Publications by authors named "Catherine Meyer-Schwesinger"

80 Publications

ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage.

J Am Soc Nephrol 2021 Mar 30. Epub 2021 Mar 30.

Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background: Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury.

Methods: Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, and , in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice.

Results: ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner.

Conclusions: ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.
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http://dx.doi.org/10.1681/ASN.2020081213DOI Listing
March 2021

ADP-Ribosylation Regulates the Signaling Function of IFN-γ.

Front Immunol 2021 8;12:642545. Epub 2021 Mar 8.

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.
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http://dx.doi.org/10.3389/fimmu.2021.642545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983947PMC
March 2021

Lysosome function in glomerular health and disease.

Cell Tissue Res 2021 Jan 12. Epub 2021 Jan 12.

Institute for Cellular and Integrative Physiology, University Medical Center Hamburg Eppendorf, Hamburg, Germany.

The lysosome represents an important regulatory platform within numerous vesicle trafficking pathways including the endocytic, phagocytic, and autophagic pathways. Its ability to fuse with endosomes, phagosomes, and autophagosomes enables the lysosome to break down a wide range of both endogenous and exogenous cargo, including macromolecules, certain pathogens, and old or damaged organelles. Due to its center position in an intricate network of trafficking events, the lysosome has emerged as a central signaling node for sensing and orchestrating the cells metabolism and immune response, for inter-organelle and inter-cellular signaling and in membrane repair. This review highlights the current knowledge of general lysosome function and discusses these findings in their implication for renal glomerular cell types in health and disease including the involvement of glomerular cells in lysosomal storage diseases and the role of lysosomes in nongenetic glomerular injuries.
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http://dx.doi.org/10.1007/s00441-020-03375-7DOI Listing
January 2021

The ins-and-outs of podocyte lipid metabolism.

Kidney Int 2020 11;98(5):1087-1090

Institute for Cellular and Integrative Physiology, University Clinic Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

Obesity and hyperlipidemia are the most prevalent independent risk factors of ESRD, suggesting that lipid accumulation is detrimental to renal function. The origin of lipid accumulation (a common feature in podocyte injury) and its pathophysiological relevance are unknown. This commentary discusses the finding by Liu et al. that deficiency of the endoplasmic reticulum enzyme SOAT1, which metabolizes cholesterol to cholesterol esters, attenuates renal/podocyte injury in murine models of diabetes and Alport's syndrome.
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http://dx.doi.org/10.1016/j.kint.2020.07.008DOI Listing
November 2020

Podocytes Produce and Secrete Functional Complement C3 and Complement Factor H.

Front Immunol 2020 14;11:1833. Epub 2020 Aug 14.

University Children's Research@Kinder-UKE, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Podocytes are an important part of the glomerular filtration barrier and the key player in the development of proteinuria, which is an early feature of complement mediated renal diseases. Complement factors are mainly liver-born and present in circulation. Nevertheless, there is a growing body of evidence for additional sites of complement protein synthesis, including various cell types in the kidney. We hypothesized that podocytes are able to produce complement components and contribute to the local balance of complement activation and regulation. To investigate the relevant balance between inhibiting and activating sides, our studies focused on complement factor H (CFH), an important complement regulator, and on C3, the early key component for complement activation. We characterized human cultured podocytes for the expression and secretion of activating and regulating complement factors, and analyzed the secretion pathway and functional activity. We studied glomerular CFH and C3 expression in puromycin aminonucleoside (PAN) -treated rats, a model for proteinuria, and the physiological mRNA-expression of both factors in murine kidneys. We found, that C3 and CFH were expressed in cultured podocytes and expression levels differed from those in cultivated glomerular endothelial cells. The process of secretion in podocytes was stimulated with interferon gamma and located in the Golgi apparatus. Cultured podocytes could initiate the complement cascade by the splitting of C3, which can be shown by the generation of C3a, a functional C3 split product. C3 contributed to external complement activation. Podocyte-secreted CFH, in conjunction with factor I, was able to split C3b. Podocytes derived from a patient with a CFH mutation displayed impaired cell surface complement regulation. CFH and C3 were synthesized in podocytes of healthy C57Bl/6-mice and were upregulated in podocytes of PAN treated rats. These data show that podocytes produce functionally active complement components, and could therefore influence the local glomerular complement activation and regulation. This modulating effect should therefore be considered in all diseases where glomerular complement activation occurs. Furthermore, our data indicate a potential novel role of podocytes in the innate immune system.
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http://dx.doi.org/10.3389/fimmu.2020.01833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457071PMC
April 2021

Pathogen-induced tissue-resident memory T17 (T17) cells amplify autoimmune kidney disease.

