Publications by authors named "David M Small"

23 Publications

  • Page 1 of 1

Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics.

Front Immunol 2020 4;11:92. Epub 2020 Feb 4.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, United States.

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.
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http://dx.doi.org/10.3389/fimmu.2020.00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010807PMC
March 2021

An intravital window to image the colon in real time.

Nat Commun 2019 12 11;10(1):5647. Epub 2019 Dec 11.

Department of Biomedical Engineering, Duke University, Durham, NC, 27710, USA.

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.
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http://dx.doi.org/10.1038/s41467-019-13699-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906443PMC
December 2019

Computed optical coherence microscopy of mouse brain ex vivo.

J Biomed Opt 2019 11;24(11):1-18

Cornell University, Meinig School of Biomedical Engineering, Ithaca, New York, United States.

The compromise between lateral resolution and usable imaging depth range is a bottleneck for optical coherence tomography (OCT). Existing solutions for optical coherence microscopy (OCM) suffer from either large data size and long acquisition time or a nonideal point spread function. We present volumetric OCM of mouse brain with a large depth coverage by leveraging computational adaptive optics (CAO) to significantly reduce the number of OCM volumes that need to be acquired with a Gaussian beam focused at different depths. We demonstrate volumetric reconstruction of mouse brain with lateral resolution of 2.2  μm, axial resolution of 4.7  μm, and depth range of ∼1.2  mm optical path length, using only 11 OCT data volumes acquired on a spectral-domain OCM system. Compared to focus scanning with step size equal to the Rayleigh length of the beam, this is a factor of 4 fewer datasets required for volumetric imaging. Coregistered two-photon microscopy confirmed that CAO-OCM reconstructions can visualize various tissue microstructures in the brain. Our results also highlight the limitations of CAO in highly scattering media, particularly when attempting to reconstruct far from the focal plane or when imaging deep within the sample.
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http://dx.doi.org/10.1117/1.JBO.24.11.116002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880187PMC
November 2019

Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.

Front Physiol 2018 31;9:969. Epub 2018 Jul 31.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, United States.

Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made , cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of interactions between cells and the native microenvironment are behind other organ systems. Our goal was to develop methods for intravital imaging of cardiac structural and calcium dynamics with microscopic resolution. Ventilated mice expressing GCaMP6f, a genetically encoded calcium indicator, received a thoracotomy to provide optical access to the heart. Vasculature was labeled with an injection of dextran-labeled dye. The heart was partially stabilized by a titanium probe with a glass window. Images were acquired at 30 frames per second with spontaneous heartbeat and continuously running, ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, vasculature, and GCaMP6f signal in cardiomyocytes as a function of both the cardiac and respiratory cycle. We demonstrated the capability to simultaneously measure calcium transients, vessel size, and tissue displacement in three dimensions with micrometer resolution. Reconstruction at various combinations of cardiac and respiratory phase enabled measurement of regional and single-cell cardiomyocyte calcium transients (GCaMP6f fluorescence). GCaMP6f fluorescence transients in individual, aberrantly firing cardiomyocytes were also quantified. Comparisons of calcium dynamics (rise-time and tau) at varying positions within the ventricle wall showed no significant depth dependence. This method enables studies of coupling between contraction and excitation during physiological blood perfusion and breathing at high spatiotemporal resolution. These capabilities could lead to a new understanding of normal and disease function of cardiac cells.
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http://dx.doi.org/10.3389/fphys.2018.00969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079295PMC
July 2018

Indoxyl Sulfate Induces Apoptosis and Hypertrophy in Human Kidney Proximal Tubular Cells.

Toxicol Pathol 2018 06 22;46(4):449-459. Epub 2018 Apr 22.

