA encodes an NH2-terminal truncated form of band 3 and that its 5' end differs from that of the erythrocyte band 3 mRNA (K. E. Kudrycki and G. E. Shull. J. Biol. Chem. 264: 8185-8192, 1989). To determine the genetic basis for the alternative kidney and erythroid mRNAs, we 1) isolated and characterized a rat erythroid band 3 cDNA, 2) isolated the rat band 3 gene and determined the exon/intron organization of sequences corresponding to the alternative 5' ends of the rat kidney and erythroid mRNAs, and 3) identified the transcription initiation sites of the two transcripts. The unique sequences at the 5' end of the rat erythroid mRNA are derived from exons 1-3 and are followed directly by sequences from exon 4 that are common to both mRNAs. In the kidney mRNA, sequences upstream of exon 4 are derived entirely from intron 3. Primer extension and S1 nuclease protection analyses demonstrate the presence of multiple transcription initiation sites for the rat erythroid band 3 mRNA at the beginning of exon 1, whereas the transcription initiation site for the kidney mRNA is located within intron 3. Thus two distinct promoters, separated by almost 5 kb of genomic sequence, are responsible for the highly tissue-specific transcription of the alternative rat erythroid and kidney band 3 mRNAs.
Cholesterol, macrophages, and gene expression of TGF-beta 1 and fibronectin during nephrosis
Authors: Ding G. , Pesek-Diamond I. , Diamond J. R.
Published: December 2017

Hypercholesterolemia aggravates experimental progressive glomerular injury. Evidence suggests the infiltrating glomerular macrophage (M phi) is a potential effector mechanism for the noxious effects of hypercholesterolemia. Because transforming growth factor (TGF)-beta 1 is secreted by activated M phi s and also stimulates fibronectin production by glomerular cells, we evaluated the kinetics of gene expression for these moieties in glomeruli isolated from nephrotic rats at 3, 7, 11, and 42 days after the delivery of puromycin aminonucleoside (PA). We also assessed whether cholesterol feeding, which raises the glomerular M phi number, alters the glomerular mRNA levels for TGF-beta 1 and fibronectin. Glomerular mRNA levels for TGF-beta 1 and fibronectin in nephrotic rats exhibited a biphasic temporal pattern, decreasing significantly below control at 3 and 7 days after PA but increasing significantly at 11 and 42 days after PA. The upregulated gene expression for TGF-beta 1 and fibronectin at 11 days after PA temporally corresponded to the phase of mesangial M phi infiltration in this model. Cholesterol feeding to both normal and nephrotic rats significantly increased glomerular TGF-beta 1 and fibronectin mRNA levels at 11 days after PA. Immunohistochemical labeling for M phi s and intracellular TGF-beta 1 demonstrated both mesangial and cortical interstitial localization with the TGF-beta1-positive cells possessing M phi nuclear morphology. These findings identify a novel interaction between hypercholesterolemia, augmented glomerular M phi accumulation, and upregulated glomerular TGF-beta 1 and fibronectin gene expression. These perturbations within the acutely injured glomerulus constitute an early pathobiological determinant for the later development of mesangial matrix expansion and glomerulosclerosis.
Transforming growth factor-beta inhibits phosphate transport in renal epithelial cells
Authors: Law F. , Rizzoli R. , Bonjour J. P.
Published: December 2017

The effect(s) of transforming growth factor-beta (TGF-beta) on Pi transport was investigated in confluent opossum kidney (OK) epithelial cells. TGF-beta induced a time- and concentration-dependent decrease in the initial rate of sodium-dependent Pi, but not alanine, transport. This selective inhibitory effect on Pi transport was largely reversible and was not associated with a rise in adenosine 3',5'-cyclic monophosphate production. The reduction in Pi uptake was also independent of changes in extracellular calcium concentrations and prostaglandin synthesis. TGF-beta-mediated Pi transport inhibition appeared to involve neither pertussis toxin-sensitive G protein(s) nor augmented protein kinase C activity. However, the probable role of a serine/threonine protein kinase in signal transduction was supported by the considerable attenuation of TGF-beta effect by H-7. Furthermore, the TGF-beta-induced Pi transport reduction was blunted by cycloheximide and abolished by actinomycin D. In conclusion, TGF-beta selectively inhibits the activity of the sodium-dependent Pi transport system present in the apical membrane of renal epithelial cells. This action appears to be exerted via an unprecedented inhibitory pathway that might involve a serine/threonine protein kinase and alterations in the transcriptional and translational processes.
Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium
Authors: Burns K. D. , Inagami T. , Harris R. C.
Published: December 2017

