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

† † 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 multidimensional protein identification technology detected 2775 unique proteins, of which 316 exhibited significantly 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 restriction or acute aldosterone infusion similarly increased urine exosomal gENaC[112–122] peptide concentrations 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 proteins.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%) increased and 203 (7.3%) decreased during LS diet. Hierarchical clustering demonstrated a visible distribution pattern of proteins clustered with dietary sodium intake (Figure 1). A high- resolution figure with protein ID annotation is available in the Supplemental Material.

Dietary Sodium Restriction Does Not Markedly Alter Urinary Exosomal Marker Abundance Intracellular vesicle trafficking proteins are enriched in exosomes and have been proposed as specific vesicular orexosomal markers.1,6,15 As anticipated, all of the major exosome markers, including multivesicular body marker TSG101 and several tetraspanin proteins (e.g., CD9, CD63, CD81, and CD82), along with proteins consistent with exosome biosynthesis, were detected in our preparations (Table 2). After correcting for multiple comparisons we found no statistically significant difference in these urinary exosome proteins during the LS diet, although several common exosomal proteins (CD9, charged multivesicular body protein 1a, charged multivestic- ular body protein 6) were altered in uncorrected analysis. Overall, our data suggest that dietary sodium intake does not markedly alter urinary exosomal protein markers, although the abundance of a few endosomal sorting complex required for transport III complex proteins tended to change with diet (Table 2). Therefore, we normalized protein abun- dances in each sample according to the amount of total protein injected and total spectral count from each MudPIT analysis.

Pathway Analysis Identifies Biologic Processed Altered During Dietary Sodium Restriction To examine systems associated with exosomal proteome changes during the LS diet, we performed gene ontology Figure 1. Low salt diet alters urinary exosomal protein expres- (GO) enrichment analysis for the 316 proteins with significant sion. Heat map of urine exosome protein expression during LS versus HS diet. The change in expression within each participant alterations. Figure 2 depicts the significantly enriched biologic (using LS as reference) for 316 proteins (vertical axis) from 14 processes, molecular functions, and cellular components as- paired samples demonstrates clustering during the HS versus the sociated with these proteins. Up- or downregulated proteins in LS diet. The change in expression within each participant is shown these GO categories are listed in the Supplemental Table 2. Of as a color-coded data point for each protein (green, decrease particular note is the upregulation of peptidase and protease during the HS versus LS; red, increase). A high-resolution anno- activities, as represented by upregulation by furin, elastase, tated version is available in the Supplemental Material.

