bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary NaCl modulates Na(+)-H+ antiporter activity in renal cortical apical membrane vesicles
Authors: Moe O. W. , Tejedor A. , Levi M. , Seldin D. W. , Preisig P. A. , Alpern R. J.
Published: December 2017

An apical membrane Na(+)-H+ antiporter mediates proximal tubule NaCl and NaHCO3 reabsorption. The present studies examined whether chronic changes in dietary NaCl intake lead to an adaptation of the Na(+)-H+ antiporter. Rats were maintained either on a low-salt (LS, 0% NaCl) or a high-salt (HS, 2% NaCl) diet for 4 days. Na(+)-H+ antiporter was assayed using the acridine orange fluorescence method in apical membrane vesicles prepared by Mg2+ aggregation. Rats on LS diet exhibited a higher maximal activity (Vmax) for antiporter activity (0.109 +/- 0.008 s-1) compared with rats on HS diet (0.090 +/- 0.008 s-1; P less than 0.01), whereas Na activity (KNa) was similar (13.1 +/- 1.5 and 14.2 +/- 0.8 mM in HS and LS groups, respectively). The modulation of Na(+)-H+ antiporter activity was amiloride sensitive. Proton permeability, Na(+)-dependent [3H]glucose uptake, and vesicle enrichments were similar in both groups. In addition, the dietary protocols were not associated with any systemic acid-base disturbances, K+ deficiency, or hyperfiltration, conditions that have previously been demonstrated to alter Na(+)-H+ antiporter activity. Acute differences in extracellular fluid volume, induced by 10% body weight isohydric expansion failed to affect Na(+)-H+ antiporter activity. Thus chronic alterations in dietary NaCl intake alter the maximal activity (Vmax) of the Na(+)-H+ antiporter. This adaptation may contribute to decreased NaCl reabsorption in extracellular fluid volume expansion, enhanced NaCl reabsorption in extracellular fluid volume depletion, and enhanced NaHCO3 reabsorption in chronic metabolic alkalosis associated with volume contraction.
Mechanisms of rapid desensitization to arginine vasopressin in vascular smooth muscle cells
Authors: Caramelo C. , Tsai P. , Okada K. , Briner V. A. , Schrier R. W.
Published: December 2017

This study characterized the rapid desensitization induced by arginine vasopressin (AVP) in vascular smooth muscle cells (VSMC) in culture. The Ca2+ mobilization response and, in some experiments, the intracellular pH changes were used as a probe for the desensitization phenomenon. In VSMC, AVP desensitization was homologous, concentration dependent, and occurred in less than 30 s. The desensitization was complete with 10(-7) M AVP. Receptor occupancy was a critical factor in the maintenance of desensitization, since complete hormone washing by acid glycine buffer produced an earlier (less than 5 min) recovery of the cell response, whereas partial hormone washing with saline (pH 7.4) required 15 min to produce any significant recovery. Protein kinase C activation was a significant mechanism in AVP desensitization, because protein kinase C downregulation inhibited the desensitization phenomenon. Receptor internalization was, however, not important for the desensitization phenomenon, since it still occurred at 4 degrees C. Treatment with pertussis toxin did not affect the Ca2+ mobilization response but decreased the AVP-mediated intracellular alkalinization, therefore suggesting that a Gi or Go protein may be involved in some but not all the aspects of the AVP signal transduction and the desensitization phenomena.
Transfer of Na transport inhibition in proximal tubules from saline volume-expanded to nonexpanded rats
Authors: Reddy S. , Gyory A. Z. , Bostrom T. , Cochineas C.
Published: December 2017

In dual micropuncture experiments the shrinking drop technique was used to measure volume flux with artificial tubular fluid (AF) alternating with harvested tubular fluid (HTF) during saline volume-expanded (VE) and nonexpanded (NE) periods. In VE rats, volume flux (Jv) (nl.mm-1.min-1) with AF was 1.78 +/- 0.08 (means +/- SE) during NE and was reduced to 1.39 +/- 0.09 (P = 0.01) during subsequent VE, whereas with randomly alternating HTF during VE it was 1.07 +/- 0.08 (P less than 0.0001 and less than 0.03, respectively). Jv with HTF from NE rats tested in the VE rats was 1.20 +/- 0.06, which is significantly higher than that measured with their own HTF (Wilcoxon rank, P = 0.05). In NE rats Jv was 1.65 +/- 0.10 and 1.69 +/- 0.10 with AF and HTF and was 1.28 +/- 0.07 (P less than 0.001 from both) with HTF obtained from VE rats. Elemental analysis of reaspirated tubular fluids showed no significant differences in Na or Cl concentrations among any of the fluids. It is concluded that during VE, a transferable Na transport inhibitor appears in proximal tubular fluid, which, together with a changed proximal tubular epithelium, possibly due to physical forces, accounts for proximal tubular Na transport inhibition during VE.
Site and mechanism of action of epidermal growth factor in rabbit cortical collecting duct
Authors: Muto S. , Furuya H. , Tabei K. , Asano Y.
Published: December 2017

