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Pioglitazone delays proximal tubule dysfunction andimproves cerebral vessel endothelial dysfunction innormoalbuminuric people with type 2 diabetes mellitus§
Ligia Petrica a, A. Vlad b,*, M. Petrica c, C.D. Jianu c, Gh. Gluhovschi a, Florica Gadalean a,V. Dumitrascu d, Calina Ianculescu d, Catalina Firescu e, S. Giju f, Cristina Gluhovschi a,F. Bob a, Silvia Velciov a, Gh. Bozdog a, Oana Milas a, Roxana Marian a, S. Ursoniu g
aDepartment of Nephrology, ‘Victor Babes’ University of Medicine and Pharmacy, County Emergency Hospital, Timisoara, RomaniabDepartment of Diabetes and Metabolic Diseases, ‘Victor Babes’ University of Medicine and Pharmacy, County Emergency Hospital, Timisoara,
RomaniacDepartment of Neurology, ‘Victor Babes’ University of Medicine and Pharmacy, County Emergency Hospital, Timisoara, RomaniadClinical Laboratory, Department of Blood Biochemistry, ‘Victor Babes’ University of Medicine and Pharmacy, County Emergency Hospital,
Timisoara, RomaniaeClinical Laboratory, Department of Immunology, ‘Victor Babes’ University of Medicine and Pharmacy, County Emergency Hospital, Timisoara,
RomaniafClinical Laboratory, Department of Urine Biochemistry, ‘Victor Babes’ University of Medicine and Pharmacy, County Emergency Hospital,
Timisoara, RomaniagDepartment of Public Health Medicine, ‘Victor Babes’ University of Medicine and Pharmacy, County Emergency Hospital, Timisoara, Romania
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 2
a r t i c l e i n f o
Article history:
Received 14 March 2011
Received in revised form
22 May 2011
Accepted 23 May 2011
Published on line 2 July 2011
Keywords:
Proximal tubule
Endothelial dysfunction
Albuminuria
Pioglitazone
a b s t r a c t
Aim: The renal and cerebral protective effects of pioglitazone were assessed in normoal-
buminuric patients with type 2 diabetes mellitus (DM).
Methods: A total of 68 normoalbuminuric type 2 DM patients were enrolled in a one-year
open-label randomized controlled trial: 34 patients (pioglitazone-metformin) vs. 34 patients
(glimepiride-metformin). All patients were assessed concerning urinary albumin: creatinine
ratio (UACR), urinary alpha1-microglobulin, urinary beta2-microglobulin, plasma asymmet-
ric dymethyl-arginine (ADMA), GFR, hsC-reactive protein, fibrinogen, HbA1c; pulsatility
index, resistance index in the internal carotid artery and middle cerebral artery, intima-
media thickness in the common carotid artery; cerebrovascular reactivity was evaluated
through the breath-holding test.
Results: At 1 year there were differences between groups regarding ADMA, urinary beta2-
microglobulin, urinary alpha1-microglobulin, parameters of inflammation, serum creati-
nine, GFR, UACR, the cerebral haemodynamic indices. Significant correlations were found
between alpha 1-microglobulin-UACR (R2 = 0.143; P = 0.001) and GFR (R2 = 0.081; P = 0.01);
beta2-microglobulin-UACR (R2 = 0.241; P = 0.0001) and GFR (R2 = 0.064; P = 0.036); ADMA–GFR
C o n t e n t s l i s t s a v a i l a b l e a t S c i e n c e D i r e c t
Diabetes Researchand Clinical Practice
journal homepage: www.elsevier .com/locate/diabres
, pa
(R2 = 0.338; P = 0.0001)§ This work was performed in the Department of Nephrology, DeparNeurology, County Emergency Hospital, 10, I. Bulbuca Str., 300736 Tim
* Corresponding author at: Str. Ciprian Porumbescu Nr.71 A, Ap7, 300E-mail addresses: ligiapetrica@rdslink.ro, ligia_petrica@yahoo.co.uk
(M. Petrica), dcjianu@yahoo.com (C.D. Jianu), ggluhovschi@yahoo.com (vicdumi@yahoo.com (V. Dumitrascu), ninaianculescu@yahoo.com (C. Ia(S. Giju), ggluhovschi@yahoo.com (C. Gluhovschi), flaviu_bob@yahoo.co(G. Bozdog), hhuanita@yahoo.com (O. Milas), roxanam10@yahoo.co.uk
0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights
doi:10.1016/j.diabres.2011.05.032
rameters of inflammation, HbA1c, duration of DM, cerebral indices.
tment of Diabetes and Metabolic Diseases, and the Department ofisoara, Romania. Tel.: +40256463001/433; fax: +40256486956.
239 Timisoara, Romania. Tel.: +40723010065; fax: +40256486956. (L. Petrica), vladrian@hotmail.com (A. Vlad), ligiapetrica@rdslink.roG. Gluhovschi), fnnicorici@yahoo.com (F. Gadalean),nculescu), cfirescu64@yahoo.com (C. Firescu), soringiju@yahoo.comm (F. Bob), s_velciov@yahoo.com (S. Velciov), g.bozdog@yahoo.com
(R. Marian), sursoniu@yahoo.com (S. Ursoniu).
reserved.
There were no correlations between ADMA–UACR, urinary alpha1-microglobulin and beta2-
microglobulin.
Conclusion: Proximal tubule (PT) dysfunction precedes albuminuria and is dissociated from
endothelial dysfunction in patients with type 2 DM. Pioglitazone delays PT dysfunction and
improves cerebral vessels endothelial dysfunction in normoalbuminuric patients with type
2 DM. Key words: proximal tubule; endothelial dysfunction, albuminuria; pioglitazone.
# 2011 Elsevier Ireland Ltd. All rights reserved.
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 2 23
1. Introduction
Diabetes mellitus (DM) represents a major health problem
worldwide due to its deriving cardiovascular and renal
complications. Diabetic chronic kidney disease (CKD) may
be attributed to up to 20–40% of cases referred to renal
replacement therapies in the USA, Europe, and Japan [1,2].
