TY - JOUR
T1 - Determination of caveolin-1 in renal caveolar and non-caveolar fractions in experimental type 1 diabetes
AU - Demová, H.
AU - Komers, R.
N1 - Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 2009
Y1 - 2009
N2 - Caveolin-1 (CAV-1) is the main structural component of caveolae, acting as a modulator of signal transduction. CAV-1 might be involved in the pathophysiology of microvascular complications in Type 1 diabetes (DM). We sought to determine whether fractionation on sucrose gradient (SF), a method routinely utilized for isolation of caveolar fractions in homogenous cell lines, is applicable for CAV-1-related studies in tissues with multiple cell types, such as the normal rat kidney cortex (C). Using this method, we also determined whether streptozotocin-induced DM in rats (4-week duration) leads to changes in renal subcellular targeting of CAV-1, and evaluated the effects of tight metabolic control (insulin, 12 IU/day) and angiotensin receptor blocker, losartan (4 weeks, 20 mg/kg/day). Immunoblotting of individual fractions obtained from C revealed CAV-1 expression in fractions 4-6 that corresponded to light scattering band that typically forms after separating cellular fractions on SF. These fractions were considered to be caveolar fractions. In C, CAV-1 was also detectable in fractions 8-10. These and all other fractions except caveolar fractions were considered to be non-caveolar fractions. A ratio of caveolar/non-caveolar expression of CAV-1 (CNCR) was computed for each renal cortex allowing comparisons of CAV-1 subcellular distribution in C and DM rats, and effects of treatments. Using this approach, DM was characterized by marked increases in CNCR as compared to C (5.54±1.56 vs. 2.65±1.33, p<0.05) that were reduced by treatment with insulin (0.78±0.24, p<0.01 vs. DM) or losartan (0.84±0.06, p<0.01 vs. DM). In summary, analysis of CAV-1 following the SF of renal cortex detected similar distribution of the protein as in homogenous cell lines, DM-induced changes in CAV-1 targeting, and the effects of pharmacological treatments. This suggests applicability of SF in studies focusing on CAV-1 targeting in organs with various cell lines in vivo.
AB - Caveolin-1 (CAV-1) is the main structural component of caveolae, acting as a modulator of signal transduction. CAV-1 might be involved in the pathophysiology of microvascular complications in Type 1 diabetes (DM). We sought to determine whether fractionation on sucrose gradient (SF), a method routinely utilized for isolation of caveolar fractions in homogenous cell lines, is applicable for CAV-1-related studies in tissues with multiple cell types, such as the normal rat kidney cortex (C). Using this method, we also determined whether streptozotocin-induced DM in rats (4-week duration) leads to changes in renal subcellular targeting of CAV-1, and evaluated the effects of tight metabolic control (insulin, 12 IU/day) and angiotensin receptor blocker, losartan (4 weeks, 20 mg/kg/day). Immunoblotting of individual fractions obtained from C revealed CAV-1 expression in fractions 4-6 that corresponded to light scattering band that typically forms after separating cellular fractions on SF. These fractions were considered to be caveolar fractions. In C, CAV-1 was also detectable in fractions 8-10. These and all other fractions except caveolar fractions were considered to be non-caveolar fractions. A ratio of caveolar/non-caveolar expression of CAV-1 (CNCR) was computed for each renal cortex allowing comparisons of CAV-1 subcellular distribution in C and DM rats, and effects of treatments. Using this approach, DM was characterized by marked increases in CNCR as compared to C (5.54±1.56 vs. 2.65±1.33, p<0.05) that were reduced by treatment with insulin (0.78±0.24, p<0.01 vs. DM) or losartan (0.84±0.06, p<0.01 vs. DM). In summary, analysis of CAV-1 following the SF of renal cortex detected similar distribution of the protein as in homogenous cell lines, DM-induced changes in CAV-1 targeting, and the effects of pharmacological treatments. This suggests applicability of SF in studies focusing on CAV-1 targeting in organs with various cell lines in vivo.
KW - Caveolin-1
KW - Insulin
KW - Kidney disease
KW - Losartan
KW - Sucrose fractionation
KW - Type 1 diabetes mellitus
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M3 - Article
C2 - 18657009
AN - SCOPUS:72549100557
SN - 0862-8408
VL - 58
SP - 563
EP - 568
JO - Physiological Research
JF - Physiological Research
IS - 4
ER -