Identity Of Renin Cells Research Articles

Abstract P215: The reninness score: integrative analysis of multi-omic data to define renin cell identity.

Renin cell (RC) descendants regain the endocrine renin phenotype to overcome homeostatic threats, a process known as recruitment. The determinants that control the identity, fate, and recruitment of RCs are not well understood. We aim to 1) define the chromatin pattern that determines RC identity; and 2) develop a computational tool to score samples by similarity to that pattern. We compared open chromatin regions (ATAC-seq) between non-renin-expressing (n = 184) and renin-expressing samples (n = 19) from three different sources and stimulation states: 1) primary RCs in basal state (WT); 2) primary RCs chronically stimulated to produce renin (Recruited); and 3) constitutively renin-expressing tumoral cells. Differential analyses between the three types of RCs and the non-RCs revealed 1,525 unique open chromatin regions shared in at least two RC groups, including regions associated with RC-specific genes: Ren1 and Akr1b7. Increased accessibility regions in RC groups are cell type specific and enriched in renin-related GO terms ( e.g., cAMP and Notch signaling pathway). The bZip family ( e.g. , Atf3, p < 10 -2147 ) was the most enriched motifs in tumoral cells, and MEF2 family ( e.g. , MEF2a, p < 10 -91 ) in primary cells. We did not find a unique set of TFs specific to the Recruited group, indicating that renin recruitment may not be regulated by a recruitment-specific factor family, but could instead be driven by post-translational modification, differential co-factors, or epigenetic modifications. Next, we used a machine-learning approach to calculate a "reninness" score by training a genomic region model with renin and non-renin bulkATAC-seq data. We identified a cluster of regions (n = 112,992) around the trained renin label, which included 89% (21,820 of 24,612) of the regions with increased accessibility from the differential analyses. We calculated the reninness score based on the distance between the trained label and the predicted bulkATA sample embeddings using the trained model. Homogeneous WT RCs had the highest score, followed by renin-expressing tumoral and primary cells. Non-RCs had the lowest scores. We also adapted bulkATAC region-embedding models to pseudo-bulk ATAC- and scATAC-seq data. The observed reninness scores show that the model can also capture differences in renin recruitment potential. In conclusion, we identified the chromatin configurations defining the identity and recruitment of RCs and developed a computational score to quantify it.

Abstract P433: Impaired Renin Activity in a Bicistronic Ren1 c -T2A-tdTomato Reporter Mouse

The study of renin cell identity and function often requires the isolation of this rare cell type –0.01% of the kidney cells. Multicistronic constructs are commonly used to express reporter genes in mice. They consist of two or more genes linked by 2A peptide sequences, which produce separate proteins through a ribosomal skipping mechanism. Here, we report on the generation of a mouse model to label renin-expressing cells with a bright fluorescent reporter for the tracking and isolation of renin cells. Ren1 ctdTomato mice were generated by inserting a bicistronic T2A-tdTomato knock-in cassette upstream of the TGA stop codon of the Ren1 c gene. Kidneys from adult heterozygous (het) Ren1 ctdTomat o mice showed tdTomato signal confined to the JG area under basal conditions, and extending along the afferent arterioles and in the intraglomerular mesangium upon treatment with captopril + low-salt diet to induce the endocrine transformation of renin cells. Unexpectedly, homozygous (homo) mice exhibited increased tdTomato signal that extended in the afferent arterioles and the mesangium even under normal physiological conditions, and progressive thickening of the kidney arterioles with age. Despite reduced Renin immunostaining in the renal cortex, homo mice exhibited significantly higher kidney Ren1 mRNA (7.9 ± 1.2 vs 1.2 ± 0.6, p< 0.0001) and circulating Renin levels (121.7 ± 44.0 vs 66.7 ± 29.4 ng/ml, p< 0.003) when compared to het controls. Moreover, homo mice showed significantly lower blood pressure measured under anesthesia (62.3 ± 5.9 vs 90.0 ± 5.7 mmHg, p< 0.0001), and Ang I plasma levels (159.3 ± 34.8 vs 500.0 ± 184.1 pmol/L, p< 0.05), indicating compromised Renin activity. The concentric arteriolar hypertrophy phenotype observed in these mice is identical to that described when RAS is genetically or pharmacologically inhibited, including the presence of mutations in the renin gene. Unlike mice with global deletion of renin, these animals did not require neonatal saline injections to survive and did not develop other kidney abnormalities, indicating that the bicistronic approach rendered a renin hypomorphic mouse. Ren1 ctdTomato mice constitute an excellent model for the labeling of renin-expressing cells and for the study of the mechanisms involved in the development of concentric vascular hypertrophy under RAS inhibition. In addition, this model may provide a better understanding of factors controlling renin protein folding, stability, packaging, and release.

