Browsing by Author "Wang, Exing"

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
    (Taylor & Francis Online, 2014-03-01) Sandoval, Ruben M.; Wang, Exing; Molitoris, Bruce A.; Department of Medicine, School of Medicine
    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.
  • Thumbnail Image
    Item
    Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance
    (American Physiological Society, 2021) Yadav, Shiv Pratap S.; Sandoval, Ruben M.; Zhao, Jingfu; Huang, Yifan; Wang, Exing; Kumar, Sudhanshu; Campos-Bilderback, Silvia B.; Rhodes, George; Mechref, Yehia; Molitoris, Bruce A.; Wagner, Mark C.; Medicine, School of Medicine
    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.
  • Thumbnail Image
    Item
    Optical Aberrations and Objective Choice in Multicolor Confocal Microscopy
    (Future Science, 2000-03) Dunn, Kenneth W.; Wang, Exing; Medicine, School of Medicine
    Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in wide-field microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy.
  • Thumbnail Image
    Item
    A portable fiberoptic ratiometric fluorescence analyzer provides rapid point-of-care determination of glomerular filtration rate in large animals
    (Nature Publishing group, 2012-01) Wang, Exing; Meier, Daniel J.; Sandoval, Ruben M.; Von Hendy-Willson, Vanessa E.; Pressler, Barrak M.; Bunch, Robert M.; Alloosh, Mouhamad; Sturek, Michael S.; Schwartz, George J.; Molitoris, Bruce A.; Medicine, School of Medicine
    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.
  • Thumbnail Image
    Item
    Sepsis-Induced Glomerular Endothelial Dysfunction Mediates Reductions in GFR and Increases in Protein Filtration
    (Office of the Vice Chancellor for Research, 2012-04-13) Sandoval, Ruben M.; Rhodes, George; Wang, Exing; Camposbilderback, Silvia B.; Wean, Sarah E.; Molitoris, Bruce A.
    Background: Sepsis is now the leading cause of acute kidney injury (AKI) known to decrease Glomerular filtration rate (GFR) and increase proteinuria. There also exists a discrepancy between renal perfusion and GFR. Methods: To evaluate the potential role of the glomerulus in the overall pathogenesis of these abnormalities, we studied surface glomeruli in 8-10 week old Munich Wistar Frmter rats using intravital 2-photon microscopy in a cecal ligation and puncture (CLP) model of sepsis to ask targeted questions and compare the metric of measured GFR to serum creatinine changes at 24 hours post CLP. Results: Male rats undergoing CLP showed an increase in serum creatinine from 0.23 +/- 0.06 mg/dl to 0.80 +/-0.17 (P0.01) and a decrease in real time GFR from 0.69 +/- 0.06 ml/min/100gm body wt to 0.34 +/-0.15 (P0.01). Hemodynamic monitoring revealed normal and hyperdynamic cardiac status within the CLP group. Quantitative analysis of 15 glomeruli in three CLP septic rats revealed a reduction in red blood cell flow rates within capillary loops from 1,771 +/- 467 to 576 +/- 327 um/sec (P0.01); an increase in WBC adherence to glomerular capillary endothelial cells from 0.42 +/-0.33 to 7.25 +/- 5.82 WBC's/standardized glomerular volume (P0.05) in CLP rats; and an increase in the glomerular sieving coefficient (GSC) of a 150kD dextran from 0.007 +/- 0.003 to 0.097 +/- 0.046 (P0.05). Rouleaux formations were seen only in septic rats. Conclusions: These data indicate glomerular endothelial-WBC interactions during sepsis, in part, explain the reduction in GFR and increased filtration of large molecular weight proteins. The results from real time GFR accurately detected the drop in renal function for this model of sepsis.