IU Indianapolis ScholarWorks :: Browsing by Author "Wagner, Mark C."

Browsing by Author "Wagner, Mark C."

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Albumin uptake and processing by the proximal tubule: physiological, pathological, and therapeutic implications
(American Physiological Society, 2022) Molitoris, Bruce A.; Sandoval, Ruben M.; Yadav, Shiv Pratap S.; Wagner, Mark C.; Medicine, School of Medicine
For nearly 50 years the proximal tubule (PT) has been known to reabsorb, process, and either catabolize or transcytose albumin from the glomerular filtrate. Innovative techniques and approaches have provided insights into these processes. Several genetic diseases, nonselective PT cell defects, chronic kidney disease (CKD), and acute PT injury lead to significant albuminuria, reaching nephrotic range. Albumin is also known to stimulate PT injury cascades. Thus, the mechanisms of albumin reabsorption, catabolism, and transcytosis are being reexamined with the use of techniques that allow for novel molecular and cellular discoveries. Megalin, a scavenger receptor, cubilin, amnionless, and Dab2 form a nonselective multireceptor complex that mediates albumin binding and uptake and directs proteins for lysosomal degradation after endocytosis. Albumin transcytosis is mediated by a pH-dependent binding affinity to the neonatal Fc receptor (FcRn) in the endosomal compartments. This reclamation pathway rescues albumin from urinary losses and cellular catabolism, extending its serum half-life. Albumin that has been altered by oxidation, glycation, or carbamylation or because of other bound ligands that do not bind to FcRn traffics to the lysosome. This molecular sorting mechanism reclaims physiological albumin and eliminates potentially toxic albumin. The clinical importance of PT albumin metabolism has also increased as albumin is now being used to bind therapeutic agents to extend their half-life and minimize filtration and kidney injury. The purpose of this review is to update and integrate evolving information regarding the reabsorption and processing of albumin by proximal tubule cells including discussion of genetic disorders and therapeutic considerations.
Altered O-glycomes of Renal Brush-Border Membrane in Model Rats with Chronic Kidney Diseases
(MDPI, 2021-10-21) Yu, Aiying; Zhao, Jingfu; Zhong, Jieqiang; Wang, Junyao; Yadav, Shiv Pratap S.; Molitoris, Bruce A.; Wagner, Mark C.; Mechref, Yehia; Medicine, School of Medicine
Chronic kidney disease (CKD) is defined as a decrease in renal function or glomerular filtration rate (GFR), and proteinuria is often present. Proteinuria increases with age and can be caused by glomerular and/or proximal tubule (PT) alterations. PT cells have an apical brush border membrane (BBM), which is a highly dynamic, organized, and specialized membrane region containing multiple glycoproteins required for its functions including regulating uptake, secretion, and signaling dependent upon the physiologic state. PT disorders contribute to the dysfunction observed in CKD. Many glycoprotein functions have been attributed to their N- and O-glycans, which are highly regulated and complex. In this study, the O-glycans present in rat BBMs from animals with different levels of kidney disease and proteinuria were characterized and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A principal component analysis (PCA) documented that each group has distinct O-glycan distributions. Higher fucosylation levels were observed in the CKD and diabetic groups, which may contribute to PT dysfunction by altering physiologic glycoprotein interactions. Fucosylated O-glycans such as 1-1-1-0 exhibited higher abundance in the severe proteinuric groups. These glycomic results revealed that differential O-glycan expressions in CKD progressions has the potential to define the mechanism of proteinuria in kidney disease and to identify potential therapeutic interventions.
Changes in the Expression of Renal Brush Border Membrane N-Glycome in Model Rats with Chronic Kidney Diseases
(MDPI, 2021-11-11) Yu, Aiying; Zhao, Jingfu; Yadav, Shiv Pratap S.; Molitoris, Bruce A.; Wagner, Mark C.; Mechref, Yehia; Medicine, School of Medicine
Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments.
Gentamicin Induced Intracellular Toxicity in Saccharomyces cerevisiae
(2011-02) Lin, Lin; Goebl, Mark, 1958-; Harrington, Maureen A.; Bauer, Margaret E.; Wagner, Mark C.; Molitoris, Bruce A.
At the present time, gentamicin is used in the treatment of both Gram-negative and Gram-positive bacterial infections. However, the poorly understood side effect of nephrotoxicity is a serious problem and is one of the dose-limiting factors in the use of gentamicin. In our model system, Saccharomyces cerevisiae, which is relatively resistant to gentamicin, at least 20 genes are required for gentamicin resistance. Inspection of the physical and genetic interactions of the gentamicin sensitive mutants reveals a network centered on the ARF pathway which plays a key role in the regulation of retrograde trafficking. Our studies show that arf1ts arf1Δ arf2Δ cells, gea1ts gea1Δ gea2Δ cells, and gcs1ts gcs1Δ glo3Δ cells are all hypersensitive to gentamicin which indicates that impaired Arf1 function causes yeast cells to become hypersensitive to gentamicin. As evidence, cellular CPY trafficking and processing are blocked by the presence