Laminin and fibronectin content of mouse glomerular and tubula basement membrane

D. K. Brees, R. C. Ogle, James Williams

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1% deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 ± 0.001 μg/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 ± 0.1 μg/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 ± 0.8 and 7.7 ± 1.0 μg/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalRenal Physiology and Biochemistry
Volume18
Issue number1
StatePublished - 1995
Externally publishedYes

Fingerprint

Glomerular Basement Membrane
Laminin
Fibronectins
Basement Membrane
Deoxycholic Acid
Guanidine
Weights and Measures
Permeability
Kidney
Proteins
Sonication
Complex Mixtures
Centrifugation
Detergents
Sucrose
Electrophoresis
Enzyme-Linked Immunosorbent Assay

Keywords

  • Detergent
  • Isolation
  • Kidney
  • Solubilization

ASJC Scopus subject areas

  • Physiology
  • Nephrology

Cite this

Laminin and fibronectin content of mouse glomerular and tubula basement membrane. / Brees, D. K.; Ogle, R. C.; Williams, James.

In: Renal Physiology and Biochemistry, Vol. 18, No. 1, 1995, p. 1-11.

Research output: Contribution to journalArticle

@article{328d6ef395e14d3db775a3aa2cb4878e,
title = "Laminin and fibronectin content of mouse glomerular and tubula basement membrane",
abstract = "Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1{\%} deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 ± 0.001 μg/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 ± 0.1 μg/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 ± 0.8 and 7.7 ± 1.0 μg/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components.",
keywords = "Detergent, Isolation, Kidney, Solubilization",
author = "Brees, {D. K.} and Ogle, {R. C.} and James Williams",
year = "1995",
language = "English (US)",
volume = "18",
pages = "1--11",
journal = "Kidney and Blood Pressure Research",
issn = "1420-4096",
publisher = "S. Karger AG",
number = "1",

}

TY - JOUR

T1 - Laminin and fibronectin content of mouse glomerular and tubula basement membrane

AU - Brees, D. K.

AU - Ogle, R. C.

AU - Williams, James

PY - 1995

Y1 - 1995

N2 - Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1% deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 ± 0.001 μg/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 ± 0.1 μg/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 ± 0.8 and 7.7 ± 1.0 μg/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components.

AB - Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1% deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 ± 0.001 μg/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 ± 0.1 μg/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 ± 0.8 and 7.7 ± 1.0 μg/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components.

KW - Detergent

KW - Isolation

KW - Kidney

KW - Solubilization

UR - http://www.scopus.com/inward/record.url?scp=0028831520&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028831520&partnerID=8YFLogxK

M3 - Article

C2 - 7533313

AN - SCOPUS:0028831520

VL - 18

SP - 1

EP - 11

JO - Kidney and Blood Pressure Research

JF - Kidney and Blood Pressure Research

SN - 1420-4096

IS - 1

ER -