Renin Transcription Research Articles

Inhibition of Renin Expression Is Regulated by an Epigenetic Switch From an Active to a Poised State.

Renin-expressing cells are myoendocrine cells crucial for the maintenance of homeostasis. Renin is regulated by cAMP, p300 (histone acetyltransferase p300)/CBP (CREB-binding protein), and Brd4 (bromodomain-containing protein 4) proteins and associated pathways. However, the specific regulatory changes that occur following inhibition of these pathways are not clear. We treated As4.1 cells (tumoral cells derived from mouse juxtaglomerular cells that constitutively express renin) with 3 inhibitors that target different factors required for renin transcription: H-89-dihydrochloride, PKA (protein kinase A) inhibitor; JQ1, Brd4 bromodomain inhibitor; and A-485, p300/CBP inhibitor. We performed assay for transposase-accessible chromatin with sequencing (ATAC-seq), single-cell RNA sequencing, cleavage under targets and tagmentation (CUT&Tag), and chromatin immunoprecipitation sequencing for H3K27ac (acetylation of lysine 27 of the histone H3 protein) and p300 binding on biological replicates of treated and control As4.1 cells. In response to each inhibitor, Ren1 expression was significantly reduced and reversible upon washout. Chromatin accessibility at the Ren1 locus did not markedly change but was globally reduced at distal elements. Inhibition of PKA led to significant reductions in H3K27ac and p300 binding specifically within the Ren1 super-enhancer region. Further, we identified enriched TF (transcription factor) motifs shared across each inhibitory treatment. Finally, we identified a set of 9 genes with putative roles across each of the 3 renin regulatory pathways and observed that each displayed differentially accessible chromatin, gene expression, H3K27ac, and p300 binding at their respective loci. Inhibition of renin expression in cells that constitutively synthesize and release renin is regulated by an epigenetic switch from an active to poised state associated with decreased cell-cell communication and an epithelial-mesenchymal transition. This work highlights and helps define the factors necessary for renin cells to alternate between myoendocrine and contractile phenotypes.

Kidney Renin Release under Hypoxia and Its Potential Link with Nitric Oxide: A Narrative Review.

The renin-angiotensin system (RAS) and hypoxia have a complex interaction: RAS is activated under hypoxia and activated RAS aggravates hypoxia in reverse. Renin is an aspartyl protease that catalyzes the first step of RAS and tightly regulates RAS activation. Here, we outline kidney renin expression and release under hypoxia and discuss the putative mechanisms involved. It is important that renin generally increases in response to acute hypoxemic hypoxia and intermittent hypoxemic hypoxia, but not under chronic hypoxemic hypoxia. The increase in renin activity can also be observed in anemic hypoxia and carbon monoxide-induced histotoxic hypoxia. The increased renin is contributed to by juxtaglomerular cells and the recruitment of renin lineage cells. Potential mechanisms regulating hypoxic renin expression involve hypoxia-inducible factor signaling, natriuretic peptides, nitric oxide, and Notch signaling-induced renin transcription.

Abstract P153: Protein Kinase A Inhibition Epigenetically Silences Ren1

Renin-expressing cells are myoendocrine cells crucial for survival which detect changes in blood pressure and release renin to maintain homeostasis. One of the pathways responsible for renin expression includes cAMP as a central factor. cAMP binds to subunits of protein kinase A (PKA), ultimately recruiting both CBP and p300. Binding to the cAMP-responsive element in the renin enhancer region thus amplifies renin transcription. To evaluate transcriptomic and epigenomic changes occurring at the renin locus via the cAMP pathway, we treated As4.1 cells (a tumoral cell line that constitutively expresses renin) with the PKA inhibitor H89 (treated) or DMSO (control). Using scRNA-seq, we confirm that PKA inhibition leads to significant decreases in Ren1 abundance (p=3.18e-25), and Ccl8 , Cilp , Cdkn1a , Lox , Ly6c1 , Ccl7 , Cryab are all significantly increased. Analysis of bulk ATAC-seq of treated and control As4.1 cells revealed 527 significantly increased regions of accessible chromatin and 1523 significantly decreased. Within the increased accessibility regions, motifs for the transcription factors Bach1 , Fos , Fosb , Trp73 , and Gli1 are overrepresented. Within the regions with decreased accessibility are enriched motifs for Zfp161 , Esr2 , Zfa , Maz , Phf21a , Hlf , and Tcf7 . We further identified a cluster of differentially accessible regions composed of 172 chromatin regions that shift concurrently in accessibility following PKA inhibition. Within these regions, the KLF (p= 7.5e-11) and AP-1 (p= 1.4e-5) families of transcription factors are overrepresented. Interestingly, open chromatin around the Ren1 locus is not differentially accessible following H89 treatment. To better understand the effect of PKA inhibition, we evaluated H3K27Ac and P300 binding via ChIP-seq. H3K27Ac is significantly reduced at the renin locus (p=1.99e-08), and P300 binding is also significantly reduced (p=1.79e-13). Taken together, the data suggests that cAMP pathway inhibition controls renin expression through a reduction not in accessibility, but via a switch from an active to poised state of epigenetic control and the rise in immediate early genes regulating cellular gene expression.

