Alberto Giannoni

Michele Emdin

Roberta Poletti

Francesca Bramanti

Concetta Prontera

Massimo Piepoli

MD Claudio Passino
email: passino@ifc.cnr.it

neurohormonal derangement, Cheyne-Stokes respiration and arrhythmias

Keywords: chemoreflex, norepinephrine, natriuretic peptides, heart failure, Cheyne-Stokes respiration, arrhythmias

INTRODUCTION

Despite the advances in the treatment of chronic heart failure (HF), its epidemiological relevance is still increasing 1 . Ventricular remodelling, symptoms and final outcome are highly influenced by neurohormonal derangement 2 , characterized by the activation of the sympathetic 3 and the renin-angiotensin-aldosterone system 4 , and by the enhanced secretion of natriuretic peptides, whose plasma levels hold diagnostic and prognostic value 5 . Increased sympathetic activity, one of the major determinants of the evolution of the disease and of lifethreatening events, is elicited by changes in autonomic afferent feed-back, via baroreceptor desensitisation 6 , ergoreceptor 7 , and chemoreceptor sensitisation 8 .

In particular, increased chemosensitivity has been also associated with several markers of worse clinical status and prognosis in HF patients, such as ventilatory response to exercise 9 or night-time Cheyne-Stokes respiration (CSR) 10, 11 . Recently, increased peripheral chemosensitivity has been pointed out as independent prognostic factor in HF 12 .

However, the importance of increased sensitivity to either hypoxia or hypercapnia and the possible role of their combination in the pathophysiology of heart failure have not been fully investigated. Moreover, there are few data on the impact of chemosensitivity on other neurohormonal axes than the adrenergic one, including renin-angiotensin-aldosterone and cardiac natriuretic peptide systems.

Our hypothesis is that chemosensitivity might play a central role in influencing neurohormonal activation and the overall clinical picture of HF patients, even on optimal pharmacological treatment.

The purpose of our study was to evaluate in a prospective cohort of patients with chronic HF (optimally treated by a complete neuro-hormonal antagonist drug approach) the actual prevalence of enhanced chemosensitivity to both hypoxia and hypercapnia and its significance with regard to neuro-hormonal activation, including cardiac endocrine function, occurrence of CSR, clinical status, and response to exercise.

METHODS

Subjects and study design 3

From 2005 to 2006 we screened 84 consecutive HF patients coming from our outpatient clinic, with echocardiographic evidence of impaired left ventricular systolic function (ejection fraction -EF-<45%). Exclusion criteria were New York Heart Association (NYHA) class IV, acute coronary syndrome within 6 months before examination, severe renal dysfunction (i.e creatinine clearance < 35 ml•min -1 ), pulmonary disease (vital capacity and total lung capacity < 50% of predicted value, FEV1 < 50% of predicted value, and FEV1/FVC < 70%), obstructive sleep apnoea syndrome (as determined by a preliminary polysomnography), and treatment with morphine or derivates, theophylline, oxygen, benzodiazepines or acetazolamide. Sixty patients matched these criteria and were enrolled in the study (Table 1): all were on stable (i.e., > 1 month) optimal pharmacological treatment, with restriction of water-sodium intake. The study of the World Medical Association, and has been approved by the Institutional Ethics Committee;

design
informed consent was obtained from all subjects enrolled in the study.

Chemosensitivity evaluation

Chemoceptive sensitivity was assessed using the rebreathing technique 13, 14 to assess.

Subjects were examined in standardized conditions, in a quiet room at a confortable temperature, while seated and connected to a rebreathing circuit through a mouthpiece. They were not allowed to smoke or to drink alcohol or caffeine-containing beverages in the 12 h preceding the study.

Electrocardiogram, airway flow, and respiratory gases were recorded continuously through a breath-by-breath gas analyzer (Vmax, Sensormedics, Yorba Linda, CA, USA), and oxygen

Day-time cardio-respiratory recording and polysomnography

All subjects underwent a 20-minute recording while awake and spontaneously breathing in a supine position, as previously described 16 . We recorded a two lead electrocardiogram, chest

RESULTS

On the whole, 60% of HF patients showed increased hypoxic and/or hypercapnic chemosensitivity, when compared to controls. Isolated increased HVR and HCVR were found in 13% in 20% of patients, respectively, whereas a combined increase in both HVR and HCVR was present in 27% of the patients (Table 2).

Patients with increased chemosensitivity to hypoxia and/or hypercapnia did not differ as concerns to age, gender, BMI, left ventricular dimensions and function, renal and pulmonary function and arterial gas analysis values, as compared to patients with normal chemosensitivity (Table 2). Enhanced HVR and/or HCVR were associated with worse clinical severity, as expressed by percentage of patients in NYHA class III (Table 2).

Chemosensitivity and neurohormonal activation

Patients with combined enhancement of HVR and HCVR, as compared with patients with normal chemosensitivity, showed a significant increase of plasma norepinephrine concentration, and the highest plasma levels of BNP and NT-proBNP, despite a similar degree of left ventricular systolic dysfunction. No significant differences were found with regard to cortisol, PRA, aldosterone and thyroid profile (Table 3).

A significant relationship was found between HVR or HCVR, plasma norepinephrine level (R 0.40 and R 0.37 respectively, both P< 0.01) and B-type natriuretic peptide expression (BNP: both R 0.40, P<0.01; NT-proBNP: R 0.37 and R 0.41 respectively, both P<0.01) (Figure 1).

Chemosensitivity and Cheyne-Stokes respiration

Day-time CSR was found in 17 patients (28%). All HF patients with preserved chemoceptive sensitivity had a normal breathing pattern, whereas day-time CSR occurrence increased progressively when enhanced HVR or HCVR were present, and increased significantly (P < 0.001) when combined (Figure 2). At multivariate analysis HCVR and plasma BNP level resulted the only independent predictors of diurnal CSR (HCVR: = 9.2, BNP: = 3. 3).

As regards nocturnal CSR, patients with combined enhancement of HVR and HCVR presented the highest AHI, at polysomnography (Figure 2). Moreover, a significant positive correlation between HVR/HCVR and AHI was found (R= 0.57 and R=0.58, respectively, both P< 0.001).

Finally, HCVR ( = 4.4, p< 0.05) was the only independent predictor of night-time CSR (defined by an AHI> 10), among all univariate predictors (HVR, HCVR, VE/VCO 2 slope, norepinephrine, BNP and NT-proBNP), with significant findings at ROC analysis (AUC 0.87 ± 0.04, cut-off value 0.76 l•min -1 •mmHg -1 , sensitivity 81.1%, specificity 73.2%, P< 0.001).

Chemosensitivity and response to exercise

Patients with enhanced chemosensitivity both to hypoxia and hypercapnia showed lower ventilatory efficiency, as expressed by VE/VCO 2 slope at cardiopulmonary test, with a non significant trend towards lower peak VO 2 /kg and maximum exercise workload (Table 2).

Moreover, the degree of ventilatory inefficiency was related to the level of chemosensitivity to both hypoxia (R= 0.42, P< 0.01) and hypercapnia (R= 0.50, P < 0.001) (Figure 4).

Chemosensitivity, arrhythmias and heart rate variability

Fifteen patients (25%) were on chronic atrial fibrillation at 24 h electrocardiographic recording, while 25 (42%) presented at least one episode of nonsustained ventricular tachycardia (more than or equal to three consecutive ventricular complexes, at a rate of more than 100 beats•min -1 , lasting for less than 30 s). Patients with a combined enhancement of chemosensitivity to hypoxia and hypercapnia showed a higher incidence of both atrial fibrillation (P < 0.01) and nonsustained ventricular tachycardia (P < 0.001), when compared to patients with normal chemosensitivity (Table 3), in spite of similar pharmacological treatment. 8

Furthermore, we also analysed heart rate variability in patients in sinus rhythm (n° 34): patients with combined enhancement of HVR and HCVR presented a significant reduction of SDANN, among time domain heart rate variability, with a non significant trend for SDRR, likely due to the small sample size (as shown in table 3). There were no differences among groups with regard to both RMSSD and pNN50 (data not shown).

DISCUSSION

Our study shows that increased chemosensitivity is frequent in mild to moderate chronic HF, regardless of the optimized medical treatment (including beta-blockade, anti-aldosterone drugs, and ACE/Angiotensin II inhibition). Moreover, increased chemosensitivity (particularly when combined to both hypoxia and hypercapnia) enhances sympathetic activation and is associated with depressed heart rate variability and augmented plasma concentration of BNP and NT-proBNP. Finally, it is associated with the occurrence of respiratory abnormalities, such as altered ventilatory response to exercise, either day-time or night-time CSR, with increased incidence of atrial fibrillation and ventricular arrhythmias and worse clinical status, independently of the degree of left ventricular systolic dysfunction.

