Mounsif Haloui

Ubiquitous [Na+]i/[K+]i-Sensitive Transcriptome in Mammalian Cells: Evidence for Ca2+i-Independent Excitation-Transcription Coupling

Stimulus-dependent elevation of intracellular Ca2+ ([Ca2+]i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na+]i trigger c-Fos expression via a novel Ca2+ i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na+]i/[K+]i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na+]i and reduce [K+]i, cells were treated for 3 hrs with the Na+,K+-ATPase inhibitor ouabain or placed for the same time in the K+-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p<0.0001; R2>0.62). Among these Na+ i/K+ i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca2+]i, we performed identical experiments in Ca2+-free media supplemented with extracellular and intracellular Ca2+ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na+ i/K+ i-sensitive genes. Among the ubiquitous Na+ i/K+ i-sensitive genes whose expression was regulated independently of the presence of Ca2+ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca2+-depleted cells. Overall, our findings indicate that Ca2+ i-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na+]i/[K+]i ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders.

Transcriptomic Changes Triggered by Hypoxia: Evidence for HIF-1α -Independent, [Na + ] i /[K + ] i -Mediated, Excitation-Transcription Coupling

This study examines the relative impact of canonical hypoxia-inducible factor-1alpha- (HIF-1α and Na+ i/K+ i-mediated signaling on transcriptomic changes evoked by hypoxia and glucose deprivation. Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na+]i and 2-fold reduction of [K+]i. Using global gene expression profiling we found that Na+,K+-ATPase inhibition by ouabain or K+-free medium in rat aortic vascular smooth muscle cells (RASMC) led to the differential expression of dozens of genes whose altered expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion. For further investigations, we selected Cyp1a1, Fos, Atf3, Klf10, Ptgs2, Nr4a1, Per2 and Hes1, i.e. genes possessing the highest increments of expression under sustained Na+,K+-ATPase inhibition and whose implication in the pathogenesis of hypoxia was proved in previous studies. In ouabain-treated RASMC, low-Na+, high-K+ medium abolished amplification of the [Na+]i/[K+]i ratio as well as the increased expression of all tested genes. In cells subjected to hypoxia and glucose deprivation, dissipation of the transmembrane gradient of Na+ and K+ completely eliminated increment of Fos, Atf3, Ptgs2 and Per2 mRNAs and sharply diminished augmentation expression of Klf10, Edn1, Nr4a1 and Hes1. In contrast to low-Na+, high-K+ medium, RASMC transfection with Hif-1a siRNA attenuated increments of Vegfa, Edn1, Klf10 and Nr4a1 mRNAs triggered by hypoxia but did not impact Fos, Atf3, Ptgs2 and Per2 expression. Thus, our investigation demonstrates, for the first time, that Na+ i/K+ i-mediated, Hif-1α- -independent excitation-transcription coupling contributes to transcriptomic changes evoked in RASMC by hypoxia and glucose deprivation.

[Na+]i‐induced c‐Fos expression is not mediated by activation of the 5′‐promoter containing known transcriptional elements

In vascular smooth muscle cells and several other cell types, inhibition of Na+/K+‐ATPase leads to the expression of early response genes, including c‐Fos. We designed this study to examine whether or not a putative Na+i/K+i‐sensitive element is located within the c‐Fos 5′‐UTR from − 650 to + 103 containing all known response elements activated by ‘classic’ stimuli, such as growth factors and Ca2+i‐raising compounds. In HeLa cells, the highest increment of c‐Fos mRNA content was noted after 6 h of Na+/K+‐ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c‐Fos protein accumulation in ouabain‐treated cells correlated with a gain of Na+i and loss of K+i. Augmented c‐Fos expression was also observed under inhibition of Na+/K+‐ATPase in K+‐free medium and in the presence of the Na+ ionophore monensin. The effect of ouabain on c‐Fos expression was sharply attenuated under dissipation of the transmembrane Na+ gradient, but was preserved in the presence of Ca2+ chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na+i‐mediated, Ca2+i‐ and extracellular regulated kinase‐independent mechanism of gene expression. In contrast to massive c‐Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c‐Fos 5′‐UTR. Negative results were also obtained in ouabain‐treated vascular smooth muscle cells and C11 Madin–Darby canine kidney cells possessing augmented c‐Fos expression. Our results reveal that Na+i‐induced c‐Fos expression is not mediated by the 5′‐UTR containing transcriptional elements activated by growth factors and other ‘classic stimuli’.