1 Centre for Kidney Disease Research, Translational Research Institute, University of Queensland, Brisbane, Australia.

Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with declining kidney function. Although generally thought of as a consequence of declining kidney function, emerging evidence demonstrates direct cytotoxic role of IS on endothelial cells and cardiomyocytes, largely through the expression of pro-inflammatory and pro-fibrotic factors. The direct toxicity of IS on human kidney proximal tubular epithelial cells (PTECs) remains a matter of debate. The current study explored the effect of IS on primary cultures of human PTECs and HK-2, an immortalized human PTEC line. Pathologically relevant concentrations of IS induced apoptosis and increased the expression of the proapoptotic molecule Bax in both cell types. IS impaired mitochondrial metabolic activity and induced cellular hypertrophy. Furthermore, statistically significant upregulation of pro-fibrotic (transforming growth factor-β, fibronectin) and pro-inflammatory molecules (interleukin-6, interleukin-8, and tumor necrosis factor-α) in response to IS was observed. Albumin had no influence on the toxicity of IS. The results of this study suggest that IS directly induced a pro-inflammatory and pro-fibrotic phenotype in proximal tubular cells. In light of the associated apoptosis, hypertrophy, and metabolic dysfunction, this study demonstrates that IS may play a role in the progression of chronic kidney disease.
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http://dx.doi.org/10.1177/0192623318768171DOI Listing
June 2018

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy.

Biomed Opt Express 2018 01 13;9(1):214-229. Epub 2017 Dec 13.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, 526 N. Campus Rd., Ithaca, NY 14853, USA.

Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool.
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http://dx.doi.org/10.1364/BOE.9.000214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772576PMC
January 2018

N-acetyl-cysteine increases cellular dysfunction in progressive chronic kidney damage after acute kidney injury by dampening endogenous antioxidant responses.

Am J Physiol Renal Physiol 2018 05 10;314(5):F956-F968. Epub 2018 Jan 10.

Centre for Kidney Disease Research, Faculty of Medicine, Translational Research Institute, University of Queensland , Brisbane , Australia.

Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-β1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.
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http://dx.doi.org/10.1152/ajprenal.00057.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842871PMC
May 2018

Effects of exercise and lifestyle intervention on oxidative stress in chronic kidney disease.

Redox Rep 2017 May 11;22(3):127-136. Epub 2017 Jan 11.

b School of Human Movement and Nutrition Sciences , The University of Queensland , Brisbane , Australia.

Objectives: Determine the effects of a 12-month exercise and lifestyle intervention program on changes in plasma biomarkers of oxidative stress in pre-dialysis chronic kidney disease (CKD) patients.

Methods: A total of 136 stage 3-4 CKD patients were randomized to receive standard nephrological care with (N = 72) or without (N = 64) a lifestyle and exercise intervention for 12 months. Plasma total F-isoprostanes (IsoP), glutathione peroxidase (GPX) activity, total antioxidant capacity (TAC), anthropometric and biochemical data were collected at baseline and at 12 months.

Results: There were no significant differences between groups at baseline. There were no significant differences in changes for standard care and lifestyle intervention, respectively, in IsoP (p = 0.88), GPX (p = 0.87), or TAC (p = 0.56). Patients identified as having high IsoP at baseline (>250 pg/mL) had a greater decrease in IsoP with lifestyle intervention compared to standard care; however, the difference was not statistically significant (p = 0.06). There was no difference in the change in kidney function (eGFR) between standard care and lifestyle intervention (p = 0.33).

Discussion: Exercise and lifestyle modification in stage 3-4 CKD did not produce changes in systemic biomarkers of oxidative stress over a 12-month period, but patients with high IsoP may benefit most from the addition of intervention to standard care.
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http://dx.doi.org/10.1080/13510002.2016.1276314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837678PMC
May 2017

Simultaneous optical and electrical in vivo analysis of the enteric nervous system.

Nat Commun 2016 06 7;7:11800. Epub 2016 Jun 7.

School of Electrical and Computer Engineering, Cornell University, Ithaca, New York 14853, USA.

The enteric nervous system (ENS) is a major division of the nervous system and vital to the gastrointestinal (GI) tract and its communication with the rest of the body. Unlike the brain and spinal cord, relatively little is known about the ENS in part because of the inability to directly monitor its activity in live animals. Here, we integrate a transparent graphene sensor with a customized abdominal window for simultaneous optical and electrical recording of the ENS in vivo. The implanted device captures ENS responses to neurotransmitters, drugs and optogenetic manipulation in real time.
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http://dx.doi.org/10.1038/ncomms11800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899629PMC
June 2016

Decreased apoptosis repressor with caspase recruitment domain confers resistance to sunitinib in renal cell carcinoma through alternate angiogenesis pathways.

