BackgroundUrinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear.MethodsUrine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization.ResultsUrine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47–0.95) and patients (R2 0.41–0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers.Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment–specific EVs.

Because of the presence of the blood-brain barrier, brain renin-angiotensin system activity should depend on local (pro)renin synthesis. Indeed, an intracellular form of renin has been described in the brain, but whether it displays angiotensin (Ang) I-generating activity (AGA) is unknown. Here, we quantified brain (pro)renin, before and after buffer perfusion of the brain, in wild-type mice, renin knockout mice, deoxycorticosterone acetate salt-treated mice, and Ang II-infused mice. Brain regions were homogenized and incubated with excess angiotensinogen to detect AGA, before and after prorenin activation, using a renin inhibitor to correct for nonrenin-mediated AGA. Renin-dependent AGA was readily detectable in brain regions, the highest AGA being present in brain stem (>thalamus=cerebellum=striatum=midbrain>hippocampus=cortex). Brain AGA increased marginally after prorenin activation, suggesting that brain prorenin is low. Buffer perfusion reduced AGA in all brain areas by >60%. Plasma renin (per mL) was 40× to 800× higher than brain renin (per gram). Renin was undetectable in plasma and brain of renin knockout mice. Deoxycorticosterone acetate salt and Ang II suppressed plasma renin and brain renin in parallel, without upregulating brain prorenin. Finally, Ang I was undetectable in brains of spontaneously hypertensive rats, while their brain/plasma Ang II concentration ratio decreased by 80% after Ang II type 1 receptor blockade. In conclusion, brain renin levels (per gram) correspond with the amount of renin present in 1 to 20 μL of plasma. Brain renin disappears after buffer perfusion and varies in association with plasma renin. This indicates that brain renin represents trapped plasma renin. Brain Ang II represents Ang II taken up from blood rather than locally synthesized Ang II.

Fluid management has been a vital part of routine clinical care for more than 180 years. The increasing number of available fluids has generated controversy about the optimal choice of resuscitation fluid. In this review, we provide a critical overview of the different fluids available, their composition, the relevant physiology as well as the published evidence on clinical outcomes to guide their use. Commonly used infusion fluids include semisynthetic colloids and crystalloids; the latter comprises both normal saline (NaCl 0.9%) and the more chloride-restricted 'balanced' crystalloids. Despite their significantly greater intravascular persistence, semisynthetic colloids have an importantly adverse safety profile and are associated with greater incidence of renal failure and increased mortality; their use should be restricted. To date, evidence for clinical benefits associated with albumin solutions is generally lacking; its merits in specific clinical situations are the subject of further investigation. Infusion of normal saline, with its supraphysiological chloride content, is associated with higher serum chloride concentrations and metabolic acidosis, as well as renal vasoconstriction in animal and human models. Infusion of 'balanced' crystalloids is not linked to such changes. Although data on clinical outcomes associated with crystalloid infusion are heterogeneous, advantages of balanced salt solutions might include a lower need of blood products, and lower incidence of renal replacement therapy, hyperkalaemia and postoperative infections. Taken together, a critical appraisal of the data suggests that balanced salt solutions deserve consideration as infusates of first choice.

Urinary extracellular vesicles (uEVs) are emerging as non‐invasive biomarkers for various kidney diseases, but it is unknown how differences in nephron mass impact uEV excretion. To address this, uEV excretion was measured before and after human kidney donor nephrectomy and rat nephrectomy. In male and female donors, uEVs were quantified in cell‐free spot and 24‐h urine samples using nanoparticle tracking analysis (NTA), EVQuant, and CD9‐time‐resolved fluorescence immunoassay. Female donors had significantly lower total kidney volume (TKV) and excreted 49% fewer uEVs than male donors. uEV excretion correlated positively with estimated glomerular filtration rate (eGFR), creatinine clearance, and TKV (R's between 0.6 and 0.7). uEV excretion rate could also be predicted from spot urines after multiplying spot uEV/creatinine by 24‐h urine creatinine. Donor nephrectomy reduced eGFR by 36% ± 10%, but the excretion of uEVs by only 16% (CD9+ uEVs ‐37%, CD9‐ uEVs no decrease). Donor nephrectomy increased the podocyte marker WT‐1 and the proximal tubule markers NHE3, NaPi‐IIa, and cubilin in uEVs two‐ to four‐fold when correcting for the nephrectomy. In rats, the changes in GFR and kidney weight correlated with the changes in uEV excretion rate (R = 0.46 and 0.60, P < 0.01). Furthermore, the estimated degree of hypertrophy matched the change in uEV excretion rate (1.4‐ to 1.5‐fold after uninephrectomy and four‐fold after 5/6th nephrectomy). Taken together, our data show that uEV excretion depends on nephron mass, and that nephrectomy reduces uEV excretion less than expected based on nephron loss due to compensatory hypertrophy. The major implication of our findings is that a measure for nephron mass or uEV excretion rate should be included when comparing uEV biomarkers between individuals.