systolic hypertension, progressive proteinuria, and finally marked focal and segmental glomerulosclerosis (GS). In this model van Dokkum et al observed an.
Reprogramming development of experimental hereditary hypertension
Maarten Koeners
Financial support by the Dutch Kidney Foundation (Nierstichting Nederland) and the J.E. Jurriaanse Stichting for the publication of this thesis is gratefully acknowledged.
Printed by: Cover design: ISBN:
Gildeprint Drukkerijen BV Artistic impression of “reprogramming” by Michel Cecalovic, www.cekalovic.com 978-90-393-4659-4
Reprogramming development of experimental hereditary hypertension Het herprogrammeren van de ontwikkeling van experimentele erfelijke hypertensie (met een samenvatting in het Nederlands)
Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. W.H. Gispen, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 4 oktober 2007 des middags te 2.30 uur
door Maarten Paulus Maria Koeners geboren op 3 april 1979, te Leiderdorp
Promoter:
Prof.dr. M.A Vos
Co-promotoren:
Dr. J.A. Joles Dr. B. Braam
The studies described in this thesis were supported by the Dutch Kidney Foundation (Nierstichting Nederland) (C03.2039 and NS 6013)
“begin en eind zijn wel familie, maar het begin is blind, het eind ziet” (Friedrich Rückert)
Voor iedereen die ik lief heb en Anouska, natuurlijk
Contents List of Abbreviations
7 8
Chapter 1 Chapter 2
9 23
General introduction Outline and questions of the thesis
Part I - Investigation of the development of hypertension Chapter 3 The renal transcriptome from birth to old age in Spontaneously Hypertensive Rats Chapter 4 Detection of basal NO production in rat tissues using iron-dithiocarbamate complexes Part II - Perinatal treatments NO/ROS balance as a starting point: Chapter 5 Nitric oxide, superoxide and renal blood flow autoregulation in SHR after perinatal L-arginine and antioxidants Chapter 6 Perinatal nitric oxide supplements increase renal Vascular resistance and ameliorate hypertension and Glomerular injury in adult Fawn-Hooded Hypertensive rats Chapter 7 Perinatal inhibition of NF-kappaB has long-term Antihypertensive effects in both Spontaneously and Fawn-Hooded Hypertensive Rats Transcriptional response as a starting point: Chapter 8 Maternal supplementation with citrulline has long-term antihypertensive effects in Spontaneously Hypertensive Rats Chapter 9 Role of soluble epoxide hydrolase in (Ephx2) in the generation and maintenance of high blood pressure in Spontaneously Hypertensive Rats Chapter 10 Summary and answers to questions of the thesis Chapter 11 General Discussion Algemene Samenvatting in het Nederlands List of References Acknowledgments Curriculum Vitae Appendices
27 51
69
89
111
131
151
165 171 179 191 205 208 209
List of abbreviations ANG ASL ASS ATCE AUDA DETC EET Ephx2 EPR ERPF FeK FeNa FF FHH GFR GS L-NNA MAP MNIC NF-ĸB NO NOS PDTC QTL RBF ROS RPP RVR SBP SHR SOD TBARS Tempol UkV UnaV WKY
angiotensin argininosuccinate lyase argininosuccinate synthase arginine, taurine, vitamine c and E 12-(3-adamantan-1-ureido)-dodecanoic acid diethyldithiocarbamate epoxyeicosatrienoic acid soluble epoxide hydrolase electron paramagnetic resonance effective renal plasma flow fractional excretion potassium fractional excretion sodium filtration fraction fawn-hooded hypertensive rat glomerular filtration rate glomerulosclerosis Nω-Nitro-L-arginine mean arterial pressure mononitrosyl iron-dithiocarbamate nuclear factor-kappa B nitric oxide nitric oxide synthase pyrrolidine di thio carbamate quantative trait locus renal blood flow reactive oxygen species renal perfusion pressure renal vascular resistance systolic blood pressure spontaneously hypertensive rat superoxide dismutase thiobarbituric acid reactive substances 4-hydroxy-2,2,6,6-tetrametyl piperidine-1-oxyl urinary excretion of potassium urinary excretion of sodium Wistar-Kyoto rat
Chapter 1 General introduction
≈ “Personalized medicine: elusive dream or imminent reality? In summary, it is both. The elusive dream is to eventually have a treatment custom matched for you, as a patient, based on your individual genetic profile, demographics, and environmental factors. The imminent reality is that we are not there yet. Personalized medicine will finally become reality when medicine no longer needs to be called personalized medicine to indicate that prescriptions are routinely written for patients based on the unique genetic patterns of polymorphisms in their genome – it will simply be called medicine.” 1
≈
Chapter 1 Hypertension Hypertension is a highly complex disorder at both the genetic and physiological level. The hereditary nature of this disorder has been well established2, 3, and many genes can contribute to the development of a high blood pressure. However, there are many non-genetic factors, like high sodium intake or obesity, which are closely associated with hypertension.4, 5 With the exception of distinct monogenetic hypertension, it is impossible to separate discrete genetic and environmental components. Therefore, when we speak of the development of hypertension, we consider the complex interaction between environmental factors and genetic traits. Because regulation of blood pressure is the effectuation of a hierarchically organized, highly integrated cardiovascular system, it is of no surprise that hypertension is a major risk factor for cardiovascular diseases. Approximately 1 billion individuals worldwide have hypertension and the relationship between blood pressure and risk for cardiovascular disease events is consistent and is independent of other risk factors. The higher the blood pressure, the greater the chance of myocardial infarction, heart failure, stroke, and kidney disease.6 Unfortunately, the