Bettina Scharner

Witten/Herdecke University

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Frank Thévenod

Witten/Herdecke University

Wing‐Kee Lee

Bielefeld University

Stephanie Probst

Witten/Herdecke University

Christian Langelueddecke

Witten/Herdecke University

Johannes Fels

Witten/Herdecke University

Natascha A. Wolff

Witten/Herdecke University

Bayram Edemir

Martin Luther University Halle-Wittenberg

Richard Betten

Witten/Herdecke University

Timm Schreiber

University of Duisburg-Essen

Eleni Roussa

University of Freiburg

Cooperative Institutions

Witten/Herdecke University

Czech Academy of Sciences, Institute of Physiology

University of Manchester

University of Göttingen

Bielefeld University

Marien Hospital Witten

Saarland University

Beth Israel Deaconess Medical Center

Ludwig-Maximilians-Universität München

Center for Research and Advanced Studies of the National Polytechnic Institute

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Tonicity inversely modulates lipocalin-2 (Lcn2/24p3/NGAL) receptor (SLC22A17) and Lcn2 expression via Wnt/β-catenin signaling in renal inner medullary collecting duct cells: implications for cell fate and bacterial infection

Cell Communication and Signaling (2018)

R. Betten Bettina Scharner Stephanie Probst Bayram Edemir Natascha A. Wolff

We have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/β-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe3+-free (apo-)Lcn2. To determine the effects of osmolarity/tonicity changes, Wnt/β-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells. Normosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. β-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS). Hyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/β-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, β-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability. Lcn2-R upregulation and Lcn2 downregulation via Wnt/β-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.

10.1186/s12964-018-0285-3

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Citations (20)

Inverse Regulation of Lipocalin-2/24p3 Receptor/Slc22a17 and Lipocalin-2 Expression by Tonicity, Nfat5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance

Stephanie Probst Bettina Scharner Wing‐Kee Lee Frank Thévenod

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (Lcn2) receptor (Slc22a17) apically to possibly mediate high-affinity reabsorption of filtered proteins by endocytosis, yet its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates Slc22a17 in cultured mouse inner medullary CD cells, whereas activation of toll-like receptor 4 (TLR4) via bacterial lipopolysaccharides (LPS) downregulates Slc22a17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP) and downregulation by TLR4 signaling that occur via the transcription factors Nfat5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity via Nfat5, AVP via CREB and TLR4 signaling on the expression of Slc22a17 and its ligand Lcn2 in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity was 300 mosmol/l whereas addition of 50-100 mmol/l NaCl for up to 72 h induced hyperosmolarity/-tonicity (400-500 mosmol/l). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17 and Lcn2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 occurred with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17 via activation of Nfat5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17. Conversely, though Nfat5 mediated hyperosmolarity/-tonicity induced downregulation of Lcn2 expression, AVP reduced Lcn2 expression and predominantly apical Lcn2 secretion evoked by LPS, but through a posttranslational mode of action that was independent of cAMP signaling. In conclusion, the hyperosmotic/-tonic upregulation of Slc22a17 in mCCD(cl.1) cells via Nfat5 and by AVP via CREB suggests a contribution of Slc22a17 to adaptive osmotolerance, whereas Lcn2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.

Osmotic concentration

10.20944/preprints201909.0047.v1

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Citations (4)

Inverse Regulation of Lipocalin-2/24p3 Receptor/SLC22A17 and Lipocalin-2 Expression by Tonicity, NFAT5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance

International Journal of Molecular Sciences (2019)

Stephanie Probst Bettina Scharner Ruairi McErlean Wing‐Kee Lee Frank Thévenod

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50-100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400-500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17/SLC22A17 and Lcn2/LCN2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17/SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17/SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2/LCN2 expression, AVP reduced Lcn2/LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.

