A portable fiberoptic ratiometric fluorescence analyzer provides rapid point-of-care determination of glomerular filtration rate in large animals.

Measurement of the glomerular filtration rate (GFR) is the gold standard for precise assessment of kidney function. A rapid, point-of-care determination of the GFR may provide advantages in the clinical setting over currently available assays. Here we demonstrate a proof of principle for such an approach in a pig and dogs, two species that approximate the vascular access and GFR results expected in humans. In both animal models, a sub-millimeter optical fiber that delivered excitation light and collected fluorescent emissions was inserted into a peripheral vein (dog) or central venous access (pig) by means of commercial intravenous catheters. A mixture of fluorescent chimeras of a small freely filterable reporter and large non-filterable plasma volume marker were infused as a bolus, excited by light-emitting diodes, and the in vivo signals detected and quantified by photomultiplier tubes in both species in less than 60 min. Concurrent standardized 6-h iohexol plasma kidney clearances validated the accuracy of our results for both physiologic and a chronic kidney disease setting. Thus, our ratiometric technique allows for both measurement of plasma vascular volume and highly accurate real-time GFR determinations, enabling clinical decision making in real time.

Fetuin-A uptake in bovine vascular smooth muscle cells is calcium dependent and mediated by annexins.

Fetuin-A is a known inhibitor of vascular calcification in vitro. In arteries with calcification, there is increased immunostaining for fetuin-A. However, vascular smooth muscle cells (VSMC) do not synthesize fetuin-A, suggesting fetuin-A may be endocytosed to exert its inhibitory effects. To examine the mechanism by which fetuin-A is taken up in bovine VSMC (BVSMC), we examined living cells by confocal microscopy and determined the uptake of Cy5-labeled fetuin-A. The results demonstrated that fetuin-A was taken up in BVSMC only in the presence of extracellular calcium, whereas phosphorus had no effect. Additional studies demonstrated the calcium-dependent uptake was specific for fetuin-A and only observed in BVSMC and osteoblasts, but not epithelial, endothelial, or adipose cells. The uptake was dose dependent, but could not be inhibited by excess unlabeled fetuin-A, suggesting a fluid phase rather than a receptor-mediated process. Fetuin-A also induced a sustained increase in intracellular calcium in BVSMC in the presence of extracellular calcium, whereas there was no increase in the absence of extracellular calcium. To further characterize the uptake, we utilized an inhibitor of annexin calcium channel activity, demonstrating inhibition of both fetuin-A uptake and intracellular calcium increase. Finally, we demonstrate that fetuin-A binds to annexin II at the cell membrane of BVSMC. In summary, our study demonstrates calcium- and annexin-dependent uptake of fetuin-A that leads to a sustained rise in intracellular calcium. This regulated uptake may be a mechanism by which fetuin-A inhibits VSMC calcification in the presence of excess calcium.

Lipoproteins during the estrous cycle in swine.

The purpose of this study was to examine lipoprotein values at high versus low 17beta-estradiol (E2) concentrations in Yucatan miniature swine. Estrous cycles were measured by heat checking the female on a daily basis using a boar. All swine were fed a 1,050-g low-fat, standard chow diet (8% kcal from fat) once per day. Fasted (24 hours) blood samples were collected during low (early luteal, day 5) and high (late follicular, day 18) E2 concentrations to determine differences in concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein cholesterol (HDL-C), and subfractions. Concentrations of E2 differed significantly from day 5 (3.5 +/- 0.7 pg/mL) to day 18 (14.2 +/- 1.8 pg/mL) of the estrous cycle. Except for HDL(3)-C, all lipoprotein parameters examined were significantly elevated during high E2 versus low E2. TC/HDL-C and LDL-C/HDL-C ratios were significantly lower during the high E2 phase. These results suggest that lipoprotein concentrations fluctuate during the estrous cycle of swine, with high E2 concentrations associated with elevated lipoprotein concentrations.

Exercise training does not reduce hyperlipidemia in pigs fed a high-fat diet.

The pig is often used as a model for studying lipoprotein metabolism as it relates to human atherosclerosis, but few studies have examined the complete lipoprotein profile and related enzymes in swine ingesting an atherogenic diet. We examined whether exercise training would moderate the effects of an atherogenic diet on lipoproteins and lipoprotein lipase (LPL) activity in miniature swine. Male (n = 30) and female (n = 32) swine were initially divided into 2 dietary groups: one consumed low-fat (8%) pig chow, and one consumed pig chow supplemented with 2% cholesterol, 17.1% coconut oil, 2.3% corn oil, and.7% sodium cholate (46% kcal from fat). Following 30 days on the diets, pigs from each diet group were further divided into sedentary and exercise trained subgroups, each cell with 6 to 8 pigs. Training occurred 5 days per week on a treadmill in which the intensity and duration were progressively increased during the 16- to 20-week training period to 75 minutes of aerobic running per session. A 4-way analysis of variance (ANOVA) with repeated measures on time indicated that at the conclusion of the study the atherogenic diet caused significantly (P <.05) increased cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and subfractions, low-density lipoprotein cholesterol (LDL-C) and subfractions, and LPL activity in both genders. For cholesterol, TG, HDL-C, HDL(2)-C, LDL-C, LDL(1&2)-C, and hepatic lipase, the female response to the diet was exaggerated compared to the male response. Exercise training produced no group differences or interactions on any lipoprotein variable. These results suggest that an atherogenic diet has a greater impact on the lipoproteins of female miniature swine than males. Furthermore, under the conditions of this study, exercise training does not moderate the effects of an atherogenic diet on lipoproteins.