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.
17 Related JoVE Articles!
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
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
5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat
Institutions: University Medical Center Utrecht.
Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1
), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.
Medicine, Issue 77, Anatomy, Physiology, Biomedical Engineering, Surgery, Nephrology Kidney Diseases, Glomerular Filtration Rate, Hemodynamics, Surgical Procedures, Operative, Chronic kidney disease, remnant kidney, chronic renal diseases, kidney, Nitric Oxide depletion, NO depletion, high salt diet, proteinuria, uremia, glomerulosclerosis, transgenic rat, animal model
Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats
Institutions: Indiana University School of Medicine.
Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4
. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo
. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6
. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2
. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.
Medicine, Issue 74, Biomedical Engineering, Molecular Biology, Cellular Biology, Anatomy, Physiology, Surgery, Nephrology, Kidney Diseases, Two-photon microscopy, Kidney, Glomerulus, Glomerular Sieving Coefficient (GSC), Permeability, Proximal Tubule, Proteinuria, macromolecules, 2 Photon, microscopy, intravital imaging, munich wistar rat, animal model
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
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)
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)
Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2
. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5
. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6
. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7
. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9
. Bevacizumab however failed to prolong overall survival in a recent phase III trial26
. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12
, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
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
Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE
Institutions: University of Cambridge, UK, University of Cambridge, UK.
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via
functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v
.) or intracerebroventricular (i.c.v.
) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.
) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo
Here we describe the methods that we have developed for the i.v
. and i.c.v.
delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
Immunology, Issue 86, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
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
Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity
Institutions: University of New South Wales , University of New South Wales , University of New South Wales .
Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2
. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3
. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4
. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6
. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo
in large myelinated human axons7-9
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13
. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ
in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+
channelopathy in motor and sensory axons10-13
. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11
. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10
. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10
. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.
Neuroscience, Issue 62, Chemotherapy, Neurotoxicity, Neuropathy, Nerve excitability, Ion channel function, Oxaliplatin, oncology, medicine
A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice
Institutions: University of California, San Diego , San Diego VA Healthcare System.
The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3
H or 14
C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 μl of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e.
doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.
Medicine, Issue 75, Anatomy, Physiology, Biomedical Engineering, Molecular Biology, Nephrology, Kidney Function Tests, Glomerular filtration rate, rats, mice, conscious, creatinine, inulin, Jaffe, hypertension, HPLC, animal model
Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation
Institutions: Piedmont Heart Institute, Medtronic Inc..
Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.
Medicine, Issue 72, Anatomy, Physiology, Cardiology, Biomedical Engineering, Surgery, Cardiovascular System, Cardiovascular Diseases, Surgical Procedures, Operative, Investigative Techniques, Atrial fibrillation, Cryoballoon Ablation, Pulmonary Vein Occlusion, Pulmonary Vein Isolation, electrophysiology, catheterizatoin, heart, vein, clinical, surgical device, surgical techniques
Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
Institutions: University of Illinois Chicago - UIC.
Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa
, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa
are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8
. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11
. In this example, we use an RND protein, the P. aeruginosa
MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.
Cellular Biology, Issue 46, multidrug resistance, membrane protein, purification, transmembrane transport, MexB, detergent solubilization, protein detergent complex