The Role of Gata3 in Renin Cell Identity

The Role of Gata3 in Renin Cell Identity

Abstract P224: The Role Of Gata3 In Renin Cell Identity

Renin cells act as precursors cells of the kidney vasculature and show high plasticity in postnatal life in response to challenges to homeostasis. Our scRNA-seq studies revealed that the dual-zinc finger transcription factor Gata3, important for cell lineage commitment and differentiation inseveral tissues, is expressed in mouse renin cells. We found an enhancer associated with Gata3 in renin cells leading us to hypothesize that GATA3 is important for renin cell identity. We studied 60 and 120-day-old mice carrying a conditional deletion of Gata3 in renin cells: Gata3 fl/fl ;Ren1 dCre/+ ( Gata3-cKO ) and control Gata3 fl/fl ;Ren1 d+/+ . BUN and plasma renin levels were measured from blood samples collected by cardiac puncture. Ren1, Acta2, and Gata3 were visualized in kidney sections by immunostaining. Histological analysis was performed with H&E, PAS, or Masson’s Trichrome chemical reactions. The juxtaglomerular area (JGA) index was calculated as a ratio of renin-positive JGAs per the total number of glomeruli. Gata3-cKO mice have: 1) a marked reduction in Gata3 staining; 2) a marked reduction of Ren1 (1.78 ± 0.51 (n=5) vs. 12.96 ± 2.29 (n=6), p=.0006) and Akr1b7 (2.12 ± 0.35 (n=5) vs. 19.75 ± 5.43 (n=7), p<0.0001) mRNAs compared to controls under threat; 3) a significant decrease in circulating plasma renin compared to controls under basal conditions (15609 ± 14281 pg/ml (n=5) vs. 78778 ± 33925 pg/ml (n=7), p=.0039) and physiological threat (265981 ± 79431 vs. 62347 ± 75631 pg/ml (n=5), p=.0032); 4) a significantly reduced JGA index (28.47 ± 2.32 (n=5) vs. 44.42 ± 2.72 (n=5), p=.0001); 5) flattened JG cells with misplaced renin staining in the Bowman’s capsule; 6) renal abnormalities with evidence of glomerular sclerosis/fibrosis, hemorrhage with aneurysms, and evidence of inflammatory cell infiltration; 7) misplaced Acta2 staining in the intraglomerular mesangium, interstitium, and Bowman’s capsule. Our results suggest a role of Gata3 in the identity of cells of the renin lineage.

Abstract P225: The Reninness Score: Integrative Analysis Of Multi-omic Data To Define Renin Cell Identity.