Due to the relationship between microalbuminuria and
endothelial dysfunction, diabetic atherosclerosis and micro-
angiopathy parallel diabetic nephropathy (DN), and thus
albuminuria may be considered as a very powerful risk
marker for cardiovascular and cerebrovascular diseases in
both type 1 and type 2 DM [3,4]. However, multiple markers of
endothelial dysfunction have also been documented in
normoalbuminuric patients with type 2 DM, suggesting that
the diabetic vasculopathy may occur before the development
of microalbuminuria [5].
In spite of structural similarities of the endothelium in
various vascular segments, such as the kidney and the brain,
there are highly significant differences with regard to the
behaviour of the endothelium in these endothelial territories,
in a particular sequenciality and independently of several
factors, within different time frames in the course of DM.
To date, there is a paradigm shifting from the glomerular
theory to the tubular theory concerning the mechanisms of
albuminuria in early DN. It has been forwarded the hypothesis
according to which albuminuria is caused primarily by
impaired uptake of albumin by the proximal tubule (PT),
rather than by an increased leakiness of the glomerular
filtration barrier [6,7]. In previous works performed by us in
normoalbuminuric patients with type 2 DM we demonstrated
that PT dysfunction precedes the occurrence of albuminuria.
Furthermore, we showed that glomerular endothelial dys-
function is dissociated from endothelial dysfunction in the
brain, thus explaining why patients with type 2 DM may
develop structural and functional modifications in cerebral
vessels while remaining normoalbuminuric [8,9].
These data maintain why multitarget therapies should be
considered in patients with type 2 DM. Pioglitazone, a
thiazolidinedione compound, exerts nephro- and neuropro-
tective effects in patients with type 2 DM by significantly
decreasing albuminuria, markers of inflammation, endotheli-
al dysfunction, and the risk of first and recurrent fatal and
non-fatal stroke [10,11].
The aim of our work was to validate our previous
observations in a longitudinal study and to demonstrate that
PT dysfunction occurs before the stage of albuminuria.
Moreover, we attempted to document the different patterns
of endothelial behaviour in two distinct vascular segments,
the kidney and the brain. In addition, we assessed the renal
and cerebral protective effects of pioglitazone versus glime-
piride, a sulphonylurea compound, in normoalbuminuric
patients with type 2 DM.
2. Subjects, materials and methods
A total of 68 normoalbuminuric type 2 DM patients attending
the Department of Diabetes and Metabolic Diseases were
enrolled in a prospective 1-year open-label randomized
controlled trial. Inclusion criteria consisted of long-standing
DM (>5 years), normoalbuminuria at the time of enrollment,
absence of microangiopathic complications, no chronic
kidney disease of non-diabetic origin, and poor glycaemic
control (HbA1c > 7%) with previous medication (stable thera-
py with metformin for at least 6 months), a fact which required
association of other antidiabetic agents. Exclusion criteria
were symptoms and/or history of cerebrovascular disease
(transient ischaemic attack, stroke), and micro/macroalbumi-
nuria.
Patients were randomly assigned to pioglitazone plus
metformin (group A: 12 males; 22 females) or glimepiride
plus metformin (group B: 13 males; 21 females). Group A
consisted of 34 patients treated with pioglitazone 30 mg/day
plus metformin 1700 mg/day. Group B consisted of 34 patients
treated with glimepiride 4 mg/day plus metformin 1700 mg/
day. Glimepiride is the sulfonylurea compound most utilized
in our country as second-step therapy in the treatment of
type2 DM. Patients in both groups were on angiotensin-
converting enzyme inhibitors or angiotensin-receptor block-
ers, and statins. The Hospital Ethics Committee approved the
protocol, and every patient provided written informed consent
before enrollment. In the flow-chart are provided data
concerning enrollment and outcomes of participants in the
study (Fig. 1).
All randomized patients were assessed at the initiation, at 6
months and by the end of the study concerning plasma
asymmetric dymethyl-arginine (ADMA), serum creatinine,
glomerular filtration rate (GFR), high sensitive C-reactive
protein (hsCRP), fibrinogen, glycaemia, glycated haemoglobin
(HbA1c), cholesterol, triglycerides, haemoglobin, urine albu-
min:creatinine ratio (UACR), urinary beta2-microglobulin, and
urinary alpha1-microglobulin. Serum and urinary biomarkers
were determined in specimens frozen at �80 8C and thawed
before assay. The laboratory staff who performed the
assessments were blinded to the medical treatment of the
patients enrolled in the study.
Patients scree ned (n= 124)
Excluded (n=46)
Not meeti ng incl usion criter ia (n=30)
Refused to participate (n=6)
Analyzed (n= 34)
Discontinued in terve ntion (n=3) (complained of weight gain, oedema) Lost to fol low-u p (n= 2) (refused to continue the study)
Assigned to pioglitazone+metformi n
(n=39)Received allocated interventi on
(n=39)
Discontinued int erventi on (n=4)(complained of weight gain) Lost to follow-up (n = 1) (refused to continue the study)
Assigned to gli mepiri de+m etfor min (n= 39)
Received allocate d interventi on (n= 39)
Analyzed (n= 34)
Allocation
Analysis
Follow-Up
Patientsrandomized (n=78)
Fig. 1 – Flow-chart concerning enrollment and outcomes of study participants.
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 224
Plasma ADMA was evaluated by the ELISA method with
K7828 ADMA ELISA (Immunodiagnostik AG, Bensheim,
Germany). The reference interval was 0.45 � 0.19 mmol/l.