Decipher the complexity of cis-regulatory regions by a modified Cas9.

Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity of renin transcription in its native genomic context. With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models. SAM complexes induced activation of renin in our cells after renin specific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data. By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.

Renin Gene Editing in Zebrafish

Recent studies in zebrafish show that the renin‐angiotensin system (RAS) is highly conserved across vertebrates. In common with mammals, the zebrafish kidney contains specialised renin‐expressing mural cells throughout the kidney micro‐vasculature that contain acidic granules indicative of the processing and regulated secretion of active renin. Zebrafish are a unique model organism suitable for developmental studies and due to the translucent nature of zebrafish larvae, the functional embryonic pronephros is readily accessible for high‐resolution in vivo imaging and high‐throughput genetic and chemical screens. To investigate the role of renin and the RAS in zebrafish we designed guide RNAs to target CRISPR‐Cas9 to renin exon 2. We identified an 8bp deletion resulting in a frameshift mutation and early termination of renin transcription. Adult ren−/− knockouts, identified by sequencing, were phenotypically screened using the automated screening system (VAST), which permits the imaging of large numbers of live fish with reproducible precision. Knockout fish are viable but exhibit delayed growth during early stages of development. Imaging revealed a delayed appearance and enlargement of the swim bladder and reduced fish length was indicative of an overall developmental delay. The knockout fish were further crossed with already existing renal reporter lines to investigate further phenotypes at a cellular level. When ren−/− knockout fish were crossed with wt1b:GFP fish a delay in glomerular fusion of the pronephric kidney was observed, whilst a cross of ren−/− knockout with ren:RFP fish revealed a dramatic increase in renin cell number along the renal vasculature, suggesting a continued but non‐critical requirement for renin in the adult fish.Support or Funding InformationBHF Centre of Research Excellence Award EPSRC/MRC CDT (Optima) AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The PPAR-gamma-binding sequence Pal3 is necessary for basal but dispensable for high-fat diet regulated human renin expression in the kidney.

We reported earlier that PPAR-gamma regulates renin transcription through a human-specific atypical binding sequence termed hRen-Pal3. Here we developed a mouse model to investigate the functional relevance of the hRen-Pal3 sequence in vivo since it might be responsible for the increased renin production in obesity and thus for the development of accompanying arterial hypertension. We used bacterial artificial chromosome construct and co-placement strategy to generate two transgenic mouse lines expressing the human renin gene from identical genomic locus without affecting the intrinsic mouse renin expression. One line carried a wild-type hRen-Pal3 in the transgene (Pal3wt strain) and the other a mutated non-functional Pal3 (Pal3mut strain). Human renin expression was correctly targeted to the renin-producing juxtaglomerular (JG) cells of kidney in both lines. However, Pal3mut mice had lower basal human renin expression. Since human renin does not recognize mouse angiotensinogen as substrate, the blood pressure was not different between the strains. Stimulation of renin production with the angiotensin-converting enzyme inhibitor enalapril equipotentially stimulated the human renin expression in Pal3wt and Pal3mut mice. High-fat diet for 10weeks which is known to activate PPAR-gamma failed to increase human renin mRNA in kidneys of either strain. These findings showed that the human renin PPAR-gamma-binding sequence hRen-Pal3 is essential for basal renin expression but dispensable for the cell-specific and high-fat diet regulated renin expression in the kidney.

