Publications by authors named "Andrew M Hall"

56 Publications

Live Imaging of Mitochondria in Kidney Tissue.

Methods Mol Biol 2021 ;2275:393-402

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

Kidneys are highly aerobic organs and their function is tightly coupled to mitochondrial energy production. Renal tubular cells, particularly the proximal tubule (PT), require an abundance of mitochondria to provide sufficient energy for regulating fluid and electrolyte balance. Meanwhile, mitochondrial defects are implicated in a range of different kidney diseases. Multiphoton microscopy (MP) is a powerful tool that allows detailed study of mitochondrial morphology, dynamics, and function in kidney tissue. Here, we describe how MP can be used to image mitochondria in kidney tubular cells, either ex vivo in tissue slices or in vivo in living rodents, using both endogenous and exogenous fluorescent molecules. Moreover, changes in mitochondrial signals can be followed in real time in response to different insults or stimuli, in parallel with other important readouts of kidney tubular function, such as solute uptake and trafficking.
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http://dx.doi.org/10.1007/978-1-0716-1262-0_25DOI Listing
January 2021

Drug toxicity in the proximal tubule: new models, methods and mechanisms.

Pediatr Nephrol 2021 May 28. Epub 2021 May 28.

Department of Renal Medicine, University College London, London, UK.

The proximal tubule (PT) reabsorbs most of the glomerular filtrate and plays an important role in the uptake, metabolism and excretion of xenobiotics. Some therapeutic drugs are harmful to the PT, and resulting nephrotoxicity is thought to be responsible for approximately 1 in 6 of cases of children hospitalized with acute kidney injury (AKI). Clinically, PT dysfunction leads to urinary wasting of important solutes normally reabsorbed by this nephron segment, leading to systemic complications such as bone demineralization and a clinical scenario known as the renal Fanconi syndrome (RFS). While PT defects can be diagnosed using a combination of blood and urine markers, including urinary excretion of low molecular weight proteins (LMWP), standardized definitions of what constitutes clinically significant toxicity are lacking, and identifying which patients will go on to develop progressive loss of kidney function remains a major challenge. In addition, much of our understanding of cellular mechanisms of drug toxicity is still limited, partly due to the constraints of available cell and animal models. However, advances in new and more sophisticated in vitro models of the PT, along with the application of high-content analytical methods that can provide readouts more relevant to the clinical manifestations of nephrotoxicity, are beginning to extend our knowledge. Such technical progress should help in discovering new biomarkers that can better detect nephrotoxicity earlier and predict its long-term consequences, and herald a new era of more personalized medicine.
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http://dx.doi.org/10.1007/s00467-021-05121-9DOI Listing
May 2021

Intravital kidney microscopy: entering a new era.

Kidney Int 2021 May 17. Epub 2021 May 17.

Institute of Anatomy, University of Zurich, Zurich, Switzerland; Department of Nephrology, University Hospital Zurich, Zurich, Switzerland. Electronic address:

The development of intravital imaging with multiphoton microscopy has had a major impact on kidney research. It provides the unique opportunity to visualize dynamic behavior of cells and organelles in their native environment and to relate this to the complex 3-dimensional structure of the organ. Moreover, changes in cell/organelle function can be followed in real time in response to physiological interventions or disease-causing insults. However, realizing the enormous potential of this exciting approach has necessitated overcoming several substantial practical hurdles. In this article, we outline the nature of these challenges and how a variety of technical advances have provided effective solutions. In particular, improvements in laser/microscope technology, fluorescent probes, transgenic animals, and abdominal windows are collectively making previously opaque processes visible. Meanwhile, the rise of machine learning-based image analysis is facilitating the rapid generation of large amounts of quantitative data, amenable to deeper statistical interrogation. Taken together, the increased capabilities of multiphoton imaging are opening up huge new possibilities to study structure-function relationships in the kidney in unprecedented detail. In addition, they are yielding important new insights into cellular mechanisms of tissue damage, repair, and adaptive remodeling during disease states. Thus, intravital microscopy is truly entering an exciting new era in translational kidney research.
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http://dx.doi.org/10.1016/j.kint.2021.02.042DOI Listing
May 2021

Nuclear Spin Relaxation of Longitudinal and Singlet Order in Liquid-CO Solutions.

