High Blood Press Cardiovasc Prev DOI 10.1007/s40292-015-0082-7
REVIEW ARTICLE
Angiotensin II and Cardiovascular-Renal Remodelling in Hypertension: Insights from a Human Model Opposite to Hypertension Verdiana Ravarotto1 • Elisa Pagnin1 • Antonio Fragasso1 • Giuseppe Maiolino1 Lorenzo A. Calo`1
•
Received: 7 January 2015 / Accepted: 4 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Insights into the Angiotensin II (Ang II) signalling pathways have been provided by extensive studies using Bartter’s/Gitelman’s syndromes patients. These syndromes are characterized by activation of the reninangiotensin-aldosterone system but do not develop hypertension and cardiovascular remodelling, therefore represent a mirror image of hypertension and clinically manifest themselves as the opposite of hypertension. The short and the long-term signalling of Ang II remain an important matter of investigation to shed light on mechanisms responsible for the pathophysiology of hypertension and its long-term complications, such as cardiovascular remodelling and atherogenesis. In particular the long-term signalling of Ang II is involved in the pathophysiology of cardiovascular-renal remodelling, inflammatory and hypertrophic responses in which the relationship between RhoA/Rho kinase pathway and NO system plays a crucial role. This review reports the results of our studies in Bartter’s and Gitelman’s syndromes to get better insight these processes and the role of Ang II signaling. The information obtained from the studies in Bartter’s/Gitelman’s patients can, in fact, clarify, confirm or be used to gather more general data on the biochemical mechanisms responsible for the pathophysiology of hypertension and its long-term complications and could contribute to identify additional potential significant targets of therapy.
& Lorenzo A. Calo` [email protected] 1
Department of Medicine, Nephrology and Hypertension, University of Padova, Via Giustiniani, 2, 35128 Padua, Italy
Keywords Rho kinase Nitric oxide Angiotensin II signaling Gitelman’s syndrome Bartter’s syndrome Cardiovascular-renal remodeling
1 Introduction Angiotensin II (Ang II) signaling is central in the control and regulation of vascular tone and, in general, in cardiovascular-renal physiology and pathophysiology [1–3]. Persistent activation of the renin-angiotensin-aldosterone system (RAAS) in healthy individuals leads to hypertension and target organ damage [4]. Despite the increases in the signaling molecules associated with activation of the pressor systems that induce hypertension and its long term complications, Bartter’s and Gitelman’s patients exhibit normotension or hypotension. Bartter’s and Gitelman’s patients have normal Ang II receptor number and affinity [5, 6] and activated RAAS, but show clinical signs related to abnormal RAAS activation, suggesting that in these patients Ang II signaling is blocked or interrupted at postreceptor level. This further suggests a condition wherein the countervailing signals that oppose RAAS are, in Bartter’s and Gitelman’s patients, activated and thus are an in-vivo human model of endogenous antagonism of Ang II signaling via Ang II type 1 receptors (AT1R) [7]. Thus, Bartter’s and Gitelman’s patients offer a unique opportunity to explore the mechanisms controlled by Ang II signaling responsible for controlling the vascular tone and cardiovascular-renal remodeling in humans. This article reports the results of our studies in Bartter’s and Gitelman’s syndromes to gain better insight the role of Ang II signaling in the processes involved in particular in the long term signalling of Ang II leading to cardiovascular-renal remodeling, which involve RhoA/Rho kinase
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pathway and NO systems and the importance of the balance between these systems.
2 Bartter’s and Gitelman’s Syndromes Bartter and Gitelman’s syndromes are rare autosomal recessive diseases characterized by electrolytic alterations due to monogenetic mutations in different genes encoding for renal tubular transporters or channels (Fig. 1). The two syndromes differ by the symptoms onset timing and for the genes involved, but both are characterized by the hypokalemia, metabolic alkalosis, activation of RAAS yet normotension or hypotension. Bartter [8] first described in 1962 two cases of young patients presenting hypokalemic alkalosis, aldosteronism, high levels of angiotensin yet normotension. Bartter’s patients have also salt wasting and hyper or normocalciuria. Typical clinical features are present in childhood with muscle weakness, anorexia, polydipsia, polyuria, failure to thrive and growth retardation. Classic Bartter’s syndrome reveals in childhood or adolescence but the syndrome has a neonatal form. Patients show activation of the RAAS with high plasma renin activity, Ang II levels and hyperaldosteronism, yet normotension or hypotension. A hallmark of this syndrome is salt-wasting and hyporesponsiveness to pressor agents [9] High plasma level of prostaglandins and hyperprostaglandinuria are also present. The electrolyte abnormalities of Bartter’s syndrome are similar to those induced by treatment with furosemide or
A
other drugs that inhibit the Na-K-2Cl cotransporter of the thick ascending limb of Henle’s loop (TAL), which prompted investigations of the gene NKCC2/SLC12A1 encoding the Na-K-2Cl cotransporter [10]. In affected patients as a consequence of the mutations, there is a loss of cotransporter function, which induces Na and K wasting in the TAL. NKCC2 mutation classifies Bartter’s syndrome type I. Other mutations, are found in genes involved in the regulation of the NKCC2 activity. In particular, alterations in the apical ATP-sensitive K channel (renal outer medullary potassium channel, ROMK) identify Bartter’s syndrome type II. The ROMK channels recycles K? from the cell back into the lumen, and as a consequence, the luminal K? falling shuts down NKCC2 activity and results in salt wasting [11]. Mutations in the chloride channel CLCNKb (which mediates Cl- reabsorption) might induce intracellular chloride accumulation, which inhibits Na-K-2Cl cotransporter activity. Experimental evidence demonstrated mutations in CLCNKb in affected patients resulting in salt wasting and hypovolaemia. The presence of this mutation identifies Bartter’s syndrome type III [12]. The Bartter’s syndrome type IV is a result of a mutation in the gene encoding the regulatory protein Barttin (BSND), which is required for location of the basolateral membrane Cl channels CLCNKb and CLCNKa. As in the Bartter’s syndrome type III, the consequence is an accumulation of chloride, which affects NCCK2 activity once more with salt wasting [9]. Another phenotype described, Bartter’s syndrome type V, is due a mutation in the Ca2?sensing receptor (CaSR). The gain-of-function of the CaSR
B
Lumen
Blood
Lumen
Blood Gitelman’s Syndrome
ATP
K+ 2ClNa+
Barer’s Syndrome Type I ROMK Barer’s Syndrome Type II
Ca2+ Mg2+
NCCT
Na+
NKCC2
K+
ClC-Kb Cl-
Barer Syndrome’s Type III
CaSR
Cl-
Barer’s Syndrome Type IV
ClC-Kb ClNa+ ATP K+
Gitelman’s Syndrome
Barn K+
Na+
Na +
Ca2+
Ca2+ Na +
Mg2+
Mg2+
Barer’s Syndrome Type V
Fig. 1 Transport pathways in the thick ascending limb of Henle’s loop depicting the five (I–V) types of Bartter syndrome (a). Transport pathways in the distal convoluted tubule depicting the abnormality of Gitelman’s syndrome (b)
Angiotensin II and Cardiovascular-Renal Remodelling
in the parathyroid controls the response of the gland to the extracellular Ca2? levels and thus hormone release. This channel also present in the basolateral membrane of the TAL inhibits NKCC2 activation contributing to the furosemide-like action [9]. Gitelman syndrome [13] was first described in 1966 as a variant of Bartter’s syndrome. It has a clinical presentation less severe than Barrter’s syndrome and present symptoms in adulthood. It is characterized by hypokalemia, metabolic alkalosis, salt wasting, hypomagnesemia and hypocalciuria. The biochemical hallmark of Gitelman’s syndrome is the co-existence of hypokalemia, hypomagnesaemia and hypocalciuria. Marked symptoms are muscle weakness, cramps, joint pain, polydipsia, polyuria, hypotension, saltcraving, dizziness and a prolongation of QT interval on electrocardiogram [14, 15]. Patients can also have calcium pyrophosphate crystal deposition or chondrocalcinosis associated to arthropathy [16]. To understand the pathogenesis of this syndrome, investigations prompted attention to an association with the side effects of the thiazide diuretics. The target of this type of diuretics is the sodium-chloride cotransporter (NCC) present in the distal convoluted tubule. The cloning and characterization of the gene encoding this channel, the SLC12A3 gene, demonstrated in Gitelman’s syndrome to be present numerous mutations leading to malfunction of the cotransporter, with consequent hypokalemia, sodium wasting and metabolic alkalosis [17] (Fig. 1). In both Bartter’s and Gitelman’s syndromes is characteristically present marked RAAS stimulation with elevated plasma renin activity, high plasma Ang II level and hyperaldosteronism. Notwithstanding the activation of this pathway, patients show reduced peripheral resistance and normal or low blood pressure and resistance to the pressor effect of vasoconstrictors as Ang II and norepinephrine [7].