648 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH

Table 2. Effect of diet sodium on common exosome trafﬁcking proteins UniProtB UniProtB Corrected Protein Description Accession NSC HS NSC LS Ratio LS/HS P Value Entry Name P Value Number Tetraspanins CD9 antigen P21926 CD9 4638.32 8156.91 1.76 0.02 0.09 CD63 antigen P08962 CD63 705.26 564.59 0.80 0.89 0.96 CD81 antigen P60033 CD81 875.83 1380.07 1.58 0.15 0.36 CD82 antigen P27701 CD82 135.01 142.70 1.06 0.49 0.71 Programmed cell death 6-interacting protein Q8WUM4 PDC6I 9920.87 14,056.16 1.42 0.03 0.15 Programmed cell death protein 10 Q9BUL8 PDC10 206.55 209.94 1.02 0.78 0.90 Programmed cell death protein 6 O75340 PDCD6 1068.33 795.78 0.74 0.51 0.72 CD151 antigen P48509 CD151 90.33 40.86 0.45 0.05 0.19 ESCRT I complex Tumor susceptibility gene 101 protein Q99816 TS101 1641.37 1335.51 0.81 0.67 0.84 Vacuolar protein sorting-associated protein 28 Q9UK41 VPS28 1054.33 1314.67 1.25 0.34 0.58 homolog Vacuolar protein sorting-associated protein 37B Q9H9H4 VP37B 427.54 564.71 1.32 0.34 0.59 Vacuolar protein sorting-associated protein 37C A5D8V6 VP37C 116.08 144.65 1.25 0.34 0.59 Vacuolar protein sorting-associated protein 37D Q86XT2 VP37D 271.12 344.92 1.27 0.20 0.44 ESCRT II complex Vacuolar protein-sorting-associated protein 25 Q9BRG1 VPS25 248.01 242.40 0.98 0.90 0.96 Vacuolar protein-sorting-associated protein 36 Q86VN1 VPS36 444.21 508.17 1.14 0.33 0.57 Vacuolar-sorting protein SNF8 Q96H20 SNF8 154.65 201.85 1.31 0.16 0.38 ESCRT III complex Charged multivesicular body protein 1a Q9HD42 CHM1A 121.00 45.91 0.38 0.008 0.06 Charged multivesicular body protein 1b Q7LBR1 CHM1B 502.96 284.11 0.56 0.11 0.30 Charged multivesicular body protein 2a O43633 CHM2A 1085.69 1077.26 0.99 0.91 0.96 Charged multivesicular body protein 2b Q9UQN3 CHM2B 448.23 272.71 0.61 0.26 0.51 Charged multivesicular body protein 3 Q9Y3E7 CHMP3 104.94 195.58 1.86 0.25 0.50 Charged multivesicular body protein 4a Q9BY43 CHM4A 57.63 30.33 0.53 0.04 0.17 Charged multivesicular body protein 4b Q9H444 CHM4B 335.18 231.16 0.69 0.45 0.67 Charged multivesicular body protein 4c Q96CF2 CHM4C 59.69 79.18 1.33 0.15 0.37 Charged multivesicular body protein 5 Q9NZZ3 CHMP5 749.17 892.09 1.19 0.53 0.75 Charged multivesicular body protein 6 Q96FZ7 CHMP6 85.29 163.88 1.92 0.01 0.08 NSC, normalized spectral count; ESCT, endosomal sorting complex required for transport.

Dietary Sodium Restriction Alters Abundance of Solute (e.g., SGK-1, GILZ1, and Nedd4-2)16 were not detected in Transporter Proteins Relevant to Sodium and our MudPIT analysis. Multiple serine proteases known to Electrolyte Transport proteolytically activate ENaC (furin, neutrophil elastase, Proteins specific to each nephron segment of the nephron and kallikrein-10)16 were detectable, and all significantly were readily detectable.5 Selected proteins with functional increased during the LS diet, whereas prostasin was un- relevance to renal electrolyte transport are presented in Ta- changed (Table 4). Plasmin/plasminogen also tended to in- ble 3. Expression of proteins specifictotheproximaltubule crease, although the peptides identified do not differentiate was relatively unchanged during the LS diet, except for in- between the proenzyme and active form. Among all signifi- creased carbonic anhydrase IV abundance. Several notable cantly altered proteins, mannan-binding lectin serine proteins related to distal nephron segments were signifi- protease 2 (MASP2) was most markedly increased, al- cantly affected. During the HS diet a, b,andgENaC subu- though this protein has no previously known role in sodium nits were detected in low abundance in MudPIT analysis, homeostasis. with total spectral counts ,28 for all 28 samples. The abundance of a, b, and gENaC subunits increased during the LS Validation of Exosomal Protein Expression Using diet compared with the HS diet, whereas aquaporin 2 was Multiple-Reaction Monitoring Mass Spectrometry unchanged (corrected P=0.23). The thiazide-sensitive so- Because ENaC and NCC are known to be activated by RAAS dium chloride cotransporter (NCC) also increased to a lesser activation and aldosterone administration in rodents and in extent during the LS diet. Other aldosterone-responsive pro- vitro, we developed targeted multiple reaction monitoring teins known to regulate ENaC trafficking and activation (MRM) mass spectrometry assays for these solute transporters

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Figure 2. Low salt diet alters multiple pathways as determined by WebGestalt GO enrichment analysis. GO enrichment is presented for upregulated (A) and downregulated proteins (B) by three terms: biologic processes, molecular functions,andcellular components.