The rabbit proximal tubule (PT) has been widely utilized to study the direct effects of angiotensin II (ANG II) on PT function. The purpose of the present study was to characterize the binding properties of PT ANG II receptors, using nonpeptide antagonists, and to clone a rabbit PT ANG II receptor. In rat and rabbit kidney cortical brush-border and basolateral membranes, specific binding of 125I-ANG II was inhibited by the AT1 ANG II-receptor antagonist DuP 753, but not by the AT2 antagonist PD 123319. Using a rabbit kidney cortex cDNA library, we isolated cDNA encoding an ANG II receptor, with an open-reading frame sharing a high degree of sequence homology to previously cloned AT1 ANG II receptors. In transfected COS-1 cells, this rabbit ANG II receptor had properties of the AT1 class. Northern analysis revealed high levels of mRNA expression for this receptor in rabbit kidney cortex and adrenal gland. Within the kidney, message was detected in primary cultures of rabbit PT cells, as well as in freshly isolated rabbit PT segments. Message was also present in cells of the mouse PT line, MCT, and in rat glomerular mesangial cells. Utilizing polymerase chain reaction (PCR) with primers derived from the 1st and 4th transmembrane domains of the rat AT1A ANG II receptor, a 279-bp DNA fragment was amplified from reverse-transcribed RNA from rabbit PT cells. This DNA encoded an amino acid sequence identical to that encoded by the rabbit kidney cDNA clone in the corresponding region and differed by a single base substitution. Southern analysis of rabbit genomic DNA restriction digests with the rabbit ANG II receptor probe revealed hybridization to a single band in each lane. These results indicate that an AT1 ANG II receptor is present in the PT and that a single gene codes for the AT1 receptor in rabbit. The clone isolated in the present study should provide a useful tool with which to study the regulation of the PT renin-angiotensin system.
Substrate activation of mechanosensitive, whole cell currents in renal proximal tubule
Authors: Cemerikic D. , Sackin H.
Published: December 2017

Isolated, polarized, proximal tubule cells of Rana pipiens were voltage clamped and examined for both single-channel and whole cell currents. Barium-sensitive whole cell conductances were calculated from the difference in slopes of the current-voltage relations before and after 5 mM external barium. In 11 voltage-clamped cells with high K in the pipette (and cell), isosmotic addition of 40 mM glucose to the bathing solution increased cell volume by 23 +/- 4% within 2-3 min and increased barium-sensitive conductance by 40 +/- 10% from 0.5 to 0.7 nS (P < 0.005, with each cell as its own control). Isosmotic addition of nonmetabolizable methyl-alpha-D-glucopyranoside, which enters with Na across the apical membrane, produced a similar increase in barium-sensitive conductance (30 +/- 13%). In contrast, 3-O-methyl-D-glucopyranose, which is not cotransported with Na, did not alter either cell volume or barium-sensitive conductance. Isosmotic addition of 40 mM phenylalanine (Phe) increased cell volume by 21 +/- 3% and increased barium-sensitive conductance by 36 +/- 19% from 1.1 to 1.5 nS (P < 0.005, with each cell as its own control; n = 8). All K channels observed at the basolateral membrane of these amphibian cells were found to be activated by pipette suction (stretch) and inhibited by 5 mM external barium (outside-out patches). Hence, stretch-activated (SA) K channels must be mediating the macroscopic increase in whole cell K conductance (GK) after isosmotic addition of glucose and Phe. The process does not seem to involve changes in ATP, because Phe increased GK even more when cytosolic ATP was maintained at high levels (10(-4) M extracellular ouabain and 5 mM intracellular ATP). It is also unlikely that changes in cell pH or calcium mediate the increase in GK, because the bulk composition of the cell is "clamped" by the pipette solution in these experiments (1-micron tip patch pipettes). Consequently, the substrate-induced increase in GK probably arises from a swelling-associated deformation of the submembane cytoskeleton or a direct change in membrane tension. In either case, SA channels would play a physiological role in proximal tubule K homeostasis during sugar and amino acid reabsorption in the proximal tubule of the kidney.
Attenuation of immune-mediated glomerulonephritis with an anti-CD11b monoclonal antibody
Authors: Wu X. , Pippin J. , Lefkowith J. B.
Published: December 2017