The activation of mechanisms involved in the reduction of the blood pressure despite an activation of the RAAS, make Bartter’s and Gitelman’s syndromes a ‘‘mirror image’’ of hypertension and a human model of an endogenous antagonism of Ang II signaling via AT1R (Table 1). Insights in the mechanisms involved upon Ang II receptors stimulation could provide the identification of novel potential targets of therapy in diseases such as hypertension in which treatments consist in the direct inhibition of Ang II synthesis or activity. ACE inhibitors, Ang II AT1R blockers and direct renin inhibitors are, in fact, currently used as antihypertensive drugs. However, the potential of these treatments goes far beyond the blood pressure reduction with evidence demonstrating amelioration of cardiovascular diseases morbidity and mortality and slowing the progression of kidney diseases. This evidence correlates with a complex Ang II activity, which has clearly a pleiotropic nature confirmed by the presence of numerous enzymes, peptides and receptors [18, 19]. The multi-effects function of Ang II is exerted trough its short or long-term signaling. The specific intracellular pathways of the short-term signaling are mediated by monomeric and heterotrimeric G proteins and phospholipase Cb (PLCb) thus leading the release of intracellular messengers inositol trisphosphate (IP3) and Ca2?, generation of superoxide and the activation of protein kinase C (PKC) with ensuing vascular smooth muscle contraction [9]. Noteworthy, a counterbalancing system is represented by the nitric oxide (NO) system, which has vasodilatory and antiproliferative activity. NO is released by endothelial
Table 1 Ang II short and long term signaling opposite effects described in Bartter’s/Gitelman’s syndrome and essential hypertension (references in the text) Ang II short and long term signaling effects
Bartter’s/ Gitelman’s syndrome
Hypertension
3 Angiotensin II and the Control of Vascular Tone
Intracellular Ca2? release
Diminished
Increased
Intracellular IP3 level
Diminished
Increased
Peculiar characteristics in the pathophysiology of Bartter’s and Gitelman’s syndromes may provide insights useful for the knowledge of the mechanisms involved in controlling and regulating vascular tone and blood pressure in humans. In healthy individuals Ang II is directly involved in the activation of RAAS signaling leading to increased peripheral resistance and hypertension. Studies from our laboratory provided evidence in Bartter’s and Gitelman’s patients of a blunted signaling of Ang II despite higher level of the hormone and a normal Ang II receptor number and affinity [7]. This suggests that Ang II signaling is interrupted at post receptor level or very close to the central switch controlling Ang II signals.
PKC expression and activity
Diminished
Increased
NO system
Upregulated
Downregulated
ecNOS expression
Increased
Diminished
NO dependent relaxation
Increased
Diminished
Gaq expression
Diminished
Increased
RGS-2 expression
Increased
Diminished
Rho kinase expression/ activity
Diminished
Increased
Oxidative stress Inflammatory state
Diminished Diminished
Increased Increased
Insulin resistance
Diminished
Increased
Cardiovascular-renal target organ damage
Lack
Present
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NO synthase (eNOS) and is negatively regulated by PKC, therefore, the net effect of Ang II on vascular function and structure is the result of the balance of signaling molecules, oxidative stress and gasomessengers as nitric oxide [20, 21]. Ang II promotes short-term effects by acting directly on its receptors on the cells membranes, but also has indirect long-term effects mediated by other factors leading to proliferation and remodeling [22]. The binding of Ang II with its heterotrimeric G-proteincoupled receptors promotes the increase of free intracellular Ca2? concentration and the activation of the RhoA/ Rho kinase pathway with subsequent vasoconstriction. In hypertension Gq and Gi proteins are involved to mediate the activation of the PLC. The Ang II-AT1R complex couples with PLCb and promotes activation of PKC and phosphorylation of the regulatory chain of myosin II. In addition, the activation of the monomeric G-protein RhoA and its effector Rho kinase modulates the phosphorylation state of the regulatory chain of myosin II, mainly through inhibition of the myosin phosphatase target protein-1 (MYPT-1). These Ang II mediated pathways have the same final outcome to induce vasoconstriction and increased peripheral resistance, while at the same time NO pathway is reduced [7] (Table 1). Bartter’s and Gitelman’s syndromes, despite the higher Ang II plasma levels and the activation of the RAAS, have decreased gene and protein expression of the a subunit of Gq protein and blunted downstream intracellular events that promote Ca2? release and PKC activation [7, 23, 24]. In addition, in these patients the evaluation of the NO system via the endothelial nitric oxide synthase mRNA levels, urinary excretion of NO metabolites and NO mediated vasodilation showed a significant increase of eNOS expression [25, 26], an increased urinary excretion of NO metabolites, which positively correlated with urinary excretion of cyclic guanosine monophosphate (cGMP), the NO second messenger [25, 27, 28], and an increased NO mediated vasodilation compared with hypertensive patients [29] (Table 1). To these characteristics, which also underlie an antioxidant potential in these patients, it has to be added the demonstration of augmented expression of heme oxygenase 1 (HO-1) [30, 31], known antioxidant and antiinflammatory enzyme. The regulation of G protein-coupled receptors (GPCR) activity is critical for the integration of information and subsequent downstream signaling. Regulators of G protein signaling (RGS) proteins are essential for this purpose [32]. RGS proteins can regulate many different effector proteins acting as GTPase-activating proteins for Ga subunits and also competitively inhibit Ga binding to PLC [33]. Both NO and its second messenger, cGMP, increase the RGS-2 guanosine triphosphatase activity of Gq protein,
which leads to dephosphorylation of myosin light chain (MLC) and induces vascular smooth muscle cell relaxation. Collectively, these findings suggest that RGS-2 is central for the vasorelaxing activity of NO [34]. The relevance of RGS proteins in hypertension has been demonstrated in RGS-2 knockout mice, which showed persistent vasoconstriction and hypertension [34]. The characteristic normotension or hypotension of Bartter’s and Gitelman’s syndromes, upon Ang II stimulation, suggests that abnormalities in the GPCR complex and in its regulator RGS-2 might be involved [1]. Patients with Bartter’s and Gitelman’s syndrome have, in fact, an increased gene and protein expression of RGS-2 [35], which is the opposite of the decrease seen in hypertensive patients [36] (Table 1). The increased RGS-2 expression in Bartter’s and Gitelman’s patients may explain their downregulation of Gq protein signaling [23–25] and the reduced peripheral resistance, vascular hyporeactivity, and normotension or hypotension, typical of these patients. Silencing RGS-2 in Bartter’s and Gitelman’s patients fibroblasts, in fact, produces effects [37] that coincide with the results in knockout mice and humans [34, 36, 38].