Abstract P282: Use of a CRISPR/Cas9 System for Specification of the Renin Cell Phenotype

Renin is a key enzyme/hormone that controls blood pressure and fluid-electrolyte homeostasis. Renin transcription is subject to complex developmental, physiological, and pathological regulation. We have identified the cAMP pathway and associated epigenetic marks as crucial regulators of renin expression. However, those studies involved the use of cAMP activators or analogues or histone deacetylase inhibitors that alter the transcriptome and epigenome of the whole cell without exclusively targeting the renin locus. The CRISPR/Cas9 Type II bacterial immune system has been modified for genome editing in eukaryotes. This system consists of a CRISPR-associated endonuclease (Cas9) and single guide RNA (sgRNA) to target specific genomic areas. Modifications to the Cas9 enzyme have allowed the use of CRISPR to regulate gene expression by targeted modification of epigenetic marks. In this study we tested whether renin expression can be induced by a nuclease-null dCas9 protein fused to the catalytic core of the acetyltransferase p300 (dCas9p300) and sgRNAs directed to the renin promoter and enhancer. We used cultured arteriolar smooth muscle cells of the renin lineage that constitutively express CFP (a renin lineage marker) and YFP only when the renin gene is turned on. Cells were transfected with a dCas9p300 and sgRNA expression vectors and analyzed for YFP expression 48h after transfection. We tested four sgRNAs targeting the renin enhancer and five sgRNAs targeting the renin promoter either individually or in combination. We found few YFP+ cells when all 4 enhancer sgRNAs or 5 promoter sgRNAs were simultaneously used. The highest YFP expression (32±9 cells/well) was observed when two enhancer sgRNAs (at positions -2,757 and -2,631) and three promoter sgRNAs (at positions -719, -50 and -25) were simultaneously added to the cells. We did not find any YFP+ cells when the dCas9p300 plasmid was transfected without sgRNAs or when cells were transfected with a control plasmid. Our data support targeted acetylation as a causal mechanism of renin transactivation. CRISPR/Cas9 provides a tool to study the regulation of renin expression by targeting epigenetic marks in the promoter and enhancer of renin.

Renin expression in developing zebrafish is associated with angiogenesis and requires the Notch pathway and endothelium.

Although renin is a critical regulatory enzyme of the cardiovascular system, its roles in organogenesis and the establishment of cardiovascular homeostasis remain unclear. Mammalian renin-expressing cells are widespread in embryonic kidneys but are highly restricted, specialized endocrine cells in adults. With a functional pronephros, embryonic zebrafish are ideal for delineating the developmental functions of renin-expressing cells and the mechanisms governing renin transcription. Larval zebrafish renin expression originates in the mural cells of the juxtaglomerular anterior mesenteric artery and subsequently at extrarenal sites. The role of renin was determined by assessing responses to renin-angiotensin system blockade, salinity variation, and renal perfusion ablation. Renin expression did not respond to renal flow ablation but was modulated by inhibition of angiotensin-converting enzyme and altered salinity. Our data in larval fish are consistent with conservation of renin's physiological functions. Using transgenic renin reporter fish, with mindbomb and cloche mutants, we show that Notch signaling and the endothelium are essential for developmental renin expression. After inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts. Arising from separate lineages, but relying on mutual interplay with endothelial cells, renin-expressing cells are among the earliest mural cells observed in larval fish, performing both endocrine and paracrine functions.

Normal 25-Hydroxyvitamin D Levels Are Associated with Less Proteinuria and Attenuate Renal Failure Progression in Children with CKD.