Sci Immunol 2020 08;5(50)

Hamburg Center for Translational Immunology (HCTI), University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (T) cells persist in peripheral organs and provide immune protection against reinfection. However, whether T cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4 T cells with a T17 signature (termed T17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal T17 cells were induced by pathogens infecting the kidney, such as , , and uropathogenic , and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney T17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced T17 cells have a previously unrecognized function in aggravating autoimmune disease.
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http://dx.doi.org/10.1126/sciimmunol.aba4163DOI Listing
August 2020

Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria.

J Am Soc Nephrol 2020 08 8;31(8):1796-1814. Epub 2020 Jul 8.

Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background: The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms.

Methods: MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis.

Results: Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis.

Conclusions: Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.
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http://dx.doi.org/10.1681/ASN.2019090960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460914PMC
August 2020

Interleukin-9 protects from early podocyte injury and progressive glomerulosclerosis in Adriamycin-induced nephropathy.

Kidney Int 2020 09 21;98(3):615-629. Epub 2020 May 21.

III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

A wide spectrum of immunological functions has been attributed to Interleukin 9 (IL-9), including effects on the survival and proliferation of immune and parenchymal cells. In recent years, emerging evidence suggests that IL-9 expression can promote tissue repair in inflammatory conditions. However, data about the involvement of IL-9 in kidney tissue protection is very limited. Here, we investigated the role of IL-9 in Adriamycin-induced nephropathy (AN), a mouse model for proteinuric chronic kidney disease. Compared to wild type mice, IL-9 knockout (Il9) mice with AN displayed accelerated development of proteinuria, aggravated glomerulosclerosis and deterioration of kidney function. At an early stage of disease, the Il9 mice already displayed a higher extent of glomerular podocyte injury and loss of podocyte number compared to wild type mice. In the kidney, T cells and innate lymphoid cells produced IL-9. However, selective deficiency of IL-9 in the innate immune system in Il9Rag2 mice that lack T and B cells did not alter the outcome of AN, indicating that IL-9 derived from the adaptive immune system was the major driver of tissue protection in this model. Mechanistically, we could show that podocytes expressed the IL-9 receptor in vivo and that IL-9 signaling protects podocytes from Adriamycin-induced apoptosis in vitro. Finally, in vivo treatment with IL-9 effectively protected wild type mice from glomerulosclerosis and kidney failure in the AN model. The detection of increased serum IL-9 levels in patients with primary focal and segmental glomerulosclerosis further suggests that IL-9 production is induced by glomerular injury in humans. Thus, IL-9 confers protection against experimental glomerulosclerosis, identifying the IL-9 pathway as a potential therapeutic target in proteinuric chronic kidney disease.
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http://dx.doi.org/10.1016/j.kint.2020.04.036DOI Listing
September 2020

Inhibition of mTOR delayed but could not prevent experimental collapsing focal segmental glomerulosclerosis.

Sci Rep 2020 05 22;10(1):8580. Epub 2020 May 22.

Department of pathology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.

Anti-Thy1.1 transgenic mice develop glomerular lesions that mimic collapsing focal segmental glomerulosclerosis (FSGS) in humans with collapse of the glomerular tuft and marked hyperplasia of the parietal epithelial cells (PECs). Immunostaining of phosphor-S6 ribosomal protein (pS6RP) revealed high mTOR activity in PECs of the FSGS lesions of these mice. In this study we questioned whether the mTOR inhibitor rapamycin (sirolimus) could attenuate the development and progression of glomerulosclerotic lesions in the anti-Thy1.1 transgenic mice. We observed reduced mTOR signalling and proliferation in human parietal epithelial cells after rapamycin treatment. Experiments with anti-Thy1.1. mice showed that early treatment with sirolimus reduced the development of glomerular lesions and glomerular cell proliferation at day 4. Levels of albuminuria, podocyte injury and podocyte number were similar in the sirolimus and vehicle treated groups. The initial beneficial effects of sirolimus treatment were not observed at day 7. Late sirolimus treatment did not reduce albuminuria or the progression of glomerulosclerosis. Taken together, rapamycin attenuated PEC proliferation and the formation of early FSGS lesions in experimental FSGS and reduced human PEC proliferation in vitro. However, the initial inhibition of PEC proliferation did not translate into a decline of albuminuria nor in a sustained reduction in sclerotic lesions.
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http://dx.doi.org/10.1038/s41598-020-65352-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244565PMC
May 2020

