bluuebunny/crossref_metadata_2025 huggingface dataset:
Acidosis inhibits osteoblastic and stimulates osteoclastic activity in vitro
Authors: Krieger N. S. , Sessler N. E. , Bushinsky D. A.
Published: December 2017

Metabolic acidosis induces net calcium flux (JCa) from cultured neonatal mouse calvariae through physicochemical and cell-mediated mechanisms. To determine the role of osteoblasts in acid-induced JCa, collagen synthesis and alkaline phosphatase activity were assessed in calvariae incubated in reduced pH and bicarbonate medium, a model of metabolic acidosis (Met), and compared with controls (Ctl). Collagen synthesis fell from 30.5 +/- 1.1 in Ctl to 25.1 +/- 0.4% with Met, and alkaline phosphatase decreased from 403 +/- 25 in Ctl to 298 +/- 21 nmol Pi.min-1.mg protein-1 with Met. During acidosis JCa was correlated inversely with percent collagen synthesis (r = -0.743, n = 11, P = 0.009) and with alkaline phosphatase activity (r = -0.453, n = 22, P = 0.034). To determine the role of osteoclasts in acid-induced JCa, osteoclastic beta-glucuronidase activity was determined in Ctl and Met in the absence or presence of the osteoclastic inhibitor calcitonin (CT, 3 x 10(-9) M). Met increased beta-glucuronidase (5.9 +/- 0.2) compared with Ctl (4.6 +/- 0.3 micrograms phenolphthalein released.bone-1.h-1), whereas CT inhibited beta-glucuronidase in both Ctl and Met (3.1 +/- 0.2 and 3.5 +/- 0.3, respectively). During acidosis JCa was correlated directly with beta-glucuronidase activity (r = 0.683, n = 42, P less than 0.001). Thus the cell-mediated component of JCa during acidosis in vitro appears to result from a combination of inhibited osteoblastic and stimulated osteoclastic activity.
Vanadate causes hypokalemic distal renal tubular acidosis
Authors: Dafnis E. , Spohn M. , Lonis B. , Kurtzman N. A. , Sabatini S.
Published: December 2017

Considerable evidence supports the presence of an H(+)-K(+)-ATPase along the mammalian nephron. Inhibition of this enzyme might be expected to reduce acid excretion while increasing potassium excretion, thus causing hypokalemic distal renal tubular acidosis (RTA). In this study we administered vanadate at a dose of 5 mg/kg ip for 10 days to rats. These animals developed hypokalemic distal RTA with a blood pH of 7.22 +/- 0.01, a plasma bicarbonate of 15.2 +/- 0.6 meq/l, and a plasma potassium of 3.28 +/- 0.06 meq/l. The vanadate-treated animals had a urine pH of 6.70 +/- 0.09, a value significantly higher than NH4Cl-treated animals with the same degree of acidemia (urine pH = 5.25 +/- 0.04). When cortical collecting tubules (CCT) from these animals were microdissected and H(+)-K(+)-ATPase was measured, it was decreased by approximately 75% (P less than 0.001); but H(+)-ATPase was no different from control. In medullary collecting tubule, H(+)-K(+)-ATPase was also decreased but less than in CCT. Muscle potassium concentration in the vanadate-treated animals was significantly lower than in controls. These results demonstrate that vanadate causes hypokalemic distal RTA in association with inhibition of collecting tubule H(+)-K(+)-ATPase activity.
Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI
Authors: Kasner S. E. , Ganz M. B.
Published: December 2017

We investigated the regulatory transport processes that maintain intracellular K+ homeostasis in cultured rat glomerular mesangial cells (MCs). Intracellular K+ concentration ([K+]i) of quiescent MCs, passages 3-8, grown to subconfluence on glass cover slips, was assessed by spectrofluorometry using the K(+)-sensitive dye, K(+)-binding benzofuran isophthalate (PBFI). Serum-starved MCs were incubated at 37 degrees C in 5 microM PBFI for 90 min. Excitation ratios of luminescences at 340 and 380 nm, measured at a constant emission at 500 nm, were used to determine [K+]i. Ionophores valinomycin and nigericin were used to clamp [K+]i to known [K+]o and thereby obtain an intracellular calibration of dye. Dependence of fluorescence ratio on [K+]i conformed to Michaelis-Menten behavior, with a Km of 113 mM (n = 40). PBFI retains its sensitivity to alterations in [K+]i with pH change (pHi from 6.5 to 7.5) but is relatively insensitive when intracellular Na+ is greater than 75 mM and cell osmolarity exceeds 500 mM. Normal resting [K+]i for all experiments was determined in MCs to be 102 +/- 7 mM (n = 81) in a HCO3(-)-free HEPES-buffered solution. When MCs were exposed to ouabain, [K+]i fell to 48 +/- 6 mM and did not recover, suggesting presence of Na(+)-K(+)-ATPase. When MCs were exposed to furosemide, [K+]i transiently declined to 58 +/- 11 mM, which was followed by a rapid recovery to near steady state, indicating additional presence of Na(+)-K(+)-Cl- cotransporter. Recovery was completely abolished when MCs were exposed to ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)
Active potassium absorption by the renal distal tubule
Authors: Okusa M. D. , Unwin R. J. , Velazquez H. , Giebisch G. , Wright F. S.
Published: December 2017

