ATP releasing connexin 30 hemichannels mediate flow-induced calcium signaling in the collecting duct

ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y(2) receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(−/−) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca(2+)](i)) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30(−/−) mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca(2+)](i) in wild type CCDs. This response was blunted in Cx30(−/−) CCDs ([Ca(2+)](i) increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca(2+)](i) imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca(2+)](i) oscillations in free-flowing CDs of wild type but not Cx30(−/−) mice. The [Ca(2+)](i) oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

An estimate of extracellular vesicle secretion rates of human blood cells

Abstract Extracellular vesicles (EVs) have been implicated in the intercellular transfer of RNA and proteins through cellular secretion into the extracellular space. In blood plasma, circulating EVs are mainly derived from blood cells; however, factors that control plasma EV abundance are largely unknown. Here, we estimate the EV secretion rates for blood cell types using reported values for cell‐specific plasma EV abundances and their parental cell's ubiquity in healthy humans. While we found that plasma contains on average ∼2 plasma EVs/cell, the cell‐specific EV‐to‐cell ratio spanned four orders of magnitude from 0.13 ± 0.1 erythrocyte‐derived EVs/erythrocyte to (1.9 ± 1.3) × 103 monocyte‐derived EVs/monocyte. The steady‐state plasma EV level was maintained by an estimated plasma EV secretion rate of (1.5 ± 0.4) ×  1012 EVs/min. The cell‐specific secretion rate estimates were highest for monocytes (45 ± 21 EVs/cell/min) and lowest for erythrocytes ((3.2 ± 3.0) ×  10−3 EVs/cell/min). The estimated basal cell‐specific EV secretion rates were not significantly correlated to the cell's lifespan or size; however, we observed a highly significant correlation to cellular mitochondrial enzyme activities. Together, our analysis indicates that cell‐specific mitochondrial metabolism, for example, via reactive oxygen species, affects plasma EV abundance through increased secretion rates, and the results provide a resource for understanding EV function in human health and disease

Plasmin activity assay based on luciferase activity

<p>Pilot experiment with the Plasmin-Glo probe</p