Specific effect of maleate on an apical membrane glycoprotein (gp330) in proximal tubule of rat kidneys

1996 ◽  
Vol 271 (4) ◽  
pp. F908-F916 ◽  
Author(s):  
M. Bergeron ◽  
P. Mayers ◽  
D. Brown
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Fanconi Syndrome ◽  
Apical Membrane ◽  
Helix Pomatia ◽  
Specific Effect ◽  
Staining Intensity ◽  
Membrane Glycoprotein ◽  
Coated Pits ◽  
Immunofluorescence Labeling

Maleate treatment of rats induces transport defects similar to those seen in the Fanconi syndrome (glycosuria, aminoaciduria, phosphaturia, proteinuria, etc.) and causes an accumulation of apical vesicles in proximal tubule epithelial cells. Because the apical membrane glycoprotein, gp330, is a receptor associated with the apical endocytotic and recycling apparatus in these cells, we examined the effect of maleate on the distribution of this protein and other brush border markers. Rats received sodium maleate (400 mg/kg ip) and were killed at various times between 45 min and 3 h; kidneys were perfusion fixed with paraformaldehyde-lysine-periodate before processing for immunofluorescence and immunoelectron microscopy. In control rats, staining with a polyclonal or monoclonal gp330 antibody showed a uniform distribution on the brush border and in coated pits of all proximal tubule cells. In the S3 segments, the immunofluorescence labeling of the microvilli was generally uniform but at times showed spike labeling, suggesting that gp330 sheds easily from the apical membrane. After maleate treatment, the staining intensity of the brush border was decreased in all proximal tubule segments, and cytoplasmic streaks as well as an intense vacuolar staining were seen. In the S3 segment, a remarkable mosaic pattern of staining was observed, with the brush border of some cells being completely negative, while adjacent cells showed an apparently normal staining pattern. These results were confirmed at the electron microscope level, using the protein A-gold technique. Maleate had no effect on the distribution or staining intensity of four other brush border markers, dipeptidyl peptidase IV, and various lectins (Helix pomatia lectin, peanut lectin, elderberry bark lectin). The urinary excretion of gp330 occurs in normal rats and was already increased as early as 1 h after maleate injection and remained at a twofold increment between 6 and 24 h. These data suggest that the generalized membrane transport derangement seen in this experimental Fanconi syndrome could occur via a specific effect on gp330, which seems to block endocytosis and the recycling apparatus at the late endosome level and inhibits the formation of new dense apical tubules.

Assembly of distinctive coated pit and microvillar microdomains in the renal brush border

1992 ◽  
Vol 262 (1) ◽  
pp. F55-F67 ◽  
Author(s):  
D. Biemesderfer ◽  
G. Dekan ◽  
P. S. Aronson ◽  
M. G. Farquhar
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Apical Membrane ◽  
Stage Iv ◽  
Cytoskeletal Proteins ◽  
Neonatal Rat ◽  
Stage Iii ◽  
Coated Pits ◽  
Marker Proteins ◽  
Renal Brush Border

The plasma membrane of the kidney brush border is composed of two compositionally distinct microdomains, microvilli and clathrin-coated pits. To study their assembly we have immunolocalized brush border marker proteins in the developing proximal tubule epithelium of the neonatal rat and compared their time and site of appearance with those of basolateral markers, Na-K-ATPase and fodrin. The proteins studied were dipeptidyl peptidase IV (DPPIV) (microvilli), actin and villin (microvillar cytoskeletal proteins), glycoprotein 330 (gp330) (coated pits), and clathrin (coated pit cytoskeleton). Although apical microvilli and coated pits were first seen in the stage III nephron, many brush border markers including DPPIV, actin, and clathrin appeared earlier in the development and initially were not polarized. Only during stage III did they become concentrated at the apical membrane. Villin first appeared in the stage III proximal tubule where it was located diffusely in the cytoplasm and in lysosomes as well as along the apical membrane. It did not completely colocalize with actin until stage IV. Gp330 first appeared during stage III and from the beginning was restricted to the apical clathrin-coated membrane domains and endosomes. The results demonstrate that 1) the expression of renal brush border proteins during development is asynchronous, and 2) unlike the basolateral plasmalemmal domain, which is established early in nephrogenesis, brush border assembly occurs later, approximately coinciding with the onset of glomerular filtration.

