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NDT Advance Access originally published online on March 30, 2006
Nephrology Dialysis Transplantation 2006 21(6):1697-1701; doi:10.1093/ndt/gfl112
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Short Communication

Early markers of inflammation in a high angiotensin II state—results of studies in Bartter's/Gitelman's syndromes

Paul A. Davis1, Michele Mussap2, Elisa Pagnin3, Lara Bertipaglia3, Vincenzo Savica4, Andrea Semplicini3 and Lorenzo A. Calò3

1 Department of Nutrition, University of California, Davis, 2 Department of Laboratory Medicine, 3 Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova and 4 Division of Nephrology, University of Messina, Italy

Correspondence and offprint requests to: Lorenzo A. Calò, MD, PhD, Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova, Via Giustiniani, 2, 35128 Padova, Italy. Email: renzcalo{at}unipd.it



   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Inflammation has been increasingly recognized as playing a critical role in hypertension and atherosclerosis as reflected by overexpression and increased production of a variety of pro-inflammatory mediators. As angiotensin II (Ang II) also plays a major role in these diseases, the relationship between inflammation and Ang II has drawn increasing scrutiny. This study explores Ang II effects in Bartter's and Gitelman's syndromes (BS/GS) which do not develop hypertension and related cardiovascular remodelling and atherosclerosis, in spite of high Ang II levels and activation of the renin–angiotensin–aldosterone system while the NO system is up-regulated.

Methods. We evaluated the plasma levels of inflammation-associated markers, C-reactive protein (CRP), serum amyloid A (SAA), vascular cell adhesion molecules (VCAM) and intercellular adhesion molecules (ICAM), and the inflammation-related cytokines interleukin-6 (IL-6) and tumour necrosis factor-{alpha} (TNF-{alpha}) using immunonephelometric and ELISA-based assays.

Results. The study demonstrated that all markers of inflammation except TNF-{alpha}, were unchanged in BS/GS (2.51±0.62 mg/l in BS/GS vs 1.7±0.6 in controls for CRP; 4.56±1.09 mg/l in BS/GS vs 4.51±1.0 for SAA; 1.84±0.27 ng/l in BS/GS vs 2.1±0.3 for IL-6; 449±83 ng/ml in BS/GS vs 410±92 for VCAM and 234±26 ng/ml in BS/GS vs 185±22 for ICAM), while TNF-{alpha} was increased (10.5±2.03 vs 3.68±0.2, P = 0.0001).

Conclusions. The results of this study stress the critical role played by Ang II in controlling vascular biology including inflammation-related processes as well as highlighting the utility of BS/GS in investigating these pathways.

Keywords: angiotensin II; atherosclerosis; Bartter's syndrome; Gitelman's syndrome; hypertension; inflammation



   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Hypertension and atherosclerosis are increasingly viewed as diseases in which inflammation plays a critical pathogenetic role, and this has led to a heightened interest in studying the role of inflammatory factors [1,2]. These studies have shown that development and progression of these diseases are associated with increased expression and production of a variety of pro-inflammatory mediators including cytokines (leucocyte adhesion molecules, intercellular adhesion molecules (ICAM-1) and vascular cell adhesion molecules (VCAM-1) [1,2], chemotactic proteins (MCP-1) [1], nuclear transcription factor (NF-{kappa}B) [1–3] and growth factors (tumour necrosis factor-alpha, TNF-{alpha} and transforming growth factor, TGF-ß) [1–4] in a variety of different cell types.

Given the involvement and the critical role played by inflammation and Ang II in the pathogenesis of a wide spectrum of cardiovascular and renal diseases [1,5,6], the relationship between inflammation and Ang II has drawn increasing scrutiny. In fact, increased local or tissue Ang II formation in target organs induces inflammation [1,5,6] as Ang II is itself a powerful pro-inflammatory cytokine and growth factor. There is accumulating evidence that Ang II activates NF-{kappa}B [1,5,6], the key nuclear transcription factor in inflammatory and fibrotic diseases and this activation leads to transcription of numerous inflammatory genes, including interleukin-6 (IL-6), TNF-{alpha} and TGF-ß [3,4]. In addition, Ang II activates NADPH oxidase [7] with an increase of reactive oxygen species (ROS) and induction of oxidative stress, which is tightly linked with inflammation [5,6,8,9].