To examine the exact target cell and mechanism of action of epidermal growth factor (EGF) in the isolated cortical collecting duct from rabbit kidney, we compared electrical properties of collecting duct (CD) cells (principal cells) and intercalated (IC) cells in absence and presence of EGF at 10(-8) M. Differentiation of CD and IC cells was based on values of basolateral membrane voltage (Vb) and fractional apical membrane resistance (fRa). In CD cells, upon addition of EGF to bath, lumen-negative transepithelial voltage (VT) was decreased from -8.0 +/- 1.9 to -2.4 +/- 1.3 mV (n = 22, P less than 0.001), but Vb was little changed (from -85.1 +/- 2.8 to -83.1 +/- 2.7 mV, n = 19), indicating that EGF in bath mainly caused changes in apical membrane voltage. In addition, peritubular EGF increased transepithelial resistance (RT) from 132.9 +/- 15.8 to 153.8 +/- 18.4 omega.cm2 (n = 16, P less than 0.001) as well as fRa from 0.31 +/- 0.06 to 0.39 +/- 0.07 (n = 12, P less than 0.01). These actions of EGF were prevented by pretreatment with 50 microM luminal amiloride. Luminal EGF had no effects on VT, Vb, RT, or fRa of CD cells. In IC cells, upon addition of EGF to bath, neither Vb nor fRa was affected. From these results, we conclude that EGF acts on the CD cell at the basolateral border and inhibits mainly the amiloride-sensitive Na+ conductance in the apical membrane.
Isolated MTAL cells produce an inhibitor of ouabain-sensitive oxygen consumption
Authors: Cantley L. G. , Fuhro R. , Silva P.
Published: December 2017

Dispersed medullary thick ascending limb (MTAL) cells of the rabbit kidney produce an inhibitor of transport-related oxygen consumption when incubated at 37 degrees C. Appearance of this inhibitor is enhanced by incubation with arachidonic acid. Its appearance can be prevented by either 10(-6) M nordihydroguaiaretic acid or 10(-4) M indomethacin, but not by 10(-6) M indomethacin. These results suggest that the transport inhibitor is an eicosanoid produced by a pathway other than that catalyzed by cyclooxygenase. It inhibits ouabain-sensitive respiration in intact cells treated with amphotericin, suggesting direct inhibition of the Na(+)-K(+)-ATPase.
Effect of V1/V2-receptor antagonism on renal function and response to vasopressin in conscious dogs
Authors: Rose C. E. , Rose K. Y. , Kinter L. B.
Published: December 2017

To characterize effects of V1- and V2-receptor stimulation on renal function, eight conscious mongrel female dogs were studied in four separate studies greater than or equal to 2 wk apart during the following six consecutive 20-min periods: 1) intrarenal administration of the full V1/V2-receptor antagonist SKF 105494 (100 ng.kg-1.min-1) during basal circulating vasopressin (VP) levels (n = 3), 2) elevation of renal arterial plasma VP concentrations by intrarenal administration of exogenous arginine VP (0.05 mU.kg-1.min-1, n = 5), 3) simultaneous administration of SKF 105494 at (100 ng.kg-1.min-1) with intrarenal administration of exogenous VP (0.05 mU.kg-1.min-1; n = 5), and 4) intrarenal vehicle alone (n = 5). When administered during conditions of basal circulating endogenous VP, the full receptor antagonist effects were limited to opposition of hydrosmotic effects of VP. Elevation of renal arterial plasma VP levels through infusion of exogenous VP resulted in decreased renal plasma flow, glomerular filtration rate, osmolar clearance, urinary flow rate, and free water clearance and increased urine osmolality. These effects were all abolished by simultaneous administration of V1/V2-receptor antagonist. These data suggest that, under basal low levels of circulating VP, VP only influences renal water excretion. However, when plasma VP concentrations are elevated, VP may contribute to renal vasoconstriction and secondarily to reduced solute excretion, in addition to its effects on free water clearance.
Suitability of tritiated inulin for determination of glomerular filtration rate
Authors: Shalmi M. , Lunau H. E. , Petersen J. S. , Bak M. , Christensen S.
Published: December 2017

Purity of different batches of [3H]inulin delivered from leading manufacturers was elevated with a chromatographic method (Sephadex G-25 column) that allowed simultaneous analysis of cold inulin, [3H]inulin, and [14C]inulin in the same run. Among four batches of [3H]inulin received within 5 mo, two were found relatively pure, whereas two were partly decomposed to lower-molecular-weight fragments. The chromatographic profile of pure isotopes was not significantly affected by redistribution and freeze drying, nor by subsequent storage in the freeze-dried state at -20 degrees C for up to 5 mo, nor by incubation in aqueous solution at 37 degrees C for 24 h. Three batches of [3H]inulin with different grades of decomposition (noninulin percentages 13%, 38%, and 61%, respectively) were selected for clearance experiments and infused simultaneously with cold and undecomposed [14C]inulin to conscious rats. [14C]inulin had a significantly higher clearance than cold inulin (+7.6 +/- 0.6%) and relatively pure [3H]inulin (+12.4 +/- 0.4%). Decomposed [3H]inulin isotopes progressively underestimated clearance of cold inulin to an extent related to the degree of decomposition. Thus at the end of the 5-h clearance experiment, ratios between clearance of tracer and of cold inulin were 0.92, 0.71, and 0.60 for the three 3H isotopes, respectively. This study indicates that [3H]inulin delivered from leading manufacturers may be decomposed to an extent that invalidates its use as a marker for glomerular filtration rate (GFR). It is thus necessary to check the purity routinely before use. Within the same rat, clearance of undecomposed [3H]inulin and [14C]inulin may differ by 12%, and for this reason they should not be used interchangeably as GFR markers.
Electrophysiological characterization of upper portion of descending limb of long-looped nephron
Authors: Yoshitomi K. , Imai M.
Published: December 2017

