scholarly journals THE POLARITY OF THE PROXIMAL TUBULE CELL IN RAT KIDNEY

1972 ◽  
Vol 54 (2) ◽  
pp. 232-245 ◽  
Author(s):  
Hans-G Heidrich ◽  
Rolf Kinne ◽  
Eva Kinne-Saffran ◽  
Kurt Hannig
Keyword(s):  
Proximal Tubule ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Rat Kidney ◽  
High Specific Activity ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Surface Charges ◽  
Tubule Cell ◽  
Rat Kidney Cortex

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.

Epidermal growth factor receptor regulation in rat kidney: two models of renal growth

1989 ◽  
Vol 257 (6) ◽  
pp. F1059-F1064 ◽  
Author(s):  
M. T. Behrens ◽  
A. L. Corbin ◽  
M. K. Hise
Keyword(s):  
Folic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Rat Kidney Cortex ◽  
Epidermal Growth

The binding of 125I-labeled epidermal growth factor (EGF) to plasma membranes prepared from rat kidney cortex was studied following unilateral nephrectomy, a model of proximal tubule cell hypertrophy, and following the administration of folic acid, a model of proximal tubule cell hyperplasia. Binding of 125I-EGF was a linear function of basolateral membrane protein content and time of incubation. Specific binding to luminal brush-border membranes was not evident in these studies. Neither insulin nor insulin-like growth factor I could displace EGF binding, indicating that binding was specific. Scatchard analysis revealed a single binding site. The KD in sham-operated animals 48 h after surgery was 11.2 +/- 1.4 nM, whereas Bmax averaged 95.2 +/- 4.1 fmol/mg protein (n = 3). Similar values were obtained in nephrectomized animals. The Bmax of folic acid-untreated animals averaged 212.5 +/- 6.9 fmol/mg 48 h after administration, whereas that of vehicle-injected controls averaged 85.4 +/- 9.2 (n = 3, P less than 0.001). Differences in binding were not related to changes in affinity, ligand degradation by the preparations, or receptor binding of endogenous EGF. These data indicate that regeneration following folic acid administration is associated with an upregulation of proximal nephron EGF receptors that may play an important role in the mitogenic response.

scholarly journals Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell.

1984 ◽  
Vol 98 (5) ◽  
pp. 1630-1636 ◽  
Author(s):  
J S Rodman ◽  
D Kerjaschki ◽  
E Merisko ◽  
M G Farquhar
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Specific Binding ◽  
Rat Kidney ◽  
Light Chains ◽  
Kidney Cortex ◽  
Immunoperoxidase Staining ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Kidney Proximal Tubule

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule intermicrovillar apical invaginations is composed of clathrin heavy and light chains, and (b) that the differential distribution of proteins in these two brush border microdomains is maintained in appropriately prepared brush border fractions.

Identification of an apical \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger in rat kidney proximal tubule

2003 ◽  
Vol 285 (3) ◽  
pp. C608-C617 ◽  
Author(s):  
Snezana Petrovic ◽  
Liyun Ma ◽  
Zhaohui Wang ◽  
Manoocher Soleimani
Keyword(s):  
Proximal Tubule ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Immunocytochemical Staining ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Kidney Proximal Tubule ◽  
Anion Transporter