Increased Renal Epithelial Na Channel Expression and Activity Correlate With Elevation of Blood Pressure in Spontaneously Hypertensive Rats

Elevation of blood pressure with age is one of the hallmarks of hypertension in both males and females. This study examined transcriptomic profiles in the kidney of 12-, 40-, and 80-week-old spontaneously hypertensive rats and 4 recombinant inbred strains in search for functional genetic elements supporting temporal dynamics of blood pressure elevation. We found that both in males and females of spontaneously hypertensive rats and hypertensive recombinant inbred strains age-dependent blood pressure increment was accompanied by 50% heightened expression of epithelial sodium channel &bgr;- and &ggr;-subunits. Epithelial sodium channel subunit expression correlated positively with blood pressure but correlated negatively with renin expression. Increased epithelial sodium channel activity was observed in cultured epithelial cells isolated from the kidney medulla of 80-week-old spontaneously hypertensive rats but not in age-matched normotensive Wistar Kyoto. This difference remained evident after 24-hour treatment with aldosterone. 22Na uptake in the perfused kidney medulla was increased whereas the urinary Na/K ratio was decreased in old spontaneously hypertensive rats compared with normotensive controls. The difference was eliminated by the administration of epithelial sodium channel inhibitor benzamil. Observations in recombinant inbred strains representing various mixtures of parental hypertensive and normotensive genomes suggest that Scnn1g and Scnn1b genes themselves are not implicated in heightened expression and that the increased expression is neither secondary nor required for a partial elevation of blood pressure in contrast to spontaneously hypertensive rats. We suggest that spontaneously hypertensive rats display an intact negative feed-back between renin-angiotensin-system and epithelial Na channel activity whose upregulated expression is supported by a yet unknown mechanism.

PROX1 gene CC genotype as a major determinant of early onset of type 2 diabetes in slavic study participants from Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation study

Background: The prevalence of diabetic nephropathy varies according to ethnicity. Environmental as well as genetic factors contribute to the heterogeneity in the presentation of diabetic nephropathy. Our objective was to evaluate this heterogeneity within the Caucasian population. Methods: The geo-ethnic origin of the 3409 genotyped Caucasian type 2 diabetes (T2D) patients of Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation was determined using principal component analysis. Genome-wide association studies analyses of age of onset of T2D were performed for geo-ethnic groups separately and combined. Results: The first principal component separated the Caucasian study participants into Slavic and Celtic ethnic origins. Age of onset of diabetes was significantly lower in Slavic patients (P = 7.3 × 10−20), whereas the prevalence of hypertension (P = 4.9 × 10−31) and albuminuria (5.1 × 10−9) were significantly higher. Age of onset of T2D and albuminuria appear to have an important genetic component as the values of these traits were also different between Slavic and Celtic individuals living in the same countries. Common and geo-ethnic-specific loci were found to be associated to age of onset of diabetes. Among the latter, the PROX1/PROX1-AS1 genes (rs340841) had the highest impact. Single-nucleotide polymorphism rs340841 CC genotype was associated with a 4.4 year earlier onset of T2D in Slavic patients living or not in countries with predominant Slavic populations. Conclusion: These results reveal the presence of distinct genetic architectures between Caucasian ethnic groups that likely have clinical relevance, among them PROX1 gene is a strong candidate of early onset of diabetes with variations depending on ethnicity.

OS 05-01 PROX1 GENE CC GENOTYPE AS A MAJOR DETERMINANT OF EARLY ONSET OF T2D AND CARDIOVASCULAR COMPLICATIONS IN SLAVIC SUBJECTS FROM ADVANCE STUDY.