The upper portion of the descending limb of long-looped nephron (LDLu) of the hamster is characterized by high water and ion permeabilities. Although the paracellular route is considered to be the major pathway representing cation permselectivity of this segment, ion transport mechanisms through the transcellular pathway are unknown. To study this issue; we applied cable analysis and conventional microelectrode technique to the hamster LDLu perfused in vitro. The transmural voltage (VT) was not different from zero, and transmural resistance (RT) was very low, 18.3 +/- 2.0 omega.cm2 (n = 12). The basolateral membrane voltage was -80 +/- 2 mV (n = 55), and fractional apical membrane resistance was 0.92 +/- 0.23 (n = 5). Ouabain (0.1 mM) in the bath decreased basolateral membrane voltage (VB) by 23 +/- 3 mV (n = 6, P less than 0.001). Increase in K+ concentration in bath and in lumen from 5 to 50 mM decreased VB by 39 +/- 2 (n = 7, P less than 0.01) and apical membrane voltage (VA) by 10 +/- 1 mV (n = 7, P less than 0.001), respectively. Addition of 2 mM Ba2+ to bath and to lumen decreased VB by -47 +/- 2 (n = 11, P less than 0.001) and decreased VA by 8 +/- 1 mV, respectively. Reduction of HCO3- in bath from 25 to 2.5 mM decreased VB by 4 +/- 1 mV (n = 7, P less than 0.005). Reduction of bath Cl- did not cause any rapid deflection of VB. No appreciable Na+ conductance was detected in the apical membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Three-dimensional anatomy and renal concentrating mechanism. II. Sensitivity results
Authors: Wexler A. S. , Kalaba R. E. , Marsh D. J.
Published: December 2017

A mathematical model has been developed to simulate hypertonic urine formation in the renal medulla. The model uses published values of membrane transport parameters, as have other models, but is unique in its representation of the three-dimensional anatomy of the medulla. The model successfully predicts measured fluid flows, osmolarities, and NaCl and urea concentrations. The model results are presented in the companion to this paper [A. S. Wexler, R. E. Kalaba, D. J. Marsh. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F368-F383, 1991.]. In this paper we provide tests of the sensitivity of model performance to variations in the description of the anatomy and in membrane transport parameters. From these studies we conclude that 1) strict counterflow arrangements are required in the outer stripe to prevent loss of NaCl to the systemic circulation, 2) the radial organization in the inner stripe materially improves performance of the inner medulla, 3) radial organization of the inner medulla is essential to hypertonic urine formation there, 4) the model is most sensitive to variation in collecting duct parameters, and 5) reabsorption of urea in the distal tubule improves system performance. The results support the claim that the three-dimensional structure, as captured in the model, provides a crucial framework for the production of hypertonic urine.
Relationships between intracellular amino acid levels and protection against injury to isolated proximal tubules
Authors: Weinberg J. M. , Nissim I. , Roeser N. F. , Davis J. A. , Schultz S. , Nissim I.
Published: December 2017

Metabolism and cellular levels of glycine, alanine, and other relevant amino acids in proximal tubules were studied during models of acute injury and protection by glycine. Freeze-clamped, normal rabbit renal cortex was very rich in glycine (66.8 nmol/mg protein) and glutamate and also had substantial levels of taurine, alanine, glutamine, serine, and aspartate. Isolated proximal tubules were severely depleted of all these amino acids (glycine, 2.1 nmol/mg protein). During 37 degrees C incubation in presence of alanine, tubules recovered only glutamate to a level approximating that in vivo (38.8 nmol/mg protein, 15.2 mM). Glycine added to medium at levels ranging from 0.25 to 2 mM was actively concentrated four- to sixfold by tubule cells. Two millimolar glycine potently protected tubules from lethal cell injury induced by hypoxia, antimycin A, or ouabain. Glycine levels of injured tubules rapidly equilibrated with medium, irrespective of whether glycine was loaded by preincubation or was added concomitantly with the injury maneuver. Metabolism of glycine during protection, assessed by changes in total levels, gas chromatography-mass spectroscopy determination of the fate of [13C]glycine, and redistribution of label from [3H]glycine was minimal. The data suggest that glycine plays an essential, constitutive role in maintenance of tubule cell structural integrity independently of common metabolic pathways. Intracellular amino acid content is sufficiently labile for depletion of structurally essential amino acids to potentially occur in a variety of settings, but, even with severe ATP depletion or Na+ pump inhibition, supplemental glycine is readily available to intracellular sites of action.
Effect of Na-channel blockers and lumen Ca on K secretion by rat renal distal tubule
Authors: Okusa M. D. , Velazquez H. , Wright F. S.
Published: December 2017

In previous studies the effectiveness of amiloride (AML) in reducing K secretion has been variable. Based on studies by Cuthbert and Wong (Mol. Pharmacol. 8: 222-229, 1972) in which the Na-channel-blocking action of AML in frog skin was found to require the availability of Ca ions in extracellular fluid, we postulated that the ability of AML and its analogue, benzamil (BZA), to inhibit distal tubule K secretion depends on the presence of Ca in luminal fluid. We found that addition of Ca to a perfusion solution containing 50 microM BZA did reduce K secretion more than BZA alone. Maximal inhibition was observed with 2.5 mM free ionic Ca. Graded increases in luminal Ca in presence of AML or BZA reduced K transport in a dose-dependent manner. The decrease in K secretion with increasing luminal Ca was paralleled by a decrease in transepithelial voltage. These results support our hypothesis that the effectiveness of Na-channel blockers to reduce K secretion by the rat distal tubule depends on presence of luminal Ca and suggest an interaction between luminal Ca and Na-channel blockers on the Na channel.
Accelerator mass spectrometry: application to study of aluminum kinetics in the rat
Authors: Meirav O. , Sutton R. A. , Fink D. , Middleton R. , Klein J. , Walker V. R. , Halabe A. , Vetterli D. , Johnson R. R.
Published: December 2017