Angiotensin-converting enzyme inhibitors (ACEi) for renin-angiotensin-aldosterone system (RAAS) blockade are routinely used to slow CKD progression. However, vitamin D may also promote renoprotection by suppressing renin transcription through cross-talk between RAAS and vitamin D-fibroblast growth factor-23 (FGF-23)-Klotho pathways. To determine whether vitamin D levels influence proteinuria and CKD progression in children, we performed a post hoc analysis of the Effect of Strict Blood Pressure Control and ACE Inhibition on Progression of CKD in Pediatric Patients (ESCAPE) cohort. In 167 children (median eGFR 51 ml/min per 1.73 m(2)), serum 25-hydroxyvitamin D (25(OH)D), FGF-23, and Klotho levels were measured at baseline and after a median 8 months on ACEi. Children with lower 25(OH)D levels had higher urinary protein/creatinine ratios at baseline (P=0.03) and at follow-up (P=0.006). Levels of 25(OH)D and serum vitamin D-binding protein were not associated, but 25(OH)D ≤50 nmol/L associated with higher diastolic BP (P=0.004). ACEi therapy also associated with increased Klotho levels (P<0.001). The annualized loss of eGFR was inversely associated with baseline 25(OH)D level (P<0.001, r=0.32). Five-year renal survival was 75% in patients with baseline 25(OH)D ≥50 nmol/L and 50% in those with lower 25(OH)D levels (P<0.001). This renoprotective effect remained significant but attenuated with ACEi therapy (P=0.05). Renal survival increased 8.2% per 10 nmol/L increase in 25(OH)D (P=0.03), independent of eGFR; proteinuria, BP, and FGF-23 levels; and underlying renal diagnosis. In children with CKD, 25(OH)D ≥50 nmol/L was associated with greater preservation of renal function. This effect was present but attenuated with concomitant ACEi therapy.

Identification of bona fide alternative renin transcripts expressed along cortical tubules and potential roles in promoting insulin resistance in vivo without significant plasma renin activity elevation.

Renin belongs to a family of aspartyl proteases and is the rate-limiting enzyme in the synthesis of the potent vasoactive peptide angiotensin II. Processing of renal renin has been extensively investigated in juxtaglomerular granular cells, in which prorenin and active renin are present in secretory condensed granules. Previous studies demonstrated alternative renin transcription in rat adrenal glands. Different studies reported novel intracellular forms of renin deduced from novel 5' variants derived from renin mRNA in both mice and humans. Comprehensive detailed studies in genetically engineered mice showed that both a secreted and an intracellular form of renin plays divergent mechanism regulating fluid intake and metabolism by the brain renin-angiotensin system; however, the presence, regulation, and functions of these renin isoforms in kidney and adrenal gland are not fully understood in mice. To investigate the characteristics of renin isoforms in mice, we performed a systematic inventory of renin transcripts of mice with and without a duplication of the renin gene alternatively from previous studies. We discovered a novel isoform of renin of the Ren2 gene, which conserved functionally important residues of the prosegment and incomplete isoforms of the Ren1C/D gene lacking a pre-pro segment. In situ hybridization assays revealed alternative renin isoforms expressed along cortical tubules. Newly generated transgenic mice with systemic overexpression of alternative renin transcript showed enhanced local angiotensin II generation without elevation of plasma renin activity and systemic insulin resistance in vivo, providing new pathophysiological insights into insulin resistance exaggerated by bona fide renin isoform.

English

Renin distal enhancer plays a pivotal role in renin gene expression, and the genetic variants C-5312T of renin enhancer can affect renin gene transcription level. However, the mechanism associated with the transcription level changes remains unknown. Therefore, it is of interest to investigate the possible role of distal enhancer in regulating the expression of renin gene. Single nucleotide polymorphism in renin distal enhancer was identified in 34 hypertensive patients by automatic sequencing. The data showed that the renin enhancer from the patients have genetic variants C-5312T or C-5312T SNP. Hence, the functionality of the renin enhancer and influence of the genetic variants C-5312T on binding to Sp1 is studied. These results from the binding study suggested that Sp1 binds to the DNA in GC rich region. Thus, the genetic variant C-5312T has changed the binding pattern of Sp1 to renin enhancer. This is likely to influence Sp1 activity to stimulate the expression of renin gene. The binding of Sp1 to the cis-element will enhance transcription of renin gene. Thus, polymorphism within C-5312T might contribute to the reduction of renin transcription.