4 Angiotensin II and Cardiovascular-Renal Remodeling in Hypertension Long-term signaling of Ang II promotes changes in the cell oxidative state and leads to cardiovascular and renal remodeling in hypertension, atherosclerosis, heart and kidney failure [1, 39, 40]. Oxidative stress is due to an imbalance between oxidant and antioxidant agents and the loss of redox homeostasis due to increased pro-oxidant and pro-thrombotic activities, causes harmful effects, in terms of free radicals and nitroxidative stress. There are many sources of reactive oxygen species: NAD(P)H oxidases, Xantine oxidases, P-450 monoxygenases, Lipoxygenases and cycloxygenases. All these enzymes produce superoxide anion, which is an intermediate product of oxygen reduction. Its pathophysiological relevance is the induction of high reactive compounds as hydroxyl radical (OH) and peroxynitrite (OONO-). The main oxidative stress inducer is NAD(P)H oxidase (Nox) that catalyzes the production of superoxide from oxygen and NAD(P)H [41]. Noxs are characterized by a catalytic subunit and a little p22phox subunit able to form the heterodimeric cytochrome b558. Once p22phox and Nox are associated, phosphorylation cascades involving cytosolic subunits induce translations to the membrane and conformational changes in both the enzyme and the cellular membrane [41]. Specific agonists induce the activation of Nox to promote its
Angiotensin II and Cardiovascular-Renal Remodelling
signaling sequelae including the ability to produce superoxide anion (O2-) through oxygen reduction. The generated O2- initiate the production of free radicals and reactive oxidants, which are involved in atherosclerotic lesions and cardiovascular and renal remodeling [40, 41]. In Bartter’s and Gitelman’s syndromes the response of the NAD(P)H oxidase to Ang II is reduced. Ang II stimulation produces in these patients a reduced oxidative state (Table 1) in terms of decreased p22phox gene expression [30]. The decrease of the oxidant potential was related to a decrease in the OONO- [30] and to an increase in the HO-1 gene expression [30], known antioxidant enzyme. In addition, Bartter’s and Gitelman’s patients shows a reduced susceptibility of the low density lipoprotein (LDL) to oxidation [31]. Oxidated LDL are deeply involved in the progression of the atherosclerotic lesions both in the cardiovascular and renal systems and the increased NO production in these patients has also a role in the protection of LDL from oxidation [31]. Furthermore, the reduced production of O2- results in these patients in an increased bioavailability of NO, which is spared from a reaction with O2- to produce OONO- [30]. Another effect related with the reduction of oxidative stress is, in Bartter’s and Gitelman’s patients, the reduced gene and protein expression of the atherothrombogenic factor plasminogen activator inhibitor-1 (PAI-1) upon the Ang II challenge [42]. Ang II stimulation promotes long-term fibrotic outcomes, particularly involving cytokines. The profibrotic cytokine transforming growth factor b (TGFb) controls proliferation, cell differentiation and in the kidney is cause of fibrosis. Bartter’s and Gitelman’s patients exhibit a reduced gene expression of TGFb [30].
5 The RhoA/Rho Kinase/NO System Relationship The activation of the RhoA/Rho kinase pathway, downstream of Ang II AT1R stimulation, in addition to lead to vasoconstriction via agonist-induced Ca2? sensitization in smooth muscle contraction [43], is a key mechanism for the induction of cardiovascular-renal remodeling [43–46] via modulation of the phosphorylation state of the regulatory chain of myosin II, mainly through inhibition of MYPT-1. In these effects a very important role is played by the downregulation of eNOS activity and the existence of an inverse relationship between RhoA/Rho kinase expression and activity with NO bioavailability [47, 48]. Basal release of NO from the endothelium, in fact, prevents the activation of RhoA/Rho kinase in smooth muscle through protein kinase G-dependent inhibitory phosphorylation of RhoA [47, 48]. On the other hand, excessive production of reactive oxygen species inactivate NO, leading to a blunted
NO bioactivity and increased vascular tone and cardiovascular-renal remodeling, at least in part through elevated RhoA–Rho kinase activity [47–49]. The importance of the balance between the RhoA/Rhokinase pathway and the NO system is crucial in cardiovascular-renal pathophysiology being involved in: (i) changes in cardiovascular-renal structure (remodeling) and induction of atherosclerosis [50–53]; (ii) involvement in the pathophysiological relationships between inflammation and hypertension [54]; (iii) and in those between hypertension, glucose metabolism and insulin resistance [55]. The involvement of RhoA/Rho-kinase in these processes is based on: (i) the RhoA/Rho-kinase activation of NAD(P)H oxidase [51], with the induction of oxidative stress; (ii) RhoA/Rho-kinase control of the atherothrombogenetic PAI-1 [56]; (iii) the suppression of vascular neointimal formation by the inhibition of Rho-kinase [57]; (iv) the demonstration of RhoA/Rho-kinase signaling involvement in C-reactive protein (CRP)-induced atherothrombogenesis through the CRP-mediated RhoA/ Rhokinase- induced increased nuclear factor kappa B (NFjB) activity, resulting in increase of the PAI-1 [58] and (v) activation of the phosphoinositol 3 kinase (PI3K)/Akt activation of eNOS by Rho-kinase inhibition, which leads to cardiovascular-renal protection and prevention of atherogenesis [59]; (vi) an abnormality in glucose transport and metabolism leading to insulin resistance by the angiotensin II-induced [55] and RhoA/Rho-kinase mediated inhibition of PI3K/Akt [59]. The activation of RhoA is upregulated by Rho guanine nucleotide exchange factors (RhoGEFs) that catalyze the exchange of GDP to GTP [60]. This exchange leads to a conformational change of the G protein, allowing regulation of downstream effectors. In addition, they can act as sensors for signals coming from activated G-protein-coupled receptors, including those coming from Ang II AT1R stimulation, that are coupled to the heterotrimeric Gq protein [61]. In spontaneously hypertensive rats RhoGEFs are overexpressed [62] and p63RhoGEF plays a mayor role in the Ang-II-mediated early RhoA/Rho kinase activation [63, 64]. This, in addition to the contribution of Ang-II-mediated RhoA activation by p115RhoGEF [65], leads to vascular contraction, cell proliferation and cardiovascular remodeling [43]. In Bartter’s and Gitelman’s patients the activity of RhoA/Rho kinase pathway is blunted (Table 1). We have shown in these patients a reduced expression and response of Rho kinase to Ang II challenge, which indicates downregulation of RhoA/Rho kinase pathway [42, 43]. Rho kinase downregulation in Bartter’s and Gitelman’s patients was associated with upregulation of NO system and increased NO-mediated vasodilation (Table 1), partly