The advent of accelerator mass spectrometry (AMS) now permits the ultrasensitive detection of extremely long-lived isotopes, including 14C, 26Al, and 41Ca. Until now, tracer studies of aluminum kinetics have not been possible because aluminum has only two isotopes, with half-lives of 6.5 min (29Al) and 7 x 10(5) yr (26Al), neither of which is suitable for conventional studies. In a novel experiment we have employed AMS to study aluminum kinetics in a normal rat and a 5/6-nephrectomized rat over a 3-wk period of intravenous injection of a tracer dose of 26Al. Kinetics were similar in the two animals; approximately 75% of intravenously injected tracer 26Al was excreted in the urine in the first 24 h as was approximately 80% after 3 wk. Renal clearance of 26Al was approximately 0.75 ml.min-1.kg body wt-1 in both rats. The results clearly demonstrate the potential of this technique for isotope tracer studies in animals as well as in humans.
Effects of AVP and deoxycorticosterone on Na+ and water transport in the Dahl salt-sensitive rat CCD
Authors: Hawk C. T. , Schafer J. A.
Published: December 2017

Cortical collecting ducts (CCD) from inbred Dahl salt-sensitive rats were perfused in vitro to study effects of arginine vasopressin (AVP, present in the bath) and deoxycorticosterone pivalate (DOC) pretreatment on lumen-to-bath and bath-to-lumen fluxes of 22Na+ (J1----b and Jb----1 in pmol.min-1.mm-1, respectively), hydraulic conductivity (Pf, microns/s), and transepithelial voltage (VT, mV). J1----b was 37.1 +/- 5.3 (mean +/- SE) in untreated rats and increased to 83.2 +/- 15.9 with AVP. VT increased from -0.3 +/- 0.6 to -7.0 +/- 2.0. In DOC-pretreated rat CCDs, baseline J1----b was higher (85.1 +/- 7.6) as was VT (-11.3 +/- 1.7); J1b----b and VT were doubled with AVP addition (185.6 +/- 18.6 and -21.7 +/- 2.3, respectively). Thus J1----b in AVP-stimulated CCDs from untreated rats was not significantly different from control (no AVP) J1----b from DOC-pretreated rats; however, AVP produced a greater J1----b increase in the latter CCDs. Neither AVP nor DOC had an effect on Jb----1, which ranged from 25 to 50. Benzamil reduced J1----b to values not significantly different from Jb----1, and VT became zero in CCDs treated with both AVP and DOC, indicating Na+ transport stimulated by both hormones occurs through apical membrane Na+ channels. Pf increased from 59 +/- 80 to 1,072 +/- 176 with AVP addition to untreated rat CCDs and was unaltered by DOC or benzamil. Thus the Dahl rat CCD exhibits a transport response to AVP and DOC that is indistinguishable from that observed in Sprague-Dawley rats, as previously reported by this laboratory.
Intracellular betaine substitutes for sorbitol in protecting renal medullary cells from hypertonicity
Authors: Moriyama T. , Garcia-Perez A. , Olson A. D. , Burg M. B.
Published: December 2017

Renal medullary cells are normally exposed to a variably high extracellular NaCl concentration. They compensate by accumulating large amounts of organic osmolytes, including sorbitol and betaine. The sorbitol is synthesized from glucose, catalyzed by aldose reductase. Previously, inhibition of aldose reductase activity was noted to greatly reduce renal medullary cell survival and growth (measured by cloning efficiency) in tissue cultures of renal medullary cells in hypertonic medium. In contrast, inhibition of aldose reductase and renal medullary sorbitol accumulation is not associated with kidney damage in vivo. In the present experiments we find that addition of betaine to the medium, and its resultant uptake by the cells, largely replaces the decrease in sorbitol caused by aldose reductase inhibitors and restores the cloning efficiency. We presume that in vivo uptake of betaine by renal medullary cells similarly protects them from harm when aldose reductase inhibitors lower sorbitol. The results also demonstrate that one organic osmolyte can substitute for another in protecting cells from hypertonicity, consistent with the compatible osmolytes hypothesis.
Axial heterogeneity of rabbit cortical collecting duct
Authors: Emmons C. L. , Matsuzaki K. , Stokes J. B. , Schuster V. L.
Published: December 2017

The rabbit cortical collecting duct (CCD) consists of three major cell types: principal cells transport K+, beta-intercalated cells absorb Cl-, and alpha-intercalated cells secrete H+. We used functional and histological methods to assess axial distribution of these cell types along rabbit CCD. In perfused CCDs, lumen-to-bath Rb+ rate coefficient (an index of principal cell K+ transport) was not different in tubules from outer cortex (1 mm from renal surface) compared with those from inner cortex (2 mm from renal surface), suggesting that principal cell function is homogeneous along the CCD. In contrast, Cl- rate coefficient (a measure of beta-intercalated cell function) was twice as high in CCDs from outer compared with inner cortex, suggesting heterogeneity of beta-intercalated cells along the CCD. To further investigate these regional differences, we fixed and embedded kidneys and identified three cell types in CCD cross sections using carbonic anhydrase staining and peanut lectin binding. Comparing tubule cross sections from outer with those from inner cortex, we found no axial difference in the fraction of cells that were either principal cells (64%) or total (lectin binding and nonlectin binding) intercalated cells (36%). However, the lectin-binding intercalated cell subset was significantly increased in outer compared with inner cortex. We conclude that there is not heterogeneity of principal cells along the rabbit CCD; however, beta-cell number and function are increased in outer CCD. Collecting duct heterogeneity begins within the cortical segment.
Renal adaptation to dietary phosphate deprivation: role of proximal tubule brush-border membrane fluidity
Authors: Levine B. S. , Knibloe K. A. , Golchini K. , Hashimoto S. , Kurtz I.
Published: December 2017

