bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium
Authors: Ayo S. H. , Radnik R. A. , Glass W. F. , Garoni J. A. , Rampt E. R. , Appling D. R. , Kreisberg J. I.
Published: December 2017

Nodular expansion of glomerular mesangium with increased amounts of extracellular matrix (ECM) material is pathognomic of diabetic nephropathy. The precise mechanisms involved in this accumulation are unknown. Recently, we reported using a solid-phase enzyme-linked immunosorbent assay (ELISA) technique that glomerular mesangial cells, the principal cell type residing in glomerular mesangium, accumulate 50–60% more fibronectin (FN), laminin (LM), and type IV collagen (T-IV) when cultured in medium containing high glucose (30 mM) (S. H. Ayo, R. A. Rodnik, J. Garoni, W. F. Glass II, and J. I. Kreiberg. Am. J. Pathol. 136: 1339-1348, 1990). ECM assembly is controlled by its rate of synthesis and degradation, as well as its binding and rate of incorporation into the ECM. To elucidate the mechanisms involved, pulse-chase experiments were designed to estimate ECM protein synthesis from the incorporation of Trans-35S [( 35S]methionine, [35S]cysteine) into immunoprecipitated FN, LM, and T-IV. mRNA levels were examined, and degradation rates were estimated from the disappearance of radioactivity from matrix proteins in mesangial cells previously incubated with Trans-35S. One week of growth in 30 mM glucose resulted in approximately 40–50% increase in the synthesis of all three matrix proteins compared with 10 mM glucose-grown cells. This was accompanied by a significant increase in the transcripts for all three matrix proteins (approximately twofold). The specific activity of the radiolabel in trichloroacetic acid-precipitable cell protein showed no difference between cells grown in 10 or 30 mM glucose, indicating that total protein synthesis was unchanged. After 1 wk, the rate of FN, LM, and T-IV collagen degradation was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
Isolation of nephrocalcin from kidney tissue of nine vertebrate species
Authors: Nakagawa Y. , Renz C. L. , Ahmed M. , Coe F. L.
Published: December 2017

Nephrocalcin (NC), a urinary calcium oxalate monohydrate (COM) crystal growth inhibitor, was purified from kidneys of nine vertebrate species including humans. All isolates were glycoproteins with high contents of aspartic and glutamic acids and small amounts of basic and aromatic amino acids. Carbohydrate contents ranged from 4 to 16 wt% among the different species, but all contained fucose, mannose, galactose, glucose, galactosamine, glucosamine, and N-acetylneuraminic acid. Although amino acid and carbohydrate compositions were similar, dissociation constants derived from calcium oxalate monohydrate inhibition varied between 10(-7) and 10(-8) M, and highest affinity could be related to highest ability of the kidney of origin to concentrate urine. Using an antibody raised against NC from human kidney tissue culture medium, we found strong immunoreactivity with two species, pigs and sheep. Thus far all vertebrate kidneys possess COM growth-inhibiting material that seems to be of glycoprotein character, and all the glycoproteins isolated so far share similar features, suggesting NC-like proteins are a well-conserved trait.
Peptide YY luminal processing and axial heterogeneity of basolateral binding in renal proximal tubules
Authors: Nielsen S. , Sheikh S. P. , Sheikh M. I. , Christensen E. I.
Published: December 2017

This study investigates the definite location of peptide YY (PYY) binding sites on the basolateral membranes in proximal tubules. S1, S2, and S3 segments were dissected, perfused in vitro, and exposed to [125I-Tyr36]monoiodo-PYY either in the bath fluid or in the perfusate. S1 segments exposed to [125I-Tyr36]PYY in the bath fluid were fixed and prepared for electron microscope autoradiography. The results demonstrated a high degree of axial heterogeneity of basolateral binding of PYY, since only S1 bound PYY, 0.59 +/- 0.09 pg/mm after 15 min; 89.1% could be displaced with unlabeled PYY. PYY was not internalized, 90% of the grains were associated with the basolateral membranes, and no accumulation of grains was observed over the vacuolar apparatus. After luminal perfusion with PYY, 79.3 +/- 7.2% was processed, 61.7 +/- 6.3% was degraded at the brush border, and no tubular accumulation was detected. Thus PYY is not taken up by endocytosis. Unexpectedly, a very large fraction of processed PYY was transported from lumen to bath as trichloroacetic acid (TCA)-precipitable label constituting 41.6 +/- 4.7%. There was no axial heterogeneity in the luminal handling of PYY. In conclusion, this study reveals a high density of PYY binding sites at the basolateral membranes from S1 segments, indicating a selective function of S1 segments on stimulation with PYY. In contrast to other proteins PYY was not internalized from the basolateral membranes.
Reversed polarity of Na(+) -K(+) -ATPase: mislocation to apical plasma membranes in polycystic kidney disease epithelia
Authors: Wilson P. D. , Sherwood A. C. , Palla K. , Du J. , Watson R. , Norman J. T.
Published: December 2017