prevalence of hypertension is still increasing, possibly due to aging of the population and excessive calorie and sodium intake that accompanies modern lifestyle.6 Antihypertensive drugs are increasingly prescribed and commonly have to be taken for the rest of ones life. Evidently, a better understanding of the underlining mechanisms of hypertension development is necessary to develop new strategies to treat or, if possible, prevent this increasingly prevalent disorder. The development of hereditary hypertension As stated above the development of hypertension encompasses a complex interaction between environmental factors and genetic traits that undermine normal blood pressure regulation. However, it is well known that the contribution of the kidneys is paramount. For instance transplantation studies have demonstrated that blood pressure “follows” the kidney7 and all monogenetic (Mendelian) forms of hypertension (and hypotension) affect blood pressure by changing net renal sodium balance.2 In addition Guyton et al inextricably linked long-term blood pressure regulation to renal excretory function8, therefore it is commonly believed that hypertension only occurs when renal water and sodium handling is altered. Together this underlines the dominant role of the kidney in long-term blood pressure regulation. To date genetic studies of the general population have thus far been disappointing, with no variants identified that can account for a substantial effect on blood pressure.2 Given the polygenic and diverse nature of hypertension it remains a major challenge to link genetic traits with renal water and sodium handling. Studying the development of hypertension from the very beginning, or
11
General introduction even before its onset, might provide new insights. Four pivotal components can be identified in the development and progression of high blood pressure (figure 1A). 1) The interaction between environmental factors and hereditary traits is most likely the first component, which will lead to an impaired blood pressure regulation. Specific initiating factors might vary per individual and are usually very hard to identify. 2) This interaction largely defines the transcriptome, giving rise to many cascades. 3) These cascades can both impair renal hemodynamics and alter renal structure. 4) Because these alterations and the interaction between hemodynamics and renal structure the kidneys will fail to regulate natriuresis and blood pressure properly and shift their regulatory set-points to accommodate higher pressures. The impaired natriuresis and the increase in blood pressure can derange the transcriptome, impair renal hemodynamics and alter renal structure giving rise to a vicious cycle leading to full-blown hypertension. A Development of hereditary hypertension I
Hereditary traits
II
III
Impaired renal hemodynamics
IV
Deranged transcriptome
Impaired Natriuresis
Hypertension
Altered renal structure
Environmental factors
B Programming blood pressure regulation during perinatal development I
II Nutrition Maternal behavior Maternal conditions Temperature Oxygenation Utero-placental circulation Exogenous agents
III
Transciptome
Regulatory set-points Organogenisis Embryonic and fetal growth Placental growth
Adult life IV
Blood pressure regulation
Figure 1: The proposed four components of development of hereditary hypertension (a) and how during perinatal development blood pressure regulation could be programmed (b).
12
Chapter 1 Gene-environment interactions The genome of an individual contains all the genetic information needed for a whole life span. The genome is programmed to actively express specific genes at particular time points; from embryogenesis to adult life. This timed expression of specific genetic information is the sum of complex gene-gene and geneenvironment interactions. Identifying the genes for a complex disorder as hypertension has had limited success so far2, 9, suggesting that hypertension is not only polygenic but also under strong influence of the environment. Thus, the sum of (abnormal) inherited genetic information and/or distinct aberrant environmental factors can give rise to a deranged transcriptome.. This is especially true during the developmental phase, which is exceptionally sensitive to environmental factors. The initiation and development of hypertension is a form of such a developmental plasticity10 (see “programming paragraph” and BOX 2). How can environmental stimuli have an impact on development via the genetic make-up of an individual that is already determined during conception? Epigenetic control of gene expression contributes to the developmental processes (which is defined as regulation of gene activity without an alteration of the nucleotide sequence of DNA, see BOX 1). Therefore if an environmental stimuli can change epigenetic control of gene expression it will be able to derange the transcriptome without having effect on the genome. A
Environment A
Genome A
STATE I
Environment B
STATE II
Phenotype A
B
Genome C
Environment C
STATE III
Phenotype B
Figure 2: Schematic representation how a different environment can generate different phenotypes. Another possibility is that one genome can give rise to different “states” of gene expressions resulting in similar or different phenotypes. The interaction between a specific genome (Genome A) and different environmental conditions (Environment A and B) determines in which state an individual genome will be expressed (State I or II, respectively). These states are discrete and usually will result in the same phenotype (Phenotype A, figure 2A). Analogously, 4 is the sum of 2 plus 2 but also 1 plus 3, meaning that different combinations can result in the same outcome.