Isolated or combined enhancement of chemosensitivity to hypoxia and hypercapnia are present in 60% of our patients, in spite of optimal treatment. When considered separately, the overall prevalence of increased chemosensitivity to hypoxia (40%) is similar to that previously reported 12 . On the other hand, increased chemosensitivity to hypercapnia occurred less frequently in our series (47%), if compared with previous observations (76%) in patients with mild systolic heart failure 18 . This might be explained by a blunting effect of beta-blocker administration (more frequent in our series: 92% vs 17% of patients) on the sympathetic efferent arm, with a possible prevalent effect on central chemoreflex.

In regard to neuro-hormonal derangement, our findings support the hypothesis that chronic To our knowledge, this is the first study which has evidenced the effect of chemoreflex on cardiac endocrine function in humans. We found that patients with combined enhancement of chemosensitivity were characterized by higher plasma levels of B-type natriuretic peptides, as compared to patients with normal chemosensitivity, despite a similar degree of left ventricular systolic dysfunction. In this subset, beyond haemodynamic stress, the production and secretion of natriuretic peptides could be elicited by a higher sympathetic drive and chronic periodical occurrence of hypoxia during CSR 24, 25 , two recognized additional stimuli for the release of natriuretic peptides 26 . Plasma concentration of B-type natriuretic peptides, would be confirmed not merely as a marker of myocardial dysfunction, but rather as an index of overall neuro-hormonal activation, contributing to its diagnostic and prognostic value in HF. support this hypothesis, extending this concept to the day-time period. Indeed, all patients with normal chemosensitivity presented a normal breathing pattern, whereas there was a progressive increase in CSR occurrence from patients with isolated to those with combined chemosensitivity enhancement.

At multivariate analysis, increased chemosensitivity to hypercapnia was an independent predictor of both day-time and night-time CSR, suggesting that CO 2 changes could be more relevant than O 2 variations for the genesis of CSR, as previously proposed 38, 39 . This provides further support to the concept that the two entities share a common pathophysiological basis. BNP level resulted an independent predictor only for day-time CSR, suggesting a stronger influence of haemodynamic or neuro-hormonal factors during wakefulness.

Combined enhancement of hypoxic and hypercapnic ventilatory response was associated with an increased ventilatory response to exercise, as expressed by the VE/VCO 2 slope, a recognized 10 prognostic marker in HF 40, 41 . The level of chemosensitivity activation was inversely related to ventilatory efficiency, thus confirming the powerful influence of chemoreceptors on ventilation during exercise 9, 41, 42 . As a matter of fact, patients with altered chemoreflex presented a worse clinical status, as expressed by higher NYHA class, independently from the degree of left ventricular dysfunction. The lack of a significant association between chemoreflex and peak-VO 2 in this series suggests a secondary role of chemoreflex on functional capacity on effort, mainly dependent on altered haemodynamics 43 and loss of skeletal muscle mass and function 44 .

Finally, the combined enhancement of HVR and HCVR was associated with a higher incidence of both chronic atrial fibrillation and nonsustained ventricular tachycardias at 24 h electrocardiographic recording, likely to be related to the increased sympathetic tone caused by the reflex alteration, supporting previous observations 45 .

In conclusion, despite optimal pharmacological treatment, increased chemosensitivity to hypoxia and hypercapnia influences several pathophysiological pathways associated with disease

  included a standard clinical evaluation and: a) the detection of chemosensitivity to hypoxia and to hypercapnia, by assessment of the individual hypoxic-normocapnic (HVR) and normoxic-hypercapnic ventilatory response (HCVR); b) neuro-hormonal evaluation; c) echocardiography; d) arterial blood gas analysis; e) cardio-pulmonary exercise testing; g) 24hour electrocardiographic recording; h) 20-minute day-time polygraphy and nocturnal polisonnography for CSR assessment. The entire protocol was completed for each patient within 3 days. The investigation was carried out in accordance with the Declaration of Helsinki (2000)

  ) and ventilatory efficiency (slope of the ventilation versus carbon dioxide production relation in its linear part, VE/VCO 2 ) were determined. All CPTs and echocardiographic studies were performed by a same physician blinded to the blood sampling results. The echocardiographic studies were performed by the same physician. Twenty-four hour electrocardiographic recording was obtained by a three-lead (precordial, posterior, inferior leads) digital system (Elamedical, France). In patients with sinus rhythm, from 24-hour electrocardiographic recordings we also computed 24-h average values of normal RR intervals (RR), standard deviation of all RR (SDNN), standard deviation of 5 min mean values of RR (SDANN), square root of the mean of the sum of the squares of differences between adjacent RR (RMSSD), and the number of adjacent RR differing by more than 50 ms, as percent of the total number of RR (pNN50).

  wall and abdominal movements by electrical inductance, oronasal airflow by nasal pressure transducers, beat-to-beat blood pressure (Colin® tonometry, San Antonio, TX, USA), SaO 2 (Pulse Oxymeter Pulsox-7, Minolta®) and end-tidal pressure of the CO 2 signal (Cosmoplus®, Novametrics). All patients also underwent nocturnal continuous polygraphic recording by conventional polysomnography (PSG, E-series 2, Compumedics) within the same day of the short-term recording. An episode of apnoea was defined as the cessation of inspiratory airflow for at least 10 seconds, whereas a hypopnoea was defined as a reduction in airflow (> 50% of tidal volume) lasting 10 seconds or more and associated with at least a 4 percent decrease in arterial oxyhemoglobin saturation 17 . Apnoea and hypopnoea were considered as central or obstructive by the absence or presence of rib-cage and abdominal excursions, respectively. As regards night-time poligraphy, the severity of apnoeas was quantified by means of the apnoeahypopnoea index (AHI = the number of episodes of apnoea and hypopnoea per hour, cut-off for diagnosis of CSR = 10).Statistical analysisStatistical analysis was performed by the SPSS 13.0 program (1989-2004, LEAD technologies Inc., USA). Norepinephrine, aldosterone, plasma renin activity, BNP and NT-proBNP were logarithmically transformed to correct for a skewed distribution. Mean differences among groups were evaluated through analysis of variance. Discrete variables where compared by chi-square test with Yate's correction, or Fisher's exact test when appropriate. Post hoc testing was performed using the Bonferroni correction. The Spearman rank correlation was used to determine the direct relationship between different numerical variables. The predictive power of 6 a variable was quantified in terms of the area under the receiver operating curve (ROC) and the statistical significance of AUC difference from that of the line of "no information" was evaluated by Mann-Whitney U-Statistics. Multiple logistic regression analysis was employed in order to evaluate the influence of different variables on CSR occurrence. Values are presented as mean S.D.; P value < 0.05 was considered significant.

  enhancement of chemosensitivity might play a central role in sustaining adrenergic activation, possibly by the direct stimulation of sympathetic centers from chemoreceptors 8,19 , or by inducing CSR-related periodic hypoxemia 20, 21 . A role of chemoreflex in eliciting adrenergic activation in HF 22 , is confirmed by its persistent action on the sympathetic axis in patients following cardiac transplant 23 . Furthermore, enhanced chemosensitivity seems also to be associated with abnormal heart rate variability, indicating an autonomic imbalance at sinus node level.

  The observation made byCheyne and Stokes two centuries ago on periodic breathing at daytime in awake HF patients 27, 28 , has been recently confirmed and associated with sleep-time respiratory abnormalities 29, 30 . Day-time 31, 32 and night-time CSR 33, 34 have been associated with poor prognosis in HF patients. Increased chemosensitivity, in particular to hypercapnia, has been suggested to play a central pathophysiological role in the onset CSR 10, 11, 35, 36 , together with altered haemodynamics (or delayed circulatory time) 37 . Our results

  Relationship between enhanced chemosensitivity to hypoxia (HVR) and to hypercapnia (HCVR) with norepinephrine (panel (a) and (b), respectively) and with B-type natriuretic peptide level (BNP, panel (c) and (d), respectively). The graph represents all 60 subjects studied.

Figure 2 Figure 3

 23 Figure 2 Prevalence of diurnal Cheyne-Stokes respiration at short-term polygraphy (CSR, left Y axis) and nocturnal apnoea-hypopnoea index at polisomnography (AHI, right Y axis) among patients with preserved chemoreflex, with isolated or combined enhanced chemosensitivity to hypoxia (HVR) and hypercapnia (HCVR). * and †= p<0.001 vs normal chemoreflex

Figure 4

 4 Figure 4Relationship between enhanced chemosensitivity to hypoxia (HVR) and to hypercapnia (HCVR) with peak oxygen uptake (VO 2/kg , panel (a) and (b), respectively) and with VE/VCO 2 slope (panel (c) and (d), respectively). The graph represents all 60 subjects studied.