Biochem Biophys Res Commun 2016 Apr 17;473(1):47-53. Epub 2016 Mar 17.

Centre for Kidney Disease Research, School of Medicine, Translational Research Institute, The University of Queensland, Brisbane, Queensland, 4102, Australia. Electronic address:

Apoptosis repressor with caspase recruitment domain (ARC), an endogenous inhibitor of apoptosis, is upregulated in a number of human cancers, thereby conferring drug resistance and giving a rationale for the inhibition of ARC to overcome drug resistance. Our hypothesis was that ARC would be similarly upregulated and targetable for therapy in renal cell carcinoma (RCC). Expression of ARC was assessed in 85 human RCC samples and paired non-neoplastic kidney by qPCR and immunohistochemistry, as well as in four RCC cell lines by qPCR, Western immunoblot and confocal microscopy. Contrary to expectations, ARC was significantly decreased in the majority of clear cell RCC and in three (ACHN, Caki-1 and 786-0) of the four RCC cell lines compared with the HK-2 non-cancerous human proximal tubular epithelial cell line. Inhibition of ARC with shRNA in the RCC cell line (SN12K1) that had shown increased ARC expression conferred resistance to Sunitinib, and upregulated interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). We therefore propose that decreased ARC, particularly in clear cell RCC, confers resistance to targeted therapy through restoration of tyrosine kinase-independent alternate angiogenesis pathways. Although the results are contrary to expectations from other cancer studies, they were confirmed here with multiple analytical methods. We believe the highly heterogeneous nature of cancers like RCC predicate that expression patterns of molecules must be interpreted in relation to respective matched non-neoplastic regions. In the current study, this procedure indicated that ARC is decreased in RCC.
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http://dx.doi.org/10.1016/j.bbrc.2016.03.048DOI Listing
April 2016

Protection against oxidative stress-induced apoptosis in kidney epithelium by Angelica and Astragalus.

J Ethnopharmacol 2016 Feb 21;179:412-9. Epub 2015 Dec 21.

Centre for Kidney Disease Research, School of Medicine, University of Queensland, Translational Research Institute, Brisbane, Australia. Electronic address:

Ethnopharmacological Relevance: Astragalus membranaceus either alone or in combination with Angelica sinensis has been used traditionally for kidney disease in East Asia and China for thousands of years. Previous studies using in vivo animal models have shown the benefits of these medicinal herbs in kidney diseases that involve oxidative stress. However, the mechanisms by which these medicinal herbs protect kidney cells remain largely unknown.

Aim Of The Study: To investigate the mechanisms by which ethanol, methanol and aqueous crude extracts of roots of A. membranaceus and A. sinensis afford protection to human kidney proximal tubular epithelial cells, using an in vitro model of oxidative stress.

Materials And Methods: Ethanol, methanol and aqueous extracts of roots of A. membranaceus and A. sinensis were prepared by a three-solvent sequential process. HK2 human kidney proximal tubular epithelial cells were treated with H2O2 alone (0.5mM) or in combination with different concentrations of extracts. Cell mitosis and death (microscopy) and cell viability (MTT assay) were compared. Western immunoblot was used to study expression of apoptosis-related proteins (pro-apoptotic Bax andanti-apoptotic Bcl-XL), and cell survival (NFκB subunits p65 and p50), pro-inflammatory (TNF-α) and protective (TGFβ1) proteins.

Results: H2O2-induced oxidative stress significantly increased apoptosis and reduced cell survival; upregulated pro-apoptotic and down-regulated Bcl-XL; increased NFκB (p65, p50); increased TNFα and decreased TGFβ1. All changes indicated kidney damage and dysfunction. All were modulated by all extracts of both plant species, except for NFκB which was only modulated by extracts of A. membranaceus.

Conclusions: In conclusion, in a model of oxidative stress that might occur after nephrotoxicity, the plant extracts were protective via anti-apoptotic and anti-inflammatory mechanisms.
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http://dx.doi.org/10.1016/j.jep.2015.12.027DOI Listing
February 2016

Intravital Multiphoton Imaging of the Kidney: Tubular Structure and Metabolism.