The intra-assay precision was 6.2–10.5%, while the inter-assay
precision was 6.2–7.9%.
Alpha 1-microglobulin was evaluated in the second morning
urine specimen with N a1-Microglobulin Kit (Siemens Health-
care Diagnostics, Marburg, Germany) by means of particle-
enhanced immunonephelometry using the BN ProSpec Sys-
tem. The reference interval was 12 mg/l or 0.07–5 mg/g
creatinine. The intra-assay precision was 2.9–5.2% CV, while
the inter-assay precision was 7.4–13.2% CV. Alpha1-micro-
globulin is a marker of early proximal tubular dysfunction and
is used in several renal diseases, including DN. It is a low-
molecular-weight protein found in blood in a free or unbound
form, and in complexes with IgA and albumin; it is freely
filtered by the glomeruli and is reabsorbed by the PTs.
Beta 2-microglobulin was assessed in the second morning
urine specimen with N Latex b2-Microglobulin Kit (Siemens
Healthcare Diagnostics, Marburg, Germany) by means of
particle-enhanced immunonephelometry using the BN Pro-
Spec System. The reference interval was <0.2 mg/l. The
precision of the assay was measured using two different
concentrations of control material and three different con-
centrations each of pooled urine. The results yielded a total
coefficient of variation of 4.5–5.3% CV. Beta2-microglobulin is a
low-molecule weight protein filtered freely by the glomeruli
and reabsorbed almost entirely by the proximal tubule. It is
utilized as an indicator of impairment of glomerular filtration
and tubular reabsorption.
Albuminuria was measured by means of immunonephelo-
metry on the BNProSpec System, with N Antiserum to Human
Albumin (Siemens Healthcare Diagnostics, Marburg,
Germany). The reference interval was 30 mg/l or 17 mg/g
creatinine in males and 25 mg/g creatinine in females. The N
Antiserum to Human Albumin was evaluated for the assay of
urine on a BN System and yielded a Within-Run CV of 2.2% and
a total CV of 2.6% with a mean of 79 mg/l. The results (ten runs
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 2 25
with four determinations per run) were evaluated by analysis
of variance. Urine cultures were negative for bacteriuria in all
patients.
The cut-off values for glycaemia were �108 mg/dl,
HbA1c � 7%, cholesterol � 190 mg/dl, triglycerides � 150 mg/
dl, serum creatinine in males � 1.4 mg/dl and in fema-
les � 1.3 mg/dl, fibrinogen � 400 mg/dl, hsCRP � 1.2 mg/l,
GFR � 90 ml/min/1.73 m2, systolic blood pressure
(SBP) � 130 mmHg, diastolic blood pressure (DBP) � 80 mmHg
[12].
CKD was defined as either kidney damage (pathological
abnormalities or markers of damage including abnormalities
in blood or urine samples) or GFR < 60 ml/min/1.73 m2 for over
3 months (estimated GFR-MDRD4 equation formula: 186 � (SC
in mg/dl)�1.154 � (age in years)�0.203 � (0.742 if female) – Kidney
Disease Outcome Quality Initiative 2002-K/DOQI 2002; Kidney
Disease Improving Global Outcomes-KDIGO 2005) [13].
2.1. The cerebrovascular ultrasound measurements
Measurement of carotid intima-media thickness (IMT) was per-
formed in the common carotid arteries (CCAs), bilaterally, by
means of a high-resolution carotid artery ultrasound equip-
ment (MYLAB 50-ESAOTE, Italy), provided with PW and CW
Doppler, color and power Doppler and a linear transducer of 5–
7.5 MHz. All scanning throughout the study was performed by
two experienced neurologists at the same location using the
same equipment. The mean inter-observer difference was
0.002 (SD, 0.049) mm. The ultrasound beam was adjusted to
obtain longitudinal scans of the right and the left CCA to
assess two parallel echogenic lines corresponding to the
blood-intima and media-adventitia interface on the posterior
wall of the artery. The localized thickness of more than 2 mm
was excluded as plaque lesion. IMT values were averaged on
three determinations for both CCAs, and the greater value of
averaged IMT was considered significant for each measure-
ment (normal range values – up to 1 mm). The coefficient of
variation of the repeated IMT measurement was 1% [14]. Both
neurologists were blinded to medical treatment in the study
patients.
The pulsatility indices (PIs) and resistance indices (RIs) in the
internal carotid arteries (ICAs) were assessed by extracranial
Doppler ultrasound (ECD) with fast-Fourier spectral transfor-
mation analysis (Explorer CVC-Montpellier, France); a contin-
uous wave (4MHz-CW) probe was utilized for ECD exploration
of the ICAs, bilaterally, with regard to their patency, the
Gosling’s pulsatility index (PI) (systolic flow velocity � diasto-
diastolic flow velocity/mean flow velocity; normal range
values PI < 1), and the Pourcelot’s resistance index (RI) in
the ICAs, which evaluates the distensibility and compliance of
the sonorized arteries (systolic flow velocity � diastolic flow
velocity/systolic flow velocity) (normal range values RI < 0.7); a
pulsed-wave (2 MHz-PW) probe was utilized for transcranial
Doppler (TCD) ultrasound of the middle cerebral arteries
(MCAs), bilaterally, through the transtemporal window at a
depth of 50 mm. The patency of the vessels, the PIs and the RIs
were estimated for each MCA as described above [14].
