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

Prostasin-dependent activation of epithelial Na+ channels by low plasmin concentrations

Udgivelsesdato: 2009-Sep-30Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial Na(+) channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity, and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the gamma-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 microg/ml). At a high plasmin concentration of 30 microg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, gammaENaC and prostasin were co-localized. A monoclonal antibody directed against the inhibitory peptide of gammaENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the gamma-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC. Key words: Serine proteases, Sodium retention, Kidney, M-1 cells

Adipocyte-Endothelium Crosstalk in Obesity

Obesity is characterized by pathological adipose tissue (AT) expansion. While healthy AT expansion enhances systemic insulin sensitivity, unhealthy AT expansion through increased adipocyte size is associated with insulin resistance, fibrosis, hypoxia, and reduced adipose-derived adiponectin secretion. The mechanisms causing the unhealthy AT expansion are not fully elucidated; yet, dysregulated crosstalk between cells within the AT is an important contributor. Evidence from animal and human studies suggests a crucial role of the crosstalk between vascular endothelium (the innermost cell type in blood vessels) and adipocytes for metabolic homeostasis. Arterial endothelial cells are directly involved in maintaining normal organ functions through local blood flow regulation. The endothelial-dependent regulation of blood flow in AT is hampered in obesity, which negatively affects the adipocyte. Moreover, endothelial cells secrete extracellular vesicles (EVs) that target adipocytes in vivo. The endothelial EVs secretion is hampered in obesity and may be affected by the adipocyte-derived adipokine adiponectin. Adiponectin targets the vascular endothelium, eliciting organ-protective functions through binding to T-cadherin. The reduced obesity-induced adiponectin binding of T-cadherin reduces endothelial EV secretion. This affects endothelial health and cell-cell communication between AT cells and distant organs, influencing systemic energy homeostasis. This review focuses on the current understanding of endothelial and adipocyte crosstalk. We will discuss how obesity changes the AT environment and how these changes contribute to obesity-associated metabolic disease in humans. Particularly, we will describe and discuss the EV-dependent communication and regulation between adipocytes, adiponectin, and the endothelial cells regulating systemic energy homeostasis in health and metabolic disease in humans.</p

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.</p

Dietary Na⁺ intake in healthy humans changes the urine extracellular vesicle prostasin abundance while the vesicle excretion rate, NCC, and ENaC are not altered

Low Na+ intake activates aldosterone signaling, which increases renal Na+ reabsorption through increased apical activity of the NaCl cotransporter (NCC) and the epithelial Na+ channel (ENaC). Na+ transporter proteins are excreted in urine as an integral part of cell-derived extracellular vesicles (uEVs). It was hypothesized that Na+ transport protein levels in uEVs from healthy humans reflect their physiological regulation by aldosterone. Urine and plasma samples from 10 healthy men (median age: 22.8 yr) were collected after 5 days on a low-Na+ (70 mmol/day) diet and 5 days on a high-Na+ (250 mmol/day) diet. uEVs were isolated by ultracentrifugation and analyzed by Western blot analysis for EV markers (CD9, CD63, and ALIX), transport proteins (Na+-K+-ATPase α1-subunit, NCC, ENaC α- and γ-subunits, and aquaporin 2), and the ENaC-cleaving protease prostasin. Plasma renin and aldosterone concentrations increased during the low-Na+ diet. uEV size and concentration were not different between diets by tunable resistive pulse sensing. EV markers ALIX and CD9 increased with the low-Na+ diet, whereas CD63 and aquaporin 2 excretion were unchanged. Full-length ENaC γ-subunits were generally not detectable in uEVs, whereas ENaC α-subunits, NCC, and phosphorylated NCC were consistently detected but not changed by Na+ intake. Prostasin increased with low Na+ in uEVs. uEV excretion of transporters was not correlated with blood pressure, urinary Na+ and K+ excretion, plasma renin, or aldosterone. In conclusion, apical Na+ transporter proteins and proteases were excreted in uEVs, and while the excretion rate and size of uEVs were not affected, EV markers and prostasin increased in response to the low-Na+ diet.</p