Front Chem 2021 26;9:668044. Epub 2021 Apr 26.

School of Chemistry, University of Southampton, Southampton, United Kingdom.

Hyperpolarization techniques can enormously enhance the NMR signal thus allowing the exploitation of hyperpolarized substrates for MRI applications. The short lifetime of hyperpolarized spin order poses significant limitations in such applications. Spin order storage can be prolonged through the use of long-lived spin states. Additionally, the storage of spin polarization-either in the form of longitudinal or singlet order-can be prolonged in low viscosity solutions. Here, we report the use of low viscosity liquid-CO solutions to store nuclear spin polarization in the form of longitudinal and singlet order for extended periods. Our results demonstrate that this storage time can be considerably sustained in liquid-CO solutions in comparison to other low viscosity solvents, opening up the possibility of new, exciting storage experiments in the future.
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http://dx.doi.org/10.3389/fchem.2021.668044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107397PMC
April 2021

The proximal tubule, protein uptake, and the riddle of the segments.

Kidney Int 2021 04;99(4):803-805

Mechanisms of Inherited Kidney Disorders Group, Institute of Physiology, University of Zurich, Zurich, Switzerland.

The proximal tubule is divided anatomically into 3 distinct segments-S1 to S3-on the basis of differences in cellular ultrastructure, but the functional processes that define and shape these remain elusive. In a new study, Christensen used 3-dimensional nephron reconstruction, electron microscopy, and antibody staining to precisely map protein uptake to the structure of the proximal tubule. They reported striking axial patterns in endocytosis along the segments, which showed substantial plasticity in disease states.
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http://dx.doi.org/10.1016/j.kint.2020.12.031DOI Listing
April 2021

The red blood cell as a novel regulator of human B-cell activation.

Immunology 2021 Mar 17. Epub 2021 Mar 17.

Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK.

Non-immune cells are increasingly recognized as important in regulating immunity, but the role of red blood cells (RBC) remains relatively unexplored, despite their abundance in the circulation and a cell surface rich in potential ligands. Here, we determine whether RBC influence the activation state of human B cells. Separation of RBC from peripheral blood mononuclear cells increased B-cell expression of HLA-DR/DP/DQ, whilst reconstitution reduced the levels of B-cell activation markers HLA-DR/DP/DQ, CD86, CD69 and CD40, as well as decreasing proliferative responses and IgM secretion. Inhibition of B cells required contact with RBC and was abrogated by either removal of sialic acids from RBC or blocking the corresponding lectin receptor CD22 on B cells. Chronic lymphocytic leukaemia B cells express low levels of CD22 and were less susceptible to inhibition by RBC, which may contribute to their activated phenotype. Taken together, the results identify a novel mechanism that may suppress inappropriate responsiveness of healthy B cells whilst circulating in the bloodstream.
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http://dx.doi.org/10.1111/imm.13327DOI Listing
March 2021

Changes in NAD and Lipid Metabolism Drive Acidosis-Induced Acute Kidney Injury.

J Am Soc Nephrol 2021 Feb 21;32(2):342-356. Epub 2021 Jan 21.

Institute of Anatomy, University of Zurich, Zurich, Switzerland

Background: The kidney plays an important role in maintaining normal blood pH. Metabolic acidosis (MA) upregulates the pathway that mitochondria in the proximal tubule (PT) use to produce ammonia and bicarbonate from glutamine, and is associated with AKI. However, the extent to which MA causes AKI, and thus whether treating MA would be beneficial, is unclear.

Methods: Gavage with ammonium chloride induced acute MA. Multiphoton imaging of mitochondria (NADH/membrane potential) and transport function (dextran/albumin uptake), oxygen consumption rate (OCR) measurements in isolated tubules, histologic analysis, and electron microscopy in fixed tissue, and urinary biomarkers (KIM-1/clara cell 16) assessed tubular cell structure and function in mouse kidney cortex.