Renin cell (RC) descendants regain the endocrine renin phenotype to overcome homeostatic threats, a process known as recruitment. The determinants that control the identity, fate, and recruitment of RCs are not well understood. We aim to 1) define the chromatin pattern that determines RC identity; and 2) develop a computational tool to score samples by similarity to that pattern. We compared open chromatin regions (ATAC-seq) between non-renin-expressing (n = 184) and renin-expressing samples (n = 19) from three different sources and stimulation states: 1) primary RCs in basal state (WT); 2) primary RCs chronically stimulated to produce renin (recruited); and 3) constitutively renin-expressing tumoral cells. Differential analyses between the three types of RCs and the non-RCs revealed 1,525 unique open chromatin regions shared in at least two RC groups, including regions associated with RC-specific genes: Ren1 and Akr1b7. Increased accessibility regions in RC groups are enriched in renin-related GO terms ( e.g., cAMP and Notch signaling pathway). The bZip family ( e.g. , Atf3, p < 10 -2147 ) was the most enriched motifs in tumoral cells, and MEF2 family ( e.g. , MEF2a, p < 10 -91 ) in primary cells. Co-enriched motifs (e.g., MEIS1, p < 10 -10 ; NR4A2, p < 10 -9 ) in Ren1 and Akr1b7 regions suggest a group of TFs govern their co-expression. In addition, we identified unique open chromatin regions and enriched motifs ( e.g., E2F4, p < 10 -9 ; SP2, p < 10 -7 ) in recruited RCs, suggesting associated TFs involved in the recruitment.Next, we used a machine-learning approach to calculate a "reninness" score by training a genomic region model with renin and non-renin ATAC-seq data. We identified a cluster of regions (n = 112,992) around the trained renin label, which included 89% (21,820 of 24,612) of the increased accessibility regions from the differential analyses. We calculated the reninness score based on the distance between the trained label embeddings and predicted samples using the trained model. Homogeneous WT RCs had the highest score, followed by renin-expressing tumoral and primary cells. Non-RCs had the lowest score. In conclusion, we identified the chromatin configurations defining the identity and recruitment of RCs and developed a computational score to quantify it.

The role of Gata3 in renin cell identity.

Renin cells are precursors for other cell types in the kidney and show high plasticity in postnatal life in response to challenges to homeostasis. Our previous single-cell RNA-sequencing studies revealed that the dual zinc-finger transcription factor Gata3, which is important for cell lineage commitment and differentiation, is expressed in mouse renin cells under normal conditions and homeostatic threats. We identified a potential Gata3-binding site upstream of the renin gene leading us to hypothesize that Gata3 is essential for renin cell identity. We studied adult mice with conditional deletion of Gata3 in renin cells: Gata3fl/fl;Ren1dCre/+ (Gata3-cKO) and control Gata3fl/fl;Ren1d+/+ counterparts. Gata3 immunostaining revealed that Gata3-cKO mice had significantly reduced Gata3 expression in juxtaglomerular, mesangial, and smooth muscle cells, indicating a high degree of deletion of Gata3 in renin lineage cells. Gata3-cKO mice exhibited a significant increase in blood urea nitrogen, suggesting hypovolemia and/or compromised renal function. By immunostaining, renin-expressing cells appeared very thin compared with their normal plump shape in control mice. Renin cells were ectopically localized to Bowman's capsule in some glomeruli, and there was aberrant expression of actin-α2 signals in the mesangium, interstitium, and Bowman's capsule in Gata3-cKO mice. Distal tubules showed dilated morphology with visible intraluminal casts. Under physiological threat, Gata3-cKO mice exhibited a lower increase in mRNA levels than controls. Hematoxylin-eosin, periodic acid-Schiff, and Masson's trichrome staining showed increased glomerular fusion, absent cubical epithelial cells in Bowman's capsule, intraglomerular aneurysms, and tubular dilation. In conclusion, our results indicate that Gata3 is crucial to the identity of cells of the renin lineage.NEW & NOTEWORTHY Gata3, a dual zinc-finger transcription factor, is responsible for the identity and localization of renin cells in the kidney. Mice with a conditional deletion of Gata3 in renin lineage cells have abnormal kidneys with juxtaglomerular cells that lose their characteristic location and are misplaced outside and around arterioles and glomeruli. The fundamental role of Gata3 in renin cell development offers a new model to understand how transcription factors control cell location, function, and pathology.

Abstract P116: Specific Chromatin Configurations Across The Genome Determine The Renin Cell Identity

