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Activation of the Endogenous Renin-Angiotensin- Aldosterone System Or Aldosterone Administration Increases Urinary Exosomal Sodium Channel Excretion

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Activation of the Endogenous Renin-Angiotensin- Aldosterone System Or Aldosterone Administration Increases Urinary Exosomal Sodium Channel Excretion CLINICAL RESEARCH www.jasn.org Activation of the Endogenous Renin-Angiotensin- Aldosterone System or Aldosterone Administration Increases Urinary Exosomal Sodium Channel Excretion † † Ying Qi,* Xiaojing Wang, Kristie L. Rose,* W. Hayes MacDonald,* Bing Zhang, ‡ | Kevin L. Schey,* and James M. Luther § Departments of *Biochemistry, †Bioinformatics, ‡Division of Clinical Pharmacology, Department of Medicine, §Division of Nephrology, Department of Medicine, and |Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee ABSTRACT Urinary exosomes secreted by multiple cell types in the kidney may participate in intercellular signaling and provide an enriched source of kidney-specific proteins for biomarker discovery. Factors that alter the exosomal protein content remain unknown. To determine whether endogenous and exogenous hormones modify urinary exosomal protein content, we analyzed samples from 14 mildly hypertensive patients in a crossover study during a high-sodium (HS, 160 mmol/d) diet and low-sodium (LS, 20 mmol/d) diet to activate the endogenous renin-angiotensin-aldosterone system. We further analyzed selected exosomal protein content in a separate cohort of healthy persons receiving intravenous aldosterone (0.7 mg/kg per hour for 10 hours) versus vehicle infusion. The LS diet increased plasma renin activity and aldosterone concentration, whereas aldosterone infusion increased only aldosterone concentration. Protein analysis of paired urine exosome samples by liquid chromatography-tandem mass spectrometry–based multidimen- sional protein identification technology detected 2775 unique proteins, of which 316 exhibited signifi- cantly altered abundance during LS diet. Sodium chloride cotransporter (NCC) and a-andg-epithelial sodium channel (ENaC) subunits from the discovery set were verified using targeted multiple reaction monitoring mass spectrometry quantified with isotope-labeled peptide standards. Dietary sodium restric- tion or acute aldosterone infusion similarly increased urine exosomal gENaC[112–122] peptide concentra- tions nearly 20-fold, which correlated with plasma aldosterone concentration and urinary Na/K ratio. Urine exosomal NCC and aENaC concentrations were relatively unchanged during these interventions. We conclude that urinary exosome content is altered by renin-angiotensin-aldosterone system activation. Urinary measurement of exosomal gENaC[112–122] concentration may provide a useful biomarker of ENaC activation in future clinical studies. J Am Soc Nephrol 27: 646–656, 2016. doi: 10.1681/ASN.2014111137 Urinary exosomes are small (approximately 100 nm activity are unknown. Further characterization of diameter) vesicles excreted by multiple cell types the urinary exosome protein response to these along the nephron and urogenital tract that provide a unique source of kidney-enriched pro- teins.1,2 Urinary exosomes contain RNA and pro- Received November 25, 2014. Accepted April 22, 2015. teins, including multiple sodium channels and Published online ahead of print. Publication date available at transporters, and they may contribute to physio- www.jasn.org. 1–10 logic processes in the kidney. Whether proteins Correspondence: Dr. James M. Luther, Vanderbilt University within urinary exosomes are altered by dietary fac- Medical Center2200 Pierce Avenue, 560 RRB, Nashville, TN tors or exogenous hormones and whether these 37232-6602. Email: [email protected] changes are useful in determining physiologic Copyright © 2016 by the American Society of Nephrology 646 ISSN : 1046-6673/2702-646 J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH modifying factors would inform their use in future biomarker We previously identified .3000 unique proteins from hu- discovery efforts. man urinary exosomes using multidimensional protein iden- Urinary exosomes are formed in cells lining the nephron by tification technology (MudPIT).5 In the present study, we formation of endocytic vesicles that may eventually be released tested the hypothesis that RAAS activation during a low- into the urinary space by the process of exocytosis.11 The exo- sodium (LS) diet and during exogenous aldosterone infusion some content includes membrane and soluble proteins as well alters the urinary exosome proteomic profile. We extended these as RNA, all of which may be relatively protected from degra- findings using a more targeted, sensitive, and quantitative dation in the urine by the vesicular lipid bilayer. The role of approach to investigate the profile of ENaC and NCC, which exosomes in human physiology is an area of intense investi- are known to play an essential role in renal sodium and po- gation, but within the kidney they may transport their con- tassium homeostasis. tents intercellularly, signal fibrotic responses, and perform innate immune functions.7–9 Much interest has focused on urinary exosomes