Results: MA induces an acute change in NAD redox state (toward oxidation) in PT mitochondria, without changing the mitochondrial energization state. This change is associated with a switch toward complex I activity and decreased maximal OCR, and a major alteration in normal lipid metabolism, resulting in marked lipid accumulation in PTs and the formation of large multilamellar bodies. These changes, in turn, lead to acute tubular damage and a severe defect in solute uptake. Increasing blood pH with intravenous bicarbonate substantially improves tubular function, whereas preinjection with the NAD precursor nicotinamide (NAM) is highly protective.

Conclusions: MA induces AKI changes in PT NAD and lipid metabolism, which can be reversed or prevented by treatment strategies that are viable in humans. These findings might also help to explain why MA accelerates decline in function in CKD.
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http://dx.doi.org/10.1681/ASN.2020071003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054907PMC
February 2021

A temperature-controlled sample shuttle for field-cycling NMR.

J Magn Reson 2020 Aug 27;317:106778. Epub 2020 Jun 27.

University of Southampton, Highfield Campus, Southampton SO17 1BJ, United Kingdom. Electronic address:

We present a design for a temperature-controlled sample shuttle for use in NMR measurements at variable magnetic field strength. Accurate temperature control was achieved using a mixture of water-ethylene glycol as a heat transfer fluid, reducing temperature gradients across the sample to < 0.05 °C and minimising convection. Using the sample shuttle, we show how the longitudinal (T) and singlet order (T) relaxation time constants were measured for two molecules capable of supporting long-lived states, with new record lifetimes observed at low field and above ambient temperatures.
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http://dx.doi.org/10.1016/j.jmr.2020.106778DOI Listing
August 2020

Quantitative intravital Ca imaging maps single cell behavior to kidney tubular structure.

Am J Physiol Renal Physiol 2020 08 22;319(2):F245-F255. Epub 2020 Jun 22.

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

Ca is an important second messenger that translates extracellular stimuli into intracellular responses. Although there has been significant progress in understanding Ca dynamics in organs such as the brain, the nature of Ca signals in the kidney is still poorly understood. Here, we show that by using a genetically expressed highly sensitive reporter (GCaMP6s), it is possible to perform imaging of Ca signals at high resolution in the mouse kidney in vivo. Moreover, by applying machine learning-based automated analysis using a Ca-independent signal, quantitative data can be extracted in an unbiased manner. By projecting the resulting data onto the structure of the kidney, we show that different tubular segments display highly distinct spatiotemporal patterns of Ca signals. Furthermore, we provide evidence that Ca activity in the proximal tubule decreases with increasing distance from the glomerulus. Finally, we demonstrate that substantial changes in intracellular Ca can be detected in proximal tubules in a cisplatin model of acute kidney injury, which can be linked to alterations in cell structure and transport function. In summary, we describe a powerful new tool to investigate how single cell behavior is integrated with whole organ structure and function and how it is altered in disease states relevant to humans.
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http://dx.doi.org/10.1152/ajprenal.00052.2020DOI Listing
August 2020

Multiparametric imaging reveals that mitochondria-rich intercalated cells in the kidney collecting duct have a very high glycolytic capacity.

FASEB J 2020 06 4;34(6):8510-8525. Epub 2020 May 4.

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

Alpha intercalated cells (αICs) in the kidney collecting duct (CD) belong to a family of mitochondria rich cells (MRCs) and have a crucial role in acidifying the urine via apical V-ATPase pumps. The nature of metabolism in αICs and its relationship to transport was not well-understood. Here, using multiphoton live cell imaging in mouse kidney tissue, FIB-SEM, and other complementary techniques, we provide new insights into mitochondrial structure and function in αICs. We show that αIC mitochondria have a rounded structure and are not located in close proximity to V-ATPase containing vesicles. They display a bright NAD(P)H fluorescence signal and low uptake of voltage-dependent dyes, but are energized by a pH gradient. However, expression of complex V (ATP synthase) is relatively low in αICs, even when stimulated by metabolic acidosis. In contrast, anaerobic glycolytic capacity is surprisingly high, and sufficient to maintain intracellular calcium homeostasis in the presence of complete aerobic inhibition. Moreover, glycolysis is essential for V-ATPase-mediated proton pumping. Key findings were replicated in narrow/clear cells in the epididymis, also part of the MRC family. In summary, using a range of cutting-edge techniques to investigate αIC metabolism in situ, we have discovered that these mitochondria dense cells have a high glycolytic capacity.
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http://dx.doi.org/10.1096/fj.202000273RDOI Listing
June 2020

The iron chelator Deferasirox causes severe mitochondrial swelling without depolarization due to a specific effect on inner membrane permeability.