The cerebrovascular reactivity (CVR) was evaluated in the
MCAs (with the same TCD equipment as described above) by
utilizing the breath-holding test (BHT). Both MCAs were
examined separately by evaluating the mean flow velocity
(MFV), systolic flow velocity (SFV) and diastolic flow velocity
(DFV) at rest (normal respiration). The CVR was assessed
during the BHT, such as follows: the patient breathes normally
by inhaling the air from the examination room, holding breath
thereafter for 20 s by the end of a normal inspiration. The MFV,
SFV and DFV were monitored for each patient in both MCAs at
rest (normal respiration – normocapnia), during the manoeu-
ver of breath-holding and by the end of the BHT, when the flow
velocities reached their maximal values (hypercapnia). The
CVR was estimated in relationship with the increase in the
MFV in both MCAs during hypercapnia as compared to the
basal velocity in cm/s and in % increase: normal CVR-%
increase >15%; diminished CVR-% increase = 5–10%;
exhausted CVR-% increase <5% [15].
2.2. Statistical analysis
Clinical, biological and cerebral haemodynamic indices are
presented as means, Standard Deviations (SD) and propor-
tions. The Wilcoxon matched-pairs signed-rank test was used
to compare the values within groups and Mann–Whitney U-
test to compare values between groups at baseline, at 6
months and 12 months follow-ups. A pooled univariate
regression analysis (Pearson’s correlation analysis) was
carried out to evaluate the significance of the relation between
continuous variables. Only significant variables yielded by
univariate regression analysis were introduced in the models
in multivariate regression analysis (Cox & Snell R square). The
relation between variables was assessed using R2 values. The P
values for all hypothesis tests were two-sided, and statistical
significance was set at P < 0.05. All analyses were conducted
with Stata 9.2 (Statacorp, Texas, USA).
3. Results
3.1. Characteristics of the study patients
Characteristics of the study patients (clinical, biological, and
cerebrovascular data) at baseline and during the follow-up
period are presented in Table 1.
3.2. Plasma ADMA, UACR, Urinary a1-, and urinary b2-microglobulin
The levels of ADMA at baseline were increased in a small
number of patients in both groups (17.64%), with a slight
increase by the end of the study in 29.41% of patients in group
B, but still within normal range. The results of the pooled
univariate regression analysis concerning ADMA are pre-
sented in Table 2 and of multivariate regression analysis in
Table 4.
The levels of urinary alpha 1-microglobulin were increased
at baseline in 26.47% of patients in group A and in 29.41% of
patients in group B. At 6 months, urinary alpha 1-micro-
globulin increased in 29.41% of patients in group A vs. 44.11%
in group B and there were no correlations between alpha 1-
microglobulin and plasma ADMA, HbA1c, duration of DM,
UACR, and GFR. Of the 79.42% patients in group B who were
Table 1 – Clinical and biological data of the patients studied.
Parameter Baseline 6 months 12 months
Group A Group B P Group A Group B P Group A Group B P
Nr. of subjects 34 34 34 34 34 34
Age (years) 56.88 � 6.44 58.82 � 7.78 0.17
DM-duration (years) 10 � 3.48 10.17 � 5.26 0.57
Body mass index 33.71 � 6.37 32.10 � 5.95 0.34 33.79 � 6 32.18 � 5.50 0.25 33.80 � 5.95 32.38 � 5.24 0.32
SBP (mmHg) 136.76 � 12.24 134.55 � 10.02 0.40 134.85 � 7.92 136.17 � 8.07 0.41 134.41 � 9.67 137.79 � 10.16 0.13
DBP (mmHg) 78.08 � 7.28 77.35 � 8.63 0.63 77.20 � 6.76 77.50 � 7.30 0.68 76.17 � 5.78 77.79 � 8.27 0.27
Hb (g/dl) 12.87 � 0.92 13.21 � 0.79 0.19 12.92 � 0.78 13.13 � 0.73 0.31 12.97 � 0.75 12.98 � 0.68 0.93
Serum creatinine (mg/dl) 0.97 � 0.17 0.94 � 0.16 0.31 0.95 � 0.16 1.01 � 0.20 0.26 0.95 � 0.17 1.20 � 0.21 <0.0001
GFR (ml/min/1.73 m2) 72.25 � 13.35 75.43 � 14.93 0.37 73.52 � 13.51 69.48 � 15.80 0.21 73.76 � 14.78 56.90 � 12.20 <0.0001
Glycaemia (mg/dl) 180.61 � 56.38 200.97 � 65.80 0.19 163.76 � 46.66 174.05 � 54.38 0.28 139.35 � 37.34 151.26 � 55.15 0.38
HbA1c (%) 7.7 � 0.81 7.49 � 1.03 0.21 7.27 � 0.69 7.39 � 0.97 0.94 6.85 � 0.73 7.19 � 1.12 0.40
Serum cholesterol (mg/dl) 208.82 � 39.11 224.76 � 43.41 0.10 209.94 � 36.22 216.88 � 20.73 0.12 209.82 � 28.98 212.91 � 29.04 0.73
Triglycerides (mg/dl) 160.82 � 55.14 165.70 � 53.76 0.65 146.58 � 39.41 152.88 � 39.42 0.44 135.97 � 26.86 146.52 � 30.72 0.13
Fibrinogen (mg/dl) 577.67 � 104.54 545.41 � 89.75 0.07 472.88 � 113.99 550.02 � 82.95 0.001 405.73 � 98.37 552.58 � 71.96 <0.0001