(Supplemental Table 1). Protein expression levels in urine including ACE1, ACE2, and angiotensinogen, did not change exosomes were quantified using stable isotope-labeled peptide in abundance (data not shown). standards for these proteins. Four sample pairs during the HS/LS diets and four sample pairs during vehicle/aldosterone infusion were used in the DISCUSSION MRM validation study (participant characteristics shown in Table 1). The selected bENaC target peptide was not reliably Urine exosomal proteins provide a rich source of potential detected in the validation sample set. Although subject-to- biomarkers, but the effect of activation of the endogenous subject variability was significant, most results agreed with RAAS on exosomal protein cargo has not been previously MudPIT spectral counting results. The target peptide for investigated in humans. We now demonstrate that RAAS NCC[200–209] was unchanged during the LS diet or aldosterone activation modifies urinary exosome protein cargo using mass infusion (Figure 3A). The abundance of aENaC tended to in- spectrometry–based methods. Furthermore, urinary biomark- crease during the LS diet and aldosterone infusion, although ers for renal sodium channel and transporter activity in humans this effect was not statistically significant (Figure 3B). Dietary are lacking. We developed a sensitive and specific MRM mass sodium restriction significantly increased the gENaC[112–122] spectrometry method to measure a gENaC peptide which is peptide (Figure 3C), which also correlated positively with markedly altered under conditions of avid renal sodium reab- plasma aldosterone levels (Figure 3D). Other target peptides, sorption, including LS diet and aldosterone infusion.

650 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH

Table 3. Selected proteins of potential relevance to renal electrolyte transport UniProtB Region/Cell Type UniProtB Corrected Accession NSC HS NSC LS Ratio LS/HS P Value Protein Description Entry Name P Value Number mTAL Solute carrier family 12 member 1 (NKCC2) Q13621 S12A1 3909.2 4059.4 1.04 0.56 0.76 DCT Solute carrier family 12 member 3 (NCC) P55017 S12A3 4836.0 7431.4 1.54 0.09 0.26 Serine/threonine-protein kinase OSR1 O95747 OXSR1 127.5 109.0 0.86 0.76 0.89 Transient receptor potential cation channel subfamily Q9NQA5 TRPV5 97.8 80.9 0.83 0.73 0.87 V member 5 Transient receptor potential cation channel subfamily Q9H1D0 TRPV6 80.3 91.5 1.14 0.16 0.39 V member 6 CD-PC Amiloride-sensitive sodium channel, a subunit (aENaC) P37088 SCNNA 41.7 49.0 1.18 0.003 0.03 Amiloride-sensitive sodium channel, b subunit (bENaC) P51168 SCNNB 41.7 47.1 1.13 0.04 0.17 Amiloride-sensitive sodium channel, g subunit (gENaC) P51170 SCNNG 43.6 60.7 1.39 0.008 0.06 Aquaporin-2 P41181 AQP2 184.5 85.5 0.46 0.07 0.23 Solute carrier family 12 member 2 (NKCC1) P55011 S12A2 451.4 231.0 0.51 0.04 0.15 Sodium/potassium-transporting ATPase subunit a-1 P05023 AT1A1 1615.0 1469.3 0.91 0.89 0.96 Sodium/potassium-transporting ATPase subunit a-3 P13637 AT1A3 825.2 801.5 0.97 0.62 0.81 Sodium/potassium-transporting ATPase subunit b-1 P05026 AT1B1 297.2 335.8 1.13 0.20 0.44 14–3-3 protein b/a P31946 1433B 727.4 623.4 0.86 0.39 0.62 14–3-3 protein « P62258 1433E 1270.9 1399.8 1.10 0.30 0.55 CD-IC Sodium-independent sulfate anion transporter Q86WA9 S2611 62.5 66.9 1.07 0.35 0.59 Solute carrier family 12 member 2 (NKCC1) P55011 S12A2 451.4 231.0 0.51 0.04 0.15 Band 3 anion transport protein P02730 B3AT 100.6 66.0 0.66 0.36 0.60 Pendrin O43511 S26A4 1023.6 2925.8 2.86 0.004 0.04 Sodium-independent sulfate anion transporter Q86WA9 S2611 62.5 66.9 1.07 0.35 0.59 NSC, normalized spectral count; mTAL, medullary thick ascending limb; DCT, distal convoluted tubule; CD-PC, collecting duct- principal cell; CD-IC, collecting duct-intercalated cell.

