scholarly journals In vivo measurements of kidney glomerular number and size in healthy and Os/+ mice using MRI

2019 ◽  
Vol 317 (4) ◽  
pp. F865-F873 ◽  
Author(s):  
Edwin J. Baldelomar ◽  
Jennifer R. Charlton ◽  
Kimberly A. deRonde ◽  
Kevin M. Bennett
Keyword(s):  
Ex Vivo ◽  
Nephron Number ◽  
Wild Type ◽  
Resonance Imaging ◽  
Renal Hypoplasia ◽  
Nephron Endowment ◽  
In Vivo Measurements ◽  
Glomerular Number ◽  
Direct Measurements

The development of chronic kidney disease (CKD) is associated with the loss of functional nephrons. However, there are no methods to directly measure nephron number in living subjects. Thus, there are no methods to track the early stages of progressive CKD before changes in total renal function. In this work, we used cationic ferritin-enhanced magnetic resonance imaging (CFE-MRI) to enable measurements of glomerular number ( Nglom) and apparent glomerular volume (aVglom) in vivo in healthy wild-type (WT) mice ( n = 4) and mice with oligosyndactylism (Os/+; n = 4), a model of congenital renal hypoplasia leading to nephron reduction. We validated in vivo measurements of Nglom and aVglom by high-resolution ex vivo MRI. CFE-MRI measured a mean Nglom of 12,220 ± 2,028 and 6,848 ± 1,676 (means ± SD) for WT and Os/+ mouse kidneys in vivo, respectively. Nglom measured in all mice in vivo using CFE-MRI varied by an average 15% from Nglom measured ex vivo in the same kidney (α = 0.05, P = 0.67). To confirm that CFE-MRI can also be used to track nephron endowment longitudinally, a WT mouse was imaged three times by CFE-MRI over 2 wk. Values of Nglom measured in vivo in the same kidney varied within ~3%. Values of aVglom calculated from CFE-MRI in vivo were significantly different (~15% on average, P < 0.01) from those measured ex vivo, warranting further investigation. This is the first report of direct measurements of Nglom and aVglom in healthy and diseased mice in vivo.

Normal variation in nephron numbers

2015 ◽  
Author(s):  
Michiel F. Schreuder
Keyword(s):  
Kidney Development ◽  
Ex Vivo ◽  
Imaging Techniques ◽  
Nutritional Deficiencies ◽  
Resonance Imaging ◽  
Intrauterine Growth ◽  
Nephron Endowment ◽  
Magnetic Resonance Imaging Techniques ◽  
Renal Size

Kidney development includes the formation of nephrons, which ceases around the 36th week of gestation. At that time, around 900,000 nephrons are formed, but with a 10-fold variation (from 200,000 to over 2 million). Many factors have been described to influence the number of nephrons per individual, such as genetic variations, intrauterine growth and prematurity, maternal diseases and (nutritional) deficiencies, and drugs used during nephrogenesis. Counting nephrons is currently only possible ex vivo, even though magnetic resonance imaging techniques are getting to the stage that in vivo estimations using stereology (the gold standard methodology) can be expected to become available in the next decade. In the meantime, renal size is often used as a marker for nephron endowment.

scholarly journals Measuring rat kidney glomerular number and size in vivo with MRI

2018 ◽  
Vol 314 (3) ◽  
pp. F399-F406 ◽  
Author(s):  
Edwin J. Baldelomar ◽  
Jennifer R. Charlton ◽  
Scott C. Beeman ◽  
Kevin M. Bennett
Keyword(s):  
Ex Vivo ◽  
Rat Kidney ◽  
Male Rats ◽  
Cationized Ferritin ◽  
Nephron Number ◽  
Renal Disease Progression ◽  
Glomerular Volume ◽  
Glomerular Number ◽  
The Individual