Renin cell descendants, such as smooth muscle cells, regain the endocrine renin phenotype to overcome homeostatic threats. The mechanism involved and the determinants of the renin cell identity are not well understood. We hypothesize that renin cell identity is governed by specific chromatin domain configurations genome wide. We compared open chromatin regions between renin- and non-renin-expressing cells. Non-renin control ATAC-seq data were mainly from ENCODE, containing 178 samples of 26 cell types. The differential analyses with DESeq2 between three renin groups (renin-expressing tumoral cell lines, native and recruited renin cells) and non-renin control group revealed that the chromatin configurations are different between the tumoral cell line and the primary kidney cells (native and recruited) in 1) number of differential regions (22,321 increased accessibility regions in the tumoral cell lines and 3,964 in the primary kidney renin cells); 2) enriched GO terms, and 3) distribution pattern over the genome. Within a small proportion of overlapped differential accessibility regions, all three groups have accessibility regions associated with known renin-specific genes: Ren1 and Akr1b7. The motif enrichment analyses with the MEME suite on the increased accessibility regions show different sets of enriched transcription factor (TF) motifs between the tumoral cell line and primary cells. The top enriched motifs in the tumoral cell line are the bZip family, including Atf3 (q-value: 2.56e-2147), Fos (q-value: 1.01e-2406), and JunB (q-value: 8.73e-2432). The top enriched motifs in the primary cells are the MEF2 family, including MEF2a (q-value: 2.18e-91), MEF2c (q-value: 1.28e-80), and MEF2d (q-value: 1.28e-80), consistent with unpublished work in our lab. In addition, we found four topmost enriched motifs (MEIS1, NR4A2, FOXJ3, and NR4A1, with q-values of 8.24e-10, 9.78e-9, 3.96e-7, and 2.67e-6) co-enriched in regions associated with Ren1 and Akr1b7. This indicates that a set of TFs regulates the co-expressing of Ren1 and Akr1b7. The chromatin domains and TF we described are likely to control renin cell identity and may serve as therapeutic targets for kidney/vascular diseases and hypertension.

Abstract 011: Epigenetic Determinants Of Renin Cell Identity

Renin secreting cells are restricted in adult mammals within the walls of renal arterioles near the entrance to the glomeruli and are therefore known as juxtaglomerular (JG) cells. These JG cells are critical for survival through the maintenance of homeostasis via the release of the peptide hormone renin in response to changes in blood pressure. By performing independently paired scATAC-seq and scRNA-seq during mouse kidney development, we sought to identify epigenetic markers of renin cell identity. While we lack spatial information, we identified putative JG cells through the identification of cells enriched in canonical markers (Ren1 and Akr1b7) of JG cell identity using both expression and accessibility derived measures of gene activity. We constructed a pseudotime trajectory of cells that lead to the mature JG population and performed pairwise comparisons of cell populations to identify the genomic regions and transcription factors (TFs) contributing to JG cell identity. As JG cells form along this trajectory, significantly different (Log2FC>0.5 and FDR<0.1) regions of accessible chromatin define the JG cell population. Within these open regions, the MEF2 family of TF motifs are significantly enriched (log10Padj > 20). We further identify a specific region of accessible chromatin (chr1:133345385-133345885) unique to the initial population of JG cells located within a Ren1 super enhancer previously identified by our group. This region is co-accessible with several regions of open chromatin within the greater Ren1 gene region (correlation > 0.75) where we identify putative binding sites for Mef2b, 2c, and 2d at these co-accessible regions. As the mature renin-expressing cells further differentiate into terminally differentiated cell populations including pericytes and mesangial cells, we continue to see significant enrichment of the MEF2 TF family within the JG cell population compared to downstream populations (log10Padj > 646.6891). Overall, we have presented the first investigation and finding using single-cell -omics of the JG cell population and uncover the importance of the MEF2 family of TFs as significant drivers of JG cell formation and identity.