Urinary exosomes are an attractive source of renal bio- The observed urinary exosome protein content in the marker proteins because they are noninvasively obtained present study generally reflects anticipated protein expression and the isolation process concentrates the proteins and changes in the nephron, with some exceptions. In particular, reduces potential interfering substances commonly encoun- the gENaC[112–122] peptide abundance increased markedly tered in urinary proteomics. The relationship between during the LS diet and after aldosterone infusion, whereas exosomal protein expression and renal protein expression the increase in aENaC was not significant and NCC expres- remains undefined and cannot be directly addressed in the sion did not change appreciably. Aldosterone can increase re- present study. The analysis of ENaC and NCC proteins may nal ENaC activity by increasing a-subunit expression and provide some insight, however, because RAAS activation is increasing cell surface localization. The aENaC and gENaC known to increase protein expression of aENaC and subunits can also be activated via protease-dependent re- NCC.16,17 moval of inhibitory peptides, and the cleaved forms increase

Table 4. Proteins and proteases potentially involved in sodium channel activation UniProt UniProt Corrected Protein Description HS NSC LS NSC Ratio LS/HS P Value Accession Number Entry Name P Value 14–3-3 protein sigma P31947 1433S 1417.8 630.3 0.44 6.4E-4 0.01 Prostasin Q16651 PRSS8 361.9 315.5 0.87 0.97 0.99 Furin P09958 FURIN 45.8 66.3 1.45 1.5E-3 0.02 Neutrophil elastase P08246 ELNE 610.3 1144.4 1.88 2.8E-3 0.03 Mannan-binding lectin serine protease 2 O00187 MASP2 432.5 2352.2 5.44 5.4E-8 4.8E-5 Kallikrein-10 O43240 KLK10 64.0 108.5 1.70 2.4E-3 0.03 NSC, normalized spectral count.

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proteases, MASP2 increased most significantly. MASP2 has been identified as 1 of 13 active serine hydrolases in human urine but has not been linked to activation of sodium channels.22 MASP2 activates the innate immune system by associating with mannan-binding lectin and activating the terminal complement cascade, and MASP2 deficiency in humans is associated with severe pneumococcal infection and autoimmune disease.23,24 Because the RAAS also increases inflammatory and profibrotic mediators such as plasminogen activator inhibitor-1,25 MASP2 may provide an additional link between the RAAS and inflammatory pathways. In patients with hyperaldosteronism, NCC and phosphorylated NCC are increased in urinaryexosomes, and in rats acute aldosterone administration increases phosphorylated NCC, NCC, and prostasin excretion.21 In our study, NCC abundance increased during the LS diet in our MudPITanalysis, although this finding was not validated using MRM methods or during aldosterone infusion. We also did not observe any change in urinary prostasin during LS diet. Although we searched for Figure 3. Endogenous renin-angiotensin-aldosterone system NCC phosphopeptides in the MudPIT analysis, none were activation and exogenous aldosterone increase urinary exosomal detected. Identification of phosphorylated peptides gENaC excretion. Effect of LS and HS diet on urinary exosome requires a much larger starting urine volume, urine collection peptide abundance for NCC (A), aENaC (B), and gENaC (C). with a phosphatase inhibitor, and phosphoenrichment to in- gENaC abundance in urinary exosomes correlated strongly with crease the sensitivity of this method.1 Our model of RAAS , plasma aldosterone. P 0.001 for linear correlation (D). Aldo, al- activation differs significantly from primary aldosteronism dosterone. with regard to species and pathophysiology and duration of activation, which could explain differences in NCC or prosta- in abundance during aldosterone administration.17–19 The sin. For example, short-term RAAS activation during periods gENaC[112–122] amino acid sequence assayed in this study is of dietary sodium restriction (secondary aldosteronism) pre- located in the extracellular N-terminal domain of gENaC, vents hypotension, whereas prolonged aldosterone excess near a putative cleavage site. Cleavage and removal of the (primary aldosteronism) produces hypertension. inhibitory peptide by proteases, such as furin, prostasin, The urinary exosomal gENaC[112–122] biomarker could kallikrein, plasmin, elastase, or CAP2, maximally activate provide a useful estimate of ENaC activation in future clinical murine ENaC in vitro, although it is not clear from the pres- studies. The urinary sodium-to-potassium ratio has been pre- ent studies if cleavage directly affects gENaC[112–122] abun- viously used in clinical studies to estimate ENaC activity, but dance.16,20 Other potential explanations for the increased this measure is not specific for ENaC and can be affected by abundance of this gENaC[112–122] peptide include increased other variables, such as altered potassium intake. Nasal poten- absolute gENaC production and urinary excretion, or more tial difference has also been used to estimate ENaC activity, efficient trypsin digestion after gENaC is proteolytically although it has not been proven to reflect renal ENaC activ- 26 cleaved in vivo. Although this gENaC[112–122] biomarker ity. In animal studies, Western blot analysis of ENaC subu- may be useful in humans, this peptide region is variable nits in kidney tissue has been extensively used to measure across species, so an analogous peptide sequence would be expression, but obtaining adequate renal tissue for this mea- needed in rodents. Further studies under different treatment surement in clinical studies is impractical. Western blot anal- conditions are warranted to determine the source of the in- ysis for ENaC has been performed on urinary exosomal and crease in target peptide levels and to explore the clinical total urinary protein in prior clinical studies,27,28 but these utility of this measure. have reported low sensitivity and none have demonstrated a Multiple serine proteases known to cleave gENaC were significant response during RAAS activation. The gENaC[112–122] readily detectable within urinary exosomes by MudPIT anal- biomarker identified in the present study was detected using a ysis, including plasmin, urokinase, kallikreins, furin, and more sensitive and specificMRMmethod,increasedmarkedly prostasin. Although prostasin is increased by aldosterone in during exogenous and endogenous RAAS activation, and in- rodents and has previously been identified as a marker of creased within 1 day to 1 week of stimulation. hyperaldosteronism in humans,21 we observed no significant Although unbiased MudPIT proteomic analysis has the increase during the LS diet. The proteases furin, neutrophil advantage of discovering previously unknown relationships, elastase, and kallikrein-10 were significantly increased by the this approach also requires validation to verify significant LS diet, even after correction for multiple testing. Among all relationships. We performed validation studies of the relevant