Sci Rep 2020 01 31;10(1):1577. Epub 2020 Jan 31.

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.
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http://dx.doi.org/10.1038/s41598-020-58386-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994599PMC
January 2020

Targeting glycolysis in proliferative kidney diseases.

Am J Physiol Renal Physiol 2019 12 11;317(6):F1531-F1535. Epub 2019 Nov 11.

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

Glycolytic activity is increased in proliferating cells, leading to the concept that glycolysis could be a therapeutic target in cystic diseases and kidney cancer. Preclinical studies using the glucose analog 2-deoxy-d-glucose have shown promise; however, inhibiting glycolysis in humans is unlikely to be without risks. While proximal tubules are predominantly aerobic, later segments are more glycolytic. Understanding exactly where and why glycolysis is important in the physiology of the distal nephron is thus crucial in predicting potential adverse effects of glycolysis inhibitors. Live imaging techniques could play an important role in the process of characterizing cellular metabolism in the functioning kidney. The goal of this review is to briefly summarize recent findings on targeting glycolysis in proliferative kidney diseases and to highlight the necessity for future research focusing on glycolysis in the healthy kidney.
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http://dx.doi.org/10.1152/ajprenal.00460.2019DOI Listing
December 2019

Axial differences in endocytosis along the kidney proximal tubule.

Am J Physiol Renal Physiol 2019 12 28;317(6):F1526-F1530. Epub 2019 Oct 28.

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

The proximal tubule (PT) reabsorbs filtered proteins via receptor-mediated endocytosis to prevent energetically inefficient wasting in the urine. Recent intravital imaging studies have suggested that protein reabsorption occurs in early (S1) segments, which have a very high capacity. In contrast, uptake of fluid phase substrates also occurs in distal (S2) segments. In this article, we will review these findings and their implications for understanding integrated proximal tubular function, patterns of damage caused by endocytosed toxins, and the origins of proteinuria. We will also discuss whether compensatory downstream increases in protein uptake might occur in disease states, and the environmental factors that could drive these changes.
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http://dx.doi.org/10.1152/ajprenal.00459.2019DOI Listing
December 2019

A Case of Drug-Induced Proximal Tubular Dysfunction.

Clin J Am Soc Nephrol 2019 09 10;14(9):1384-1387. Epub 2019 Jun 10.

Department of Renal Medicine, University College London, London, United Kingdom; and.

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http://dx.doi.org/10.2215/CJN.01430219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730515PMC
September 2019

Combination peptide immunotherapy suppresses antibody and helper T-cell responses to the major human platelet autoantigen glycoprotein IIb/IIIa in HLA-transgenic mice.

Haematologica 2019 05 4;104(5):1074-1082. Epub 2018 Dec 4.