Maintenance of potassium homeostasis during potassium depletion appears to involve an active potassium absorptive mechanism in the distal nephron. Direct demonstration of such a pathway in the distal tubule of the rat has been lacking. The purpose of the current study was to examine the hypothesis that an ATP-dependent active transport mechanism plays a role in potassium absorption by the rat distal tubule. We utilized in vivo microperfusion techniques in Sprague-Dawley rats maintained on a regular diet of low-potassium diet for 3-4 wk. The effect of a selective inhibitor of the gastric H-K-adenosinetriphosphatase (ATPase) (Sch 28080, 0.1 mM) was tested in distal tubules of both groups of rats. Distal tubules of normal rats secreted potassium. Sch 28080 had no effect on this net potassium flux. In contrast, distal tubules of potassium-deficient rats absorbed potassium. Sch 28080 abolished this potassium absorption and produced a small hyperpolarization of the lumen-negative transepithelial voltage (VTE). The change in VTE can be explained by a concomitant increase in potassium concentration in the late distal tubule. These results are consistent with the presence of an H-K-ATPase in the distal tubule of potassium-deficient rats.
Growth factors and metanephrogenesis
Authors: Hammerman M. R. , Rogers S. A. , Ryan G.
Published: December 2017

The formation of all organs during embryogenesis, including kidney, is dependent on the timed and sequential expression of a number of polypeptide growth factors. Synthesis and actions of one or more members of the insulin-like growth factor, epidermal growth factor/transforming growth factor-alpha, transforming growth factor-beta, platelet-derived growth factor, fibroblast growth factor, and nerve growth factor families have been characterized in the developing metanephric kidney. Studies originating from a number of laboratories have defined the localization of growth factor mRNAs, receptors and peptides, have delineated patterns of growth factor synthesis, and have established the growth factor dependency of embryonic kidney development. The results of these investigations will be summarized in this editorial review and integrated within the broader context of growth factor cellular physiology and growth factor expression in nonrenal systems.
Triiodothyronine enhances renal tubule cell replication by stimulating EGF receptor gene expression
Authors: Humes H. D. , Cieslinski D. A. , Johnson L. B. , Sanchez I. O.
Published: December 2017

Thyroid hormone is known to accelerate renal function recovery following toxic acute renal failure. Because epidermal growth factor (EGF)-receptor activation is most likely critical in renal replicative repair, these studies were undertaken to assess whether triiodothyronine (T3), the most active form of thyroid hormone, may modulate EGF-induced renal proximal tubule cell proliferation by an effect on the EGF receptor. Rabbit renal proximal tubular cells were grown in primary culture and treated with or without T3 (0.1 or 1.0 nM) for 24 to 48 h. Compared with nontreated controls, T3 exposure led to significant increases in EGF-promoted DNA synthesis, as measured by [3H]thymidine incorporation, in renal tubule cells. Furthermore, T3 treatment resulted in increases in EGF receptor mRNA in proximal tubule cells compared with nontreated cells and was associated with elevated numbers of EGF receptors on the cell surface of proximal tubule cells. EGF binding studies demonstrated that T3 treatment had only modest effects on Kd values of both the high-affinity binding site (0.19 nM) and the low-affinity binding site (3.7 nM) but substantially increased the maximal number of high-affinity sites from 3.8 x 10(3) to 9.4 x 10(3) receptors/cell and the maximal number of low-affinity sites from 132 x 10(3) to 199 x 10(3) receptors/cell. These findings suggest that a T3 effect to increase EGF receptor gene expression with resulting increases in the number of cell surface EGF receptors on renal proximal tubule cells and a potentiated mitogenic response to EGF may be a mechanism for thyroid hormone to enhance renal function recovery following toxic acute renal failure.
Pharmacological characterization of V1a vasopressin receptors in the rat cortical collecting duct
Authors: Ammar A. , Roseau S. , Butlen D.
Published: December 2017

Vasopressin receptors in distal segments of the rat nephron were identified in isolated tubules using two labeled ligands: the [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine,4-threonine,8-ornithine,9-125I-tyrosylamide]- vasotocin [125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT] and the linear analogue, Phaa1,D-Tyr(Me)2,Phe3,Gln4,Asn5,Arg6, Pro7,Arg8,125I-Tyr-NH2(9) [125I-Tyr-NH2(9)-linear antagonist (LA)-V1a)]. Specific 125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-OVT binding to cortical collecting ducts (CCD) was saturable with incubation time and dose, reversible after elimination of free ligand, and characterized by the following rank order for recognition of vasopressin analogues: desGly9-d-(CH2)5-[Tyr(Et)2,Val4]arginine vasopressin (AVP) greater than or equal to d(CH2)5[Tyr-(ET)2,Val4]AVP greater than or equal to AVP greater than or equal to d(CH2)5[Tyr(Me)2]AVP = 1-desamino-8-D-arginine vasopressin (DDAVP) greater than or equal to Tyr-NH2(9)-LA-V1a greater than [8-arginine]vasotocin (AVT) greater than d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT greater than oxytocin (OT) greater than [Phe2,Orn8]VT much greater than [Thr4,Gly7]-OT. Scatchard plots of dose-dependent 125I-Tyr-NH2(9)-LA-V1a binding to medullary thick ascending limbs (MTAL), CCD, and outer medullary collecting ducts (OMCD) revealed the presence of high- and low-affinity binding sites corresponding to V1a and V2 vasopressin receptors, respectively; the densities of V1a receptors are approximately 20% of the total number of vasopressin receptors in CCD and 5% in MTAL and OMCD.
Regulation of the renal Na-HCO3 cotransporter by cAMP and Ca-dependent protein kinases
Authors: Ruiz O. S. , Arruda J. A.
Published: December 2017

