University of Southern Denmark

Albuminuria in kidney transplant recipients is associated with increased urinary serineproteases and activation of the epithelial sodium channel

Hinrichs, Gitte Rye; Michelsen, Jannie Solmunde; Zachar, Rikke; Friis, Ulla Glenert;Svenningsen, Per; Birn, Henrik; Bistrup, Claus; Jensen, Boye LPublished in:American Journal of Physiology: Renal Physiology

DOI:10.1152/ajprenal.00545.2017

Publication date:2018

Document versionAccepted manuscript

Citation for pulished version (APA):Hinrichs, G. R., Michelsen, J. S., Zachar, R., Friis, U. G., Svenningsen, P., Birn, H., Bistrup, C., & Jensen, B. L.(2018). Albuminuria in kidney transplant recipients is associated with increased urinary serine proteases andactivation of the epithelial sodium channel. American Journal of Physiology: Renal Physiology, 315(1), F151-F160. https://doi.org/10.1152/ajprenal.00545.2017

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1

ALBUMINURIA IN KIDNEY TRANSPLANT RECIPIENTS IS ASSOCIATED WITH 1

INCREASED URINARY SERINE PROTEASES AND ACTIVATION OF THE EPITHELIAL 2

SODIUM CHANNEL 3

AUTHORS 4

Gitte R. Hinrichs1, Jannie S. Michelsen2, Rikke Zachar1, Ulla G. Friis1, Per Svenningsen1, Henrik Birn3, 5

Claus Bistrup2, Boye L. Jensen1 6

AFFILIATIONS 7

1University of Southern Denmark, Department of Cardiovascular and Renal Research, Odense, Denmark 8

2Odense University Hospital, Department of Nephrology, Odense, Denmark 9

3Aarhus University, Department of Biomedicine, and Aarhus University Hospital, Department of Renal 10

Medicine, Aarhus, Denmark 11

AUTHORS CONTRIBUTIONS 12

Gitte R. Hinrichs, Claus Bistrup and Boye L Jensen: Participated to the conception and design of the work, 13

performance of the research, data analysis, the interpretation of the data, drafting of the work and writing of 14

the manuscript. Jannie S. Michelsen: Participated in performance of the research and data collection. 15

Rikke Zachar and Per Svenningsen: Contributed with data analysis, in particular the exosome analysis and 16

writing of the manuscript. Ulla G. Friis: Contributed with patch clamp experiment and drafting of the 17

manuscript. Henrik Birn: Contributed to interpretation of the data and writing of the manuscript. 18

19

RUNNING HEAD 20

ENaC activity in albuminuric kidney transplant recipients 21

22

2

CORRESPONDING AUTHOR 23

Gitte Rye Hinrichs MD 24

Department of Cardiovascular and Renal Research 25

Institute of Molecular Medicine 26

University of Southern Denmark 27

J. B. Winsløws Vej 21,3., DK-5000 Odense C, Denmark. 28

Telephone: +45 6550 3796. Fax: +45 6613 3479 29

E-mail: ghinrichs@health.sdu.dk 30

31

3

ABSTRACT 32

Albuminuria predicts adverse renal outcome in kidney transplant recipients. The present study addressed the 33

hypothesis that albuminuria is associated with increased urine serine proteases with the ability to activate the 34

epithelial sodium channel (ENaC) and with greater extracellular volume and higher blood pressure. In a 35

cross-sectional design, kidney transplant recipients with (n=18) and without (n=19) albuminuria were 36

included for office blood pressure measurements, estimation of volume status by bioimpedance, and 37

collection of spot urine and plasma samples. Urine was analyzed for serine proteases and for ability to 38

activate ENaC current in vitro. Urine exosome protein was immunoblotted for prostasin and γ-ENaC protein. 39

In present study it was found, that compared to non-albuminuria (8.8 mg/g creatinine), albuminuric 40

(1722mg/g creatinine) kidney transplant recipients had a higher systolic and diastolic blood pressure, despite 41

receiving significantly more antihypertensives, and a greater urinary total plasminogen, active plasmin, 42

active urokinase-type plasminogen activator and prostasin protein abundance which correlated significantly 43

with u-albumin. Fluid overload correlated with systolic blood pressure, urinary albumin/creatinine and 44

plasminogen/creatinine. Urine from albuminuric kidney transplant recipients evoked a greater amiloride- and 45

aprotinin-sensitive inward current in single collecting duct cells (murine cell line M1). γENaC subunits at 50 46

and 75 kDa showed increased abundance in urine exosomes from albuminuric kidney transplant recipients 47

when compared to controls. These findings show, that albuminuria in kidney transplant recipients is 48

associated with hypertension, ability of urine to proteolytically activate ENaC current and increased 49

abundance of γENaC. ENaC activity could contribute to hypertension and adverse outcome in posttransplant 50

proteinuria. 51

52

KEYWORDS: Aldosterone, allograft, exosome, hypertension, proteinuria 53

4

INTRODUCTION 54

Kidney transplantation is a superior treatment in patients with end stage renal disease due to survival - and 55

quality of life benefits (18, 24, 44, 56). Chronic allograft dysfunction is a multifactorial process associated 56

with interstitial fibrosis, renal tubular atrophy and graft loss. The most important non-immunological factors 57

that may adversely impact long-term renal graft function are hypertension (21, 26, 33, 34) and proteinuria (1, 58

14, 16, 17, 25, 30) both being strong predictors of graft dysfunction. Both proteinuria and hypertension are 59

modifiable risk factors, and reduction in hypertension has been associated with improved graft survival (27, 60

33). High salt intake is positively associated with blood pressure (BP) in kidney transplant recipients (KTRs) 61

(42, 53) and thus an independent risk factor, suggesting hypertension is salt-sensitive in KTRs. Studies have 62

implied a significant association between dietary salt intake and proteinuria (57); however, the mechanisms 63

underlying this association are not fully understood. Interestingly, the urinary albumin excretion is greater in 64

salt-sensitive hypertensive patients when compared to salt resistant hypertensive patients (31). Evidence for a 65

pathophysiological connection between proteinuria and salt-sensitive BP comes from studies in several 66

proteinuric conditions demonstrating a concurrent aberrant filtration of plasma serine proteases, in particular 67

urokinase-type plasminogen activator (uPA), the zymogen plasminogen, and prostasin into the tubular fluid. 68