hsC-reactive protein (mg/dl) 14.55 � 2.42 13.87 � 1.62 0.19 8.40 � 2.28 14.13 � 1.86 <0.0001 5.37 � 2.17 14.06 � 1.93 <0.0001
Urine albumin/creat (mg/g) 17.59 � 3.99 16.56 � 3.89 0.23 17.03 � 3.60 18.42 � 4.30 0.23 16.91 � 4.80 24.22 � 10.59 0.002
Urine beta2microglob (mg/l) 0.14 � 0.04 0.14 � 0.04 0.91 0.12 � 0.05 0.15 � 0.05 0.007 0.12 � 0.06 0.18 � 0.05 <0.0001
Urine alpha1/creat (mg/g) 3.40 � 1.22 3.43 � 1.23 0.86 3.34 � 1.26 3.93 � 1.35 0.009 3.34 � 1.32 5.76 � 2.46 <0.0001
ADMA (mmol/l) 0.42 � 0.18 0.39 � 0.19 0.23 0.39 � 0.18 0.55 � 0.22 <0.0001 0.37 � 0.18 0.63 � 0.28 <0.0001
RI-ICA 0.86 � 0.03 0.85 � 0.03 0.19 0.81 � 0.04 0.85 � 0.04 0.0007 0.71 � 0.05 0.84 � 0.07 <0.0001
PI-ICA 1.27 � 0.14 1.23 � 0.12 0.07 1.10 � 0.15 1.24 � 0.12 0.0002 0.88 � 0.13 1.25 � 0.17 <0.0001
RI-MCA 0.85 � 0.05 0.85 � 0.05 0.56 0.73 � 0.07 0.85 � 0.06 <0.0001 0.62 � 0.07 0.82 � 0.15 <0.0001
PI-MCA 1.27 � 0.07 1.23 � 0.07 0.07 1.02 � 0.10 1.24 � 0.08 <0.0001 0.78 � 0.10 1.26 � 0.26 <0.0001
IMT-CCA 1.00 � 0.09 0.97 � 0.08 0.07 0.85 � 0.11 0.98 � 0.09 <0.0001 0.80 � 0.12 1.05 � 0.18 <0.0001
MFV-basal 41.52 � 5.24 43.97 � 5.69 0.06 45.44 � 5.41 42.41 � 5.75 0.028 50.71 � 6.23 41.36 � 5.62 <0.0001
MFV-BHT% 7.33 � 3.67 7.94 � 2.51 0.42 10.33 � 3.79 6.60 � 3.84 0.0001 13.21 � 4.28 7.20 � 4.90 <0.0001
DM, diabetes mellitus; SBP, systolic blood pressure; DBP, diastolic blood pressure; Hb, haemoglobin; GFR, glomerular filtration rate; HbA1c, glycated haemoglobin; ADMA, asymmetric dymethyl-
arginine; RI, resistance index (normal range values < 0.7); PI, pulsatility index (normal range values < 1); IMT, intima-media thickness (normal range values < 1 mm); CCA, common carotid artery; ICA,
internal carotid artery; MCA, middle cerebral artery (Mann–Whitney U-test; P < 0.05).
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rati
on
DM
0.0
66
�0.3
18
0.0
34
0.0
69
0.0
12
0.0
30
0.0
61
0.0
05
0.0
42
0.0
64
0.0
13
0.0
36
0.0
89
0.0
10
0.0
13
0.0
58
0.0
17
0.0
47
GFR
0.3
19
0.1
94
<0.0
01
0.2
98
�0.0
06
<0.0
01
0.3
01
�0.0
03
<0.0
01
0.2
70
�0.0
07
<0.0
01
0.3
26
�0.0
05
<0.0
01
0.3
07
�0.0
10
<0.0
01
Hb
A1c
0.0
98
�1.7
91
0.0
09
0.1
05
0.0
68
0.0
07
0.0
69
0.0
25
0.0
29
0.0
74
0.0
68
0.0
24
0.0
79
0.0
46
0.0
20
0.1
52
0.1
27
0.0
01
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.
Table 2 – Univariate regression analysis for ADMA.
Variable Parameter
ADMA
R2 Coef b P
hsCRP 0.364 0.033 <0.001
Fibrinogen 0.409 0.001 <0.001
Duration DM 0.251 0.030 <0.001
GFR 0.348 �0.010 <0.001
HbA1c 0.232 0.136 <0.001
Urine albumin/creat 0.005 0.002 0.539
Urine a1 microglob 0.016 0.015 0.296
Urine b2 microglob 0.006 0.356 0.510
ADMA, asymmetric dymethyl-arginine; hsCRP, high-sensitive C-
reactive protein; DM, diabetes mellitus; GFR, glomerular filtration
rate; HbA1c, glycated haemoglobin.
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 2 27
still normoalbuminuric at 6 months, 48.14% presented with
increased levels of urinary alpha1-microglobulin. By the end of
the study the levels of urinary alpha1-microglobulin were
increased in group A in 32.35% of patients vs. 61.76% of
patients in group B and correlated significantly with UACR
(R2 = 0.143; P = 0.001; b = 0.097) and GFR (R2 = 0.081; P = 0.01;
b = �0.042). It is worth pointing out that by the end of the
follow-up period, in group B, 47.05% of patients became
microalbuminuric vs. none of the patients in group A.
The levels of urinary beta2-microglobulin at baseline were
increased in 20.58% of patients in group A vs. 26.47% of
patients in group B. At 6 months, urinary beta 2-microglobulin
increased in 23.52% of patients in group A vs. 38.23% in group B
and there was no correlation with plasma ADMA, HbA1c,
duration of DM, UACR, and GFR. Of note, in group B, of the
79.42% patients who remained normoalbuminuric at 6
months, urinary beta2-microglobulin was increased in
40.74% of cases. At 12 months the levels of urinary beta2-
microglobulin increased in group A in 26.47% of patients vs.
55.88% in group B and correlated significantly with UACR
(R2 = 0.241; P = 0.0001; b = 0.003) and GFR (R2 = 0.064; P = 0.036;
b = �0.0009).
3.3. Cerebral ultrasound data
A pooled univariate regression analysis performed at 12
months between the cerebral haemodynamic indices and
the biological variables showed significant correlations be-
tween IMT-CCA, RI-ICA, PI-ICA, RI-MCA, PI-MCA and MFV-
BHT, and plasma ADMA, hsCRP, fibrinogen, duration of DM,
HbA1c, and GFR (Table 3). Independent variables were entered
into the models for multivariate regression analysis, the
results of which are presented in Table 4.