Institute of Medical Sciences, Ashgrove Road West, University of Aberdeen

Platelet destruction in immune thrombocytopenia is caused by autoreactive antibody and T-cell responses, most commonly directed against platelet glycoprotein IIb/IIIa. Loss of self-tolerance in the disease is also associated with deficient activity of regulatory T cells. Having previously mapped seven major epitopes on platelet glycoprotein IIIa that are recognized by helper T cells from patients with immune thrombocytopenia, the aim was to test whether peptide therapy with any of these sequences, alone or in combination, could inhibit responses to the antigen in humanized mice expressing HLA-DR15. None of the individual peptides, delivered by a putative tolerogenic regimen, consistently suppressed the antibody response to subsequent immunization with human platelet glycoprotein IIb/IIIa. However, the combination of glycoprotein IIIa peptides aa6-20 and aa711-725, which contain the predominant helper epitopes in patients and elicited the strongest trends to suppress when used individually, did abrogate this response. The peptide combination also blunted, but did not reverse, the ongoing antibody response when given after immunization. Suppression of antibody was associated with reduced splenocyte T-cell responsiveness to the antigen, and with the induction of a regulatory T-cell population that is more responsive to the peptides than to purified platelet glycoprotein IIb/IIIa. Overall, these data demonstrate that combinations of peptides containing helper epitopes, such as platelet glycoprotein IIIa aa6-20 and aa711-725, can promote suppression of responses to the major antigen implicated in immune thrombocytopenia. The approach offers a promising therapeutic option to boost T-cell regulation, which should be taken forward to clinical trials.
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http://dx.doi.org/10.3324/haematol.2017.179424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518892PMC
May 2019

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

J Am Soc Nephrol 2018 11 9;29(11):2696-2712. Epub 2018 Oct 9.

Institute of Anatomy,

Background: The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown.

Methods: To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach.

Results: Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology.

Conclusions: Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.
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http://dx.doi.org/10.1681/ASN.2018050522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218873PMC
November 2018

Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule.

Am J Physiol Renal Physiol 2018 12 22;315(6):F1613-F1625. Epub 2018 Aug 22.

Institute of Anatomy, University of Zurich , Zurich , Switzerland.

Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.
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http://dx.doi.org/10.1152/ajprenal.00165.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336998PMC
December 2018

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure.

J Am Soc Nephrol 2018 07 13;29(7):1849-1858. Epub 2018 Apr 13.

Centre for Nephrology and.

For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure. We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations. The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. analysis showed that the particular mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death. In this novel genetic disorder, fully penetrant heterozygous missense mutations in trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.
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http://dx.doi.org/10.1681/ASN.2017111179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050927PMC
July 2018

The targeted anti-oxidant MitoQ causes mitochondrial swelling and depolarization in kidney tissue.

Physiol Rep 2018 04;6(7):e13667

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is implicated in the pathogenesis of numerous kidney diseases. Damaged mitochondria are thought to produce excess reactive oxygen species (ROS), which can lead to oxidative stress and activation of cell death pathways. MitoQ is a mitochondrial targeted anti-oxidant that has shown promise in preclinical models of renal diseases. However, recent studies in nonkidney cells have suggested that MitoQ might also have adverse effects. Here, using a live imaging approach, and both in vitro and ex vivo models, we show that MitoQ induces rapid swelling and depolarization of mitochondria in PT cells, but these effects were not observed with SS-31, another targeted anti-oxidant. MitoQ consists of a lipophilic cation (Tetraphenylphosphonium [TPP]) joined to an anti-oxidant component (quinone) by a 10-carbon alkyl chain, which is thought to insert into the inner mitochondrial membrane (IMM). We found that mitochondrial swelling and depolarization was also induced by dodecyltriphenylphosphomium (DTPP), which consists of TPP and the alkyl chain, but not by TPP alone. Surprisingly, MitoQ-induced mitochondrial swelling occurred in the absence of a decrease in oxygen consumption rate. We also found that DTPP directly increased the permeability of artificial liposomes with a cardiolipin content similar to that of the IMM. In summary, MitoQ causes mitochondrial swelling and depolarization in PT cells by a mechanism unrelated to anti-oxidant activity, most likely because of increased IMM permeability due to insertion of the alkyl chain.
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http://dx.doi.org/10.14814/phy2.13667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880956PMC
April 2018

Renal Tubular Cell Mitochondrial Dysfunction Occurs Despite Preserved Renal Oxygen Delivery in Experimental Septic Acute Kidney Injury.

Crit Care Med 2018 04;46(4):e318-e325

Bloomsbury Institute of Intensive Care Medicine, Division of Medicine, University College London, London, United Kingdom.

Objective: To explain the paradigm of significant renal functional impairment despite preserved hemodynamics and histology in sepsis-induced acute kidney injury.

Design: Prospective observational animal study.

Setting: University research laboratory.