These serine proteases can activate the amiloride-sensitive epithelial sodium channel (ENaC) in the 69

collecting ducts by extracellular proteolytic release of an inhibitory peptide tract from the γ-subunit (2, 4, 7, 70

8, 47, 49). Soluble serine proteases, plasmin, prostasin and uPA in the tubular fluid, may turn ENaC from 71

“moderate” to a highly active state in albuminuria and enhance sodium and water reuptake (6, 20, 23, 38, 39, 72

49, 52). While this pathophysiological mechanism cannot explain the occurrence of albuminuria, it could 73

potentially maintain a vicious cycle once albuminuria manifests in post-transplant nephropathy with 74

increased sodium reabsorption leading to an expansion of the extracellular volume (ECV) and hypertension 75

with further aggravation of albuminuria. The present study was designed to address the hypothesis that post-76

transplant nephropathy is associated with aberrant presence of proteases in urine with ability to activate 77

ENaC current and that the amounts of proteases would relate directly to BP and fluid overload. The 78

hypothesis was addressed in a cross-sectional observational study design where urine samples from KTRs 79

5

with or without albuminuria were compared to identify urine serine proteases in albuminuric KTRs and to 80

demonstrate their in vitro effect on ENaC. Secondary outcomes were to analyze the association between 81

urinary serine proteases, fluid overload and BP in KTRs. 82

83

MATERIAL AND METHODS 84

Study design 85

The study was an investigator-initiated, cross-sectional, observational study conducted at the outpatient 86

clinic at the Department of Nephrology, Odense University Hospital, Denmark. The study was approved by 87

The Ethics Committee of The Region of Southern Denmark (Project-ID: S-20150015) and the Danish Data 88

Protection Agency (ID: 2008-58-0035). All patients received oral and written information about the study 89

and gave written informed consent to participate. The study was performed in accordance with the Helsinki 90

Declaration. 91

Study population 92

The study included stable ambulatory KTRs presenting consecutively for a routine follow-up at the 93

outpatient clinic at the Department of Nephrology, Odense University Hospital between February 2016 to 94

November 2016. The KTRs were recruited according to the following inclusion criteria: (I) age between 18-95

75 years, (II) kidney transplantation > 1 year ago, (III) follow at the outpatient clinic, (IV) severe 96

albuminuria (urine albumin/creatinine-ratio >300mg/g) or normoalbuminuria (urine albumin/creatinine-ratio 97

<30mg/g) as per KDIGO guidelines. The exclusion criteria were: (I) lack of compliance or understanding of 98

the study, (II) clinical relevant organic or systemic disease including malignancy or (III) treatment with anti-99

mineralocorticoids, including amiloride or aldosterone antagonists. 100

Data collection 101

Blood pressure recordings Office BP was recorded three times by a validated automatic oscillometric 102

device (OMRON, model HBP – 1300) (9) after 30 min of rest in the sitting position. The average of the 103

measurements was recorded. 104

6

Evaluation of fluid status Body composition was evaluated using a Body Composition Monitor (BCM) 105

(Fresenius Medical Care Deutschland GmbH), applied to the patient after 30 min of rest. Electrodes were 106

placed on one hand and one foot with the patient in a supine position. The height and weight of the patients 107

were registered, and the measurements were initiated. The BCM device uses bioimpedance spectroscopy 108

with a spectrum of 50 frequencies between 5 and 1000kHz, to measure EVC, intracellular (ICV), total body 109

water and calculate fluid overload (overhydration (OH)). The values obtained were normalized to body 110

surface area of 1,73m2 (10, 29). 111

Blood and urine sampling Blood samples were obtained after 30 min of rest in a sitting position, 112

centrifuged at 1500g at 4˚C for 15 min and the plasma was frozen and stored at -80°C. Immediately upon 113

voiding, two tablets of complete (protease inhibitor cocktail, Cat; 11836145001, Sigma Aldrich, St. Louis, 114

Missouri, USA) were added to 100 ml of urine which was frozen at -80°C. Other spot urine samples were 115

centrifuged at 13.000 rpm for 1 min and the supernatant were frozen in -80°C as aliquots. 116

Analyses 117

Urine and plasma analysis Urine electrolytes, creatinine, albumin and plasma-sodium, potassium, albumin 118

and eGFR were analyzed at the Department of Biochemistry and Pharmacology, Odense University Hospital 119

using automated, standardized assays. Urinary osmolality was analyzed by osmometer (The Advanced TM 120

OSMOMETER Model 3D3, Advanced Instruments, INC). Urine total plasmin(ogen) (referred to 121

plasminogen and plasmin concentration) and plasma aldosterone were analyzed using commercial ELISA 122

Kits (Human Plasminogen Total Antigen, IHPLGKT-TOT, Innovative Research, Inc., Novi, Michigan, 123

U.S.A and MS E-5200, Labor Diagnostika Nord GmbH & Co. KG, Germany, respectively) in accordance 124

with the manufactures provisions. EDTA-plasma (100 μl) incubated with plasma from a nephrectomized 125

sheep for 3 h and plasma renin concentration (PRC) was measured by radioimmunoassay of ANGI through 126

the antibody-trapping method of Poulsen and Jørgensen as previously described (41). Concentrations were 127

measured by the rate of ANGI formation and standardized in terms of international units per liter (IU∙l−1) by 128

the activity of the WHO International Standard (ref. no. 68-356; National Institute for Biological Standards 129

7

and Control, Hertfordshire, UK) of which samples of 0.05 IU/l were included in every run of the renin assay. 130

In the period of measurement, 1 IU of the WHO standard corresponded to 32 ± 5 ng AngI per hour. 131

