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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. annexins; cellular uptake; intracellular calcium; vascular calcification

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [[Ca.sup.2+]].sub.i] transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [[Ca.sup.2+]].sub.i] transients were recorded at depths [less than or equal to] 100 [micro]m from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [[Ca.sup.2+]].sub.i] transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% ([t.sub.90-50%]) and from 50 to 10% ([t.sub.50-10%]) of the peak [[Ca.sup.2+]].sub.i] were (means [+ or -] SE) 73 [+ or -] 4 and 126 [+ or -] 10 ms, respectively, and at 2 Hz, 62 [+ or -] 3 and 94 [+ or -] 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [[Ca.sup.2+]].sub.i] decay was markedly slower in fura-2-loaded hearts ([t.sub.90-50%] at 1 Hz, 128 [+ or -] 9 ms and at 2 Hz, 88 [+ or -] 5 ms; [t.sub.50-10%] at 1 Hz, 214 [+ or -] 18 ms and at 2 Hz, 163 [+ or -] 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [[Ca.sup.2+]].sub.i] decline resulted from increased cytosolic [Ca.sup.2+] buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the [Ca.sup.2+] chelator BAPTA. Propagating calcium waves and [[Ca.sup.2+]].sub.i] amplitude alternans were readily detected in paced hearts. This approach should be of genera] utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging. rhod-2; fura-2; BAPTA; 2,3-butanedione monoxime; cytochalasin D

A model system composed of phospholipid vesicles with Quin-2 was utilized to evaluate the transport selectivity of Ca2+ ionophores such as A237187, ionomycin and 4-BrA23187. Ionomycin and A23187 ionophore exhibited high selectivity to Zn2+, Mn2+, Ca2+, Co2+, Ni2+ and Sr2+, respectively. Furthermore, Zn2+ and Mn2+ were transported by complexes that are composed of equal amounts of A23187 and 4-BrA23187 carboxylic acid ionophores.

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca[sup 2+]][sub i] transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca[sup 2+]][sub i] transients were recorded at depths ≤100 µm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca[sup 2+]][sub i] transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t[sub 90-50%]) and from 50 to 10% (t[sub 50-10%]) of the peak [Ca[sup 2+]][sub i] were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2Hz, 62 ± 3 and 94 ± 6 ms(n = 19, P<0.05vs. 1Hz) in rhod-2-1oaded cardiomyocytes. [Ca[sup 2+]][sub i] decay was markedly slower in fura-2-1oaded hearts (t[sub 90-50%] at I Hz, 128 ± 9 ms and at 2 Hz, 88 ± 5 ms; t[sub 50-l0%] at 1 Hz, 214 ± 18 ms and at 2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2induced deceleration of [Ca[sup 2+]][sub i] decline resulted from increased cytosolic Ca[sup 2+] buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca[sup 2+] chelator BAPTA. Propagating calcium waves and [Ca[sup 2+]][sub i] amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging. [ABSTRACT FROM AUTHOR]

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required.

The rapid diagnosis and quantification of acute kidney injury (AKI) severity remain high clinical priorities. By combining intravital fluorescent ratiometric two-photon kidney imaging and the two-compartment pharmacokinetics model, we demonstrate that rapid quantification of glomerular filtration rate (GFR) can be achieved in physiologic and AKI rat kidney models. Using a bolus infusion of a mixture of FITC-inulin and a 500-kDa Texas Red dextran, a full spectrum of GFR values, ranging from 0.17 to 1.12 ml·min(-1)·100 g(-1), was obtained. The GFR values thus determined correlated well with values obtained by the standard 2-h inulin infusion clearance method with a Pearson's correlation coefficient of 0.85. In addition, postischemia deterioration was studied by measuring GFR using the two-photon approach during 24 h following a 45-min bilateral ischemia clamp model. The GFR was found to decline sharply during the initial 4 h followed by a nadir with little sign of rising over the ensuing 24-h period. Moreover, a FITC-labeled 5-kDa dextran was identified as having nearly identical filtration characteristics as FITC-inulin, but had markedly increased fluorescent intensity, thus minimizing the quantity needed for individual studies. The technique reported allows for very rapid GFR determinations, within 10-15 min, based on plasma clearance of a freely filtered fluorescence probe, instead of a prolonged one-compartment interstitial space reporter molecule clearance employed by other technologies.