13
General introduction However, certain specific combinations of genome and environment (Genome C and Environment C) can give rise to another phenotype than common in the general population, e.g. disease (Phenotype B, figure 1B). Deranged transcriptome Proteins or transcription factors, which are translated from expressed genes, can activate more genes giving rise to many cascades, which finally will determine the phenotype. Many of these cascades are redox-sensitive or can be activated by stimuli e.g. Angiotensin II. Therefore, environmental factors, which modulate such stimuli, can contribute to the so-called “transcriptional response” both directly and indirectly. Because hypertension is clearly polygenetic, it is of major interest, especially with perinatal reprogramming in mind (see “reprogramming”), whether there are genes that are consistently regulated by this transcriptional response in hypertensive individuals, especially during early development. If so, one could use these genes as potential targets to prevent hypertension. BOX 1 Epigenetics A term coined by Conrad Waddington in the 1940s. Originally, epigenetics referred to the study of the way genes and their products bring the phenotype into being. Today, it is primarily concerned with the mechanisms through which cells become committed to a particular form or function and through which that functional or structural state is then transmitted in cell lineages. Modern epigenetics is important not only because it has practical significance for medicine, agriculture, and species conservation, but also because it has implications for the way in which we should view heredity and evolution. Epigenetic control of gene expression An epigenetic change is a change in the state of expression of a gene that does not involve a mutation in the DNA sequence. Epigenetic control of gene expression occurs in two main ways: either the DNA itself is chemically altered, or the histones that package DNA into chromatin are modified. Histones determine whether the chromatin is tightly packed, in which case gene expression is shut down (or silenced), or relaxed, in which case gene expression is active. The first kind of alteration takes the form of methyl groups added to the DNA by DNA methylation. The methyl group can be sensed by proteins that turn gene expression on or off through regulating chromatin structure. The second, more complex kind of alteration involves changes to the histones around which chromosomal DNA is wrapped. Each histone has a protruding 'tail' to which more than 20 chemical tags can attach. Some of these tags, or certain combinations of them, give rise to relaxedchromatin; others have the opposite effect. 11-13
Modified from
14
Chapter 1 Renal hemodynamics and structure An increase in blood pressure, which is not corrected by one of the many interacting control mechanisms, can initiate and drive a vicious cycle that includes a deranged transcriptional response14-19, impaired or shifted renal hemodynamics 20-25 , and stimulated renal reactive oxygen species (ROS) production.26-29 Although the time course can vary, in the long run this will often cause renal injury. Both the architecture and function of the kidney determines long-term blood pressure regulation. The tightly controlled and fine-tuned vascular constrictor-dilator balance is believed to be vital for blood pressure regulation. A shift between this vascular constrictor-dilator balance can cause an impaired renal function, i.e. too much dilation can cause renal damage due to an increase of transmitted pressures an tomuch constriction impaired pressure natriuresis. Natriuresis driven by pressure provides a primary and powerful means of stabilizing total body sodium and blood pressure over a wide range of sodium intakes, a hypothesis first proposed by Guyton.8 In fact it is conceivable that hypertension cannot occur when there is no shift in the pressure-natriuresis curve to higher pressures. When renal hemodynamics are impaired or shifted to other set points, e.g. increased resistance or diminished RBF autoregulation, this can affect the renal structure. When the transmission of pressure to the glomerulus increases capillary ballooning starts to occur, finally resulting in adhesions and sclerosis.30 In addition an altered renal structure, e.g. less nephrons, can lead to glomerular hypertension.31 Once again especially during the developmental phase when renal ontogeny is not yet complete the kidney is very susceptible to environmental stimuli determining renal function and structure.32 Renal injury associated with hypertension can lead to end-stage renal disease (ESRD), however of interest is that only a fraction of individuals of untreated hypertension develop such renal complications.33 Loutzenhiser et al hypothesized that the divergence in susceptibility of the kidney to hypertensive injury lies in the regulation of pre-glomerular resistance.34 Normally this mechanism dampens the transmission of elevated blood pressure into the glomerulus. Reprogramming development of hypertension Programming Because the developmental phase is exceptionally sensitive to environmental factors it is possible that adult blood pressure regulation is programmed due to gene-environment interactions is this critical period, a hypothesis first proposed by Dr. David Barker. Barker found an association between low birth weight, due to aberrant perinatal development, and cardiovascular disease later in life.35 Since
15