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explained by increased expression of the eNOS, compared with both hypertensive as well as healthy normotensive individuals [7, 25, 29]. These findings in humans parallel the upregulation of NO system upon Rho kinase inhibition found in vitro and in vivo in hypertensive rats, and suggested to lead to cardiovascular protection [59, 66, 67]. The increased expression in Bartter’s and Gitelman’s patients [30], of the antioxidant HO-1 [68] fits with the downregulation of RhoA/Rho kinase pathway in these patients given that the activation of Akt pathway, which induces HO-1 expression [69], is reported after Rho kinase inhibition [59]. Finally the downregulation RhoA/Rho kinase pathway [43] is supported in these patients by the reduced mRNA and protein expression of the Ang-II-induced p115RhoGEF [70] and, more importantly, of p63RhoGEF [71]. Moreover these patients have decreased phosphorylation of MYPT-1 [71], a marker of Rho kinase activity [44], which conversely is increased in hypertensive patients [71]. Rho kinase, in fact, via an inhibitory phosphorylation of MYPT-1, increases the activity of MLC kinase, leading to smooth muscle contraction and cardiovascular and renal remodeling [72]. In Bartter’s and Gitelman’s patients Ang II signaling via AT2R is activated (Table 1), as evidenced by increased Ang-II-induced expression of the mitogen-activated protein kinase phosphatase-1, a main effector of AT2R signaling [6]. AT2R stimulation evokes opposing effects on vascular tone compared to those mediated by AT1R stimulation, such as vasodilation and triggers antiproliferative signals. The activation of Ang II signaling via AT2R in these patients likely plays a major role in the mechanism(s) that produce the blunted Ang II signaling via AT1R and the related pathways, and offers additional support regarding the role of Ang II AT2R signaling in the inhibition of Ang II-mediated, p63RhoGEF/Gaq-induced RhoA/Rho kinase activation, for the cardiovascular protective effects of AT1R blockers beyond AT1R blockade [73]. Finally, the lack of cardiovascular remodeling in terms left ventricular hypertrophy [74] and carotid artery intima media thickness [29] in these patients despite high Ang II, also fits with the reduced RhoA/Rho kinase activity and with the activation of NO system [7]. The nature of all these responses in these patients may provide additional evidence for AT2R signaling’s counter-regulatory roles of AT1R signaling, including the regulation of the balance between RhoA/Rho kinase activity and NO system [75, 76]. Not only decreased oxidant potential, but also anti-inflammatory potential is another characteristic of Bartter’s and Gitelman’s syndromes, likely as consequence of the blunted Ang II signaling via AT1R, the activation of AT2R signaling, the reduced RhoA/Rho kinase activity and the upregulation of NO system (Table 1). These patients, despite elevated Ang II, have unchanged levels of C-reactive
protein (CRP) and other inflammatory mediators, such as vascular cell adhesion molecule (VCAM), intracellular cell adhesion molecule (ICAM), interleukin (IL)-6 [77], whose expression depends on NFjB activity [78, 79]. Inflammation has a key role in hypertension, atherosclerosis and cardiovascular and renal diseases and their progression is mediated by many pro-inflammatory elements and cytokines, most of which are induced by Ang II which is itself a pro-inflammatory molecule [80]. Rho kinase signaling is activated by CRP, increases NFjB activity [58, 81] and blocking Rho kinase pathway inactivates NFjB induced inflammation [82]. Compared with normotensive healthy individuals, Bartter’s and Gitelman’s patients have unchanged NFjB levels but likely reduced activity due to the increased IjB expression, the inhibitory subunit of NFjB. An increased activity of IjB would therefore lead to reduced transcription of gene related to inflammation and remodeling [83].
6 Conclusions Bartter’s and Gitelman’s patients are a human model of endogenously blunted and blocked Ang II signaling, which causes these patients to present with findings and processes that are the exact opposite of those described in hypertension (Table 1). The extensive series of studies from our laboratory on Ang II signaling using Bartter’s and Gitelman’s patients, have traced key regulatory elements of this signaling such as RGS-2, Gaq protein, NO system and Rho kinase pathways and the findings from these studies have confirmed in this human model their importance in vascular tone regulation and cardiovascular-renal remodeling. The importance of the RhoA/Rho-kinase pathway and its close relationships with Ang II and NO bioavailability in cardiovascular-renal biology, make this pathway of essential importance in a wide spectrum of conditions in cardiovascular-renal pathophysiology. An imbalance of the RhoA/Rho-kinase and NO systems towards an increase of the RhoA/Rho-kinase pathway is determinant not only for the induction of hypertension and for conditions such as insulin resistance and glucose intolerance, known often to be associated with hypertension, but also with long-term complications of hypertension such as cardiovascular-renal remodeling and atherogenesis. For these important consequences of RhoA/Rho-kinase activation and reduction of NO system, RhoA/Rho kinase pathway is considered a potential target for cardiovascular-renal protection [45]. The overall clinical, biochemical and molecular picture of Bartter’s and Gitelman’s syndromes may therefore contribute to understanding the pathophysiological mechanisms in humans involved in cardiovascular-renal remodeling and linking RhoA/Rho-kinase signaling and the
Angiotensin II and Cardiovascular-Renal Remodelling
NO system with hypertension and its relationships with inflammation, insulin resistance, atherogenesis and remodeling. The insights into mechanisms responsible for the control of vascular tone and cardiovascular-renal remodeling derived by studies in this human model opposite to hypertension, in particular those on RhoA/Rho kinase pathway/NO system/oxidative stress relationships, have identified, confirmed and extended the knowledge of the processes evoked by Ang II signaling, highlighted their crucial importance in mediating Ang II effects and could contribute to identify additional potential significant targets of therapy.