652 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH sodium channels using MRM analysis and stable isotope- the study, as described previously.29 We provided participants with a labeled standards in a separate cohort of individuals who diet containing 10 mmol (LS) or 200 mmol (HS) sodium/day for 7 received aldosterone infusion or LS diet. Targeted MRM days prepared by the Vanderbilt clinical research center kitchen under analysis is less costly, less time-consuming, and more sensitive the guidance of a registered dietitian. After presenting to the clinical than MudPIT analysis. Analysis with stable isotope-labeled research center at 7 A.M., urine samples were collected immediately standards allows accurate identification and quantification of into a protease inhibitor cocktail as previously described,4,5 and sam- target peptides and also overcomes the difficulty encountered ples were stored at 280°C until processing. Fourteen paired samples with antibody-based methods due to nonspecific binding. were available for analysis by MudPIT and four for MRM. Although it is not feasible to perform such targeted analysis of all proteins, this general approach can be followed in future Aldosterone Infusion studies to generate custom MS assays targeting pathways of Urine exosome samples were collected from healthy participants who interest, potentially in a single multiplex assay. Another were recruited and enrolled as previously reported.14 Participants limitation of the MudPIT analysis is the high inter-subject reported to the Vanderbilt Clinical Research Center on the fifth and variation for individual protein expression, which greatly seventh evenings of a 160-mmol/day sodium controlled diet. Partici- reduces the ability to detect meaningful changes. This limi- pants were administered aldosterone (0.7 mg/kg per hour in 5% dex- tation was overcome in the present study by using a cross-over trose; Professional Compounding Corporation of America) or vehicle design in which paired samples were used to eliminate the by intravenous infusion between 10:00 P.M. and 08:00 A.M. as previously between-subject variation. For future studies, a similar ap- described and returned for the second study day for the remaining proach may be used to identify significantly altered proteins infusion.14 The order of drug administration was randomized and dou- and to develop sensitive and specific MRM methods before ble-blinded by Vanderbilt Investigational Drug Services. BP was mon- widespread application in heterogeneous populations. itored hourly during drug infusion (Dinamap, GE Medical). Serum The present study provides a proof-of-principle concept potassium was monitored throughout the drug infusion, and oral po- that urinaryexosome protein expression is altered by the renin- tassium chloride was administered as needed to maintain serum potas- angiotensin-aldosterone system, with a significant increase in sium at a level of $3.8 mEq/L. We collected all urine after 3 hours of gENaC[112–122] peptide and urinary proteases. Thus, appro- drug infusion (between 1:00 A.M. and 7:00 A.M.) into a protease inhibitor priate selection of urinary exosomal protein biomarkers may cocktail as described previously and stored at 280°C until processing.2 provide insight into renal physiology. Selection of specificpep- Four paired urine samples were available for analysis. tide targets within individual proteins, such as peptides adja- cent to proteolytic cleavage sites or phosphopeptides may Laboratory Assays provide a more sensitive measure than overall protein expres- Screening electrolytes and lipid panels were performed in the sion alone. We propose that exosomal gENaC[112–122] is a bio- Vanderbilt clinical laboratory. Plasma aldosterone and plasma renin marker of proteolytically activated ENaC. Identification of activity were measured by radioimmunoassay as described pre- additional specific biomarkers may require identification of viously.25,29 Urine sodium and potassium concentrations were mea- modified peptides, such as phosphorylated forms, which is sured by flame photometry and creatinine by the sodium picrate technically more difficult. Our study also demonstrates the method.25,29 importance of controlling for dietary sodium intake when analyzing urinary biomarkers, which may prove difficult in Urine Exosome Isolation translating urinary biomarker discovery into broader clinical Urine samples were thawed and exosomes isolated as previously applications. described.1 Briefly, urine specimens were thawed with running tap water, separated into aliquots, and centrifuged at 17,000 g for 20 minutes at 4°C. The 17,000 g supernatant was ultracentrifuged at CONCISE METHODS 200,000 g for 1 hour at 24°C and the pellet was saved. The centrifugation steps were repeated on each aliquot until all of the urine was Clinical Study Protocols processed. The pellets from all aliquots of the same patient sample All studies were approved by the Vanderbilt University Institutional were pooled together and resuspended in isolation solution (10 mM Review Board and conducted in accordance with the Declaration of triethanolamine and 250 mM sucrose). To denature and remove ex- Helsinki. Informed consent was obtained, and participants cess uromodulin (Tamm-Horsfall protein),which can co-sediment underwent a screening history and physical before study enrollment. with exosomes in the 200,000 g centrifugation step, the resuspended Participants with a history of coronary artery disease, diabetes, renal pellet was mixed with 200 mg/ml dithiothreitol (DTT) and incubated insufficiency (eGFR,60 ml/min per 1.73 m2), anemia, major med- at 95°C for 2 minutes, then diluted 1:20 with isolation solution.15 The ical problems, or inability to comply with the protocol were excluded. sample was centrifuged at 17,000 g for 20 minutes at 4°C, and the resulting supernatant was ultracentrifuged at 200,000 g for 1 hour at fi Dietary Sodium Restriction 24°C. The nal pellet was suspended in 100 ml of HPLC-grade H2O Participants with mild-to-moderate hypertension were recruited and and frozen at 280°C. The protein concentration of exosome prepa- washed out from antihypertensive medications for $3 weeks before rations was measured with the BCA protein assay kit (Pierce).