as a source for biomarker discovery in hu- RESULTS mans due to the relative enrichment of membrane proteins.11 Validation of a urinary biomarker would be supported by Participant Characteristics and Effects of Dietary predictable alterations during physiologic stimulation or in- Sodium Restriction hibition. Potential approaches in humans could use dietary, We assessed the urinary exosome protein cargo using matched pharmacologic, or hormonal modification. urine samples from 14 patients during a high-sodium (HS) The renin-angiotensin-aldosterone system (RAAS) is acti- and a low-sodium (LS) diet to activate the endogenous RAAS vated in response to dietary sodium restriction, which helps and increase renal sodium reabsorption. In a separate crossover maintain long-term BP by modifying renal sodium and water study, aldosterone (0.7 mg/kg per hour) and vehicle were in- handling.12 The RAAS stimulates sodium reabsorption in part fused intravenously overnight (10 pm–8 am) as described pre- via aldosterone, leading to epithelial sodium channel (ENaC) viously,14 and urine was collected from 1 am to 7 am for and sodium chloride cotransporter (NCC) activation, which exosome isolation and analysis. Participant characteristics can be inhibited by potassium sparing (e.g., amiloride) and are presented in Table 1. During the LS diet, plasma renin thiazide diuretics, respectively.13 Because no direct measures activity and plasma aldosterone increased and urinary so- of renal ENaC activity exist in humans, it has been estimated dium excretion (198.5621.0 for HS diet versus 18.662.1 by urinary sodium-to-potassium ratio in prior studies. So- mmol/d for LS diet; P,0.001) and urinary sodium-to-potas- dium channel peptides and novel sodium channel phosphor- sium ratio (2.2060.18 versus 0.2960.05, respectively; ylation sites have been identified by proteomic analysis of P,0.001) decreased as anticipated. Urinary creatinine con- urinary exosomal proteins,1 but no studies have investigated centration (0.8460.12 mg/ml for HS diet versus 0.9560.17 their physiologic role or the dynamic changes during RAAS mg/ml for LS diet; P=0.24), creatinine excretion rate (1.596 activation in humans. 0.10 g/d versus 1.6360.13 g/d; P=0.77), urinary exosomal Table 1. Participant characteristics and physiologic effects of dietary sodium restriction Dietary Study Aldosterone Characteristic P Value P Value HS LS HS+Vehicle HS+Aldosterone Screening measurements Age (yr) 42.963.0 44.967.8 Men/women (n/n)7/72/2 Race (white/black) (n/n) 10/4 4/0 Height (m) 1.7460.033 1.7165.5 Weight (kg) 90.967.8 98.366.4 Body mass index (kg/m2) 29.561.6 33.862.1 Creatinine (mg/dl) 0.8960.04 0.7960.09 Serum sodium (mEq/L) 139.260.49 139.560.65 Serum potassium (mEq/L) 4.060.08 3.860.10 Pre- and post-dietary measures Systolic BP (mmHg) 136.064.1 131.463.4 0.32 111.865.5 108.668.2 0.56 Diastolic BP (mmHg) 81.563.3 79.162.0 0.36 60.563.9 58.163.4 0.47 Heart rate (beats/min) 64.562.4 67.862.8 0.004 60.365.5 57.964.7 0.27 Plasma aldosterone (ng/dl) 7.6960.81 16.161.8 ,0.001 7.561.2 83.5627.9 0.06 Plasma renin activity (ng AngI/ml per hour 0.8760.20 2.9260.49 ,0.001 1.0860.22 1.8760.31 0.04 Unless otherwise noted, values are the mean6SD. J Am Soc Nephrol 27: 646–656, 2016 RAAS Alters Urinary Exosomes 647 CLINICAL RESEARCH www.jasn.org protein excretion (50.467.2 mg protein versus 54.266.2 mg and kallikrein-related peptidase 10, among others. These pro- protein; P=0.58), diastolic BP, and systolic BP were not sig- teins are responsible for the enriched GO terms, extracellular nificantly changed by LS diet. matrix organization, extracellular structure organization, and regulation of membrane protein ectodomain proteolysis in Dietary Sodium Restriction Alters Urinary Exosomal the biologic process category; serine-type peptidase activity, Protein Expression extracellular matrix structural constituent, serine hydrolase Paired urine exosome samples from 14 patients during the LS activity, and serine-type endopeptidase activity in the molec- and HS diets were analyzed by MudPIT. We identified a total of ular function category; and extracellular-related terms in the 1,514,909 tryptic peptides, representing 34,208 unique pep- cellular component category. For the downregulated proteins, tides with scores above the minimum peptide identity thresh- the abundance of ribosomal proteins observed at lower levels olds (see Concise Methods section). After removal of proteins in the HS samples are responsible for the endoplasmic retic- with low total spectral counts (,10 spectral counts across 28 ulum (ER)- and translation-related GO terms and the macro- samples), 2775 proteins remained (Supplemental Table 1). molecular complex and non-membrane bound organelle Dietary sodium restriction significantly altered the expression terms in Figure 2. of 316 of 2775 (11.4%) urinary exosome proteins after adjust- ment for multiple comparisons. Of these, 113 (4.1%) in- creased and 203 (7.3%) decreased during LS diet. Hierarchical clustering demonstrated a visible distribution pattern of pro- teins clustered with dietary sodium intake (Figure 1). A high- resolution figure with protein ID annotation is available in the Supplemental Material.
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