Changes in the activity of the brush-border Na-H antiporter are accompanied by parallel changes in the activity of the Na-HCO3 cotransporter. Adenosine 3',5'-cyclic monophosphate (cAMP) and calmodulin inhibit the Na-H antiporter, whereas protein kinase C (PKC) stimulates it. We hypothesized that cAMP, calmodulin, and PKC should have similar effects on the Na-HCO3 cotransporter activity. Phosphorylated renal basolateral membranes were treated with either cAMP, calmodulin, or phorbol ester. cAMP, 1 microM, inhibited HCO3-dependent 22Na uptake without affecting 22Na uptake in presence of gluconate, suggesting that cAMP inhibits Na-HCO3 cotransporter activity without altering diffusive 22Na uptake. The effect of cAMP to inhibit the Na-HCO3 cotransporter could also be elicited by the catalytic subunit of cAMP, and this inhibitory effect was prevented by the protein kinase A (PKA) inhibitor. Calmodulin (1 microM), in presence of Ca, also inhibited HCO3-dependent 22Na uptake in presence of HCO3, whereas 22Na uptake in the presence of gluconate was unchanged. The inhibitory effect of calmodulin on HCO3-dependent 22Na uptake was prevented by N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide (W-13), an inhibitor of calmodulin. Phorbol 12-myristate 13-acetate and PKC stimulated Na-HCO3 cotransporter activity, whereas the inactive analogue, 4 alpha-phorbol, failed to elicit such a stimulation. Basolateral membranes displayed cAMP-dependent and Ca-dependent protein kinase activities. Thus PKA and Ca-dependent protein kinases regulate the activity of the Na-HCO3 cotransporter and suggest that hormones that act through these systems modulate the activity of the Na-HCO3 cotransporter.
Proximal tubular dopamine production regulates basolateral Na-K-ATPase
Authors: Baines A. D. , Ho P. , Drangova R.
Published: December 2017

Regulation of proximal tubular Na-K-adenosine-triphosphatase (ATPase), brush-border membrane Na(+)-H+ antiporter and Na(+)-Pi symporter activity by endogenously produced dopamine was examined in Wistar rats. Na-K-ATPase was measured in basolateral membrane (BLM) fractions permeabilized with alamethicin or sodium dodecyl sulfate (SDS). Carbidopa (5 mg/kg) injected 18 h before removal of kidneys increased maximal activity (Vmax) noncompetitively in cortical BLM but not in other membrane fractions or outer medullary BLM (-2 +/- 4%). Chronic renal denervation did not alter the response. Carbidopa stimulated Na-K-ATPase in cortical BLM from rats eating a normal salt diet with and without 1% saline to drink (+18 +/- 4% and +22 +/- 4%, respectively; P greater than 0.001). Carbidopa did not increase Vmax of BLM Na-K-ATPase from rats eating a low-salt diet (+1.5 +/- 4%); however, when the low-salt diet was supplemented with 1 mM dihydroxyphenylalanine (dopa) to drink for 1 day carbidopa, increased Vmax by 18 +/- 3% (P = 0.018). Carbidopa did not alter the Michaelis constant (Km) for Na or K or inhibitory constant (Ki) for ouabain. Injection of the DA1 antagonist Sch 23390 (2 mg/kg) also increased Na-K-ATPase (18 +/- 4%; P = 0.014). Western blots using a monoclonal alpha-subunit antibody revealed a 22 +/- 8% increase following carbidopa treatment (P = 0.033; n = 19 pairs). Carbidopa had no effect on Na(+)-H+ antiporter activity (22Na uptake) or on Na(+)-32Pi cotransport in brush-border membrane vesicles. These results indicate that dopamine produced in proximal tubules tonically reduces Na-K-ATPase Vmax by decreasing the number of alpha-subunits associated with the BLM.
Involvement of C3 exotoxin-sensitive G proteins (rho/rac) in PTH signal transduction in OK cells
Authors: Reshkin S. J. , Murer H.
Published: December 2017

Parathyroid hormone (PTH) in opossum kidney (OK) cells leads to inhibition of Na-Pi cotransport, to the generation of inositol trisphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) and to a phosphorylation of proteins present in an enriched apical membrane fraction (27, 28; for review see Ref. 23). In the present report we have identified two of these phosphoproteins with molecular weights of approximately 22,000 and approximately 24,000, respectively, as guanosine 5'-triphosphate (GTP)-binding proteins, ADP-ribosylated by the Clostridium botulinum exotoxin C3 and recognized by an anti-rho polyclonal antibody but not by pan-ras monoclonal antibody; as suggested by Western-blot analysis the content of the proteins recognized by the anti-rho antibody did not alter in the membrane fraction as a function of treatment with PTH. Transient permeabilization of OK cells using streptolysin O and including the C3 exotoxin attenuated PTH-dependent inhibition of Na-Pi cotransport at hormone concentrations higher than 10(-10) M; residual PTH-dependent inhibition is equal to that observed after pharmacological activation of protein kinase A and protein kinase C, respectively. C3 exotoxin did not alter PTH-dependent generation of cAMP but modified production of IP3; it was increased at 10(-11) M and reduced at 10(-8) M PTH, respectively. It is suggested that protein kinase A may be involved in the phosphorylation of C3 exotoxin-sensitive G proteins (rho/rac). These proteins could be involved in PTH signal transduction.
Conductive properties of a rabbit cortical collecting duct cell line: regulation by isoproterenol
Authors: Dietl P. , Kizer N. , Stanton B. A.
Published: December 2017