J Am Soc Nephrol 27: 646–656, 2016 RAAS Alters Urinary Exosomes 653 CLINICAL RESEARCH www.jasn.org

Liquid Chromatography-Tandem Mass Spectometry precursor ion mass-to-charge ratio and all other spectra were pro- Analysis cessed to form .dta files for both doubly and triply charged precursor Exosome protein from each sample (25 mg) was dried using a Speed- ions. The .dta files were searched against a human subset of the Uni- Vac and reduced with 20 mM DTT in 50 mM NH4HCO3,50%tri- prot KB protein database (June 2012 release) with a total of 20,360 fluoroethanol (TFE) at 56°C for 45 minutes, followed by alkylation protein entries using the Sequest algorithm.31 The search was trypsin with 40 mM iodoacetamide in the dark for 1 hour at room temper- strict and a precursor mass tolerance of 62.5 Da was used. The ature. DTT, 50 mmol, was added to the sample for another hour in searches were performed allowing for the following differential mod- the dark to destroy excess iodoacetamide. The samples were diluted ifications: +57 on cysteine (for carboxyamidomethylation) and +16 5-fold with 50 mM NH4HCO3 to reduce the TFE concentration to on methionine (oxidation). A concatenated database of forward and 5% and were subjected to enzymatic digestion for 12–16 hours at reverse sequences was constructed to allow determination of false 37°C using trypsin gold (1:25 trypsin-to-exosome protein ratio; discovery rates. The search results were imported into ProteoIQ soft- Promega, Madison, WI). The digestion was stopped by adding 2 ml ware (Premier Biosoft, Palo Alto, CA) for comparative proteomics of 100% formic acid. The trypsin-digested samples were centrifuged analysis. Results were filtered using minimum Xcorr scores of 1.5 for at 20,000 g for 20 minutes at 4°C, the supernatant was saved, and the +1 precursor ions, 2.5 for +2 precursor ions, and 3.5 for $+3 pre- pellets were extracted twice with 50 ml of 50% acetonitrile/0.1% for- cursor ions, respectively, a minimum of two peptides per protein, an mic acid. Extracts and the supernatant were pooled and dried by average of two spectral counts per protein, and an overall maximum SpeedVac. Dried samples were reconstituted with 100 mlofHPLC false discovery rate of ,0.05 in each sample. water with 0.1% formic acid (FA), centrifuged at 215,000 g for 20 minutes at 4°C, and the supernatant containing tryptic peptides MRM Analysis 2 was collected and stored at 80°C until liquid chromatography-mass For MRM analysis, 10 mg of exosomal protein, isolated from each spectometry analysis. patient urine sample, was placed in 20 mM Tris, 40% TFE, 18.75 mM The tryptic peptides were analyzed using an LTQ Velos mass tris(2-carboxyethyl)phosphine) for reduction at room temperature fi spectrometer (Thermo Fisher Scienti c) with a 12-step MudPIT for 1 hour, followed by alkylation using 75 mM iodoacetamide in the 5 fl analysis as previously described. Brie y, tryptic peptides were loaded dark for 45 minutes. Samples were diluted by adding 3 volumes of 3 onto a 4 cm 150 mm internal diameter (ID) microcapillary fused 100 mM Tris and digested with 1 mgoftrypsinat37°Covernight. silica pre-column packed in-house with C18 resin (Jupiter C18, 5-mm Tryptic peptides were dried by SpeedVac. Isotope-labeled peptide particle size, 300-Å pore size) followed by 6 cm of strong cation- standards and three other external control peptides were spiked into exchange resin (Luna SCX, 5-mm particle size, 100-Å pore size). The the samples in