Between-assay coefficient of variation was 15%. 132

Urinary exosomes Frozen urine samples were thawed overnight at 4°C and vortexed thoroughly. Seventy ml 133

of the samples were centrifuged at 3,000 g and 4°C for 30 min (Sorvall RC 26 Plus). Exosomes were isolated 134

from the supernatant by ultracentrifugation at 45,000 rpm (220,000 g, 4°C) for 100 min (Beckman 135

Ultracentrifuge L-70). The pellet was resuspended in 2 x 100 µl resuspension buffer (sucrose: 0.3 M; 136

imidazole: 25 mM; EDTA-disodium salt: 1 mM; pH 7.2; cOmplete mini tablet (Roche)) and stored at -80°C 137

until western blot-analysis were performed. 138

Western blotting Crude urine samples or exosomes were subjected to western blotting. The loaded amount 139

was normalized to urinary creatinine. The urine was mixed with 4x SDS sample buffer (NuPAGE, 140

invitrogen) and 10 X reducing buffer (NuPAGE, invitrogen) and heated for 5 min. Samples were loaded onto 141

NuPAGE 4-12% Bis-Tris Gel, separated by electrophoresis, and subsequently transferred to Immobilon-P 142

transfer membrane (Merck Milipore). The membrane was blocked for one hour with 5% milk or 3% BSA in 143

TRIS-buffered saline with Tween 20 (TBST). The membrane was incubated with primary antibody: anti-144

prostasin (cat. No. 15527-1-AP; Proteintech Group, IL, USA, 1:1000); anti-plasminogen (ab 6189-100; 145

Abcam, MA, USA, 1:5000); anti-urokinase (ab8473; Lot. No. GR106505-1, Abcam, MA, USA, 1:2000); 146

anti- apoptosis-linked gene 2 interacting protein (ALIX; ab 53538, Santa Cruz Biotechnology, Dallas, Texas, 147

USA, 1: 1:500), anti-aquaporin 2((AQP2), C-17)(ab 9882, Santa Cruz, 1:2000), or anti γ-ENaC (in house, 148

mAb 4C11; 1:1000) (38) for one hour at room temperature or overnight at 4°C. Following wash 2 x 5 min 149

and 1 x 15 min with TBST the membrane was incubated with HRP-conjugated secondary antibodies (Dako, 150

Denmark 1:2000). After repeated wash in TBST, the signals were developed by enhanced 151

chemiluminescence (PerkinElmer, Inc, Waltham, MA, USA) and recorded using Molecular Imager 152

ChemiDocTMXRS+ (Bio-Rad). For densitometry, band density was analyzed using Image Lab software (Bio-153

rad). The obtained values were compared to the positive control or to the mean of the control group. 154

8

Whole cell patch-clamp experiments Murine cortical collecting ducts cells (M1, ATCC, Boras, Sweden) 155

were used for whole-cell patch clamp experiments as previously described (27). Briefly, M1 cells were 156

maintained in DMEM: F12 (Life Technologies, Taastrup, Denmark) with 5 µmol/L dexamethasone (Sigma, 157

ST. Louis, Mo) and 5% FCS (Life Technologies), and incubated at 37˚C / 5% CO2. Experiments were 158

conducted at room temperature in the tight-seal whole-cell configuration 24-30 h after seeding the cells. The 159

patch pipettes were heat-polished with resistances of 5-7 MΩ. Seal resistance range was 1-15 GΩ. High 160

resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier (HEKA) controlled by 161

pulse v8.11 software on a Power Macintosh G3 computer. The current was monitored by the response to a 162

voltage step of -160 mV for 200 ms from a holding potential of -60 mV. This pulse was repeated every 3 s 163

throughout the entire experiment. After 30-60 s, the cell was gently flushed with urine and the current 164

monitored. 165

Urinary protease activity Urinary protease activity was measured using a quantitative protease assay 166

(EnzChek Peptidase/protease Assay Kit (33758)) as previously described (7). Urine was subjected to 167

aprotinin-coated sepharose beads to enrich for serine proteases (49). Pure human plasmin was used to 168

produce a standard curve. Fifty μL of eluate was used for the fluorescence assay. 169

Statistical evaluation Normally distributed data (D'Agostino & Pearson omnibus or KS normality test) were 170

presented as mean ± SEM. Data that were not normally distributed were log-transformed and presented in 171

semi-logarithmic diagrams with geometric means. The albuminuric group and controls were compared by 172

unpaired Student´s T-test. If log-transformed data were not normally distributed, the statistical analyses were 173

performed using non-parametric correlation (Spearman) and nonparametric t-test (Mann-Whitney test). 174

Correlations were evaluated using Pearson correlation. One-way analysis of variance (ANOVA) followed by 175

Bonferroni´s Multiple Comparison post-hoc test was used when comparing means of three groups. P < 0.05 176

was considered statistically significant. GraphPad Prism 6,07 for Windows was used to produce graphic 177

presentations. 178

RESULTS 179

9

Patient characteristics KTRs were classified as albuminuric (albumin-creatinine ratio (ACR) > 300 mg/g) 180

or non-albuminuric controls (ACR < 30mg/g) (Table 1). There was no significant difference between the 181

groups with respect to age, gender distribution, time from transplantation or numbers of patients receiving 182

calcineurin inhibitors. In the albuminuria group, the majority of grafts originated from deceased donors 183

whereas in the control group, the majority received kidneys from living donors. KTRs with albuminuria had 184

significantly higher systolic- and diastolic BP despite significantly more antihypertensive medications (Table 185

1) and displayed reduced p-albumin concentration (Table 1). There were no significant differences in eGFR, 186

plasma electrolytes,plasma aldosterone and renin concentration between groups (Table 1). The mean weight, 187

body mass index, extracellular water (ECW), intracellular water (ICW), ECV/ICV ratio (E/I) and degree of 188

volume expansion did not differ significantly between groups although there was a trend towards greater 189

volume expansion in albuminuric KTRs (P = 0.08). 190

191

Albuminuria correlated with augmented urine excretion of serine proteases 192

The total urinary plasminogen/creatinine ratio was significantly higher in the albuminuric KTRs compared to 193

controls (Figure 1A). Urinary plasmin(ogen) excretion related directly and significantly with urinary albumin 194

excretion (P=0.001, R=0.69, Figure 1B). The relationship between the degree of albuminuria and urinary 195

plasminogen levels after excluding the control patients was also significant (data not shown, P=0.001, 196