Neuro-hormonal assay, cardio-pulmonary exercise test, echocardiographic study and 24- hour eletrocardiographic recording.

	value, by passing a portion of the expired air into a scrubbing circuit before returning it to a 5l
	rebreathing bag. Conversely, during the progressive normoxic hypercapnic trial (from resting
	end-tidal CO 2 values until 50 mmHg or an increase 10 mmHg from the basal values, according
	to individual tolerance), inspired partial pressure of O 2 was kept at baseline value by adding
	oxygen to the circuit. The two trials were performed in a random order. All signals were
	digitized on-line (National Instruments, USA, 500 sample•s -1 ) and analyzed to derive respiratory
	rate, breath-to-breath tidal volume (Vt), and minute ventilation (VE), as well as SaO 2 and end-
	tidal pressure of CO 2 .
	HVR was expressed by the linear regression slope between minute ventilation and SaO 2 ,
	during a hypoxic-normocapnic trial. HCVR was expressed by the linear regression slope
	between minute ventilation and end-tidal pressure of CO 2 , during the hypercapnic-normoxic
	trial. The average HVR values obtained in a group of 12 age-and gender-matched healthy
	subjects (Table 1) were 0.35	0.21 l•min -1 •%SaO 2 -1 , and HCVR values 0.31	0.24 l•min -
	1 •mmHg -1 (mean S.D.). As cut-off value for defining increased chemosensitivity, we considered
	0.77 l•min -1 •%SaO 2 -1 as to HVR and 0.79 l•min -1 •mmHg -1 as to HCVR (i.e. two-fold the S.D.
	value of the control group).
	Plasma BNP, catecholamines, aldosterone level and renin activity, and thyroid profile were
	assayed as described in detail elsewhere 15 ; NT-proBNP was measured with an automated
	electrochemiluminescent immunoassay (Roche diagnostics, Germany). Patients underwent a
	symptom-limited cardio-pulmonary exercise test on a bicycle ergometer according to a ramp
	protocol with increments of 10 W•min -1 (Vmax, Sensormedics, Yorba Linda, CA, USA). Peak
	oxygen uptake (VO 2 , the highest value at peak-exercise, over a 20-second average
	saturation (Masimo SET Radical, Pulse Oximeter). A 4-minute baseline recording was
	performed during spontaneous breathing. The mean SaO 2 and CO 2 -et during this recording were
	assumed as subject resting values. During the progressive isocapnic hypoxia trial (from resting
	SaO 2 values to 70-80%, according to individual tolerance), end-tidal CO 2 was kept at baseline

  5, P< 0.05) among all univariate predictors (HVR, HCVR, VE/VCO 2 slope, norepinephrine, BNP and NT-proBNP). The strong ability of chemosensitivity to hypercapnia and BNP plasma level in predicting CSR occurrence was confirmed by ROC analysis (HCVR: AUC 0.92 ± 0.04, cut-off value 0.88 l•min -1 •mmHg -1 , sensitivity 88.2%, specificity 83.7%; BNP: AUC 0.86 ± 0.05, cut-off value 169 ng•l -1 , sensitivity 94.1%, specificity 70.4%, both P< 0.001; figure

  progression in HF, such as neuro-hormonal activation, control of respiration at rest and during effort, and ventricular arrhythmias. Hence, we suggest that the assessment of chemoreflex sensitivity may contribute to the diagnostic definition of high risk HF patients 12 and that treating abnormal chemoreflex, either by optimization of conventional treatment or by specific intervention 42, 46 , could be considered a novel therapeutic target in heart failure. 8. Narkiewicz, K., Pesek, C.A., van de Borne, P.J., Kato, M., Somers, V.K. (1999) Enhanced sympathetic and ventilatory responses to central chemoreflex activation in heart failure.

	Clinical Science Immediate Publication. Published on 26 Oct 2007 as manuscript CS20070292
	Circulation. 100, 262-267 9. Chua, T.P., Clark, A.L., Amadi, A.A., Coats, A.J. (1996) The relationship between chemosensitivity and the ventilatory response to exercise in chronic heart failure. J Am Coll Cardiol. 27, 650-657 10. Javaheri, S. A mechanism of central sleep apnea in patients with heart failure. (1999) N Engl J Med. 341, 949-954 11. Solin, P., Roebuck, T., Johns, D.P, Walters, E.H., Naughton, M.T. (2000) Peripheral and central ventilatory responses in central sleep apnea with and without congestive heart failure. Am J Respir Crit Care Med. 162, 2194-2200 12. Ponikowski, P., Chua, T.P., Anker, S.D., et al. (2001) Peripheral chemoreceptor hypersensitivity: an ominous sign in patients with chronic heart failure. Circulation.104, 544-549 13. Read, DJ. (1967) A clinical method for assessing the ventilatory response to carbon dioxide. Australas Ann Med. 16, 20-32 14. Rebuck, A.S., Campbell, E.J. (1974) A clinical method for assessing the ventilatory response to hypoxia. Am Rev Respir Dis. 109, 345-350 2 ( a ) P O S T -P R I N T	THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070292
	15. Emdin, M., Passino, C., Prontera, C., et al. (2004) Cardiac natriuretic hormones, neuro-hormones, thyroid hormones and cytokines in normal subjects and patients with heart failure. Clin Chem Lab Med 42, 627-636 16. Mortara, A., Sleight, P., Pinna, G.D., et al. (1997) Abnormal awake respiratory patterns are common in chronic heart failure and may prevent evaluation of autonomic tone by measures of heart rate variability. Circulation. 96, 246-252 S t a g e
	13

Table 1

 1 Characteristics of control subjects and HF patients.

	Values are expressed by mean SEM, except for categorical data expressed by %.

19 S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 26 Oct 2007 as manuscript CS20070292 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070292

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 .

 2 Clinical characteristics, cardiac, renal and pulmonary function according to Values are expressed by mean SEM, except for categorical data expressed by %. * = P< 0.001 vs normal HVR and HCVR; † = P < 0.05 vs increased HCVR alone. BMI: body mass index; NYHA= New York Heart association; EF: left ventricular ejection fraction; HVR: chemosensitivity to hypoxia; HCVR: chemosensitivity to hypercapnia; LV-EDVi= left ventricular end diastolic volume index; LV-ESVi= left ventricular end systolic volume index; Creatinine cl.= creatinine clearance; FEV1= forced expiratory volume in 1 second; VC= vital capacity; PAO 2 = arterial partial pressure of oxygen; PACO 2 = arterial partial pressure of carbon dioxide.

	chemosensitivity
		Normal	Increased	Increased	Increased
		HVR and HCVR	HVR	HCVR	HVR and HCVR
	N (%)	24 (40)	8 (13)	12 (20)	16 (27)
	Age (yrs)	64 2	64 2	68 5	69 3
	Male (n, %)	20 (83%)	7 (88%)	11 (92%)	15 (93%)
	BMI (kg•m -2 )	27.9 0.9	28.8 1.2	26.2 0.9	27.7 0.6
	NYHA class III (%)	16	50*	50*	56*
	LVEF (%)	31.4 1.4	28.3 2.9	29.8 2.3	31.4 2.0
	LV-EDVi (ml•m -2 )	135.1 7.2	136.3 16.4	146.9 10.1	140.4 9.2
	LV-ESDi (ml•m -2 )	86.4 6.8	96.6 13.1	94.1 11.9	89.1 6.9
	Creatinine cl. (ml•min -1 )	84.0 6.6	84.8 10.1	80.8 7.7	74.1 5.3
	FEV 1 (L)	2.64 0.24	2.61 0.35	2.27 0.23	2.56 0.19
	FEV1/VC	72.3 1.9	78.7 3.2	73.3 4.9	68.5 2.8
	PH	7.44 0.01	7.45 0.01	7.44 0.01	7.46 0.01
	paO 2 (mmHg)	80.2 1.5	82.8 1.9	78.0 3.3	78.9 2.1
	paCO 2 (mmHg)	36.5 1.0	35.1 0.3	35.8 0.9	33.3 0.9
	HVR (l•min -1 •%SaO 2 -1 )	0.40 0.04	1.12 0.13*	0.55 0.05	1.17 0.07*
	HCVR (l•min -1 •mmHg -1 )	0.47 0.04	0.66 0.06	0.99 0.04*	1.29 0.09* †

Table 3 :

 3 Neurohormonal profile, functional capacity and ventilatory efficiency and arrhythmias according to chemosensitivity .

	Normal	Increased	Increased	Increased
	HVR and HCVR	HVR	HCVR	HVR and HCVR

( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 26 Oct 2007 as manuscript CS20070292 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070292 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGMENTS


Angela Schulz

Maria Schlesener

Judith Weiss

Jonna Hänsch

Norbert Wendt

Peter Kossmehl

Daniela Grimm

Roland Vetter

MD, FAHA Reinhold Kreutz
email: reinhold.kreutz@charite.de

The

Keywords: gender, genetics, kidney, albuminuria, hypertension, rat

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Currently, it is becoming increasingly evident that the development, manifestation and progression of cardiovascular and renal diseases differ between women and men [START_REF] Denton | Physiological and molecular mechanisms governing sexual dimorphism of kidney, cardiac, and vascular function[END_REF]. With regard to renal disease a large body of evidence obtained in various animal models indicated that the progression of renal injury and failure is more pronounced in males compared to females [5;19]. A previous meta-analysis involving 11,345 patients with nondiabetic renal disease confirmed these experimental observations by showing that progression of chronic renal disease is slower in female than in male patients [START_REF] Neugarten | Effect of gender on the progression of nondiabetic renal disease: a meta-analysis[END_REF]. However, another meta-analysis enrolling a significant smaller number of only 1,860 patients suggested that the rate of renal disease progression may be even faster in women compared to men if the analysis accounted for other factors that are associated with a faster rate of progression [START_REF] Jafar | The rate of progression of renal disease may not be slower in women compared with men: a patient-level meta-analysis[END_REF]. In diabetic nephropathy, i.e. the major single cause of end-stage renal disease, it remains unclear whether gender status affects the progression of the disease [2;5;31].