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REVIEW ARTICLE
Angiotensin II and Cardiovascular-Renal Remodelling in Hypertension: Insights from a Human Model Opposite to Hypertension Verdiana Ravarotto1 • Elisa Pagnin1 • Antonio Fragasso1 • Giuseppe Maiolino1 Lorenzo A. Calo`1
•
Received: 7 January 2015 / Accepted: 4 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Insights into the Angiotensin II (Ang II) signalling pathways have been provided by extensive studies using Bartter’s/Gitelman’s syndromes patients. These syndromes are characterized by activation of the reninangiotensin-aldosterone system but do not develop hypertension and cardiovascular remodelling, therefore represent a mirror image of hypertension and clinically manifest themselves as the opposite of hypertension. The short and the long-term signalling of Ang II remain an important matter of investigation to shed light on mechanisms responsible for the pathophysiology of hypertension and its long-term complications, such as cardiovascular remodelling and atherogenesis. In particular the long-term signalling of Ang II is involved in the pathophysiology of cardiovascular-renal remodelling, inflammatory and hypertrophic responses in which the relationship between RhoA/Rho kinase pathway and NO system plays a crucial role. This review reports the results of our studies in Bartter’s and Gitelman’s syndromes to get better insight these processes and the role of Ang II signaling. The information obtained from the studies in Bartter’s/Gitelman’s patients can, in fact, clarify, confirm or be used to gather more general data on the biochemical mechanisms responsible for the pathophysiology of hypertension and its long-term complications and could contribute to identify additional potential significant targets of therapy.
& Lorenzo A. Calo` [email protected] 1
Department of Medicine, Nephrology and Hypertension, University of Padova, Via Giustiniani, 2, 35128 Padua, Italy
Keywords Rho kinase Nitric oxide Angiotensin II signaling Gitelman’s syndrome Bartter’s syndrome Cardiovascular-renal remodeling
1 Introduction Angiotensin II (Ang II) signaling is central in the control and regulation of vascular tone and, in general, in cardiovascular-renal physiology and pathophysiology [1–3]. Persistent activation of the renin-angiotensin-aldosterone system (RAAS) in healthy individuals leads to hypertension and target organ damage [4]. Despite the increases in the signaling molecules associated with activation of the pressor systems that induce hypertension and its long term complications, Bartter’s and Gitelman’s patients exhibit normotension or hypotension. Bartter’s and Gitelman’s patients have normal Ang II receptor number and affinity [5, 6] and activated RAAS, but show clinical signs related to abnormal RAAS activation, suggesting that in these patients Ang II signaling is blocked or interrupted at postreceptor level. This further suggests a condition wherein the countervailing signals that oppose RAAS are, in Bartter’s and Gitelman’s patients, activated and thus are an in-vivo human model of endogenous antagonism of Ang II signaling via Ang II type 1 receptors (AT1R) [7]. Thus, Bartter’s and Gitelman’s patients offer a unique opportunity to explore the mechanisms controlled by Ang II signaling responsible for controlling the vascular tone and cardiovascular-renal remodeling in humans. This article reports the results of our studies in Bartter’s and Gitelman’s syndromes to gain better insight the role of Ang II signaling in the processes involved in particular in the long term signalling of Ang II leading to cardiovascular-renal remodeling, which involve RhoA/Rho kinase
V. Ravarotto et al.
pathway and NO systems and the importance of the balance between these systems.
2 Bartter’s and Gitelman’s Syndromes Bartter and Gitelman’s syndromes are rare autosomal recessive diseases characterized by electrolytic alterations due to monogenetic mutations in different genes encoding for renal tubular transporters or channels (Fig. 1). The two syndromes differ by the symptoms onset timing and for the genes involved, but both are characterized by the hypokalemia, metabolic alkalosis, activation of RAAS yet normotension or hypotension. Bartter [8] first described in 1962 two cases of young patients presenting hypokalemic alkalosis, aldosteronism, high levels of angiotensin yet normotension. Bartter’s patients have also salt wasting and hyper or normocalciuria. Typical clinical features are present in childhood with muscle weakness, anorexia, polydipsia, polyuria, failure to thrive and growth retardation. Classic Bartter’s syndrome reveals in childhood or adolescence but the syndrome has a neonatal form. Patients show activation of the RAAS with high plasma renin activity, Ang II levels and hyperaldosteronism, yet normotension or hypotension. A hallmark of this syndrome is salt-wasting and hyporesponsiveness to pressor agents [9] High plasma level of prostaglandins and hyperprostaglandinuria are also present. The electrolyte abnormalities of Bartter’s syndrome are similar to those induced by treatment with furosemide or
A
other drugs that inhibit the Na-K-2Cl cotransporter of the thick ascending limb of Henle’s loop (TAL), which prompted investigations of the gene NKCC2/SLC12A1 encoding the Na-K-2Cl cotransporter [10]. In affected patients as a consequence of the mutations, there is a loss of cotransporter function, which induces Na and K wasting in the TAL. NKCC2 mutation classifies Bartter’s syndrome type I. Other mutations, are found in genes involved in the regulation of the NKCC2 activity. In particular, alterations in the apical ATP-sensitive K channel (renal outer medullary potassium channel, ROMK) identify Bartter’s syndrome type II. The ROMK channels recycles K? from the cell back into the lumen, and as a consequence, the luminal K? falling shuts down NKCC2 activity and results in salt wasting [11]. Mutations in the chloride channel CLCNKb (which mediates Cl- reabsorption) might induce intracellular chloride accumulation, which inhibits Na-K-2Cl cotransporter activity. Experimental evidence demonstrated mutations in CLCNKb in affected patients resulting in salt wasting and hypovolaemia. The presence of this mutation identifies Bartter’s syndrome type III [12]. The Bartter’s syndrome type IV is a result of a mutation in the gene encoding the regulatory protein Barttin (BSND), which is required for location of the basolateral membrane Cl channels CLCNKb and CLCNKa. As in the Bartter’s syndrome type III, the consequence is an accumulation of chloride, which affects NCCK2 activity once more with salt wasting [9]. Another phenotype described, Bartter’s syndrome type V, is due a mutation in the Ca2?sensing receptor (CaSR). The gain-of-function of the CaSR
B
Lumen
Blood
Lumen
Blood Gitelman’s Syndrome
ATP
K+ 2ClNa+
Barer’s Syndrome Type I ROMK Barer’s Syndrome Type II
Ca2+ Mg2+
NCCT
Na+
NKCC2
K+
ClC-Kb Cl-
Barer Syndrome’s Type III
CaSR
Cl-
Barer’s Syndrome Type IV
ClC-Kb ClNa+ ATP K+
Gitelman’s Syndrome
Barn K+
Na+
Na +
Ca2+
Ca2+ Na +
Mg2+
Mg2+
Barer’s Syndrome Type V
Fig. 1 Transport pathways in the thick ascending limb of Henle’s loop depicting the five (I–V) types of Bartter syndrome (a). Transport pathways in the distal convoluted tubule depicting the abnormality of Gitelman’s syndrome (b)
Angiotensin II and Cardiovascular-Renal Remodelling
in the parathyroid controls the response of the gland to the extracellular Ca2? levels and thus hormone release. This channel also present in the basolateral membrane of the TAL inhibits NKCC2 activation contributing to the furosemide-like action [9]. Gitelman syndrome [13] was first described in 1966 as a variant of Bartter’s syndrome. It has a clinical presentation less severe than Barrter’s syndrome and present symptoms in adulthood. It is characterized by hypokalemia, metabolic alkalosis, salt wasting, hypomagnesemia and hypocalciuria. The biochemical hallmark of Gitelman’s syndrome is the co-existence of hypokalemia, hypomagnesaemia and hypocalciuria. Marked symptoms are muscle weakness, cramps, joint pain, polydipsia, polyuria, hypotension, saltcraving, dizziness and a prolongation of QT interval on electrocardiogram [14, 15]. Patients can also have calcium pyrophosphate crystal deposition or chondrocalcinosis associated to arthropathy [16]. To understand the pathogenesis of this syndrome, investigations prompted attention to an association with the side effects of the thiazide diuretics. The target of this type of diuretics is the sodium-chloride cotransporter (NCC) present in the distal convoluted tubule. The cloning and characterization of the gene encoding this channel, the SLC12A3 gene, demonstrated in Gitelman’s syndrome to be present numerous mutations leading to malfunction of the cotransporter, with consequent hypokalemia, sodium wasting and metabolic alkalosis [17] (Fig. 1). In both Bartter’s and Gitelman’s syndromes is characteristically present marked RAAS stimulation with elevated plasma renin activity, high plasma Ang II level and hyperaldosteronism. Notwithstanding the activation of this pathway, patients show reduced peripheral resistance and normal or low blood pressure and resistance to the pressor effect of vasoconstrictors as Ang II and norepinephrine [7].