Nephrotoxic nephritis (NTN), a model of autoimmune glomerulonephritis, is characterized by glomerular inflammation, which results in both proteinuria and an increase in eicosanoid production. In light of the ability of CD18 integrins to participate in leukocyte adherence (and thereby migration), we examined the role of the integrin CD11b/CD18 in NTN using OX42, a monoclonal antibody directed against rat CD11b. Administration of OX42 30 min before induction of NTN decreased proteinuria (by 50%) but did not affect the number of leukocytes found in the glomerulus or the accompanying increase in glomerular eicosanoid production. Administration of OX42 16 h before disease induction led to a more substantial decrease in proteinuria (80%) and, in contrast to 30 min pretreatment, decreased the number of neutrophils found in the glomerulus and the accompanying increase in glomerular eicosanoid production (both by 50%). OX42 pretreatment had no effect on the number of macrophages found in glomeruli. Circulating leukocytes from animals treated with OX42 in vivo showed saturating surface levels of antibody by fluorescence-activated cell sorting (FACS) analysis and normal upregulation of CD11b by pharmacological activation. Sixteen hours after in vivo injection of OX42, 50% more peripheral leukocytes were labeled relative to control leukocytes labeled with OX42 ex vivo. Glomerular leukocytes in NTN exhibited upregulated expression of CD11b relative to peripheral leukocytes. These data show that CD11b/CD18 may participate in the acute expression of glomerular damage in NTN in a fashion not wholly dependent on blocking neutrophil migration into glomeruli. Blockade of surface receptors (as opposed to inhibition of upregulation) is sufficient to obtain this effect.
Dopamine antagonizes the actions of angiotensin II in renal brush-border membrane
Authors: Sheikh-Hamad D. , Wang Y. P. , Jo O. D. , Yanagawa N.
Published: December 2017

In the present study, we examined the effects of dopamine and angiotensin II (ANG II) in renal brush-border membrane (BBM). With isolated BBM vesicles, dopamine (> 10(-4) M) directly inhibited BBM 22Na+ uptake and activated phospholipase C (PLC). These effects were mimicked by DA1 agonist but not DA2 agonist and were prevented by DA1 antagonist but not DA2 antagonist, indicating the involvement of DA1 receptors. In contrast to dopamine, ANG II directly stimulated BBM 22Na+ uptake and activated BBM phospholipase A2 (PLA2). Neither dopamine nor ANG II altered BBM adenosine 3',5'-cyclic monophosphate content. In the presence of dopamine, ANG II failed to stimulate BBM Na+ uptake and PLA2. However, both DA1 and DA2 agonists similarly abrogated the actions of ANG II, and both DA1 and DA2 antagonists were required to restore ANG II actions in the presence of dopamine, indicating the involvement of both DA1 and DA2 receptors in the antagonistic effect of dopamine. Dopamine, as well as DA1 or DA2 agonists, also lowered 125I-ANG II BBM binding. In summary, these results show that, in renal BBM, dopamine impedes ANG II receptor binding and antagonizes the stimulatory effects of ANG II on Na+ uptake and PLA2. This occurred through both DA1 and DA2 receptors and independent of DA1 effects on BBM Na+ uptake or PLC.
Calcitonin stimulates calcium transport in distal convoluted tubule cells
Authors: Gesek F. A. , Friedman P. A.
Published: December 2017