In addition to gender, experimental and clinical studies carried out in the last decade documented a role for genetic factors contributing to the development of both diabetic and nondiabetic renal disease [26;29]. An increase of urinary albumin excretion (UAE) is an important predictor for the development of chronic nephropathy and already a slight increase of UAE represents an independent risk factor for cardiovascular events and mortality not only in arterial hypertension and diabetes but also in the general population [4;29]. Recently, several quantitative trait loci (QTL) have been identified by genome-wide linkage analyses for UAE in patients with diabetes or hypertension and in the general population [8;9;14] and in several hypertensive genetic rat models [3;10;22;27;28;30;33]. In accordance with other rat models of renal damage the Munich Wistar Frömter rat (MWF) exhibits a marked sexual dimorphism regarding the progression of renal injury [5;7;19]. Thus, male MWF rats exhibit spontaneous hypertension and increased UAE leading to overt proteinuria and progressive glomerulosclerosis with age [START_REF] Fassi | Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit[END_REF]. In contrast, female rats develop lower blood pressures, only mild albuminuria and are protected from glomerulosclerosis [7;13;24]. We therefore focussed in our previous genetic studies aiming to dissect the genetic basis of UAE in the MWF strain only on male animals [START_REF] Schulz | Genetic dissection of increased urinary albumin excretion in the munich wistar fromter rat[END_REF][START_REF] Schulz | A major gene locus links early onset albuminuria with renal interstitial fibrosis in the MWF rat with polygenetic albuminuria[END_REF][START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF]. We recently demonstrated that although albuminuria in male MWF is determined by a complex interplay of several quantitative trait loci (QTL) genetic exchange of one major locus on rat chromosome 6 (RNO6) leads to marked suppression of early onset albuminuria and subsequent renal damage in male animals [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF]. This was achieved by targeted breeding and characterization of consomic MWF-6 SHR rats in which RNO6 from the albuminuria resistant spontaneously hypertensive rat (SHR) [22;25] was introgressed into the isogenic background of MWF. However, the relevance of genetic factors for the modulation of mild albuminuria observed in female MWF animals has not been studied so far. We therefore asked the question whether genetic factors, i.e. QTL on RNO6, are also capable of modulating low grade albuminuria in female MWF that are protected from overt renal disease by their gender status.

Methods

Animals and experimental groups

All animals from MWF/Rkb, SHR/Rkb and our recently generated consomic MWF-6 SHR strain [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF] were obtained from our colonies (laboratory code Rkb) at the Charité, Campus Benjamin Franklin, Germany. The consomic strain MWF-6 SHR was derived from MWF/Rkb and SHR/Rkb and was generated by sequential marker assisted backcrossing transferring the whole RNO6 from SHR into the MWF background as recently described [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF]. Rats were grouped under conditions of regular 12h diurnal cycles using an automated light switching device and climate-controlled conditions at a room temperature of 22°C. The rats were fed a normal diet containing 0.2% NaCl and had free access to food and water.

Study A. In study A we first evaluated the sexual dimorphism in adult SHR and MWF rats at 24 weeks of age. For this we selected male and female SHR (n=10 each) and MWF rats (n=18-19 each) and studied systolic blood pressure (SBP), urinary albumin excretion (UAE) and performed renal histology analysis by determination of glomerulosclerosis and tubulointerstitial damage indices. Study B. The aim of study B was to evaluate the role of RNO6 for the development of albuminuria and renal damage in female MWF rats. We therefore compared female consomic MWF-6 SHR (n=20) rats with female MWF (n=18) and female SHR (n=10) with low UAE. In these animals we performed in accordance with previous studies [START_REF] Schulz | Genetic dissection of increased urinary albumin excretion in the munich wistar fromter rat[END_REF][START_REF] Schulz | A major gene locus links early onset albuminuria with renal interstitial fibrosis in the MWF rat with polygenetic albuminuria[END_REF][START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF] time-course analysis for UAE at 8, 14, 18, and 24 weeks of age. In addition, we determined body and organ weights of heart and kidney, plasma concentrations of creatinine and urea, creatinine clearance, renal structural changes and renal collagen III mRNA expression.

In study A and B animals were killed after measuring UAE and systolic blood pressure at the age of 24 week.

Study C. Since male MWF rats develop progressive overt albuminuria with age we followed a subgroup of female MWF and consomic MWF-6 SHR animals (n=8, each) from study B to investigate the development of UAE in older female animals at 32 weeks. We compared the results with the data obtained in age-matched male animals analyzed in a recent report [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF].

Laboratory measurements

For urine analysis animals were placed in metabolic cages for 2 days. The first day was used for adaptation and urine was collected for the last 24h for biochemistry analysis. UAE as an indicator for renal damage was measured by a rat-specific ELISA-technique as previously described [START_REF] Kreutz | Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number[END_REF].

Systolic blood pressure was measured at 24 weeks of age by a noninvasive tail-cuff method in awake animals using a computer-assisted oscillatory detection device (TSE, Bad Homburg, Germany) as previously described [START_REF] Kreutz | Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number[END_REF]. These measurements involved 2 training sessions on 2 days followed by up to 18 (minimum 12) recordings in awake rats on 3 consecutive days was performed as previously reported [START_REF] Kreutz | Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number[END_REF]. Blood was drawn from the aorta for the determination of serum creatinine and urea with standard methods. Both kidneys and the heart were excised. The body, total kidney and heart weights were determined, respectively. For light microscopy evaluation the left kidney was fixed and embedded in paraffin for histology studies including determination of glomerulosclerosis index (GSI), tubulointerstitial damage index (TDI), and quantification of renal interstitial fibrosis (RIF) as reported [START_REF] Schulz | Genetic dissection of increased urinary albumin excretion in the munich wistar fromter rat[END_REF].

RNA analysis

RNA was isolated from kidneys by the TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions and was resuspended in DEPC-treated H 2 O. First strand cDNA synthesis was carried out on 2 µg of total RNA in a 20 µl reaction using the First Strand cDNA Synthesis Kit (Fermentas Life Sciences, St. Leon-Rot, Germany) following the manufacturer's recommendations. To quantify mRNA expression of collagen III in kidney we employed a real-time quantitative RT ("TaqMan") PCR method as reported [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF]. To normalize our expression data we used porphobilinogen deaminase (PBGD) as a housekeeping gene (GenBank accession no. X06827) as previously reported [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF].

Statistical analysis

Statistical analysis was performed using two-way ANOVA followed by Bonferroni´s adjustment and by non-parametric Mann-Whitney U-test. Data are presented as means ± SEM and p-values <0.05 were considered significant.

Results

Study A. Evaluation of sexual dimorphism in adult SHR and MWF rats.

At the age of 24 weeks systolic blood pressure values were significantly lower in both adult male and female parental MWF rats compared to male and female SHR rats (p<0.0001; Figure 1A). A sexual dimorphism with higher systolic blood pressure in males compared to females (p<0.01, respectively) was observed in SHR, whereas the blood pressure difference in MWF animals with somewhat lower values in females were not significant. In contrast to blood pressures UAE values are overall markedly elevated in MWF compared to SHR (Figure 1 B, p<0.0001). Both male and female SHR animals demonstrated comparable low UAE values of ~ 2 mg/24h, while MWF animals exhibited a striking sexual dimorphism with ~7fold higher values in male (162.6±15.9 mg/24h) compared to female (24.0±5.5 mg/24h) animals (Figure 1B, p=0.0003). Analysis of structural changes in the kidney of these animals at 24 weeks of age demonstrated a significant increase of GSI and TDI, i.e. glomerular and tubulointerstitial damage, in male MWF rats compared to female MWF and SHR animals of both sex (Figure 2, p<0.0001, respectively). Representative pictures of this histology analysis performed after PAS staining are shown in Figure 3.

Study B. Evaluation of the role of rat chromosome 6 in female consomic MWF-6 SHR rats.