Given the emerging role of Ang II as a common pathway in these various diseases, attention has been directed towards systems that allow Ang II effects to be explored and understood. We have focused on Bartter's/Gitelman's syndromes (BS/GS) as potentially very helpful models in the aspect of Ang II biology. The clinical picture of BS/GS is characterized by hypokalaemia, sodium depletion, activation of the renin–angiotensin–aldosterone system with increased plasma levels of Ang II, yet normo-hypotension, reduced peripheral resistance and hyporesponsiveness to pressor agents [10–12]. These findings are the results of specific genetic defects of kidney transporters and ion channels. However, our exploration of these findings has provided explanations for these findings and has led us to propose that BS/GS is a good human model to explore the mechanisms responsible for Ang II signalling [10,11,13]. The fact that BS/GS do not develop hypertension and related diseases such as atherosclerosis in spite of high Ang II level and activation of the RAAS, suggests that understanding how this occurs in these patients should shed light on the cellular basis of Ang II action. Our extensive studies in BS/GS have provided important insights into the mechanistic details of Ang II-related pathways for vascular tone regulation [10,11]. To increase our understanding of this model and further extend its utility in understanding Ang II-related mechanisms, we have evaluated the level and status of a variety of inflammation-associated markers, thought to be involved in the actions of Ang II acting as a pro-inflammatory agent. We have measured C-reactive protein (CRP), the best known and the most commonly used indicator of inflammation together with acute phase reactants such as serum amyloid A (SAA), soluble VCAM and ICAM, and inflammatory processes-related cytokines such as interleukin-6 (IL-6) and TNF-{alpha}.



   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
We recruited 12 patients (seven males and five females, age range 18–54) with either BS (n = 2) or GS (n = 10) from our cohort of BS/GS patients, the same evaluated in previous studies [10,11]; all have a full biochemical characterization with seven having undergone full genetic analysis and five awaiting the results of the genetic screenings. Twenty normotensive healthy subjects (12 males and eight females, age range 24–56), from the staff of the Department of Clinical and Experimental Medicine, University of Padova, were used as control group.

Table 1 shows clinical and laboratory data of the patients and controls included in the study.


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  		Table 1. Clinical and laboratory data of the patients included in the study

 
The study protocol was approved by our institutional authorities and informed consent was obtained from all the study participants. None of the patients or controls have been taking drugs for at least 2 weeks prior to the study and all the subjects abstained from food, alcohol and caffeine-containing drinks for at least 12 h prior to the study. All subjects were reported to be consuming a normal Italian diet, which contains approximately 150 mmol of sodium/day.

C-reactive protein (CRP)
The CRP was measured by highly sensitive particle-enhanced nephelometric immunoassay (Dade Behring, Milan, Italy). The method is based on monoclonal antibodies to CRP with a detection limit of 0.175 mg/l and analytical sensitivity of 0.04 mg/l.

Serum amyloid A (SAA) protein
The SAA was measured by a particle-enhanced nephelometric immunoassay (Dade Behring, Milan, Italy). The method is based on sheep polyclonal antibodies to human SAA (SAA type 2) and levels are based on 6 min data collection in a fixed time mode by the fully automated BN II system (Dade Behring, Milan, Italy). The coefficient of variation (CV,%) for within-run and between-run was 3.35–5.05% and 3.07–6.90%, respectively

Interleukin-6 (IL-6)
Quantitative measurement of IL-6 was done by a solid-phase, enzyme-labelled, chemiluminescent sequential immunometric assay on the Immulite 1000 analyser (Medical System, Genova, Italy). The CV ranges were from 5.1 to 7.5%, with an absolute range of 0–5.9 ng/l.

Tumour necrosis factor-{alpha} (TNF-{alpha})
The TNF-{alpha} was measured by an immunometric assay on the Immulite 1000 analyser (Medical System, Genova, Italy). Imprecision of the method was expressed as CV ranges 2.6–3.6% (within-run) and 4.0–6.5% (between-run).