scholarly journals GAIP, GIPC and Gαi3 are Concentrated in Endocytic Compartments of Proximal Tubule Cells: Putative Role in Regulating Megalin’s Function

2002 ◽  
Vol 13 (4) ◽  
pp. 918-927 ◽  
Author(s):  
Xiaojing Lou ◽  
Tammie McQuistan ◽  
Robert A. Orlando ◽  
Marilyn Gist Farquhar
Keyword(s):  
Proximal Tubule ◽  
G Protein ◽  
Brush Border ◽  
Border Region ◽  
Cell Fractionation ◽  
Coated Pits ◽  
Proximal Tubule Cells ◽  
G Protein Alpha Subunit ◽  
Endocytic Compartments ◽  
Protein Alpha Subunit

ABSTRACT. Megalin is the most abundant endocytic receptor in the proximal tubule epithelium (PTE), where it is concentrated in clathrin-coated pits (CCPs) and vesicles in the brush border region. The heterotrimeric G protein alpha subunit, Gαi3, has also been localized to the brush border region of PTE. By immunofluorescence GIPC and GAIP, components of G protein-mediated signaling pathways, are also concentrated in the brush border region of PTE and are present in megalin-expressing cell lines. By cell fractionation, these signaling molecules cosediment with megalin in brush border and microvillar fractions. GAIP is found by immunoelectron microscopy in CCPs, and GIPC is found in CCPs and apical tubules of endocytic compartments in the renal brush border. In precipitation assays, GST-GIPC specifically binds megalin. The concentration of Gαi3, GIPC, and GAIP with megalin in endocytic compartments of the proximal tubule, where extensive endocytosis occurs, and the interaction between GIPC and the cytoplasmic tail of megalin suggest a model whereby G protein-mediated signaling may regulate megalin’s endocytic function and/or trafficking.

Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells

2001 ◽  
Vol 280 (5) ◽  
pp. F886-F894 ◽  
Author(s):  
Sharon L. Ashworth ◽  
Ruben M. Sandoval ◽  
Melanie Hosford ◽  
James R. Bamburg ◽  
Bruce A. Molitoris
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Apical Membrane ◽  
Critical Role ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Actin Binding ◽  
Proximal Tubule Cell ◽  
Apical Domain

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544–F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.

Colchicine-induced redistribution of an apical membrane glycoprotein (gp330) in proximal tubules

1989 ◽  
Vol 257 (2) ◽  
pp. C397-C407 ◽  
Author(s):  
E. J. Gutmann ◽  
J. L. Niles ◽  
R. T. McCluskey ◽  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Colchicine Treatment ◽  
Basement Membranes ◽  
Apical Plasma Membrane ◽  
Membrane Glycoprotein ◽  
Proximal Tubule Cells ◽  
Basolateral Plasma Membrane

Factors governing the selective, polarized insertion of membrane proteins are poorly understood, but some studies have suggested that microtubules are involved in the generation and maintenance of cell polarity. We have examined by immunocytochemistry the effect of the microtubule-disrupting agent, colchicine, on the cellular distribution of an endogenous glycoprotein, gp330, which is normally inserted only into the apical plasma membrane of proximal tubule epithelial cells. In control rats, gp330 was localized in the brush border and in apical invaginations and vesicles. Six hours after injection of colchicine, however, vesicles containing gp330 were dispersed throughout the entire cytoplasm of the cell. Many vesicles were packed into basolateral infoldings, close to the plasma membrane, but there was no significant insertion of gp330 into the basolateral membrane. When rabbit anti-gp330 antiserum was injected intravenously into colchicine-treated rats, immune complexes appeared in the glomerular basement membrane but could not be detected in peritubular basement membranes. This supports the conclusion that colchicine treatment does not result in the insertion of gp330 into the basolateral plasma membrane of proximal tubule cells. Our results indicate that although microtubules are involved in the accumulation of gp330-containing vesicles at the apical pole of the cell, other factors must be required for fusion with the plasma membrane to occur.

scholarly journals Redistribution of villin to proximal tubule basolateral membranes after ischemia and reperfusion

1997 ◽  
Vol 273 (6) ◽  
pp. F1003-F1012 ◽  
Author(s):  
Dennis Brown ◽  
Richard Lee ◽  
Joseph V. Bonventre
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Actin Binding ◽  
Ischemia And Reperfusion ◽  
Proximal Tubule Cells ◽  
Basolateral Plasma Membrane ◽  
Tubule Cells ◽  
Renal Tubular