The activation of mechanisms involved in the reduction of the blood pressure despite an activation of the RAAS, make Bartter’s and Gitelman’s syndromes a ‘‘mirror image’’ of hypertension and a human model of an endogenous antagonism of Ang II signaling via AT1R (Table 1). Insights in the mechanisms involved upon Ang II receptors stimulation could provide the identification of novel potential targets of therapy in diseases such as hypertension in which treatments consist in the direct inhibition of Ang II synthesis or activity. ACE inhibitors, Ang II AT1R blockers and direct renin inhibitors are, in fact, currently used as antihypertensive drugs. However, the potential of these treatments goes far beyond the blood pressure reduction with evidence demonstrating amelioration of cardiovascular diseases morbidity and mortality and slowing the progression of kidney diseases. This evidence correlates with a complex Ang II activity, which has clearly a pleiotropic nature confirmed by the presence of numerous enzymes, peptides and receptors [18, 19]. The multi-effects function of Ang II is exerted trough its short or long-term signaling. The specific intracellular pathways of the short-term signaling are mediated by monomeric and heterotrimeric G proteins and phospholipase Cb (PLCb) thus leading the release of intracellular messengers inositol trisphosphate (IP3) and Ca2?, generation of superoxide and the activation of protein kinase C (PKC) with ensuing vascular smooth muscle contraction [9]. Noteworthy, a counterbalancing system is represented by the nitric oxide (NO) system, which has vasodilatory and antiproliferative activity. NO is released by endothelial
Table 1 Ang II short and long term signaling opposite effects described in Bartter’s/Gitelman’s syndrome and essential hypertension (references in the text) Ang II short and long term signaling effects
Bartter’s/ Gitelman’s syndrome
Hypertension
3 Angiotensin II and the Control of Vascular Tone
Intracellular Ca2? release
Diminished
Increased
Intracellular IP3 level
Diminished
Increased
Peculiar characteristics in the pathophysiology of Bartter’s and Gitelman’s syndromes may provide insights useful for the knowledge of the mechanisms involved in controlling and regulating vascular tone and blood pressure in humans. In healthy individuals Ang II is directly involved in the activation of RAAS signaling leading to increased peripheral resistance and hypertension. Studies from our laboratory provided evidence in Bartter’s and Gitelman’s patients of a blunted signaling of Ang II despite higher level of the hormone and a normal Ang II receptor number and affinity [7]. This suggests that Ang II signaling is interrupted at post receptor level or very close to the central switch controlling Ang II signals.
PKC expression and activity
Diminished
Increased
NO system
Upregulated
Downregulated
ecNOS expression
Increased
Diminished
NO dependent relaxation
Increased
Diminished
Gaq expression
Diminished
Increased
RGS-2 expression
Increased
Diminished
Rho kinase expression/ activity
Diminished
Increased
Oxidative stress Inflammatory state
Diminished Diminished
Increased Increased
Insulin resistance
Diminished
Increased
Cardiovascular-renal target organ damage
Lack
Present
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NO synthase (eNOS) and is negatively regulated by PKC, therefore, the net effect of Ang II on vascular function and structure is the result of the balance of signaling molecules, oxidative stress and gasomessengers as nitric oxide [20, 21]. Ang II promotes short-term effects by acting directly on its receptors on the cells membranes, but also has indirect long-term effects mediated by other factors leading to proliferation and remodeling [22]. The binding of Ang II with its heterotrimeric G-proteincoupled receptors promotes the increase of free intracellular Ca2? concentration and the activation of the RhoA/ Rho kinase pathway with subsequent vasoconstriction. In hypertension Gq and Gi proteins are involved to mediate the activation of the PLC. The Ang II-AT1R complex couples with PLCb and promotes activation of PKC and phosphorylation of the regulatory chain of myosin II. In addition, the activation of the monomeric G-protein RhoA and its effector Rho kinase modulates the phosphorylation state of the regulatory chain of myosin II, mainly through inhibition of the myosin phosphatase target protein-1 (MYPT-1). These Ang II mediated pathways have the same final outcome to induce vasoconstriction and increased peripheral resistance, while at the same time NO pathway is reduced [7] (Table 1). Bartter’s and Gitelman’s syndromes, despite the higher Ang II plasma levels and the activation of the RAAS, have decreased gene and protein expression of the a subunit of Gq protein and blunted downstream intracellular events that promote Ca2? release and PKC activation [7, 23, 24]. In addition, in these patients the evaluation of the NO system via the endothelial nitric oxide synthase mRNA levels, urinary excretion of NO metabolites and NO mediated vasodilation showed a significant increase of eNOS expression [25, 26], an increased urinary excretion of NO metabolites, which positively correlated with urinary excretion of cyclic guanosine monophosphate (cGMP), the NO second messenger [25, 27, 28], and an increased NO mediated vasodilation compared with hypertensive patients [29] (Table 1). To these characteristics, which also underlie an antioxidant potential in these patients, it has to be added the demonstration of augmented expression of heme oxygenase 1 (HO-1) [30, 31], known antioxidant and antiinflammatory enzyme. The regulation of G protein-coupled receptors (GPCR) activity is critical for the integration of information and subsequent downstream signaling. Regulators of G protein signaling (RGS) proteins are essential for this purpose [32]. RGS proteins can regulate many different effector proteins acting as GTPase-activating proteins for Ga subunits and also competitively inhibit Ga binding to PLC [33]. Both NO and its second messenger, cGMP, increase the RGS-2 guanosine triphosphatase activity of Gq protein,
which leads to dephosphorylation of myosin light chain (MLC) and induces vascular smooth muscle cell relaxation. Collectively, these findings suggest that RGS-2 is central for the vasorelaxing activity of NO [34]. The relevance of RGS proteins in hypertension has been demonstrated in RGS-2 knockout mice, which showed persistent vasoconstriction and hypertension [34]. The characteristic normotension or hypotension of Bartter’s and Gitelman’s syndromes, upon Ang II stimulation, suggests that abnormalities in the GPCR complex and in its regulator RGS-2 might be involved [1]. Patients with Bartter’s and Gitelman’s syndrome have, in fact, an increased gene and protein expression of RGS-2 [35], which is the opposite of the decrease seen in hypertensive patients [36] (Table 1). The increased RGS-2 expression in Bartter’s and Gitelman’s patients may explain their downregulation of Gq protein signaling [23–25] and the reduced peripheral resistance, vascular hyporeactivity, and normotension or hypotension, typical of these patients. Silencing RGS-2 in Bartter’s and Gitelman’s patients fibroblasts, in fact, produces effects [37] that coincide with the results in knockout mice and humans [34, 36, 38].