With dietary phosphate (Pi) restriction, fluidity of renal proximal tubule brush-border membranes (BBM) and Na-dependent Pi transport (Na-Pi) are increased, suggesting that changes in BBM fluidity are critical for adaptation to Pi restriction. To test this hypothesis, the temporal relationship between Na-Pi transport and changes in BBM fluidity was assessed after Pi deprivation in rats. Renal cortex was obtained from rats fed either a 0.03% (-P) or a 0.6% (+P) Pi diet for 4 h or 7 days, and BBM were prepared. Na-Pi uptake by BBM was measured by use of rapid filtration, and BBM fluidity was assessed by use of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). After 4 h on the diets, Na-Pi uptake was 439 +/- 142 (SD) and 984 +/- 184 pmol.mg protein-1.5 s-1 in +P and -P, respectively (P less than 0.01, n, = 8). Na-dependent proline uptake was unchanged. DPH anisotropy and total cholesterol were similar between groups: 0.204 +/- 0.025 and 0.401 +/- 0.047 nmol/mg protein, respectively, in +P and 0.205 +/- 0.015 and 0.392 +/- 0.037 in -P (P greater than 0.05, n = 8-10). After 7 days, Na-Pi uptake was 841 +/- 291 in +P and 2,168 +/- 848 pmol.mg protein-1.5 s-1 in -P, P less than 0.01, n = 8. DPH anisotropy and BBM cholesterol were 0.175 +/- 0.019 and 443 +/- 132 nmol/mg protein, respectively, in +P and 0.162 +/- 0.020 (n = 8) and 341 +/- 128 (n = 3) in -P (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Extent of glomerular injury in active and resolving lupus nephritis: a theoretical analysis
Authors: Myers B. D. , Chagnac A. , Golbetz H. , Newton L. , Strober S. , Sibley R. K.
Published: December 2017

Patients with diffuse, proliferative lupus nephritis (DPLN) were subjected to differential solute clearances (n = 22) and serial renal biopsy (n = 11) before and again after 6-12 mo of immunosuppressive therapy. Glomerular sieving of dextrans of graded size was analyzed with a heteroporous membrane model. This revealed active DPLN to be associated with 1) a reduction of overall pore density accompanied by a 53% depression of glomerular filtration rate (GFR), and 2) appearance of a subset of large, nondiscriminatory pores, which accounted for the observed nephrotic level of proteinuria. Morphometric analysis of biopsy tissue provided evidence of reduced filtration surface area due to global or segmental occlusion of capillary loops in glomerular tufts. Activity of DPLN resolved posttreatment. A computed increase in pore density was associated with a 24% increment in GFR; a marked reduction in the fraction of shuntlike pores was accompanied by a parallel reduction of proteinuria into a subnephrotic range. Repeat biopsy revealed diminished glomerular cellularity, fewer immune deposits, and an ensuing increase in the fraction of tuft area occupied by patent loops. Epithelial filtration slit frequency also increased. Neither functional nor structural recovery was complete, however. Residual pore density approximated only 23-35% of that in healthy controls, and corresponding shuntlike pores were threefold more prominent. We conclude that severe DPLN is only partially reversible by current modalities of treatment and that the ensuing residual injury is far more severe than suggested by conventional tests of renal function.
Distribution of luminal carbonic anhydrase activity along rat inner medullary collecting duct
Authors: Wall S. M. , Flessner M. F. , Knepper M. A.
Published: December 2017

The isolated perfused tubule technique was utilized to determine whether endogenous luminal carbonic anhydrase is present in the initial or terminal parts of the inner medullary collecting duct (IMCD) of the rat. This was accomplished by measuring the luminal disequilibrium pH in the presence of a large luminal proton source created by perfusing the lumen with a solution containing 10 mM NH4Cl. (NH3 efflux causes H+ to be released from NH+4 in the lumen). The disequilibrium pH was calculated by subtracting the equilibrium pH from the measured pH at the end of the tubule lumen. The end-luminal equilibrium pH was calculated from the total CO2 concentration in the collected fluid, as measured by microcalorimetry. The end-luminal pH was determined by measuring the fluorescent signal from the the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), which was added to the luminal perfusate in its nonesterified form. In the initial IMCD, there was no measurable disequilibrium pH. With the addition of the carbonic anhydrase inhibitor acetazolamide to the luminal fluid, a significant acidic pH disequilibrium was elicited. In the terminal IMCD under control conditions a statistically significant acidic disequilibrium pH was measured. The disequilibrium was obliterated when exogenous carbonic anhydrase was added to the luminal perfusate. These findings were verified by measuring total ammonia flux by ultramicrofluorometry. The results demonstrate endogenous luminal carbonic anhydrase activity in the initial IMCD but a lack of enzyme activity in the terminal IMCD.
Vitamin D3 metabolites increase [Ca2+]i in rabbit renal proximal straight tubule cells
Authors: Suzuki M. , Kurihara S. , Kawaguchi Y. , Sakai O.
Published: December 2017