Subjects: Male Wistar rats.

Intervention: Using a fluid-resuscitated sublethal rat model of fecal peritonitis, changes in renal function were characterized in relation to global and renal hemodynamics, and histology at 6 and 24 hours (n = 6-10). Sham-operated animals were used as comparison (n = 8). Tubular cell mitochondrial function was assessed using multiphoton confocal imaging of live kidney slices incubated in septic serum.

Measurements And Main Results: By 24 hours, serum creatinine was significantly elevated with a concurrent decrease in renal lactate clearance in septic animals compared with sham-operated and 6-hour septic animals. Renal uncoupling protein-2 was elevated in septic animals at 24 hours although tubular cell injury was minimal and mitochondrial ultrastructure in renal proximal tubular cells preserved. There was no significant change in global or renal hemodynamics and oxygen delivery/consumption between sham-operated and septic animals at both 6- and 24-hour timepoints. In the live kidney slice model, mitochondrial dysfunction was seen in proximal tubular epithelial cells incubated with septic serum with increased production of reactive oxygen species, and decreases in nicotinamide adenine dinucleotide and mitochondrial membrane potential. These effects were prevented by coincubation with the reactive oxygen species scavenger, 4-hydroxy-2,2,6,6-tetramethyl-piperidin-1-oxyl.

Conclusions: Renal dysfunction in sepsis occurs independently of hemodynamic instability or structural damage. Mitochondrial dysfunction mediated by circulating mediators that induce local oxidative stress may represent an important pathophysiologic mechanism.
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http://dx.doi.org/10.1097/CCM.0000000000002937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856355PMC
April 2018

Online monitoring of a photocatalytic reaction by real-time high resolution FlowNMR spectroscopy.

Chem Commun (Camb) 2017 Dec;54(1):30-33

Centre for Sustainable Chemical Technologies, University of Bath, Claverton Down, Bath, BA2 7AY, UK.

We demonstrate how FlowNMR spectroscopy can readily be applied to investigate photochemical reactions that require sustained input of light and air to yield mechanistic insight under realistic conditions. The Eosin Y mediated photo-oxidation of N-allylbenzylamine is shown to produce imines as primary reaction products from which undesired aldehydes form after longer reaction times. Facile variation of reaction conditions during the reaction in flow allows for probe experiments that give information about the mode of action of the photocatalyst.
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http://dx.doi.org/10.1039/c7cc07059dDOI Listing
December 2017

New frontiers in intravital microscopy of the kidney.

Curr Opin Nephrol Hypertens 2017 05;26(3):172-178

aInstitute of Anatomy, University of Zurich bDepartment of Nephrology, University Hospital Zurich cCenter for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland.

Purpose Of Review: Intravital imaging with multiphoton microscopy enables the detailed study of dynamic cellular processes within functioning organs in living animals, in ways that would not otherwise be possible. It therefore represents a powerful tool in translational kidney research. In this article, we will discuss several new technical developments that have significantly increased the capabilities of kidney imaging.

Recent Findings: Important contemporary advances in biomedical imaging technology include longer wavelength excitation lasers, far-red emitting fluorescent reporters, highly sensitive detectors, fluorescence lifetime measurements, adaptive optics, microendoscopes, high-throughput automated analysis algorithms and tissue clearing techniques. Several recent studies have utilized intravital microscopy to gain valuable new insights into important physiological and pathophysiological processes in the kidney, such as renal handling of albumin and the cellular pathogenesis of acute kidney injury in sepsis.

Summary: Major technological advances are rapidly expanding the frontiers of intravital microscopy, which is likely to play an increasingly important role in preclinical kidney research in the coming years.
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http://dx.doi.org/10.1097/MNH.0000000000000313DOI Listing
May 2017

Effective antigen presentation to helper T cells by human eosinophils.

Immunology 2016 Dec 20;149(4):413-422. Epub 2016 Sep 20.

Immunity, Infection and Inflammation Programme, Institute of Medical Sciences, School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, UK.

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.
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http://dx.doi.org/10.1111/imm.12658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095493PMC
December 2016

Mitochondria as therapeutic targets in acute kidney injury.