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder in which renal tubules become enormously enlarged due to fluid accumulation. Na(+) -K(+) -ATPase was compared in normal and cystic regions of whole kidneys and in confluent primary cultures of microdissected renal tubule and cyst-lining epithelia. Immunostaining with antibodies directed against the Na(+) -K(+) -ATPase catalytic alpha-subunit was confined to apical, luminal plasma membranes of ADPKD epithelia, which was a complete reversal of the normal renal tubule polarized location in basolateral membranes. Mislocated Na(+) -K(+) -ATPase was shown to be functionally active, because identical intense apical staining was observed by use of a cytochemical assay. In addition, biochemical assays showed a significant increase in these ouabain-inhibitable Na(+) -K(+) -ATPase specific activity levels in ADPKD kidneys compared with age-matched normal kidneys. Specific binding of [3H] ouabain was not only increased but also confined to the apical membrane vesicles prepared from cystic regions of ADPKD kidneys compared with normal age-matched controls, in which binding was confined to basolateral membrane vesicles. Although steady-state levels of Na(+) -K(+) -ATPase alpha- and beta-subunit in mRNAs were increased somewhat in ADPKD kidneys, this alone was not sufficient to account for the observed activation. Confluent ADPKD epithelia grown on dual-chamber, permeable membrane supports also showed reversed polarity of 22NaCl vectorial transport, because this was from basal to apical media compartments. Because this transport could also be blocked by ouabain, this suggested apical Na(+) -K(+) -ATPase was responsible and implicated altered polarity of Na(+) -K(+) -ATPase and resultant Na+ secretion as a mechanism for cyst formation in ADPKD. Because no reversal of polarity of other basolateral or apical membrane proteins was detected, an intracellular sorting defect specific for Na(+) -K(+) -ATPase is proposed.
Carbonic anhydrase in turtle bladder mitochondrial-rich luminal and subluminal cells
Authors: Fritsche C. , Kleinman J. G. , Bain J. L. , Heinen R. R. , Riley D. A.
Published: December 2017

Bladders from March-April turtles were processed for carbonic anhydrase (CA) cytochemically using the method of D.A. Riley, S. Ellis, and J. Bain (Neuroscience 13: 189, 1984). CA-positive cells comprised 11.1 +/- 0.7% of mucosal epithelial cells. Microplicated (MP) cells comprised 47.2 +/- 1.8% of CA-positive cells and displayed at least two distinct staining patterns: the first was characterized by reaction product that filled the luminal one-third, including the terminal web and microplicae. These cells possessed extensive microplicae, a morphological feature of ongoing H+ secretion. The second was characterized by reaction product distributed throughout cells, excluding the terminal web and microplicae, with greatest intensity in the luminal one-third below the terminal web. These cells possessed flattened microplicae, a morphological feature of diminished H+ secretion. Microvillated (MV) cells comprised 6.0 +/- 1.0% of CA-reactive cells. The basal layer was occupied by 46.8 +/- 1.7% of CA-positive cells, which were termed subluminal (SL) cells. SL cells were mitochondrial rich and did not contact the lumen. Extracellular CA staining was common between the lateral margins of contiguous mitochondrial-rich or non-mitochondrial-rich cells.
Renin release from isolated juxtaglomerular apparatus depends on macula densa chloride transport
Authors: Lorenz J. N. , Weihprecht H. , Schnermann J. , Skott O. , Briggs J. P.
Published: December 2017

Transport inhibitor and ion substitution studies were performed using perfused, superfused preparations of the isolated rabbit juxtaglomerular apparatus to investigate transport dependency of macula densa-mediated renin secretion. In the first experimental series, tubular perfusion with a high-NaCl solution containing 10(-6) M bumetanide increased renin secretion compared with perfusion with high NaCl alone from 8.7 to 24.6 nano-Goldblatt hog units (nGU)/min. Bath addition of 10(-6) M bumetanide had no effect on renin release. The second series tested ability of luminal addition of 54 mmol/l Na or Cl salts to inhibit renin secretion, starting from a stimulated value produced by low-NaCl perfusion. Perfusion with a high-NaCl solution decreased renin secretion from 58.9 to 14.8 nGU/min, which served as a positive control. Addition of choline chloride decreased renin secretion from 42.7 to 16.6 nGU/min, and RbCl decreased renin secretion from 54.9 to 17.0 nGU/min. In contrast, addition of two different Na salts had no effect on renin release (from 41.7 to 31.6 nGU/min with sodium isethionate and from 14.1 to 13.5 nGU/min with sodium acetate). Also, in the presence of 26 mmol/l Cl, addition of 54 mmol/l Na had no effect on renin secretion (29.9-36.8 nGU/min). These data demonstrate that renin secretion is directly stimulated by luminal application of transport blockers and can be inhibited by increases in Cl concentration at the macula densa but not by changes in Na concentration. These results support the hypothesis that the initiating signal for macula densa control of renin secretion is an inverse change in transport rate via the luminal Na(+)-K(+)-2Cl- cotransporter.
Colocalization of H(+)-ATPase and band 3 anion exchanger in rabbit collecting duct intercalated cells
Authors: Schuster V. L. , Fejes-Toth G. , Naray-Fejes-Toth A. , Gluck S.
Published: December 2017

Two major types of intercalated cells (IC) have been previously defined in rabbit collecting duct: alpha-cells have a basolateral band 3-like anion exchanger and secrete H+, whereas beta-cells bind peanut agglutinin (PNA) apically and are believed to secrete HCO3-. To further define IC types, we double-labeled kidney sections with anti-H(+) -ATPase antibodies and with either an anti-band 3 antibody or PNA. We found four patterns of staining: 1) IC with apical H(+)-ATPase and basal band 3, a configuration consistent with ongoing H+ secretion, which prevailed in the inner stripe of outer medulla (OMCDi); 2) diffuse H(+)-ATPase labeling across the cell and basal band 3, which was most numerous in the outer stripe of outer medulla (OMCDo); 3) IC with "bright" apical peanut lectin, diffuse H(+)-ATPase, and no band 3, which was abundant in the cortical collecting duct (CCD) and probably represents HCO3(-)-secreting cells; and 4) "hybrid" cells with various staining combinations (e.g., apical lectin binding and apical H(+)-ATPase), which although they are uncommon, were seen in the CCD. Consistent with this immunocytochemical finding of hybrid cells, cell-sorting studies on isolated CCD IC showed that 6-18% of PNA-positive cells also stained positively for band 3. We conclude that 1) band 3-positive IC in the OMCD vary axially. Most OMCDi IC are probably active proton secretors, whereas up to one-half of OMCDo IC may be latent H+ secretors. 2) The diffuse H(+)-ATPase pattern in putative beta-cells differs from comparable results in the rat and is not consistent with a "reversed" alpha-cell. HCO3- secretion by beta-cells may be driven by an H+ extrusion mechanism other than the alpha-cell pump re-sorted to the basolateral membrane. 3) The possibility of hybrid cells that might combine alpha- and beta-cell transport proteins suggests a mechanism for functional reversal of collecting duct IC polarity.
A novel metalloproteinase present in freshly isolated rat glomeruli
Authors: Le Q. , Shah S. , Nguyen H. , Cortez S. , Baricos W.
Published: December 2017