Methods Mol Biol 2016 ;1397:155-172

Centre for Kidney Disease Research, School of Medicine, Translational Research Institute, The University of Queensland, 37 Kent Street, Woolloongabba, Brisbane, QLD, 4102, Australia.

Multiphoton microscopy (MPM) allows the visualization of dynamic pathophysiological events in real time in live animals. Intravital imaging can be applied to investigate novel mechanisms and treatments of different forms of kidney disease as well as improve our understanding of normal kidney physiology. Using rodent models, in conjunction with endogenous fluorescence and infused exogenous fluorescent dyes, measurement can be made of renal processes such as glomerular permeability, juxtaglomerular apparatus function, interactions of the tubulointerstitium, tubulovascular interactions, vascular flow rate, and the renin-angiotensin-aldosterone system. Subcellular processes including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes of apoptosis and necrosis can also be seen and measured by MPM. The current methods chapter presents an overview of MPM with a focus on techniques for intravital kidney imaging and gives examples of instances where intravital MPM has been utilized to study renal pathophysiology. Suggestions are provided for MPM methods within the confines of intravital microscopy and selected kidney structure. MPM is undoubtedly a powerful new technique for application in experimental nephrology, and we believe it will continue to create new paradigms for understanding and treating kidney disease.
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http://dx.doi.org/10.1007/978-1-4939-3353-2_12DOI Listing
October 2016

Indoxyl sulphate and kidney disease: Causes, consequences and interventions.

Nephrology (Carlton) 2016 Mar;21(3):170-7

Centre for Kidney Disease Research, Translational Research Institute, School of Medicine, University of Queensland, Brisbane, Queensland, Australia.

In the last decade, chronic kidney disease (CKD), defined as reduced renal function (glomerular filtration rate (GFR) < 60 mL/min per 1.73 m(2) ) and/or evidence of kidney damage (typically manifested as albuminuria) for at least 3 months, has become one of the fastest-growing public health concerns worldwide. CKD is characterized by reduced clearance and increased serum accumulation of metabolic waste products (uremic retention solutes). At least 152 uremic retention solutes have been reported. This review focuses on indoxyl sulphate (IS), a protein-bound, tryptophan-derived metabolite that is generated by intestinal micro-organisms (microbiota). Animal studies have demonstrated an association between IS accumulation and increased fibrosis, and oxidative stress. This has been mirrored by in vitro studies, many of which report cytotoxic effects in kidney proximal tubular cells following IS exposure. Clinical studies have associated IS accumulation with deleterious effects, such as kidney functional decline and adverse cardiovascular events, although causality has not been conclusively established. The aims of this review are to: (i) establish factors associated with increased serum accumulation of IS; (ii) report effects of IS accumulation in clinical studies; (iii) critique the reported effects of IS in the kidney, when administered both in vivo and in vitro; and (iv) summarize both established and hypothetical therapeutic options for reducing serum IS or antagonizing its reported downstream effects in the kidney.
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http://dx.doi.org/10.1111/nep.12580DOI Listing
March 2016

O-GlcNAc protein modification in C2C12 myoblasts exposed to oxidative stress indicates parallels with endogenous antioxidant defense.

Biochem Cell Biol 2015 Feb 8;93(1):63-73. Epub 2014 Oct 8.

a Antioxidant Research Group, School of Human Movement Studies, The University of Queensland, Brisbane, QLD, Australia.

A growing body of evidence demonstrates the involvement of protein modification with O-linked β-N-acetylglucosamine (O-GlcNAc) in the stress response and its beneficial effects on cell survival. Here we investigated protein O-GlcNAcylation in skeletal muscle cells exposed to oxidative stress and the crosstalk with endogenous antioxidant system. The study focused on antioxidant enzymes superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase 1 (GPX1), and transcriptional regulators proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and forkhead box protein O1 (FOXO1), which play important roles in oxidative stress response and are known to be O-GlcNAc-modified. C2C12 myoblasts were subjected to 24 h incubation with different reagents, including hydrogen peroxide, diethyl maleate, high glucose, and glucosamine, and the inhibitors of O-GlcNAc cycling enzymes. Surprisingly, O-GlcNAc levels were significantly increased only with glucosamine, whilst other treatments showed no effect. Significant changes at the mRNA level were observed with concomitant upregulation of the genes for O-GlcNAc enzymes and stress-related proteins with oxidizing agents and downregulation of these genes with agents promoting O-GlcNAcylation. Our findings suggest a role of O-GlcNAc in the stress response and indicate an inhibitory mechanism controlling O-GlcNAc levels in the muscle cells. This could represent an important homeostatic regulation of the cellular defense system.
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http://dx.doi.org/10.1139/bcb-2014-0106DOI Listing
February 2015

Oxidative stress contributes to muscle atrophy in chronic kidney disease patients.