Osmotic concentration

10.3390/ijms20215398

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Citations (13)

Distinct concentration-dependent oxidative stress profiles by cadmium in a rat kidney proximal tubule cell line

Archives of Toxicology (2024)

Wing‐Kee Lee Stephanie Probst Bettina Scharner Timo Deba Faouzi Dahdouh

Levels and chemical species of reactive oxygen/nitrogen species (ROS/RNS) determine oxidative eustress and distress. Abundance of uptake pathways and high oxygen consumption for ATP-dependent transport makes the renal proximal tubule particularly susceptible to cadmium (Cd

NOX4

Viability assay

TBARS

10.1007/s00204-023-03677-z

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Citations (8)

Chronic cadmium exposure induces transcriptional activation of the Wnt pathway and upregulation of epithelial-to-mesenchymal transition markers in mouse kidney

Toxicology Letters (2010)

Prabir K. Chakraborty Bettina Scharner Jasna Jurasović Barbara Meßner David Bernhard

LRP5

LRP6

WNT3A

10.1016/j.toxlet.2010.05.007

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Insoluble HIFa protein aggregates by cadmium disrupt hypoxia-prolyl hydroxylase (PHD)-hypoxia inducible factor (HIFa) signaling in renal epithelial (NRK-52E) and interstitial (FAIK3-5) cells

BioMetals (2024)

Timm Schreiber Bettina Scharner Frank Thévenod

Hypoxia-Inducible Factors

Hypoxia

10.1007/s10534-024-00631-z

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Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium

Archives of Toxicology (2021)

Stephanie Probst Johannes Fels Bettina Scharner Natascha A. Wolff Eleni Roussa

Abstract The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe 2+ overload. The nephrotoxic non-essential metal ion Cd 2+ can displace Fe 2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe 2+ and Cd 2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD 3 ] collecting duct cell lines. Cells were exposed to equipotent Cd 2+ (0.5–5 μmol/l) and/or Fe 2+ (50–100 μmol/l) for 4–24 h. Hepcidin ( Hamp1 ) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe 2+ -induced mIMCD 3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd 2+ -induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe 2+ prevented Cd 2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe 2+ with desferrioxamine augmented Cd 2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe 2+ , but not Cd 2+ . Because Fe 2+ and Cd 2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

10.1007/s00204-021-03106-z

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Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule

International Journal of Molecular Sciences (2019)

Johannes Fels Bettina Scharner Ralf Zarbock Itzel Pamela Zavala-Guevara Wing‐Kee Lee

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, β2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, β2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-β2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.

Metallothionein

10.3390/ijms20102379

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Citations (33)

Regulation of 24p3 Receptor (24p3R/SLC22A17) Expression by Tonicity in Rodent Renal Inner Medullary Collecting Duct (IMCD) Cells

The FASEB Journal (2016)

Frank Thévenod Christian Langelueddecke Stephanie Probst Bettina Scharner Richard Betten

Background and Aims The 24p3/NGAL/lipocalin‐2 receptor (24p3R/SLC22A17) is apically expressed in rodent collecting ducts, including IMCD, where it may mediate protein endocytosis (Langelueddecke C. et al. J. Biol. Chem. 287 :159–169, 2012) and induce pro‐inflammatory and pro‐fibrotic signalling in response to albuminuria (Dizin E. et al. Am. J. Physiol. Renal Physiol. 305 :F1053‐F1063, 2013). Cells of the inner medulla are exposed in vivo to a hyperosmotic environment, which requires an adaptive response for survival. Interestingly, we have identified a putative TonEBP DNA consensus motif in the 24p3R promoter sequence. Here we investigated the expression of 24p3R in cultured IMCD cells that were exposed to increased osmolarity. Methods Cultured mouse IMCD cells (mIMCD3) and rat primary IMCD cells were cultured in 300 – 900 mOsm/kg for 1–6 days. Hypertonicity was induced by equimolar addition of NaCl + urea. 24p3R expression and localization were determined by qPCR, immunoblotting, surface biotinylation, flow cytometry and immunofluorescence microscopy. Results Hypertonicity decreased cell proliferation and was accompanied by suppression of the cell proliferation genes c‐myc and cyclin‐D1 in mIMCD3 cells, which may reflect cellular stress and damage. Hypertonicity increased 24p3R mRNA and 24p3R protein expression and localization in plasma membranes of mIMCD3 and primary IMCD cells. Furthermore, plasma membrane localization of 24p3R was associated with increased dimerization of the protein. Conclusions Hyperosmotic environment decreases proliferation of IMCD cells but increases plasma membrane expression and dimerization of 24p3R, which may be mediated by TonEBP and contribute to adaptation of IMCD cells to osmotic stress. Support or Funding Information Funded by DFG TH345/11‐1 and ZBAF

Osmotic concentration

Renal medulla

10.1096/fasebj.30.1_supplement.741.2

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