SLC26A6 (or putative anion transporter 1, PAT1) is located on the apical membrane of mouse kidney proximal tubule and mediates [Formula: see text] exchange in in vitro expression systems. We hypothesized that PAT1 along with a [Formula: see text] exchange is present in apical membranes of rat kidney proximal tubules. Northern hybridizations indicated the exclusive expression of SLC26A6 (PAT1 or CFEX) in rat kidney cortex, and immunocytochemical staining localized SLC26A6 on the apical membrane of proximal tubules, with complete prevention of the labeling with the preadsorbed serum. To examine the functional presence of apical [Formula: see text] exchanger, proximal tubules were isolated, microperfused, loaded with the pH-sensitive dye BCPCF-AM, and examined by digital ratiometric imaging. The pH of the perfusate and bath was kept at 7.4. Buffering capacity was measured, and transport rates were calculated as equivalent base flux. The results showed that in the presence of basolateral DIDS (to inhibit [Formula: see text] cotransporter 1) and apical EIPA (to inhibit Na+/H+ exchanger 3), the magnitude of cell acidification in response to addition of luminal Cl– was ∼5.0-fold higher in the presence than in the absence of [Formula: see text]. The Cl–-dependent base transport was inhibited by ∼61% in the presence of 0.5 mM luminal DIDS. The presence of physiological concentrations of oxalate in the lumen (200 μM) did not affect the [Formula: see text] exchange activity. These results are consistent with the presence of SLC26A6 (PAT1) and [Formula: see text] exchanger activity in the apical membrane of rat kidney proximal tubule. We propose that SLC26A6 is likely responsible for the apical [Formula: see text] (and Cl–/OH–) exchanger activities in kidney proximal tubule.

scholarly journals Immunoelectron Microscopic Localization of the Electrogenic Na/HCO3 Cotransporter in Rat and Ambystoma Kidney

2000 ◽  
Vol 11 (12) ◽  
pp. 2179-2189
Author(s):  
ARVID B. MAUNSBACH ◽  
HENRIK VORUM ◽  
TAE-HWAN KWON ◽  
SØREN NIELSEN ◽  
BRIAN SIMONSEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Rat Kidney Cortex ◽  
C Terminus ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Abstract. Immunofluorescence analysis has revealed that electrogenic Na+/HCO3- (NBC1) is expressed in the proximal tubule of rat kidney and in the proximal and distal tubules of the salamander Ambystoma tigrinum kidney. The present study was undertaken to define the detailed subcellular localization of the NBC1 in rat and Ambystoma kidney using high-resolution immunoelectron microscopy. For this purpose, two rabbit polyclonal antibodies raised against amino acids 928 to 1035 and amino acids 1021 to 1035 of the C-terminus of rat kidney (rkNBC1) were developed. The affinity-purified antibodies revealed a strong band of approximately 140 kD in immunoblots of membranes from rat kidney cortex but no signal in membranes isolated from outer and inner medulla. Deglycosylation reduced the apparent molecular weight to approximately 120 kD, corresponding to the predicted molecular weight. A similar but weaker band was also present in membranes isolated from the lateral part of Ambystoma kidney. In rat kidney, immunohistochemistry confirmed the presence of rkNBC1 in convoluted segments of the proximal tubules. In ultrathin cryosections or Lowicryl HM20 sections from rat kidney cortex, distinct immunogold labeling was associated with the basolateral plasma membrane of segments S1 and S2 of proximal tubules, whereas in S3 no labeling was observed. The labeling density was similar at the basal and lateral plasma membrane and was specifically associated with the inner surface of the membrane consistent with the internal position of the C-terminus of the transporter. In contrast, rkNBC1 was absent from the apical plasma membrane and not observed in intracellular vesicles, including those closely associated with basolateral plasma membrane. In Ambystoma kidney, a weak labeling was present in the basolateral membrane of the proximal tubule and stronger labeling was observed in the late distal segment. The results demonstrate that rkNBC1 is expressed only in segment S1 and segment S2 of rat proximal tubule as well as Ambystoma proximal and late distal tubule and that rkNBC1 is present in both basal and lateral plasma membranes and absent in intracellular vesicles of the apical plasma membrane.