Mechanisms of sodium retention in nephrotic syndrome

PURPOSE OF REVIEW: Proteinuria in nephrotic syndrome is associated with sodium retention and edema. Recent studies from mice, rats and humans have shown that the sodium retention depends on urinary serine proteases and that it can be mitigated by blockers (amiloride, triamterene) of the epithelial sodium channel ENaC. The present review outlines the mechanisms of protease-stimulated sodium retention during proteinuric diseases.RECENT FINDINGS: Inhibition of protease activity in nephrotic mice using aprotinin alleviates sodium retention. From both human and mice studies, an increased proteolytic cleavage of the γENaC subunit plays a role in ENaC activation. In animal models, urokinase-plasmin contributes but not as sole mediators of sodium retention. Across experimental models, human case reports and small intervention trials, amiloride alleviates nephrotic sodium retention and low-renin hypertension with high efficacy.SUMMARY: Although the exact mechanisms for proteolytic ENaC activation are not resolved, multiple, redundant proteases are involved. Experimental and clinical evidence indicate that aberrant proteolytic ENaC activation is a primary driver of sodium retention in nephrotic syndrome and contributes to hypertension in conditions with low-grade proteinuria. Thus, we foresee increased and personalized use of amiloride treatment of nephrotic and other proteinuric disease patients with associated sodium retention and hypertension.</p

Benchmarking transcriptome deconvolution methods for estimating tissue- and cell-type-specific extracellular vesicle abundances

Extracellular vesicles (EVs) contain cell-derived lipids, proteins and RNAs; however, determining the tissue- and cell-type-specific EV abundances in body fluids remains a significant hurdle for our understanding of EV biology. While tissue- and cell-type-specific EV abundances can be estimated by matching the EV's transcriptome to a tissue's/cell type's expression signature using deconvolutional methods, a comparative assessment of deconvolution methods' performance on EV transcriptome data is currently lacking. We benchmarked 11 deconvolution methods using data from four cell lines and their EVs, in silico mixtures, 118 human plasma and 88 urine EVs. We identified deconvolution methods that estimated cell type-specific abundances of pure and in silico mixed cell line-derived EV samples with high accuracy. Using data from two urine EV cohorts with different EV isolation procedures, four deconvolution methods produced highly similar results. The three methods were also concordant in their tissue- and cell-type-specific plasma EV abundance estimates. We identified driving factors for deconvolution accuracy and highlighted the importance of implementing biological knowledge in creating the tissue/cell type signature. Overall, our analyses demonstrate that the deconvolution algorithms DWLS and CIBERSORTx produce highly similar and accurate estimates of tissue- and cell-type-specific EV abundances in biological fluids.</p

Unlocking the potential of extracellular vesicles in nephrology:what does MISEV2023 add?

Extracellular vesicles, small membrane-bound packages secreted by virtually all cells of the body, have become a focus of interest in nephrology over the recent years. After the first characterization of their proteomic and transcriptomic content, scientific attention shifted toward their potential as biomarkers for kidney diseases both as diagnostic and monitoring tools. More recently, researchers have begun exploring whether extracellular vesicles mediate intercellular signaling inside the nephron and between the kidney and other organs throughout the body. Nevertheless, the field of extracellular vesicle research has struggled to translate major findings to the clinical context due to numerous methods to separate extracellular vesicles, yielding fractions of different sizes and varying purity, unclear terminology, and, hence, limitations concerning reproducibility. The International Society of Extracellular Vesicles, therefore, has striven to reduce these barriers by an ongoing initiative to increase rigor and standardization of extracellular vesicle research. The “Minimal Information for Studies of Extracellular Vesicles” guideline is the result of this initiative and, in its now third iteration, provides the most concise suggestions for investigating extracellular vesicles to date. This mini review illustrates the advances made in extracellular vesicle research in nephrology so far using informative examples, outlines the advances made by the former Minimal Information for Studies of Extracellular Vesicles guidelines, and shows what potential using the latest iteration holds.</p