Curr Opin Nephrol Hypertens 2016 07;25(4):355-62

Institute of Anatomy, University of Zurich, Zurich, Switzerland.

Purpose Of Review: Mitochondria are complex intracellular organelles with a variety of important functions. The kidney tubule is densely packed with mitochondria, and mitochondrial dysfunction is thought to be central to the pathogenesis of acute kidney injury (AKI). Mitochondria therefore represent potential targets for novel therapeutic interventions in AKI.

Recent Findings: Several mitochondrial targeted approaches have shown promise in recent preclinical studies of AKI, including measures to: reduce oxidative stress within mitochondria; prevent mitochondrial fission and activation of cell death pathways; enhance recycling of damaged mitochondria via autophagy and mitophagy; and accelerate mitochondrial biogenesis postinsult.

Summary: Recent studies show that it is now eminently feasible to pharmacologically manipulate various key aspects of mitochondrial biology in the kidney, and this has much potential for the future treatment of AKI. However, significant hurdles will have to be overcome in the translational pathway for these strategies to successfully migrate to the clinic.
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http://dx.doi.org/10.1097/MNH.0000000000000228DOI Listing
July 2016

Cytotoxicity and apoptotic gene expression in an in vitro model of the blood-brain barrier following exposure to poly(butylcyanoacrylate) nanoparticles.

J Drug Target 2016 08 5;24(7):635-44. Epub 2016 Feb 5.

a Department of Biological Sciences , Northern Kentucky University , Highland Heights , KY , USA ;

Background Poly(butylcyanoacrylate) (PBCA) nanoparticles (NPs) loaded with doxorubicin (DOX) and coated with polysorbate 80 (PS80) have shown efficacy in the treatment of rat glioblastoma. However, cytotoxicity of this treatment remains unclear. Purpose The purpose of this study was to investigate cytotoxicity and apoptotic gene expression using a proven in vitro co-culture model of the blood-brain barrier. Methods The co-cultures were exposed to uncoated PBCA NPs, PBCA-PS80 NPs or PBCA-PS80-DOX NPs at varying concentrations and evaluated using a resazurin-based cytotoxicity assay and an 84-gene apoptosis RT-PCR array. Results The cytotoxicity assays showed PBCA-PS80-DOX NPs exhibited a decrease in metabolic function at lower concentrations than uncoated PBCA NPs and PBCA-PS80 NPs. The apoptosis arrays showed differential expression of 18 genes in PBCA-PS80-DOX treated cells compared to the untreated control. Discussion As expected, the cytotoxicity assays demonstrated enhanced dose-dependent toxicity in the DOX loaded NPs. The differentially expressed apoptotic genes participate in both the tumor necrosis factor receptor-1 and mitochondria-associated apoptotic pathways implicated in current DOX chemotherapeutic toxicity. Conclusion The following data suggest that the cytotoxic effect may be attributed to DOX and not the NPs themselves, further supporting the use of PBCA-PS80 NPs as an effective drug delivery vehicle for treating central nervous system conditions.
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http://dx.doi.org/10.3109/1061186X.2015.1132222DOI Listing
August 2016

Long wavelength multiphoton excitation is advantageous for intravital kidney imaging.

Kidney Int 2016 Mar;89(3):712-9

Intravital multiphoton microscopy is a powerful tool to study kidney physiology in living animals. However, certain technical issues have curbed its usage to date, including limited depth of tissue penetration and high background emission of endogenous signals. Most previous studies have used the excitation range 700–1000 nm. Since newer longer wavelength excitation lasers may provide solutions to these problems we constructed a microscope coupled to a laser tunable up to 1300 nm and optimized for kidney imaging. This set-up offers substantial advantages for intravital studies, especially when coupled with newly available far-red probes. First, the background at longer wavelengths is markedly reduced, thus increasing the signal to background ratio. Second, the depth of tissue penetration is significantly increased, enabling detailed imaging of previously inaccessible structures, such as deeper glomeruli. Third, using a combination of two- and three-photon excitation, multiple different fluorescent probes can be imaged simultaneously in the same animal, with clear spectral separation. Application of these techniques helped visualize pathological aspects of tubular cell function in a well-established model of acute kidney injury (maleate toxicity). Thus, utilizing long wavelength excitation offers substantial advantages for intravital kidney imaging, which together enhance the capabilities of this powerful and increasingly used research technique.
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http://dx.doi.org/10.1038/ki.2015.323DOI Listing
March 2016

The urinary proteome and metabonome differ from normal in adults with mitochondrial disease.