R=0,67). Plasma plasminogen was significantly higher in the albuminuric group compared to controls 197

(Figure 1C). Urinary protease activity was analyzed in random subsets of aprotinin-affinity-purified urine 198

samples from both groups (n=12) and compared to a pure plasmin standard curve. There was no significant 199

difference in protease activity between the albuminuric group compared to control KTRs (Figure1D, P = 200

0.29, Students t test). Western blotting identified plasminogen in urine samples from albuminuric KTRs 201

displaying bands consistent with intact plasminogen (≈100 kDa), plasmin heavy A chain (≈ 60 kDa) and the 202

active plasmin B light chain (≈ 26 kDa). No plasminogen, heavy A or B light chains were detected in 203

controls (Figure 2A). Purified human active plasmin served as a positive control and displayed bands at ≈ 60 204

10

and ≈ 26 kDa while human plasma displayed predominantly the intact zymogen plasminogen that migrated 205

just below ≈ 100 kDa (Figure 2A). Semi-quantitative analysis by densitometry showed significantly higher 206

densities of the ≈ 100 kDa bands in the albuminuric group when compared to controls (Figure 2B). Western 207

blotting identified uPA in urine from albuminuric KTRs with bands at ≈ 60, ≈ 50- and ≈ 25 kDa compatible 208

with pro-uPA zymogen (≈ 60 kDa), two-chain active uPA (≈ 50 kDa) and low molecular weight uPA (≈ 30 209

kDa), while no uPA was identified in urine from control patients (Figure 2C). When quantitated by 210

densitometry the excretion of the active two-chain uPA was significantly greater in albuminuric KTRs 211

(Figure 2D). Western blotting for prostasin displayed a ≈ 40 kDa band in urine from albuminuric KTRs 212

(n=11) and in 5 out of 13 KTRs from the control group (Figure 2E). The excretion of prostasin was higher in 213

urine from albuminuric KTRs when compared to controls (Figure 2F) and correlated significantly with 214

urinary albumin excretion (data not shown, P = 0.0004, R = 0.44). 215

216

Urine from albuminuria transplant recipients activates ENaC in vitro 217

In vitro activation of inward current likely carried by ENaC, was demonstrated by the detection of an 218

increase in whole-cell inward current in single M1-cells when exposed to urine from KTRs (Figure 3). Urine 219

from albuminuric KTRs (n=5) resulted in ≈10 times increase in inward current (P< 0.0001) while no 220

significant increase was observed with urine from control KTRs. The increase in inward current with urine 221

from albuminuric KTRs was abolished with amiloride (2 µmol/L), Figure 3B) and aprotinin (700µg/ml), 222

Figure 3B). 223

Urine exosome γ-ENaC protein abundance and proteolysis 224

The purified exosomal urine fractions were all positive for the exosome marker ALIX with no difference 225

between groups (Figure 4A and 4E). A homogenate of a human kidney cortex pool (HCP) also displayed 226

ALIX (Figure 4A). Principal-specific AQP2 was identified in the exosomal fraction with bands migrating at 227

25 and 37 kDa corresponding to unglycosylated and mature AQP2, respectively, and with no significant 228

difference in abundance between the albuminuric KTRs and controls (Figure 4B and 4E). Western blotting 229

11

for prostasin revealed a ≈40 kDa band with similar intensity in all KTRs, and an additional 50 kDa band in 230

albuminuric KTRs and in HCP with higher abundance in the albuminuric KTRs when compared to controls 231

(Figure 4C, and 4E, densitometry of 50 kDa band not shown). Immunoblotting of human kidney cortex 232

tissue homogenate pool (HCP) for γENaC with an antibody directed against the inhibitory peptide tract (58) 233

resulted in four distinct bands at ~90, 75, 50 and 37kDa likely corresponding to full length intact and 234

glycosylated γENaC, furin-cleaved γENaC, and γENaC cleaved by a distally acting protease only (Figure 235

4D). No full-length γENaC was detected in the exosome fractions from the albuminuric or the control KTRs. 236

Exosomal fractions revealed a 75 kDa band corresponding to furin-cleaved ENaC, the abundance of this 237

band was significantly higher in the albuminuric KTRs (Figure 4E). A 50 kDa band was observed 238

consistently and with a significantly higher abundance in the albuminuric KTRs (Figure 4E). The 37 kDa 239

band seen in kidney cortex tissue was inconsistently observed in exosomes with faint signal in some of the 240

albuminuric KTRs. 241

242

Albuminuria/protease activity and relation with extracellular fluid volume and blood pressure 243

Pre-hoc hypotheses were analyzed regarding a direct relation between urinary serine protease protein and 244

fluid overload and hypertension. Significant positive correlations were observed between fluid overload and 245

the urine albumin/creatinine-ratio (P = 0.01; r=0.21) (Figure 5A) as well as between fluid overload and urine 246

plasminogen/creatinine ratio (P=0.02; r=0.16) (Figure 5B). Fluid overload also correlated significantly with 247

systolic BP (P=0.0006; r=0.31) (Figure 5C). 248

249

12

DISCUSSION 250

The present study was designed to test whether albuminuria in KTRs was associated with urinary serine 251

proteases, gain of protease activity and ability of urine to activate ENaC current in vitro. Subsequent aim was 252

to examine a potential association between urinary serine proteases and in vivo water accumulation and 253

hypertension. The data showed (I) significantly higher systolic and diastolic BP and greater use of 254

antihypertensive medication in KTRs with albuminuria; (II) a direct significant relation between systolic BP 255

and fluid overload, u-albumin/creatinine and urine plasminogen/creatinine in KTRs; (III) an increased 256

urinary excretion of the serine proteases plasmin, uPA, and prostasin in albuminuric KTRs that correlated 257

with u-albumin; (IV) activation of amiloride- and aprotinin-sensitive inward current in collecting duct cells 258

by urine from albuminuric KTRs only; and (V) increased abundance of active, cleaved γENaC protein in 259

urinary exosomes from albuminuric KTRs. These results are consistent with proteolytic activation of ENaC 260

leading to impaired Na+ excretion, fluid retention, and hypertension in albuminuric KTRs. 261