General introduction then a large body of experimental and epidemiological studies supports this concept36-44 and the hypothesis of developmental plasticity was developed (BOX 2).45, 46 The complex interactions among genes and environmental factors like nutrition, maternal behavior and conditions, temperature, oxygenation, uteroplacental circulation and exogenous agents will give rise to a unique and specifically timed transcriptome which will determine and interact with renal regulatory set-points, organogenesis and embryonic, fetal and placental growth (figure 1A+B). This will generate the fundaments of coordinated systems regulating cardiovascular homeostasis and thus determine blood pressure regulation in adult life.38 Most of these studies focus on how aberrant perinatal factors increase the occurrence of pathologies later in life. Our idea is that when such factors are superimposed on a specific background of inherited traits, like hypertension, this will affect the complex developmental gene expression programs, consequently determining fetal development. Hence, programming may also have beneficial long-lasting affects against an abnormal genetic background. Accordingly, if the perinatal factors are advantageous they may be able to prevent or correct abnormal development. Several studies by Racasan et al support this concept of an inverted Barker phenomenon or reprogramming.47, 48 Reprogramming Because hypertension is associated with a deranged transcriptome, impaired hemodynamics, and an altered renal structure, it is plausible that factors that correct these components in the developmental phase, could program development beneficially (reprogramming). To be able to correct one or more of these components during early development one needs potential targets that can be manipulated by exogenous agents. Because the balance between nitric oxide (NO) and ROS plays a role in many blood pressure regulating systems and is found to be ‘out of balance’ even before hypertension is present, manipulating the NO/ROS balance in early development might be a useful strategy for reprogramming the complex dynamics of developmental gene expression programs and fetal development. This might lead to normalization of blood pressure in adult life with, although not necessarily, persistently corrected renal characteristics including a shift in the transcriptional response, preserved nephrogeneisis, and adequate renal sodium excretion and renal blood flow autoregulation. Perinatal interventions that improve NO/ROS balance may also correct NO/ROS balance in later life, but, because of the complex interactions, cause and effect will be hard to dissect in this respect.
16
Chapter 1 NO/ROS balance Vascular structure and function is dependent on both NO and ROS and the balance between them. The main sources of superoxide, and hence responsible for ROS generation, are NADPH oxidase, the mitochondrial respiratory chain, xanthine oxidase and uncoupled NOS. ROS have important biological functions e.g. the regulation of redox-sensitive transcription factors, redox-sensitive transduction pathways, direct interaction with various molecules and as a weapon against invading microorganisms. Oxidative stress occurs when ROS generation exceeds endogenous antioxidant levels. Oxidative stress is invariably present in animal models of induced and hereditary hypertension, e.g. angiotensin (ANG) II induced hypertension, the Spontaneously Hypertensive Rat (SHR) and the Dahl salt-sensitive rat, and antioxidant treatment in these models is beneficial. In concordance, inducing oxidative stress can cause hypertension49 and a high blood pressure per se can cause oxidative stress.50 Interestingly, both NO synthase (NOS) inhibition as well as glutathione depletion (which will increase ROS) resulted in partially overlapping adaptations in gene expression programs in the heart before hypertrophy.51 Taken together, it is conceivable that a shift in the (renal) redox balance of an individual can start or drive the development of hypertension. Oxidative stress is often caused or maintained by a self-perpetuating cycle, consisting of a deficiency of NO and an increased ROS production.26, 52 NO is synthesized by NOS by converting arginine to citrulline. In general NO has a vasodilatory and natriuretic property. Induced NO deficiency by inhibition of NOS has been shown to cause marked and persistent hypertension.53-55 There are multiple causes of a reduced or deficient NO generation including reduced availability of NOS, or the NOS substrate L-Arginine. Because Vaziri et al reported an abundance of NOS in SHR56 it is likely that inappropriate arginine availability can cause NO deficiency. Taken together the balance between NO and ROS seems to be pivotal the development and progression of hypertension. The determination and especially quantification of oxidative stress is particularly difficult because NO and ROS are highly reactive substances. One can measure urinary excretion of NO metabolites, nitrite plus nitrate, or thiobarbituric reactive substances (TBARS), however these are indirect measurements. To investigate NO content in the kidney directly we developed a new method to trap NO in vivo with which we could quantify NO with electron paramagnetic resonance in tissues of very young rats (Chapter 4).
17
General introduction BOX 2 Developmental plasticity A critical period when a system is plastic and sensitive to the environment, followed by loss of plasticity and a fixed functional capacity. For most organs and systems the critical period occurs in utero. There are good reasons why it may be advantageous in evolutionary terms for the body to remain plastic during development. It enables the production of phenotypes that are better matched to their environment than would be possible if the same phenotype was produced in all environments. Developmental plasticity is defined as the phenomenon by which one genotype can give rise to a range of different physiological or morphological states in response to different environmental conditions during development. Reprogramming When, during the plastic phase of development and against an aberrant genetic background, superimposed factors affect the complex developmental gene expression programs eventuating in beneficial long-lasting effects. 45
Modified from
Reprogramming hypertension with perinatal treatments This thesis describes five different perinatal treatments, which are capable of ameliorating, in one way or the other, the development of hypertension in two distinct models of hypertension, namely SHR and Fawn-Hooded Hypertensive (FHH) rats (table 1). Two strategies were used to design these studies. The first strategy started with the hypothesis that a shift in the perinatal NO/ROS balance, due to a deranged transcriptome, is capable of impairing renal function, e.g. hemodynamics or pressure-natriuresis, thereby significantly contributing to the development of hypertension. Therefore we used several agents to correct NO/ROS balance or prevent down stream signaling cascades. The second strategy started with the investigation of the transcriptional response of the SHR throughout life, which suggested two candidate pathways that were potentially eligible for perinatal intervention. Figures 3 summarize all the perinatal interventions, which are introduced in more detail below.
