Browsing by Author "Rhodes, George"

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    Effects of biomechanical forces on signaling in the cortical collecting duct (CCD)
    (American Physiological Society (APS), 2014-07-15) Carrisoza-Gaytan, Rolando; Liu, Yu; Flores, Daniel; Else, Cindy; Lee, Heon Goo; Rhodes, George; Sandoval, Ruben M.; Kleyman, Thomas R.; Lee, Francis Young-In; Molitoris, Bruce; Satlin, Lisa M.; Rohatgi, Rajeev; Department of Medicine, IU School of Medicine
    An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm2 of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.
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    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.
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    Mechanism of increased clearance of glycated albumin by proximal tubule cells
    (The American Physiological Society, 2016-05-01) Wagner, Mark C.; Myslinski, Jered; Pratap, Shiv; Flores, Brittany; Rhodes, George; Campos-Bilderback, Silvia B.; Sandoval, Ruben M.; Kumar, Sudhanshu; Patel, Monika; Ashish; Molitoris, Bruce A.; Medicine, School of Medicine
    Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn)-mediated transcytosis by many cell types, including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of the present study was to address the impact of specific known biological albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it after transcytosis at physiological pH. Equilibrium dissociation constants were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin, we studied glucose and methylgloxal modified albumins (21 days). All had reduced affinity to FcRn at pH 6.0, suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address why modified albumin has reduced affinity, we analyzed the structure of the modified albumins using small-angle X-ray scattering. This analysis showed significant structural changes occurring to albumin with glycation, particularly in the FcRn-binding region, which could explain the reduced affinity to FcRn. These results offer an explanation for enhanced proximal tubule-mediated sorting and clearance of abnormal albumins.
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    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.