Determination of caveolin-1 in renal caveolar and non-caveolar fractions in experimental type 1 diabetes.
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.
Demová
H
H
Diabetes Center, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
Komers
R
R
D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism*; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism*; D008297:Male; D051381:Rats; D017208:Rats, Wistar
D051242:Caveolin 1