Tyrosine protein phosphorylation in plasma membranes of rat kidney cortex

1994 ◽  
Vol 267 (3) ◽  
pp. F415-F422
Author(s):  
L. Tremblay ◽  
R. Beliveau
Keyword(s):  
Protein Phosphorylation ◽  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Specific Protein ◽  
Ammonium Molybdate ◽  
Kidney Cortex ◽  
Protein Kinase Activity ◽  
Rat Kidney Cortex ◽  
Endogenous Substrates

The endogenous tyrosine protein kinase activity (TPKA) associated with brush-border (BBM) and basolateral (BLM) membranes of rat kidney cortex was studied with an anti-phosphotyrosine monoclonal antibody (PY20). Distinct major phosphotyrosine-containing proteins were associated with BBM (50, 54, and 120 kDa) and BLM (37, 90, 130, and 170 kDa). For both plasma membranes, tyrosine phosphorylation leveled off after 10 min of incubation. Endogenous phosphotyrosine-specific protein phosphatases (PT-Pases) were active in both membranes, since the presence of sodium vanadate or ammonium molybdate, which are inhibitors of PTPases, was essential to detect endogenous phosphorylation. Substrates and/or tyrosine protein kinases (TPKs) seem to be differently distributed in these plasma membranes, since phosphorylation of endogenous substrates in BLM and BBM was differently sensitive to competitive inhibitors of TPKs. Moreover, insulin- and insulin-like growth factor I-stimulated tyrosine phosphorylation of a 90-kDa substrate was only observed in solubilized BLM proteins. However, similar p60v-src-related TPKs appear to be present in the BBM and BLM, since an antibody raised against p60v-src recognized proteins of 52, 58, and 75 kDa by immunoblotting and could immunoprecipitate the TPKs associated with both plasma membranes. These data provide evidence that the endogenous tyrosine protein phosphorylation observed in the BLM is catalyzed by nonreceptor TPKs as well as receptor TPKs, whereas that observed in the BBM is exclusively due to nonreceptor TPKs.

Subcellular distribution of renal tripeptide-releasing exopeptidases active on collagen-like sequences

1987 ◽  
Vol 252 (5) ◽  
pp. F890-F898
Author(s):  
K. J. Andersen ◽  
J. K. McDonald
Keyword(s):  
Subcellular Distribution ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Rat Kidney ◽  
Broad Band ◽  
Distribution Patterns ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Tripeptidyl Peptidase ◽  
N Terminus

The rat kidney cortex was found to contain two N-terminal exopeptidases of the tripeptidyl peptidase (TPP) class. Each required a free N-terminus to catalyze the release of collagen-related (Gly-Pro-X) "triplets." In accordance with their apparent pH optima, activities were routinely determined fluorimetrically at pH 4.0 (TPP 4) and at pH 7.0 (TPP 7) on Gly-Pro-Met-2-naphthylamide. The specific activity in both the homogenate and the classical subfractions was much greater at pH 7 than at pH 4. Subfractionation of the microsomal fraction by equilibrium banding in sucrose did not separate the TPP 4 and TPP 7 activities. The banding density (1.18 g/ml) and the distribution patterns for TPP 7 in the microsomal subfractions, and also in the subfractions of the small lysosomes in the mitochondrial-lysosomal (ML) fraction, demonstrate that TPP 7 is associated with smooth membranes. The TPP 4 and TPP 7 activities were clearly separated during subfractionation of the ML fraction. Rate sedimentation demonstrated that TPP 4 was present in the large, fast-sedimenting lysosomes (protein droplets) and in a heterogeneous broad band of smaller lysosomes. Equilibrium banding of the small lysosomes gave two distinct TPP 4-containing populations at densities 1.20 and 1.235 g/ml. Notably, dipeptidyl peptidase II (DPP II) gave identical banding densities and showed distributions very similar to TPP 4.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Inositol lipid signalling occurs in brush-border membranes during initiation of compensatory renal growth in the rat

1993 ◽  
Vol 295 (2) ◽  
pp. 599-605 ◽  
Author(s):  
H Banfić ◽  
M Vuica ◽  
M Knotek ◽  
S Moslavac ◽  
N Divecha
Keyword(s):  
Brush Border ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Unilateral Nephrectomy ◽  
Kidney Cortex ◽  
Compensatory Renal Growth ◽  
Rat Kidney Cortex ◽  
Contralateral Kidney ◽  
Inositol Lipids ◽  
Stimulation Of