Vitamin D metabolites exert both acute and chronic influences on proximal tubule function. To further evaluate vitamin D action on the kidney, we examined the immediate effects of vitamin D metabolites on cytoplasmic calcium ion concentration [( Ca2+]i), using fura-2 and patch-clamp method in cultured proximal straight tubule cells of rabbit kidney. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and 25-hydroxyvitamin D3 [25(OH)D3] evoked a transient rise in [Ca2+]i, and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] caused a sustained rise in [Ca2+]i; all effects were dose dependent. [Ca2+]i transient, evoked by 1,25(OH)2D3 alone, was abolished in Ca(2+)-free media. Pretreatment of cells in Ca(2+)-free media with caffeine (4 mM) or ryanodine (1 microM) to deplete Ca2+ store of endoplasmic reticulum or with TMB-8 (5 mM) to block Ca2+ release from storage blunted the effect of 25(OH)D3 on [Ca2+]i but not of 24,25(OH)2D3. Data were also supported by activities of Ca-dependent K channel and show that these three vitamin D metabolites in pharmacological doses increase [Ca2+]i of proximal tubule cells from different sources.
Locally formed dopamine stimulates cAMP accumulation in LLC-PK1 cells via a DA1 dopamine receptor
Authors: Grenader A. , Healy D. P.
Published: December 2017

Proximal tubules have been shown to produce dopamine (DA) from (-)-3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) and to express DA1 dopamine (DA1) receptors linked to inhibition of sodium transport. The LLC-PK1 renal epithelial cell line expresses proximal tubule cell-like properties in vitro. Here, we sought to determine whether the LLC-PK1 cell line would be a useful model system to study dopaminergic mechanisms in vitro. LLC-PK1 cells contained high levels of aromatic L-amino acid decarboxylase (AADC) (Km 0.19 +/- 0.08 mM, Vmax 3.69 +/- 0.57 nmol.mg-1.min-1) and converted L-dopa to DA in a nonsaturable fashion up to 1 mM L-dopa. DA production was blocked by the AADC inhibitor carbidopa. Dopamine stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in LLC-PK1 cells in a dose-dependent manner (50% effective concentration, 1.53 +/- 0.38 microM; maximal stimulation, 46.6 +/- 10.88 pmol/mg protein); this effect was blocked by addition of DA1-receptor antagonists. L-Dopa also stimulated cAMP accumulation, and this effect was attenuated by an equimolar concentration of carbidopa and blocked by the DA1 antagonist Sch 23390. These results indicate that LLC-PK1 cells convert L-dopa to DA, which then stimulates cAMP via a DA1 receptor coupled to activation of adenylate cyclase. Moreover, the demonstration that locally formed DA can act as an autocrine/paracrine substance in LLC-PK1 cells in vitro is consistent with a role for DA as an autocrine/paracrine substance in vivo.
Desensitization of DA1 dopamine receptors coupled to adenylyl cyclase in opossum kidney cells
Authors: Bates M. D. , Caron M. G. , Raymond J. R.
Published: December 2017

Peripheral dopamine receptors are classified as DA1 and DA2 receptors, similar to but distinct from central D1 and D2 receptors. Here we report the characterization of DA1 dopamine receptors in the opossum kidney (OK) cell line, which possesses properties of renal proximal tubule cells. OK cell membranes contain 248 +/- 12 fmol [125I]Sch 23982 binding sites/mg protein, which possess pharmacological properties appropriate for a DA1 receptor. Dopamine stimulates adenylyl cyclase via these receptors 4.3 +/- 0.4-fold (50% effective concentration = 4.0 +/- 0.7 microM). The responsiveness of this signaling system is regulated by agonist exposure. Exposure of these cells to dopamine leads to a rapid and profound desensitization of DA1-receptor-stimulated adenylyl cyclase that appears to be independent of the slower downregulation of DA1 receptors. Treatment of cells with 8-bromoadenosine 3',5'-cyclic monophosphate also desensitizes dopamine-stimulated adenylyl cyclase but in a fashion qualitatively and quantitatively distinct from that induced by agonist exposure. These data suggest that the cellular machinery for both homologous and heterologous desensitization of the DA1-receptor response exists in OK cells. Thus OK cells provide a model system for the study of the peripheral actions of dopamine at DA1 receptors and the regulation of these receptors.
Cholesterol esterase in preglomerular microvessels from normal and cholesterol-fed rabbits
Authors: Kamanna V. S. , Saied S. I. , Evans-Hexdall L. , Kirschenbaum M. A.
Published: December 2017

Feeding cholesterol to rabbits produces an atherosclerotic model sharing common metabolic features with the disease in humans, including the vascular lipid accumulation. In coronary vascular cells, this lipid accumulation has been associated with decreased prostacyclin (PGI2) biosynthesis and acid cholesterol esterase activity. Unlike the coronary vascular bed, renal microvasculature appears relatively resistant to atherosclerotic injury. This study examined whether renal microvessels from cholesterol-fed rabbits demonstrated similar metabolic changes in coronary vascular cells. Rabbits were fed either a 0% or 2% cholesterol diet for 1 mo. Similar to coronary vascular cells, in renal microvessels from cholesterol-fed rabbits PGI2 biosynthesis decreased and tissue concentrations of cholesterol and cholesteryl esters increased. However, unlike coronary vascular cells, renal microvascular cholesterol esterase activity increased. Light and electron microscopy revealed sporadic lipid deposits in renal microvessels from cholesterol-fed rabbits and no foam cells or occlusive lesions. In vitro addition of prostanoids to normal renal microvessels had no effect on cholesterol esterase activity. It is inviting to speculate that the increased acid cholesterol esterase activity in renal microvessels from cholesterol-fed rabbits protected them from developing extensive microvascular lesions. These biochemical events may explain the relative resistance of human renal microvessels to the development of occlusive atherosclerotic microvascular lesions.
Interaction of trichlormethiazide or amiloride with PTH in stimulating Ca2+ absorption in rabbit CNT
Authors: Shimizu T. , Nakamura M. , Yoshitomi K. , Imai M.
Published: December 2017

