CNP is present in canine renal tubular cells and secreted by cultured opossum kidney cells

A. Nir, K. W. Beers, A. L. Clavell, C. M. Wei, D. M. Heublein, T. P. Dousa, John C Jr. Burnett

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

C-type natriuretic peptide (CNP) is a vasoactive and antimitogenic peptide that is structurally similar but genetically distinct from atrial natriuretic peptide. While first discovered in the brain, CNP has been shown to be produced by endothelial cells and may function in a paracrine and autocrine fashion in the control of vascular tone. Recently, CNP immunoreactivity and B-type natriuretic peptide receptors (NPR-B), for which CNP is a specific ligand, have been identified in the kidney. The present study was designed to determine whether renal epithelial cells produce and secrete CNP and whether CNP immunoreactivity is present in canine kidney. Opossum kidney (OK) cells that express proximal tubular cell characteristics were incubated for 6 h in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). CNP immunoreactivity was measured in the preincubation and 6-h conditioned media by radioimmunoassay (RIA) using a specific antibody to CNP-22. Furthermore the molecular form of this CNP-like protein was determined by reverse-phase high-performance liquid chromatography (HPLC), and intracellular localization of the CNP immunoreactivity was determined by immunohistochemical staining. CNP immunoreactivity was also determined in renal tissue from dogs subjected to saline or endothelin infusion. Six-hour incubation in DMEM resulted in accumulation of CNP immunoreactivity (baseline below detection level vs. 6 h = 117.3 ± 8.3 pg/ml, P < 0.001). Intracellular CNP concentration determined after sonication was 1.9 ± 0.2 μg/g protein, and immunohistochemical staining for CNP was markedly positive in the cytoplasm. HPLC demonstrated that the CNP immunoreactivity in the conditioned media corresponded to the CNP-53 molecular form. CNP immunohistochemical staining was mild in renal tubular cells of saline-infused dogs and intense in renal tubular cells of endothelin-infused dogs. This study demonstrates that CNP-like peptide is present in renal tubular cells in vivo and is produced and secreted by cultured renal epithelial cells in vitro. These findings suggest that CNP may play a role in the paracrine and/or autocrine regulation of renal function.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Regulatory Integrative and Comparative Physiology
Volume267
Issue number6 36-6
StatePublished - Dec 1 1994
Externally publishedYes

Fingerprint

C-Type Natriuretic Peptide
Opossums
Canidae
Cultured Cells
Kidney
Eagles
Brain Natriuretic Peptide
Endothelins
Dogs
Staining and Labeling
Conditioned Culture Medium
Epithelial Cells
High Pressure Liquid Chromatography

Keywords

  • C-type natriuretic peptide
  • cell culture
  • opossum renal cells
  • radioimmunoassay

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

CNP is present in canine renal tubular cells and secreted by cultured opossum kidney cells. / Nir, A.; Beers, K. W.; Clavell, A. L.; Wei, C. M.; Heublein, D. M.; Dousa, T. P.; Burnett, John C Jr.

In: American Journal of Physiology - Regulatory Integrative and Comparative Physiology, Vol. 267, No. 6 36-6, 01.12.1994.

Research output: Contribution to journalArticle

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abstract = "C-type natriuretic peptide (CNP) is a vasoactive and antimitogenic peptide that is structurally similar but genetically distinct from atrial natriuretic peptide. While first discovered in the brain, CNP has been shown to be produced by endothelial cells and may function in a paracrine and autocrine fashion in the control of vascular tone. Recently, CNP immunoreactivity and B-type natriuretic peptide receptors (NPR-B), for which CNP is a specific ligand, have been identified in the kidney. The present study was designed to determine whether renal epithelial cells produce and secrete CNP and whether CNP immunoreactivity is present in canine kidney. Opossum kidney (OK) cells that express proximal tubular cell characteristics were incubated for 6 h in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). CNP immunoreactivity was measured in the preincubation and 6-h conditioned media by radioimmunoassay (RIA) using a specific antibody to CNP-22. Furthermore the molecular form of this CNP-like protein was determined by reverse-phase high-performance liquid chromatography (HPLC), and intracellular localization of the CNP immunoreactivity was determined by immunohistochemical staining. CNP immunoreactivity was also determined in renal tissue from dogs subjected to saline or endothelin infusion. Six-hour incubation in DMEM resulted in accumulation of CNP immunoreactivity (baseline below detection level vs. 6 h = 117.3 ± 8.3 pg/ml, P < 0.001). Intracellular CNP concentration determined after sonication was 1.9 ± 0.2 μg/g protein, and immunohistochemical staining for CNP was markedly positive in the cytoplasm. HPLC demonstrated that the CNP immunoreactivity in the conditioned media corresponded to the CNP-53 molecular form. CNP immunohistochemical staining was mild in renal tubular cells of saline-infused dogs and intense in renal tubular cells of endothelin-infused dogs. This study demonstrates that CNP-like peptide is present in renal tubular cells in vivo and is produced and secreted by cultured renal epithelial cells in vitro. These findings suggest that CNP may play a role in the paracrine and/or autocrine regulation of renal function.",
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T1 - CNP is present in canine renal tubular cells and secreted by cultured opossum kidney cells

AU - Nir, A.

