Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells

L. Vergara, Xiaoyong Bao, M. Cooper, E. Bello-Reuss, L. Reuss

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca2+ removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.

Original languageEnglish (US)
Pages (from-to)173-184
Number of pages12
JournalJournal of Membrane Biology
Volume196
Issue number3
DOIs
StatePublished - Dec 1 2003

Fingerprint

Proximal Kidney Tubule
Adenosine Triphosphate
Gap Junctions
Connexin 43
Divalent Cations
Kidney
Intercellular Junctions
Primary Cell Culture
Propidium
Wounds and Injuries
Gadolinium
Endocytosis
Fluorescein
Fluorescent Antibody Technique
Homeostasis
Necrosis
Coloring Agents
Ischemia
Western Blotting
Epithelial Cells

Keywords

  • Cell injury
  • Connexin 43
  • Human kidney
  • Hypoxia
  • Ischemia

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells. / Vergara, L.; Bao, Xiaoyong; Cooper, M.; Bello-Reuss, E.; Reuss, L.

In: Journal of Membrane Biology, Vol. 196, No. 3, 01.12.2003, p. 173-184.

Research output: Contribution to journalArticle

Vergara, L. ; Bao, Xiaoyong ; Cooper, M. ; Bello-Reuss, E. ; Reuss, L. / Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells. In: Journal of Membrane Biology. 2003 ; Vol. 196, No. 3. pp. 173-184.
@article{6c428c0aed2747abaca52b000d4a14fb,
title = "Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells",
abstract = "We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca2+ removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.",
keywords = "Cell injury, Connexin 43, Human kidney, Hypoxia, Ischemia",
author = "L. Vergara and Xiaoyong Bao and M. Cooper and E. Bello-Reuss and L. Reuss",
year = "2003",
month = "12",
day = "1",
doi = "10.1007/s00232-003-0636-9",
language = "English (US)",
volume = "196",
pages = "173--184",
journal = "Journal of Membrane Biology",
issn = "0022-2631",
publisher = "Springer New York",
number = "3",

}

TY - JOUR

T1 - Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells

AU - Vergara, L.

AU - Bao, Xiaoyong

AU - Cooper, M.

AU - Bello-Reuss, E.

AU - Reuss, L.

PY - 2003/12/1

Y1 - 2003/12/1

N2 - We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca2+ removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.

AB - We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca2+ removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.

KW - Cell injury

KW - Connexin 43

KW - Human kidney

KW - Hypoxia

KW - Ischemia

UR - http://www.scopus.com/inward/record.url?scp=0742325002&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0742325002&partnerID=8YFLogxK

U2 - 10.1007/s00232-003-0636-9

DO - 10.1007/s00232-003-0636-9

M3 - Article

VL - 196

SP - 173

EP - 184

JO - Journal of Membrane Biology

JF - Journal of Membrane Biology

SN - 0022-2631

IS - 3

ER -