Abstract P091: Role Of Gata3 In Renin Cell Identity

Renin cells act as precursors for other cell types in the kidney vasculature and show high plasticity in postnatal life in response to challenges to homeostasis. Our scRNA-seq studies revealed that the dual-zinc finger transcription factor (TF) Gata3, important for cell lineage commitment and differentiation in several tissues, is expressed in mouse renin cells under normal conditions and homeostatic threat. In addition, we found an enhancer associated with Gata3 in renin cells leading us to hypothesize that GATA3 is important for renin cell identity. We studied 60 and 76 day-old mice carrying a conditional deletion of Gata3 in renin cells: Gata3fl/fl;Ren1dCre/+ (Gata3 cKO), and control Gata3fl/fl;Ren1+/+ counterparts. Gata3 cKO mice exhibited increased BUN (43±4.6mg/dl) vs. control mice (BUN: 21±2mg/dl), suggestive of hypovolemia and/or compromised renal function. By immunostaining, renin expressing cells appeared very thin compared to the normal plump shape in control mice. Renin signal was abnormally localized to the Bowman’s capsule in some glomeruli, and there was an atypical strong Acta2 signal in the mesangium and Bowman’s capsule in Gata3 cKO mice. Distal tubules showed dilated morphology without visible intraluminal casts whereas proximal tubules appeared normal. Gata3 cKO recruited mice exhibited marked reductions in Ren1+ cells compared to controls by an 87% reduction of Ren1 mRNA levels by RT-qPCR (1.68 ± 0.11 vs 12.98 ± 0.98, p=0.0006). H&E, PAS, and Mason’s Trichrome reactions demonstrated increased glomerular fusion, absent cubical epithelial cells in Bowman’s capsule, and intraglomerular aneurysms. In summary, we found abnormal expression and localization of Ren1 and Acta2 and abnormal morphology of kidneys in Gata3 cKO mice. Our results suggest a role of Gata3 in the identity of cells of the renin lineage. Future studies will investigate the role of Gata3 during development and in older animals.

Abstract MP12: The Mef2c Transcription Factor Regulates The Renin Cell Identity

Introduction: The Renin-Angiotensin-System is essential to maintain blood pressure and fluid electrolyte homeostasis. Because precise regulation of expression and release of renin is critical for survival, understanding the molecular regulation of the renin cell identity is a vital area of study. Advances in epigenetics have enabled finer dissection of chromatin factors which maintain the identity of the renin cell. By studying genes with heightened accessibility profiles that are unique to the JG cell, we now have the capacity to unravel the determinants of the renin cell identity. Hypothesis: That transcription factors central to the governance of renin cell identity can be identified through the Assay for Transposase Accessible Chromatin (ATAC-seq) differential accessibility analysis. Methods: Native renin cell ATAC-seq was compared to existing ENCODE ATAC-seq datasets from 40 other cell types to define regions/peaks which characterize the JG program. Peaks with high intensity and ≥2-fold increase in signal were selected for Motif analysis to search for transcription factors (TFs) whose consensus sequence is enriched in those regions. Identified TFs were then selected for validation by in-situ hybridization and conditional deletion in renin cells. Results: 1) The Mef2c transcription factor was identified as having a consensus sequence in regulatory regions unique to the JG cell. It has clear expression in RNA-seq of renin cells (65 transcripts per million, n=3) and a predicted binding site in the renin gene. These results were validated by in-situ hybridization where signal localized at the JG area was detected in concordance with our in-silico results. 2) We generated Mef2c conditional knockout animals using our Ren1d-Cre mouse to study the effect in renin expression and identity. These mice displayed reduced renin immunostaining at the JG area and a 40% reduction in renin mRNA expression by qPCR from kidney cortices relative to wild-type (n=2, preliminary data). Conclusions: Our studies identified Mef2c as a TF target which likely has an essential role in maintaining and preserving renin cell identity. Experiments involving transcriptomics and epigenomics are ongoing to understand the changes wrought by Mef2c deletion in renin cells.

Renin-Expressing Cells Require β1-Integrin for Survival and for Development and Maintenance of the Renal Vasculature.

Juxtaglomerular cells are crucial for blood pressure and fluid-electrolyte homeostasis. The factors that maintain the life of renin cells are unknown. In vivo, renin cells receive constant cell-to-cell, mechanical, and neurohumoral stimulation that maintain their identity and function. Whether the presence of this niche is crucial for the vitality of the juxtaglomerular cells is unknown. Integrins are the largest family of cell adhesion molecules that mediate cell-to-cell and cell-to-matrix interactions. Of those, β1-integrin is the most abundant in juxtaglomerular cells. However, its role in renin cell identity and function has not been ascertained. To test the hypothesis that cell-matrix interactions are fundamental not only to maintain the identity and function of juxtaglomerular cells but also to keep them alive, we deleted β1-integrin in vivo in cells of the renin lineage. In mutant mice, renin cells died by apoptosis, resulting in decreased circulating renin, hypotension, severe renal-vascular abnormalities, and renal failure. Results indicate that cell-to-cell and cell-to-matrix interactions via β1-integrin is essential for juxtaglomerular cells survival, suggesting that the juxtaglomerular niche is crucial not only for the tight regulation of renin release but also for juxtaglomerular cell survival-a sine qua non condition to maintain homeostasis.