Using highly specific mass assays, concentrations of inositol lipids and 1,2-diacylglycerol (DAG) were determined in plasma membranes isolated from rat kidney cortex. Significantly higher concentrations of inositol lipids were determined in brush-border (BBM) than in basal-lateral (BLM) plasma membranes, although DAG concentrations were similar in both. After unilateral nephrectomy, a decrease in PtdIns(4,5)P2 and PtdIns4P, with a concomitant increase in DAG and translocation of protein kinase C (PKC), were observed in BBM but not in BLM isolated from the remaining kidney. On the other hand, stimulation of renal cortical slices with insulin-like growth factor II (IGF-II) or phenylephrine caused similar effects in BLM but not in BBM. Stimulation of phospholipase C activity with translocation of PKC only to BBM in one kidney was also induced by occlusion of blood flow through the contralateral kidney for 15 min. At 30 min after the occlusion was removed and reflow established, DAG concentration and the amount of PKC in BBM returned to control values. These results suggest that an early signal after unilateral nephrectomy is transmitted to cells through BBM and can be switched on and off by blood occlusion and reflow through the contralateral kidney, while hormonal signals caused by IGF-II and phenylephrine are transmitted to cells through BLM.

scholarly journals Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes

2006 ◽  
Vol 290 (6) ◽  
pp. F1525-F1533 ◽  
Author(s):  
Marouan Abouhamed ◽  
Jakub Gburek ◽  
Wei Liu ◽  
Blazej Torchalski ◽  
Andreas Wilhelm ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Rt Pcr ◽  
Divalent Metal Transporter 1 ◽  
Rat Kidney Cortex ◽  
Divalent Metal Transporter ◽  
Alexa Fluor ◽  
Metal Transporter ◽  
Late Endosomes

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.

In vivo generation of hydrogen peroxide by rat kidney cortex and glomeruli

1989 ◽  
Vol 256 (1) ◽  
pp. F158-F164 ◽  
Author(s):  
B. R. Guidet ◽  
S. V. Shah
Keyword(s):  
Hydrogen Peroxide ◽  
Catalase Activity ◽  
Renal Cortex ◽  
Specific Activity ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Compound I ◽  
Rat Kidney Cortex ◽  
Hydrogen Peroxide Generation

The purpose of this study was to demonstrate in vivo generation of hydrogen peroxide by rat renal cortex and glomeruli. Aminotriazole irreversibly inactivates catalase only in the presence of hydrogen peroxide, and previous studies have shown that aminotriazole-mediated inhibition of catalase is a measure of in vivo changes in the hydrogen peroxide generation. Aminotriazole injected intraperitoneally caused a dose-dependent (0.1-1 g/kg) and a time-dependent (15, 30, 60, 90, 120 min) inhibition of the catalase activity in renal cortex. We confirmed that catalase inactivation by aminotriazole was due to formation of a catalase-hydrogen peroxide intermediate (compound I) because catalase inactivation was prevented by ethanol (2 g/kg), a competitive substrate for compound I. The specific activity of catalase in the glomeruli [0.27 +/- 0.026 k/mg protein (where k is the first-order reaction rate constant), n = 5] was significantly lower than the specific activity in the tubules (1.04 +/- 0.15 k/mg protein, n = 5) obtained from the same rats. The residual catalase activity (RCA) in the glomeruli (0.05 +/- 0.01 k/mg protein) was 19% of control values at 90 min after aminotriazole injection (1 g/kg). Taken together these data provide evidence for in vivo generation of hydrogen peroxide by rat renal cortex and glomeruli under normal conditions. Aminotriazole-mediated inhibition of catalase has been used in previous studies as a measure of in vivo changes in the hydrogen peroxide generation.(ABSTRACT TRUNCATED AT 250 WORDS)

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