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      The Indiana O’Brien Center for Advanced Renal Microscopic Analysis

      Author(s): 1 , 1 , 1
      Publication date Created:
      Publication date (Print):
      Journal: American Journal of Physiology-Renal Physiology
      Publisher: American Physiological Society

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          Abstract

          The Indiana O’Brien Center for Advanced Microscopic Analysis is a National Institutes of Health (NIH) P30-funded research center dedicated to the development and dissemination of advanced methods of optical microscopy to support renal researchers throughout the world. The Indiana O’Brien Center was founded in 2002 as an NIH P-50 project with the original goal of helping researchers realize the potential of intravital multiphoton microscopy as a tool for understanding renal physiology and pathophysiology. The center has since expanded into the development and implementation of large-scale, high-content tissue cytometry. The advanced imaging capabilities of the center are made available to renal researchers worldwide via collaborations and a unique fellowship program. Center outreach is accomplished through an enrichment core that oversees a seminar series, an informational website, and a biennial workshop featuring hands-on training from members of the Indiana O’Brien Center and imaging experts from around the world.

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          Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes.

          Michael Y. Gerner, Wolfgang Kastenmuller, Ina Ifrim … (2012)
          Flow cytometry allows highly quantitative analysis of complex dissociated populations at the cost of neglecting their tissue localization. In contrast, conventional microscopy methods provide spatial information, but visualization and quantification of cellular subsets defined by complex phenotypic marker combinations is challenging. Here, we describe an analytical microscopy method, "histo-cytometry," for visualizing and quantifying phenotypically complex cell populations directly in tissue sections. This technology is based on multiplexed antibody staining, tiled high-resolution confocal microscopy, voxel gating, volumetric cell rendering, and quantitative analysis. We have tested this technology on various innate and adaptive immune populations in murine lymph nodes (LNs) and were able to identify complex cellular subsets and phenotypes, achieving quantitatively similar results to flow cytometry, while also gathering cellular positional information. Here, we employ histo-cytometry to describe the spatial segregation of resident and migratory dendritic cell subsets into specialized microanatomical domains, suggesting an unexpected LN demarcation into discrete functional compartments. Copyright © 2012 Elsevier Inc. All rights reserved.
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            siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury.

            A Faerman, Guilherme Campos, Bruce Molitoris … (2009)
            Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.
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              In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury.

              Bruce Molitoris, Nicholas Sandoval, Kenneth Rhodes … (2012)
              Mitochondrial dysfunction has been implicated in the pathogenesis of acute kidney injury due to ischemia and toxic drugs. Methods for imaging mitochondrial function in cells using confocal microscopy are well established; more recently, it was shown that these techniques can be utilized in ex vivo kidney tissue using multiphoton microscopy. We extended this approach in vivo and found that kidney mitochondrial structure and function can be imaged in anesthetized rodents using multiphoton excitation of endogenous and exogenous fluorophores. Mitochondrial nicotinamide adenine dinucleotide increased markedly in rat kidneys in response to ischemia. Following intravenous injection, the mitochondrial membrane potential-dependent dye TMRM was taken up by proximal tubules; in response to ischemia, the membrane potential dissipated rapidly and mitochondria became shortened and fragmented in proximal tubules. In contrast, the mitochondrial membrane potential and structure were better maintained in distal tubules. Changes in mitochondrial structure, nicotinamide adenine dinucleotide, and membrane potential were found in the proximal, but not distal, tubules after gentamicin exposure. These changes were sporadic, highly variable among animals, and were preceded by changes in non-mitochondrial structures. Thus, real-time changes in mitochondrial structure and function can be imaged in rodent kidneys in vivo using multiphoton excitation of endogenous and exogenous fluorophores in response to ischemia-reperfusion injury or drug toxicity.
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                Author and article information

                Journal
                Title: American Journal of Physiology-Renal Physiology
                Abbreviated Title: American Journal of Physiology-Renal Physiology
                Publisher: American Physiological Society
                ISSN (Print): 1931-857X
                ISSN (Electronic): 1522-1466
                Publication date Created: May 01 2021
                Publication date (Print): May 01 2021
                Volume: 320
                Issue: 5
                Pages: F671-F682
                Affiliations
                [1 ]Division of Nephrology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana
                Article
                DOI: 10.1152/ajprenal.00007.2021
                PubMed ID: 33682441
                SO-VID: c282a938-dea8-499b-9a70-a93c784b8036
                Copyright © © 2021
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