Department of Medicine, Duke University, Durham, North Carolina, USA. Department of Medicine, Durham VA Medical Centers, Durham, North Carolina, USA.
Address correspondence to: Steven D. Crowley, Division of Nephrology, Department of Medicine, Durham VA, and Duke University Medical Centers, DUMC Box 103015, Durham, North Carolina 27710, USA. Phone: 919.684.9788; Email: firstname.lastname@example.org.
Published October 2, 2018 - More info
Renin cells are crucial for survival — they control fluid-electrolyte and blood pressure homeostasis, vascular development, regeneration, and oxygen delivery to tissues. During embryonic development, renin cells are progenitors for multiple cell types that retain the memory of the renin phenotype. When there is a threat to survival, those descendants are transformed and reenact the renin phenotype to restore homeostasis. We tested the hypothesis that the molecular memory of the renin phenotype resides in unique regions and states of these cells’ chromatin. Using renin cells at various stages of stimulation, we identified regions in the genome where the chromatin is open for transcription, mapped histone modifications characteristic of active enhancers such as H3K27ac, and tracked deposition of transcriptional activators such as Med1, whose deletion results in ablation of renin expression and low blood pressure. Using the rank ordering of super-enhancers, epigenetic rewriting, and enhancer deletion analysis, we found that renin cells harbor a unique set of super-enhancers that determine their identity. The most prominent renin super-enhancer may act as a chromatin sensor of signals that convey the physiologic status of the organism, and is responsible for the transformation of renin cell descendants to the renin phenotype, a fundamental process to ensure homeostasis.
Maria Florencia Martinez, Silvia Medrano, Evan A. Brown, Turan Tufan, Stephen Shang, Nadia Bertoncello, Omar Guessoum, Mazhar Adli, Brian C. Belyea, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
Renin-expressing cells have been conserved through evolution and maintain blood pressure and fluid homeostasis. Lack of availability of tools to study the specifics of renin regulation has limited advances in this field. In the current issue of the Journal of Clinical Investigation, Martinez and colleagues used the genome-wide assessment of the chromatin status of cells and uncovered a unique set of super-enhancers that determine the identity of renin cells. The renin super-enhancers play a key role in the molecular memory of renin cell function, a mechanism at the core of preserving homeostasis.
As the rate-limiting protease that catalyzes the initial step in the activation of the renin angiotensin system (RAS) proteolytic cascade, renin is a master regulator of blood pressure and fluid homeostasis. Renin cleaves angiotensinogen to angiotensin I (Ang I), which is in turn converted by angiotensin-converting enzyme (ACE) to Ang II, the primary effector molecule of the RAS. Ang II then stimulates type 1 angiotensin receptors to execute several RAS functions that prevent circulatory collapse, including systemic vasoconstriction and salt and water reabsorption in the kidney nephron (1). Over the past 30 years, other constituents of the RAS cascade have been identified that temper the prohypertensive effects of AT1 receptors, including the enzyme ACE2, the effector molecule Ang 1–7, and the type 2 Ang II receptor (2–4). Moreover, the RAS is now known to play additional vital roles in biological functions as diverse as development, oncogenesis, inflammation, and even dementia (5–10). Nevertheless, despite the increasing complexity of the RAS, renin remains the ultimate driver of RAS activation with all of its pleiotropic functions.
Renin is highly conserved across multiple species from humans to Zebrafish (1). In humans, the renin gene rests on chromosome 1 and encodes a 406 amino acid renin precursor that is cleaved at the C-terminus to yield prorenin and then at the N-terminus to form active renin (11). In recent years, a distinct receptor for prorenin (PRR) has been identified that highlights the capacity of the RAS to regulate development, blood pressure, and water homeostasis independently of its traditional effector molecule Ang II (12–15). While renin is expressed in several tissues (16, 17), renin derived from the kidney is thought to direct most of the key functions of the intrarenal RAS. In the healthy adult, renin is expressed in specialized cells termed juxtaglomerular (JG) cells, as they rest within the wall of the afferent arteriole where it abuts the kidney glomerulus. Sensitive methods have also detected renin expression at lower levels in the collecting tubule (18). Following a hypotensive insult, renin expression upregulates dramatically to activate the RAS (19, 20), thereby restoring blood pressure via preservation of intravascular volume and enhancement of vascular reactivity. This induction of renin relies in part on the “memory” of cells in the kidney that produced renin during development and can reinitiate renin transcription outside of the juxtaglomerular apparatus in response to a stressor. Nevertheless, although renin regulates a multitude of functions in the healthy and diseased organism, surprisingly little has been known to date regarding how cells with renin memory are invited to again produce renin when an organism experiencing hemorrhage or some other form of hypovolemia requires vigorous RAS activation for its survival. Indeed, research in this area was limited in the past because the tools to incisively explore the regulation of renin induction were not available.