To determine the renal tubular site and mechanism of the anticalciuric effects of trichloromethiazide (TCM) and amiloride, we studied the effects of these diuretics on net Ca2+ absorption (JCa) in isolated rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). TCM (100 microM) in the lumen increased JCa in the CNT without affecting lumen-negative transepithelial voltage (VT). This effect was dependent on the amount of parathyroid hormone (PTH, 0.1 or 1 nM) in the bath. TCM had no detectable effect on JCa in the absence of PTH. Addition of 100 microM ouabain to the bath decreased PTH-stimulated JCa and abolished the TCM-stimulated JCa. Elimination of Na+ from the lumen increased PTH-stimulated JCa. Under this condition the ability of TCM to increase JCa was abolished, suggesting that the inhibition of Na+ entry from the lumen with TCM may account for the increase in PTH-stimulated JCa. Elimination of Na+ from the bath in the presence of PTH decreased JCa and abolished the stimulatory effect of TCM on JCa in the presence of PTH. Changes in VT caused by amiloride may not account for the increase in JCa, because JCa was not changed when VT was deflected to more positive direction by increasing bath K+ from 5 to 15 mM. Neither TCM nor amiloride affected JCa in the CCD. From these observations, we speculate that the inhibition of Na+ entry across the apical cell membrane by either thiazides or amiloride may stimulate PTH-induced JCa. The intact Na(+)-Ca2+ antiporter in the basolateral cell membrane is essential for the anticalciuric effect of thiazides and amiloride.
Effects of dopamine-receptor antagonists and renal denervation on amino acid-induced hyperfiltration
Authors: Mendez R. E. , Lopez R. , Lopez G. , Marti M. S. , Martinez-Maldonado M.
Published: December 2017

The mechanism whereby an infusion of amino acids (AA) leads to increments in glomerular filtration rate (GFR) and renal plasma flow (RPF) is incompletely understood. Dopamine (DA) is a catecholamine in which known actions at low doses include the ability to increase both GFR and RPF. Furthermore, urinary DA excretion has been shown to be augmented after an oral protein load. We therefore studied the renal hemodynamic response to intravenous infusion of a 10% mixed AA solution in anesthetized euvolemic Wistar-Furth rats in the presence or absence of specific DA1 [Sch 23390 (SCH)] and DA2 [S-sulpiride (S-SP)] receptor antagonists. Infusion of AA in vehicle-pretreated rats resulted in a 28 +/- 8% increase in GFR and a 29 +/- 6% increase in effective ERPF over baseline values. Administration of AA in the presence of SCH also resulted in elevations in both GFR and ERPF by 23 +/- 3% and 26 +/- 6%, respectively. In contrast, when AA were given in the presence of S-SP, the rise in both GFR and ERPF was completely prevented. To examine whether the AA-induced hyperfiltration was due to DA release from renal nerves or enhanced renal tubule DA synthesis, we administered AA to rats in which the left kidney had been chronically denervated while the right kidney remained intact. Infusion of AA led to significant increments in GFR (33 +/- 4%) and ERPF (34 +/- 7%) only in the intact control kidney, whereas GFR and ERPF remained unaltered in the denervated kidney.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of leukocytes in metabolic and functional derangements of experimental glomerulonephritis
Authors: Lefkowith J. B. , Nagamatsu T. , Pippin J. , Schreiner G. F.
Published: December 2017

Nephrotoxic nephritis (NTN) is characterized by an influx of leukocytes into the glomerulus, with accompanying glomerular dysfunction and a marked increase in glomerular eicosanoid production. We examined the relationship between the glomerular inflammatory cell infiltrate and the concomitant metabolic/functional changes in this model of renal disease using a combination of in vivo immunologic strategies to both decrease [X-irradiation and cobra venom factor (CVF)] and increase (preimmunization with rabbit immunoglobulin G or accelerated NTN) the inflammatory cell infiltrate. With the use of these manipulations, a close correlation between glomerular leukocytes and the proteinuria of NTN was observed. The ablative strategies (X-irradiation and CVF) also attenuated the increase in leukotriene B4 (LTB4) generation seen with NTN and virtually completely prevented the increase in thromboxane B2 (TxB2) production (basal and angiotensin II elicited). Accelerated NTN, in contrast, increased and prolonged the rise in glomerular LTB4 production and exacerbated the increase in TxB2 production. Glomerular prostaglandin E2 production was not altered by the induction of nephritis nor any of the aforementioned immunologic manipulations. Regression analysis established that glomerular TxB2 production correlated significantly with the leukocyte influx at both 3 and 24 h. Glomerular LTB4 production correlated only with the presence of leukocytes at 3 h. Both glomerular TxB2 and LTB4 production were closely correlated with the renal dysfunction as assessed by proteinuria. These data suggest that leukocytes play a direct critical role in both the functional and metabolic alterations that occur in the setting of NTN. They further imply that leukocytes are crucial to the observed increase in glomerular eicosanoid production.(ABSTRACT TRUNCATED AT 250 WORDS)
Mechanism of enhanced eicosanoid production by isolated glomeruli from rats with bilateral ureteral obstruction
Authors: Yanagisawa H. , Morrissey J. , Klahr S.
Published: December 2017