After ischemia and reperfusion, severe alterations in the cytoskeletal organization of renal tubular epithelial cells have been reported. These effects, accompanied by a modification in the polarized distribution of some membrane transport proteins, are especially evident in the proximal tubule. In normal proximal tubule cells, actin is concentrated in apical brush border microvilli, along with the actin-binding protein villin. Because villin plays an important role in actin bundling and in microvillar assembly but can also act as an actin-fragmenting protein at higher calcium concentrations, we examined the effects of ischemic injury and reperfusion on the distribution of villin and actin in proximal tubule cells of rat kidney. Using specific antibodies against villin and actin, we show that these proteins redistribute in parallel from the apical to the basolateral plasma membrane within 1 h of reperfusion after ischemia. Ischemia alone had no effect on the staining pattern. Repolarization of villin to the apical membrane begins within hours after reperfusion with enhanced apical localization over time during the period of regeneration. This apical repolarization of villin is accompanied by the migration of actin back to the apical membrane. These results show not only that villin may be involved in the initial disruption of the actin cytoskeleton during reperfusion injury but also that its migration back to the apical domain of these cells accompanies the reestablishment of a normal actin distribution in the brush border.

scholarly journals Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

2014 ◽  
Vol 306 (9) ◽  
pp. F981-F995 ◽  
Author(s):  
Mohammad M. Al-bataineh ◽  
Fan Gong ◽  
Allison L. Marciszyn ◽  
Michael M. Myerburg ◽  
Núria M. Pastor-Soler
Keyword(s):  
Protein Kinase ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Phosphodiesterase Inhibitor ◽  
Cell Monolayers ◽  
Apical Pole ◽  
Kidney Slices ◽  
Immunofluorescence Labeling ◽  
Amp Activated Protein Kinase

The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress.

scholarly journals Proximal tubule transferrin uptake is modulated by cellular iron and mediated by apical membrane megalin–cubilin complex and transferrin receptor 1

2019 ◽  
Vol 294 (17) ◽  
pp. 7025-7036 ◽  
Author(s):  
Craig P. Smith ◽  
Wing-Kee Lee ◽  
Matthew Haley ◽  
Søren B. Poulsen ◽  
Frank Thévenod ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Transferrin Receptor ◽  
Apical Membrane ◽  
Cellular Iron ◽  
Transferrin Receptor 1 ◽  
Transferrin Uptake

Luminal flow rate regulates proximal tubule H-HCO3 transporters

1992 ◽  
Vol 262 (1) ◽  
pp. F47-F54 ◽  
Author(s):  
P. A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Flow Rate ◽  
Initial Rate ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Rate Dependence ◽  
Unstirred Layer ◽  
Disulfonic Acid ◽  
Luminal Flow

In vivo microperfusion was used to examine the mechanism of luminal flow rate dependence of proximal tubule acidification. Luminal flow rate was acutely changed between 5 and 40 nl/min, while luminal and peritubular capillary composition were held constant. With inhibition of basolateral membrane base transport by peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), cell pH (pHi) provides a sensitive index of apical membrane H secretory activity. At a luminal perfusate [HCO3] of 25 mM, progressive increases in luminal flow rate (5----15----25----40 nl/min) caused progressive increases in pHi. This effect was of a smaller magnitude with a luminal perfusate [HCO3] of 60 mM and was further decreased at a luminal perfusate [HCO3] of 100 mM. This pattern of diminished flow rate dependence at higher luminal [HCO3] is consistent with the presence of a luminal unstirred layer, whose composition can be modified by luminal flow rate. The activity of the apical membrane Na-H antiporter, assayed as the initial rate of pHi recovery from an acid load in the presence of peritubular DIDS, was faster at 40 compared with 5 nl/min. Basolateral membrane Na-3HCO3 symporter activity, assayed as the initial rate of pHi recovery from an alkali load in the absence of luminal and peritubular chloride, was faster at 40 compared with 5 nl/min. This effect was eliminated by luminal amiloride, suggesting an indirect effect of flow mediated by changes in pHi secondary to flow rate-dependent changes in apical membrane Na-H antiporter activity. In summary, increases in luminal flow rate directly increase apical membrane H secretion, possibly by modification of a luminal unstirred layer.(ABSTRACT TRUNCATED AT 250 WORDS)

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