ONLINE SUPPLEMENT
PLASMIN IN URINE FROM PATIENTS WITH TYPE 2 DIABETES AND TREATMENTRESISTANT HYPERTENSION ACTIVATES ENAC IN VITRO
Kristian B. BUHLa, Christina S. OXLUNDb, Ulla G. FRIISa, Per SVENNINGSENa, Claus
BISTRUPc, Ib A. JACOBSENb, Boye L. JENSENa
a
Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of
Southern Denmark, Odense, Denmark.
b
Research Unit for Cardiovascular and Metabolic Prevention, Department of Endocrinology,
Odense University Hospital, Odense, Denmark.
c
Department of Nephrology, Odense University Hospital, Odense, Denmark.
The reported study was a substudy to a published randomized intervention study on the effect of
spironolactone on blood pressure in patients with type 2 diabetes and treatment resistant
hypertension [1]. Power calculations with a minimal relevant blood pressure difference of 7 mmHg
in response to spironolactone, yielded a number of participants to be ~110 [1]. 119 patients
participated in the intervention study; of those 119, full set of urine was available on 113, which are
the reported number in the present study. The present investigation was a cross-sectional study on
the urine and plasma sample taken at inclusion.
Inclusion criteria for patients
Patients entered the study following an assessment denoted visit -1 ensuring inclusion criteria were
met. Medical history was obtained using study protocol and blood and spot urine samples were
collected and patients performed the 24 hour ambulatory blood pressure measurement at home
collectively denoted as visit 0. In this study treatment resistant hypertension was defined as: systolic
blood pressure above 130mmHg and/or diastolic blood pressure above 80 mmHg, despite the use of
minimum 3 relevant antihypertensive drugs in relevant dose. One of the antihypertensive drugs
mandatory had to be an ACE-inhibitor or Angiotensin II receptor antagonist. And it was also
mandatory the patients were treated with a diuretic (Thiazide, Indapamid or loop diuretics). Patients
had to be younger than 75 years and diagnosed with Type 2 diabetes. The debut of diabetes had to
be after the age of 30 years and in addition not requiring insulin the first year after diagnosis. At
inclusion and during the study, fertile women had to use anti-conception either; birth control pills,
intrauterine spiral (IUC) or gestagen contraceptive implant.
Patients with systolic blood pressures above 180mmHg (office BP) or above 175mmHg in daytime
average ABPM and diastolic blood pressures above 110 mmHg (office BP) or above 85 mmHg in
daytime average ABPM, were excluded from the study. Same goes for the patients that had more
than 10% changes in the anti-diabetic treatment 1 month prior to the trial. Other exclusion criterions
were: pregnancy, type 1 diabetes, total cholesterol > 10mmolL or/and Triglyceride > 8 mmol/L,
NYHA class III or IV, mental illness or clinically relevant co-morbidity incl. malignancy. A great
effort was done to eliminate diagnosed secondary hypertension hence patients diagnosed with:
obstructive sleep apnea, primary hyperaldosteronism (incl. Pheochromocytoma, Cushing's
Syndrome), chronic kidney disease defined by eGFR < 50ml/min, coactatio aortae and stenosis of
the kidney artery were excluded from the study. Because patients assigned to this study later were
administered spironolactone and amiloride, the patients receiving these specific drugs was excluded
from the study. Also patients with spironolactone intolerance had to be excluded from the study.
Patients receiving prednisolone or NSAID treatment could not enter the study. Finally, patients
from whom aliquots of urine collection were not available were excluded from the study.
Extended Materials and methods
Urine creatinine and albumin concentrations were measured acutely at the department of
Clinical Biochemistry and Pharmacology, Odense University Hospital. Remaining aliquots of the
spot urine samples were stored at -80˚C in 4.5ml Nunc® CryoTubes®. Prior to analysis, 4.5 ml urine
aliquots were thawed on ice bath and centrifuged at 9500 g for 30 seconds. The supernatant was
used for the experiments.
Urinary plasminogen concentration was analyzed using a commercial ELISA kit (Human
Plasminogen Total Antigen Assay (Cat# IHPLGKT-TOT, Innovative Research, Novi, Michigan,
USA) following the manufacturer’s protocol. The ELISA detects plasminogen, plasmin and antiplasmin-plasmin-complexes. Inter-assay variation measuring plasminogen in urine samples was
15.3%, n=13. Samples were diluted allowing measurements in the range of 0.1-20.0 ng/ml
plasmin(ogen). Undiluted samples displaying values below measuring range were given the value
0.1 ng/ml (lower detection limit) plasmin(ogen) for analyses.
Western immunoblotting was done using crude urine samples loading 10µL of urine to each well.