The present study was carried out to characterize the membrane conductive properties of RCCT-28A cells, a continuous cell line derived from rabbit cortical collecting duct (CCD). RCCT-28A cells have many phenotypic properties of acid-secreting intercalated cells (A-IC). Using the whole cell patch-clamp technique, we found that the cells are conductive to Cl-, but not to Na+ or K+. The beta-adrenergic agonists isoproterenol (2 x 10(-6) M) and adenosine 3',5'-cyclic monophosphate (cAMP, 10(-4) M) increased the whole cell Cl- conductance. Protein kinase A (150 nM) in the patch pipette (i.e., intracellular solution) also increased whole cell Cl- conductance. Because isoproterenol increases cAMP levels in these cells, we conclude that isoproterenol stimulates the Cl- conductance by increasing cell cAMP, which in turn activates protein kinase A. In contrast, vasopressin does not increase cAMP in these cells and did not increase the Cl- conductance. In conclusion, these experiments show that RCCT-28A cells, like A-IC, are conductive only to Cl-. Thus RCCT-28A cells are a good model with which to study Cl- channels in the collecting duct.
Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells
Authors: Cybulsky A. V. , Goodyer P. R. , Cyr M. D. , McTavish A. J.
Published: December 2017

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.
H(+)-K(+)-ATPase activity in rat collecting duct segments
Authors: Gifford J. D. , Rome L. , Galla J. H.
Published: December 2017

Previous studies have suggested the presence of an H(+)-K(+)-ATPase in rat cortical and medullary intercalated cells with similar properties to the gastric proton pump. The purpose of this study was to determine the functional contribution of an H(+)-K(+)-adenosinetriphosphatase(ATPase) to total CO2 (tCO2) transport along the rat collecting duct. After baseline determination of tCO2 transport in isolated perfused collecting duct segments, Sch 28080 (10 microM) was added to either the perfusate or bath. When Sch 28080 was added to the perfusate, there was no effect in the cortical collecting duct (CCD, 20.8 +/- 6.7 vs. 25.3 + 3.0 pmol.mm-1.min-1), but a marked decrease in tCO2 absorption was effected in both the outer medullary (OMCD, 37.6 + 6.2 vs. 10.7 +/- 4.1 pmol.mm-1.min-1) and initial inner medullary collecting duct (IMCD1, 34.4 +/- 8.1 vs. 16.2 +/- 5.6 pmol.mm-1.min-1). In the CCD from rats with acute alkalosis in vivo, Sch 28080 added to the bath inhibited tCO2 secretion in the CCD (-17.1 +/- 4.4 vs 3.5 + 3.3 pmol.mm-1.min-1). These findings suggest that 1) H(+)-K(+)-ATPase is important in tCO2 absorption in the OMCD and IMCD1 and in tCO2 secretion in the CCD, 2) HCO3(-)-absorbing intercalated cells differ functionally in the cortex and medulla, 3) HCO3- secretion is not the reverse process of HCO3- absorption in the CCD, and 4) H(+)-K(+)-ATPase is important in distal acidification under normal and altered acid-base conditions.
Cysteine oxidation by the postischemic rat kidney
Authors: Scaduto R. C. , Grotyohann L. W.
Published: December 2017

Renal levels of glutathione are markedly decreased during periods of renal ischemia due to catabolism to cysteine. We previously demonstrated that cysteine accumulates in the tissue as the thiol during ischemia, and resumption of blood flow causes a transient elevation of cysteine levels in the renal venous effluent and return of tissue cysteine levels to control values. In this study, the oxidation state of renal venous cyst(e)ine was determined. Although cysteine accumulated as the reduced thiol during ischemia, cysteine released into the renal vein upon blood reflow was found to be almost entirely in the disulfide form. To distinguish between oxidation of arterial cysteine and renal cysteine formed from ischemia-induced reduced glutathione (GSH) catabolism, a labeling procedure was developed to label kidney GSH with 35S without significant labeling of arterial plasma cyst(e)ine. With this procedure, the source of oxidized cysteine that appeared in the renal venous plasma after ischemia was identified as resulting from renal GSH catabolism. The data indicate that a rapid oxidative process occurs during the initial period of blood reflow to the postischemic kidney. After 35 min of ischemia, 3 mumol cysteine/g dry wt were released from the kidney and oxidized. Cysteine oxidation is also expected to generate oxygen-centered free radicals. Pretreatment of animals with deferoxamine, a iron chelator, was without effect on the relative amount of venous cysteine in the oxidized form, arguing against a role for free iron in this oxidative process.(ABSTRACT TRUNCATED AT 250 WORDS)
Glucose transporters of rat proximal tubule: differential expression and subcellular distribution
Authors: Dominguez J. H. , Camp K. , Maianu L. , Garvey W. T.
Published: December 2017

In the late proximal tubule, glucose reabsorption progressively lowers the concentration of luminal glucose, and concentrative glucose influx increases to ensure complete glucose reabsorption. The change in glucose influx is effected by luminal Na(+)-dependent glucose transporters (Na(+)-GLUT), which exhibit higher Na(+)-to-glucose stoichiometric ratios in the late proximal tubule. In this work, the corresponding changes in the axial distribution of basolateral glucose efflux transporters (GLUTs) were examined. mRNAs encoding high-affinity facilitative basolateral transporter GLUT1, low-affinity GLUT2, and apical Na(+)-GLUT were identified in mixed populations of proximal convoluted and straight tubules. The organization of the cognate proteins was also appraised on Western blots. GLUT1 was present in glomeruli, proximal convoluted, and straight tubules, GLUT2 was only expressed in the proximal convoluted tubule, and Na(+)-GLUT was present in both proximal convoluted and straight segments. GLUT1 and GLUT2 were confined to the basolateral membrane, whereas Na(+)-GLUT was preferentially localized to the brush-border membrane. These data are consistent with the idea that glucose influx in early and late proximal tubule is achieved through Na(+)-GLUT, that GLUT1 and GLUT2 are responsible for glucose efflux in the early proximal tubule, and that in the late proximal tubule, where transcellular glucose flux is lower, only GLUT1 mediates glucose efflux.
Osmolytes in renal medulla during rapid changes in papillary tonicity
Authors: Beck F. X. , Schmolke M. , Guder W. G. , Dorge A. , Thurau K.
Published: December 2017