a final volume of 20 ml. The samples were diluted into fl 2D trap column was coupled to a nano ow capillary analytical col- 140 ml of 0.1% trifluoroacetic acid (TFA) and acidified to final pH#3 umn (100 mm ID) packed with 10 cm of 3-mm C18 reverse-phase using 50% TFA followed by a clean-up step using homemade mini- resin (Jupiter C18, 3-mm particle size, 300-Å pore size) constructed C18 cartridges. In brief, a disc of C18 filter membrane (2215-C18 solid with a laser-pulled electrospray emitter tip. Multidimensional sepa- phase extraction, Disk Empore; Chrom Tech Inc.) was cored with a rations were performed using 5-mlpulsesofammoniumacetate 16-gauge needle and the cored piece was fitted tightly into a 200-ml (0, 25, 50, 75, 100, 150, 200, 250, 300, 500, 750, and 1000 mM pipette tip. Three milligrams of C18 (Jupiter C18, 5-mm particle size, ammonium acetate) in 0.1% FA delivered by an autosampler. After 300-Å pore size) resin, suspended in 200 ml of methanol, was loaded each salt pulse, the peptides were eluted with a 105-minute reverse phase into the pipette tip and spun at 3600 g for 1 minute at room temper- solvent gradient from 2% acetonitrile (ACN)/0.1% FA to 45% ACN/ ature to form a mini-C18 cartridge. The mini-C18 cartridges were fi 0.1% FA for the rst 10 salt pulses and a reverse solvent gradient from equilibrated with 600 ml 0.1% TFA in HPLC water. Five micrograms 2% ACN/0.1% FA to 95% ACN/ 0.1% FA for 1 M salt pulse at a 0.5-ml of digested urine exosome protein was loaded on the cartridge, fl per minute ow rate. Gradient-eluted peptides were introduced into the centrifuged at 3600 g for 1.5 minutes at room temperature, and LTQ Velos instrument via a nano-electrospray ionization source. washed three times with 0.1% TFA. The sample was eluted twice The LTQ Velos mass spectrometer was operated in data-dependent with 300 ml of 80% ACN/0.1% TFA. All eluted peptides were pooled fi mode in which the rst an initial full MS scan recorded the mass-to- and dried by SpeedVac. The sample was solubilized in 20 ml of 0.1% – charge ratios of ions over the mass-to-charge range of 300 2000, and vol/vol formic acid before MRM analysis. fi the ve most abundant ions were automatically selected for subse- Proteotypic peptides detected in MudPIT analysis were selected quent collision-induced dissociation. Dynamic exclusion (repeat through bioinformatics analysis as guided by Kuzyk et al.32 C-terminal count 1, exclusion list size 150, and exclusion duration 60 seconds) [13C]/[15N] labeled heavy peptides (AQUA) were synthesized and was enabled to allow detection of less abundant ions. purified by Sigma-Aldrich. Upon receipt, the quantity and quality of each isotope labeled peptide were evaluated by matrix-assisted Data Analysis of MudPIT laser desorption/ionization and LC-MS/MS analysis. The following Liquid chromatography-tandem mass spectometry (LC-MS/MS) raw isotopically labeled internal control peptides were included: SGGTY- files were converted into .dta files by the ScanSifter algorithm.30 Spec- FLISR (NCC) at 50 fmol, HLLADLEQETR (gENaC) at 50 fmol, tra that contained ,25 peaks or that had ,2e1 measured total ion AEQNDFIPLLSTVTGAR (aENaC) at 250 fmol, and three other ex- current were not converted. The .dta files for singly charged precursor ternal control peptides at 10 fmol (SSAAPPPPPR, TASEFDSAIAQDK, ions were created if 90% of the total ion current occurred below the and LTILEELR). A list of MRM transitions for the selected target