4. Discussion
The aim of our longitudinal study was to validate our previous
observations and to demonstrate that in type 2 DM, PT
dysfunction occurs before the stage of albuminuria in early
DN. Furthermore, we evaluated the different patterns of
endothelial dysfunction in two distinct vascular territories,
the kidney and the brain, both affected by microangiopathic
Table 4 – Multivariate regression analysis for the cerebral haemodynamic indices and ADMA.
Parameter Variable
Coef b P 95% CI F Prob > F R2
PI-MCA Constant 0.473 <0.001 0.365 to 0.582 62.51 <0.001 0.657
hsCRP 0.032 <0.001 0.020 to 0.043
ADMA 0.472 <0.001 0.262 to 0.682
RI-MCA Constant 0.452 <0.001 0.395 to 0.509 56.79 <0.001 0.636
hsCRP 0.012 <0.001 0.005 to 0.018
ADMA 0.302 <0.001 0.193 to 0.412
IMT-CCA Constant 0.581 <0.001 0.507 to 0.656 51.68 <0.001 0.613
hsCRP 0.021 <0.001 0.013 to 0.029
ADMA 0.268 <0.001 0.124 to 0.412
VMF-BHT% Constant 18.846 <0.001 16.693 to 20.999 41.23 <0.001 0.559
hsCRP �0.304 0.012 �0.538 to �0.069
ADMA �11.208 <0.001 �15.377 to �7.038
ADMA Constant 0.182 0.001 �0.178 to 0.544 33.32 <0.001 0.609
Fibrinogen 0.0009 <0.001 0.0005 to 0.0014
GFR �0.005 0.001 �0.008 to �0.002
DM-duration 0.019 0.001 0.009 to 0.029
hsCRP, high-sensitive C-reactive protein; DM, diabetes mellitus; GFR, glomerular filtration rate; HbA1c, glycated haemoglobin; ADMA,
asymmetric dimethyl-arginine; RI, resistance index; PI, pulsatility index; ICA, internal carotid artery; MCA, middle cerebral artery; IMT, intima-
media thickness; CCA, common carotid artery; MFV, mean flow velocity; BHT, breath-holding test; MFV-BHT%, percentage increase of MFV after
BHT as compared to basal MFV; 95% CI, confidence interval.
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 228
complications in the course of type 2 DM. The nephro- and
cerebroprotective effects of pioglitazone were also studied.
We found that PT dysfunction precedes glomerular
endothelial dysfunction and is a critical component in the
mechanisms of albuminuria in early DN. Moreover, glomeru-
lar endothelial dysfunction is dissociated from endothelial
dysfunction in the brain in normoalbuminuric patients with
type 2 DM. Pioglitazone delayed PT dysfunction and improved
cerebral vessels endothelial dysfunction, effects which were
unrelated to metabolic control.
4.1. PT dysfunction does not parallel endothelialdysfunction in early DN
Current mechanisms of proteinuria in the course of DM are
mainly explained by alterations in the podocyte biology and
glomerular endothelial dysfunction [16]. The classical concept
concerning albuminuria states that albuminuria is related to
the severity of diabetic glomerular lesions, but this correlation
is not strict in type 2 DM due to the fact that these lesions may
precede the onset of albuminuria [17]. Endothelial dysfunction
may also occur in patients with type 2 DM even when the
patients are still normoalbuminuric, a fact demonstrated by
markers of endothelial dysfunction which may be elevated
years before any evidence of microangiopathic complications
in both types of diabetes [18].
NO is synthesized by the vascular endothelium from the
amino acid L-arginine by constitutive and inducible NO
synthases and plays an important role in the maintenance
of vascular homeostasis. The endogenous L-arginine me-
tabolite, ADMA, inhibits cellular L-arginine uptake and NO-
synthase activity. Circulating ADMA is elevated in patients
with overt DN, even when the GFR is still within normal
range [19].
It has been stated that in both normo- and microalbumi-
nuric diabetic patients plasma ADMA levels have prognostic
implications for the transition to a more advanced stage of
albuminuria [20,21]. In our study, however, the levels of ADMA
at baseline and by the end of the study were increased in a
small number of patients in both groups, but still within
normal range. Plasma ADMA did not correlate with UACR and
the biomarkers of PT dysfunction, but only with the
parameters of inflammation, GFR, duration of DM, and HbA1c.
These results are in contrast to similar data found in
albuminuric patients with type 2 DM [22], allowing for the
assumption that an inflammatory state at the glomerular level
may be found in normoalbuminuric patients with type 2 DM.
It has recently been recognized that structural and
functional changes in the PT cell may be more important
than abnormalities in the glomerulus with regard to the
onset of early DN. Decreased peritubular capillary flow due
to impaired endothelial function related to even slightly
increased levels of plasma ADMA may induce tubulointer-
stitial ischemia and subsequent modifications in the func-
tion of PT cells [23]. In patients with early DN diagnosed
solely on PT abnormalities and markers of tubular dysfunc-
tion, the levels of ADMA may remain within normal range
[21], as we demonstrated in a previous study [9] and in the
present work.
The tubular theory concerning the mechanisms of albu-
minuria in early DN states that there is a reduction in the
retrieval pathway of albumin in the PT which precedes
glomerular damage [7]. Severe albumin leakage can be easily
identified by massive accumulation of albumin within PT
epithelial cells, thus leading to PT cells exposure to high
albumin loads and subsequent damage [24]. Several PT injury
biomarkers have been associated with normoalbuminuria and
changes in albuminuria in diabetic patients [25–28].