The effect of acute changes in extracellular tonicity on cell electrolyte concentrations at the renal papillary tip and on organic osmolytes in different kidney zones was studied using electron microprobe analysis and high-performance liquid chromatography in four groups of rats: controls, 1- or 4-h water diuresis, and 4-h water diuresis followed by 30-min deamino-[Cys1,D-Arg8]vasopressin (ddAVP). The sum of the papillary interstitial concentrations of Na, K, and Cl was reduced from 981 mmol/kg wet wt in controls to 318 mmol/kg wet wt after 4-h diuresis and increased after ddAVP to 840 mmol/kg wet wt. In papillary collecting ducts intracellular electrolytes fell from 225 to 156 mmol/kg wet wt after 4-h diuresis and rose to 268 mmol/kg wet wt (significantly higher than control) after ddAVP. Organic osmolytes [sum of glycerophosphorylcholine (GPC), betaine, myo-inositol, and sorbitol] at the papillary tip decreased from 2,018 (control) to 1,037 mmol/kg protein after 4-h diuresis and did not increase after ddAVP. After ddAVP, cell P concentration, an index of cell GPC concentration, increased, indicating cell shrinkage. GPC concentration increased, indicating cell shrinkage. The results suggest that the concentrations of all osmoeffectors in papillary cells initially increase due to cell shrinkage in response to hypertonic stress. The higher intracellular ionic strength may be a signal for modulation of transport and metabolism of organic osmolytes.
Stretch- and volume-activated channels in isolated proximal tubule cells
Authors: Filipovic D. , Sackin H.
Published: December 2017

Apical and basolateral channels were studied in isolated proximal tubule cells of Necturus kidney. Many of these isolated cells maintained their polarity, with clearly delineated apical and basolateral regions. A 20-pS stretch-activated (SA) cation-selective channel was identified at the apical side of these cells. This channel was permeable to Ca, K, and Na but was not significantly gated by either membrane potential or cytosolic Ca. Negative pipette pressure (15 cmH2O) increased the open probability (Po) of this channel from 0.04 +/- 0.02 to 0.26 +/- 0.08 (n = 6). Two types of Ca-independent, mechanosensitive, K-selective (SAK) channels were identified at the basolateral surface of polarized proximal tubule cells, i.e., a 30-pS long-open time (50 +/- 7 ms) channel (n = 9), and a 46-pS short-open time (1.3 +/- 0.7 ms) channel (n = 10). Pipette suction (-12 cmH2O) increased the Po of the short-open time channels from 0.008 to 0.015 and increased the Po of the long-open time channel from 0.03 to 0.19. The effect of swelling was studied with isolated cells suspended at the tip of patch pipettes. A 50% dilution of the bath doubled cell volume, hyperpolarized the membrane potential by 11 +/- 0.7 mV, and increased the Po of the basolateral SAK channels. This was followed by a spontaneous regulatory volume decrease (RVD), repolarization of the membrane potential, and a decrease in Po. In contrast, isosmotic (bath side) replacement of an impermeant anion (methanesulfonate) with a permeant anion (Cl) doubled cell volume in 5 min but without a subsequent RVD. This sustained swelling hyperpolarized the cell potential by 5.5 +/- 0.7 mV (n = 16) and increased the Po of short-open time channel by a factor of 2.3 from 0.03 +/- 0.01 to 0.07 +/- 0.02 (n = 6). The increase in Po was primarily produced by a reduction in the interburst closed time, which decreased from 142 +/- 43 ms in K methanesulfonate to 36 +/- 11 ms in KCl solutions. These results are consistent with the hypothesis that cell swelling activates Ca-independent K channels at the basolateral membrane of renal proximal tubule. Efflux of K through these channels may partially mediate renal cell volume regulation.
Changes in electrical properties of guinea pig smooth muscle membrane by experimental bladder outflow obstruction
Authors: Seki N. , Karim O. M. , Mostwin J. L.
Published: December 2017

The changes in membrane electrical properties of guinea pig bladder smooth muscle following experimental bladder outflow obstruction were studied by means of an intracellular microelectrode technique. The results of this study can be summarized as follows. 1) Bladder outflow obstruction resulted in a threefold increase in bladder weight after 4-8 wk. 2) The resting membrane potential was unchanged with obstruction; however, the obstructed smooth muscle membrane was more quiescent, with less spontaneous electrical activity compared with control tissue. 3) The membrane constants, space constant and time constant, were both reduced in the obstructed bladders. 4) There was no detectable difference in membrane depolarization induced by high extracellular K+ solution between control and obstructed bladders. 5) Both the membrane depolarization induced by K(+)-free solution or ouabain-containing Krebs solution and ouabain-sensitive membrane hyperpolarization by K(+)-containing solution after application of K(+)-free solution were significantly increased in the obstructed bladders. 6) Low extracellular Cl- solution evoked greater membrane depolarization in obstructed bladders. These results suggest that bladder outflow obstruction results in suppression of the cell-to-cell transfer of electrical activity and activation of a membrane electrogenic Na(+)-K+ pump mechanism in guinea pig detrusor.
Renal effects of endogenous prostaglandin synthesis promoter ONO-3122
Authors: Takabatake T. , Hara H. , Ishida Y. , Ohta H. , Kobayashi K.
Published: December 2017