ATAC-ing the mechanisms of renin regulation.

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.

Abstract 015: Ctcf is Required for Renin Expression and Maintenance of the Structural Integrity of the Kidney

Renin is crucial for the regulation of blood pressure and fluid electrolyte homeostasis. The transcriptional machinery that regulates renin expression and thus determines the identity of renin cells is not completely understood. CCCTC-binding factor ( Ctcf ), is an important chromatin organizer of genes that confer cell identity and tissue-specificity. Because Ctcf binds to several sites in the neighborhood of the renin locus, we hypothesized that Ctcf may regulate renin expression. To test this hypothesis, we generated mice with conditional deletion of Ctcf in cells of the renin lineage ( Ctcf cKO). Ctcf cKO mice showed fewer renin-positive cells as shown by immunostaining, and a 70% reduction of renin mRNA levels when compared to control mice (0.292 ± 0.246 vs 1.003 ± 0.097, p<0.001 ). In addition, plasma renin levels were significantly decreased in Ctcf cKO versus control mice (15276.544 ± 6778.735 pg/ml vs 62321.62 ± 21881.99 pg/ml, p<0.001 ). Consistent with reduced renin levels, Ctcf cKO mice had lower mean arterial pressures (60.09 ± 3.12 mmHg vs 75.07 ± 3.06 mmHg, p<0.001 ). The kidney/body weight ratio in Ctcf cKO mice was markedly reduced (1.05 ± 0.228 % vs 1.29 ± 0.074 %, p<0.05 ), indicating a more pronounced effect in kidney than in somatic growth (20.09 ± 1.47 g vs 22.95 ± 2.95 g, p<0.05 ). Masson’s trichrome staining revealed interstitial fibrosis coinciding with cortical depressions in Ctcf cKO kidneys. Moreover, PAS staining of Ctcf cKO kidneys showed dilated tubules with intraluminal casts, and areas with crowded sclerotic and crescent glomeruli surrounded by disorganized packed cells. Finally, Ctcf cKO mice exhibited renal failure evidenced by increased BUN (44.29 ± 17.62 mg/dL vs 26 ± 3.39 mg/dL, p<0.05) , and inability to concentrate their urine (448 ± 85.57 mOsm/kg vs 1519.33 ± 382.39 mOsm/kg, p<0.001 ). In summary, deletion of Ctcf in cells from the renin lineage leads to decreased endowment of renin-expressing cells accompanied by decreased circulating renin, hypotension, severe morphological abnormalities of the kidney and ultimately renal failure. We conclude that Ctcf is necessary for the appropriate expression of renin, control of renin cell number and structural integrity of the kidney.

Fate of Renin Cells During Development and Disease.

Although renin cells are crucial for blood pressure homeostasis, little is known about their nature. We now know that renin cells are precursors that appear early and in multiple tissues during embryonic development. They participate in morphogenetic events, vascular development and injury, tissue repair, and regeneration. When confronted to a homeostatic threat, renin cell descendants have the capability to switch the renin gene on or off. This poorly understood switch or molecular memory enables the organism to maintain constancy of the internal milieu and tissue perfusion. Here, we discuss briefly the major events that govern the acquisition and maintenance of renin cell identity and how manipulations that alter the fate of renin cells can lead to serious disease. We also advance the concept that renin cells are at the center of an ancestral system of defense linking the endocrine, the immune, and the repair responses of the organism. The renin–angiotensin system (RAS) is crucial in the regulation of blood pressure and fluid–electrolyte homeostasis.1,2 In the traditional view of the RAS, renin is released by the kidney juxtaglomerular cells, and on reaching the circulation, it acts on its only known substrate, angiotensinogen, produced mainly in the liver to yield angiotensin I (Ang I), a decapeptide and Des-Ang I-angiotensinogen, a large molecule of unclear function. Thereafter, Ang I is hydrolyzed by angiotensin-converting enzyme to yield the octapeptide Ang II, a fast acting and powerful vasoconstrictor that regulates peripheral vascular resistance, renal hemodynamics, and sodium reabsorption via several mechanisms, including the stimulation of aldosterone secretion by the adrenal glands. Most of the known cardiovascular and renal actions of the RAS are achieved by the actions of Ang II on its receptors, mainly AT1 receptors. It should be noted that for the system to operate properly, it needs to respond accurately …