The prevalence of albuminuria/proteinuria in KTRs is high and a well-established, independent, prognostic 262

marker for progressive chronic allograft dysfunction, graft loss, and cardiovascular disease (CVD), being the 263

main causes of transplant failure (46). Post-transplant proteinuria has been attributed to glomerulonephritis, 264

nephrosclerosis, renal vein thrombosis, chronic rejection, allograft glomerulopathy and reflux nephropathy 265

(15, 40, 55). Hypertension is another important prognostic marker of chronic allograft dysfunction and CVD 266

(22, 33) with a prevalence of 50-90% (21, 32, 34, 35). These observations were corroborated in the present 267

study that included KTRs by the degree of albuminuria and showed that albuminuric KTRs received 268

significantly more antihypertensive medication and had higher BP levels compared to normo-albuminuric 269

KTRs. The pathogenesis of posttransplant hypertension is multifactorial but data support, that impaired renal 270

sodium excretion contributes. Calcineurin inhibitors (CNIs) increased NCC activity (19) and NCC 271

abundance and this correlated to blood pressure response of thiazide use (50). To our knowledge, 272

involvement of ENaC and effect of ENaC blockers has not previously been investigated. In the present 273

study, the majority of albuminuric KTRs had received grafts from deceased donor which is in accord with 274

the well-known adverse impact on graft kidney function. However, eGFR was not significantly different 275

13

between groups. Is posttransplant hypertension sodium dependent? In a cross-sectional study, there was a 276

positive association between sodium intake and blood pressure (53) and dietary sodium restriction reduced 277

blood pressure and albuminuria in KTRs (13). Moreover, extracellular volume expansion in KTRs was 278

associated with increased blood pressure and increased sodium intake in agreement with a contribution from 279

enhanced renal sodium and water reabsorption (11). In the non-transplant setting, patients with salt-sensitive 280

hypertension display greater urinary albumin excretion which is in agreement with a general coupling. 281

Together the evidence supports that hypertension is sodium dependent (5, 13, 53). In the present study, there 282

was no significant difference in fluid overload between albuminuric KTRs and controls, however, it should 283

be emphasized, that usual diuretic and antihypertensive treatment were maintained. Nevertheless, there was a 284

direct relation between fluid overload and systolic BP, and between fluid load and urinary albumin/creatinine 285

and plasminogen/creatinine ratios. Albuminuria was associated with an increased excretion of serine 286

proteases uPA, plasmin and prostasin which have the ability to induce “second hit” cleavage of γENaC 287

causing the putative release of an inhibitory peptide tract. In non-transplant CKD patients, there was a 288

correlation between body water and urinary plasminogen and proteinuria (45) consistent with impaired 289

sodium excretion and a possible involvement of ENaC. We have previously demonstrated a correlation 290

between proteinuria and urinary serine proteases in preeclampsia (7), type 2 diabetes (8), nephrotic syndrome 291

in adult, (49) and pediatric patients (4, 47). Moreover, amiloride was an efficient add on to achieve blood 292

pressure control in subjects with type 2 diabetes (37), and increased natriuresis in response to 2 days high 293

doses of amiloride (3). However, the potential risk of hyperkalemia is evident with reduced kidney function 294

(51) The findings in KTRs are in line with a similar effect from aberrant filtration of enzymes as a 295

consequence of a damaged glomerular filtration barrier. The proteases may mutually activate, leading to an 296

amplified enzymatic activity as the ultrafiltrate is concentrated along the nephron and collecting ducts (48). 297

Soluble prostasin was present also in the urine of some normo-albuminuric KTRs. This is likely due to the 298

physiological expression of prostasin in principal cells leading to its release with exosomes (2, 12, 43, 54). 299

Such interpretation was supported by a significant increase in total urinary prostasin in albuminuric KTRs, 300

while the abundance of exosome-associated prostasin was not significantly different between groups, 301

suggesting constant membrane abundance and shedding rate of GPI-anchored prostasin (12). Aldosterone 302

14

has little impact on kidney prostasin abundance (2, 36), and plasma aldosterone was not different between 303

groups. Therefore, aldosterone does not account for differences in exosomal γ-ENaC abundance (28). 304

Assuming that the increased exosomal excretion of cleaved γENaC moieties reflects principal cell surface 305

abundance, this indicates an increased collecting duct ENaC abundance. Of note, the detected γENaC 306

products contain the inhibitory peptide tract and are not equivalent to full channel activation which would 307

remove the epitope and yield a lower signal abundance. As the excretions of the exosomal marker ALIX as 308

well as AQP2 were similar between groups, the increase in exosomal γENaC in albuminuric KTRs was not 309

related to a difference in overall and in principal cell exosome release rate. The present findings imply that 310

distally acting K+-sparing diuretics such as amiloride and spironolactone should be tested for superior effect 311

in KTRs. No published studies have specifically addressed differential natriuretic potencies of diuretic 312

classes in KTRs. 313

Summary and perspective 314

Findings in albuminuric KTRs are consistent with pathophysiological activation of ENaC, impaired Na+ 315

excretion, fluid retention and hypertension. The study is limited by the cross-sectional design and the small 316

number of patients, but it provides a tentative explanation for sodium sensitive hypertension in albuminuric 317

KTRs which may be tested in future, interventional trials. 318

ACKNOWLEDGEMENT 319

The authors thank Gitte Kitlen, Mie Rytz Hansen, Kristoffer Rosenstand and Liselotte Sommer for skillful 320

technical assistance. 321

GRANTS 322

This work was supported by Odense University Hospital, Region of Southern Denmark, Danish Research 323