Kidney Int 2015 Mar 10;87(3):610-22. Epub 2014 Sep 10.

Medical Research Council Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, University College London Institute of Neurology, London, UK.

We studied the extent and nature of renal involvement in a cohort of 117 adult patients with mitochondrial disease, by measuring urinary retinol-binding protein (RBP) and albumin; established markers of tubular and glomerular dysfunction, respectively. Seventy-five patients had the m.3243A>G mutation and the most frequent phenotypes within the entire cohort were 14 with MELAS, 33 with MIDD, and 17 with MERRF. Urinary RBP was increased in 29 of 75 of m.3243A>G patients, whereas albumin was increased in 23 of the 75. The corresponding numbers were 16 and 14, respectively, in the 42 non-m.3243A>G patients. RBP and albumin were higher in diabetic m.3243A>G patients than in nondiabetics, but there were no significant differences across the three major clinical phenotypes. The urine proteome (mass spectrometry) and metabonome (nuclear magnetic resonance) in a subset of the m.3243A>G patients were markedly different from controls, with the most significant alterations occurring in lysosomal proteins, calcium-binding proteins, and antioxidant defenses. Differences were also found between asymptomatic m.3243A>G carriers and controls. No patients had an elevated serum creatinine level, but 14% had hyponatremia, 10% had hypophosphatemia, and 14% had hypomagnesemia. Thus, abnormalities in kidney function are common in adults with mitochondrial disease, exist in the absence of elevated serum creatinine, and are not solely explained by diabetes.
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http://dx.doi.org/10.1038/ki.2014.297DOI Listing
March 2015

Dynamic multiphoton microscopy: focusing light on acute kidney injury.

Physiology (Bethesda) 2014 Sep;29(5):334-42

Indiana University School of Medicine, Indiana Center for Biological Microscopy, Indianapolis, Indiana.

Acute kidney injury (AKI) is a major global health problem; much research has been conducted on AKI, and numerous agents have shown benefit in animal studies, but none have translated into treatments. There is, therefore, a pressing unmet need to increase knowledge of the pathophysiology of AKI. Multiphoton microscopy (MPM) provides a tool to non-invasively visualize dynamic events in real time and at high resolution in rodent kidneys, and in this article we review its application to study novel mechanisms and treatments in different forms of AKI.
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http://dx.doi.org/10.1152/physiol.00010.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214830PMC
September 2014

Combination peptide immunotherapy suppresses antibody and helper T-cell responses to the RhD protein in HLA-transgenic mice.

Haematologica 2014 Mar 17;99(3):588-96. Epub 2014 Jan 17.

The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.
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http://dx.doi.org/10.3324/haematol.2012.082081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943325PMC
March 2014

Expansion of Foxp3(+) T-cell populations by Candida albicans enhances both Th17-cell responses and fungal dissemination after intravenous challenge.

Eur J Immunol 2014 Apr 13;44(4):1069-83. Epub 2014 Feb 13.

Division of Applied Medicine, University of Aberdeen, Aberdeen, UK; Aberdeen Fungal Group, University of Aberdeen, Aberdeen, UK; Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, USA.

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3(+) regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3(+) Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4(+) Foxp3(+) population that correlated positively with fungal burden. Depletion from Foxp3(hCD2) reporter mice in vivo confirmed that Foxp3(+) cells exacerbated fungal burden and inflammatory renal disease. The CD4(+) Foxp3(+) population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3(-) cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt, and secreting IL-17A. The expanded Foxp3(+) T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3(+) T cells promotes Th17-cell responses that drive pathology.
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http://dx.doi.org/10.1002/eji.201343604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992851PMC
April 2014