Short-term vitamin D3 supplementation lowers plasma renin activity in patients with stable chronic heart failure: An open-label, blinded end point, randomized prospective trial (VitD-CHF trial)

Many chronic heart failure (CHF) patients have low vitamin D (VitD) and high plasma renin activity (PRA), which are both associated with poor prognosis. Vitamin D may inhibit renin transcription and lower PRA. We investigated whether vitamin D3 (VitD3) supplementation lowers PRA in CHF patients. We conducted a single-center, open-label, blinded end point trial in 101 stable CHF patients with reduced left ventricular ejection fraction. Patients were randomized to 6 weeks of 2,000 IU oral VitD3 daily or control. At baseline, mean age was 64 ± 10 years, 93% male, left ventricular ejection fraction 35% ± 8%, and 56% had VitD deficiency. The geometric mean (95% CI) of 25-hydroxyvitamin D3 increased from 48 nmol/L (43-54) at baseline to 80 nmol/L (75-87) after 6 weeks in the VitD3 treatment group and decreased from 47 nmol/L (42-53) to 44 nmol/L (39-49) in the control group (P < .001). The primary outcome PRA decreased from 6.5 ng/mL per hour (3.8-11.2) to 5.2 ng/mL per hour (2.9-9.5) in the VitD3 treatment group and increased from 4.9 ng/mL per hour (2.9-8.5) to 7.3 ng/mL per hour (4.5-11.8) in the control group (P = .002). This was paralleled by a larger decrease in plasma renin concentration in the VitD3 treatment group compared to control (P = .020). No significant changes were observed in secondary outcome parameters, including N-terminal pro-B-type natriuretic peptide natriuretic peptide and fibrosis markers. Most CHF patients had VitD deficiency and high PRA levels. Six weeks of supplementation with 2,000 IU VitD3 increased 25-hydroxyvitamin D3 levels and decreased PRA and plasma renin concentration.

Identification of ATF2 as a transcriptional regulator of renin gene

The cAMP response element (enhCRE) in the distal enhancer regulatory region of renin gene is believed to play a major role in the control of renin transcription. enhCRE binds the CRE-binding protein (CREB), which is the main transcription factor target of cAMP signaling. Using the mouse renin-producing cell line As4.1 we found that activating transcription factor-2 (ATF2) also binds to enhCRE. N-terminal phosphorylation of ATF2, which controls its transactivation, is associated with downregulation of renin gene expression by the cytokine tumor necrosis factor-α (TNFα). The ubiquitin proteasome inhibitor MG132 also phosphorylates ATF2 and inhibits renin expression. Knockdown of ATF2 attenuated the suppression of renin gene expression by MG132, thus demonstrating that ATF2 mediates the inhibitory effect of MG132. In addition, MG132 increased the DNA-binding of ATF2 as well as the ratio of bound ATF2 to CREB. Using ATF2- and CREB-Gal4 fusion protein constructs coupled with luciferase reporter system we showed that ATF2 has a weaker transactivating capacity than CREB. These data suggest that ATF2 represses renin expression by drifting the transcriptional control of renin gene away from CREB. Accordingly, TNFα completely abrogated the cAMP-dependent stimulation of renin gene expression.

Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription

BackgroundThe Rasd1 protein is a dexamethasone induced monomeric Ras-like G protein that oscillates in the suprachiasmatic nucleus (SCN). Previous studies have shown that Rasd1 modulates multiple signaling cascades. However, it is still unclear exactly how Rasd1 carries out its function. Studying protein-protein interactions involving Rasd1 may provide insights into its biological functions in different contexts.ResultsTo further explore the molecular function of Rasd1, we performed a yeast two-hybrid screen and identified Ear2, a negative regulator of renin transcription, as an interaction partner of Rasd1. We validated the interaction in vitro and in transfected COS-7 cells. We further confirmed the interaction of endogenous Rasd1 and Ear2 from HEK293T cell and mouse brain extract. Rasd1 inhibited transcriptional repression by Ear2 on a renin promoter-luciferase reporter construct both in the presence and absence of all-trans-retinoic acid. Moreover, real-time RT-PCR showed upregulation of endogenous renin transcription in As4.1 cells over-expressing Rasd1. We demonstrated that the ligand binding domain of Ear2 is required for physical and functional interaction between the two proteins. In addition, we demonstrated that shRNA-mediated knockdown of Rasd1 results in further repression of Ear2-mediated renin transcription, whereas induction of Rasd1 by dexamethasone counteracts the effects of shRNA-mediated Rasd1 knockdown. Finally, our study showed that Rasd1 missense mutations not only attenuate their physical interaction with Ear2 but also abolish their ability to counteract repression of renin transcription mediated by Ear2.ConclusionsOur study provides evidence for physical and functional interactions between Rasd1 and Ear2. The results suggest that their interactions are involved in renin transcriptional regulation. These findings not only reveal a novel role for Rasd1-medated signaling but also provide the basis for potential intervention of renin expression.