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 2 29
In our study, the levels of urinary alpha 1-microglobulin
increased significantly and progressively at 6 and 12 months in
group B vs. group A. This trend was paralleled by the increase
in the UACR at 12 months, as reported previously [8].
In a study performed in type 2 DM patients, among patients
with normoalbuminuria, 33.6% had raised urinary alpha 1-
microglobulin. Interestingly, in patients with normal urinary
alpha 1-microglobulin, 27.6% had albuminuria. It was con-
cluded that although urinary alpha 1-microglobulin and
albumin are related, one may be present in the absence of
the other in the course of early DN. The data provided by our
study is in contrast to the above-mentioned results obtained
by Hong et al., who found a strong correlation between urinary
alpha 1-microglobulin and HbA1c [27]. Our results, however,
are in keeping with those reported in type 1 DM patients [25],
as well as in type 2 diabetics with normoalbuminuria [8,9].
Early PT dysfunction in subjects with type 2 DM may be also
characterized by increased urinary beta2-microglobulin excre-
tion [26]. In our study, the levels of urinary beta2-microglobulin
followed the same pattern as those of urinary alpha 1-
microglobulin. These facts are highly indicative of PT dysfunc-
tion which occurs before the stage of microalbuminuria.
It is worth underlining that in our study urinary alpha 1-
microglobulin and urinary beta2-microglobulin did not corre-
late with plasma ADMA during the follow-up period, thus
pointing to the fact that glomerular endothelial dysfunction
does not parallel PT dysfunction in type 2 DM, similarly to
previous results [9].
4.2. Pioglitazone delays PT dysfunction innormoalbuminuric patients with type 2 DM
Thiazolidinediones exert nephroprotective effects in patients
with type 2 DM by significantly decreasing UACR, markers of
inflammation, and endothelial dysfunction [11]. Pioglitazone
displays its protective effects at the level of the endothelium
through an increase in NO production and availability by
decreasing systemic ADMA. This mechanism constitutes a
direct action of pioglitazone on PT cells [29].
In our patients treated with pioglitazone and metformin,
plasma ADMA presented roughly stable levels by the end of
the follow-up period. A small number of patients had
increased urinary a1-microglobulin and urinary b2-micro-
globulin, while all patients remained normoalbuminuric, as
we demonstrated previously for rosiglitazone [8]. By contrast,
in the group treated with glimepiride and metformin, despite
the fact that plasma ADMA showed only slight increases, the
PT biomarkers displayed increased levels in a significant
number of patients, of which 47.05% became microalbumi-
nuric. This data is in keeping with the results reported by
Scherntharner et al., who demonstrated that UACR decreased
by 19% in patients treated with pioglitazone versus patients
treated with metformin, in a 1-year, randomized controlled
trial [30]. Since in our study there were no changes in HbA1c
levels in both groups, we assume that the renal effects of
pioglitazone were independent of glycaemic control.
Another important issue is the decline of GFR recorded in
normoalbuminuric patients with type 1 DM [31,32] and type 2
DM, with early kidney and vascular damage [33,34]. In our
study, the GFR decreased by the end of the follow-up period in
the group treated with glimepiride and metformin despite the
fact that only a small number of patients developed micro-
albuminuria, while the others remained normoalbuminuric. It
has been shown that reduced GFR may correlate with the
degree of albuminuria within the normal range [8,35].
Presumably, in our patients PT dysfunction and not endothe-
lial dysfunction is responsible for the decline in GFR, as it
derives from the correlations between the PT dysfunction
biomarkers and GFR.
4.3. Endothelial dysfunction plays a pivotal role in thebrain vasculature and is improved by pioglitazone
In view of our results which lack the evidence of glomerular
endothelial dysfunction, we assumed that in the course of
type 2 DM there may be various patterns of endothelial
behaviour in different vascular territories. In order to clarify
this hypothesis, we evaluated endothelial dysfunction in the
brain vasculature, which shares structural and functional
similarities with the renal vascularization.
4.4. Carotid artery intima-media thickness
Carotid artery IMT is a parameter that evaluates atheroscle-
rotic remodeling of cerebral vessels and its increased values
reflect vascular stiffness and are considered a surrogate
marker for cardiovascular and cerebrovascular risk [36].
ADMA has a very narrow range of normal concentrations.
Even when these concentrations are only slightly increased,
ADMA is associated with subclinical atherosclerosis in the
carotid artery and with high cardiovascular and cerebrovas-
cular risk in both type 1 and type 2 DM [37].
In our study, CCA-IMT remained unmodified in the
pioglitazone-metformin-treated patients, while in the glime-
piride-metformin-treated group this parameter increased by
the end of the follow-up period. Recent trials concerning the
vasculoprotective effects of pioglitazone have substantiated the
impact of pioglitazone on reduction of carotid IMT [10,38,39].
Pioglitazone slowed progression of IMT vs. glimepiride in type 2
DM patients, effects which were unrelated to the HbA1c levels
[38,40]. In our patients, similar data are supported by the results
of multivariate regression analysis which show a lack of
correlation between CCA-IMT and HbA1c.
Pioglitazone-induced reduction of IMT was paralleled by
decrease in parameters of inflammation, as shown in other
studies [41]. There has been demonstrated a negative
correlation between the carotid IMT and eGFR [42] and this
observation was also valid in normoalbuminuric patients with
type 2 DM [35], as was the case with our patients too.
In a study performed in normotensive type 2 DM patients it
has been shown that pioglitazone, but not glibenclamide or
voglibose, was effective in reducing UACR, IMT, and pulse-
wave velocity [43]. Our results are consistent with the above-
mentioned study, also showing concomitant nephro- and
cerebroprotective effects of pioglitazone.