Time-course analysis for UAE between week 8 and 24 in female consomic MWF-6 SHR as compared to parental MWF and SHR strains is shown in Figure 4. In contrast to SHR female MWF rats demonstrated already a significant increase of UAE at 8 weeks of age (0.1±0.01 vs. 5.2±0.8 mg/24h, p<0.0001, Figure 4). UAE progressed with age in female MWF and demonstrated mean values of 24.0 mg/24h at 24 weeks. This increase was fully suppressed in female consomic animals at all time points investigated (p<0.0001, Figure 4); they only showed a slight increase with values up to 1.7 mg/24h at 24 weeks of age. Overall, no significant differences in UAE levels between female consomic MWF-6 SHR and female SHR animals were observed. At 24 weeks of age analysis of variance revealed no significant differences for body weights and kidney weights between female rats of the three strains (Table 1). Determination of systolic blood pressure in consomic rats demonstrated values similar to female MWF, thus systolic blood pressure was lower in both strains compared to SHR (p<0.0001, histology analysis in female parental MWF and SHR rats, GSI and TDI were also low in female MWF-6 SHR and not significantly different from female rats of the other two strains (not shown). However, the quantitative analysis of RIF in female MWF revealed a significant albeit slight increase in fibrosis compared to SHR (p=0.04), while consomic animals exhibited intermediate levels (Table 1). In addition, collagen III mRNA expression was significantly increased in female MWF compared to SHR animals (p=0.0002), whereas consomic animals demonstrated significantly lower expression levels compared to MWF (p=0.04) and were not statistically different from SHR (Table 1).

Study C. Role of rat chromosome 6 in aging female MWF and comparison with male MWF rats.

In contrast to old male animals, which develop according to our previous analysis mean UAE levels of up to ~ 355 mg/24h (range 206-561 mg/24h) [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF], female MWF demonstrated only UAE levels of 18.6±3.0 mg/24h at 32 weeks of age. The latter values are comparable to the UAE rates observed in female MWF at 24 weeks of age (Figure 4). Thus, female MWF exhibit no significant progression of UAE between week 24 and 32. Despite the striking sexual dimorphism in absolute amounts of UAE between male and female MWF the relative suppression of UAE conferred by transfer of RNO6 in consomic MWF-6 SHR animals appears similar in males and females as summarized in Figure 5 (about 85-95% reduction compared to parental MWF of the same sex).

Discussion

The MWF rat represents a valuable model to study the mechanisms involved in progressive glomerular injury and renal failure [START_REF] Macconi | Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury[END_REF]. This inbred rat strain develops mild arterial hypertension, albuminuria, glomerulosclerosis and shows a reduction of nephron number [7;29]. In the present study we investigated the role of a previously identified important albuminuria QTL on RNO6 [START_REF] Schulz | A major gene locus links early onset albuminuria with renal interstitial fibrosis in the MWF rat with polygenetic albuminuria[END_REF] for the development of albuminuria in female rats, which show in contrast to male rats only low grad albuminuria. Our time-course analysis of UAE in female consomic MWF-6 SHR rats demonstrated that the mild increase of UAE observed in female MWF was fully suppressed by exchange of rat chromosome 6. Taken together with previous studies in males [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF] we show that RNO6 protects against the increase of albuminuria with age in both female and male MWF. than in males [5;19;20;31]. However, the biological basis for this phenomenon is not fully understood. In MWF animals, both females and males share the same reduction in nephron number as demonstrated by Fassi et al. [START_REF] Fassi | Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit[END_REF]. Thus, the lower UAE and the protection from overt proteinuria and structural damage of the kidney in female MWF rats are probably not attributable to differences in nephron development. Our previous studies in male MWF animals indicated that the increased UAE in MWF is a polygenetic trait that is influenced by up to 11 UAE QTL that are not linked to blood pressure [27;28]. Although the genetic studies in male MWF indicated that the increase in UAE is independent from blood pressure the possibility that the somewhat lower blood pressures observed in female MWF are contributing to the lower UAE levels in female animals could not be excluded.

On the other hand it appears possible that gender specific QTL effects [START_REF] Mcbride | Genetic and gender determinants of cerebrovascular disease[END_REF] may explain in part the sexual dimorphism in UAE in the MWF strain. However, this question has not been addressed in previous QTL mapping studies, which included only male MWF animals because of their more pronounced phenotypes [27;28]. Similarly, QTL mapping studies for UAE phenotypes in the Dahl salt-sensitive were also restricted to male animals and led to the identification of multiple QTL in male rats only [10;22]. However, in a subsequent study by Garrett et al. the confirmation of UAE QTL in congenic strains derived from Dahl saltsensitive rats was reported to be similar in male and female animals although no data for female rats were shown [START_REF] Garrett | Genetic linkage of urinary albumin excretion in Dahl salt-sensitive rats: influence of dietary salt and confirmation using congenic strains[END_REF].

As a result of the studies in male MWF we recently confirmed an important role of RNO6 for albuminuria development in male animals [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF]. In the current study we show for the first time that genetic factors, i.e. QTL on RNO6, influence also the development of albuminuria in female MWF rats. Thus, transfer of RNO6 from SHR in the MWF background is protective in both sexes.

Comparative mapping analysis [START_REF] Stoll | New target regions for human hypertension via comparative genomics[END_REF] between the 99% and 95% confidence intervals for placement of the albuminuria QTL on RNO6 with the human genome maps this region to human chromosome 14q23.1-14q31.3. This region contains 146 annotated genes. Currently, we are not able to give any indication which gene(s) within the RNO6 region is (are) responsible for onset of albuminuria in MWF. However, the data in both female and male MWF-6 SHR validate this region and provide the rationale to further develop congenic rats carrying smaller intervals, thereby reducing the number of genes within the QTL for further investigation. It has been shown already that morphologic changes of the glomerular basement membrane or ultrastructure of the podocytes are not responsible for the development of albuminuria in the MWF model [START_REF] Macconi | Effect of angiotensin-converting enzyme inhibition on glomerular basement membrane permeability and distribution of zonula occludens-1 in MWF rats[END_REF]. More recent data demonstrated that progressive proteinuria in male animals is associated with damage and loss of podocytes [START_REF] Macconi | Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury[END_REF]. Since renal damage is only mild in female compared to male MWF animals no major differences in renal function and renal structural damage were detected in consomic animals as expected, except that serum urea and renal collagen III expression were significantly reduced compared to MWF parental animals.

Previously, Fassi et al. provided evidence for glomerular hyperfiltration in MWF animals by

showing a twofold increase of single nephron glomerular filtration rate in strain [START_REF] Fassi | Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit[END_REF]. In addition, they demonstrated that in comparison to normal Wistar rats both male and female MWF animals demonstrated a similar ~50% reduction in total glomeruli number [START_REF] Fassi | Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit[END_REF].

Consequently, in the latter study total glomerular filtration rate was not significantly different between sex-matched MWF and Wistar rats at an early adult age of 12-14 weeks. The data obtained in the current study indicate that renal function as determined by serum creatinine concentrations and creatinine-clearance is impaired in female MWF and consomic MWF-6 SHR compared to SHR at 24 weeks of age. It is well established that renal function is progressively declining in aging male MWF rats with progressive proteinuria probably due to severe structural damage [23;29]. Although our current data would suggest that overall glomerular filtration rate is also lower in older female MWF compared to SHR it appears possible that single glomeruli of female MWF are hyperfiltrating, since we have recently shown that the total number of glomeruli in the MWF strain is also significantly reduced (~-30%) when compared to the SHR strain [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF]. On the other hand, our findings document that the lower creatinine-clearance in MWF and consomic animals can not be related to differences in albuminuria, which was similar between consomic animals and SHR, or to differences in renal structural damage, which was low and similar in females of all three strains. To obtain further insights into the renal function of female MWF and consomic animals would require additional studies investigating renal and glomerular hemodynamics in relation to total number of glomeruli in these animals and in comparison to female SHR using appropriate methods, e.g. inulin-clearance measurements for the determination of glomerular filtration rate [START_REF] Fassi | Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit[END_REF]. Systolic blood pressures were similar in female MWF and consomic animals thus clearly ruling out a role of blood pressure differences as an explanation for the suppressed albuminuria in MWF-6 SHR .

