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 Vacuolar H+-ATPase apical accumulation in kidney intercalated cells is regulated by PKA and AMP-activated protein kinase

2010 ◽  
Vol 298 (5) ◽  
pp. F1162-F1169 ◽  
Author(s):  
Fan Gong ◽  
Rodrigo Alzamora ◽  
Christy Smolak ◽  
Hui Li ◽  
Sajid Naveed ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Apical Membrane ◽  
Ex Vivo ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Kidney Tissue ◽  
Intercalated Cells ◽  
Clear Cells ◽  
Kidney Slices ◽  
Immunofluorescence Labeling

The vacuolar H+-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N6-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.

scholarly journals AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells

2009 ◽  
Vol 296 (4) ◽  
pp. C672-C681 ◽  
Author(s):  
Kenneth R. Hallows ◽  
Rodrigo Alzamora ◽  
Hui Li ◽  
Fan Gong ◽  
Christy Smolak ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Apical Membrane ◽  
Alkaline Ph ◽  
Clear Cells ◽  
A Subunit ◽  
Luminal Ph ◽  
Immunofluorescence Labeling ◽  
Amp Activated Protein Kinase

Acidic luminal pH and low [HCO3−] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO3−, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N 6-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [32P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.

scholarly journals Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

2010 ◽  
Vol 299 (1) ◽  
pp. F167-F177 ◽  
Author(s):  
Hui Li ◽  
Ramon F. Thali ◽  
Christy Smolak ◽  
Fan Gong ◽  
Rodrigo Alzamora ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
High Energy ◽  
Whole Body ◽  
Creatine Transporter ◽  
Creatine Uptake ◽  
Kidney Epithelial Cells ◽  
Amp Activated Protein Kinase

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na+-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten Vmax parameter. [14C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress.

Effects of endothelin on rat renal proximal tubule Na(+)-Pi cotransport and Na+/H+ exchange

1994 ◽  
Vol 266 (4) ◽  
pp. F658-F666 ◽  
Author(s):  
J. Guntupalli ◽  
T. D. DuBose
Keyword(s):  
Protein Kinase ◽  
Proximal Tubule ◽  
Collecting Duct ◽  
Second Messengers ◽  
Indirect Evidence ◽  
Low Concentrations ◽  
Proximal Tubular ◽  
Dependent Protein ◽  
Medullary Collecting Duct

Endothelin (ET), a powerful vasoconstrictive peptide, is distributed ubiquitously in various organs, including the vascular endothelium and tubules of the kidney. Although localized more abundantly to the glomerulus and inner medullary collecting duct, ET receptors have been identified in the proximal tubule. The possible effects of ET on proximal tubule transport and the potential role of second messengers in this process have not been described fully. To define the role of ET in proximal tubule transport, renal cortical slices were incubated for 3 min in the presence of various concentrations of ET. Incubation with low concentrations of ET-1 (1 x 10(-9) to 1 x 10(-11) M) within the physiological range stimulated both Na(+)-Pi cotransport and Na+/H+ exchange. Pretreatment with staurosporine (0.6 microM) for 25 min abolished completely the ET-induced effects on Na(+)-Pi cotransport and Na+/H+ exchange. Similarly, preincubation with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (200 nM) also abolished the effects of ET on these transporters. Incubation with ET decreased significantly intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Intravenous administration of pertussis toxin for 2 days prevented the ET-induced decrease in cAMP and abolished the stimulatory effects of ET on Na(+)-Pi cotransport and Na+/H+ exchange. These findings provide indirect evidence that ET participates in the regulation of proximal tubular Pi and bicarbonate homeostasis. These effects of ET are mediated by activation of protein kinase C and cAMP-dependent protein kinase A.

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.

scholarly journals Regulation of Megalin membrane traffic by AMP‐activated protein kinase in kidney proximal tubule epithelial cells

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Nikol Leshchyshyn ◽  
Laura Orofiamma ◽  
Christian Delos Santos ◽  
Sadia Rahmani ◽  
Costin Antonescu
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Proximal Tubule ◽  
Membrane Traffic ◽  
Kidney Proximal Tubule ◽  
Amp Activated Protein Kinase

Avian renal proximal tubule urate secretion is inhibited by cellular stress-induced AMP-activated protein kinase

2011 ◽  
Vol 300 (6) ◽  
pp. F1327-F1338 ◽  
Author(s):  
Amy M. Bataille ◽  
Carla L. Maffeo ◽  
J. Larry Renfro
Keyword(s):  
Protein Kinase ◽  
Proximal Tubule ◽  
Cellular Stress ◽  
Renal Proximal Tubule ◽  
Cellular Stress Response ◽  
Cancer Resistance ◽  
Compound C ◽  
Resistance Protein ◽  
Amp Activated Protein Kinase ◽  
Urate Secretion

Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein ( ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5′-aminoimidazole-4-carboxamide 1-β-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion.

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