B6.Rag1 Knockout Mice Generated at the Jackson Laboratory in 2009 Show a Robust Wild-Type Hypertensive Phenotype in Response to Ang II (Angiotensin II).

Hypertension 2020 04 10;75(4):1110-1116. Epub 2020 Feb 10.

From the Institute of Cellular and Integrative Physiology (A. Seniuk, J.L.T., A. Stubbe, P.O., C.M.-S., H.E.), University Medical Center Hamburg, Germany.

A key finding supporting a causal role of the immune system in the pathogenesis of hypertension is the observation that knockout mice on a C57Bl/6J background (B6.Rag1), which lack functional B and T cells, develop a much milder hypertensive response to Ang II (angiotensin II) than control C57Bl/6J mice. Here, we report that we never observed any Ang II resistance of B6.Rag1 mice purchased directly from the Jackson Laboratory as early as 2009. B6.Rag1 mice displayed nearly identical blood pressure increases monitored via radiotelemetry and hypertensive end-organ damage in response to different doses of Ang II and different levels of salt intake (0.02%, 0.3%, and 3% NaCl diet). Similarly, restoration of T-cell immunity by adoptive cell transfer did not affect the blood pressure response to Ang II in B6.Rag1 mice. Full development of the hypertension-resistant phenotype in B6.Rag1 mice appears to depend on the action of yet unidentified nongenetic modifiers in addition to the absence of functional T cells.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.13773DOI Listing
April 2020

A novel mouse model of phospholipase A2 receptor 1-associated membranous nephropathy mimics podocyte injury in patients.

Kidney Int 2020 05 9;97(5):913-919. Epub 2019 Nov 9.

III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

The phospholipase A2 receptor 1 (PLA2R1) is the major autoantigen in patients suffering from membranous nephropathy. To date, the lack of endogenous glomerular expression of PLA2R1 in mice and rats has impeded the establishment of PLA2R1-dependent animal models of this disease. Here, we generated a transgenic mouse line expressing murine full-length PLA2R1 in podocytes. Furthermore, expression of murine PLA2R1 did not result in any morphological disturbance as high-resolution confocal microscopy demonstrated an intact nephrin distribution with normal foot processes. Transfer of rabbit anti-mPLA2R1 antibodies to these mice induced nephrotic range proteinuria, hypercholesterolemia, and histomorphological signs of membranous nephropathy. Immunohistochemical and immunofluorescence analyses revealed enhanced staining for murine PLA2R1 in the presence of unaffected staining for murine thrombospondin type-1 domain-containing 7A in the diseased mice, resembling what is classically found in patients with PLA2R1-associated membranous nephropathy Thus, our mouse model of membranous nephropathy will allow investigation of PLA2R1-specific pathomechanisms and may help to develop and assess antigen-specific treatments in vivo.
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http://dx.doi.org/10.1016/j.kint.2019.10.022DOI Listing
May 2020

Enzyme replacement therapy in mice lacking arylsulfatase B targets bone-remodeling cells, but not chondrocytes.