VCAM-1 and ICAM-1
Plasma levels of VCAM-1 and ICAM-1 were measured by ELISA assay kits (Bender MedSystems, Wien, Austria). Detection limit was 0.9 ng/ml with a CV 3.1% (within-run) and 5.2% (between-run) for VCAM. Detection limit was 3.3 ng/ml CV 4.1% (within-run) and 7.6% (between-run) for ICAM.

Statistical analysis
Data were evaluated on a Macintosh G5 computer (Apple Computer, USA) using the Statview II statistical package (BrainPower Inc., USA). The normal distribution of the data has been established using the Kolmogorov–Smirnov test. A non-normal distribution resulted only for TNF-{alpha}. Data are expressed as mean±SE and they were analysed using Student's t-test for unpaired data and, for TNF-{alpha}, the non-parametric Wilcoxon–Mann–Whitney rank sum test. Values at a 5% level or less (P<0.05) were considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
The results of the evaluation in BS/GS patients of the different markers of inflammation that we considered in our study are shown in Table 2.


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  		Table 2. Plasma level of early markers of inflammation and cytokines in patients with Bartter's and Gitelman's syndromes (BS/GS) and in normotensive healthy subjects

 
In BS/GS CPR, SAA, IL-6, VCAM and ICAM plasma levels were not different from values obtained in healthy subjects (2.51±0.62 vs 1.7±0.6 mg/l for CPR; 4.56±1.09 vs 4.51±1.0 mg/l for SAA; 1.84±0.27 vs 2.1±0.3 ng/l for IL-6; 449±83 vs 410±92 ng/ml for VCAM and 234±26 vs 185±22 ng/ml for ICAM), while TNF-{alpha} was increased (10.5±2.03 ng/l in BS/GS vs 3.68±0.2, P = 0.0001).



   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
Inflammation is increasingly viewed as a common pathway underlying a variety of diseases. An understanding of the various aspects and mechanisms that comprise inflammation has become therefore of heightened interest. Ang II has been increasingly reported as a pro-inflammatory mediator [1]. As a result of this evidence, we have used our cohort of BS/GS patients characterized by activation of the renin–angiotensin system and high levels of Ang II to explore pathways linked to Ang II signalling. We have earlier documented in BS/GS that the short-term Ang II signalling which mediates the endocrine and haemodynamic effects of Ang II is blunted [10,11] and likely represents an important mechanism responsible for their characteristic pattern of clinical findings, i.e. reduced peripheral resistance, vascular hyporeactivity and normo-hypotension [10,11]. However, noting the growing connection of Ang II to inflammation, we felt that documenting the effects of elevated Ang II levels found in BS/GS with respect to inflammation would provide additional insights. The current study documents our findings in BS/GS patients with respect to SAA, IL6 as well as CRP, as considerable inflammatory markers of much current interest. We found that, despite elevated Ang II levels, this group of inflammatory markers were all unchanged in BS/GS patients when compared with those found in normal subjects. These results add yet more details to our earlier findings, all of which show that despite high Ang II, BS/GS patients do not exhibit the expected Ang II-related inflammatory mediated cardiovascular remodelling. These results, moreover, do suggest the involvement of NF-{kappa}B and the possibility of an impaired Ang II-mediated NF-{kappa}B induction in BS/GS [14], which, however, remains to be demonstrated in these patients.

In contrast to the unchanged levels of SAA, IL-6 and CRP compared with those of the normal healthy controls, TNF-{alpha} was elevated while ICAM and VCAM levels did not show any difference in BS/GS compared with normal subjects. The most plausible, although indirect, and speculative explanation for this increased TNF-{alpha}, could come by the presence of elevated levels of NO that we have shown to be characteristic of these patients [15, 17]. Increased NO has been reported to affect TNF-{alpha} activity- as Cauwels et al. [18] have documented that endogenous NO plays an important role in curbing TNF-related pro-oxidative activity. TNF-related systems might react to an NO-induced decline in this TNF-related pro-oxidative activity by increasing the TNF level. However, whatever the mechanisms for this increase in TNF-{alpha} in BS/GS, they are not reflected by increases in either ICAM and VCAM noted in the current study, although TNF-{alpha} is a regulator of their expression [19]. In addition, the elevations of TNF-{alpha} are not reflected in any endothelial function changes nor increased heart disease risk, as studies in our laboratory have documented unimpaired endothelial function in these patients by means of direct plethysmographic evaluations of vascular tone, such as forearm blood flow [17].