Objective: We have previously reported distinct genetic architectures of renal impairment in T2D patients of Slavic and Celtic origins participating in the ADVANCE trial (J Hypertens. 2015 Jun;33 Suppl 1:e3). Further analysis suggests that the major driver of the difference in the prevalence of T2D complications between Slavic and Celtic groups is due to an earlier onset of diabetes in Slavic patients. In an attempt to distinguish between environmental and genetic factors on age of onset of diabetes, we have determined the age of onset of T2D in Slavic subjects living in Celtic countries and confirmed the same earlier onset (-2 years) in these subjects, notwithstanding their living environment. Design and Method: We performed GWAS analyses of age of onset of T2D in 3500 T2D patients from ADVANCE trial. Analyses were done for Celtic and Slavic groups separately and combined. Results: 7 loci are associated to age of onset of T2D in patients of both Celtic and Slavic origins, including HDAC9 gene (rs1128745). 9 loci are selectively associated to this phenotype in patients of Celtic origin, including the Il23R (rs1273974) while 9 different loci are associated to it in Slavic patients only. Among the latter, PROX1/PROX1-AS1 genes (rs340841) has the highest effect size. SNP rs 340841 homozygous CC genotype is associated with 2 years earlier onset of T2D in Slavic patients living in Slavic countries or in Celtic countries. This locus is also associated with eGFR decline in Slavic, with macroalbuminuria and hypertension in all ADVANCE subjects of Caucasian origin and with Interleukin-6 levels at baseline. In recent literature search we found that PROX1 gene has been associated with abnormalities of glucose metabolism and risk of diabetes with variations depending on ethnicity. Conclusions: We conclude that fine granularity of distinction in geo-ethic background assist in resolution of clinically relevant genetic contribution to cardiovascular complication in T2D.

1A.09: DISTINCT GENETIC ARCHITECTURE OF RENAL IMPAIRMENT COMPONENTS IN TYPE 2 DIABETES WITHIN CAUCASIAN POPULATIONS OF CELTO-GERMANIC AND SLAVIC ORIGINS.

Objective: The genetic architecture of type 2 diabetes (T2D) has been reported to be different between Asian and Caucasian populations (BBRC 2014;452:213–220). It is also well recognized that renal complications of T2D start earlier and are more severe in Asian subjects. Our objective was to determine whether such heterogeneity exists within the Caucasian population with respect to phenotypic and genomic determinants of renal complications in T2D. Design and method: We analyzed two major aspects of renal impairment: increase of albuminuria as UACR and decline of estimated glomerular filtration rate as log(eGFR) in Caucasian patients during the 5 year period of the ADVANCE trial (NEJM 2014;371:1392–406). Celto-Germanic and Slavic origins of 3449 genotyped subjects were determined by principal component analysis with Eigenstrat software. The first principal component separated the 3449 individuals along a geographical gradient from East/West Europe: 1133 T2D patients were Slavic and 2316 were Celto-Germanic. Phenotypic analyses and Genome Wide Association Studies (GWAS) were performed in the two groups separately. Results: The prevalence of hypertension was significantly higher (p = 1.7x10–32) in ADVANCE Slavic subjects. The prevalence of albuminuria and UACR levels were significantly higher (p = 10–4 and 9.5x10–5, respectively) at baseline and its progression over the 5-year period was steeper (p = 6.2x10–4) in patients of Slavic origin, contrasting with a more significant decline of eGFR in Celto-Germanic subjects (p = 4.9x10–21). Other T2D outcomes (myocardial infarction and stroke) did not exhibit such a difference between East and West Europe. GWAS analyses of eGFR decline did not reveal any associated SNPs (threshold p-value of < 10–3) in common between the two geo-ethnic groups and only 6% of associated genes were shared. Similarly, GWAS of UACR progression showed that only 0.1% of SNPs were common and 7% of genes were shared between the two groups. This was very different for stroke: 25% of SNPs and more than 50% of genes were common. Conclusions: Genetic analyses have to consider geo-ethnic characteristics even within Caucasians, demonstrated here for cardinal features of renal impairment in T2D. Our data suggest that distinct understanding of genomic architectures is important to ascertain clinical utility.