Redox Rep 2015 May 13;20(3):126-32. Epub 2014 Nov 13.

Objectives: Patients with chronic kidney disease have impaired muscle metabolism, resulting in muscle atrophy. Oxidative stress has previously been identified as a significant contributor to muscle atrophy in other populations, but the contribution in chronic kidney disease is unknown. The aim of this study was to investigate the association between oxidative stress, grip strength, and lean mass in patients with chronic kidney disease.

Methods: This is a cross-sectional study of 152 participants with stage 3 or 4 chronic kidney disease. Outcome measures include grip strength, lean mass, plasma total F2-isoprostanes, inflammation, peak oxygen uptake, and standard clinical measures.

Results: Thirty four (22.4%) chronic kidney disease patients had elevated oxidative stress levels (plasma F2-isoprostanes >250 pg/ml), with 82% of patients below age-predicted grip strength normative values. There was a significant negative association between plasma F2-isoprostanes and grip strength (r = -0.251) and lean mass (r = -0.243). There were no associations with inflammation markers. Multiple linear regression identified plasma F2-isoprostanes as a significant predictor of grip strength independent of other predictors: sex, diabetes status, body mass index, body fat percent, and phosphate (adjusted r(2) = 69.5, P < 0.001).

Discussion: Plasma F2-isoprostanes were independently associated with reduced strength in chronic kidney disease patients.
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http://dx.doi.org/10.1179/1351000214Y.0000000114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837381PMC
May 2015

Oxidative stress-induced alterations in PPAR-γ and associated mitochondrial destabilization contribute to kidney cell apoptosis.

Am J Physiol Renal Physiol 2014 Oct 13;307(7):F814-22. Epub 2014 Aug 13.

Centre for Kidney Disease Research, School of Medicine, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia;

The mechanism(s) underlying renoprotection by peroxisome proliferator-activated receptor (PPAR)-γ agonists in diabetic and nondiabetic kidney disease are not well understood. Mitochondrial dysfunction and oxidative stress contribute to kidney disease. PPAR-γ upregulates proteins required for mitochondrial biogenesis. Our aim was to determine whether PPAR-γ has a role in protecting the kidney proximal tubular epithelium (PTE) against mitochondrial destabilisation and oxidative stress. HK-2 PTE cells were subjected to oxidative stress (0.2-1.0 mM H₂O₂) for 2 and 18 h and compared with untreated cells for apoptosis, mitosis (morphology/biomarkers), cell viability (MTT), superoxide (dihydroethidium), mitochondrial function (MitoTracker red and JC-1), ATP (luminescence), and mitochondrial ultrastructure. PPAR-γ, phospho-PPAR-γ, PPAR-γ coactivator (PGC)-1α, Parkin (Park2), p62, and light chain (LC)3β were investigated using Western blots. PPAR-γ was modulated using the agonists rosiglitazone, pioglitazone, and troglitazone. Mitochondrial destabilization increased with H₂O₂concentration, ATP decreased (2 and 18 h; P < 0.05), Mitotracker red and JC-1 fluorescence indicated loss of mitochondrial membrane potential, and superoxide increased (18 h, P < 0.05). Electron microscopy indicated sparse mitochondria, with disrupted cristae. Mitophagy was evident at 2 h (Park2 and LC3β increased; p62 decreased). Impaired mitophagy was indicated by p62 accumulation at 18 h (P < 0.05). PPAR-γ expression decreased, phospho-PPAR-γ increased, and PGC-1α decreased (2 h), indicating aberrant PPAR-γ activation and reduced mitochondrial biogenesis. Cell viability decreased (2 and 18 h, P < 0.05). PPAR-γ agonists promoted further apoptosis. In summary, oxidative stress promoted mitochondrial destabilisation in kidney PTE, in association with increased PPAR-γ phosphorylation. PPAR-γ agonists failed to protect PTE. Despite positive effects in other tissues, PPAR-γ activation appears to be detrimental to kidney PTE health when oxidative stress induces damage.
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http://dx.doi.org/10.1152/ajprenal.00205.2014DOI Listing
October 2014

Thimerosal induces apoptotic and fibrotic changes to kidney epithelial cells in vitro.