Calcitonin increases Ca2+ absorption by renal distal tubules. The mechanism underlying this action is not known. Calcitonin effects on Ca2+ uptake, intracellular Ca2+ activity, and membrane voltage were determined in transformed mouse distal convoluted tubule (DCT) cells. Intracellular Ca2+, measured in single cells grown on cover slips and loaded with fura-2, averaged 102 nM. Maximal increases of intracellular Ca2+ to 312 nM were elicited by 1 nM calcitonin. Calcitonin stimulated 45Ca2+ influx by 45% from control rates of 2.8 nmol.min-1 x mg protein-1. Membrane voltage was measured in single cells loaded with the voltage-sensitive dye 3,3'-dihexyloxacarbocyanine iodide. Resting membrane voltage averaged 69 mV. Calcitonin maximally hyperpolarized cells by 18 mV. The Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid, abolished calcitonin-induced membrane hyperpolarization and Ca2+ influx. Calcitonin increased halide ion permeability, estimated from the rate of 125I- efflux, by 160%. The Ca2+ channel blocker nifedipine abolished calcitonin-stimulated 45Ca2+ uptake and the increase of intracellular Ca2+. Combined exposure to calcitonin and parathyroid hormone (PTH) had no additional effect on 45Ca2+ influx or intracellular Ca2+. In contrast to the long latency (8 min) before onset of PTH effects, calcitonin increased 45Ca2+ uptake and intracellular Ca2+ concentration within 2 min. Blockade of protein synthesis with cycloheximide inhibited Ca2+ uptake induced by PTH but not that stimulated by calcitonin. We conclude that calcitonin increases Cl- conductance in DCT cells, resulting in membrane hyperpolarization and activation of Ca2+ entry through dihydropyridine-sensitive Ca2+ channels. The shorter onset of calcitonin action compared with PTH may reflect differences in the signaling mechanisms between the two hormones.
Decrease in Gs protein expression may impair adenylate cyclase activation in old kidneys
Authors: Liang C. T. , Barnes J. , Hanai H. , Levine M. A.
Published: December 2017

The possibility that alteration in stimulatory guanine nucleotide-binding protein (Gs) expression may contribute to the blunting of renal parathyroid hormone (PTH)-stimulated adenylate cyclase in aged rats was examined. Using rat cDNA probe, we identified a Gs alpha-subunit (Gs alpha) of 1.9 kb. Age did not alter the size of Gs alpha mRNA. The level of Gs alpha mRNA [normalized to poly(A)+ RNA] was decreased 23%, which was consistent with our previous report that Gs alpha protein decreased in senescence. In contrast, mRNA level of Gi alpha 2 increased with age. Level of beta-actin mRNA did not change with age. Nuclear runoff assay was performed to determine the transcription rate of Gs mRNA. Synthesis of poly(A)+ RNA and total RNA was reduced 39% and 37%, respectively, in nuclei prepared from old kidney, which suggested a general decline in RNA synthesis capacity in old rats. Our results also showed the transcription rate of Gs alpha mRNA in aged rats was reduced 89%, a decrease far exceeding that observed for total RNA or poly(A)+ RNA. We concluded that the decrease in steady-state level of Gs alpha mRNA was specific and probably was due to a reduction in the transcription activity. Thus alteration in Gs transcription may contribute, at least in part, to the impaired renal adenylate cyclase activation in aged rats.
LPS-induced MCP-1, IL-1 beta, and TNF-alpha mRNA expression in isolated erythrocyte-perfused rat kidney
Authors: Xia Y. , Feng L. , Yoshimura T. , Wilson C. B.
Published: December 2017

The capacity of the lipopolysaccharide (LPS)-stimulated isolated erythrocyte-perfused rat kidney (IEPK) to produce monocyte chemoattractant protein-1 (MCP-1), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) mRNA was investigated. The IEPK was chosen to exclude the influence of circulating neutrophils and monocytes that can produce both these mediators when exposed to LPS. The control minimal LPS group (LPS < 10 pg/ml) showed a small increase in mRNA expression for MCP-1, IL-1 beta, and TNF-alpha in the cortex and medulla after 80 min of perfusion when compared with the unperfused left kidney in which no IL-1 beta or TNF-alpha mRNA and only minimal amounts of MCP-1 mRNA were detected. LPS stimulation (1 microgram/ml for 40 or 80 min) increased MCP-1, IL-1 beta, and TNF-alpha mRNA expression, which was found predominately in peritubular capillary endothelial cells by in situ hybridization. The changes were not due to a marked perturbation of LPS on renal hemodynamics. The renal vascular resistance (RVR) remained constant (40 min LPS exposure) or increased only slightly during the last 5-10 min (80 min LPS exposure) compared with a progressive increase in RVR of the minimal LPS group. The hemodynamic effects of LPS on the IEPK appear to counteract the gradual increase in RVR seen in the minimal LPS group.
Effect of plasma prolactin on sweat rate and sweat composition during exercise in men
Authors: Boisvert P. , Brisson G. R. , Peronnet F.
Published: December 2017