We have utilized [3H] gelatin to document high activity of a metalloproteinase present in freshly isolated rat glomeruli. [3H] gelatin degradation by glomeruli was markedly inhibited by EDTA (10 mM: -89 +/- 2.3%) and o-phenanthroline (2 mM: -72 +/- 0.1%), inhibitors of metalloproteinases. No significant inhibition of [3H]gelatin degradation was observed with inhibitors of serine or cysteine proteinases. Most (greater than 80%) of the glomerular metalloproteinase (GLOMP) activity was associated with the pellet after centrifugation of sonicated glomeruli at 100,000 g for 90 min. The pH optimum for gelatin degradation by sonicated glomeruli was approximately pH 8.5. Sodium dodecyl sulfate substrate (gelatin)-polyacrylamide gel electrophoresis revealed a single major band of EDTA-inhibitable gelatin-degrading activity with a molecular mass of approximately 116-125 kDa. The GLOMP activity was not inhibited by tissue inhibitors of metalloproteinases, did not appear to be latent, and was not activated by organomercurial activators of several latent metalloproteinases. GLOMP activity was increased 3.4-fold after incubation with trypsin (20 micrograms/ml, 25 min, 22 degrees C). These data indicate that GLOMP is distinct from the previously described matrix metalloproteinases, as well as other metalloproteinases present in the kidney, including the gelatinase secreted by cultured mesangial cells, Meprin, and endopeptidase 24.11 (enkephalinase, EC 3.4.24.11).
Myo-inositol uptake by rat cultured inner medullary collecting tubule cells: effect of osmolality
Authors: Veis J. H. , Molitoris B. A. , Teitelbaum I. , Mansour J. A. , Berl T.
Published: December 2017

Myo-inositol (MI) is involved in the adaptation to hyperosmolality. Its uptake by rat inner medullary collecting duct (RIMCD) cells has not been studied. Compared with cells grown in isotonic media, those grown in hyperosmolality display marked enhancement in [3H]MI uptake [counts/min (cpm).microgram protein-1.2 h-1] from 217 +/- 23 to 718 +/- 64, P less than 0.001. This is mimicked by the supplementation with 300 mM mannitol (638 +/- 59, P less than 0.001) but not by 300 mM urea. The increment in [3H]MI is observed at 37 degrees C but not at 4 degrees C. MI uptake is Na+ dependent in cells grown both in hyperosmolal or isotonic media. At least 12 h of hyperosmolality are needed to enhance MI uptake, and reexposure to isotonic media for at least 24 h is required for the enhancement to reverse. The effects of the microtubular inhibitor, nocodazole (10 micrograms/ml), and the protein synthesis inhibitor, cycloheximide (30 micrograms/ml), were studied. Cells grown with nocodazole show unimpaired enhancement of MI uptake. Cycloheximide exposure (16 h) does not affect MI uptake in isotonic media (182 +/- 23 vs. 191 +/- 15), but inhibited enhanced MI uptake in hyperosmolality (822 +/- 53 in the absence vs. 331 +/- 24 in the presence of cycloheximide, P less than 0.001). We conclude that hyperosmolality stimulates the synthesis of a protein, most likely an Na-MI cotransporter, that markedly enhances MI uptake. This process may be critical to the osmoregulation of RIMCD cells.
Ischemia-induced loss of epithelial polarity: potential role of the actin cytoskeleton
Authors: Molitoris B. A.
Published: December 2017

Proximal tubule cells play a major role in the reabsorption of ions, water, and solutes from the glomerular filtrate. This is accomplished, in large part, by a surface membrane polarized into structurally, biochemically, and physiologically distinct apical and basolateral membrane domains separated by cellular junctional complexes. Establishment and maintenance of these unique membrane domains is essential for the normal functioning of proximal tubular cells and is dependent on cortical actin cytoskeletal-surface membrane interactions. Ischemia results in the duration-dependent loss of apical and basolateral surface membrane lipid and protein polarity. This loss of surface membrane polarity is associated with disruption of the cortical actin microfilament network and the opening of cellular tight junctions. Surface membrane lipids and proteins are then free to diffuse laterally within the membrane bilayer into the alternate membrane domain. Functionally, ischemia-induced loss of epithelial polarity is, in part, responsible for reduced sodium and glucose reabsorption. With recovery, proximal tubule cells undergo remodeling of the surface membrane such that the unique apical and basolateral membrane domains are reestablished allowing normal cellular function to return.
Biochemical and functional characterization of H(+)-K(+)-ATPase in distal amphibian nephron
Authors: Planelles G. , Anagnostopoulos T. , Cheval L. , Doucet A.
Published: December 2017

Because proton secretion and K+ reabsorption in the late distal tubule of amphibians are active, we evaluated whether these processes could be mediated by an H(+)-K(+)-ATPase similar to the gastric H(+)-K+ pump and to the K(+)-ATPase previously described in the terminal segments of the mammalian nephron. K(+)-stimulated ATPase activity was detected in microdissected segments of frog and Necturus nephron: its activity was high in the late distal and collecting tubules, whereas it was undetectable in the proximal convoluted tubule and early distal tubule. In frog collecting tubule, K(+)-ATPase had a high affinity for K+ (Km approximately 0.30 mM), was inhibited by vanadate, omeprazole, and the imidazopyridine Sch 28080, and was insensitive to ouabain. Furthermore, in vivo administration of Sch 28080 to anesthetized Necturus induced a significant rise of the steadystate intratubular pH in the late distal tubule, demonstrating that this drug inhibited tubular fluid acidification. It is suggested that K(+)-ATPase present in the terminal segments of amphibian nephron is similar to the gastric H(+)-K+ pump and is involved in urinary acidification.
Primary response gene expression in renal hypertrophy and hyperplasia: evidence for different growth initiation processes
Authors: Kujubu D. A. , Norman J. T. , Herschman H. R. , Fine L. G.
Published: December 2017

