Pioglitazone delays proximal tubule dysfunction and improves cerebral vessel endothelial dysfunction...

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: [email protected], [email protected]

(M. Petrica), [email protected] (C.D. Jianu), [email protected] ([email protected] (V. Dumitrascu), [email protected] (C. Ia(S. Giju), [email protected] (C. Gluhovschi), [email protected](G. Bozdog), [email protected] (O. Milas), [email protected]

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), [email protected] (A. Vlad), [email protected]. Gluhovschi), [email protected] (F. Gadalean),nculescu), [email protected] (C. Firescu), [email protected] (F. Bob), [email protected] (S. Velciov), [email protected]

(R. Marian), [email protected] (S. Ursoniu).

reserved.

http://dx.doi.org/10.1016/j.diabres.2011.05.032

mailto:[email protected]


















http://dx.doi.org/10.1016/j.diabres.2011.05.032

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.


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


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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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

CR

P,

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ea

ctiv

ep

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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.


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.


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].


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-


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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Pioglitazone delays proximal tubule dysfunction and improves cerebral vessel endothelial dysfunction...

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