Abstract 066: Enhancer Repertoires That Define Renin Cell Identity

Control of the renin cell phenotype is crucial for the regulation of blood pressure and fluid- electrolyte homeostasis. Enhancers are cis -acting DNA sequences that harbor distinct chromatin features and regulate gene expression in an orientation-independent manner. Recently, clusters of enhancers or super-enhancers (SE) highly enriched with master transcription factors, possessing open chromatin configuration and in close proximity to cell-identity genes have been proposed. We tested the hypothesis that renin cells have unique repertoires of enhancers and super-enhancers, distinct from other cell types. Those regulatory clusters may in turn confer the identity of renin cells. To define the genome-wide enhancer landscape characteristic of renin cells, we studied As4.1 cells, kidney tumor cells that express renin constitutively, and native renin cells sorted from the kidneys of Ren1cKO-YFP + mice. In these mice, the renin promoter drives YFP expression thus marking the renin cells. We used genome-wide ChIP-Seq for Med1 (subunit 1 of the Mediator complex), H3K27Ac (active enhancers) and Pol II (to visualize putative genomic areas undergoing transcription). The ROSE algorithm we used to ascertain super-enhancers. Chromatin accessibility genome-wide was assessed using ATAC-Seq. The results were compared to twenty-one other cell types that do not express renin. In As4.1 cells, we identified 14,871 enhancers based on H3K27Ac. Of those, 888 were classified as super-enhancers. The Med1 signal in As4.1 cells showed a SE localized 5kb upstream the Ren1 gene, which was ranked at position 25 among other SEs. The H3K27Ac signal showed highest occupancy in the same region. ChIP-Seq for H3K27Ac in YFP + cells showed 211 SEs of 2,987 peaks. The SE for the renin gene possessed the highest signal and ranked number 1, indicating its importance in renin cells. One hundred and thirteen SEs were unique to renin cells, including the SE associated with the renin gene. ATAC-Seq signals overlapped with the renin SE and the classical enhancer indicating that the chromatin was accessible for transcription. In summary, renin-expressing cells possess distinct repertoires of unique enhancers and super-enhancers that acting in concert are likely to determine the renin phenotype.

Fate and plasticity of renin precursors in development and disease

Renin-expressing cells appear early in the embryo and are distributed broadly throughout the body as organogenesis ensues. Their appearance in the metanephric kidney is a relatively late event in comparison with other organs such as the fetal adrenal gland. The functions of renin cells in extra renal tissues remain to be investigated. In the kidney, they participate locally in the assembly and branching of the renal arterial tree and later in the endocrine control of blood pressure and fluid-electrolyte homeostasis. Interestingly, this endocrine function is accomplished by the remarkable plasticity of renin cell descendants along the kidney arterioles and glomeruli which are capable of reacquiring the renin phenotype in response to physiological demands, increasing circulating renin and maintaining homeostasis. Given that renin cells are sensors of the status of the extracellular fluid and perfusion pressure, several signaling mechanisms (β-adrenergic receptors, Notch pathway, gap junctions and the renal baroreceptor) must be coordinated to ensure the maintenance of renin phenotype--and ultimately the availability of renin--during basal conditions and in response to homeostatic threats. Notably, key transcriptional (Creb/CBP/p300, RBP-J) and posttranscriptional (miR-330, miR125b-5p) effectors of those signaling pathways are prominent in the regulation of renin cell identity. The next challenge, it seems, would be to understand how those factors coordinate their efforts to control the endocrine and contractile phenotypes of the myoepithelioid granulated renin-expressing cell.

Histone acetyl transferases CBP and p300 are necessary for maintenance of renin cell identity and transformation of smooth muscle cells to the renin phenotype.