Unilateral nephrectomy is followed by compensatory renal hypertrophy, a response in which the cells of the contralateral kidney increase in size and protein content without synthesizing DNA or dividing. To determine whether the earliest phase of the hypertrophic response has features similar to mitogenic or differentiation responses, we have characterized the expression of several primary-response genes, the 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequences (TIS) genes, which are rapidly and transiently induced in the absence of intervening protein synthesis, in a variety of mitogenic and differentiation cell systems. TIS gene induction was studied in the contralateral kidney of uninephrectomized and sham-operated mice, as well as in the kidneys of mice in which renal cell proliferation was induced by folic acid injection. Induction of TIS 1, TIS 8, and TIS 11 mRNA levels following folic acid administration peaked at 2 to 4 h and persisted up to 6 to 12 h after mitogenic stimulation. In contrast, a qualitatively different pattern was observed after both uninephrectomy and sham operation; a short-lived increase (up to 1 h) in mRNA levels occurred for the three TIS genes. This pattern was qualitatively similar to that observed in the sham-operated animals. We conclude that renal hypertrophy induced by unilateral nephrectomy is a distinct cellular response, distinguishable at the earliest transcriptional level from a mitogenic response and from the responses that characterize several pathways of cell differentiation.
Medium-term cyclosporin renal dysfunction and its reversibility in rats
Authors: Sabbatini M. , De Nicola L. , Uccello F. , Romano G. , Papaccio G. , Esposito V. , Sepe V. , Conte G. , Fuiano G.
Published: December 2017

Renal dynamics and morphology were investigated by metabolic, renal micropuncture (RM), and electron microscopy (EM) studies in 50 female rats treated with cyclosporin A (CsA, 40 mg.kg body wt-1.48 h-1) for either 10, 20, or 30 days (groups CsA 10, CsA 20, and CsA 30, respectively); control rats received olive oil (group N). Body weight gain, sodium metabolism, and plasma volume were not altered by CsA administration in any group. GFR was decreased in group CsA 10 vs. group N (-10%, P less than 0.05) and was further impaired in groups CsA 20 and CsA 30 (-45%, P less than 0.01 vs. group N). RM studies showed a significant decrease of single-nephron glomerular filtration rate (SNGFR) in group CsA 30 vs. group N (-26%, P less than 0.01) after the fall of glomerular capillary pressure (Pgc;-8%, P less than 0.05), the increase of afferent arteriole resistance (Ra; +40%, P less than 0.05), and the consequent decrease of glomerular plasma flow (GPF; -28%, P less than 0.05). Thirty days after CsA withdrawal, SNGFR returned to normal values (P less than 0.01 vs. group CsA) as a result of the normalization of Pgc, Ra, and GPF.EM showed only a progressive vacuolation of proximal and distal tubular cells. These data suggest that medium-term administration of CsA is associated with reversible changes in glomerular dynamics and only mild histological lesions.
Impaired renal hemodynamic response to protein feeding in dogs with experimental Fanconi syndrome
Authors: Woods L. L. , Young E. W.
Published: December 2017

These studies were designed to test the hypothesis that intact proximal tubular function is required for protein-stimulated renal vasodilation. In normal chronically instrumented conscious dogs, a meal of raw beef (10 g/kg) caused glomerular filtration rate (GFR) to increase significantly from 63 +/- 5 to 94 +/- 10 ml/min after 90 min, while plasma alpha-amino nitrogen rose from 3.9 +/- 0.2 to 6.7 +/- 0.6 mg/dl. In another group of dogs experimental Fanconi syndrome (generalized proximal tubular dysfunction) was induced with maleic acid (25 mg/kg iv, pH 7.3). GFR fell slightly but significantly from 91 +/- 18 to 66 +/- 9 ml/min after maleic acid, while Na+ excretion rose from 24 +/- 8 to 176 +/- 24 mu eq/min, alpha-amino nitrogen excretion rose from 82 +/- 40 to 148 +/- 47 micrograms/min, and glucose excretion rose from 0.2 +/- 0.1 to 6.1 +/- 1.0 mg/min. In response to a subsequent meat meal, plasma alpha-amino nitrogen rose significantly from 3.8 +/- 0.4 to 6.2 +/- 0.5 mg/dl, but GFR did not change, averaging 66 +/- 9 ml/min over the next 120 min. These results suggest that normal proximal tubular function is necessary for protein-stimulated renal vasodilation to occur. This finding is consistent with the hypothesis that the tubuloglomerular feedback mechanism may be involved in mediating the normal renal hemodynamic response to protein feeding.
Aldosterone metabolism in combined isolated perfused rat liver and kidney
Authors: Egfjord Martin , Daugaard Henrik , Olgaard Klaus
Published: December 2017

Pages 536–548: Martin Egfjord, Henrik Daugaard, and Klaus Olgaard. “Aldosterone metabolism in combined isolated perfused rat liver and kidney.” Page 545, Table 2: braces in columns 1, 2, and 6 were inadvertently omitted in the final printing.
Renal sympathetic nerves modulate glomerular ANP receptors and filtration
Authors: Awazu M. , Kon V. , Harris R. C. , Imada T. , Inagami T. , Ichikawa I.
Published: December 2017