Hum Mol Genet 2020 03;29(5):803-816

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Mucopolysaccharidosis type VI (MPS-VI), caused by mutational inactivation of the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), is a lysosomal storage disorder primarily affecting the skeleton. We have previously reported that Arsb-deficient mice display high trabecular bone mass and impaired skeletal growth. In the present study, we treated them by weekly injection of recombinant human ARSB (rhARSB) to analyze the impact of enzyme replacement therapy (ERT) on skeletal growth and bone remodeling. We found that all bone-remodeling abnormalities of Arsb-deficient mice were prevented by ERT, whereas chondrocyte defects were not. Likewise, histologic analysis of the surgically removed femoral head from an ERT-treated MPS-VI patient revealed that only chondrocytes were pathologically affected. Remarkably, a side-by-side comparison with other cell types demonstrated that chondrocytes have substantially reduced capacity to endocytose rhARSB, together with low expression of the mannose receptor. We finally took advantage of Arsb-deficient mice to establish quantification of chondroitin sulfation for treatment monitoring. Our data demonstrate that bone-remodeling cell types are accessible to systemically delivered rhARSB, whereas the uptake into chondrocytes is inefficient.
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http://dx.doi.org/10.1093/hmg/ddaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104678PMC
March 2020

Deubiquitinating Enzyme UCH-L1 Promotes Dendritic Cell Antigen Cross-Presentation by Favoring Recycling of MHC Class I Molecules.

J Immunol 2019 10 6;203(7):1730-1742. Epub 2019 Sep 6.

Institute for Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany;

The deubiquitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) is required for the maintenance of axonal integrity in neurons and is thought to regulate the intracellular pool of ubiquitin in the brain. In this study, we show that UCH-L1 has an immunological function in dendritic cell (DC) Ag cross-presentation. UCH-L1 is expressed in mouse kidney, spleen, and bone marrow-derived DCs, and its expression and activity are regulated by the immune stimuli LPS and IFN-γ. UCH-L1-deficient mice have significantly reduced ability to cross-prime CD8 T cells in vivo and in vitro because of a reduced ability of DCs to generate MHC class I (MHC I) peptide complexes for cross-presented Ags. Mechanistically, Ag uptake by phagocytosis and receptor-mediated endocytosis as well as phagosome maturation are unaffected by loss of UCH-L1 in DCs. Rather, MHC I recycling is reduced by loss of UCH-L1, which affects the colocalization of intracellular MHC I with late endosomal/lysosomal compartments necessary for cross-presentation of Ag. These results demonstrate a hitherto unrecognized role of the deubiquitinating enzyme UCH-L1 in DC Ag processing.
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http://dx.doi.org/10.4049/jimmunol.1801133DOI Listing
October 2019

The ubiquitin-proteasome system in kidney physiology and disease.

Nat Rev Nephrol 2019 07;15(7):393-411

Institute of Cellular and Integrative Physiology, University Clinic Hamburg-Eppendorf, Hamburg, Germany.

Intracellular proteins continuously turn over by degradation and synthesis in all organ tissues. Owing to its irreversible nature, protein degradation is a highly selective process to avoid irreparable breakdown of cellular constituents, thereby disrupting cellular stability, integrity and signalling. The majority of intracellular proteins are degraded by the ubiquitin-proteasome system (UPS), a multi-enzyme process that involves the covalent conjugation of ubiquitin to a substrate protein and its recognition and degradation by the core multicomponent proteolytic complex of the UPS, the proteasome. In addition to labelling misfolded, damaged, aggregation-prone and intact but unneeded proteins for proteasomal degradation, ubiquitylation regulates a multitude of cellular processes, such as transcription, translation, endocytosis, and receptor activity and subcellular localization. In addition, the proteasome generates peptides for antigen presentation in the immune system and for further degradation by peptidases to provide amino acids for protein biosynthesis and gluconeogenesis. Alterations of the UPS or of protein substrates that render them more or less susceptible to degradation are responsible for disorders associated with renal cell dysfunction. In this Review, we provide insight into the elegant and complex nature of UPS-mediated proteostasis and focus on its established and potential roles in renal cell physiology and pathophysiology.
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http://dx.doi.org/10.1038/s41581-019-0148-1DOI Listing
July 2019

Renal proximal tubular epithelial cells exert immunomodulatory function by driving inflammatory CD4 T cell responses.

Am J Physiol Renal Physiol 2019 07 24;317(1):F77-F89. Epub 2019 Apr 24.

Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf , Hamburg , Germany.