S t a g e 2 ( a ) P O S T -P R I N T

It is well established that aging male animals of some strains of rats develop spontaneous proteinuria and glomerulosclerosis, while treatment with estrogens or orchiectomy protects against renal damage in older age [START_REF] Neugarten | Gender and the progression of renal disease[END_REF]. In addition, investigations in various renal disease models indicated that progression of renal injury can be modulated by surgical or pharmacological change of the sex hormonal status in both male and female rats [START_REF] Neugarten | Gender and the progression of renal disease[END_REF]. These data support the overall concept that androgens are deleterious and estrogens are protective for renal disease progression in experimental models. Moreover, gender-specific differences of important factors regulating renal function such as the renin-angiotensin-system [5;18;21] and the renal nitric oxide synthase isoforms [6;34] have been implicated in the sexual dimorphism of renal disease progression. Previous studies performed in Munich Wistar rats, i.e. the genetic background of MWF animals, indicated that the presence of androgens in male rats rather than the absence of estrogens provide the risk factor for development of agedependent proteinuria and glomerular damage in this strain [START_REF] Baylis | Age-dependent glomerular damage in the rat. Dissociation between glomerular injury and both glomerular hypertension and hypertrophy. Male gender as a primary risk factor[END_REF]. The current study demonstrates that early onset of albuminuria at 8 weeks of age in female MWF is comparable to the early increase of UAE in male rats [START_REF] Schulz | Development of overt proteinuria in the Munich Wistar Fromter rat is suppressed by replacement of chromosome 6 in a consomic rat strain[END_REF]. Subsequently, female rats develop only a slight increase of UAE after maturation reaching a plateau between week 24 and 32, while males show a marked progression with age. The similar suppression of albuminuria in female and male rats induced by exchange of RNO6 QTL supports the conclusion that the same genetic mechanisms are responsible for the onset of albuminuria in both male and female MWF rats, while the sexual dimorphism is probably attributable to non-genetic factors such as the influence of sex hormones. Future studies may aim to characterize how changes in sex hormonal status affect the genotype-phenotype affects in albuminuria.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 Oct 2007 as manuscript CS20070300 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070300 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 Oct 2007 as manuscript CS20070300 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070300 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 Oct 2007 as manuscript CS20070300 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070300 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  The sexual dimorphism of renal disease phenotypes observed in this model reflects clinical findings in nondiabetic renal disease indicating that disease progression is lower in females S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 Oct 2007 as manuscript CS20070300 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070300 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 Oct 2007 as manuscript CS20070300 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070300 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 .

 1 Figure 1. Systolic blood pressure (SBP) in panel A and urinary albumin excretion (UAE) in panel B of female (open bars) and male (solid bars) spontaneously hypertensive rats (SHR) and Munich Wistar Frömter rats (MWF) at 24 weeks of age. * P<0.0001 vs. SHR same gender; # P<0.01 vs. males same strain.

Figure 2 .

 2 Figure 2. Glomerulosclerosis index (GSI) in panel A and tubulointerstitial damage index (TDI) in panel B of female (open bars) and male (solid bars) spontaneously hypertensive rats (SHR) and Munich Wistar Frömter rats (MWF) at 24 weeks of age. * P<0.0001 compared to other groups, respectively.

Figure 3 .

 3 Figure 3.Histopathology of the kidney in rat strains at 24 weeks of age, respectively. Paraffin sections were stained with periodic acid-Schiff reagent (PAS). Panels A, B, and C represent female rats, respectively, of spontaneously hypertensive rat (SHR) in A, Munich Wistar Frömter rat (MWF) in B, and consomic MWF-6 SHR rat in C. Panels D, E, and F represent the corresponding male animals. All female animals (panels A, B, and C) and male SHR (panel D) demonstrate no severe abnormalities. In contrast, male MWF (panel E) show significant glomerulosclerosis in two glomeruli, tubulointerstitial infiltration (arrow), and tubular casts (*), while male consomic MWF-6 SHR (panel F) show intermediate glomerulosclerosis.

Figure 4 .

 4 Figure 4. Time course-analysis of urinary albumin excretion (UAE) of female spontaneously hypertensive rats (SHR) in white bars, consomic MWF-6 SHR in grey bars, Munich Wistar Frömter rats (MWF) in black bars at 8, 14, 18, and 24 weeks of age. * P<0.0001 compared to SHR and MWF-6 SHR , respectively.

Figure 5 .

 5 Figure 5. Urinary albumin excretion (UAE) in male and female consomic MWF-6 SHR (grey bars) and Munich Wistar Frömter (MWF) rats (black bars) at 32 wk of age. * P<0.0001 compared to S t a g e 2 ( a ) P O S T -P R I N T

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 Oct 2007 as manuscript CS20070300 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070300 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 SBP

 1 Figure 1

  Figure 2

Figure 3 S

 3 Figure 3

Figure 4 UAE

 4 Figure 4

  Table. 1). Replacement of RNO6 in the consomic strain resulted in lower serum urea concentrations compared to female parental MWF animals (p<0.0001, Table 1), while heart weight, serum creatinine and creatinine clearance at 24 weeks of age were not affected as summarized in Table 1. In addition, as expected from the data obtained for kidney

Table 1 .

 1 Overall characteristics of female parental MWF, SHR, and consomic MWF-6 SHR rats at 24 weeks of age.

	SHR	MWF-6 SHR	MWF	P -ANOVA
			Overall	MWF vs. SHR	MWF vs. MWF-6 SHR	SHR vs. MWF-6 SHR

Acknowledgements

We acknowledge the contributions of Sabine Wunderlich and Claudia Plum for laboratory assistance and of Bettina Bublath for support in animal breeding. This study was supported by grants from the DFG KR1152-3-1. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 Oct 2007 as manuscript CS20070300 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070300 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society


Patrizia Ferroni

Maria Teresa Guagnano

Angela Falco

Vincenzo Paoletti

Maria Rosaria Manigrasso

Noemi Michetti

Francesca Santilli

Fiorella Guadagni

Stefania Basili

MD. Giovanni Davì

Cent

Association of low-grade inflammation and platelet activation in hypertensive patients with microalbuminuria

Keywords: Inflammation, hypertension, platelet activation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The CD40 ligand (CD40L) is a transmembrane protein structurally related to tumor necrosis factor-(TNF-) that has been first identified on CD4+ T cells [START_REF] Mach | Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implication for CD40-CD40 ligand signaling in atherosclerosis[END_REF][START_REF] Van Kooten | Immune regulation by CD40-CD40L interactions[END_REF]. Subsequently, CD40L has been identified on the surface membrane of activated platelets [START_REF] Henn | CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells[END_REF] from where a soluble form (sCD40L) can be released into the circulation [START_REF] Aukrust | Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina. Possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes[END_REF]. Both membrane-bound and sCD40L interact with CD40 expressed on vascular cells, resulting in inflammatory and prothrombotic responses [START_REF] Henn | CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells[END_REF][START_REF] Lindmark | Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression[END_REF]. Engagement of CD40L with CD40 on endothelial cells, in fact, results in phenotypic changes that are similar to those induced by TNF-, i.e., increased expression of adhesion molecules and secretion of inflammatory cytokines [START_REF] Granger | Modulation of the inflammatory response in cardiovascular disease[END_REF]. In addition, sCD40L can act as a ligand of the platelet fibrinogen receptor glycoprotein IIb/IIIa and is involved in thrombus stabilization and platelet activation [START_REF] Prasad | The platelet CD40L/GP IIb-IIIa axis in atherothrombotic disease[END_REF]. Thus, sCD40L may represent a key molecule linking inflammation and prothrombotic state in human diseases.

To date, it is estimated that more than 95% of plasma sCD40L is derived from platelets [START_REF] Freedman | CD40-CD40L and platelet function. Beyond hemostasis[END_REF]. Accordingly, increased levels of sCD40L have been associated with enhanced in vivo platelet activation in various clinical conditions [START_REF] Cipollone | Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolemia: effects of statin therapy[END_REF][START_REF] Garlichs | Upregulation of CD40-CD40 ligand (CD154) in patients with acute cerebral ischemia[END_REF][START_REF] Danese | Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients[END_REF][START_REF] Lim | Soluble CD40 ligand, soluble P-selectin, interleukin-6, and tissue factor in diabetes mellitus: relationships to cardiovascular disease and risk factor intervention[END_REF][START_REF] Falco | Increased soluble CD40 ligand levels in cystic fibrosis[END_REF][START_REF] Roselli | Soluble CD40 ligand plasma levels in lung cancer[END_REF][START_REF] Novo | Soluble CD40L and Cardiovascular Risk in Asymptomatic Low-Grade Carotid Stenosis[END_REF]. Meanwhile, it has become evident that sCD40L could be regarded as a predictive marker of cardiovascular events in healthy individuals [START_REF] Novo | Soluble CD40L and Cardiovascular Risk in Asymptomatic Low-Grade Carotid Stenosis[END_REF][START_REF] Schonbeck | Soluble CD40L and cardiovascular risk in women[END_REF], which may not only indicate active inflammation and platelet activation, but may also exhibit direct prothrombotic properties that mediate early atherogenesis, plaque rupture, and thrombosis [START_REF] Mo | Individualizing therapy in acute coronary syndromes: using a multiple biomarker approach for diagnosis, risk stratification, and guidance of therapy[END_REF].