18
Chapter 1 Table 1: Perinatal treatments NO/ROS balance as a starting point SHR + ATCE FHH + ATCE FHH + molsidomine FHH + PDTC SHR + PDTC
(Chapter 5) (Chapter 6) (Chapter 6) (Chapter 7) (Chapter 7)
Transcriptome as starting point SHR + citrulline SHR + AUDA
(Chapter 8) (Chapter 9)
Perinatal treatment: NO/ROS balance as a starting point In previous studies we perinatally manipulated the NO/ROS balance in SHR, a model of essential hypertension with increased Angiotensin II sensitivity and a high preglomerular resistance resulting in a high blood pressure with almost no renal injury. We perinatally supplemented pregnant and lactating SHR and offspring up to 4 weeks of age with ATCE, a combination of L-arginine and antioxidants (taurine, vitamins C and E), which resulted in SHR offspring that had persistently lower blood pressure without an effect on nephron number.48 Perinatal treatment of SHR with the NO donor molsidomine also persistently lowered blood pressure, suggesting that in SHR different maneuvers, which either change NO/ROS balance or solely increase NO availability, can persistently reduce blood pressure.47 However, some issues still need to be addressed. To evaluate if perinatal ATCE treatment affected renal hemodynamics we investigated the NO- and superoxide dependency of RBF autoregulation in SHR perinatally treated with ATCE (Chapter 5). In SHR glomerular sclerosis is absent57 and there is no elevation of glomerular pressure because preglomerular resistance is high and renal autoregulation efficient.58 Thus the question remains whether perinatally improving NO/ROS balance or solely NO availability can be renoprotective. Additionally, how these perinatal treatments can suppress hypertension remains unclear. Whether the underlying mechanism includes persistently improved NO availability, sodium handling or renal hemodynamics needs to be investigated. The FHH is a genetic model of hypertension with renal injury characterized by a mild hypertension and marked proteinuria, albuminuria, and focal and segmental glomerulosclerosis (GS) at a relatively young age. Chronic NO synthase inhibition
19
General introduction (L-NAME) leads to accelerated development of GS resulting in renal injury at a younger age59 revealing a partial NO-dependency of FHH phenotype development. In Chapter 6 we address whether supporting NO availability in a model with a low preglomerular resistance and glomerular injury will paradoxically lead to better regulation of renal hemodynamics and less glomerular injury. This would indicate that the consequences of perinatal treatment on the NO system extend beyond a direct vasodilator action on the preglomerular renal vasculature. Therefore, we studied the FHH perinatally supplemented with ATCE or molsidomine (Chapter 6). When NO is deficient and ROS production excessive it is likely that ROS can excessively activate several redox-sensitive signal transduction pathways such as nuclear factor-kappa B (NF-ĸB). NF-κB serves as the general transcription factor for pro-inflammatory signals and its participation in renal disease is well recognized.60 Inflammation is proposed to be a underlying mechanism, which can perpetuate hypertension development.61 Several models of hereditary and induced hypertension display an infiltration of inflammatory cells, and treatment with immune suppressive agents or antioxidants prevent both inflammation and saltsensitive hypertension.61-63 A possible mechanism by which inflammation can contribute to the development of hypertension might be the sodium-retaining effects of intrarenal Ang II activity induced by accumulation of immune cells. This can result in a decrease in GFR, increase in tubular reabsorption, and impaired pressure natriuresis.64 Alternatively IL6 production by immune cells may contribute to the impairment of the renal levels of the vasodilator epoxyeicosatrienoic acid (EET).65 It is conceivable that inhibiting NF-κB dependent pro-inflammatory pathways could suppress or even prevent development of the hypertensive phenotype. Indeed, treatment with an NF-κB inhibitor (Pyrrolidine Di Thio Carbamate, PDTC) reduced both monocyte/macrophage infiltration and blood pressure in DOCA-salt, AngII-induced hypertension and SHR without an effect in any of the normotensive controls.66-69 Both inflammation and upregulation of NF-κB activation are already present in 3-wk-old pre-hypertensive SHR, and NF-κB inhibition from 7 wks to 25 wks of age completely abrogated the development of hypertension.70 Therefore, we studied SHR and FHH perinatally treated with PDTC (Chapter 7). Perinatal treatment: transcriptional response as a starting point To further investigate how we could prevent the development of hypertension due to a brief perinatal intervention we performed microarray studies during the development of SHR and SHR perinatally treated with ATCE (Chapter 3). With microarray it is possible to analyse the expression levels of thousands of genes simultaneously and with oligonucleotides the technique can be used to identify