Increased Renin Production in Mice With Deletion of Peroxisome Proliferator-Activated Receptor-γ in Juxtaglomerular Cells

We recently found that endogenous (free fatty acids) and pharmacological (thiazolidinediones) agonists of nuclear receptor Peroxisome proliferator-activated receptor (PPAR)gamma stimulate renin transcription. In addition, the renin gene was identified as a direct target of PPARgamma. The mouse renin gene is regulated by PPARgamma through a distal enhancer direct repeat closely related to consensus PPAR response element (PPRE). In vitro studies demonstrated that PPARgamma knockdown stimulated PPRE-driven transcription. These data predicted that deficiency of PPARgamma would upregulate mouse renin expression. Consistent with these observations knockdown of PPARgamma increased the transcription of a reporter gene driven by the mouse renin PPRE-like motif in vitro. To study the impact of PPARgamma on renin production in vivo, we used a cre/lox system to generate double-transgenic mice with disrupted PPARgamma locus in renin-producing juxtaglomerular (JG) cells of the kidney (RC-PPARgamma(fl/fl) mice). We provide evidence that PPARgamma expression was effectively reduced in JG cells of RC-PPARgamma(fl/fl) mice. Fluorescent immunohistochemistry showed stronger renin signal in RC-PPARgamma(fl/fl) than in littermate control RC-PPARgamma(wt/wt) mice. Renin mRNA levels and plasma renin concentration in RC-PPARgamma(fl/fl) mice were almost 2-fold higher than in littermate controls. Arterial blood pressure and pressure control of renal vascular resistance, which play decisive roles in the regulation of renin production were indistinguishable between RC-PPARgamma(wt/wt) and RC-PPARgamma(fl/fl) mice. These data demonstrate that the JG-specific PPARgamma deficiency results in increased mouse renin expression in vivo thus corroborating earlier in vitro results. PPARgamma appears to be a relevant transcription factor for the control of renin gene in JG cells.

Vitamin D: a new hope for chronic kidney disease?

Vitamin D analogs may slow the progression of chronic kidney disease. Tan et al. report that paricalcitol has additive effects with an angiotensin-converting enzyme inhibitor (ACEI) in suppressing extracellular matrix expression, interstitial inflammation, and myofibroblast activation in obstructive nephropathy. Paricalcitol inhibited renin transcription, countering the compensatory ACEI-induced rise in renin, thus achieving a more complete blockade of the renin-angiotensin system (RAS). Vitamin D analogs may enhance the renoprotective effects of RAS inhibitors.

Fatty acid dependent regulation of renin transcription by nuclear hormone receptor HNF‐4

Production and release of renin are pivotal in controlling the Renin‐angiotensin‐system (RAAS), a main long‐term regulator of blood pressure and electrolyte homeostasis. In this context, the regulation of renin transcription by hormonal and nutritional agents is of fundamental interest.Fatty acid dependent transcription factors LXRalpha and PPARgamma have already been shown to affect renin gene transcription. We focussed our attention on another member of the nuclear hormone receptor family, HNF‐4, which is bound by fatty acyl CoA modulating its transcriptional activity. In co‐transfection experiments, HNF‐4 activated a luciferase reporter construct containing 5797bp upstream the transcriptional start site of the renin gene more than five‐fold. This effect could be completely abolished by the addition of omega3 fatty acids at a concentration of 500μM. Using computational methods, we identified five potential binding sites for HNF‐4 in this sequence. There is first evidence that HNF‐4 binds to these sites depending on the presence of fatty acyl CoAs.As HNF‐4 is expressed abundantly in proximal tubular cells our results could shed light on the regulation of local proximal tubular RAAS by fatty acids, indicating a new potential role of HNF‐4 in the kidney. Down‐regulation of renin gene expression may contribute to the antihypertensive effect of fish oil omega3 fatty acids.

