Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC γ subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.

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) × 10 ³ monocyte‐derived EVs/monocyte. The steady‐state plasma EV level was maintained by an estimated plasma EV secretion rate of (1.5 ± 0.4) × 10 ¹² 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.

Vitamin D has cardiovascular protective effects besides regulating calcium homeostasis. To examine the chronic in vivo effect of a physiological dose of 1,25-dihydroxyvitamin D₃on the occurrence of endothelium-dependent contractions, spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were treated with the vitamin D derivative for 6 wk. The serum 1,25-dihydroxyvitamin D₃level of both treated WKY and SHR was significantly higher than in untreated rats while the mean arterial blood pressure of the treated SHR was significantly lower than that of control SHR. Aortic rings with or without endothelium were studied in conventional organ chambers for isometric force measurement. Confocal microscopy was used to measure the cytosolic free calcium concentration (with the fluorescent dye fluo 4) and reactive oxygen species (ROS; with dichlorodihydrofluorescein diacetate). Reverse transcription PCR and Western blotting were used to determine the mRNA and protein expression level of cyclooxygenase-1 (COX-1), prostacyclin synthase, and thromboxane synthase. The endothelium-dependent concentration-contraction curves to both acetylcholine- and A-23187-induced contractions were shifted to the right in aortas from treated SHR but not from treated WKY. The chronic treatment normalized the relaxations of contracted preparations to acetylcholine. There were no significant differences in the increases in cytosolic free calcium concentration evoked by acetylcholine and A-23187 between control and treated groups. The endothelial ROS level was higher in SHR than WKY aortas and reduced by the chronic treatment. The gene and protein expression studies indicated that the overexpression of COX-1 observed in SHR aorta was reduced by the chronic treatment. These results demonstrate that chronic treatment with 1,25-dihydroxyvitamin D₃modulates vascular tone and this modulation is accompanied by a lowered blood pressure, reduced expression of COX-1 mRNA and protein, and reduced ROS level in SHR. The reduction in endothelium-dependent contractions does not involve the surge in endothelial cytosolic calcium concentration that initiates the contractions.

Acute kidney injury (AKI) is often caused by ischemia-reperfusion injury (IRI). IRI significantly affects kidney metabolism, which elicits pro-inflammatory responses and kidney injury. The ischemia/reperfusion of the kidney is associated with transient high mitochondrial-derived reactive oxygen species (ROS) production rates. Excessive mitochondrial-derived ROS damages cellular components and, together with other pathogenic mechanisms, elicits a range of acute injury mechanisms that impair kidney function. Mitochondrial-derived ROS production also stimulates epithelial cell secretion of extracellular vesicles (EVs) containing RNAs, lipids, and proteins, suggesting that EVs are involved in AKI pathogenesis. This literature review focuses on how EV secretion is stimulated during ischemia/reperfusion and how cell-specific EVs and their molecular cargo may modify the IRI process. Moreover, critical pitfalls in the analysis of kidney epithelial-derived EVs are described. In particular, we will focus on how the release of kidney epithelial EVs is affected during tissue analyses and how this may confound data on cell-to-cell signaling. By increasing awareness of methodological pitfalls in renal EV research, the risk of false negatives can be mitigated. This will improve future EV data interpretation regarding EVs contribution to AKI pathogenesis and their potential as biomarkers or treatments for AKI.

Diabetic nephropathy (DN) is associated with hypertension, expanded extracellular volume and impaired renal Na⁺ excretion. It was hypothesized that aberrant glomerular filtration of serine proteases in DN causes proteolytic activation of the epithelial sodium channel (ENaC) in the kidney by excision of an inhibitory peptide tract from the γ subunit. In a cross-sectional design, urine, plasma and clinical data were collected from type 1 diabetic patients with DN (n = 19) and matched normoalbuminuric type 1 diabetics (controls, n = 20). Urine was examined for proteases by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the ‘inhibitory’ peptide tract. Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion of plasmin(ogen), prostasin and urokinase that correlated directly with urine albumin. Western blotting confirmed plasmin, prostasin and urokinase in urine from the DN group predominantly. Urine from DN evoked a significantly larger amiloride-sensitive inward current in single collecting duct cells compared with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact γENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin was observed in DN samples predominantly. DN is associated with increased urinary excretion of plasmin, prostasin and urokinase and proteolytic activation of ENaC that might contribute to impaired renal Na⁺ excretion and hypertension.