The renal effects of a prostaglandin synthesis agonist, 1-iodo-3-aminomethyl-5,6,7,8-tetrahydro-2-naphthol (ONO-3122), were investigated in anesthetized rats. ONO-3122 (0.3 mg/kg + 0.3 mg.kg-1.h-1 iv) doubled the urinary excretion of the main metabolites of prostaglandin F, and induced transient increases in renal blood flow and glomerular filtration rate (GFR) with a marked, stable natriuresis. Indomethacin suppressed the natriuresis. When the diuretic fluid losses were replaced, micropuncture showed an unaltered reabsorption of sodium in the proximal tubule but reductions in the loop of Henle (86 +/- 1 vs. 76 +/- 1%) and in the more distal segments (98 +/- 1 vs. 83 +/- 3%) with comparable reductions in water reabsorption. Potassium secretion was seen in the distal and collecting tubules. Without fluid replacement, sodium reabsorption was reduced in the loop and more distal nephron but increased in the proximal tubule. Differences between proximal and distal nephron GFR were unaffected by systemic ONO-3122. Loop perfusion with ONO-3122 did not change tubuloglomerular feedback responses, which were, however, completely suppressed by furosemide. It is concluded that ONO-3122 stimulates renal prostaglandin biosynthesis, transiently dilates renal vasculature, and induces natriuresis mainly by suppressing sodium and water reabsorption in the loop of Henle and the more distal nephron. Luminal ONO-3122 does not affect the tubuloglomerular feedback.
Role of paraventricular nucleus in control of blood pressure and drinking in rats
Authors: Jensen L. L. , Harding J. W. , Wright J. W.
Published: December 2017

The present investigation examined the abilities of angiotensin (ANG) II and III to produce increases in blood pressure and drinking when microinfused into the paraventricular nucleus (PVN) of the hypothalamus of the Sprague-Dawley rat. Dose-dependent elevations in systemic blood pressure and heart rate were measured to both ANG II and III in the anesthetized rat, with ANG II more potent than ANG III at the two highest doses examined. Pretreatment with the specific ANG receptor antagonist [Sar1,Thr8]ANG II (sarthran), blocked subsequent ANG II- and III-induced elevations in blood pressure, suggesting that these responses were dependent on the activation of ANG receptors. A similar analysis in awake rats yielded nearly equivalent results. A final experiment demonstrated that microinfusions of ANG II and III into the PVN produced drinking in a dose-dependent manner, with greater consumption to ANG II than ANG III. Again, sarthran was found to block the dipsogenic response. Histological examination revealed that the location of the injection site was linked to the character of the ANG-dependent response. These data suggest that the PVN may play a critical role in mediating central ANG effects on body water homeostasis and blood pressure regulation. Furthermore, it appears that subnuclei of the PVN may participate differentially in ANG-mediated actions.
Nephrotoxic antiserum identifies a beta 1-integrin on rat glomerular epithelial cells
Authors: O'Meara Y. M. , Natori Y. , Minto A. W. , Goldstein D. J. , Manning E. C. , Salant D. J.
Published: December 2017

A postulated mechanism of immune glomerular injury is a direct interaction between antibody and glomerular epithelial cell (GEC) surface antigens. To explore this hypothesis, we examined the interaction of the noncomplement-fixing gamma 2-subclass of sheep anti-rat nephrotoxic serum (NTS), which causes immediate complement- and neutrophil-independent proteinuria in vivo, with rat GECs in culture. Reactivity of NTS with GEC surface antigens was determined by positive immunofluorescence of GEC plasma membranes and by the ability of NTS-coated tissue culture wells to provide an adhesive substrate for GECs. NTS immunoprecipitated two proteins (135 and 118 kDa) from surface-labeled GECs. Proteins of similar molecular mass were precipitated by a polyclonal rabbit antibody that identifies the beta 1-integrin chain of the mouse fibronectin receptor (anti-FnR). In addition, NTS identified similarly sized bands on Western blot analysis of cell membranes from isolated rat glomeruli. Similar reactivity was eluted from the glomeruli of proteinuric rats injected with NTS. NTS significantly inhibited GEC adhesion to laminin, types I and IV collagen, and fibronectin and prevented GEC spreading on types I and IV collagen. Anti-FnR similarly inhibited GEC adhesion. Cell viability was not affected. These results show that NTS recognizes a pair of GEC surface proteins that have the characteristics of an alpha- and beta 1-integrin and, at low concentrations, disrupt cell-matrix interactions.
Glomerular basement membrane: in vitro studies of water and protein permeability
Authors: Daniels B. S. , Hauser E. B. , Deen W. M. , Hostetter T. H.
Published: December 2017

The glomerular basement membrane (GBM) is an integral structural component of the glomerular filter, but its contribution to the hydraulic and macromolecular permeability properties of the glomerulus has been the subject of much controversy. We have modified previously reported methods to develop a technique with which to study filtration properties of microgram quantities of isolated GBM in vitro at physiological pressures. Rat glomeruli were sieved, and cells were removed with N-laurylsarcosine and DNase. GBM (150 micrograms; greater than 95% pure) were added to a mini-ultrafiltration cell and consolidated under pressure to form a continuous filter at the base of the cell. Water flux was identical to inulin clearance at applied pressures less than 150 mmHg and increased with progressive increments in the transmembrane pressure. Hydraulic conductivity of GBM was inversely related to the prevailing transmembrane pressure gradient. The hydraulic conductivity depended on albumin concentration in a manner that was not monotonic, with the conductivity being lower at 4 g/dl albumin than at 0 or 8 g/dl. When plasma was utilized as the retentate, the fractional clearance of albumin was over twice that of immunoglobulin G, and the fractional clearance of each protein was much higher than that in the intact glomerulus. On the basis of these results, both the hydraulic and macromolecular permeability of an individual layer of GBM are much greater than that reported for the intact glomerulus. The large quantitative differences between GBM permeability and that of intact glomeruli suggest a major contribution of cellular elements to glomerular permeability properties.
Endothelium modulates renal blood flow but not autoregulation
Authors: Beierwaltes W. H. , Sigmon D. H. , Carretero O. A.
Published: December 2017