4 Angiotensin II and Cardiovascular-Renal Remodeling in Hypertension Long-term signaling of Ang II promotes changes in the cell oxidative state and leads to cardiovascular and renal remodeling in hypertension, atherosclerosis, heart and kidney failure [1, 39, 40]. Oxidative stress is due to an imbalance between oxidant and antioxidant agents and the loss of redox homeostasis due to increased pro-oxidant and pro-thrombotic activities, causes harmful effects, in terms of free radicals and nitroxidative stress. There are many sources of reactive oxygen species: NAD(P)H oxidases, Xantine oxidases, P-450 monoxygenases, Lipoxygenases and cycloxygenases. All these enzymes produce superoxide anion, which is an intermediate product of oxygen reduction. Its pathophysiological relevance is the induction of high reactive compounds as hydroxyl radical (OH) and peroxynitrite (OONO-). The main oxidative stress inducer is NAD(P)H oxidase (Nox) that catalyzes the production of superoxide from oxygen and NAD(P)H [41]. Noxs are characterized by a catalytic subunit and a little p22phox subunit able to form the heterodimeric cytochrome b558. Once p22phox and Nox are associated, phosphorylation cascades involving cytosolic subunits induce translations to the membrane and conformational changes in both the enzyme and the cellular membrane [41]. Specific agonists induce the activation of Nox to promote its
Angiotensin II and Cardiovascular-Renal Remodelling
signaling sequelae including the ability to produce superoxide anion (O2-) through oxygen reduction. The generated O2- initiate the production of free radicals and reactive oxidants, which are involved in atherosclerotic lesions and cardiovascular and renal remodeling [40, 41]. In Bartter’s and Gitelman’s syndromes the response of the NAD(P)H oxidase to Ang II is reduced. Ang II stimulation produces in these patients a reduced oxidative state (Table 1) in terms of decreased p22phox gene expression [30]. The decrease of the oxidant potential was related to a decrease in the OONO- [30] and to an increase in the HO-1 gene expression [30], known antioxidant enzyme. In addition, Bartter’s and Gitelman’s patients shows a reduced susceptibility of the low density lipoprotein (LDL) to oxidation [31]. Oxidated LDL are deeply involved in the progression of the atherosclerotic lesions both in the cardiovascular and renal systems and the increased NO production in these patients has also a role in the protection of LDL from oxidation [31]. Furthermore, the reduced production of O2- results in these patients in an increased bioavailability of NO, which is spared from a reaction with O2- to produce OONO- [30]. Another effect related with the reduction of oxidative stress is, in Bartter’s and Gitelman’s patients, the reduced gene and protein expression of the atherothrombogenic factor plasminogen activator inhibitor-1 (PAI-1) upon the Ang II challenge [42]. Ang II stimulation promotes long-term fibrotic outcomes, particularly involving cytokines. The profibrotic cytokine transforming growth factor b (TGFb) controls proliferation, cell differentiation and in the kidney is cause of fibrosis. Bartter’s and Gitelman’s patients exhibit a reduced gene expression of TGFb [30].
5 The RhoA/Rho Kinase/NO System Relationship The activation of the RhoA/Rho kinase pathway, downstream of Ang II AT1R stimulation, in addition to lead to vasoconstriction via agonist-induced Ca2? sensitization in smooth muscle contraction [43], is a key mechanism for the induction of cardiovascular-renal remodeling [43–46] via modulation of the phosphorylation state of the regulatory chain of myosin II, mainly through inhibition of MYPT-1. In these effects a very important role is played by the downregulation of eNOS activity and the existence of an inverse relationship between RhoA/Rho kinase expression and activity with NO bioavailability [47, 48]. Basal release of NO from the endothelium, in fact, prevents the activation of RhoA/Rho kinase in smooth muscle through protein kinase G-dependent inhibitory phosphorylation of RhoA [47, 48]. On the other hand, excessive production of reactive oxygen species inactivate NO, leading to a blunted
NO bioactivity and increased vascular tone and cardiovascular-renal remodeling, at least in part through elevated RhoA–Rho kinase activity [47–49]. The importance of the balance between the RhoA/Rhokinase pathway and the NO system is crucial in cardiovascular-renal pathophysiology being involved in: (i) changes in cardiovascular-renal structure (remodeling) and induction of atherosclerosis [50–53]; (ii) involvement in the pathophysiological relationships between inflammation and hypertension [54]; (iii) and in those between hypertension, glucose metabolism and insulin resistance [55]. The involvement of RhoA/Rho-kinase in these processes is based on: (i) the RhoA/Rho-kinase activation of NAD(P)H oxidase [51], with the induction of oxidative stress; (ii) RhoA/Rho-kinase control of the atherothrombogenetic PAI-1 [56]; (iii) the suppression of vascular neointimal formation by the inhibition of Rho-kinase [57]; (iv) the demonstration of RhoA/Rho-kinase signaling involvement in C-reactive protein (CRP)-induced atherothrombogenesis through the CRP-mediated RhoA/ Rhokinase- induced increased nuclear factor kappa B (NFjB) activity, resulting in increase of the PAI-1 [58] and (v) activation of the phosphoinositol 3 kinase (PI3K)/Akt activation of eNOS by Rho-kinase inhibition, which leads to cardiovascular-renal protection and prevention of atherogenesis [59]; (vi) an abnormality in glucose transport and metabolism leading to insulin resistance by the angiotensin II-induced [55] and RhoA/Rho-kinase mediated inhibition of PI3K/Akt [59]. The activation of RhoA is upregulated by Rho guanine nucleotide exchange factors (RhoGEFs) that catalyze the exchange of GDP to GTP [60]. This exchange leads to a conformational change of the G protein, allowing regulation of downstream effectors. In addition, they can act as sensors for signals coming from activated G-protein-coupled receptors, including those coming from Ang II AT1R stimulation, that are coupled to the heterotrimeric Gq protein [61]. In spontaneously hypertensive rats RhoGEFs are overexpressed [62] and p63RhoGEF plays a mayor role in the Ang-II-mediated early RhoA/Rho kinase activation [63, 64]. This, in addition to the contribution of Ang-II-mediated RhoA activation by p115RhoGEF [65], leads to vascular contraction, cell proliferation and cardiovascular remodeling [43]. In Bartter’s and Gitelman’s patients the activity of RhoA/Rho kinase pathway is blunted (Table 1). We have shown in these patients a reduced expression and response of Rho kinase to Ang II challenge, which indicates downregulation of RhoA/Rho kinase pathway [42, 43]. Rho kinase downregulation in Bartter’s and Gitelman’s patients was associated with upregulation of NO system and increased NO-mediated vasodilation (Table 1), partly