Persistent platelet activation has been demonstrated in patients with essential hypertension, especially those with advanced vascular lesions [START_REF] Davì | Diabetes mellitus, hypercholesterolemia, and hypertension but not vascular disease per se are associated with persistent platelet activation in vivo. Evidence derived from the study of peripheral arterial disease[END_REF][START_REF] Spencer | Von Willebrand factor, soluble P-selectin, and target organ damage in hypertension: a S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication[END_REF][START_REF] Minuz | Determinants of platelet activation in human essential hypertension[END_REF][START_REF] Nadar | Platelet indexes in relation to target organ damage in high-risk hypertensive patients: a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT)[END_REF], and tight blood pressure control may result in significant improvements in platelet function in hypertensive subjects at high risk of cardiovascular events [START_REF] Blann | Pharmacological modulation of platelet function in hypertension[END_REF]. In addition, low-grade inflammation (as evidenced by increased levels of C-reactive protein [CRP]) [START_REF] Sesso | C-Reactive protein and the risk of developing hypertension[END_REF][START_REF]Low-Grade inflammation and microalbuminuria in hypertension[END_REF] Although, several factors link platelet activation, endothelial dysfunction and low-grade inflammation with sCD40L, no information is available yet about their interrelationships with hypertension-related cardiovascular and metabolic abnormalities. For this reason, we addressed that issue in hypertensive patients with or without microalbuminuria (MA), which is currently acknowledged as a predictor of vascular disease [START_REF] Garg | Microalbuminuria: marker of vascular dysfunction, risk factor for cardiovascular disease[END_REF], and the strongest independent determinant of ischaemic heart disease among non-diabetic individuals with arterial hypertension [START_REF] Jensen | Arterial hypertension, microalbuminuria, and risk of ischemic heart disease[END_REF][START_REF] Jager | Microalbuminuria and peripheral arterial disease are independent predictors of cardiovascular and all-cause mortality, especially among hypertensive subjects: five-year follow-up of the Hoorn Study[END_REF].

METHODS

Design of the study

A cross-sectional comparison was performed in 50 patients with essential hypertension, with (MH) and without (EH) microalbuminuria, and in 25 healthy normotensive subjects (HS) enrolled at our Institutions (Table 1). Hypertensive patients with MA (n=25) were pairmatched for gender and age with hypertensive patients without MA (n=25) and HS (Table 1).

Exclusion criteria were represented by a history or evidence of atherothrombotic diseases, diabetes mellitus, cigarette smoking, dyslipidemia, obesity and metabolic syndrome in order to avoid confounding by other determinants of endothelial dysfunction and platelet activation.

None of the subjects was permitted to take vitamin supplements, nonsteroidal antiinflammatory drugs, or antiplatelet agents for at least 30 days before the study. Women who were taking hormonal replacement therapy were also excluded. Essential hypertension was clinically diagnosed according to the WHO criteria [START_REF]1999 World Health Organization International Society of Hypertension guidelines for the management of hypertension. Guidelines Subcommittee[END_REF], on 3 consecutive occasions and on the basis of negative results of investigation for secondary hypertension that included hormonal evaluation, renal scintigraphy and/or echo-doppler scan of the renal arteries. MA was defined as a urinary albumin excretion rate (AER) between 20 and 200 µg/min. Hypertensive patients were under long-term treatment with antihypertensive medication (angiotensin converting enzyme inhibitors and/or diuretics and/or, calcium antagonists). Both groups of hypertensive subjects received a standard diet containing ~150 mmol of sodium per day. The mean duration of hypertension was defined as the time between the diagnosis and inclusion in the present study. The study was performed under the appropriate institutional ethics approvals and in accordance with the principles embodied in the Declaration of Helsinki. Informed consent was obtained from each participating subject.

Biochemical analyses

The mean AER was calculated from three consecutive timed overnight urine collections.

AER was measured by nephelometry (Roche Diagnostics, France). Serum C-reactive protein (CRP) levels were measured with a highly sensitive immunoassay [START_REF] Ledue | Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum[END_REF]. Total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol concentrations were measured as previously described [START_REF] Davì | Diabetes mellitus, hypercholesterolemia, and hypertension but not vascular disease per se are associated with persistent platelet activation in vivo. Evidence derived from the study of peripheral arterial disease[END_REF].

Plasma sP-selectin, sCD40L levels (both by R&D Systems, Minneapolis, MN, USA) and vWF levels (Imubind vWF ELISA, American Diagnostica) were measured in citrated plasma samples by commercially available enzyme-immunoassays according to the manufacturers' instructions.

ADMA levels were measured in plasma samples by a commercially available enzymeimmunometric assay (DLD diagnostika, Hamburg, Germany), previously validated by comparison with gas chromatography/mass spectrometry [START_REF] Schulze | Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay[END_REF]. Plasma samples were processed promptly after collection (within 2 h) to minimize ex-vivo release of P-selectin and CD40L from platelets [START_REF] Thom | Measurement of soluble P-selectin and soluble CD40 ligand in serum and plasma[END_REF].

Statistical Analysis

Statistical analysis was performed by chiand by one-way analysis of variance with the Bonferroni adjustment to assess differences among the groups (hypertensive with and without microalbuminuria and non hypertensive subjects). When necessary appropriate noncorrelation coefficient, Kruskall-Wallis method and Mann-Whitney U test). Multiple linear regression analysis and factorial ANOVA analysis were performed to further analyze the data. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

Baseline characteristics of the three groups of subjects are summarized in Table 1. As shown, hypertensive patients with or without MA did not differ in terms of blood pressure, disease duration, creatinine serum level and serum lipid pattern. Healthy subjects had similar clinical and laboratory characteristics as compared to hypertensive subjects. Urinary AER was significantly higher in hypertensive patients with MA compared to hypertensive patients without MA and healthy subjects (p< 0.0001); normoalbuminuric hypertensive subjects had significantly higher values of urinary AER than controls (p< 0.0001).

Platelet activation, as reflected by sP-selectin levels, was significantly enhanced in microalbuminuric hypertensive patients as compared to either HS or EH patients (Table 2).

Furthermore, an impairment of endothelial function was found in hypertensive patients, as evidenced by increased plasma levels of both vWF and ADMA in hypertensive subjects with or without MA compared with HS (Table 2). Elevated ADMA (regression coefficient= 0.259, p= 0.01) or vWF (regression coefficient= 0.595, p< 0.0001) levels, low HDL-cholesterol (regression coefficient= -0.305, p= 0.001) and disease duration (regression coefficient= 0.244, p= 0.004) were all predictive of MA by multivariate analysis. Plasma sCD40L levels were significantly increased in MH patients compared to either EH or HS (Table 2), but no differences could be observed between the two latter groups (Figure 1). A highly significant correlation between plasma sCD40L and sP-selectin (Rho= 0.750, p< 0.0001), vWF (Rho= 0.658, p< 0.0001) or CRP levels (Rho= 0.42, p< 0.003) was observed in hypertensive patients. Subgroup analysis demonstrated that these associations were retained in the subgroup of MH patients (Figure 2) but not in EH patients, in whom only the correlation between sCD40L and sP-selectin (Rho=0.528, p<0.007) was retained.

Stepwise multivariate regression analysis showed that sP-selectin was the strongest To further evaluate the relationship between sCD40L and endothelial dysfunction or inflammatory status, hypertensive patients were stratified on the basis of the observed median CRP (1.22 mg/dl) or vWF (1120 IU/dl) levels. As shown in Figure 3, hypertensive patients with both vWF and CRP levels above the median had the highest sCD40L levels (median level 2.4 pg/ml) compared to patients with either vWF or CRP levels above the median (1.1 pg/ml) or patients with both variables below the median (0.65 pg/ml, H= 21.5, p< 0.0001).

These results were confirmed in the subset of MH (H= 10.7, p< 0.005), but not in EH patients.

Based on these results, we finally performed a factorial ANOVA analysis of all hypertensive patients to better characterize the interactions between sCD40L and the inflammatory status in the presence or absence of MA. The results are illustrated in Figure 4, showing that only MH patients with low-grade inflammation had elevated levels of sCD40L.

Multivariate regression analysis of this restricted subset of patients confirmed the independent prognostic role of sP-selectin in determining sCD40L levels (regression coefficient

DISCUSSION

To date, no clinical evidence is available linking platelet activation and/or inflammation to sCD40L production in hypertension. The present study is the first to demonstrate that increased levels of this cytokine may be at least in part related to the occurrence of in vivo platelet activation, as demonstrated by the significant association found between sCD40L and sP-selectin, a well recognized marker of in vivo platelet activation [START_REF] Blann | The adhesion molecule P-selectin and cardiovascular disease[END_REF][START_REF] Davì | Increased levels of soluble P-selectin in hypercholesterolemic patients[END_REF][START_REF] Andre | P-selectin in haemostasis[END_REF].

The occurrence of elevated sCD40L levels in patients with hypertension is still a debated issue. In fact, Tsioufis et al. recently reported that MA is not accompanied by an increase of sCD40L concentrations in essential hypertensive subjects and hypothesized an activation of different inflammatory pathways in the progression of renal and cardiovascular atherosclerotic disease in these patients [38]. However, the study group used in this study was restricted to non-smoker hypertensive subjects without other risk factors (i.e. obesity).

