Browsing by Author "Molitoris, Bruce"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    Selective Plane Illumination Microscopy, A New Imaging Modality Available at the Indiana Center for Biological Microscopy
    (Office of the Vice Chancellor for Research, 2016-04-08) Winfree, Seth; Smith, Nathaniel; Dunn, Ken; Kamocka, Malgorzata; Molitoris, Bruce
    Microscopy is a primary tool for studying 3D tissue models. Microscopy provides the only means of distinguishing the behaviors of individual cells in a heterogeneous context that obscures biochemical assays. SPIM (Selective Plane Illumination Microscopy) is new approach that is ideally suited to the unique problems involved in high-resolution imaging of 3D tissue models. In the simplest form of SPIM, a cylindrical lens is used to generate a thin lightsheet (1-10 microns) that illuminates a sample. An imaging objective lens, placed orthogonal to this lightsheet is used to collect an image of fluorescence that is selectively excited in this single illuminated plane. The sample is then rotated, and the process is repeated until a multiview dataset of the entire sample is collected. These cross-section images are then assembled to give a complete 3D image of the sample. This approach offers several advantages over conventional methods of imaging thick tissues. First, SPIM provides superior axial resolution for large field-of-view images, deconvolved SPIM volumes have isotropic 3D resolution. Second, SPIM is a “gentle” imaging approach and is better suited to imaging living tissues than either confocal or multiphoton microscopy, supporting studies of cell migration, development, signaling and physiology. Third, imaging speeds can be 30 to 200 fold faster than scanning confocal or multiphoton systems, enabling resolution of dynamic events, and rapid collection of large image datasets. We describe the assembly and customization of an OpenSPIM based lightsheet microscope (IU OpenSPIM) as a platform for developing new imaging technologies. To this end we have implemented software and hardware for multi-channel laser control and temperature and perfusion control. We present examples of highresolution, live and high speed imaging, demonstrating these capabilities. The IU OpenSPIM is a centerpiece in the development of new software for 3D tissue cytometry and a novel screening platform.
  • Thumbnail Image
    Item
    Teprasiran, a Small Interfering RNA, for the Prevention of Acute Kidney Injury in High-Risk Patients Undergoing Cardiac Surgery
    (Wolters Kluwer, 2021-10-05) Thielmann, Matthias; Corteville, David; Szabo, Gabor; Swaminathan, Madhav; Lamy, Andre; Lehner, Lukas J.; Brown, Craig D.; Noiseux, Nicolas; Atta, Mohamed G.; Squiers, Elizabeth C.; Erlich, Shai; Rothenstein, Daniel; Molitoris, Bruce; Mazer, C. David; Medicine, School of Medicine
    Background: Acute kidney injury (AKI) affects up to 30% of patients undergoing cardiac surgery, leading to increased in-hospital and long-term morbidity and mortality. Teprasiran is a novel small interfering RNA that temporarily inhibits p53-mediated cell death that underlies AKI. Methods: This prospective, multicenter, double-blind, randomized, controlled phase 2 trial evaluated the efficacy and safety of a single 10 mg/kg dose of teprasiran versus placebo (1:1), in reducing the incidence, severity, and duration of AKI after cardiac surgery in high-risk patients. The primary end point was the proportion of patients who developed AKI determined by serum creatinine by postoperative day 5. Other end points included AKI severity and duration using various prespecified criteria. To inform future clinical development, a composite end point of major adverse kidney events at day 90, including death, renal replacement therapy, and ≥25% reduction of estimated glomerular filtration rate was assessed. Both serum creatinine and serum cystatin-C were used for estimated glomerular filtration rate assessments. Results: A total of 360 patients were randomly assigned in 41 centers; 341 dosed patients were 73±7.5 years of age (mean±SD), 72% were men, and median European System for Cardiac Operative Risk Evaluation score was 2.6%. Demographics and surgical parameters were similar between groups. AKI incidence was 37% for teprasiran- versus 50% for placebo-treated patients, a 12.8% absolute risk reduction, P=0.02; odds ratio, 0.58 (95% CI, 0.37-0.92). AKI severity and duration were also improved with teprasiran: 2.5% of teprasiran- versus 6.7% of placebo-treated patients had grade 3 AKI; 7% teprasiran- versus 13% placebo-treated patients had AKI lasting for 5 days. No significant difference was observed for the major adverse kidney events at day 90 composite in the overall population. No safety issues were identified with teprasiran treatment. Conclusions: The incidence, severity, and duration of early AKI in high-risk patients undergoing cardiac surgery were significantly reduced after teprasiran administration. A phase 3 study with a major adverse kidney event at day 90 primary outcome that has recently completed enrollment was designed on the basis of these findings (NCT03510897).