AU - Beers, K. W.

AU - Clavell, A. L.

AU - Wei, C. M.

AU - Heublein, D. M.

AU - Dousa, T. P.

AU - Burnett, John C Jr.

PY - 1994/12/1

Y1 - 1994/12/1

N2 - C-type natriuretic peptide (CNP) is a vasoactive and antimitogenic peptide that is structurally similar but genetically distinct from atrial natriuretic peptide. While first discovered in the brain, CNP has been shown to be produced by endothelial cells and may function in a paracrine and autocrine fashion in the control of vascular tone. Recently, CNP immunoreactivity and B-type natriuretic peptide receptors (NPR-B), for which CNP is a specific ligand, have been identified in the kidney. The present study was designed to determine whether renal epithelial cells produce and secrete CNP and whether CNP immunoreactivity is present in canine kidney. Opossum kidney (OK) cells that express proximal tubular cell characteristics were incubated for 6 h in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). CNP immunoreactivity was measured in the preincubation and 6-h conditioned media by radioimmunoassay (RIA) using a specific antibody to CNP-22. Furthermore the molecular form of this CNP-like protein was determined by reverse-phase high-performance liquid chromatography (HPLC), and intracellular localization of the CNP immunoreactivity was determined by immunohistochemical staining. CNP immunoreactivity was also determined in renal tissue from dogs subjected to saline or endothelin infusion. Six-hour incubation in DMEM resulted in accumulation of CNP immunoreactivity (baseline below detection level vs. 6 h = 117.3 ± 8.3 pg/ml, P < 0.001). Intracellular CNP concentration determined after sonication was 1.9 ± 0.2 μg/g protein, and immunohistochemical staining for CNP was markedly positive in the cytoplasm. HPLC demonstrated that the CNP immunoreactivity in the conditioned media corresponded to the CNP-53 molecular form. CNP immunohistochemical staining was mild in renal tubular cells of saline-infused dogs and intense in renal tubular cells of endothelin-infused dogs. This study demonstrates that CNP-like peptide is present in renal tubular cells in vivo and is produced and secreted by cultured renal epithelial cells in vitro. These findings suggest that CNP may play a role in the paracrine and/or autocrine regulation of renal function.

AB - C-type natriuretic peptide (CNP) is a vasoactive and antimitogenic peptide that is structurally similar but genetically distinct from atrial natriuretic peptide. While first discovered in the brain, CNP has been shown to be produced by endothelial cells and may function in a paracrine and autocrine fashion in the control of vascular tone. Recently, CNP immunoreactivity and B-type natriuretic peptide receptors (NPR-B), for which CNP is a specific ligand, have been identified in the kidney. The present study was designed to determine whether renal epithelial cells produce and secrete CNP and whether CNP immunoreactivity is present in canine kidney. Opossum kidney (OK) cells that express proximal tubular cell characteristics were incubated for 6 h in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). CNP immunoreactivity was measured in the preincubation and 6-h conditioned media by radioimmunoassay (RIA) using a specific antibody to CNP-22. Furthermore the molecular form of this CNP-like protein was determined by reverse-phase high-performance liquid chromatography (HPLC), and intracellular localization of the CNP immunoreactivity was determined by immunohistochemical staining. CNP immunoreactivity was also determined in renal tissue from dogs subjected to saline or endothelin infusion. Six-hour incubation in DMEM resulted in accumulation of CNP immunoreactivity (baseline below detection level vs. 6 h = 117.3 ± 8.3 pg/ml, P < 0.001). Intracellular CNP concentration determined after sonication was 1.9 ± 0.2 μg/g protein, and immunohistochemical staining for CNP was markedly positive in the cytoplasm. HPLC demonstrated that the CNP immunoreactivity in the conditioned media corresponded to the CNP-53 molecular form. CNP immunohistochemical staining was mild in renal tubular cells of saline-infused dogs and intense in renal tubular cells of endothelin-infused dogs. This study demonstrates that CNP-like peptide is present in renal tubular cells in vivo and is produced and secreted by cultured renal epithelial cells in vitro. These findings suggest that CNP may play a role in the paracrine and/or autocrine regulation of renal function.

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