Pannexin‐1 mediates fluid shear stress‐sensitive purinergic signaling and cyst growth in polycystic kidney disease

Abstract

are contributed equally to this work.Abbreviations: Abcc6, ATP binding cassette subfamily c member 6; BB-FCF, brilliant blue-FCF; Cx30, connexin30; Cx30.3, connexin30.3; Cx37, connexin37; Entpd2, ectonucleoside triphosphate diphosphohydrolase 2; Entpd3, ectonucleoside triphosphate diphosphohydrolase 3; FBS, fetal bovine serum; FSS, fluid shear stress; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; MO, translation blocking morpholino; Nt5e, ecto-5′-nucleotidase; Panx1, pannexin-1; PC1, polycystin-1; PC2, polycystin-2; PKD, polycystic kidney disease; Pkd1, polycystic kidney disease 1; Pkd2, polycystic kidney disease 2; Ptgs2, prostaglandin-endoperoxide synthase 2. AbstractTubular ATP release is regulated by mechanosensation of fluid shear stress (FSS).Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1 −/− ) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1 −/− mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1 −/− mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed.Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD. K E Y W O R D SATP, fluid shear stress, pannexin-1, polycystin-1, purinergic signaling | 6383 VERSCHUREN Et al.

Extracellular vesicles regulate purinergic signaling and epithelial sodium channel expression in renal collecting duct cells

“…Total RNA was isolated from target cells treated with EVs using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription and real‐time quantitative PCR were performed as previously described 39 . RPS11 was used as a reference gene.…”

“…Reverse transcription and real-time quantitative PCR were performed as previously described. 39 Potential miRNAs regulating ENTPD1 expression were identified by in silico analysis using TargetScan, 40 miRDB, 41 and microrna.org 42 databases and literature search for presence of the miRNA in the kidney, proximal tubule, HK2 cells and/or human urine. To experimentally address the involvement of miRNAs, small RNAs were isolated using the Nucleospin miRNA kit (Macherey-Nagel, Düren, Germany).…”

Recent advances in understanding ion transport mechanisms in polycystic kidney disease

“…This group also showed the contribution of P2X7 in cyst development via PANX1-dependent ATP release in ARPKD (PCK rats) [ 123 ]. Later, Verschuren et al also demonstrated the PANX1-mediated ATP release into the tubular lumen; this phenomenon was reported to be due to fluid shear stress, and established in two models of PKD: the Pkd1 knockout mice ( iKsp-Pkd1 −/− ) and Pkd1 −/− mouse distal convoluted tubule cells [ 124 ]. Moreover, they also found that PANX1 inhibition attenuated cyst growth in zebrafish PKD2 morphants.…”

Recent Advances in Regenerative Tissue Fabrication: Tools, Materials, and Microenvironment in Hierarchical Aspects

“…The fluid shear stress was applied to mimic the environment of stem cells and to regulate their function and fate. [ 118,119 ] Jang et al used fluid shear stress to control the microenvironment to investigate its influence on both the fluid reabsorption of aquaporin‐2 and the orientation of actin cytoskeleton. [ 120 ] They induced this stimulus by using a syringe pump to cause the medium to flow through a PDMS‐based microfluidic system that had been integrated with a microporous polyester membrane.…”