Environ Toxicol 2015 Dec 18;30(12):1423-33. Epub 2014 Jun 18.

Centre for Kidney Disease Research, School of Medicine, University of Queensland, Translational Research Institute, Kent Street, Woolloongabba, Brisbane, Queensland, Australia.

Thimerosal is an ethyl mercury-containing compound used mainly in vaccines as a bactericide. Although the kidney is a key target for mercury toxicity, thimerosal nephrotoxicity has not received the same attention as other mercury species. The aim of this study was to determine the potential cytotoxic mechanisms of thimerosal on human kidney cells. Human kidney proximal tubular epithelial (HK2) cells were exposed for 24 h to thimerosal (0-2 µM), and assessed for cell viability, apoptosis, and cell proliferation; expression of proteins Bax, nuclear factor-κB subunits, and transforming growth factor-β1 (TGFβ1); mitochondrial health (JC-1, MitoTracker Red CMXRos); and fibronectin levels (enzyme-linked immunosorbent assay). Thimerosal diminished HK2 cell viability and mitosis, promoted apoptosis, impaired the mitochondrial permeability transition, enhanced Bax and TGFβ1 expression, and augmented fibronectin secretion. This is the first report about kidney cell death and pro-fibrotic mechanisms promoted by thimerosal. Collectively, these in vitro results demonstrate that (1) thimerosal induces kidney epithelial cell apoptosis via upregulating Bax and the mitochondrial apoptotic pathway, and (2) thimerosal is a potential pro-fibrotic agent in human kidney cells. We suggest that new evidence on toxicity as well as continuous surveillance in terms of fibrogenesis is required concerning thimerosal use.
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http://dx.doi.org/10.1002/tox.22012DOI Listing
December 2015

Multiphoton fluorescence microscopy of the live kidney in health and disease.

J Biomed Opt 2014 Feb;19(2):020901

University of Queensland, Translational Research Institute, Centre for Kidney Disease Research, Brisbane 4102, Australia.

The structural and functional heterogeneity of the kidney ensures a diversity of response in health and disease. Multiphoton microscopy has improved our understanding of kidney physiology and pathophysiology by enabling the visualization of the living kidney in comparison with the static view of previous technologies. The use of multiphoton microscopy with rodent models in conjunction with endogenous fluorescence and exogenous infused dyes permits the measurement of renal processes, such as glomerular permeability, juxtaglomerular apparatus function, tubulointerstitial function, tubulovascular interactions, vascular flow rate, and the intrarenal renin-angiotensin-aldosterone system. Subcellular processes, including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes apoptosis and necrosis, can also be measured by multiphoton microscopy. This has allowed valuable insight into the pathophysiology of diabetic nephropathy, renal ischemia-reperfusion injury, hypertensive nephropathy, as well as inflammatory responses of the kidney. The current review presents an overview of multiphoton microscopy with a focus on techniques for imaging the kidney and gives examples of instances where multiphoton microscopy has been utilized to study renal pathophysiology in the living kidney. With continued advancements in the field of biological optics and increased adoption in experimental nephrology, multiphoton microscopy will undoubtedly continue to create new paradigms in kidney disease.
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http://dx.doi.org/10.1117/1.JBO.19.2.020901DOI Listing
February 2014

Fibronectin and transforming growth factor beta contribute to erythropoietin resistance and maladaptive cardiac hypertrophy.

Biochem Biophys Res Commun 2014 Feb 22;444(3):332-7. Epub 2014 Jan 22.