In summary, the results of these studies further stress the critical role of Ang II in controlling vascular biology, including inflammation-related processes as well as highlighting the utility of BS/GS as a useful human model in investigating these pathways.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Undurti N Das. Is angiotensin II an endogenous pro-inflammatory molecule? Med Sci Monit 2005; 11: 155–162
  2. Alexander RW. Hypertension and the pathogenesis of atherosclerosis: oxidative stress and the mediation of arterial inflammatory response, a new perspective. Hypertension 1995; 25: 155–161[Abstract/Free Full Text]
  3. Tham DM, Martin-McNulty B, Wang YX et al. Angiotensin II is associated with activation of NF-{kappa}B mediated genes and downregulation of PPARs. Physiol Genom 2002; 11: 21–31[Abstract/Free Full Text]
  4. Ruiz-Ortega M, Ruperez M, Lorenzo O et al. Angiotensin II regulates the synthesis of proinflammatory cytokines and chemokines in the kidney. Kidney Int 2002; 62 [Suppl 82]: 12–22[CrossRef]
  5. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: Part II: animal and human studies. Circulation 2003; 108: 2034–2040[Free Full Text]
  6. Touyz RM. Role of angiotensin II in regulating vascular structural and functional changes in hypertension. Curr Hypertens Rep 2003; 5: 155–164[Web of Science][Medline]
  7. Lassegue B, Griendling KK. Reactive oxygen species in hypertension. Am J Hypertens 2004; 17: 852–860[CrossRef][Web of Science][Medline]
  8. Nathan C. Points of control in inflammation. Nature 2002; 446–452
  9. Himmelfarb J. Linking oxidative stress and inflammation in kidney disease: which is the chicken and which is the egg? Semin Dial 2004; 17: 449–454[CrossRef][Web of Science][Medline]
  10. Calò LA, Pessina AC, Semplicini A. Angiotensin II signaling in the Bartter's and Gitelman's syndromes, a negative human model of hypertension. High Blood Press Cardiovasc Prev 2005; 12: 17–26[CrossRef]
  11. Calò LA. Vascular tone control in humans: the utility of studies in Bartter's/Gitelman's syndromes. Kidney Int 2006; 69: 963–966[CrossRef][Web of Science][Medline]
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  13. Calò L, Davis PA, Semplicini A. Bartter's/Gitelman's syndrome: a model for the relationships between hypertension, angiotensin II, oxidative stress and remodeling. Clin Nephrol 2003; 59: 393–394[Web of Science][Medline]
  14. Schmeisser A, Soehnlein O, Illmer T et al. ACE inhibition lowers angiotensin II-induced chemokine expression by reduction of NF-{kappa}B activity and AT1 receptor expression. Biochem Biophys Res Commun 2004; 325: 532–540[CrossRef][Web of Science][Medline]
  15. Calò L, Davis PA, Milani M et al. Increased endothelial nitric oxide synthase mRNA level in Bartter's and Gitelman's syndrome. Relationship to vascular reactivity. Clin Nephrol 1999; 51: 12–17[Web of Science][Medline]
  16. Calò L, D’Angelo A, Cantaro S et al. Increased urinary NO2–/NO3– and cyclic GMP levels in patients with Bartter's syndrome: relationship to vascular reactivity. Am J Kidney Dis 1996; 27: 874–879
  17. Calo L, Cantaro S, Calabro A et al. Endothelium-derived vasoactive substances in Bartter's syndrome. Angiology 1995; 46: 905–913[Medline]
  18. Cauwels A, Bultinck J, Brouckaert P. Dual role of endogenous nitric oxide in tumor necrosis factor shock: induced NO tempers oxidative stress. Cell Mol Life Sci 2005; 62: 1632–1640[CrossRef][Medline]
  19. Min JK, Kim YM, Kim SW et al. TNF-related activation-induced cytokine enhances leukocyte adhesiveness: induction of ICAM-1 and VCAM-1 via TNF receptor-associated factor and protein kinase C-dependent NF{kappa}B activation in endothelial cells. J Immunol 2005; 175: 531–540[Abstract/Free Full Text]
Received for publication: 31. 1.06
Accepted in revised form: 21. 2.06


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