Isolated glomeruli from rats with bilateral ureteral obstruction (BUO) of 24-h duration produced significantly greater amounts of prostaglandin (PG) E2 and 6-keto-PGF1 alpha in vitro than glomeruli from sham-operated control (SOC) rats. This increase was abolished by the angiotensin-converting enzyme (ACE) inhibitor, enalaprilat, given in vivo. To elucidate the mechanisms responsible for enhanced eicosanoid production by glomeruli from rats with BUO, we measured the activities of phospholipase (PL) A2 and C and cyclooxygenase in glomeruli isolated from SOC and BUO rats. L-alpha-Phosphatidylcholine (PC)-specific and L-alpha-phosphatidylethanolamine (PE)-specific PLA2 activities were significantly greater in glomerular membranes from rats with BUO than from SOC rats. Likewise, both the activity and amount of cyclooxygenase were significantly greater in glomerular membranes of rats with BUO. Cyclooxygenase and the PE-specific PLA2 in glomerular membranes of rats with BUO remained at the levels seen in SOC rats when animals were treated in vivo before BUO with the ACE inhibitor, enalaprilat, and the thromboxane synthase inhibitor, OKY-046. Thus inhibition of vasoconstrictor formation leads to subsequent inhibition of vasodilator formation. In contrast to PE-specific PLA2, PC-specific PLA2 activities were further increased in glomerular membranes from both SOC and BUO rats pretreated with the two drugs.s The activity of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PIP2 PLC) was significantly decreased in glomeruli from rats with BUO compared with SOC rats. We conclude that the increased synthesis of vasodilatory eicosanoids by glomeruli from rats with BUO may be mediated by enhanced activities of PE-specific PLA2 and cyclooxygenase, which are apparently stimulated by the vasoconstrictors angiotensin and thromboxane.
Fibroblasts of rabbit kidney in culture. I. Characterization and identification of cell-specific markers
Authors: Rodemann H. P. , Muller G. A. , Knecht A. , Norman J. T. , Fine L. G.
Published: December 2017

There is currently no information available as to whether different renal fibroblast subpopulation can be identified and whether they show differences in functional properties. We therefore compared the growth characteristics of interstitial fibroblasts derived from the rabbit renal cortex and inner medulla (papilla) and sought cell-specific markers for the two populations of cells. Analyses of the population dynamics revealed that the mitotic lifespan of papillary fibroblasts (PF) is approximately 50% longer than that of cortical fibroblasts (CF), with the former going through 20 cumulative population doublings (CPD) before transition into terminally differentiated postmitotic cells compared with 9 CPD in CF. PF and CF populations contained three types of mitotically active cells (MFI, MFII, MFIII) and three types of postmitotic cells (PMFIV, PMFV, PMFVI) differentiating along a terminal cell lineage from MFI through PMFVI. In both PF and CF cultures the percent of MF-type cells decreased and the percent of postmitotic cells increased with successive doublings. Two-dimensional polyacrylamide gel electrophoresis of uniform clonal populations of MFIII-type cells revealed two specific proteins for PF-MFIII-type cells, pf1 and pf2, and three specific proteins for CF-MFIII-type cells, cf1, cf2, and cf3. Additionally, a monoclonal antibody was raised that does not recognize CF in culture, but reacts strongly with PF. These studies demonstrate that rabbit renal PF have a pattern of growth in vitro that is distinct from that of CF and that they can be positively identified by specific immunological and protein markers in vitro.
Fibroblasts of rabbit kidney in culture. II. Paracrine stimulation of papillary fibroblasts by PDGF
Authors: Knecht A. , Fine L. G. , Kleinman K. S. , Rodemann H. P. , Muller G. A. , Woo D. D. , Norman J. T.
Published: December 2017

To examine the role of tubulointerstitial cell interaction in the regulation of fibroblast growth, fibroblasts from the rabbit renal cortex (CF) and papilla (PF) were cocultured with epithelial cells from the same tissue location. Inner medullary collecting duct epithelial cells (IMCDE) or IMCDE-conditioned medium stimulated DNA synthesis in PF, whereas proximal tubule epithelium (PTE) had no effect on the proliferation of CF. PF and CF showed a similar mitogenic response to exogenous epidermal growth factor and insulin-like growth factor 1 (IGF-I). Transforming growth factor-beta 1 inhibited growth of both cell types, and basic fibroblast growth factor (bFGF) had no effect on proliferation of either cell type. In contrast, platelet-derived growth factor (PDGF) was a potent mitogen for PF but was only weakly mitogenic for CF. Both CF and PF expressed a similar number of a single-affinity class of PDGF receptors (Kd, 2-4 x 10(-10) M). Assay for growth factor activity in conditioned medium from IMCDE and PTE showed that only IMCDE produced detectable PDGF. IMCDE-stimulated proliferation of PF was partially blocked by an antibody to PDGF, whereas antibodies to IGF-I had no neutralizing effect. The data suggest a role for PDGF in the regulation of interstitial fibroblast proliferation by IMCDE in the renal papilla. This paracrine system may be important in the pathogenesis of some forms of interstitial fibrosis of the kidney.
Analysis of pulsatile pressures and flows in glomerular filtration
Authors: Drumond M. C. , Deen W. M.
Published: December 2017

Previous mathematical models of glomerular filtration have ignored the pulsatility of the glomerular capillary pressure, using only steady-state equations and time-averaged pressures and flows. Because the actual pressure pulses are rapid and of large amplitude and because the governing equations are nonlinear, it is questionable whether the effects of the pressure pulses average out in the manner that has been assumed. We have developed a model that includes sinusoidal variations in the glomerular transcapillary hydraulic pressure (delta P) and the afferent arteriolar plasma flow rate over each cardiac cycle. The analysis suggests that the previously ignored time derivatives in the luminal mass balance equations are not negligible. The amplitude of the oscillations in delta P was found to be sufficient to reverse the direction of the transmural fluxes over part of each cardiac cycle, at the more efferent locations in a capillary. However, the time-averaged values of single-nephron glomerular filtration rate and sieving coefficients for macro-molecules from the pulsatile model differed from that for a steady-state formulation by less than 0.1 and less than or equal to 10%, respectively. We conclude that use of the usual steady-state assumption introduces negligible errors in calculating glomerular membrane parameters from experimental data.