We examined characteristics of atrial natriuretic peptide (ANP) receptors in glomeruli isolated from subacutely (3-5 days) denervated (DNX) and contralateral nondenervated (non-DNX) kidneys of normal rats (NL) and rats subjected to water deprivation for 48 h (WD). Total ANP receptor density in DNX kidneys of WD rats, measured by competitive inhibition binding between 125I-labeled ANP and ANP, was twofold higher than non-DNX kidneys (726 +/- 96 vs. 384 +/- 32 fmol/mg protein, P less than 0.05). Equilibrium association constant (Ka) was not significantly different (2.33 +/- 0.43 vs. 3.34 +/- 0.78 x 10(9) M-1). In NL rats, there was no difference in ANP receptor density between DNX and non-DNX kidneys (244 +/- 20 and 264 +/- 16 fmol/mg protein). Production of guanosine 3',5'-cyclic monophosphate (cGMP), a putative second messenger of ANP, in response to ANP (10(-7) M) in glomeruli isolated from DNX was significantly larger than non-DNX kidneys of WD rats. To determine whether these changes in ANP receptors have functional consequences in vivo, glomerular capillary ultrafiltration coefficient (Kf) was assessed by micropuncture technique in WD Munich-Wistar rats. In DNX kidneys, ANP infusion (4 micrograms.kg-1.h-1) significantly increased whole kidney glomerular filtration rate (GFR) and single-nephron (SN) GFR (0.64 +/- 0.06 to 0.89 +/- 0.17 ml/min and 25 +/- 2 to 33 +/- 2 nl/min, respectively; n = 7) and Kf (1.26 +/- 0.29 to 2.18 +/- 0.41 nl.min-1.mmHg-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Postnatal maturation of rabbit renal collecting duct. II. Morphological observations
Authors: Evan A. P. , Satlin L. M. , Gattone V. H. , Connors B. , Schwartz G. J.
Published: December 2017

The renal collecting duct exhibits cellular heterogeneity. An ultrastructural analysis of this nephron segment during maturation has been performed to determine morphological characteristics of developing principal and intercalated cells (IC). Apical surface features and cytoplasmic ultrastructure were best revealed by scanning electron microscopy and by transmission electron microscopy, respectively. Morphological maturity was assessed by comparing measurements of apical perimeter, vesicular profiles, and mitochondrial volume percent. In mature cortical collecting duct (CCD), approximately 30% of total cells were rich in mitochondria and generally dark in matrix staining, i.e., IC. Most IC in outer cortex were microvillated, whereas in inner cortex most IC were microplicated with interspersed microvilli. Only approximately 16% of cells in outer medullary collecting duct (OMCD, outer stripe) were IC, with light cytoplasm and large protruding apical surfaces covered exclusively with microplicae. Neonatal CCDs showed no IC in outer cortex; a single type of principal-like cell was found in outer cortical region. Neonatal IC in both CCDs and OMCDs showed smaller apical perimeters, vesicular volumes, and mitochondrial volume percents than did mature IC. Whereas the number of cortical IC was reduced in the newborn, IC were present in mature numbers in OMCD and appeared relatively more mature than did IC of CCD. Some (approximately 10%) of IC in neonatal CCD did not show an identifiable apical surface pattern. Principal cells from both newborn CCD and OMCD were also morphometrically immature. It is clear that the neonatal collecting duct undergoes postnatal proliferation and differentiation.
Evidence for presence of functional beta-adrenoceptor in rabbit S2 proximal straight tubules
Authors: Kudo K. , Kondo Y. , Abe K. , Igarashi Y. , Tada K. , Yoshinaga K.
Published: December 2017

The effect of isoproterenol on the electrophysiological properties of the S2 proximal segment of the rabbit was examined. Isoproterenol at 10(-8) to 10(-4) M depolarized the basolateral membrane voltage (Vb) in a dose-dependent manner. Propranolol attenuated the isoproterenol-induced depolarization. These possible mechanisms of cell depolarization were explored. The role of luminal Na(+)-organic solute cotransport was negligible, since the removal of organic solute did not change the depolarization. Basolateral Na(+)-(HCO3-) cotransport was supported by the finding that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited isoproterenol-induced depolarization. Basolateral K+ conductance was suggested by the finding that the application of Ba2+ blocked the isoproterenol-induced depolarization. Na(+)-K(+)-adenosine-triphosphatase (ATPase) was questionable. Although ouabain blocked isoproterenol-induced depolarization, the removal of Na+ did not inhibit the depolarization. Further experiment revealed that dibutylyl-adenosine 3',5'-cyclic monophosphate (cAMP), 8-bromo cAMP, and forskolin did not mimic the response of isoproterenol. These results demonstrate: 1) there is a functional beta-adrenoceptor that depolarizes Vb; 2) isoproterenol-induced depolarization is due to an inhibition of basolateral K+ channel or the activation of basolateral Na(+)-(HCO3-)n cotransport; 3) isoproteronol-induced depolarization is independent of cAMP in the rabbit proximal tubule.
Renal brush-border membrane Na(+)-sulfate cotransport: stimulation by thyroid hormone
Authors: Tenenhouse H. S. , Lee J. , Harvey N.
Published: December 2017

The present study was undertaken to examine the interaction of phosphonoformic acid (PFA) with the Na(+)-sulfate cotransporter and the effect of thyroid hormone (triiodothyronine; T3) on Na(+)-dependent sulfate transport and Na(+)-dependent PFA binding in mouse renal brush-border membrane vesicles. PFA inhibits Na(+)-dependent sulfate transport in a competitive manner [apparent inhibitory constant (Ki) = 4.3 +/- 1.1 mM]. T3 administered in pharmacological doses significantly stimulates Na(+)-dependent sulfate transport in renal brush-border membranes compared with vehicle-treated controls. Although T3 has no effect on Na(+)-dependent glucose transport, T3 also stimulates Na(+)-dependent phosphate transport. Kinetic studies demonstrate that T3 increases the apparent maximal velocity (Vmax) for Na(+)-sulfate cotransport without changing the apparent Michaelis constant (Km). T3 does not significantly affect either Na(+)-dependent PFA binding or the phosphate- and sulfate-displaceable components of Na(+)-dependent PFA binding. Finally, Na(+)-dependent brush-border membrane sulfate transport is unchanged in phosphate-deprived mice that exhibit increased Na(+)-phosphate cotransport and in X-linked Hyp mice that exhibit impaired Na(+)-phosphate cotransport. The present results demonstrate that 1) PFA is a competitive inhibitor of Na(+)-sulfate cotransport, 2) T3 stimulates Na(+)-dependent sulfate, as well as Na(+)-dependent phosphate transport, but has no effect on PFA binding, and 3) phosphate deprivation and the X-linked Hyp mutation do not influence Na(+)-sulfate cotransport.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of thiazides on colonic NaCl absorption: role of carbonic anhydrase
Authors: Goldfarb D. S. , Chan A. J. , Hernandez D. , Charney A. N.
Published: December 2017