In immune-mediated glomerular diseases like crescentic glomerulonephritis (cGN), inflammatory CD4 T cells accumulate within the tubulointerstitial compartment in close contact to proximal and distal tubular epithelial cells and drive renal inflammation and tissue damage. However, whether renal epithelial cell populations play a role in the pathogenesis of cGN by modulating CD4 T cell responses is less clear. In the present study, we aimed to investigate the potential of renal epithelial cells to function as antigen-presenting cells, thereby stimulating CD4 T cell responses. Using a FACS-based protocol that allowed comparative analysis of cortical epithelial cell populations, we showed that particularly proximal tubular epithelial cells (PTECs) express molecules linked with antigen-presenting cell function, including major histocompatibility complex class II (MHCII), CD74, CD80, and CD86 in homeostasis and nephrotoxic nephritis, a murine model of cGN. Protein expression was visualized at the PTEC single cell level by imaging flow cytometry. Interestingly, we found inflammation-dependent regulation of epithelium-expressed CD74, CD80, and CD86, whereas MHCII expression was not altered. Antigen-specific stimulation of CD4 T cells by PTECs in vitro supported CD4 T cell survival and induced CD4 T cell activation, proliferation, and inflammatory cytokine production. In patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis, MHCII and CD74 were expressed by both proximal and distal tubules, whereas CD86 was predominantly expressed by proximal tubules. Thus, particularly PTECs have the potential to induce an inflammatory phenotype in CD4 T cells in vitro, which might also play a role in the pathology of immune-mediated kidney disease.
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http://dx.doi.org/10.1152/ajprenal.00427.2018DOI Listing
July 2019

An unexpected role of steroid on podocytes: from zebrafish to human nephrotic syndrome?

Kidney Int 2019 05;95(5):1015-1017

Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

In this issue of Kidney International, Jobst-Schwan et al. developed a zebrafish model of MAGI2-deficiency, which recapitulates findings of human nephrotic syndrome due to MAGI2 mutations. The authors use this model system to screen for drugs that might target and alleviate MAGI2-associated nephrotic syndrome pathways. The scientific context of this publication and the significance of its key findings are discussed in this commentary.
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http://dx.doi.org/10.1016/j.kint.2019.01.044DOI Listing
May 2019

Thrombospondin Type 1 Domain-Containing 7A Localizes to the Slit Diaphragm and Stabilizes Membrane Dynamics of Fully Differentiated Podocytes.

J Am Soc Nephrol 2019 05 10;30(5):824-839. Epub 2019 Apr 10.

Institutes of Cellular and Integrative Physiology and

Background: About 3%-5% of adults with membranous nephropathy have autoantibodies directed against thrombospondin type 1 domain-containing 7A (THSD7A), a podocyte-expressed transmembrane protein. However, the temporal and spatial expression of THSD7A and its biologic function for podocytes are unknown, information that is needed to understand the effects of THSD7A autoantibodies in this disease.

Methods: Using a variety of microscopic techniques, we analyzed THSD7A localization in postnatal, adult, and autoantibody-injected mice as well as in human podocytes. We also analyzed THSD7A function in human podocytes using confocal microscopy; Western blotting; and adhesion and migration assays.

Results: We found that THSD7A expression begins on glomerular vascularization with slit diaphragm formation in development. THSD7A localizes to the basal aspect of foot processes, closely following the meanders of the slit diaphragm in human and mice. Autoantibodies binding to THSD7A localize to the slit diaphragm. In human podocytes, THSD7A expression is accentuated at filopodia and thin arborized protrusions, an expression pattern associated with decreased membrane activity of cytoskeletal regulators. We also found that, phenotypically, THSD7A expression in human podocytes is associated not only with increases in cell size, enhanced adhesion, and reduced detachment from collagen type IV-coated plates but also, with decreased ability to migrate.

Conclusions: Our findings suggest that THSD7A functions as a foot process protein involved in the stabilization of the slit diaphragm of mature podocytes and that autoantibodies to THSD7A, on the basis of their localization, might structurally and functionally alter the slit diaphragm's permeability to protein.
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http://dx.doi.org/10.1681/ASN.2018090941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493974PMC
May 2019

Ubiquitin C-terminal hydrolase L1 (UCH-L1) loss causes neurodegeneration by altering protein turnover in the first postnatal weeks.