In the current issue of the Journal of Clinical Investigation, Martinez and colleagues have shed considerable light on the molecular triggers of renin upregulation by applying a sophisticated and complementary set of genetic interrogation techniques to three separate sets of murine cells with an active renin promoter (Ren1c): (a) true JG cells isolated from animals expressing YFP under the control of Ren1c, (b) renin-producing tumor cells (As4.1), and (c) cells with Ren1c activation due to renin deficiency, isolated by flow from the afferent arterioles of Ren1c–/– Ren1c-YFP+ animals (termed recruited cells) (21). These studies examined regions of the chromatin that regulate the capacity of renin cell descendants to transform into renin-producing cells during pathologic stress. For example, the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) revealed regions of heightened chromatin accessibility with regulatory sequences occupied by the H3K27ac acetylated form of histone H3 as a marker of enhancer activation. As a complementary approach, ChIP-Seq analysis for the transcription coactivator mediator subunit 1 (Med1) and RNA polymerase II (RNA Pol II) identified enhancer regions that were common or unique to the three cell types. Finally, use of ranking ordering of super-enhancers (ROSE) software to identify super-enhancers isolated 211 such sequences (Figure 1). These super-enhancers are characterized by large H3cK27ac domains; 107 were exclusive to the recruited cell population, whereas 91 were restricted to JG and As4.1 cells and may therefore identify active rather than recruited renin cells. Gene ontogeny analysis of these 91 genes confirmed the importance of several genes on the Notch and cAMP pathways to renin cell identity (21), some of which had been identified by the Gomez group in previous publications (22, 23).
Strategy to identify super-enhancers via complementary methodologies. ATAC-Seq identifies regulatory sequences with heightened chromatin accessibility. Sites of association with H3K27ac histones mark regions of enhancer activation. ChIP-Seq analysis localizes sites for Med1 and RNA Pol II attachment. These characteristics are analyzed via ROSE software to identify super-enhancer regions that can be interrogated for their inclusion of novel regulatory genes that influence transcription of a putative target gene.
Collectively, these studies confirm a super-enhancer region proximal to the renin gene, and analysis of predicted binding sites within this super-enhancer region identified several transcription factors known to regulate renin or vasculogenesis but also other novel regulatory genes. Accordingly, the Martinez et al. manuscript considerably advances our knowledge of the mechanisms through which the molecular memory for renin production can be tapped in settings of hemodynamic stress (21).
More broadly, the Martinez et al. experiments illustrate the power of emerging genomic tools to precisely confirm or refute earlier biological discoveries, much like new DNA evidence at an old crime scene, but also to identify regulatory sequences impacting gene transcription that older, less-sensitive methods could not detect. Thus, while the researchers were aware that Notch and Rbpj are key regulators of renin transcription, these new studies pinpointed several new genes, such as Junb and the gene encoding thyroid hormone receptor alpha (THRA), that will require focused interrogation for their roles in renin induction. Given the recognized difficulties in reproducing and translating so many basic science findings for the benefit of patients (24), a paradigm in which new technologies verify the accuracy of previous reports while identifying novel lines of investigation is welcome and necessary.
Lastly, the studies from Martinez et al. fit nicely into a constellation of recent studies through which powerful genomic strategies are at long last revolutionizing the study of nephrology. With single-cell transcriptomics, we can now define key cell lineages and their precursors within the kidney involved in normal renal physiology and in disease (25, 26). When applied to a kidney biopsy, single-cell genomic analysis can yield information to facilitate the advent of precision medicine (27). When combined with super-enhancer analysis, single-cell transcriptomics may predict which cell lineages are primed to become involved in renal damage before severe disease emerges, thus providing a desperately needed ounce of prevention for the nephrology field. For example, since hypertension remains uncontrolled in up to 50% of patients and is associated with catastrophic complications, performing a kidney biopsy in an otherwise healthy patient with prehypertension would become considerably more relevant if it revealed whether the patient would develop high renin hypertension in the future and which genes should be targeted to prevent renin induction. Similarly, in diabetic nephropathy where RAS activation plays such a detrimental role in disease progression, the kidney biopsy could reemerge as a powerful tool if genomic tools could elucidate the mechanisms that are provoking RAS stimulation in individual patients. Accordingly, new genomic tools can transform the application of older, blunt diagnostic tests like the kidney biopsy. Accordingly, the application of complementary genomic strategies now offers the promise of long-awaited specific therapies for renal disease. Thus, one message from the studies from Martinez et al. viewed in the context of other recent investigations employing novel genomic interrogation strategies is that there has never been a more exciting time at which to enter and remain in nephrology research.
This work was supported by NIH grants DK087893 and HL128355, and by the Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development Grant BX000893.
Conflict of interest: The author has declared that no conflict of interest exists.
Reference information: J Clin Invest. 2018;128(11):4748–4750. https://doi.org/10.1172/JCI124177.
See the related article at Super-enhancers maintain renin-expressing cell identity and memory to preserve multi-system homeostasis.