We investigated the role of the exercise-induced elevation of plasma prolactin (PRL) concentration on sweat rate and composition during prolonged exercise in men. Two groups of healthy young males (20-26 yr old) showing a high (high responders; n = 8) or a low (low responders; n = 7) response of plasma PRL concentration to exercise were studied during a 60-min period of exercise on a cycle ergometer (65% maximum O2 consumption) in warm conditions (26.2 +/- 0.1 degrees C; 57 +/- 1% relative humidity), 1 h after receiving 1.25 mg bromocriptine (BRC) per os or a placebo. In high responders, administration of BRC totally abolished the threefold increase in plasma PRL observed in response to exercise with placebo [placebo, 10 +/- 2 (rest) and 30 +/- 2 micrograms/l (exercise); BRC, 9 +/- 1 (rest) and 8 +/- 1 microgram/l (exercise)]. The latter was associated with a significant decrease in sweat rate (2.7 +/- 0.5 to 1.9 +/- 0.3 microliter.cm-2.min-1) and a significant increase in sweat Na+ concentration (57 +/- 7 to 68 +/- 5 mmol/l). BRC also reduced the small response in plasma PRL concentration observed in low responders [placebo, 10 +/- 1 (rest) and 15 +/- 1 microgram/l (exercise); BRC, 9 +/- 1 (rest) and 7 +/- 1 microgram/l (exercise)], but this was not associated with any change in sweat rate (2.2 +/- 0.2 to 1.9 +/- 0.3 microliter.cm-2.min-1) or in sweat Na+ concentration (63 +/- 10 to 64 +/- 9 mmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)
Short-term effect of aldosterone on vasopressin-sensitive adenylate cyclase in rat collecting tubule
Authors: el Mernissi G. , Barlet-Bas C. , Khadouri C. , Cheval L. , Marsy S. , Doucet A.
Published: December 2017

Because previous studies indicated that, in the rat collecting tubule, vasopressin (AVP)-sensitive adenylate cyclase (AC) is controlled by mineralocorticoids in the long term, the present study was designed to investigate whether such a control also exists in the short term. Therefore, we investigated the in vivo and in vitro effects of aldosterone on AC activity in cortical and outer medullary collecting tubules (CCD and OMCD, respectively) from adrenalectomized rats. Injection of aldosterone (10 micrograms/kg body wt) to adrenalectomized rats restored within 3 h AVP-sensitive AC activity in the CCD and OMCD up to the levels observed in the corresponding segments of adrenal intact rats. Similarly, incubating CCD or OMCD from adrenalectomized rats for 2.5 h in the presence of 10(-8) M aldosterone enhanced AVP-sensitive AC activity up to values similar to those found in normal rats. In vitro stimulation of AVP-sensitive AC activity was dose dependent with regard to aldosterone [apparent affinity constant (K0.5) approximately 10(-9) M], appeared after a 30-min lag period, and reached its maximum after 2-2.5 h. In addition, it was totally abolished by the antimineralocorticoid spironolactone, whereas the specific glucocorticoid antagonist RU 38486 had no effect. Finally, actinomycin D and cycloheximide totally abolished the in vitro action of aldosterone, demonstrating the involvement of protein synthesis in that process.
Endothelin-1 and endothelin-3 binding to rat nephrons
Authors: Takemoto F. , Uchida S. , Ogata E. , Kurokawa K.
Published: December 2017