Proc Natl Acad Sci U S A 2019 04 28;116(16):7963-7972. Epub 2019 Mar 28.

Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany;

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.
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http://dx.doi.org/10.1073/pnas.1812413116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475369PMC
April 2019

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins.

J Vis Exp 2019 01 18(143). Epub 2019 Jan 18.

Department of Nephrology, Medical Faculty, Heinrich-Heine-University.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).
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http://dx.doi.org/10.3791/58542DOI Listing
January 2019

Endogenous IL-22 is dispensable for experimental glomerulonephritis.

Am J Physiol Renal Physiol 2019 04 6;316(4):F712-F722. Epub 2019 Feb 6.

Third Department of Medicine, University Medical Center Hamburg-Eppendorf , Hamburg , Germany.

In recent years, the cytokine interleukin (IL)-22 attracted considerable attention due to its important immunoregulatory function in barrier tissues, such as the gut, lung, and skin. Although a regenerative role of IL-22 in renal tubular damage has been demonstrated, the role of IL-22 in the immunopathogenesis of glomerular injury is still unknown. Here, we demonstrate that the IL-22 receptor is expressed in the glomerular compartment of the kidney and that IL-22 expression increases in the renal cortex after induction of glomerular injury in a mouse model for crescentic glomerulonephritis (cGN, nephrotoxic nephritis). We identified γδ T cells and T17 cells as major sources for IL-22 in the nephritic kidney. However, neither genetic or antibody-mediated deletion of IL-22 nor genetic deficiency in its endogenous inhibitor IL-22Rα2 (IL-22 binding protein) resulted in substantial phenotypic differences in mice with cGN with respect to crescent formation, tubulointerstitial damage, and kidney function impairment. Similarly, we did not observe significant differences between wild-type or IL-22-deficient mice in a mouse model of secondary focal and segmental glomerulosclerosis (adriamycin-induced nephropathy). As shown previously, we detected concomitant upregulation of IL-17A and IFN-γ production by T cells during the course of cGN, providing alternative cytokine pathways that mediate glomerular injury in this model. In conclusion, we show here that endogenous IL-22 expression is redundant in different forms of glomerular injury, indicating that the IL-22-directed therapies that are being tested in various human diseases might not affect the kidney in patients with glomerular disease.
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http://dx.doi.org/10.1152/ajprenal.00303.2018DOI Listing
April 2019

Protecting the kidney against autoimmunity and inflammation.

Nat Rev Nephrol 2019 02;15(2):66-68

Institute of Cellular and Integrative Physiology, University Clinic Hamburg-Eppendorf, Hamburg, Germany.

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http://dx.doi.org/10.1038/s41581-018-0097-0DOI Listing
February 2019

Disease-Linked Glutarylation Impairs Function and Interactions of Mitochondrial Proteins and Contributes to Mitochondrial Heterogeneity.

Cell Rep 2018 09;24(11):2946-2956

Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. Electronic address:

Lysine glutarylation (Kglu) of mitochondrial proteins is associated with glutaryl-CoA dehydrogenase (GCDH) deficiency, which impairs lysine/tryptophan degradation and causes destruction of striatal neurons during catabolic crisis with subsequent movement disability. By investigating the role of Kglu modifications in this disease, we compared the brain and liver glutarylomes of Gcdh-deficient mice. In the brain, we identified 73 Kglu sites on 37 mitochondrial proteins involved in various metabolic degradation pathways. Ultrastructural immunogold studies indicated that glutarylated proteins are heterogeneously distributed in mitochondria, which are exclusively localized in glial cells. In liver cells, all mitochondria contain Kglu-modified proteins. Glutarylation reduces the catalytic activities of the most abundant glutamate dehydrogenase (GDH) and the brain-specific carbonic anhydrase 5b and interferes with GDH-protein interactions. We propose that Kglu contributes to the functional heterogeneity of mitochondria and may metabolically adapt glial cells to the activity and metabolic demands of neighboring GCDH-deficient neurons.
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http://dx.doi.org/10.1016/j.celrep.2018.08.014DOI Listing
September 2018

The chemokine receptor CXCR1 reduces renal injury in mice with angiotensin II-induced hypertension.