Urine samples were mixed using NuPAGE® LDS Sample Buffer (4X) (Invitrogen™, Carlsbad,
CA, USA) and NuPAGE® Sample Reducing Agent (2X) (Invitrogen™) and run on Tris-HCl ready
gels 7.5% and 4-15% (Bio-Rad Laboratories, Hercules, CA, USA). The gel was blotted on an
Immobilon®-P Transfer Membrane pore size 0.45µm (Millipore Corporation, Billerica, MA, USA).
The membrane was subsequently blocked in TBST with 5% skimmed milk and hereafter subjected
to primary anti-plasminogen antibody (ab6189, abcam®, Cambridge, UK) followed by secondary
Polyclonal Rabbit Anti-Goat Immunoglobulins/HRP (Dako, Glostrup, DK). Blots were developed
using the ECL Prime Western Blotting Detection Reagent system (Amersham™, Buckinghamshire,
UK) with Amersham Hyperfilm™ MP (Amersham™).
Zymography was done using crude urine samples loading 10µL of urine to each well in 10%
Zymogram (gelatin) Gels (Novex®, Carlsbad, CA, USA) following manufactures protocol.
Incubation period with Zymogram Developing Buffer (10X) (Novex®) was extended to 44 hours in
37˚C.
Whole-cell patch clamp experiments using M-1 cells were performed to analyze the ability of
urine to enhance inward current in single collecting duct cells. The procedure has previously been
described in details (1). The experiments were conducted on a collecting duct cell line (M1) from
ATCC (Boras, Sweden). The current was monitored by the response to a voltage step of -160 mV
for 200 ms from a holding potential of -60 mV (this pulse was repeated every 3rd second throughout
the entire experiment). After 30-60 seconds, the cell was gently flushed with urine and the current
was monitored. The identity of the urine sample was blinded to the person doing the experiment. To
block ENaC, 2 µmol/L amiloride was added to the bath solution and the patch clamp experiments
were repeated with urine as describe above.
Blood pressure
Patient characteristics
Table 1
General characteristics of the patients participating in the study. Table displays the geometric mean
within the population. When log-normalization did not cause normal distribution of the data, a
median denominated † is shown. Plasma creatinine data were normally distributed and denominated
#
.
Range
Data available n =
ACR (mg/g)
31.7†
5 – 3126
111
Weight (kg)
98.5
66.7 – 199.4
112
Body mass index (kg/m2)
32.3
21 – 50
112
Abdominal circumference (cm)
111.8
81 – 160
112
Age (years)
64.0†
35 – 75
113
HbA1c (%)
7.35
5.1 – 11.3
113
eGFR (ml/min/1.73m2)
81.4
50 – 183
112
Plasma creatinine (µmol/l)
80.7 ± 1.7#
44 – 129
113
Duration of hypertension (years)
11.0†
1 – 41
107
Duration of diabetes (years)
10.0†
2 – 30
110
Table 2
The table displays the number of antihypertensive drugs used and includes an overview of the
antihypertensive regimen used in the study.
Number of antihypertensive drugs
Number of patients
Percent
3
62
54.9 %
4
41
36.3 %
5
7
6.2 %
6
1
0.9 %
7
2
1.8 %
ACE inhibitors
72
63.7 %
AT1 receptor blockers
51
45.1 %
Beta adrenoceptor blockers
50
44.2 %
Calcium antagonists
97
85.8 %
Alpha adrenoceptor blockers
9
8.0 %
Thiazide diuretics
96
85.0 %
Renin antagonists
1
0.9 %
Loop diuretics
17
15.0 %
Other drugs
4
3.5 %
Antihypertensive drugs used
Table 3
Sub analysis of blood pressure (BP) when subdividing the obtained 24 hour BP and pulse
measurements in night (22.00 – 7.00) and day (7.00 - 22.00) intervals. List of p values obtained in
correlation analyses between blood pressure (BP) and urinary plasmin(ogen) excretion normalized
to urine creatinine concentration (U-plg/U-crea).
Data available n =
p value
Spearman r
106
p<0.01
0.25
Day
113
p<0.01
0.23
Night
111
p<0.05
0.22
106
p<0.05
0.21
Day
113
p<0.05
0.20
Night
111
p<0.01
0.26
105
p<0.01
0.25
Day
105
p<0.01
0.25
Night
104
p<0.01
0.23
106
ns
Day
112
ns
Night
111
ns
Systolic BP 24 hour
Diastolic BP 24 hour
MABP 24 hour
Pulse 24 hour
References
1.Christina S. Oxlund, Jan E. Henriksen, Lise Tarnow, Karoline Schousboe, Jeppe Gram, Ib A.
Jacobsen. Lowdose spironolactone reduces blood pressure in patients with resistant
hypertension and type 2 diabetes mellitus: a double blind randomizedclinical trial. J
hypertens 2013;31(10):2094-2102.