20
Chapter 1 genes or whole pathways that are up/down regulated in one sample compared to another sample. In combination with physiological studies (physiological genomics) it enables one to analyse the transcriptional response to interventions and/or pinpoint genes that are important for certain pathways. Thus, using micrarray techniques one can investigate the transcriptional response to a manipulated NO/ROS balance or track down genes that cause shifts in NO/ROS balance. Chon et al found, using microarray, that both NOS inhibition and glutathione depletioninduced hypertension resulted in partially overlapping adaptations in gene expression of energy and protein metabolism in the heart prior to cardiac hypertrophy.51 In addition Wesseling et al observed that NO depletion had an effect on renal expression of genes involved in synthesis of the antioxidants glutathione and bilirubin but had no direct effects on pro-oxidant systems.15 Braam et al found that gene expression patterns of fibroblasts in culture were strongly affected by anti-oxidant treatment.71 In endothelial cells Braam et al observed NO dependent gene expressions and in identified shear-sensitive and NO-dependent transcriptional regulators.72, 73 With the study described in Chapter 3 we identified two pathways that were potential candidates for perinatal intervention, namely the citrulline-arginine pathway and the epoxyeicosatrienoic acid (EET) pathway. As stated above, there are multiple causes of a reduced or deficient NO generation in the pre-hypertensive kidney. Using microarray methodology, we identified a reduction in expression of argininosuccinate synthase (ASS) and the arginine transporter, Slc7a7, in kidneys of young SHR (Chapter 3). This led us to investigate the challenging issue whether NO deficiency precedes hypertension due to reduced arginine availability in SHR. We speculate that sufficient arginine availability to produce NO in the kidney mainly depends on both endogenous arginine synthesis and arginine reabsorption. NOS produces NO by converting arginine to citrulline. In kidney proximal tubular cells, arginine is synthesized by the sequential action of ASS and argininosuccinate lyase (ASL) that convert citrulline to argininosuccinate and arginine, respectively. Citrulline delivery to proximal tubule cells is rate-limiting for renal arginine synthesis.74, 75 A defect in this citrullinearginine pathway could reduce arginine availability and hence cause a NO deficiency in pre-hypertensive kidney. A second source of arginine in the kidney is tubular uptake. Taken together, we hypothesize that decreased arginine availability in the developing SHR kidney, which leads to NO deficiency preceding hypertension, is caused by a defect in the citrulline-arginine pathway, arginine reabsorption, or both. Hence, we measured renal and cardiac amino acid pools and NO in 2-wk-old pre-hypertensive SHR and perinatally supplemented SHR with citrulline and tracked the long term effects of perinatal citrulline on blood pressure (Chapter 8).
21
General introduction Epoxyeicosatrienoic acids (EETs) are potent vasodilator agents, which act as an endothelial-derived hyperpolarizing factor operating through calcium-activated potassium channels. Soluble epoxide hydrolase (Ephx2) metabolizes EETs to less active dihydroxyeicosatrienoic acids. Although upregulation of renal Ephx2 gene expression has been documented in SHR, it remains unclear whether Ephx2 plays a pathogenic role during early development or in response to prolonged hypertension in aged SHR. We hypothesized that enhanced Ephx2 expression in the kidneys contributes to elevated blood pressure in SHR. Therefore we investigated if renal Ephx2 gene expression was increased and if perinatal treatment with AUDA (an Ephx2 inhibitor) persistently lowers blood pressure in SHR (Chapter 9). Figure 3: Overview of perinatal treatments
Perinatal development
Deranged transcriptome
Vitamin C Vitamin E Taurine
ROS generation
Impaired arginine synthesis and reabrorption
Citrulline
NO deficiency
Arginine Molsidomine
NO/ROS balance
PDTC
NFkB induced inflammation
Impaired renal hemodynamics
Adult life
Impaired pressure natriuresis
22
Hypertension
Increased Ephx2
AUDA
Chapter 2 Outline and questions of the thesis