The existence of endothelin (ET) receptor subtypes has recently been implicated in the different biological effects of ET in various tissues. Indeed, the cDNAs for two types of ET receptors, ETA and ETB, have been cloned. To further gain insights into ET function in the kidney we examined 125I-labeled ET-1 and ET-3 binding to microdissected rat nephron segments. Specific ET-1 binding was highest in the inner medullary collecting duct, whereas the cortical and outer medullary collecting ducts as well as glomeruli showed moderate binding. There was low, although not significant, ET-1 binding to the early portion of the proximal tubule. Other nephron segments displayed little ET-1 binding. The binding profile of ET-3 along the nephron markedly resembled that of ET-1. Scatchard analyses of binding of ET-1 and ET-3 using cortical collecting ducts revealed a single class of receptor for both ET-1 and ET-3; apparent dissociation constants were 2.05 +/- 0.72 and 2.58 +/- 0.32 nM, and maximal binding capacity values were 0.408 +/- 0.058 and 0.511 +/- 0.047 fmol/mm, respectively. Displacement of 125I-ET-1 binding by unlabeled ET-3 was similar to that produced by unlabeled ET-1. Furthermore, a specific ETB agonist, BQ 3020, almost completely inhibited 125I-ET-1 binding in cortical collecting ducts, whereas a specific ETA antagonist, BQ 123, had little effect. These data indicate that cortical collecting ducts express ETB receptors, to which both ET-1 and ET-3 bind equally.
Ammonium and bicarbonate transport in isolated perfused rodent ascending limbs of the loop of Henle
Authors: Flessner M. F. , Knepper M. A.
Published: December 2017

Ammonium accumulates in the renal medullas of antidiuretic animals, presumably due to countercurrent multiplication, driven by NH4+ absorption from ascending loops of Henle in the outer and inner medulla. Active absorption of NH4+ occurs in the thick ascending limb (TAL). But the passive transport properties of NH3 in the TAL and the transport of ammonium or HCO3- in the ascending thin limb (ATL) have not been studied in rodents. To investigate the potential role of the ascending limb segments in medullary accumulation of ammonium, we perfused isolated subsegments of the chinchilla ATL and of the rat ATL and TAL. After imposing concentration gradients of total ammonia or total CO2 across ATL subsegments, we found very high rates of transfer of both substances, implying that at physiological flow rates the tubule luminal fluid quickly equilibrates with the interstitium. In the medullary TAL, we found a passive NH4+ permeability of 17 x 10(-5) cm/s but a relatively low NH3 permeability of < 0.003 cm/s. The low NH3 permeability prevents backleak of NH3 when NH4+ is actively transported from the lumen. We conclude that the ATL acts as an equilibrating segment and the TAL has special permeability properties that enhance net ammonium absorption and therefore enhance medullary ammonium accumulation.
Nucleotide receptors regulate membrane ion transport in renal epithelial cells
Authors: Middleton J. P. , Mangel A. W. , Basavappa S. , Fitz J. G.
Published: December 2017

Regulation of plasma membrane ion transport by endogenous purinergic receptors was assessed in a distal renal (A6) cell line. Nucleotide analogues stimulated Na-K-Cl cotransport activity with relative potencies of ATP > UTP > ATP gamma S > 2-methylthio-ATP = alpha,beta-methylene ATP. Activation of nucleotide receptors with extracellular ATP and nucleotide analogues increased intracellular calcium concentration ([Ca2+]i) primarily by release of intracellular calcium stores, with relative potency of agonists similar to that seen for stimulation of Na-K-Cl cotransport. Neither the change in [Ca2+]i nor the stimulation of cotransport was abolished by the adenosine receptor antagonist 8-(4-[N-(2-aminoethyl)carbamoylmethoxy]-phenyl)-1,3-dipropylxanthi ne (XAC). In contrast to the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine, nucleotide analogues had no discernible effect on cytosolic adenosine 3',5'-cyclic monophosphate levels or adenylyl cyclase activity. To address possible mechanisms for stimulation of Na-K-Cl cotransport by the nucleotide receptor, 125I efflux and patch-clamp studies were used to measure chloride secretion. ATP and ionomycin markedly enhanced 125I efflux and whole cell currents, consistent with activation of chloride conductance pathways. Diphenylamine-2-carboxylate, a chloride channel blocker, eliminated the effects of ionomycin, forskolin, adenosine, and ATP on Na-K-Cl cotransport. This study demonstrates that nucleotide receptors in this model of renal epithelium initiate distinct regulation of Na-K-Cl cotransport. Nucleotide receptors may effect their responses through primary activation of membrane chloride channels.
Proximal tubular secretion of angiotensin II in rats
Authors: Braam B. , Mitchell K. D. , Fox J. , Navar L. G.
Published: December 2017