Am J Physiol Renal Physiol 2018 12 12;315(6):F1526-F1535. Epub 2018 Sep 12.

III. Department of Medicine, University Hospital Hamburg-Eppendorf , Hamburg , Germany.

The role of CXCR1, also known as fractalkine receptor, in hypertension is unknown. The present study determined the role of the fractalkine receptor CXCR1 in hypertensive renal and cardiac injury. Expression of CXCR1 was determined using CXCR1 mice that express a green fluorescent protein (GFP) reporter in CXCR1 cells. FACS analysis of leukocytes isolated from the kidney showed that 34% of CD45 cells expressed CXCR1. Dendritic cells were the majority of positive cells (67%) followed by macrophages (10%), NK cells (6%), and T cells (10%). With the use of confocal microscopy, the receptor was detected in the kidney only on infiltrating cells but not on resident renal cells. To evaluate the role of CXCR1 in hypertensive end-organ injury, an aggravated model of hypertension was used. Unilateral nephrectomy was performed followed by infusion of angiotensin II (ANG II, 1.5 ng·g·min) and a high-salt diet in wild-type ( n = 15) and CXCR1-deficient mice ( n = 18). CXCR1 deficiency reduced the number of renal dendritic cells and increased the numbers of renal CD11b/F4/80 macrophages and CD11b/Ly6G neutrophils in ANG II-infused mice. Surprisingly, CXCR1-deficient mice exhibited increased albuminuria, glomerular injury, and reduced podocyte density in spite of similar levels of arterial hypertension. In contrast, cardiac damage as assessed by increased heart weight, cardiac fibrosis, and expression of fetal genes, and matrix components were not different between both genotypes. Our findings suggest that CXCR1 exerts protective properties by modulating the invasion of inflammatory cells in hypertensive renal injury. CXCR1 inhibition should be avoided in hypertension because it may promote hypertensive renal injury.
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http://dx.doi.org/10.1152/ajprenal.00149.2018DOI Listing
December 2018

Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition.

Am J Pathol 2018 11 8;188(11):2508-2528. Epub 2018 Sep 8.

Immunology and Inflammation Center, Feinstein Institute for Medical Research and Zucker School of Medicine at Hofstra-Northwell, Manhasset, New York. Electronic address:

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist-, miR193a inhibitor-, and interferon-γ-induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA-silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship.
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http://dx.doi.org/10.1016/j.ajpath.2018.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222279PMC
November 2018

Glomerular endothelial cell maturation depends on ADAM10, a key regulator of Notch signaling.

Angiogenesis 2018 05 3;21(2):335-347. Epub 2018 Feb 3.

Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, USA.

The principal function of glomeruli is to filter blood through a highly specialized filtration barrier consisting of a fenestrated endothelium, the glomerular basement membrane and podocyte foot processes. Previous studies have uncovered a crucial role of endothelial a disintegrin and metalloprotease 10 (ADAM10) and Notch signaling in the development of glomeruli, yet the resulting defects have not been further characterized nor understood in the context of kidney development. Here, we used several different experimental approaches to analyze the kidneys and glomeruli from mice lacking ADAM10 in endothelial cells (A10ΔEC mice). Scanning electron microscopy of glomerular casts demonstrated enlarged vascular diameter and increased intussusceptive events in A10ΔEC glomeruli compared to controls. Consistent with these findings, genes known to regulate vessel caliber (Apln, AplnR and Vegfr3) are significantly upregulated in A10ΔEC glomeruli. Moreover, transmission electron microscopy revealed the persistence of diaphragms in the fenestrae of A10ΔEC glomerular endothelial cells, which was corroborated by the elevated expression of the protein PLVAP/PV-1, an integral component of fenestral diaphragms. Analysis of gross renal vasculature by light sheet microscopy showed no major alteration of the branching pattern, indicating a localized importance of ADAM10 in the glomerular endothelium. Since intussusceptions and fenestrae with diaphragms are normally found in developing, but not mature glomeruli, our results provide the first evidence for a crucial role of endothelial ADAM10, a key regulator of Notch signaling, in promoting the development and maturation of the glomerular vasculature.
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http://dx.doi.org/10.1007/s10456-018-9599-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5878725PMC
May 2018

A Heterologous Model of Thrombospondin Type 1 Domain-Containing 7A-Associated Membranous Nephropathy.