Chapter 2 The aim of the studies described in this thesis is to investigate the development of experimental hereditary hypertension and to persistently ameliorate such hereditary hypertension by applying perinatal treatment. To elucidate the development of hypertension, the transcriptional response throughout life was investigated by microarray in SHR (Chapter 3) and to evaluate if SHR already display an NO deficiency at 2wk of age we developed a new methodology to trap and quantify renal NO in vivo (Chapter 4). To ameliorate hypertension we perinatally treated SHR (Chapter 5) and FHH (Chapter 6) with L-arginine and antioxidants, SHR and FHH with an inhibitor of the pro-inflammatory transcription factor nuclear factor kappa-B (NF-kB) (Chapter 7), and FHH with an NO donor (Chapter 6). With the microarray studies we identifed two pathways that are potential candidates for perinatal intervention, the citrulline-arginine pathway and the epoxyeicosatrienoic acid (EET) pathway. Therefore we perinatally supplemented SHR with citrulline to correct the decrease arginine synthesis and/or reabsorption (Chapter 8) or with an inhibitor of soluble epoxide hydrolase, AUDA, to increase EET (Chapter 9). Part I Investigation of the development of hypertension. - Which genes are (consistently) modulated in SHR compared to WKY? (Chapter 3) - Can we measure renal NO directly in (young) rats with electron paramagnetic resonance (EPR), and is this NO derived from NO synthase? (Chapter 4) Part II Perinatal treatments NO/ROS balance as a starting point: - Does perinatal support of NO availability in SHR restore dynamic NO release and decrease superoxide-dependency of renal autoregulation with reduction of RVR? (Chapter 5) - Which genes are consistently regulated by perinatal support of NO availability in SHR? (Chapter 3) - Does perinatally supporting NO availability in FHH beneficially affect long-term blood pressure and correct NO availability and renal hemodynamics, including intrarenal vasoreactivity, while reducing renal injury? (Chapter 6) - Does perinatal inhibition of NFkB decrease the high blood pressure and consistently affect RVR and natriuresis in SHR and FHH? (Chapter 7)
25
Outline and questions of the thesis Transcriptional response as a starting point: - Is renal NO in neonatal SHR limited by arginine availability, and if so, is decreased arginine availability caused by a defect in the citrulline-arginine pathway, arginine reabsorption, or both? (Chapter 8) - Is renal NO in neonatal SHR corrected by maternal supplementation with citrulline and does this have long-term antihypertensive effects in conjunction with a shift of the renal vascular constrictor-dilator balance towards relaxation? (Chapter 8) - Is renal soluble epoxide hydrolase (Ephx2) gene expression increased in SHR vs. WKY from birth to old age? (Chapter 9) - Does reduction of activity of Ephx2 with AUDA, either during the perinatal period or in adult life, lower blood pressure in adult SHR? (Chapter 9)
26
Chapter 3 The renal transcriptome from birth to old age in Spontaneously Hypertensive Rats
Sebastiaan Wesseling1 Maarten P. Koeners1 Farid Kantouh1 Jaap A. Joles1 Hein A. Koomans1 Branko Braam2 1
Nephrology and Hypertension, University Medical Center Utrecht, Netherlands Division of Nephrology and Immunology/Dept. Medicine, Edmonton, Alberta, Canada 2
In preparation
Abstract In essential hypertension, blood pressure steadily increases during development. In order to dissect primary from secondary changes we studied renal transcriptomes of 2-day, and 2-, 24-, and 48-week-old spontaneously hypertensive rats (SHR) vs. normotensive Wistar-Kyoto rats (WKY) using oligonucleotide chips. The two early ages span postnatal nephrogeneisis. We focused on 63 genes with consistent (consecutive or non-consecutive) regulation. Of these only one, soluble epoxide hydrolase (Ephx2), was upregulated at all four ages. Of 11 genes in SHR that were only regulated at 2d and 2w (including two vascular receptors) nine were downregulated. Quantitative PCR analysis confirmed a truncation in fatty acid translocase (Cd36) in SHR and that glutathione-S-transferase, mu1 (Gstm1) gene expression was reduced in SHR at all ages and connecting tissue growth factor (CTGF) was induced in SHR from 2w. The transcriptome of SHR treated with arginine and antioxidants from 2w before birth (via the dams) were compared to control SHR at 2d and 2w, as was the transcriptome of 48w old rats that were treated until 4w and had a persistent reduced systolic blood pressure. Although 26 genes were consistently expressed at two ages, not one was consistently expressed at all three ages, and no gene with changed expression vs. WKY was consistently corrected. We found that many genes were located within blood pressure associated QTL’s. Chromosome 4 contained 12 modulated genes, six of which were located within such QTL. In conclusion, analysis of renal transcriptome over the life span of SHR, specifically aimed at identifying genes consistently modulated at more than one age, revealed reduced expression of the majority of such genes. Perinatal supplements aimed at increasing NO and reducing oxidative stress persistently reduced blood pressure in SHR but did not correct consistent differences in gene expression between SHR and WKY.