The mechanisms by which the benzothiadiazide class of diuretics inhibit electroneutral NaCl absorption are not fully understood. We studied the mechanisms of thiazide action in perfused loops of distal colon in anesthetized male Sprague-Dawley rats. Hydroflumethiazide (1 mM) reversibly inhibited greater than 40% of Na, Cl, and water absorption. Prior exposure of the colon to the carbonic anhydrase inhibitor methazolamide (0.1 mM) prevented the effects of hydroflumethiazide and metolazone, a thiazide-like drug, on colonic absorption. In Ussing flux chambers, addition of hydroflumethiazide to both the mucosal and serosal bathing solutions (but not to the mucosal solution alone) caused marked decreases in Na and Cl absorption. Such inhibition only occurred at concentrations of hydroflumethiazide (0.1 and 1.0 mM) that inhibited greater than 90% of carbonic anhydrase activity in homogenized colonic mucosa. We conclude that an important mechanism by which thiazides inhibit NaCl absorption in the rat distal colon is by inhibition of mucosal carbonic anhydrase. In tissues containing this enzyme, this mechanism of thiazide effect on ion flux must be considered.
Glomerular stereospecific synthesis and hemodynamic actions of 8,9-epoxyeicosatrienoic acid in rat kidney
Authors: Katoh T. , Takahashi K. , Capdevila J. , Karara A. , Falck J. R. , Jacobson H. R. , Badr K. F.
Published: December 2017

Renal glomerular and cortical metabolism of endogenous arachidonic acid by cytochrome P-450 epoxygenase yields 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EET). Using gas chromatography-mass spectrometry, we measured the synthesis of 8,9-EET from an endogenous pool of arachidonic acid in normal rat kidney. The (8S,9R) isomer was favored over the (8R,9S) isomer in a ratio (%) of 59 to 41 in isolated glomeruli and 68 to 32 in cortex tissue. (8S,9R)- but not (8R,9S)-EET elicited dose-dependent vasoconstriction on intrarenal administration in the euvolemic Munich-Wistar rat. Micropuncture measurements of glomerular dynamics revealed that (8S,9R)-EET increased afferent arteriolar resistance (RA) leading to reductions in single-nephron plasma flow rate (QA), net transcapillary hydraulic pressure difference (delta P), and consequently single-nephron glomerular filtration rate (SNGFR). There was no significant change in the value of the glomerular capillary ultrafiltration coefficient (Kf). In the presence of a cyclooxygenase inhibitor, indomethacin, the effects of 8,9-EET were reversed. RA fell leading to increases in QA and delta P, with resultant augmentation of SNGFR. Under these conditions, a modest reduction if Kf was noted. Thus (8S,9R)-EET is a stereoselective renal vasoconstrictor, preferentially generated over its optical isomer, (8R,9S)-EET, suggesting that it is biologically relevant and implying specific structural requirements for EET receptor activation. The principal mechanism of action of 8,9-EET is preglomerular vasoconstriction. The vasoconstrictor effect of 8,9-EET is CO dependent.
Prostaglandin E2 production in rat IMCD cells. II. Possible role for locally formed dopamine
Authors: Huo T. L. , Grenader A. , Blandina P. , Healy D. P.
Published: December 2017

Dopamine has been proposed as an intrarenal natriuretic hormone. We reported previously that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to stimulation of prostaglandin E2 (PGE2) production. In this study we examined whether locally formed dopamine could stimulate PGE2 production in cultured IMCD cells. L-Dopa stimulated PGE2 production dose dependently in cultured IMCD cells (concentration for half-maximal stimulation, 54.3 microM; maximal stimulation, 212.7% of basal), with the maximal stimulation similar to that obtained with dopamine. This effect was blocked by aromatic L-amino acid decarboxylase (AADC) inhibitors and DA2-receptor antagonists. IMCD cells also had measurable AADC activity and produced dopamine from exogenously added L-dopa. AADC inhibitors and DA2 antagonists also lowered basal PGE2 levels, suggesting that dopamine was being formed constitutively in culture. These results suggest that cultured IMCD cells have the capacity to take up and convert L-dopa to dopamine, which then stimulates PGE2 production via DA2K receptors. These results further suggest that locally formed dopamine could act as an autocrine/paracrine hormone in the kidney inner medulla to regulate PGE2 synthesis and water and electrolyte excretion.
Multiple carriers for dipeptide transport: carrier-mediated transport of glycyl-L-proline in renal BBMV
Authors: Skopicki H. A. , Fisher K. , Zikos D. , Bloch R. , Flouret G. , Peterson D. R.
Published: December 2017