Centre for Kidney Disease Research, School of Medicine, The University of Queensland at Translational Research Institute, Brisbane, Queensland 4102, Australia. Electronic address:

The use of recombinant human erythropoietin (rhEPO) to promote repair and minimize cardiac hypertrophy after myocardial infarction has had disappointing outcomes in clinical trials. We hypothesized that the beneficial non-hematopoietic effects of rhEPO against cardiac hypertrophy could be offset by the molecular changes initiated by rhEPO itself, leading to rhEPO resistance or maladaptive hypertrophy. This hypothesis was investigated using an isoproterenol-induced model of myocardial infarct and cardiac remodelling with emphasis on hypertrophy. In h9c2 cardiomyocytes, rhEPO decreased isoproterenol-induced hypertrophy, and the expression of the pro-fibrotic factors fibronectin, alpha smooth muscle actin and transforming growth factor beta-1 (TGF-β1). In contrast, by itself, rhEPO increased the expression of fibronectin and TGF-β1. Exogenous TGF-β1 induced a significant increase in hypertrophy, which was further potentiated by rhEPO. Exogenous fibronectin not only induced hypertrophy of cardiomyocytes, but also conferred resistance to rhEPO treatment. Based on these findings we propose that the outcome of rhEPO treatment for myocardial infarction is determined by the baseline concentrations of fibronectin and TGF-β1. If endogenous fibronectin or TGF-β levels are above a certain threshold, they could cause resistance to rhEPO therapy and enhancement of cardiac hypertrophy, respectively, leading to maladaptive hypertrophy.
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http://dx.doi.org/10.1016/j.bbrc.2014.01.047DOI Listing
February 2014

Oxidative stress and cell senescence combine to cause maximal renal tubular epithelial cell dysfunction and loss in an in vitro model of kidney disease.

Nephron Exp Nephrol 2012 1;122(3-4):123-30. Epub 2013 Jun 1.

Centre for Kidney Disease Research, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Brisbane, Qld., Australia.

Background: The incidence and cost of chronic kidney disease (CKD) are increasing. Renal tubular epithelial cell dysfunction and attrition, involving increased apoptosis and cell senescence, are central to the pathogenesis of CKD. The aim here was to use an in vitro model to investigate the separate and cumulative effects of oxidative stress, mitochondrial dysfunction and cell senescence in promoting loss of renal mass.

Methods: Human kidney tubular epithelial cells (HK2) were treated with moderate hydrogen peroxide (H2O2) for oxidative stress, with or without cell cycle inhibition (apigenin, API) for cell senescence. Adenosine triphosphate (ATP) and oxidative stress were measured by ATP assay, lipid peroxidation, total antioxidant capacity, mitochondrial function with confocal microscopy, MitoTracker Red CMXRos and live cell imaging with JC-1. In parallel, cell death and injury (i.e. apoptosis and Bax/Bcl-XL expression, lactate dehydrogenase), cell senescence (SA-β-galactosidase) and renal regenerative ability (cell proliferation), and their modulation with the anti-oxidant N-acetyl-cysteine (NAC) were investigated.

Results: H2O2 and API, separately, increased oxidative stress and mitochondrial dysfunction, apoptosis and cell senescence. Although API caused cell senescence, it also induced oxidative stress at levels similar to H2O2 treatment alone, indicating that senescence and oxidative stress may be intrinsically linked. When H2O2 and API were delivered concurrently, their detrimental effects on renal cell loss were compounded. The antioxidant NAC attenuated apoptosis and senescence, and restored regenerative potential to the kidney.

Conclusion: Oxidative stress and cell senescence both cause mitochondrial destabilization and cell loss and contribute to the development of the cellular characteristics of CKD.
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http://dx.doi.org/10.1159/000350726DOI Listing
January 2014

Cytochrome c: potential as a noninvasive biomarker of drug-induced acute kidney injury.

Expert Opin Drug Metab Toxicol 2012 Jun 5;8(6):655-64. Epub 2012 Apr 5.

The University of Queensland, Princess Alexandra Hospital, School of Medicine, Centre for Kidney Disease Research, Brisbane, 4102, Australia.

