Publications by authors named "Björn Buchholz"

14 Publications

  • Page 1 of 1

Cyst growth in ADPKD is prevented by pharmacological and genetic inhibition of TMEM16A in vivo.

Nat Commun 2020 08 28;11(1):4320. Epub 2020 Aug 28.

Department of Nephrology and Hypertension, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

In autosomal dominant polycystic kidney disease (ADPKD) multiple bilateral renal cysts gradually enlarge, leading to a decline in renal function. Transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) are known to drive cyst enlargement. Here we demonstrate that loss of Pkd1 increased expression of TMEM16A and CFTR and Cl secretion in murine kidneys, with TMEM16A essentially contributing to cyst growth. Upregulated TMEM16A enhanced intracellular Ca signaling and proliferation of Pkd1-deficient renal epithelial cells. In contrast, increase in Ca signaling, cell proliferation and CFTR expression was not observed in Pkd1/Tmem16a double knockout mice. Knockout of Tmem16a or inhibition of TMEM16A in vivo by the FDA-approved drugs niclosamide and benzbromarone, as well as the TMEM16A-specific inhibitor Ani9 largely reduced cyst enlargement and abnormal cyst cell proliferation. The present data establish a therapeutic concept for the treatment of ADPKD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-18104-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455562PMC
August 2020

Ocean winter warming induced starvation of predator and prey.

Proc Biol Sci 2020 07 15;287(1931):20200970. Epub 2020 Jul 15.

Marine Ecology, GEOMAR Helmholtz Centre for Ocean Research Kiel, Hohenbergstrasse 2, 24105 Kiel, Germany.

Ocean warming impacts the fitness of marine ectothermic species, leading to poleward range shifts, re-shuffling of communities, and changes in ecosystem services. While the detrimental effects of summer heat waves have been widely studied, little is known about the impacts of winter warming on marine species in temperate regions. Many species benefit from low winter temperature-induced reductions in metabolism, as these permit conservation of energy reserves that are needed to support reproduction in spring. Here, we used a unique outdoor mesocosm system to expose a coastal predator-prey system, the sea star and the blue mussel , to different winter warming scenarios under near-natural conditions. We found that the body condition of mussels decreased in a linear fashion with increasing temperature. Sea star growth also decreased with increasing temperature, which was a function of unaltered predation rates and decreased mussel body condition. relative digestive gland mass strongly declined over the studied temperature interval ( twofold). This could have severe implications for reproductive capacity in the following spring, as digestive glands provide reserve compounds to maturing gonads. Thus, both predator and prey suffered from a mismatch of energy acquisition versus consumption in warmer winter scenarios, with pronounced consequences for food web energy transfer in future oceans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rspb.2020.0970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423679PMC
July 2020

TMEM16A drives renal cyst growth by augmenting Ca signaling in M1 cells.

J Mol Med (Berl) 2020 05 18;98(5):659-671. Epub 2020 Mar 18.

Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.

Polycystic kidney disease (PKD) leads to continuous decline of renal function by growth of renal cysts. Enhanced proliferation and transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and Ca-activated TMEM16A Cl channels is thought to cause an increase in cyst volume. Recent work shows the pro-proliferative role of the Ca activated Cl channel TMEM16A (anoctamin 1), and demonstrates the essential contribution of TMEM16A to CFTR-dependent Cl secretion. The present data demonstrate an increase in intracellular Ca ([Ca]i) signals and Cl secretion by TMEM16A, in renal collecting duct principle cells from dog (MDCK) and mouse (M1) as well as primary tubular epithelial cells from PKD1-/- knockout mice. M1 organoids proliferated, increased expression of TMEM16A, and secreted Cl upon knockdown of endogenous polycystin 1 or 2 (PKD1,2), by retroviral transfection with shPKD1 and shPKD2, respectively. Knockdown of PKD1 or PKD2 increased basal intracellular Ca levels and enhanced purinergic Ca release from endoplasmic reticulum. In contrast, ryanodine receptors were found not to be expressed in mouse renal epithelial cells and caffeine had no effects on [Ca]i. Ca signals, proliferation, and Cl secretion were largely reduced by knockdown or blockade of TMEM16A. TMEM16A may be therefore important for enhanced Ca release from IP-sensitive Ca stores in polycystic kidney disease. KEY MESSAGES: • ADPKD leads to continuous decline of renal function by growth of renal cysts. • Knockdown of PKD1 or PKD2 increases TMEM16A expression. • TMEM16A enhanced intracellular Ca signals, Cl secretion, and proliferation. • TMEM16A contributes to cyst growth in ADPKD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00109-020-01894-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220898PMC
May 2020

Lipid Peroxidation Drives Renal Cyst Growth through Activation of TMEM16A.