To determine whether multiple carriers are responsible for luminal uptake of glycyl-L-proline (Gly-Pro) in the renal proximal tubule, transport of Gly-[3H]Pro was measured in brush-border membrane vesicles (BBMV). A Line-weaver-Burk analysis of Michaelis-Menten kinetics revealed the presence of two carriers: a lower affinity, higher capacity carrier (Km = 1.3 x 10(-2) M; Vmax = 4.6 x 10(-8) mol.mg-1.min-1) and a higher affinity, lower capacity carrier (Km = 2.7 x 10(-7) M; Vmax = 7.8 x 10(-13) mol.mg-1.min-1). The dipeptides Gly-Sar, beta Ala-His, and pyroGlu-His competitively inhibited the low-affinity carrier. No effect on the Km or Vmax of Gly-Pro transport in this range was seen in the presence of the dipeptides Gly-Gly or cycloHis-Pro. The high-affinity carrier exhibited a different inhibition spectrum. Competitive inhibition of Gly-Pro transport was demonstrated for the dipeptides Gly-Gly and Gly-Sar. However, none of the other peptides tested above altered Gly-Pro transport in the high-affinity range, including pyroGlu-His, which is transported by a high-affinity carrier. At both low (4 x 10(-8) M) and high (4 x 10(-3) M) concentrations, uptake of Gly-Pro was stimulated in the presence of an inwardly directed H+ gradient but was unaffected by the presence of an inward Na+ gradient. In addition, measurements in the presence of valinomycin and an outwardly directed K+ gradient strongly suggest that H(+)-stimulated uptake at both concentrations is electrogenic.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelin 1 increases cell calcium in mouse collecting tubule cells
Authors: Naruse M. , Uchida S. , Ogata E. , Kurokawa K.
Published: December 2017

Effects of endothelin 1 (ET-1) on intracellular free calcium concentration ([Ca2+]i) were examined in superfused single-nephron segments dissected from mouse kidney. ET-1, 10(-9) to 10(-6) M, caused a biphasic increase in [Ca2+]i consisting of an initial rapid rise followed by a second more sustained elevation in [Ca2+]i in cortical collecting tubules (CCT), outer medullary CT (OMCT), and inner medullary CT (IMCT). The magnitude of the response was dose dependent and was greater in CCT than in OMCT or IMCT. Additional studies using CCT revealed that Ca2+ removal from the superfusate resulted in attenuation of the second phase of [Ca2+]i with approximately 50% reduction in the height of the initial [Ca2+]i peak in response to 10(-6) M ET-1. Ca2+ channel blocker nicardipine had little effect on ET-1-evoked changes in [Ca2+]i. BAY K 8644 and high superfusate K+ also did not affect [Ca2+]i. Addition of ET-1 and arginine vasopressin (AVP), 10(-6) M each, showed the presence of homologous desensitization but the absence of heterologous desensitization in [Ca2+]i changes. There was no additive effect of ET-1 and AVP on [Ca2+]i when they were added together. These data show that ET-1 evokes a biphasic increase in [Ca2+]i of collecting tubules and suggest that the initial peak of the ET-1-evoked rise in [Ca2+]i is largely due to cell Ca2+ release and that the second sustained rise in [Ca2+]i is largely due to increased Ca2+ influx. Data also suggest that ET-1 and AVP may act in the collecting tubules through different receptors.
Characterization of integrins in cultured human renal cortical tubule epithelial cells
Authors: Simon E. E. , Liu C. H. , Das M. , Nigam S. , Broekelmann T. J. , McDonald J. A.
Published: December 2017

We have characterized the integrins present on cultured tubule epithelial cells from human renal cortexes, enriched for proximal cells, using fluorescence microscopy, immunoprecipitation, and cell adhesion assays. By immunofluorescence, the alpha 3-integrin subunit stained most intensely and was present on all cells predominantly at cell-cell contacts. The alpha 6-subunit was present on all cells in a pattern consistent with extracellular matrix contacts. The alpha 5-subunit was present on most cells in a cell-matrix contact pattern; alpha V-subunit was weakly positive and occasionally seen in cell-matrix contacts. The alpha 2-subunit was present on clusters of distal tubule cells, predominantly at cell-cell contacts. Immunoprecipitation revealed the predominant integrin to be alpha 3 beta 1 with some alpha 2 beta 1, presumably contributed by distal cells. The alpha 5 beta 1-, alpha 6 beta 1-, alpha 6 beta 4-, and alpha V beta 3-integrins, as well as trace amounts of alpha 1 beta 1-integrins, were also present. The alpha 4 beta 1-integrin was not detected. Initial attachment to fibronectin was mediated by alpha V beta 3- and alpha 5 beta 1-integrins; initial attachment to laminin was mediated by the alpha 6 beta 1- and alpha 3 beta 1- integrins and, in some preparations, by an unidentified integrin; and initial attachment to collagen type IV was mediated by alpha V beta 3-integrin and an unidentified beta 1-integrin. After extensively immunodepleting membrane extracts with anti-alpha 1, -alpha 2, -alpha 3, -alpha 4, -alpha 5, -alpha 6, and -alpha V antibodies, an anti-beta 1 antibody still precipitated an integrin. Its electrophoretic mobility differs from the laminin-binding alpha 7 beta 1-integrin. Thus we have identified many of the integrins on cortical tubule cells and their role in mediating initial attachment to extracellular matrix. However, the cell adhesion assays and immunoprecipitations suggest the presence of an unidentified beta 1-integrin that may mediate renal tubule cell attachment to laminin and collagen.
Regulation of nitrobenzylthioninosine-sensitive adenosine uptake by cultured kidney cells
Authors: Sayos J. , Blanco J. , Ciruela F. , Canela E. I. , Mallol J. , Lluis C. , Franco R.
Published: December 2017

