RhoA distribution in renal caveolar fractions in experimental type 1 diabetes.

Caveolae act as signalling platforms serving as concentrating points for numerous signalling molecules, as well as regulating flux through many distinct signalling cascades. RhoA proteins have been identified as potential actors in the pathophysiology of the cardiovascular system. We used sucrose gradient fractionation and immunoblotting to determine caveolin-1 and RhoA presence in the kidney cortex of streptozotocin-induced T1 diabetes rats (4-week duration), and of diabetic rats treated with angiotensin receptor blocker losartan (4 weeks, 20 mg/kg/day) to retard renal hypertension. Positive RhoA/caveolin-1 co-immunoprecipitation result was detected in the caveolar fraction that corresponded to the light-scattering band obtained from diabetic rats, compared to negative co-immunoprecipitation result in the caveolar fraction obtained from control rats. The detection of RhoA protein in the caveolar fractions and the prospective RhoA/caveolin-1 association can be used to examine the role of these signalling reactions in the pathophysiology of microvascular complications in type 1 diabetes.

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.

Co-inoculation of Borrelia afzelii with tick salivary gland extract influences distribution of immunocompetent cells in the skin and lymph nodes of mice.

The impact of Ixodes ricinus salivary gland extract (SGE) on inflammatory changes in the skin and draining lymph nodes of mice, elicited by the infection with the important human pathogen, B. afzelii, was determined using flow cytometry. SGE injected together with spirochetes reduced the numbers of leukocytes and gammadelta-T lymphocytes in infected epidermis at early time-points post infection. In draining lymph nodes, the anti-inflammatory effect of SGE was manifested by the decrease of total cell count compared with that in mice treated with inactivated SGE. Changes in subpopulations of immunocompetent cells apparently reflected the effect of SGE on the proliferation of spirochetes in the host. The significance of tick saliva anti-inflammatory effect for saliva activated transmission of B. afzelii is shown.