Nephron number is highly variable in humans and is thought to play an important role in renal health. Chronic kidney disease (CKD) is the result of too few nephrons to maintain homeostasis. Currently, nephron number can only be determined invasively or as a terminal assessment. Due to a lack of tools to measure and track nephron number in the living, the early stages of CKD often go unrecognized, preventing early intervention that might halt the progression of CKD. In this work, we present a technique to directly measure glomerular number ( Nglom) and volume in vivo in the rat kidney ( n = 8) using MRI enhanced with the novel contrast agent cationized ferritin (CFE-MRI). Adult male rats were administered intravenous cationized ferritin (CF) and imaged in vivo with MRI. Glomerular number was measured and each glomerulus was spatially mapped in 3D in the image. Mean apparent glomerular volume (a Vglom) and intrarenal distribution of the individual glomerular volume (IGV), were also measured. These metrics were compared between images of the same kidneys scanned in vivo and ex vivo with CFE-MRI. In vivo Nglom and a Vglom correlated to ex vivo metrics within the same kidneys and were within 10% of Nglom and a Vglom previously validated by stereologic methods. This is the first report of direct in vivo measurements of Nglom and a Vglom, introducing an opportunity to investigate mechanisms of renal disease progression and therapeutic response over time.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Allen M Andres ◽  
Chengqun Huang ◽  
Eric P Ratliff ◽  
Genaro Hernandez ◽  
Pamela Lee ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Wild Type ◽  
Simulated Ischemia ◽  
Selective Targeting ◽  
Cardioprotective Effects ◽  
Mitochondrial Turnover ◽  
First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

scholarly journals Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity

2018 ◽  
Vol 114 (8) ◽  
pp. 1178-1188 ◽  
Author(s):  
Daniel S Gaul ◽  
Julien Weber ◽  
Lambertus J van Tits ◽  
Susanna Sluka ◽  
Lisa Pasterk ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Myocardial Infarction ◽  
Bone Marrow ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Neutrophil Extracellular Traps ◽  
Wild Type ◽  
Rotational Thromboelastometry ◽  
Extracellular Traps

AbstractAimsSirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).Methods and resultsUsing a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01).ConclusionsSirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

scholarly journals In vivo magnetic resonance imaging of orthotopic prostate cancer

BioTechniques ◽  
2020 ◽  
Vol 69 (1) ◽  
pp. 37-45
Author(s):  
Murali K Ravoori ◽  
Sheela Singh ◽  
Peiying Yang ◽  
Wei Wei ◽  
Huiqin Chen ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Mechanisms Of Action ◽  
Tumor Burden ◽  
Therapeutic Interventions ◽  
Resonance Imaging ◽  
Prostate Tumors ◽  
Tumor Weight ◽  
History Of ◽  
Old Mice

Methods for imaging orthotopic prostate tumors within the prostate or small tumors with extension outside the prostate are needed to more closely model human prostate tumors, which are most commonly located within the gland or may extend just through the gland. By comparing MR sequences, we found that the T2-based Dixon ‘water only’ sequence best visualized tumors within the prostate of mouse models in both young and old mice and that tumor weight derived from this sequence correlated highly with ex vivo tumor weight (r2 = 0.98, p < 0.001, n = 12). This should aid tumor detection, margin delineation and evaluation of tumor burden to enable studies including, but not limited to, evaluating the natural history of the disease, the mechanisms of action and the efficacy of therapeutic interventions.

Targeted inactivation of Gαi does not alter cardiac function or β-adrenergic sensitivity

2001 ◽  
Vol 280 (2) ◽  
pp. H569-H575 ◽  
Author(s):  
Mohit Jain ◽  
Chee Chew Lim ◽  
Kohzo Nagata ◽  
Vannessa M. Davis ◽  
David S. Milstone ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Ventricular Myocytes ◽  
Ex Vivo ◽  
Cardiac Response ◽  
Similar Degree ◽  
Wild Type ◽  
Isolated Hearts ◽  
Targeted Inactivation ◽  
Contractile Performance