As Bautista pointed out in his critique, although selection is sometimes used to prevent confounding, it limits generalizability and is rarely justifiable, as confounding can be controlled efficiently using multiple regression analysis [39]. In our cohort of patients, sCD40L levels were higher in MH compared to both hypertensive subjects without MA or age-and gender-matched normotensive subjects. This finding is in agreement with those by other authors suggesting that plasma sCD40L levels may contribute to the increased In the present study, the findings of increased sCD40L levels were limited to a subset of hypertensive patients with MA. The latter significantly correlated with the duration of hypertension, which is in agreement with the findings of other authors [START_REF] Garg | Microalbuminuria: marker of vascular dysfunction, risk factor for cardiovascular disease[END_REF]. Moreover, we showed that increased plasma levels of vWF and ADMA (two well recognized markers of endothelial dysfunction) were both predictive of MA. These findings are confirmatory of previous studies [START_REF]Low-Grade inflammation and microalbuminuria in hypertension[END_REF][START_REF] Pedrinelli | Microalbuminuria and endothelial dysfunction in essential hypertension[END_REF][START_REF] Taddei | Endothelial dysfunction in essential hypertension: clinical implications[END_REF][START_REF] Goonasekera | Vascular endothelial cell activation associated with increased plasma asymmetric dimethyl arginine in children and young adults with hypertension: a basis for atheroma?[END_REF][START_REF] Garg | Microalbuminuria: marker of vascular dysfunction, risk factor for cardiovascular disease[END_REF] and are in agreement with those by Pedrinelli et al., who demonstrated the presence of increased vWF levels in patients with essential hypertension and MA as well as intra-individual correlation between the two variables, suggesting that a dysfunctional glomerular endothelium may cause reduced renal function [START_REF] Pedrinelli | Microalbuminuria and endothelial dysfunction in essential hypertension[END_REF]. Along with the occurrence of endothelial dysfunction (as evidenced by increased levels both vWF and ADMA), we observed a low-grade inflammatory condition in all hypertensive patients either with MA or without MA. Indeed, circulating CRP levels were significantly higher in MH than EH patients or healthy subjects. This is not surprising if we consider a recent report by Sesso et al. [START_REF] Sesso | C-Reactive protein and the risk of developing hypertension[END_REF] demonstrating that CRP levels are associated with future development of hypertension, which suggests that hypertension is in part an inflammatory disorder.

The major conclusion that may be drawn from this study is that sCD40L levels appear to discriminate a high-risk subset of patients characterized by microalbuminuria and low-grade inflammation. This is of particular interest since several population-based studies have shown an association between MA and cardiovascular morbidity and mortality [START_REF] Garg | Microalbuminuria: marker of vascular dysfunction, risk factor for cardiovascular disease[END_REF], leading to the demonstration that MA is the strongest independent determinant of ischaemic heart disease among subjects with arterial hypertension [START_REF] Jensen | Arterial hypertension, microalbuminuria, and risk of ischemic heart disease[END_REF]. Furthermore, MA accompanied by evidence of subclinical inflammation has been proposed as a strong correlate of metabolic abnormalities in essential hypertension and might identify a patient subset at very high cardiovascular risk. In contrast, isolated microalbuminuria may represent a distinct pathophysiological condition characterized by a more benign profile and possibly a better prognosis [START_REF]Low-Grade inflammation and microalbuminuria in hypertension[END_REF]. In this context, the finding of enhanced levels of sCD40L mainly in a subgroup of MH patients with low-grade inflammation (as evidenced by elevated CRP One limitation to the study might be represented by the small number of patients.

However, we must consider that the population enrolled was highly selected, since we excluded hypertensive patients with concomitant risk factors for atherothrombosis, such as obesity, cigarette smoking, diabetes mellitus and metabolic syndrome all of which have been shown to affect significantly the levels of urinary albumin excretion and to be associated with biochemical evidence of in vivo platelet activation [9-13, 15, 42, 43]. In terms of study limitation, the current findings were limited to hypertensive patients with microalbuminuria, a surrogate of the subclinical atherosclerotic burden and low-grade inflammation limiting the generalization of the study. Nevertheless, this study may provide new insight into the pathophysiologic mechanisms linking early renal impairment with elevated cardiovascular risk.

In conclusion, these data confirm that CD40L represents a key molecule linking inflammation and thrombosis. Thus, inhibition of the CD40/CD40L system may represent a potential therapeutic target in a subgroup of hypertensive patients at high risk for cardiovascular events.

Figure 3

Box-plot analysis of plasma sCD40L levels in all hypertensive patients (n=50) with vWF and/or CRP levels below (negative) or above (positive) the median values.

Figure 4

Factorial ANOVA analysis of plasma sCD40L levels in patients with (MH, n=25) or without (EH, n=25) microalbuminuria stratified on the basis of CRP levels below (negative) or above (positive) the median value observed in hypertensive patients.

  and endothelial dysfunction (as evidenced by increased levels of von Willebrand factor [vWF] or asymmetric S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society dimethylarginine [ADMA])[24-27] have been frequently associated with essential hypertension and may help identifying patients at very high cardiovascular risk.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  All subjects were studied on an outpatient basis. Blood samples were obtained in the early morning after a 12-hour fast. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Multiple regression analysis always included, as the independent variables, male gender, age, body mass index (BMI), AER, disease duration, antihypertensive treatment, systolic and diastolic blood pressure, blood lipids and smoking habit. With 25 subjects recruited in the cross-sectional comparison, the study had a 95% power to detect a 40% difference in sCD40L serum levels between the hypertensive microalbuminuric patients and control groups with a two-tailed of 0.05. Data are presented as mean (1 SD) or median and interquartile range (IQR; 25th , 75th percentile). Only p values lower than 0.05 were regarded as statistically significant. All tests were two-tailed and analyses were performed using a computer software package (Statistica 6.0, StatSoft Inc., Tulsa, OK or Statistical Package for the Social Sciences, version 13.0, SPSS Inc., and Chicago, IL). S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society independent predictor of sCD40L levels (regression coefficient [SE]= 0.787 [0.13]; p<0.0001) in MH patients.

[

  SE]=0.838 [0.15]; p<0.0001). S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  susceptibility of hypertensive patients to develop vascular damage [40] probably through blunted angiogenesis [41]. In particular, Patel et al. suggested that sCD40L might reflect abnormal platelet activation and, thus, increased sCD40L (as a marker of CD40 ligation) may contribute to the increased cardiovascular risk in patients with hypertension [41].

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society concentrations) suggests that this cytokine might be directly responsible for prothrombotic properties that mediate thrombosis and acute vascular events.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society 38. Tsioufis, C., Dimitriadis, K., Taxiarchou, E., et al. (2006) Diverse associations of microalbuminuria with C-reactive protein, interleukin-18 and soluble CD40 ligand in male essential hypertensive subjects. Am. J. Hypertens. 19, 462-466. 39. Bautista, L.E. (2006) Associations of microalbuminuria with inflammation markers in hypertensive men. Am. J. Hypertens. 19, 467. 40. Desideri, G., Cipollone, F., Valeri, L., et al. (2007) Enhanced plasma soluble CD40 ligand levels in essential hypertensive patients with blunted nocturnal blood pressure decrease. Am. J. Hypertens. 20, 70-76. 41. Patel, J.V., Lim, H.S., Nadar, S., Tayebjee, M., Hughes, E.A., Lip, G.Y. (2006) Abnormal soluble CD40 ligand and C-reactive protein concentrations in hypertension: relationship to indices of angiogenesis. J. Hypertens. 24, 117-121. 42. Lee W.L., Lee W.J., Chen Y.T., et al. (2006) The presence of metabolic syndrome is independently associated with elevated serum CD40 ligand and disease severity in patients with symptomatic coronary artery disease. Metabolism. 55, 1029-1034. 43. Angelico F., Alessandri C., Ferro D., et al. Enhanced soluble CD40L in patients with the metabolic syndrome: Relationship with in vivo thrombin generation. Diabetologia. 49, 1169-1174. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 :

 2 Laboratory variables of hypertensive patients and healthy subjects. HS: healthy subjects; Mann-Whitney U test of healthy subjects vs. hypertensive with MA: *p< 0.0001; p< 0.001. ADMA: asymmetrical dimethylarginine. Data are presented as mean +SD or median (25th 75th percentile) S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 Nov 2007 as manuscript CS20070307 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070307 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

FIGURE LEGENDS Figure 1

 1 FIGURE LEGENDS

Figure 2

 2 Figure 2 Correlation analysis of plasma sCD40L levels and vWF (Panel A, Rho= 0.662, p< 0.001), sP-selectin (Panel B, Rho= 0.750, p< 0.0001) or C-reactive protein (Panel C, Rho= 0.419, p< 0.05) in hypertensive patients with microalbuminuria.

© 2007 The Authors Journal compilation © 2007 Biochemical Society