J Am Soc Nephrol 2019 02 3;30(2):228-242. Epub 2019 Jan 3.

Department of Physiology, University of Regensburg, Regensburg, Germany; and

Background: Transepithelial chloride secretion, through the chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1), drives cyst enlargement in polycystic kidney disease (PKD). Polycystic kidneys are hypoxic, and oxidative stress activates TMEM16A. However, mechanisms for channel activation in PKD remain obscure.

Methods: Using tissue samples from patients with autosomal dominant PKD, embryonic kidney cultures, and an MDCK cyst model, we assessed peroxidation of plasma membrane phospholipids in human and mouse polycystic kidneys. We also used electrophysiologic Ussing chamber and patch clamp experiments to analyze activation of TMEM16A and growth of renal cysts.

Results: Peroxidation of phospholipids in human and mouse kidneys as well as MDCK cysts is probably due to enhanced levels of reactive oxygen species. Lipid peroxidation correlated with increased cyst volume as shown in renal cultures and MDCK cysts in three-dimensional cultures. Reactive oxygen species and lipid peroxidation strongly activated TMEM16A, leading to depletion of calcium ion stores and store-operated calcium influx. Activation of TMEM16A- and CFTR-dependent chloride secretion strongly augmented cyst growth. Exposure to scavengers of reactive oxygen species, such as glutathione, coenzyme Q10, or idebenone (a synthetic coenzyme Q10 homolog), as well as inhibition of oxidative lipid damage by ferrostatin-1 largely reduced activation of TMEM16A. Inhibition of TMEM16A reduced proliferation and fluid secretion .

Conclusions: These findings indicate that activation of TMEM16A by lipid peroxidation drives growth of renal cysts. We propose direct inhibition of TMEM16A or inhibition of lipid peroxidation as potentially powerful therapeutic approaches to delay cyst development in PKD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2018010039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362630PMC
February 2019

A Fluorescent Benzo[g]isoquinoline-Based HIF Prolyl Hydroxylase Inhibitor for Cellular Imaging.

ChemMedChem 2019 01 21;14(1):94-99. Epub 2018 Dec 21.

Department of Chemistry and Pharmacy, Inorganic and Organometallic Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Egerlandstraße 1, 91058, Erlangen, Germany.

Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201800483DOI Listing
January 2019

Hypoxia inducible factor stabilization improves defective ischemia-induced angiogenesis in a rodent model of chronic kidney disease.

Kidney Int 2017 03 4;91(3):616-627. Epub 2016 Dec 4.

Department of Nephrology and Hypertension, University of Erlangen-Nürnberg, Erlangen, Germany. Electronic address:

Chronic kidney disease (CKD) is associated with increased risk and worse prognosis of cardiovascular disease, including peripheral artery disease. An impaired angiogenic response to ischemia may contribute to poor outcomes of peripheral artery disease in patients with CKD. Hypoxia inducible factors (HIF) are master regulators of angiogenesis and therefore represent a promising target for therapeutic intervention. To test this we induced hind-limb ischemia in rats with CKD caused by 5/6 nephrectomy and administered two different treatments known to stabilize HIF protein in vivo: carbon monoxide and a pharmacological inhibitor of prolyl hydroxylation 2-(1-chloro-4- hydroxyisoquinoline-3-carboxamido) acetate (ICA). Expression levels of pro-angiogenic HIF target genes (Vegf, Vegf-r1, Vegf-r2, Ho-1) were measured by qRT-PCR. Capillary density was measured by CD31 immunofluorescence staining and HIF expression was evaluated by immunohistochemistry. Capillary density in ischemic skeletal muscle was significantly lower in CKD animals compared to sham controls. Rats with CKD showed significantly lower expression of HIF and all measured pro-angiogenic HIF target genes, including VEGF. Both HIF stabilizing treatments rescued HIF target gene expression in animals with CKD and led to significantly higher ischemia-induced capillary sprouting compared to untreated controls. ICA was effective regardless of whether it was administered before or after induction of ischemia and led to a HIF expression in skeletal muscle. Thus, impaired ischemia-induced angiogenesis in rats with CKD can be improved by HIF stabilization, even if started after onset of ischemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.kint.2016.09.028DOI Listing
March 2017

HIF-prolyl hydroxylases in the rat kidney: physiologic expression patterns and regulation in acute kidney injury.