It is now established that all of the components necessary for the local formation of angiotensin II (ANG II) coexist in the kidney and can alter local ANG II production rate. However, data on ANG II concentrations in different compartments within the kidney are limited. Recently, proximal tubule fluid ANG II concentrations in the nanomolar range were reported. Using an ANG II radioimmunoassay procedure with enhanced sensitivity, we performed experiments to explore proximal tubular fluid ANG II levels further and to determine the source of the ANG II. Total free-flow proximal tubular fluid samples (n = 11) had an average ANG II concentration of 13 +/- 2 nM. These concentrations were similar (10 +/- 2 nM) in samples collected into pipettes containing the inhibitors enalaprilat and EDTA (n = 17). Fluid collected from blocked proximal tubules that were perfused with artificial tubular fluid showed similar ANG II concentrations both in the presence (22 +/- 3 nM) and absence (22 +/- 4 nM) of the angiotensin-converting-enzyme inhibitor, enalaprilat, in the perfusate. Plasma ANG II concentrations were much lower and averaged 155 +/- 26 pM. Isotonic saline expansion lowered plasma ANG II levels to 30 +/- 5 pM (P < 0.01) but did not significantly decrease intraluminal ANG II (8 +/- 1 nM). These data provide further evidence that intratubular ANG II concentrations are in the nanomolar range and are regulated independently of the plasma ANG II levels. The data obtained from perfused tubules indicate that the proximal tubule adds substantial amounts of ANG II or a precursor into the tubular lumen.
Enhanced HCO3 secretion by distal tubule contributes to NaCl-induced correction of chronic alkalosis
Authors: Wesson D. E. , Dolson G. M.
Published: December 2017

Free-flow micropuncture studies show depressed net HCO3 reabsorption in the surface distal tubule of rats undergoing correction of chronic metabolic alkalosis induced by NaCl infusion. The present studies used in vivo microperfusion of the rat distal tubule to investigate whether the described reduction in net HCO3 reabsorption was due to decreased luminal H+ secretion or to increased HCO3 secretion. Animals with correcting alkalosis had higher HCO3 secretion in this nephron segment than did animals with maintained alkalosis regardless of whether the perfusing solution was initially Cl free (-27.6 vs. -16.6 pmol.mm-1.min-1, P < 0.01) or contained 40 mM Cl (-31.5 vs. -23.0 pmol.mm-1.min-1, P < 0.01); H+ secretion was not different between animals with correcting and maintained alkalosis. Animals without alkalosis (control) demonstrated no differences in H+ or HCO3 secretion in response to NaCl infusion. These studies demonstrate that enhanced HCO3 secretion mediates the depressed net HCO3 reabsorption observed in the distal tubule of rats undergoing NaCl-induced correction of chronic metabolic alkalosis.
Biosynthesis of the gp330/44-kDa Heymann nephritis antigenic complex: assembly takes place in the ER
Authors: Biemesderfer D. , Dekan G. , Aronson P. S. , Farquhar M. G.
Published: December 2017