Inhibitory Gαi protein increases in the myocardium during hypertrophy and has been associated with β-adrenergic receptor (β-AR) desensitization, contractile dysfunction, and progression of cardiac disease. The role of Gαi proteins in mediating basal cardiac function and β-AR response in nonpathological myocardium, however, is uncertain. Transgenic mice with targeted inactivation of Gαi2 or Gαi3 were examined for in vivo cardiac function with the use of conscious echocardiography and for ex vivo cardiac response to inotropic stimulation with the use of Langendorff blood-perfused isolated hearts and adult ventricular cardiomyocytes. Echocardiography revealed that percent fractional shortening and heart rate were similar among wild-type, Gαi2 -null, and Gαi3 -null mice. Comparable baseline diastolic and contractile performance was also observed in isolated hearts and isolated ventricular myocytes from wild-type mice and mice lacking Gαi proteins. Isoproterenol infusion enhanced diastolic and contractile performance to a similar degree in wild-type, Gαi2 -null, and Gαi3 -null mice. These data demonstrate no observable role for inhibitory G proteins in mediating basal cardiac function or sensitivity to β-AR stimulation in nonpathological myocardium.

scholarly journals The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
V. Helfinger ◽  
K. Palfi ◽  
A. Weigert ◽  
K. Schröder
Keyword(s):  
Ex Vivo ◽  
Macrophage Polarization ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Superoxide Anions ◽  
Wild Type ◽  
Macrophage Population ◽  
The Family ◽  
High Level

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.

scholarly journals Modulation of the Effects of Lung Immune Response on Bone Marrow by Oral Antigen Exposure

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
P. Xavier-Elsas ◽  
C. L. C. A. Silva ◽  
L. Pinto ◽  
T. Queto ◽  
B. M. Vieira ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Lymphocytes ◽  
Ex Vivo ◽  
Allergen Challenge ◽  
Allergic Airway Inflammation ◽  
Oral Exposure ◽  
Wild Type ◽  
Homologous Antigen ◽  
Mutant Strains

Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.

scholarly journals Aerobactin, but Not Yersiniabactin, Salmochelin, or Enterobactin, Enables the Growth/Survival of Hypervirulent (Hypermucoviscous) Klebsiella pneumoniaeEx VivoandIn Vivo

2015 ◽  
Vol 83 (8) ◽  
pp. 3325-3333 ◽  
Author(s):  
Thomas A. Russo ◽  
Ruth Olson ◽  
Ulrike MacDonald ◽  
Janet Beanan ◽  
Bruce A. Davidson
Keyword(s):  
Virulence Factor ◽  
Human Urine ◽  
Ex Vivo ◽  
Systemic Infection ◽  
Relative Activity ◽  
Ascites Fluid ◽  
Specific Gene ◽  
Wild Type ◽  
Content Type

The siderophore aerobactin is the dominant siderophore produced by hypervirulentKlebsiella pneumoniae(hvKP) and was previously shown to be a major virulence factor in systemic infection. However, strains of hvKP commonly produce the additional siderophores yersiniabactin, salmochelin, and enterobactin. The roles of these siderophores in hvKP infection have not been optimally defined. To that end, site-specific gene disruptions were created in hvKP1 (wild type), resulting in the generation of hvKP1ΔiucA(aerobactin deficient), hvKP1ΔiroB(salmochelin deficient), hvKP1ΔentB(enterobactin and salmochelin deficient), hvKP1Δirp2(yersiniabactin deficient), and hvKP1ΔentBΔirp2(enterobactin, salmochelin, and yersiniabactin deficient). The growth/survival of these constructs was compared to that of their wild-type parent hvKP1ex vivoin human ascites fluid, human serum, and human urine andin vivoin mouse systemic infection and pulmonary challenge models. Interestingly, in contrast to aerobactin, the inability to produce enterobactin, salmochelin, or yersiniabactin individually or in combination did not decrease theex vivogrowth/survival in human ascites or serum or decrease virulence in thein vivoinfection models. Surprisingly, none of the siderophores increased growth in human urine. In human ascites fluid supplemented with exogenous siderophores, siderophores increased the growth of hvKP1ΔiucA, with the relative activity being enterobactin > aerobactin > yersiniabactin > salmochelin, suggesting that the contribution of aerobactin to virulence is dependent on both innate biologic activity and quantity produced. Taken together, these data confirm and extend a role for aerobactin as a critical virulence factor for hvKP. Since it appears that aerobactin production is a defining trait of hvKP strains, this factor is a potential antivirulence target.

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