Am J Pathol 2009 May 6;174(5):1663-74. Epub 2009 Apr 6.

Department of Nephrology and Hypertension, Friedrich-Alexander-University Erlangen-Nuremberg,Erlangen, Germany.

Hypoxia-inducible transcription factors (HIFs) play important roles in the response of the kidney to systemic and regional hypoxia. Degradation of HIFs is mediated by three oxygen-dependent HIF-prolyl hydroxylases (PHDs), which have partially overlapping characteristics. Although PHD inhibitors, which can induce HIFs in the presence of oxygen, are already in clinical development, little is known about the expression and regulation of these enzymes in the kidney. Therefore, we investigated the expression levels of the three PHDs in both isolated tubular cells and rat kidneys. All three PHDs were present in the kidney and were expressed predominantly in three different cell populations: (a) in distal convoluted tubules and collecting ducts (PHD1,2,3), (b) in glomerular podocytes (PHD1,3), and (c) in interstitial fibroblasts (PHD1,3). Higher levels of PHDs were found in tubular segments of the inner medulla where oxygen tensions are known to be physiologically low. PHD expression levels were unchanged in HIF-positive tubular and interstitial cells after induction by systemic hypoxia. In rat models of acute renal injury, changes in PHD expression levels were variable; while cisplatin and ischemia/reperfusion led to significant decreases in PHD2 and 3 expression levels, no changes were seen in a model of contrast media-induced nephropathy. These results implicate the non-uniform expression of HIF-regulating enzymes that modify the hypoxic response in the kidney under both regional and temporal conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2353/ajpath.2009.080687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671255PMC
May 2009

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum.

EMBO J 2009 Mar 15;28(5):490-9. Epub 2009 Jan 15.

Renal Division, University Hospital of Freiburg, Freiburg, Germany.

Ca(2+) is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca(2+) influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca(2+)-dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca(2+) concentration in the endoplasmic reticulum (ER). ER-resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca(2+) ATPase by increasing the ER Ca(2+) permeability. This results in diminished cytosolic and mitochondrial Ca(2+) signals upon stimulation of inositol 1,4,5-trisphosphate receptors and reduces Ca(2+) release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca(2+) release and augments sensitivity to apoptosis. Our findings indicate an important function of ER-resident TRPP2 in the modulation of intracellular Ca(2+) signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emboj.2008.307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657577PMC
March 2009

Increased expression of secreted frizzled-related protein 4 in polycystic kidneys.

J Am Soc Nephrol 2009 Jan 22;20(1):48-56. Epub 2008 Oct 22.

Renal Division, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany.

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disease associated with progressive renal failure. Although cyst growth and compression of surrounding tissue may account for some loss of renal tissue, the other factors contributing to the progressive renal failure in patients with ADPKD are incompletely understood. Here, we report that secreted frizzled-related protein 4 (sFRP4) is upregulated in human ADPKD and in four different animal models of PKD, suggesting that sFRP4 expression is triggered by a common mechanism that underlies cyst formation. Cyst fluid from ADPKD kidneys activated the sFRP4 promoter and induced production of sFRP4 protein in renal tubular epithelial cell lines. Antagonism of the vasopressin 2 receptor blocked both promoter activity and tubular sFRP4 expression. In addition, sFRP4 selectively influenced members of the canonical Wnt signaling cascade and promoted cystogenesis of the zebrafish pronephros. sFRP4 was detected in the urine of both patients and animals with PKD, suggesting that sFRP4 may be a potential biomarker for monitoring the progression of ADPKD. Taken together, these observations suggest a potential role for SFRP4 in the pathogenesis of ADPKD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2008040345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615724PMC
January 2009

TRPP2 and TRPV4 form a polymodal sensory channel complex.

J Cell Biol 2008 Aug;182(3):437-47

Renal Division, University Hospital Freiburg, 79106 Freiburg, Germany.