Inhibition of the production of the endothelium-derived relaxing factor (EDRF) nitric oxide using N omega-nitro-L-arginine methyl ester (L-NAME) increases blood pressure (BP) and decreases renal blood flow (RBF), suggesting that basal EDRF can modulate both systemic resistance and renal perfusion. We tested whether L-NAME inhibition of EDRF could also change the autoregulation of RBF. Blood pressure and RBF were measured in Inactin-anesthetized Sprague-Dawley rats. A bolus of 10 mg/kg body wt of L-NAME produced the maximum pressor response (23 +/- 3 mmHg) and blocked acetylcholine-induced renal vasodilation. In control rats, sequential changes in renal perfusion pressure showed that RBF was well autoregulated down to 95 +/- 2 mmHg. L-NAME increased BP, decreased RBF by 33% (P less than 0.005), and increased renal vascular resistance twofold. Although RBF was decreased, the kidney was still able to autoregulate RBF, although reset around the lower flow. Acute hypertension by carotid occlusion and vagotomy increased BP by 26 +/- 6 mmHg (P less than 0.005) and slightly increased RBF, while autoregulation was maintained. The pressor response to L-NAME was amplified to 38 +/- 6 mmHg (P less than 0.001), but RBF decreased by 35% (P less than 0.01). Autoregulation of RBF was maintained, although reset around the lower flow. We conclude that, although endothelial EDRF production may help maintain RBF, it does not seem to mediate the intrinsic autoregulatory responses of the renal vasculature to altered renal perfusion pressure.
Cyclic 3',5'-nucleotide diesterases in dynamics of cAMP and cGMP in rat collecting duct cells
Authors: Yamaki M. , McIntyre S. , Rassier M. E. , Schwartz J. H. , Dousa T. P.
Published: December 2017

We studied cyclic 3',5'-nucleotide phosphodiesterase (PDE) isozymes and their role in adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP metabolism in a rat inner medullary collecting duct (IMCD) cell line. The homogenized and fractionated IMCD cells of cAMP-PDE and all of cGMP-PDE activity were found in the cytosol. The majority of cytosolic cAMP-PDE (greater than 50%) was isozyme PDE-IV; the Ca(2+)-calmodulin-sensitive PDE-I was present only in cytosol. Preincubation of IMCD cells with PDE-IV inhibitor rolipram markedly (5x) enhanced levels of cAMP both basal and in the presence of [Arg8]vasopressin (AVP). Cilostamide (for PDE-III) or vinpocetine had no effect, whereas PDE-I inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MeoM-IBMX) enhanced AVP-dependent cAMP levels. Exposure of IMCD cells to 2 microM ionomycin decreased both basal and AVP-stimulated cAMP. Depletion of Ca2+ by preincubation of IMCD cells in the Ca(2+)-free medium with ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid markedly enhanced the stimulatory response of cAMP to AVP, and addition of 8-MeoM-IBMX further enhanced the AVP response. The levels of cGMP, basal or in response to atriopeptin (ANP), were not affected by PDE-V inhibitor zaprinast, but both inhibitors of PDE-I, 8-MeoM-IBMX and vinpocetine, increased basal cGMP, and 8-MeoM-IBMX also increased cGMP levels enhanced by ANP. The depletion of Ca2+ from IMCD cells alone had no effect on cGMP levels, but effects of 8-MeoM-IBMX and vinpocetine on the ANP-stimulated cGMP levels were enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)
Dynamics of TGF-initiated nephron-nephron interactions in normotensive rats and SHR
Authors: Yip K. P. , Holstein-Rathlou N. H. , Marsh D. J.
Published: December 2017

Proximal tubular pressure, glomerular filtration rate, and early distal tubule Cl- oscillate at 35 mHz in normotensive rats because of tubuloglomerular feedback (TGF); the oscillation bifurcates to chaos in spontaneously hypertensive rats (SHR). To examine the importance of TGF-initiated vascular interactions between nephrons in these dynamics, we measured tubular pressure simultaneously in two or more nephrons. The oscillations were synchronized in nephrons supplied by a common cortical radial artery. The correlation coefficient of pressure records from coupled nephrons was 0.86 +/- 0.02. Intratubular furosemide perfusion diminished the oscillation in both the perfused and the coupled nephron; total autospectral power in each of the nephrons and cross-spectral power were reduced to 45% of control. The correlation between noncoupled nephrons was not significant, and intratubular furosemide perfused in one nephron had no effect on adjacent but noncoupled nephrons. In SHR, the correlation coefficient of tubular pressure records was high from coupled nephrons only; furosemide diminished the autospectral power of pressure fluctuations to approximately 60-75% of control in both perfused and coupled nephrons, and cross-spectral power was affected by a similar amount. Nephron-nephron interactions, specific to vascular connectivity, persist in SHR and appear to be stronger than in normotensive rats.
Sites of antinatriuretic action of insulin along rat nephron
Authors: Feraille E. , Marsy S. , Cheval L. , Barlet-Bas C. , Khadouri C. , Favre H. , Doucet A.
Published: December 2017