654 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH peptides (Supplemental Table 1) was generated by Skyline software Core), UL1-RR024975 and -TR000445 from NCATS/NIH (Vander- based on product ions detected in MudPIT analyses.33 The identi- bilt Institute for Clinical and Translational Research), and the Van- ties of the target peptides were confirmed according to retention derbilt Mass Spectrometry Research Center. time equivalence and transition intensity equivalence to the isotope- This research was presented in abstract form at the American labeled peptide. Society of Nephrology Renal Week 2013. Target peptides were analyzed by a 60-minute scheduled MRM LC- MS/MS analysis. Peptides were loaded onto a 40 mm30.1 mm (Jupiter C18, 5-mm particle size, 300-Å pore size) kasil fritted trap DISCLOSURES 3 column that was connected inline to a 200 mm 0.1 mm (Jupiter None. C18, 3-mm particle size, 300-Å pore size), self-packed analytical column using a NanoAcuity HPLC system (Waters). After trapping and fl equilibration, peptides were gradient-eluted at a ow rate of 400 nl/ REFERENCES min using a 60-minute gradient from 1% ACN/0.1% FA to 45% ACN/0.1% FA followed by a 5-minute ramp to 90% ACN/0.1% FA. 1. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, The column effluent was delivered directly to a triple quadrupole Wang NS, Knepper MA: Large-scale proteomics and phosphoproteo- mass spectrometer (TSQ-Vantage; Thermo Fisher Scientific) via a mics of urinary exosomes. J Am Soc Nephrol 20: 363–379, 2009 fi fi nano-electrospray source. A scheduled MRM method was developed 2. Pisitkun T, Shen RF, Knepper MA: Identi cation and proteomic pro ling of exosomes in human urine. Proc Natl Acad Sci U S A 101: 13368– according to a series of unscheduled scouting experiments. 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