The Heymann nephritis antigenic complex (HNAC) consists of two components, i.e., 1) gp330, a large glycoprotein localized in coated pits of the proximal tubule and glomerular epithelium, and 2) a 44-kDa protein which is homologous to the human alpha 2-macroglobulin receptor-associated protein (RAP). To examine the biosynthesis and assembly of HNAC, tissue fragments prepared from collagenase-digested 1-day-old rat kidneys were radiolabeled, and gp330 and RAP were immunoprecipitated with specific antibodies. By electron microscopy the tubule organization was seen to be largely intact. Results obtained on the biosynthesis of a control brush border protein, dipeptidylpeptidase IV (DPPIV), showed that tubules prepared in this manner are capable of synthesis and posttranslational processing of brush border membrane proteins and thus are suitable for short-term (< 3 h) biosynthetic experiments in vitro. Results of pulse chase and digestion with endoglycosidase H (Endo H) indicated that the time required for newly synthesized gp330 to mature in the endoplasmic reticulum (ER) and transit the middle Golgi compartments [half time (t1/2) = 90 min] was significantly longer than that of DPPIV (t1/2 = 20 min). Coprecipitation and cosedimentation (sucrose velocity gradient centrifugation) experiments showed that gp330 associates with RAP very early after synthesis and that the 44-kDa protein remains associated with gp330 during its subsequent folding, oligomerization, and transport to the Golgi. These findings demonstrate that HNAC assembles in at least two steps. The first step is the association of gp330 with RAP forming a large (19.3S) heterodimer, which sediments with the thyroglobulin (mol wt = 669,000) standard. This step begins within 30 min of synthesis and is Ca2+ dependent. The second step, which occurs > 60 min after synthesis, is the formation of a larger heterooligomer, which results in a shift in size of the complex from 19.3 to 38.6S. Both steps occur before acquisition of Endo H resistance. These results indicate that HNAC consists of a large multimeric complex that is assembled in the rough ER.
Bicarbonate-activated adenylyl cyclase in fluid-transporting tissues
Authors: Mittag T. W. , Guo W. B. , Kobayashi K.
Published: December 2017

A bicarbonate-stimulated adenylyl cyclase (AC) activity was found in ocular ciliary processes, which secrete the aqueous humor of the eye. Other fluid-transporting tissues also showed HCO3(-)-stimulated AC activity. Relative to basal, the response to 10 mM NaHCO3 was greatest in the particulate fraction of bovine ciliary processes, followed by bovine corneal endothelium, bovine choroid plexus, and rat kidney (medulla and cortex). However, no activity was detectable in bovine retina, cerebral cortex, or cerebellum. The activity in ciliary processes was present only in the particulate fraction, was supported by Mg2+ or Mn2+, was independent of GTP, and was additive to the stimulatory G protein-dependent AC response (via beta-adrenergic receptor stimulation). The activation of AC was dose dependent up to 100 mM bicarbonate with a 50% effective concentration of 2-3 mM. The HCO3- response was unaffected by 1 mM methazolamide, a carbonic anhydrase inhibitor, but HSO3- was a partially selective inhibitor compared with its effect on forskolin-stimulated AC activities. The presence of membrane-bound bicarbonate-sensitive AC in fluid-transporting tissues suggests an autoregulatory mechanism for intracellular HCO3- concentration acting via adenosine 3',5'-cyclic monophosphate in the control of membrane ion transporters.
Endothelin modulates angiotensin II-induced mitogenesis of human mesangial cells
Authors: Bakris G. L. , Re R. N.
Published: December 2017

Angiotensin II (ANG II) elicits either a hypertrophic or hyperplastic response depending on culture conditions. Human mesangial cell (HMC)-generated endothelin (ET) plays a role in mediating the hyperplastic effects of arginine vasopressin. The interaction between ANG II and ET is not described in HMC. The present study evaluates the possible effect of ANG II on HMC production of ET, its relationship to mitogenesis, and the effect of insulin. ANG II (10(-8) M) increased [3H]thymidine incorporation in proliferative HMC at 48 h (13 +/- 1 vs. 24 +/- 1 x 10(3) counts.min-1.well-1, for control vs. ANG II; P < 0.05). Cell counts showed parallel increases [12 +/- 1 (control) vs. 18 +/- 1 x 10(3) counts/well; P < 0.05]. This mitogenic effect was attenuated by a monoclonal antibody to ET-1 or the ANG II-receptor antagonist, DuP 753. Insulin potentiated the mitogenic response of ANG II through increases in HMC ET production (69 +/- 7 vs. 189 +/- 13 pg/ml, for insulin alone vs. insulin+ANG II; P < 0.05). This study supports the concept that ANG II may act as a mitogen under certain culture conditions and its effect is, in part, mediated through ET.