This study was aimed at identifying the renal sites of the antinatriuretic action of insulin by evaluating whether this hormone may alter the function of Na-K-ATPase in specific nephron segments. For this purpose, possible actions of insulin on the rate of 86Rb uptake were evaluated in vitro on single segments of proximal convoluted tubule (PCT), thick ascending limb, and collecting tubule microdissected from collagenase-treated kidneys of normal rats. Results indicate that physiological concentrations of insulin inhibited by 44% the initial rate of ouabain-sensitive 86Rb uptake in the medullary and cortical thick ascending limb, whereas it increased it by 40% in proximal tubules and by 60% in both cortical and medullary collecting tubules. The kinetics and dose dependence of insulin actions were different in the thick ascending limb, the PCT, and the collecting tubule, with the latter less sensitive but displaying an earlier response to insulin than the PCT and the thick ascending limb.
H-K-ATPase enhancement of Rb efflux by cortical collecting duct
Authors: Zhou X. , Wingo C. S.
Published: December 2017

Previous studies suggest that enhancement of rubidium tracer (86Rb) lumen-to-bath efflux following removal of luminal Na is mediated in part by a Ba-insensitive pathway. To determine the role of a primary active K pump in this response, we examined the action of known inhibitors of H-K-ATPase (Sch 28080) and Na-K-ATPase (ouabain) on the 86Rb lumen-to-bath efflux coefficient (KRb). Luminal Sch 28080 (10 microM) significantly reduced KRb by 39 +/- 8.0% (P less than 0.05), whereas luminal ouabain (0.1 mM) reduced KRb by 10 +/- 14% (P = not significant), suggesting that a luminal H-K-ATPase mediates Rb efflux. To examine whether H-K-ATPase mediates Rb in KRb following removal of luminal Na, additional experiments were conducted to examine the effect of Sch 28080 on KRb in the presence and the absence of luminal Na. In the presence of luminal Na, 10 microM Sch 28080 reduced KRb by 15 +/- 5.0%. However, in the absence of luminal Na, 10 microM Sch 28080 decreased KRb by 48 +/- 8.2%. The percentage inhibition of KRb by Sch 28080 was significantly greater in the absence of luminal Na than in its presence (P less than 0.01), suggesting that the enhancement of KRb following removal of luminal Na is mediated in part by an H-K-ATPase pathway. In either case transepithelial voltage was not significantly altered. In contrast to the lack of effect of luminal ouabain, addition of 0.1 mM ouabain to the bath increased KRb (69.8 +/- 11.1 vs. 95.9 +/- 18.7 nm/s, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Functional and structural characterization of endosomes from toad bladder epithelial cells
Authors: Zeidel M. L. , Hammond T. , Botelho B. , Harris H. W.
Published: December 2017

Previous functional studies of toad bladder endosomes have been complicated by the presence of multiple endosome subpopulations each possessing different permeability characteristics. To identify and characterize both water channel-containing vesicles (WCV) and other endosome subpopulations, we combined flow cytometry, electron microscopy, stop-flow fluorometry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Flow cytometry of endosomes identified distinct populations of fluorescein-labeled endosomes in bladders after removal of antidiuretic hormone (ADH) stimulation (ADH withdrawal). Centrifugation separated the larger fluorescein-labeled vesicles, sedimenting at lower speed (intermediate pellet, IP), from the smaller fluorescein-labeled vesicles, sedimenting at high speed (high-speed pellet, HSP). Permeability and structural studies of these subpopulations revealed the following. 1) IP endosomes labeled 10 min after ADH withdrawal (ADH IP) represented a highly purified population of WCV with high water permeability (Pf) that exhibited a low-activation energy and sensitivity to organic mercurials. 2) IP endosomes from unstimulated bladders did not contain functional water channels. 3) HSP from either ADH withdrawal or unstimulated bladders exhibited low Pf and acidified after addition of extravesicular ATP; moreover, protein compositions of purified HSP were distinct from those of purified IP. These results suggest that HSPs represent constitutive and not ADH-sensitive endosomes. 4) High permeability to protons (PH+) was seen in ADH IP endosomes but not the other fractions, providing strong evidence that the ADH water channel conducts protons. 5) Multivesicular bodies (MVB) exhibited low Pf and PH+, indicating that they do not possess functional water channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Renal kinin antagonism does not impair pressure-induced natriuresis
Authors: Strick D. M. , Fiksen-Olsen M. J. , Carretero O. A. , Romero J. C.
Published: December 2017

We studied the contribution of the renal kallikrein-kinin system to short-term electrolyte and water balance during baseline and during acutely elevated renal perfusion pressure (RPP) in the anesthetized dog. Renal blood flow, glomerular filtration rate, urine flow rate, renin secretion rate, and urinary excretion of sodium, potassium, prostaglandin E2 (PGE2), and kinins were measured at baseline RPP during intrarenal infusion of 0.9% saline or the competitive bradykinin analogue [D-Arg0,Hyp3,Thi5,D-Phe7,Thi8]bradykinin (50 micrograms/min), which blocks vascular and interstitial kinin receptors. RPP was then raised above baseline (control group 25%; kinin analogue group 22%) by ligating the celiac artery, the superior mesenteric artery, and the aorta distal to the renal arteries. Renal parameters were again measured during infusion of saline or the kinin analogue. The analogue had no effect on renal hemodynamic or excretory parameters at baseline perfusion pressures. Increasing RPP significantly increased urine flow rates and urinary sodium excretion rates (control group, 43 mumol/min; kinin analogue group, 55 mumol/min) in both groups of animals. Increasing pressure also tended to decrease renin secretion rate in both groups of animals; however, neither increased pressure nor infusion of the analogue affected urinary excretion of PGE2 or kinins. The results suggest that intrarenal kinins are not powerful short-term regulators of electrolyte and water balance and that an intact kallikrein-kinin system is not necessary to induce pressure diuresis and natriuresis.