Peroxisome Proliferator-Activated Receptor-γ Is Involved in the Control of Renin Gene Expression

Based on the presence of a functional retinoic acid receptor/retinoid X receptor transcription factor binding sequence (hormone-responsive element) in the renin gene enhancer and on the fact that the peroxisome proliferator-activated receptors (PPARs) bind to DNA as heterodimers with retinoid X receptors, we speculated that PPARs are involved in the regulation of renin gene expression. To test this hypothesis, we used the human renin-producing cell line CaLu-6. Endogenous or pharmacological PPARgamma agonists (unsaturated fatty acids and thiazolidinediones, respectively) stimulated renin gene expression. Surprisingly, we found that PPARgamma targets a palindromic repeat with a 3-bp spacer (Pal3) in the proximal human renin promoter. Thus, renin is the first gene described with a functional Pal3 sequence. PPARgamma agonists also stimulated renin gene expression in cultured native juxtaglomerular cells, which are the main source of renin in vivo. In summary, PPARgamma was identified as a novel intracellular mediator involved in the upregulation of renin transcription.

Interleukin-1β Attenuates Renin Gene Expression Via a Mitogen-Activated Protein Kinase Kinase-Extracellular Signal-Regulated Kinase and Signal Transducer and Activator of Transcription 3-Dependent Mechanism in As4.1 Cells

The precise mechanism by which cytokines such as IL-1beta negatively modulate expression of the renin gene remains incomplete. IL-1beta can repress renin transcription under both baseline and retinoic acid-stimulated conditions in As4.1 cells, a renin-expressing cell line derived from the kidney. This repression does not require a negative regulatory element present in the renin enhancer but is optimal in the presence of the entire renin enhancer. Three tandem copies of the retinoic acid response element is sufficient to attenuate the retinoic acid-response by IL-1beta. The decrease in retinoic acid-induced renin promoter activity in response to IL-1beta was blocked with the general tyrosine kinase inhibitor Genistein. IL-1beta caused an increase in the phosphorylation of ERK, but not p38MAPK or c-Jun N-terminal kinase. PD98059, an Erk kinase inhibitor, significantly decreased IL-1beta-mediated phosphorylation of ERK1/2, and attenuated the repression of baseline renin transcription in response to IL-1beta. PD98059 partially reversed the IL-1beta effect on retinoic acid-mediated transcription. To further investigate this mechanism, we searched the downstream effectors of ERK1/2 pathway. Although there was no effect of IL-1beta on the phosphorylation of ELK, Janus kinase 2, or signal transducers and activators of transcription (STAT) 1, IL-1beta significantly increased tyrosine-phosphorylation of STAT3, an effect attenuated by PD98059. STAT3 overexpression significantly repressed transcription of the renin gene, whereas small interfering RNA-mediated knockdown of STAT3 increased renin at baseline and attenuated the IL-1beta response. We conclude that in As4.1 cells, IL-1beta down-regulates renin gene expression via a mechanism involving the Erk-STAT3 pathway.

Liver X receptors and regulate renin expression in vivo

The renin-angiotensin-aldosterone system controls blood pressure and salt-volume homeostasis. Renin, which is the first enzymatic step of the cascade, is critically regulated at the transcriptional level. In the present study, we investigated the role of liver X receptor alpha (LXR(alpha)) and LXR(beta) in the regulation of renin. In vitro, both LXRs could bind to a noncanonical responsive element in the renin promoter and regulated renin transcription. While LXR(alpha) functioned as a cAMP-activated factor, LXR(beta) was inversely affected by cAMP. In vivo, LXRs colocalized in juxtaglomerular cells, in which LXR(alpha) was specifically enriched, and interacted with the renin promoter. In mouse models, renin-angiotensin activation was associated with increased binding of LXR(alpha) to the responsive element. Moreover, acute administration of LXR agonists was followed by upregulation of renin transcription. In LXR(alpha) mice, the elevation of renin triggered by adrenergic stimulation was abolished. Untreated LXR(beta) mice exhibited reduced kidney renin mRNA levels compared with controls. LXR(alpha)LXR(beta) mice showed a combined phenotype of lower basal renin and blunted adrenergic response. In conclusion, we show herein that LXR(alpha) and LXR(beta) regulate renin expression in vivo by directly interacting with the renin promoter and that the cAMP/LXR(alpha) signaling pathway is required for the adrenergic control of the renin-angiotensin system.