Chapter 3 Introduction In essential hypertension, blood pressure, driven by complex genetic traits and environmental factors, steadily increases to reach hypertensive levels in adulthood. It is well known, mainly from transplantation studies76-78, that blood pressure “follows” the kidney and all monogenic forms of hypertension have a renal origin.2 What is less well known is which programs in the developing kidney drive the initiation of hypertension. Dissecting causality from secondary effects once the trait has come to full expression is difficult. Studies in normotensive children of patients with essential hypertension have revealed differences in plasma renin and renal hemodynamics79, the renal response to insulin80, platelet alpha 2-adrenoceptors81 and Na+/K+ fluxes in erythrocytes.82 These studies only indirectly characterize possible functional intrarenal changes based on circulating elements. The renal transcriptome has been the object of several studies in spontaneously hypertensive rats (SHR).16-19 The earliest time point studied was 3-4w of age16, 17, which is commonly termed pre-hypertensive. This is probably a misnomer due to technical inability of most investigators to detect small differences in blood pressure at this early age.83 At 2 days of age, the kidney contains at least 26 terminally differentiated cell types, recognizable by morphology, location and function.84 At 2 weeks of age, renal vascular and nephron development is complete.85, 86 By 24 weeks of age blood pressure in the SHR has stabilized.48, 87 Finally, by one year of age there is mild proteinuria in male SHR88 and even less in female SHR.89 In our previous study we succeeded to persistently reduce hypertension in offspring up to age of 48 weeks from SHR dams that was perinatally treated with a combination of L-arginine and antioxidants (taurine, vitamins C and E) in the last two weeks of pregnancy up to first 4 weeks of lactation.48 These perinatally treated rats will probably have an altered renal transcriptome compared to untreated SHR. Some of the changes may well be related to alleviation of the hypertension. In order to dissect primary from secondary changes we studied the renal transcriptome of 2-day old, and 2-, 24-, and 48-week-old SHR. In particular we focused on genes expressed at 2 days of age, and genes expressed at multiple ages. And we included the renal transcriptome of 2-day and 2- and 48-week-old SHR perinatally treated SHR as mentioned above. Furthermore we tried to link the identified genes to known genetic trait loci for blood pressure in rats, as was done previously in SHR for a more restricted age span.17
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The renal transcriptome in SHR Methods Breeding and supplement protocol Untreated SHR and normotensive Wistar-Kyoto rats (WKY), (Iffa-Credo, France), kept under standard conditions, were fed non-synthetic rodent chow (Special Diets Services, Witham, Essex, England). SHR dams were perinatally treated with LArginine, Taurine, Vitamin C and E in the last two weeks of pregnancy and the first 4 weeks of lactation in order to increase NO and reduce oxidative during nephrogenesis. Female offspring of these treated SHR dams were used in the present study (SHR+ATCE). L-Arginine, Taurine and vitamin C were dissolved in drinking water at doses of 20 g/L, 25 g/L and 594 mg/L, respectively. Vitamin E was mixed in finely grounded chow (9 g/kg) that contained 2 and 82 mg/kg vitamin C and E, respectively. No difference was found in intake of food and water between treated and untreated rats (data not shown). Sentinel animals, housed under the same conditions, were regularly monitored for infections. The Utrecht University Board for studies in experimental animals approved the protocol. Adult SHR and WKY females were mated with adult males from the same strain and supplier. At age of 2d the pups were sacrificed by decapitation and at age of 2w and older the rats were exsanguinated under pentobarbital anesthesia. Rats studied at 24w of age were purchased (Harlan). Total RNA isolation, microarray procedures and analysis Details of the procedures are available in appendix A. In short, total RNA was extracted (TRIzol™) from renal cortex cryostatically sliced off frozen kidney, dissolved in distilled H2O and stored at -80°C. For microarray analysis, total RNA was pooled per group at each age in equal amounts per subject. At 2d, 2w, 24w and 48w of age we studied WKY (n=6, n=6, n=8 and n=8, respectively), SHR (n=6, n=5, n=6 and n=12) and SHR+ATCE (n=6, n=5, none, and n=6). Total RNA was reverse transcribed (RT) with allyl-dUTP incorporation and labeled with Cy3 or Cy5 dyes (Amersham Biosciences, Piscataway, NJ). Samples were hybridized to rat 7.5k Oligo Chips manufactured in the Genomics Laboratory, containing Rat Genome Array-Ready Oligo Set Version 1.1 of Qiagen-Operon90, spotted in duplicate. Samples of WKY or SHR+ATCE were compared to age-matched SHR and a dye switch procedure was applied.91 SHR Cy3- and Cy5-labeled cDNA were also compared to allow elimination of unreliable spots. After washing, slides were stored in the dark, until scanning using Agilent Scanner (BioDiscovery, El Segundo, CA). Images were quantified using Imagene Software (BioDiscovery) and data normalized as described previously.91 Duplicates of the genes were averaged. A ratio of a spot is defined as log2(Cy5/Cy3). Microarray data of SHR-versus-SHR consisted of 6 arrays (four at 24w and two at 48w; control arrays). Average ratio’s from the 6 control arrays were used and the standard deviation (SD) of all
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Chapter 3 averaged ratio’s was 0.189. The ratios in control arrays above 3 times the SD were considered unreliable. The cutoff was set at 0.6. The following procedure was then applied: a gene with an absolute ratio >0.6 in at least 3 arrays was considered unreliable. Genes with modulation in one or two arrays were tested on outliers using Grubbs’ test. Significant outliers were removed from the data and the remaining data were retested. Out of 5889 genes, 5614 genes were considered reliable. Hence, the genes in WKY and SHR+ATCE with ratio of ≥0.6 or ≤-0.6 were considered modulated. Clustering Analysis Hierarchical clustering of microarray data was performed using the Expression Profile data CLUSTering and analysis (EPCLUST). (http://ep.ebi.ac.uk/EP/EPCLUST/) Average linkage (average distance, UPGMA) clustering based on correlation measure-based distance was performed on data for ratios >0.6 or