of gentamicin in some of these mutants. Interestingly, gentamicin can directly affect the level of the GTP-bound form of Arf1 in a cell growth phase-dependent manner; even though total Arf1 levels in S. cerevisiae are not affected. As predicted, we also find that gentamicin-bound resin can enrich both yeast Arf1-TAP protein and rat Arf1 protein in vitro. With the help of mass spectrometry, we also generated a gentamicin-binding protein list. Gentamicin hypersensitivity is also observed in S. cerevisiae double deletion strains that lack both ARF1 and ARF2 but are kept alive by the presence of hARF4 or bARF1. Increased -1 programmed ribosomal frameshifting efficiency is also observed in cells treated with gentamicin. Finally, a comparison of a gentamicin mixture and four of the gentamicin congeners reveals that gentamicin C1 is less toxic than other gentamicin congeners or the gentamicin total mixture.
Glycosylation of a key cubilin Asn residue results in reduced binding to albumin
(Elsevier, 2022) Yadav, Shiv Pratap Singh; Yu, Aiying; Zhao, Jingfu; Singh, Jasdeep; Kakkar, Saloni; Chakraborty, Srinivas; Mechref, Yehia; Molitoris, Bruce; Wagner, Mark C.; Medicine, School of Medicine
Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin's albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin-albumin interaction.
Intravital Multiphoton Microscopy as a Tool for Studying Renal Physiology, Pathophysiology and Therapeutics
(Frontiers Media, 2022-03-24) Molitoris, Bruce A.; Sandoval, Ruben M.; Wagner, Mark C.; Medicine, School of Medicine
Intravital multiphoton microscopy has empowered investigators to study dynamic cell and subcellular processes in vivo within normal and disease organs. Advances in hardware, software, optics, transgenics and fluorescent probe design and development have enabled new quantitative approaches to create a disruptive technology pioneering advances in understanding of normal biology, disease pathophysiology and therapies. Offering superior spatial and temporal resolution with high sensitivity, investigators can follow multiple processes simultaneously and observe complex interactions between different cell types, intracellular organelles, proteins and track molecules for cellular uptake, intracellular trafficking, and metabolism in a cell specific fashion. The technique has been utilized in the kidney to quantify multiple dynamic processes including capillary flow, permeability, glomerular function, proximal tubule processes and determine the effects of diseases and therapeutic mechanisms. Limitations include the depth of tissue penetration with loss of sensitivity and resolution due to scattered emitted light. Tissue clearing technology has virtually eliminated penetration issues for fixed tissue studies. Use of multiphoton microscopy in preclinical animal models offers distinct advantages resulting in new insights into physiologic processes and the pathophysiology and treatment of diseases.
Is Albumin Toxic to the Kidney?: It Depends
(Wolters Kluwer, 2023) Molitoris, Bruce A.; Wagner, Mark C.; Medicine, School of Medicine
Lrpap1 (RAP) Inhibits Proximal Tubule Clathrin Mediated and Clathrin Independent Endocytosis, Ameliorating Renal Aminoglycoside Nephrotoxicity
(Wolters Kluwer, 2023) Wagner, Mark C.; Sandoval, Ruben M.; Yadav, Shiv Pratap S.; Campos, Silvia B.; Rhodes, George J.; Phillips, Carrie L.; Molitoris, Bruce A.; Medicine, School of Medicine
Background: Proximal tubules (PTs) are exposed to many exogenous and endogenous nephrotoxins that pass through the glomerular filter. This includes many small molecules, such as aminoglycoside and myeloma light chains. These filtered molecules are rapidly endocytosed by the PTs and lead to nephrotoxicity. Methods: To investigate whether inhibition of PT uptake of filtered toxins can reduce toxicity, we evaluated the ability of Lrpap1 or receptor-associated protein (RAP) to prevent PT endocytosis. Munich Wistar Frömter rats were used since both glomerular filtration and PT uptake can be visualized and quantified. The injury model chosen was the well-established gentamicin-induced toxicity, which leads to significant reductions in GFR and serum creatinine increases. CKD was induced with a right uninephrectomy and left 40-minute pedicle clamp. Rats had 8 weeks to recover and to stabilize GFR and proteinuria. Multiphoton microscopy was used to evaluate endocytosis in vivo and serum creatinine, and 24-hour creatinine clearances were used to evaluate kidney functional changes. Results: Studies showed that preadministration of RAP significantly inhibited both albumin and dextran endocytosis in outer cortical PTs. Importantly, this inhibition was found to be rapidly reversible with time. RAP was also found to be an excellent inhibitor of PT gentamicin endocytosis. Finally, gentamicin administration for 6 days resulted in significant elevation of serum creatinine in vehicle-treated rats, but not in those receiving daily infusion of RAP before gentamicin. Conclusions: This study provides a model for the potential use of RAP to prevent, in a reversible manner, PT endocytosis of potential nephrotoxins, thus protecting the kidney from damage.
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.
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.

Browsing by Author "Wagner, Mark C."

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