The effect of nitrobenzylthioinosine (NBTI) on [3H]adenosine uptake and the characterization of the [3H]NBTI binding in cell (primary cultures and LLC-PK1 cell line) plasma membrane and brush-border membrane (BBM) vesicles from pig renal cortices and LLC-PK1 cells was analyzed. [3H]adenosine uptake was strongly inhibited by NBTI in nonconfluent cells, whereas it was totally insensitive to the reagent in BBM. The concentration dependence of [3H]adenosine uptake in BBM was linear, suggesting simple diffusion. In both cell membranes and BBM high-affinity [3H]NBTI binding was observed. [3H]NBTI binding as well as NBTI-sensitive [3H]adenosine uptake was strongly reduced when cells grew to confluence. Both reduction effects were reproduced by treatment of nonconfluent cells with chlorophenyl adenosine 3',5'-cyclic monophosphate (cAMP), which indicates that the transporter is regulated by a cAMP-dependent protein kinase. To confirm this hypothesis, the binding of [3H]NBTI was analyzed in pig kidney BBM obtained in the presence of orthovanadate and alkaline phosphatase. With respect to control membranes, BBM obtained in the presence of orthovanadate showed a lower maximum number of binding sites (Bmax), whereas those obtained in the presence of alkaline phosphatase showed a slight increase in Bmax for [3H]NBTI binding. Taken together, these results suggest that the reduction in both [3H]NBTI-binding capacity and NBTI-sensitive [3H]adenosine uptake takes place by a mechanism that involves phosphorylation of the transporter molecule or of a protein that interacts with it.
Fulminant metanephric apoptosis and abnormal kidney development in bcl-2-deficient mice
Authors: Sorenson C. M. , Rogers S. A. , Korsmeyer S. J. , Hammerman M. R.
Published: December 2017

Apoptosis of the developing metanephric kidney plays an important role in renal organogenesis. The bcl-2 is an oncogene that inhibits apoptotic cell death in a variety of settings. The bcl-2 (-/-) mice complete embryonic development but, in contrast to bcl-2 (+/-) and bcl-2 (+/+) littermates, manifest growth retardation, hypopigmentation of hair, lymphoid apoptosis, abnormal kidney morphology, and renal failure postnatally. To provide insight into the mechanism for the latter abnormalities, we examined metanephric kidneys from bcl-2 (-/-), bcl-2 (+/-), and bcl-2 (+/+) mice, as well as embryonic day 12 (E12) mouse embryos, and compared growth and development of metanephroi in vitro. Kidneys from bcl-2 (+/-) mice developed normally. In contrast, development of kidneys from bcl-2 (-/-) mice was abnormal as reflected by a marked reduction of renal size in newborns compared with kidneys of bcl-2 (+/-) littermates. In addition, kidneys from bcl-2 (-/-) mice contained far fewer nephrons and had smaller nephrogenic zones. Although metanephroi obtained from E12 bcl-2 (+/-) and bcl-2 (-/-) mouse embryos were comparable in size, apoptosis of cells within metanephric blastemas of metanephroi from E12 bcl-2 (-/-) embryos was strikingly enhanced compared with that in blastemas of metanephroi from bcl-2 (+/-) embryos. During 3 days in culture, growth and development of metanephroi from bcl-2 (-/-) embryos were visibly reduced compared with those from bcl-2 (+/-) embryos.(ABSTRACT TRUNCATED AT 250 WORDS)
Stimulation of chloride transport by cAMP in rat proximal tubules
Authors: Wang T. , Segal A. S. , Giebisch G. , Aronson P. S.
Published: December 2017

We have previously demonstrated that formate and oxalate stimulate transcellular Cl- absorption (JCl) in the rat proximal tubule by a mechanism involving DIDS-sensitive anion exchange across the luminal membrane and diphenylamine-2-carboxylate (DPC)-sensitive Cl- channels in the basolateral membrane. Recent evidence indicates cAMP activation of Cl- channels in apical and basolateral membranes of proximal tubule cells. We therefore tested the effect of cAMP on Cl- and fluid transport in rat proximal tubule studied by luminal and capillary microperfusion in situ. The luminal perfusate contained 5 mM HCO3- and 145 mM Cl-, and the capillary perfusate contained 25 mM HCO3- and 110 mM Cl-, simulating conditions in the late proximal tubule. Addition of 0.5 mM dibutyryl cAMP markedly stimulated fluid absorption (Jv) and JCl. Similar effects resulted from addition of forskolin (10 microM) to stimulate cAMP production. The increments in Jv and JCl due to dibutyryl cAMP were abolished when the Cl- channel blocker DPC (200 microM) was added to the capillary perfusate but not when it was added to the lumen. The increments in Jv and JCl due to dibutyryl cAMP were unaffected by luminal DIDS (100 microM), which abolishes the increments in Jv and JCl induced by addition of oxalate. In contrast, the increments in Jv and JCl due to dibutyryl cAMP were abolished by luminal 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; 10 microM), another Cl- channel blocker. Luminal NPPB had no effect on baseline Jv and JCl nor on the increments in Jv and JCl induced by addition of oxalate.(ABSTRACT TRUNCATED AT 250 WORDS)
Interaction between cGMP-dependent dilators and autoregulation in rat preglomerular vasculature
Authors: Bouriquet N. , Casellas D.
Published: December 2017

The influence of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent dilators on autoregulatory responses (AR) of arcuate arteries (ArcA) and afferent arterioles at early sites and at juxtaglomerular sites (JAA) was assessed by videomicroscopy using in vitro blood-perfused juxtamedullary nephron preparations. AR were quantified as fractional changes in luminal diameter induced by doubling blood perfusion pressure (60-120 mmHg). Baseline AR ranged from 17 +/- 2% to 21 +/- 2% in ArcA and from 24 +/- 2% to 34 +/- 4% in JAA. Direct perivascular applications of increasing concentrations of 8-bromo-cGMP (8-BrcGMP, 10 microM to 1 mM), of the NO donors sodium nitroprusside (10 microM to 1 mM) and 3-morpholino-sydnonimine chlorhydrate (SIN1; 10 microM to 1 mM), and of rat atrial natriuretic factor (ANF, 0.1 nM to 10 nM) dose- and pressure-dependently dilated all vessels at 60 mmHg. Concomitantly, AR values were dose-dependently reduced or reversed to pressure-induced dilations. During application of 8-BrcGMP and NO donors, the segmental gradient of sensitivity of AR was ArcA > JAA; the opposite gradient was found with ANF (i.e., JAA > ArcA). The present results demonstrate that compounds known to utilize the cGMP-signaling pathway act as modulators of AR along the juxtamedullary preglomerular vasculature.