Introduction: Acute kidney injury (AKI) in critically ill patients is closely associated with increased morbidity and mortality, yet there remains continued reliance on increased serum creatinine and blood urea nitrogen to diagnose AKI. These biomarkers increase only after significant renal structural damage has occurred. Recent research efforts have focused on discovery and validation of novel serum and urine biomarkers to detect AKI prior to extensive structural damage. Cytochrome c is best known as an indicator of cell death burden in any organ or tissue. It is released during mitochondrial damage that is associated with processing of apoptosis, cell lysis during necrosis and even reversible mitochondrial and cell injury.

Areas Covered: This article reviews the current literature on the potential for cytochrome c as an early biomarker of AKI. The article is based on PubMed searches, using the terms 'acute kidney injury,' 'renal failure,' 'biomarker,' 'toxicity' and 'cytochrome c', with a focus on experimental and clinical data.

Expert Opinion: Cytochrome c, as a biomarker, has the potential to improve outcome for AKI patients. Its release indicates mitochondrial damage, one of the earliest changes in cell injury and death. New mitochondrial-targeted therapeutics may be designed around this molecule. Its disadvantages include only transient increase at expression levels that are easily measurable and nonspecificity for kidney injury. The appropriate and optimal utilization of cytochrome c as a biomarker for AKI will be realized only after its complete characterization in experimental and clinical arenas.
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http://dx.doi.org/10.1517/17425255.2012.679657DOI Listing
June 2012

Oxidative stress, anti-oxidant therapies and chronic kidney disease.

Nephrology (Carlton) 2012 May;17(4):311-21

Centre for Kidney Disease Research, School of Medicine, The University of Queensland, Queensland, Australia.

Chronic kidney disease (CKD) is a common and serious problem that adversely affects human health, limits longevity and increases costs to health-care systems worldwide. Its increasing incidence cannot be fully explained by traditional risk factors. Oxidative stress is prevalent in CKD patients and is considered to be an important pathogenic mechanism. Oxidative stress develops from an imbalance between free radical production often increased through dysfunctional mitochondria formed with increasing age, type 2 diabetes mellitus, inflammation, and reduced anti-oxidant defences. Perturbations in cellular oxidant handling influence downstream cellular signalling and, in the kidney, promote renal cell apoptosis and senescence, decreased regenerative ability of cells, and fibrosis. These factors have a stochastic deleterious effect on kidney function. The majority of studies investigating anti-oxidant treatments in CKD patients show a reduction in oxidative stress and many show improved renal function. Despite heterogeneity in the oxidative stress levels in the CKD population, there has been little effort to measure patient oxidative stress levels before the use of any anti-oxidants therapies to optimize outcome. This review describes the development of oxidative stress, how it can be measured, the involvement of mitochondrial dysfunction and the molecular pathways that are altered, the role of oxidative stress in CKD pathogenesis and an update on the amelioration of CKD using anti-oxidant therapies.
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http://dx.doi.org/10.1111/j.1440-1797.2012.01572.xDOI Listing
May 2012

The role of readiness to change in response to treatment of adolescent depression.

J Consult Clin Psychol 2009 Jun;77(3):422-8

Department of Psychology, University of Oregon, Eugene, OR 97403, USA.

The effect of readiness to change on treatment outcome was examined among 332 adolescents (46% male, 74% Caucasian), ages 12 through 17 years (M = 14.6, SD = 1.5), with major depressive disorder who were participating in the Treatment for Adolescents With Depression Study (TADS). TADS is a randomized clinical trial comparing the effectiveness of fluoxetine (an antidepressant medication), cognitive-behavioral therapy, their combination, and a pill placebo. An abbreviated Stages of Change Questionnaire was used to obtain 4 readiness to change scores: precontemplation, contemplation, action, and maintenance. The association between each readiness score and depression severity across 12 weeks of acute treatment for depression, as measured by the Children's Depression Rating Scale--Revised, was examined. Although treatment response was not moderated by any of the readiness scores, baseline action scores predicted outcome: Higher action scores were associated with better outcome regardless of treatment modality. Furthermore, treatment effects were mediated by change in action scores during the first 6 weeks of treatment, with increases in action scores related to greater improvement in depression. Assessing readiness to change may have implications for tailoring treatments for depressed adolescents.
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http://dx.doi.org/10.1037/a0014154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822468PMC
June 2009