34 AUGMENTED RENAL ENAC EXPRESSION AND ACTIVITY CORRELATE WITH AGE-DEPENDENT DEVELOPMENT OF HYPERTENSION IN FEMALE SPONTANEOUSLY HYPERTENSIVE RATS (SHR)

Objectives: Age-dependent development of essential hypertension has a sex-specific pattern with a steeper slope of blood pressure (BP) increase in elderly women. Genomic determinants of age-dependent BP elevation remain poorly understood. Design: BP in 12-, 40- and 80-week-old female SHR and recombinant inbred strains (RIS) derived from SHR and normotensive BN-Lx rats was measured by radiotelemetry. Affymetrix rat expression assay was employed to examine gene expression profile in the whole kidney of SHR and 4 RIS selected for contrasting temporal dynamics of BP elevation. Epithelial Na channel (ENaC) activity in renal epithelial cells (REC) from kidney medulla was estimated as amiloride-sensitive 22Na uptake, whole-cell macroscopic and short circuit currents. Results: Microarray results and subsequent real-time PCR validation showed that age-dependent increment of BP in SHR and RIS was accompanied by more than 50% heightened expression of ENaC &bgr;- and &ggr;-subunits. ENaC subunit expression was positively correlated with systolic and diastolic BP but negatively with renin expression. ENaC activity was increased in REC isolated from the kidney medulla of 80-week-old hypertensive HXB3 but not in age-matched normotensive HXB13 rats. This difference persisted after 24-hr treatment of REC with aldosterone. Conclusions: BP elevation in female SHR and SHR-derived RIS exhibiting an age-dependent development of hypertension correlates with renal overexpression of ENaC &bgr;- and &ggr;-subunits and heightened activity of this channel in cultured REC. Our results suggest that augmented Na reabsorption by overexpressed ENaC may contribute to age-dependent BP elevation by a mechanism that is distinct from renin-angiotensin-aldosterone system. Supported by CIHR.

O55 Gènes communs à plusieurs complications du Diabète de Type 2

Introduction Le diabete de type 2 (DT2) et ses complications sont des traits multifactoriels a forte composante genetique et influences par des facteurs environnementaux. Les desordres micro- et macrovasculaires affectent plusieurs organes menant a des complications severes telles que la nephropathie, la neuropathie, la retinopathie et un taux d’evenements cardiovasculaires indesirables majeurs (MACE) chez environ la moitie des patients diabetiques. Materiels et methodes Nous avons etudie la cohorte ADVANCE d’un essai clinique randomise pour determiner l’effet de la diminution de la pression arterielle et du controle intensif de la glycemie chez des patients DT2 a risque eleve de complications. Le DNA genomique a ete extrait de 2 313 patients d’ADVANCE d’origine caucasienne. Les cas ayant une ou plusieurs complications du diabete etaient compares a des patients DT2 sans complications a l’entree de l’etude. Le DNA a ete genotype avec les puces Affymetrix Gene-Chipâ SNP 5.0 et 6.0 et un criblage genomique global (GWAS) a ete fait avec PLINK v1.07 afin d’identifier les SNPs associes aux traits suivants : hypertension, fibrillation auriculaire, complications micro-macrovasculaires, infarctus du myocarde et angine, MACE, MACE et albuminurie ou neuropathie. Resultats Le gene de la phosphodiesterase 4D specifique a l’AMPc (PDE4D) est commun aux 7 complications. 27 SNPs localises dans les introns de PDE4D forment 3 groupes de SNPs associes a differents phenotypes et correspondant a differents haploblocs. Nous avons identifie quatre autres genes associes a 6 de ces complications, le CUB and sushi domain-containing protein 1 precursor (CSMD1), l’ataxin2 binding protein 1 (A2BP1), la guanylate kinase2 associee a la membrane (MAGI2) et la proteine ribosomale S6, 90kd, polypeptide 2 (RBS6KA2). Conclusion Nos resultats suggerent un role pleiotropique de la PDE4D et d’autres genes dans plusieurs complications du diabete de type 2.