Council for Independent Research. The Novo Nordisk Foundation, Innovationsfonden/Strategic Research 324

Council and The Danish Society of Nephrology. 325

DISCLOSURE 326

15

The authors declare no conflicts of interest 327

REFERNCES 328

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31. Nesovic M, Stojanovic M, Nesovic MM, Ciric J, and Zarkovic M. Microalbuminuria is associated with 422 salt sensitivity in hypertensive patients. Journal of human hypertension 10: 573-576, 1996. 423 32. Ojo AO. Cardiovascular complications after renal transplantation and their prevention. Transplantation 424 82: 603-611, 2006. 425 33. Opelz G, and Dohler B. Improved long-term outcomes after renal transplantation associated with blood 426 pressure control. American journal of transplantation : official journal of the American Society of 427 Transplantation and the American Society of Transplant Surgeons 5: 2725-2731, 2005. 428 34. Opelz G, Wujciak T, and Ritz E. Association of chronic kidney graft failure with recipient blood pressure. 429 Collaborative Transplant Study. Kidney international 53: 217-222, 1998. 430 35. Opelz G, Zeier M, Laux G, Morath C, and Dohler B. No improvement of patient or graft survival in 431 transplant recipients treated with angiotensin-converting enzyme inhibitors or angiotensin II type 1 432 receptor blockers: a collaborative transplant study report. Journal of the American Society of Nephrology : 433 JASN 17: 3257-3262, 2006. 434 36. Oxlund C, Kurt B, Schwarzensteiner I, Hansen MR, Staehr M, Svenningsen P, Jacobsen IA, Hansen PB, 435 Thuesen AD, Toft A, Hinrichs GR, Bistrup C, and Jensen BL. Albuminuria is associated with an increased 436 prostasin in urine while aldosterone has no direct effect on urine and kidney tissue abundance of prostasin. 437 Pflugers Archiv : European journal of physiology 2017. 438 37. Oxlund CS, Buhl KB, Jacobsen IA, Hansen MR, Gram J, Henriksen JE, Schousboe K, Tarnow L, and 439 Jensen BL. Amiloride lowers blood pressure and attenuates urine plasminogen activation in patients with 440 treatment-resistant hypertension. Journal of the American Society of Hypertension : JASH 8: 872-881, 2014. 441 38. Passero CJ, Hughey RP, and Kleyman TR. New role for plasmin in sodium homeostasis. Current opinion 442 in nephrology and hypertension 19: 13-19, 2010. 443 39. Passero CJ, Mueller GM, Rondon-Berrios H, Tofovic SP, Hughey RP, and Kleyman TR. Plasmin activates 444 epithelial Na+ channels by cleaving the gamma subunit. The Journal of biological chemistry 283: 36586-445 36591, 2008. 446 40. Peddi VR, Dean DE, Hariharan S, Cavallo T, Schroeder TJ, and First MR. Proteinuria following renal 447 transplantation: correlation with histopathology and outcome. Transplantation proceedings 29: 101-103, 448 1997. 449 41. Poulsen K, and Jorgensen J. An easy radioimmunological microassay of renin activity, concentration and 450 substrate in human and animal plasma and tissues based on angiotensin I trapping by antibody. The Journal 451 of clinical endocrinology and metabolism 39: 816-825, 1974. 452 42. Rodrigo E, Monfa E, Albines Z, Serrano M, Fernandez-Fresnedo G, Ruiz JC, Pinera C, Palomar R, 453 Martin-Penagos L, and Arias M. Sodium Excretion Pattern at 1 Year After Kidney Transplantation and High 454 Blood Pressure. Annals of transplantation : quarterly of the Polish Transplantation Society 20: 569-575, 455 2015. 456 43. Salih M, Fenton RA, Zietse R, and Hoorn EJ. Urinary extracellular vesicles as markers to assess kidney 457 sodium transport. Current opinion in nephrology and hypertension 25: 67-72, 2016. 458 44. Schnuelle P, Lorenz D, Trede M, and Van Der Woude FJ. Impact of renal cadaveric transplantation on 459 survival in end-stage renal failure: evidence for reduced mortality risk compared with hemodialysis during 460 long-term follow-up. Journal of the American Society of Nephrology : JASN 9: 2135-2141, 1998. 461 45. Schork A, Woern M, Kalbacher H, Voelter W, Nacken R, Bertog M, Haerteis S, Korbmacher C, Heyne N, 462 Peter A, Haring HU, and Artunc F. Association of Plasminuria with Overhydration in Patients with CKD. 463 Clinical journal of the American Society of Nephrology : CJASN 11: 761-769, 2016. 464 46. Shin M, Song SH, Kim JM, Kwon CH, Joh JW, Lee SK, and Kim SJ. Clinical significance of proteinuria at 465 posttransplant year 1 in kidney transplantation. Transplantation proceedings 44: 610-615, 2012. 466 47. Staehr M, Buhl KB, Andersen RF, Svenningsen P, Nielsen F, Hinrichs GR, Bistrup C, and Jensen BL. 467 Aberrant glomerular filtration of urokinase-type plasminogen activator in nephrotic syndrome leads to 468 amiloride-sensitive plasminogen activation in urine. American journal of physiology Renal physiology 309: 469 F235-241, 2015. 470