In response to a homeostatic threat circulating renin increases by increasing the number of cells expressing renin by dedifferentiation and re-expression of renin in arteriolar smooth muscle cells (aSMCs) that descended from cells that expressed renin in early life. However, the mechanisms that govern the maintenance and reacquisition of the renin phenotype are not well understood. The cAMP pathway is important for renin synthesis and release: the transcriptional effects are mediated by binding of cAMP responsive element binding protein with its co-activators, CBP and p300, to the cAMP response element in the renin promoter. We have shown previously that mice with conditional deletion of CBP and p300 (cKO) in renin cells had severely reduced renin expression in adult life. In this study we investigated when the loss of renin-expressing cells in the cKO occurred and found that the loss of renin expression becomes evident after differentiation of the kidney is completed during postnatal life. To determine whether CBP/p300 is necessary for re-expression of renin we subjected cKO mice to low sodium diet + captopril to induce retransformation of aSMCs to the renin phenotype. The cKO mice did not increase circulating renin, their renin mRNA and protein expression were greatly diminished compared with controls, and only a few aSMCs re-expressed renin. These studies underline the crucial importance of the CREB/CBP/p300 complex for the ability of renin cells to retain their cellular memory and regain renin expression, a fundamental survival mechanism, in response to a threat to homeostasis.

Two microRNAs, miR-330 and miR-125b-5p, mark the juxtaglomerular cell and balance its smooth muscle phenotype

We have shown that microRNAs (miRNAs) are necessary for renin cell specification and kidney vascular development. Here, we used a screening strategy involving microarray and in silico analyses, along with in situ hybridization and in vitro functional assays to identify miRNAs important for renin cell identity. Microarray studies using vascular smooth muscle cells (SMCs) of the renin lineage and kidney cortex under normal conditions and after reacquisition of the renin phenotype revealed that of 599 miRNAs, 192 were expressed in SMCs and 234 in kidney cortex. In silico analysis showed that the highly conserved miR-330 and miR-125b-5p have potential binding sites in smoothelin (Smtn), calbindin 1, smooth muscle myosin heavy chain, α-smooth muscle actin, and renin genes important for the myoepithelioid phenotype of the renin cell. RT-PCR studies confirmed miR-330 and miR-125b-5p expression in kidney and SMCs. In situ hybridization revealed that under normal conditions, miR-125b-5p was expressed in arteriolar SMCs and in juxtaglomerular (JG) cells. Under conditions that induce reacquisition of the renin phenotype, miR-125b-5p was downregulated in arteriolar SMCs but remained expressed in JG cells. miR-330, normally absent, was expressed exclusively in JG cells of treated mice. In vitro functional studies showed that overexpression of miR-330 inhibited Smtn expression in SMCs. On the other hand, miR-125b-5p increased Smtn expression, whereas its inhibition reduced Smtn expression. Our results demonstrate that miR-330 and miR-125b-5p are markers of JG cells and have opposite effects on renin lineage cells: one inhibiting and the other favoring their smooth muscle phenotype.

CBP and p300 are essential for renin cell identity and morphological integrity of the kidney

The mechanisms that govern the identity of renin cells are not well understood. We and others have identified cAMP as an important pathway in the regulation of renin synthesis and release. Recently, experiments in cells from the renin lineage led us to propose that acquisition and maintenance of renin cell identity are mediated by cAMP and histone acetylation at the cAMP responsive element (CRE) of the renin gene. Ultimately, the transcriptional effects of cAMP depend on binding of the appropriate transcription factors to CRE. It has been suggested that access of transcription factors to this region of the promoter is facilitated by the coactivators CREB-binding protein (CBP) and p300, which possess histone acetyltransferase activity and may be, in turn, responsible for the remodeling of chromatin underlying expression of the renin gene. We hypothesized that CBP and p300 are therefore required for expression of the renin gene and maintenance of the renin cell. Because mice homozygous for the deletion of CBP or p300 die before kidney organogenesis begins, no data on kidney or juxtaglomerular cell development in these mice are available. Therefore, to define the role of these histone acetyltransferases in renin cell identity in vivo, we used a conditional deletion approach, in which floxed CBP and p300 mice were crossed with mice expressing cre recombinase in renin cells. Results show that the histone acetyltransferases CBP and p300 are necessary for maintenance of renin cell identity and structural integrity of the kidney.