J Am Soc Nephrol 2017 Nov 16;28(11):3262-3277. Epub 2017 Aug 16.

III. Medizinische Klinik.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target for autoimmunity in patients with membranous nephropathy (MN). Circulating autoantibodies from patients with THSD7A-associated MN have been demonstrated to cause MN in mice. However, THSD7A-associated MN is a rare disease, preventing the use of patient antibodies for larger experimental procedures. Therefore, we generated antibodies against the human and mouse orthologs of THSD7A in rabbits by coimmunization with the respective cDNAs. Injection of these anti-THSD7A antibodies into mice induced a severe nephrotic syndrome with proteinuria, weight gain, and hyperlipidemia. Immunofluorescence analyses revealed granular antigen-antibody complexes in a subepithelial location along the glomerular filtration barrier 14 days after antibody injection, and immunohistochemistry for rabbit IgG and THSD7A as well as ultrastructural analyses showed the typical characteristics of human MN. Mice injected with purified IgG from rabbit serum that was taken before immunization failed to develop any of these changes. Notably, MN developed in the absence of detectable complement activation, and disease was strain dependent. , anti-THSD7A antibodies caused cytoskeletal rearrangement and activation of focal adhesion signaling. Knockdown of the THSD7A ortholog, thsd7aa, in zebrafish larvae resulted in altered podocyte differentiation and impaired glomerular filtration barrier function, with development of pericardial edema, suggesting an important role of THSD7A in glomerular filtration barrier integrity. In summary, our study introduces a heterologous mouse model that allows further investigation of the molecular events that underlie MN.
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http://dx.doi.org/10.1681/ASN.2017010030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661286PMC
November 2017

Ubiquitin C-Terminal Hydrolase L1 is required for regulated protein degradation through the ubiquitin proteasome system in kidney.

Kidney Int 2018 01 26;93(1):110-127. Epub 2017 Jul 26.

Department of Internal Medicine, Nephrology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.
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http://dx.doi.org/10.1016/j.kint.2017.05.016DOI Listing
January 2018

A novel in vivo method to quantify slit diaphragm protein abundance in murine proteinuric kidney disease.

PLoS One 2017 12;12(6):e0179217. Epub 2017 Jun 12.

Department of Nephrology, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.

Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179217PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467901PMC
September 2017

IL-33-Mediated Expansion of Type 2 Innate Lymphoid Cells Protects from Progressive Glomerulosclerosis.

J Am Soc Nephrol 2017 Jul 2;28(7):2068-2080. Epub 2017 Feb 2.

III. Medical Clinic,

Innate lymphoid cells (ILCs) have an important role in the immune system's response to different forms of infectious and noninfectious pathologies. In particular, IL-5- and IL-13-producing type 2 ILCs (ILC2s) have been implicated in repair mechanisms that restore tissue integrity after injury. However, the presence of renal ILCs in humans has not been reported. In this study, we show that ILC populations are present in the healthy human kidney. A detailed characterization of kidney-residing ILC populations revealed that IL-33 receptor-positive ILC2s are a major ILC subtype in the kidney of humans and mice. Short-term IL-33 treatment in mice led to sustained expansion of IL-33 receptor-positive kidney ILC2s and ameliorated adriamycin-induced glomerulosclerosis. Furthermore, the expansion of ILC2s modulated the inflammatory response in the diseased kidney in favor of an anti-inflammatory milieu with a reduction of pathogenic myeloid cell infiltration and a marked accumulation of eosinophils that was required for tissue protection. In summary, kidney-residing ILC2s can be effectively expanded in the mouse kidney by IL-33 treatment and are central regulators of renal repair mechanisms. The presence of ILC2s in the human kidney tissue identifies these cells as attractive therapeutic targets for CKD in humans.
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http://dx.doi.org/10.1681/ASN.2016080877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491284PMC
July 2017

Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation.

Sci Transl Med 2016 11;8(366):366ra162

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany.

Ion channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5'-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1β (IL-1β). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1β release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeutics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7.
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http://dx.doi.org/10.1126/scitranslmed.aaf8463DOI Listing
November 2016