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48. Svenningsen P, Andersen H, Nielsen LH, and Jensen BL. Urinary serine proteases and activation of ENaC 471 in kidney--implications for physiological renal salt handling and hypertensive disorders with albuminuria. 472 Pflugers Archiv : European journal of physiology 467: 531-542, 2015. 473 49. Svenningsen P, Bistrup C, Friis UG, Bertog M, Haerteis S, Krueger B, Stubbe J, Jensen ON, Thiesson HC, 474 Uhrenholt TR, Jespersen B, Jensen BL, Korbmacher C, and Skott O. Plasmin in nephrotic urine activates the 475 epithelial sodium channel. Journal of the American Society of Nephrology : JASN 20: 299-310, 2009. 476 50. Tutakhel OAZ, Moes AD, Valdez-Flores MA, Kortenoeven MLA, Vrie MVD, Jelen S, Fenton RA, Zietse R, 477 Hoenderop JGJ, Hoorn EJ, Hilbrands L, and Bindels RJM. NaCl cotransporter abundance in urinary vesicles 478 is increased by calcineurin inhibitors and predicts thiazide sensitivity. PloS one 12: e0176220, 2017. 479 51. Unruh ML, Pankratz VS, Demko JE, Ray EC, Hughey RP, and Kleyman TR. Trial of Amiloride in Type 2 480 Diabetes with Proteinuria. Kidney international reports 2: 893-904, 2017. 481 52. Vallet V, Chraibi A, Gaeggeler HP, Horisberger JD, and Rossier BC. An epithelial serine protease 482 activates the amiloride-sensitive sodium channel. Nature 389: 607-610, 1997. 483 53. van den Berg E, Geleijnse JM, Brink EJ, van Baak MA, Homan van der Heide JJ, Gans RO, Navis G, and 484 Bakker SJ. Sodium intake and blood pressure in renal transplant recipients. Nephrology, dialysis, 485 transplantation : official publication of the European Dialysis and Transplant Association - European Renal 486 Association 27: 3352-3359, 2012. 487 54. van der Lubbe N, Jansen PM, Salih M, Fenton RA, van den Meiracker AH, Danser AH, Zietse R, and 488 Hoorn EJ. The phosphorylated sodium chloride cotransporter in urinary exosomes is superior to prostasin 489 as a marker for aldosteronism. Hypertension 60: 741-748, 2012. 490 55. Vathsala A, Verani R, Schoenberg L, Lewis RM, Van Buren CT, Kerman RH, and Kahan BD. Proteinuria 491 in cyclosporine-treated renal transplant recipients. Transplantation 49: 35-41, 1990. 492 56. Vollmer WM, Wahl PW, and Blagg CR. Survival with dialysis and transplantation in patients with end-493 stage renal disease. The New England journal of medicine 308: 1553-1558, 1983. 494 57. Weir MR, and Fink JC. Salt intake and progression of chronic kidney disease: an overlooked modifiable 495 exposure? A commentary. American journal of kidney diseases : the official journal of the National Kidney 496 Foundation 45: 176-188, 2005. 497 58. Zachar RM, Skjodt K, Marcussen N, Walter S, Toft A, Nielsen MR, Jensen BL, and Svenningsen P. The 498 epithelial sodium channel gamma-subunit is processed proteolytically in human kidney. Journal of the 499 American Society of Nephrology : JASN 26: 95-106, 2015. 500

501

502

19

LEGENDS 503

FIGURE 1 (A) Urinary total plasminogen/creatinine-ratio was significantly higher in KTRs with albuminuria 504

(n= 18) compared to normoalbuminuric transplant recipients (controls, n= 13), six values in the control 505

group were under the detection range of the assay and were not included (geometric means 1721 vs 7,66 506

µg/g, P < 0,0001 by Student t test). Since data were log-normally distributed, the Y-axis is logarithmic. (B) 507

Urinary plasmin(ogen) correlated significantly with urine albumin from spot urine samples (P=0.001, 508

R=0.69), six samples from the control group exhibited u-plasmin(ogen) values below detection range and 509

these were included in the analysis with a value of zero. (C) Plasma plasmin(ogen) concentration was 510

significantly higher in the albuminuric group (geometric means 186637 vs 61376 ng/ml, P = 0,004 by 511

Student t test). (D) Serine proteases were purified from 0,7 ml crude urine samples from 12 albuminuric 512

KTRs and 12 controls by aprotinin-coated sepharose beads. Eluate was used for fluorescence serine protease 513

activity assay (50 µL). Diluted series of pure human plasmin served as standard curve. Broken line A 514

indicates the mean of serine protease activity from the albuminuric group and B from controls (89285 vs 515

48471 Fluorescein counts). 516

FIGURE 2 Western immunoblot of spot urine samples normalized for creatinine and separated by SDS-517

PAGE from kidney transplant recipients (KTRs) with - and without albuminuria. (A) Intact plasminogen (83-518

88kDa), active plasmin (60kDa), plasmin heavy A chain (~ 60kDa) and plasmin light B chain (~26kDa) was 519

present in urine from albuminuria patients. The matched control group showed no detectable plasmin(ogen). 520

Purified human active plasmin (P1) served as a positive control and displayed bands at 60 and 26 kDa while 521

human plasma (P2) displayed predominantly the intact zymogen plasminogen that migrated just below 100 522

kDa. Plasminogen band densities were significantly higher in the albuminuric group compared to controls 523

(B) (P = 0,0007, Mann-Whitney test). (C) Urine urokinase-type plasminogen activator (uPA) was detected as 524

a band migrating at ~50kDa in samples predominantly from the albuminuric group. Omitting the primary 525

antibody and only probing with the secondary antibody did not produce any visible bands. (D) Density of the 526

50kDA band was significantly higher in the albuminuric patients (P = 0,03, Mann-Whitney test). (E) 527

Western immunoblot of urine samples normalized to creatinine for prostasin displayed band migrating at 528

20

~40kDa in all the tested albuminuric recipients (n=11) and in 5 out of 13 tested samples from the control 529

group (Figure 2E). Densitometry of prostasin protein bands was significantly higher in urine samples from 530

albuminuric KTRs (F) (P = 0,0005, Student t test). Human placenta homogenate served as positive control 531

for prostasin (P, Figure 2E). 532

FIGURE 3 Patch Clamp. A-B, Original current trace obtained in murine cortical collecting duct cells (M1) 533

by patch clamp recordings from single cells. A, The grey trace, displays a recording from a M1 cell before 534

superfusion with urine and the black trace displays a recording from the same cell after superfusion with 535

urine from an albuminuric kidney transplant recipient. B, Bar graph showing the average changes in current 536

obtained in patch clamp recordings in response to superfusion with urine from controls (black) and 537

albuminuric KTRs (grey). The same urine samples from the albuminuric KTRs were tested with separate 538

cells after addition of amiloride (dark grey) and after aprotinin (light grey). pA, picoampere; (pF values, 539

mean ± SEM: Controls 27.42 ± 4.83; albuminuric KTRs 12.63 ± 0.83; albuminuric KTRs + amiloride 13,77 540