V. Ravarotto et al.
explained by increased expression of the eNOS, compared with both hypertensive as well as healthy normotensive individuals [7, 25, 29]. These findings in humans parallel the upregulation of NO system upon Rho kinase inhibition found in vitro and in vivo in hypertensive rats, and suggested to lead to cardiovascular protection [59, 66, 67]. The increased expression in Bartter’s and Gitelman’s patients [30], of the antioxidant HO-1 [68] fits with the downregulation of RhoA/Rho kinase pathway in these patients given that the activation of Akt pathway, which induces HO-1 expression [69], is reported after Rho kinase inhibition [59]. Finally the downregulation RhoA/Rho kinase pathway [43] is supported in these patients by the reduced mRNA and protein expression of the Ang-II-induced p115RhoGEF [70] and, more importantly, of p63RhoGEF [71]. Moreover these patients have decreased phosphorylation of MYPT-1 [71], a marker of Rho kinase activity [44], which conversely is increased in hypertensive patients [71]. Rho kinase, in fact, via an inhibitory phosphorylation of MYPT-1, increases the activity of MLC kinase, leading to smooth muscle contraction and cardiovascular and renal remodeling [72]. In Bartter’s and Gitelman’s patients Ang II signaling via AT2R is activated (Table 1), as evidenced by increased Ang-II-induced expression of the mitogen-activated protein kinase phosphatase-1, a main effector of AT2R signaling [6]. AT2R stimulation evokes opposing effects on vascular tone compared to those mediated by AT1R stimulation, such as vasodilation and triggers antiproliferative signals. The activation of Ang II signaling via AT2R in these patients likely plays a major role in the mechanism(s) that produce the blunted Ang II signaling via AT1R and the related pathways, and offers additional support regarding the role of Ang II AT2R signaling in the inhibition of Ang II-mediated, p63RhoGEF/Gaq-induced RhoA/Rho kinase activation, for the cardiovascular protective effects of AT1R blockers beyond AT1R blockade [73]. Finally, the lack of cardiovascular remodeling in terms left ventricular hypertrophy [74] and carotid artery intima media thickness [29] in these patients despite high Ang II, also fits with the reduced RhoA/Rho kinase activity and with the activation of NO system [7]. The nature of all these responses in these patients may provide additional evidence for AT2R signaling’s counter-regulatory roles of AT1R signaling, including the regulation of the balance between RhoA/Rho kinase activity and NO system [75, 76]. Not only decreased oxidant potential, but also anti-inflammatory potential is another characteristic of Bartter’s and Gitelman’s syndromes, likely as consequence of the blunted Ang II signaling via AT1R, the activation of AT2R signaling, the reduced RhoA/Rho kinase activity and the upregulation of NO system (Table 1). These patients, despite elevated Ang II, have unchanged levels of C-reactive
protein (CRP) and other inflammatory mediators, such as vascular cell adhesion molecule (VCAM), intracellular cell adhesion molecule (ICAM), interleukin (IL)-6 [77], whose expression depends on NFjB activity [78, 79]. Inflammation has a key role in hypertension, atherosclerosis and cardiovascular and renal diseases and their progression is mediated by many pro-inflammatory elements and cytokines, most of which are induced by Ang II which is itself a pro-inflammatory molecule [80]. Rho kinase signaling is activated by CRP, increases NFjB activity [58, 81] and blocking Rho kinase pathway inactivates NFjB induced inflammation [82]. Compared with normotensive healthy individuals, Bartter’s and Gitelman’s patients have unchanged NFjB levels but likely reduced activity due to the increased IjB expression, the inhibitory subunit of NFjB. An increased activity of IjB would therefore lead to reduced transcription of gene related to inflammation and remodeling [83].
6 Conclusions Bartter’s and Gitelman’s patients are a human model of endogenously blunted and blocked Ang II signaling, which causes these patients to present with findings and processes that are the exact opposite of those described in hypertension (Table 1). The extensive series of studies from our laboratory on Ang II signaling using Bartter’s and Gitelman’s patients, have traced key regulatory elements of this signaling such as RGS-2, Gaq protein, NO system and Rho kinase pathways and the findings from these studies have confirmed in this human model their importance in vascular tone regulation and cardiovascular-renal remodeling. The importance of the RhoA/Rho-kinase pathway and its close relationships with Ang II and NO bioavailability in cardiovascular-renal biology, make this pathway of essential importance in a wide spectrum of conditions in cardiovascular-renal pathophysiology. An imbalance of the RhoA/Rho-kinase and NO systems towards an increase of the RhoA/Rho-kinase pathway is determinant not only for the induction of hypertension and for conditions such as insulin resistance and glucose intolerance, known often to be associated with hypertension, but also with long-term complications of hypertension such as cardiovascular-renal remodeling and atherogenesis. For these important consequences of RhoA/Rho-kinase activation and reduction of NO system, RhoA/Rho kinase pathway is considered a potential target for cardiovascular-renal protection [45]. The overall clinical, biochemical and molecular picture of Bartter’s and Gitelman’s syndromes may therefore contribute to understanding the pathophysiological mechanisms in humans involved in cardiovascular-renal remodeling and linking RhoA/Rho-kinase signaling and the
Angiotensin II and Cardiovascular-Renal Remodelling
NO system with hypertension and its relationships with inflammation, insulin resistance, atherogenesis and remodeling. The insights into mechanisms responsible for the control of vascular tone and cardiovascular-renal remodeling derived by studies in this human model opposite to hypertension, in particular those on RhoA/Rho kinase pathway/NO system/oxidative stress relationships, have identified, confirmed and extended the knowledge of the processes evoked by Ang II signaling, highlighted their crucial importance in mediating Ang II effects and could contribute to identify additional potential significant targets of therapy.
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