4.5. Diabetic cerebral microangiopathy
Diabetic cerebral microangiopathy, of which the underlying
pathological condition is arteriosclerosis, increases the resis-
d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 9 4 ( 2 0 1 1 ) 2 2 – 3 230
tance of cerebral vessels. ADMA may increase vascular
stiffness and decrease cerebral perfusion in diabetic patients
[44]. Plasma ADMA levels coordinate microangiopathy-related
cerebral damage in normoalbuminuric diabetic patients [5,45].
The PIs and RIs reflect the vascular resistance distal to the
examined artery. Thus, impairment of the small intracranial
perforating arteries, mainly involved in diabetic cerebral
microangiopathy, may be evaluated by assessment of the
PIs and RIs in the carotid, vertebrobasilar and MCAs [8,9,46–
48]. Diabetic cerebral microangiopathy is related to DN [48],
but it may also occur in normoalbuminuric patients with type
2 DM [8,9].
In our patients the PIs and RIs in the ICAs and MCAs
correlated with plasma ADMA, duration of DM, HbA1c, GFR,
and parameters of inflammation, data which is consistent
with previous studies [8,9,47,48] The PIs and RIs in the ICAs
and MCAs were increased in both groups at the beginning of
the study, but decreased significantly in the pioglitazone-
metformin group vs. the glimepiride-metformin group by the
end of the study, data comparable with the results provided
for pioglitazone, which decreased cerebrovascular resistance
in patients with type 2 DM [49] and for rosiglitazone [8].
4.6. Cerebrovascular reactivity
Cerebrovascular reactivity is a haemodynamic parameter
which represents normal cerebral artery blood flow velocity
increase in response to a vasodilatory stimulus. A decreased
CVR is indicative of preexisting vasodilation, which reflects a
reduced reserve capacity of cerebral autoregulation [15]. The
cerebral vasodilatory capacity is impaired in patients with
type 2 DM [9,50–52].
It has been demonstrated that NO is a critical regulator of
brain perfusion, a fact supported by the presence of NO
synthase in the brain and cerebral arteries [44,45]. Long-term
diabetes is associated with endothelial dysfunction which
results in a decreased release of endothelial NO and
consequently affects the ability of the cerebral vessels to
relax efficiently. CO2 vasoreactivity of cerebral vasculature is
impaired in patients with endothelial dysfunction and may
serve as a surrogate of cerebrovascular endothelial function
[53]. Due to the fact that ADMA is produced in relatively high
amounts in brain, it may be an important endogenous
modulator of cerebral vasculature tone under resting condi-
tions and in response to vasoreactive stimuli, including
hypercapnia [44].
Our study reveals decreased levels of the basal flow velocity
in the MCAs in both groups and an inappropriate increase in
the mean flow velocity during hypercapnia induced by the
BHT in normoalbuminuric patients with type 2 DM.
MFVs after BHT in the MCAs correlated with ADMA, a fact
which supports the intervention of ADMA in the impairment
of endothelial-dependent vasodilatation of cerebral vessels
during hypercapnia. Also, the impaired CVR correlated with
duration of DM, HbA1c, parameters of inflammation, and GFR,
as was reported previously [9,46,50].
Diabetic nephropathy is considered an indicator of cerebral
microangiopathy, as has been demonstrated in the studies
performed by utilizing the BHT in patients with type 1 DM [54],
and with type 2 DM [52]. In our study, however, impairment in
the CVR occurred in normoalbuminuric patients with type 2
DM, thus showing that the endothelium in the cerebral vessels
has a different behaviour in the course of long-standing DM, as
compared to the glomerular endothelium [9]. Pioglitazone
significantly improved the CVR in our patients, data which is
in keeping with other studies on vascular reactivity performed
in normoalbuminuric patients with type 2 DM [55].
It is worth underlining that the cerebrovascular modifica-
tions shown in our patients with type 2 DM and normoalbu-
minuria (mean level of UACR in the range high-normal of
17 mg/g) are consistent with the data published in other
studies which show that in both diabetic and non-diabetic
populations, a small increase in the albumin excretion rate
even in the ‘normoalbuminuric’ or ‘submicroalbuminuric’
range may act as a major independent risk factor for
cardiovascular disease [56,57].
Our study has several limitations. First, the small sample-
size of the patients studied decreases the statistical power of
the study. Second, blood CO2 levels were not assessed during
the BHT in order to increase the accuracy of the measure-
ments. Finally, the study was not double-blinded, except for
the laboratory staff and the neurologists who performed the
neurosonological evaluations who were blinded for the
medication administered.
In conclusion, inpatients with type 2 DM, PT dysfunction may
occur before the stage of albuminuria. It is therefore possible that
in early DN, sequentially, PT dysfunction precedes glomerular
endothelial dysfunction. Furthermore, glomerular endothelial
dysfunction is dissociated from endothelial dysfunction in the
brain, thus explaining why patients with type 2 DM may develop
cerebral vessels structural and functional modifications while
remaining normoalbuminuric. It may be assumed that in the
course of type 2 DM there are distinct endothelial territories, in
which endothelial dysfunction occurs within different time
frames. Moreover, it appears that endothelial dysfunction in the
cerebral vessels occurs before the endothelial impairment at the
glomerular level. A note of caution, however, is mandatory
before reaching a final conclusion due to the fact that the lack of
correlations between parameters of endothelial dysfunction and
of PT dysfunction does not necessarily prove causality, but also
does not imply independent mechanisms of albuminuria.
Pioglitazone delays PT dysfunction and improves cerebral
vessels endothelial dysfunction in normoalbuminuric patients
with type 2 DM. These nephro- and neuroprotective effects of
pioglitazone are beyond glycaemic control. Further studies on
larger cohorts and a longer follow-up period are warranted in
order to support these data.
Conflict of interest
There are no conflicts of interest.
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