The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.
17 Related JoVE Articles!
Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration
Institutions: University of Edinburgh.
Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.
Medicine, Issue 88, Murine, Acute Kidney Injury, Ischaemia, Reperfusion, Nephrectomy, Regeneration, Laparotomy
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys
Institutions: Eastern Virginia Medical School, Medical University of South Carolina, University of Michigan.
Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a
gene with wnt5a
antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP)
transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a
knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a
knockdown. Injection of mouse Wnt5a
mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a
function in the kidney.
Medicine, Issue 94, Wnt5a, polycystic kidney disease, morpholino, microinjection, zebrafish, pronephros
Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration
Institutions: University of Notre Dame .
Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1
. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1
. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2
. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2
, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3
. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2
. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5
. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6
. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8
. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.
Developmental Biology, Issue 54, kidney, zebrafish, regeneration, epithelium, acute kidney injury, ablation
Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury
Institutions: University of Pittsburgh, University of Pittsburgh, Harvard Medical School.
In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant. The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography. We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.
Developmental Biology, Issue 42, intravenous microinjection, zebrafish, gentamicin, acute kidney injury
Dissection of Organs from the Adult Zebrafish
Institutions: University of Pennsylvania-School of Medicine.
Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease. Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition. Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling. In addition, the bipotential gonad primordium develops into either testis or ovary. This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques. This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.
Developmental Biology, Issue 37, adult, zebrafish, organs, dissection, anatomy
A β-glucuronidase (GUS) Based Cell Death Assay
Institutions: Texas A&M University.
We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, β-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana
leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium
strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.
Plant Biology, Issue 51, Cell death, GUS, Transient expression, Nicotiana benthamiana.
Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice
Institutions: Vanderbilt University Medical Center.
Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.
Medicine, Issue 78, Immunology, Infection, Biomedical Engineering, Anatomy, Physiology, Kidney, Mice, Inbred Strains, Renal Insufficiency, Acute Kidney Injury, Ischemia-reperfusion, acute kidney injury, post injury fibrosis, mice, ischemia, reperfusion, fibrosis, animal model
Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization
Institutions: University of Notre Dame.
The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ
hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.
Developmental Biology, Issue 89, animals, vertebrates, fishes, zebrafish, growth and development, morphogenesis, embryonic and fetal development, organogenesis, natural science disciplines, embryo, whole mount in situ hybridization, flat mount, deyolking, imaging
A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells
Institutions: University of Massachusetts, University of Massachusetts, University of Massachusetts.
Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5
. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8
. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9
. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13
. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11
. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.
Cancer Biology, Issue 55, tumor, vascular, endothelial, tube formation, Matrigel, in vitro
Development of automated imaging and analysis for zebrafish chemical screens.
Institutions: University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, University of Pittsburgh, University of Pittsburgh.
We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1
. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2
. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3
. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.
Cellular Biology, Issue 40, Zebrafish, Chemical Screens, Cognition Network Technology, Fibroblast Growth Factor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI),Tg(dusp6:d2EGFP)
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+
release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Institutions: University of Arkansas for Medical Sciences .
The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro
model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo
. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0
-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0
-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro
Cellular Biology, Issue 71, Biochemistry, Molecular Biology, Genetics, Pharmacology, Physiology, Medicine, Protein, Mitochondrial dysfunction, mitochondria, protein kinase C, renal proximal tubular cells, reactive oxygen species, oxygen consumption, electron transport chain, respiratory complexes, ATP, adenovirus, primary culture, ischemia, cells, flow cytometry
Mouse Kidney Transplantation: Models of Allograft Rejection
Institutions: The University of Edinburgh.
Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.
Medicine, Issue 92, transplantation, mouse model, surgery, kidney, immunology, rejection
Use of a Hanging-weight System for Isolated Renal Artery Occlusion
Institutions: University of Colorado, University of Colorado, Korea University College of Medicine.
In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3
. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1
. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4
. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5
. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6
. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6
.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1
. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13
. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14
. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.
Medicine, Issue 53, targeted gene deletion, murine model, acute renal failure, ischemia, reperfusion, video demonstration