The primary cilium has evolved as a multifunctional cellular compartment that decorates most vertebrate cells. Cilia sense mechanical stimuli in various organs, but the molecular mechanisms that convert the deflection of cilia into intracellular calcium transients have remained elusive. Polycystin-2 (TRPP2), an ion channel mutated in polycystic kidney disease, is required for cilia-mediated calcium transients but lacks mechanosensitive properties. We find here that TRPP2 utilizes TRPV4 to form a mechano- and thermosensitive molecular sensor in the cilium. Depletion of TRPV4 in renal epithelial cells abolishes flow-induced calcium transients, demonstrating that TRPV4, like TRPP2, is an essential component of the ciliary mechanosensor. Because TRPV4-deficient zebrafish and mice lack renal cysts, our findings challenge the concept that defective ciliary flow sensing constitutes the fundamental mechanism of cystogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200805124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2500130PMC
August 2008

Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function.

J Immunol 2008 Apr;180(7):4697-705

Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany.

Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1alpha on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1alpha protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1alpha significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1alpha and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.180.7.4697DOI Listing
April 2008

HIF activation protects from acute kidney injury.

J Am Soc Nephrol 2008 Mar 6;19(3):486-94. Epub 2008 Feb 6.

Medical Clinic 4, Department of Nephrology and Hypertension, Friedrich-Alexander-University Erlangen Nuremberg, Krankenhausstrasse 12, D - 91054 Erlangen, Germany.

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2007040419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2391048PMC
March 2008

Trafficking of TRPP2 by PACS proteins represents a novel mechanism of ion channel regulation.

EMBO J 2005 Feb 3;24(4):705-16. Epub 2005 Feb 3.

Renal Division, University Hospital of Freiburg, Freiburg, Germany.

The trafficking of ion channels to the plasma membrane is tightly controlled to ensure the proper regulation of intracellular ion homeostasis and signal transduction. Mutations of polycystin-2, a member of the TRP family of cation channels, cause autosomal dominant polycystic kidney disease, a disorder characterized by renal cysts and progressive renal failure. Polycystin-2 functions as a calcium-permeable nonselective cation channel; however, it is disputed whether polycystin-2 resides and acts at the plasma membrane or endoplasmic reticulum (ER). We show that the subcellular localization and function of polycystin-2 are directed by phosphofurin acidic cluster sorting protein (PACS)-1 and PACS-2, two adaptor proteins that recognize an acidic cluster in the carboxy-terminal domain of polycystin-2. Binding to these adaptor proteins is regulated by the phosphorylation of polycystin-2 by the protein kinase casein kinase 2, required for the routing of polycystin-2 between ER, Golgi and plasma membrane compartments. Our paradigm that polycystin-2 is sorted to and active at both ER and plasma membrane reconciles the previously incongruent views of its localization and function. Furthermore, PACS proteins may represent a novel molecular mechanism for ion channel trafficking, directing acidic cluster-containing ion channels to distinct subcellular compartments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.emboj.7600566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC549624PMC
February 2005

An inwardly rectifying whole cell current induced by Gq-coupled receptors.

Biochem Biophys Res Commun 2004 Sep;322(1):177-85

Renal Division and Center for Clinical Research, University Hospital Freiburg, Breisacherstr. 66, 79106 Freiburg, Germany.

Ca(2+) influx across the plasma membrane after stimulation of G protein-coupled receptors is important for many physiological functions. Here we studied the regulation of an inwardly rectifying whole cell current and its putative role in Ca(2+) entry in Xenopus oocytes. Expression of P2Y(1) or M1 receptors in Xenopus oocytes elicited a characteristic inwardly rectifying current without receptor stimulation. This current displayed distinct activation and inactivation kinetics and was highly Ca(2+)-dependent. After stimulation of endogenous G(q)-coupled receptors in water-injected cells similar currents were observed. We therefore speculated that the current could be activated via Ca(2+) store depletion induced by constitutive stimulation of the IP(3) cascade in cells overexpressing G(q)-coupled receptors. Receptor-independent Ca(2+) store depletion also induced the current. In conclusion, this current is activated after store depletion suggesting a role in Ca(2+) entry after stimulation of G(q)-coupled receptors. Finally, our data do not support the proposed ionotropic properties of the P2Y(1) receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2004.07.103DOI Listing
September 2004