Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease

Autor: Claire E. Hills, Wai Han Yiu, Gareth Price, Colin R. Green, Christos E. Chadjichristos, Joe A. Potter, Eleftherios Siamantouras, Sydney C.W. Tang, Panagiotis Kavvadas, Paul E. Squires
Přispěvatelé: Chadjichristos, Christos, University of Lincoln, Des Maladies Rénales Rares aux Maladies Fréquentes, Remodelage et Réparation, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), CHU Tenon [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), The University of Hong Kong (HKU), University of Auckland [Auckland]
Jazyk: angličtina
Rok vydání: 2020
Předmět:
medicine.medical_treatment
C710 Applied Molecular Biology
Biophysics and Biochemistry
Connexin
lcsh:Medicine
Biochemistry
[SDV.MHEP.UN]Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology
Connexins
Cell Line
Adherens junction
03 medical and health sciences
chemistry.chemical_compound
Mice
0302 clinical medicine
Adenosine Triphosphate
Chronic kidney disease
medicine
Animals
Humans
lcsh:QH573-671
Renal Insufficiency
Chronic
Cell adhesion
Molecular Biology
030304 developmental biology
0303 health sciences
Tight junction
Chemistry
lcsh:Cytology
Research
Purinergic receptor
lcsh:R
Cell Biology
Middle Aged
Hemichannels
[SDV.MHEP.UN] Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology
Cell biology
ATP
Cytokine
Tubular injury
Kidney Tubules
030220 oncology & carcinogenesis
Paracellular transport
Connexin 43
C700 Molecular Biology
Biophysics and Biochemistry
cardiovascular system
Adenosine triphosphate
Zdroj: Cell Communication and Signaling : CCS
Cell Communication and Signaling, Vol 18, Iss 1, Pp 1-17 (2020)
Cell Communication and Signaling
Cell Communication and Signaling, BioMed Central, 2020, 18 (1), pp.79. ⟨10.1186/s12964-020-00558-1⟩
ISSN: 1478-811X
DOI: 10.1186/s12964-020-00558-1⟩
Popis: Background Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. Methods Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-β1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/− mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. Results Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-β1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/− mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. Conclusion Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. Graphical abstract In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-β1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP]e), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability.
Databáze: OpenAIRE
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