± 1.9; albuminuric KTRs + aprotinin 16.12 ± 4.46) 541

FIGURE 4 (A-D) Western immunoblotting of exosomes contained in urine-pellet after ultracentrifugation of 542

urine samples from kidney transplant patients with albuminuria (KTRs) and without albuminuria (Controls). 543

The loaded amount of protein was normalized for urine creatinine before SDS-PAGE. Aliquots from the 544

same human kidney cortex homogenate pool was run as a positive control on most gels (HCP) (A,E). Human 545

kidney cortex pool (HCP). The exosome marker ALIX showed a single significant band migrating at 546

approximately 100kDa, detected in all samples and with no difference between groups (P = 0,86, Student t 547

test). (B, E) Immunoblot for the principal-specific aquaporin-2 (AQP-2); there was no significant difference 548

in AQP2 abundance between the groups (P = 0,86, Student t test). (C, E) Immunoblotting for prostasin 549

demonstrated a band of the expected molecular size that migrated ~40kDa in all patients with no significant 550

difference between groups (P = 0,074, Student t test). (D) Immunoblotting for γENaC with an antibody 551

directed against the inhibitory peptide tract showed a band migrating at approximately 75kDa corresponding 552

to furin-cleaved ENaC in exosomes, whereas the intact 100 kDa protein was observed only in human kidney 553

cortex pool (HCP). The abundance of the 75kDa band was significantly higher in the albuminuric group by 554

21

densitometry (4E, lower) (P = 0,003, Mann-Whitney test). Three patients from the albuminuric group 555

displayed a band at 37kDa consistent with proteolytically cleaved γENaC. The band at 50kDa was observed 556

most consistently, and with a significantly higher abundance in the albuminuric transplant recipients (P = 557

0,001, Student t test). 558

FIGURE 5 (A-C) Fluid overload (OH) measured using body composition monitor (BCM-Fresenius) 559

correlated significantly with urinary albumin-creatinine ratio (P = 0.014; r=0.21) (7 controls had values 560

under the detection level of u-albumin, and these values are not included), plasminogen-creatinine ratio 561

(P=0.02; r=0.16), and systolic BP (P=0.0006; r=0.31). 562

563

564

22

Table 1. Baseline characteristics of albuminuric kidney transplant recipients (KTRs, ACR > 300mg/g) and 565

normoalbuminuric KTRs (controls, ACR < 30mg/g) 566

567

Albuminuric KTRs Controls P-value

Recipient characteristics

Number of participants 18 19

Sex distrubution (female/male) 11 female/7 male 10 female/9 male

Median age (years) 52.1 ± 4 54.8 ± 3 0.58 ns

Time from transplantation (months) 97.9 75.5 0.39 ns

Donor (deceased/living related) 12 deceased/6 living

8 deceased/11

living

Clinical and biochemical parameters

Systolic BP (mmHg) 147 ± 4 128 ± 2,7 0.0005 ***

Diastolic BP (mmHg) 83 ± 3 75 ± 1.9 0.05 *

Heart rate (bpm) 69 ± 3 67 ± 3 0.64 ns

Urine albumin/creatinine (mg/g) 1722 ± 243.4 8.8 ± 2.1 <0.0001 ****

Urinary Na+/K+ ratio 2.7 ± 0.5 3.0 ± 0.3 0.57 ns

Urinary osmolality (mosm/kg) 380.7 ± 33 437 ± 42 0.3 ns

Plasma albumin (g/L) 39.9 ± 0.9 42.5 ± 0.6 0.02 *

Plasma natrium (mmol/L) 139.5 141 0.38 ns

Plasma kalium (mmol/L) 4.2 ± 0.1 4.1 ± 0.1 0.91 ns

eGFR (ml/min/1,73m2) 38.1 ± 5 48.5 ± 3.3 0.09 ns

Plasma renin (mIU/L) 42.6 ± 1.2 30.4 ± 1.2 0.26 ns

Plasma aldosterone (ng/L) 124.5 ± 6.1 118.7 ± 6.8 0.53 ns

Medication

23

Calcineurin inhibitors (numbers of

patients) 13/18 17/19

Antihypertensive medications (numbers) 1 2.4 ± 0.3 1.3 ± 0.3 0.01 *

Angiotensin-converting enzyme 7/18 5/19

Angiotensin receptor blockers 7/18 2/19

Alpha- receptor blockers 2/18 2/19

Beta receptor blockers 5/18 7/19

Calcium channel blockers 10/18 5/19

Furosemide 10/18 5/19

Thiazide 3/18 1/19

Body Composition Monitor

Weight (kg) 75.8 ± 4.8 76.3 ± 3.8 0.93 ns

Body mass index (kg/m2) 26 ± 1.1 25.9 ± 0.9 0.99 ns

Total body weight (L/1,73m2) 35.3 ± 1.5 34.3 ± 1.7 0.66 ns

Extracellular volume (L/1,73m2) 16.8 ± 0.7 16.3 ± 0.8 0.61 ns

Intracellular volume (L/1,73m2) 18.5 ± 0.9 18 ± 0.9 0.72 ns

Fluid overload (L/1,73m2) 1.2 ± 0.3 0.6 ± 0.1 0.08 ns

Normally distributed data are presented as mean ± SEM; ns, non-significant. See text for details.1Numbers of

antihypertensive drugs including diuretics. See text for details

568

569

Figure 1

Figure 2

-20

0

20pA

/pF

A

B

Figure 3

0

50

100

150

Incr

ease

in in

war

d c

urr

ent

(%)

Controln=5

Albuminuric KTRs n=5

Albuminuric KTRs + Amilorid n=5

Albuminuric KTRs +Aprotinin n=4

**** ****

0.40.3s

0.20.10.0

Figure 4

HCP

HCP

HCP

HCP

HCP

HCP

Figure 5