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RationaleRenin cells are essential for survival. They control the morphogenesis of the kidney arterioles, and the composition and volume of our extracellular fluid, arterial blood pressure, tissue perfusion, and oxygen delivery. It is known that renin cells and associated arteriolar cells descend fromFoxD1+ progenitor cells, yet renin cells remain challenging to study due in no small part to their rarity within the kidney. As such, the molecular mechanisms underlying the differentiation and maintenance of these cells remain insufficiently understood.ObjectiveWe sought to comprehensively evaluate the chromatin states and transcription factors (TFs) that drive the differentiation ofFoxD1+ progenitor cells into those that compose the kidney vasculature with a focus on renin cells.Methods and ResultsWe isolated single nuclei ofFoxD1+ progenitor cells and their descendants fromFoxD1cre/+;R26R-mTmGmice at embryonic day 12 (E12) (ncells=1234), embryonic day 18 (E18) (ncells=3696), postnatal day 5 (P5) (ncells=1986), and postnatal day 30 (P30) (ncells=1196). Using integrated scRNA-seq and scATAC-seq we established the developmental trajectory that leads to the mosaic of cells that compose the kidney arterioles, and specifically identified the factors that determine the elusive, myo-endocrine adult renin-secreting juxtaglomerular (JG) cell. We confirm the role ofNfixin JG cell development and renin expression, and identified the myocyte enhancer factor-2 (MEF2) family of TFs as putative drivers of JG cell differentiation.ConclusionsWe provide the first developmental trajectory of renin cell differentiation as they become JG cells in a single-cell atlas of kidney vascular open chromatin and highlighted novel factors important for their stage-specific differentiation. This improved understanding of the regulatory landscape of renin expressing JG cells is necessary to better learn the control and function of this rare cell population as overactivation or aberrant activity of the RAS is a key factor in cardiovascular and kidney pathologies.

Rationale: Renin-expressing cells are myoendocrine cells crucial for survival. They have been postulated to possess a pressure-sensing mechanism, a baroreceptor, which can detect slight changes in blood pressure and respond with precise and synchronized amounts of renin synthesized and released to the circulation to maintain blood pressure and fluid-electrolyte balance. The location and nature of this puzzling pressure-sensing structure have remained unknown as it was originally suggested over 60 years ago. Objective: To elucidate the location and structure of the renin cell baroreceptor. Methods and Results: We used a variety of genetically modified mice whereby renin cells were exposed in vivo to either low or high arterial pressure. In addition, we applied direct mechanical stimuli, that is, pneumatic pressure or stretch, directly to renin cells cultured under different conditions and substrata. Changes in perfusion pressure and/or direct mechanical stimuli induced significant changes in renin gene expression and the phenotype of renin cells. Importantly, the experiments show that the pressure-sensing mechanism (the baroreceptor) resides in the renin cells; it requires initial extracellular sensing by integrin β1 at the renin cell membrane and is transduced to the nuclear membrane and chromatin by lamin A/C. Conclusions: These studies show that the enigmatic baroreceptor is a nuclear mechanotransducer that resides in the renin cells per se and is responsible for the sensing and transmission of extracellular physical forces directly to the chromatin of renin cells via lamin A/C to regulate renin gene expression, renin bioavailability, and homeostasis.

Renin, the rate-limiting enzyme of the renin-angiotensin system, is increased in the kidneys subjected to ureteral obstruction. We elucidated the changes and contribution of renin-producing cells (RPCs) and the cells of renin lineage (CoRL) during kidney damage and regeneration using a partial unilateral ureteral obstruction (pUUO) model in neonatal mice. Renin cells were ablated by cell-specific expression of diphtheria toxin A chain (DTA) and labeled with GFP. We compared the cell fate changes between kidneys from the wild-type (WT) and the DTA-expressing animals, using the pUUO model. The kidneys of 3W old DTA-positive Ren1 dDTA/+ ;R26R DTA/+ and Ren1 dCre/+ ;R26R DTA/+ genotypes were grossly normal. We observed no significant differences in the Myh11-positive renal vascular area between the WT and the DTA-positive mice (WT-3W: 1.72±0.10 % (n=6), Ren1 dDTA/+ ;R26R DTA/+ -3W: 1.94 ±0.123 % (n=7), Ren1 dCre/+ ;R26R DTA/+ -3W: 2.017±0.16% (n=4)). In the kidneys of the WT group, RPCs and the CoRL significantly increased at 3W post-obstruction and the distribution was similar to the sham-operated kidneys at 2W post-relief of obstruction (Renin-positive area-WT: Sham-3W: 0.57±0.02% (n=6), Obstructed-3W: 1.59±0.44% (n=4), Post-release-2W: 0.71±0.12% (n=3)). There was a significant decrease in RPCs in the DTA animals, and the reduction of RPCs and CoRL was markedly higher in Ren1 dCre/+ ;R26R DTA/+ mice in comparison to Ren1 dDTA/+ ;R26R DTA/+ group. Also, the DTA-positive animals did not respond to pUUO with a significant increase in renin as in the WT (Renin-positive area: Ren1 dDTA/+ ;R26R DTA/+ Sham-3W: 0.13±0.048% (n=5), Obstructed-3W: 0.23±0.10% (n=5), Post-release-2W: 0.14±0.04% (n=9) Ren1 dCre/+ ;R26R DTA/+ Sham: 0.010±0.004% (n=4), Obstructed: 0.07±0.004% (n=4), Post-release: 0.03±0.004% (n=3)). The renal fibrotic area and Tgfb1 mRNA levels post-obstruction and relief of obstruction were significantly high in Ren1 dCre/+ ;R26R DTA/+ group in comparison to Ren1 dDTA/+ ;R26R DTA/+ genotype due to the depletion of CoRL. Thus, our current study reveals that CoRL undergoes cell fate changes in response to obstruction and the release of obstruction, and these changes play a critical role in promoting kidney regeneration post-relief of obstruction.

Hypotension and changes in fluid-electrolyte balance pose immediate threats to survival. Juxtaglomerular cells respond to such threats by increasing the synthesis and secretion of renin. In addition, smooth muscle cells (SMCs) along the renal arterioles transform into renin cells until homeostasis has been regained. However, chronic unrelenting stimulation of renin cells leads to severe kidney damage. Here, we discuss the origin, distribution, function, and plasticity of renin cells within the kidney and immune compartments and the consequences of distorting the renin program. Understanding how chronic stimulation of these cells in the context of hypertension may lead to vascular pathology will serve as a foundation for targeted molecular therapies.

The epigenetic regulator Dot1, the only known histone H3K79 methyltransferase, has a conserved role in organismal development and homoeostasis. In yeast, Dot1 is required for telomeric silencing and genomic integrity. In Drosophila, Dot1 (Grappa) regulates homoeotic gene expression. Dysregulation of DOT1L (human homologue of Dot1) causes leukaemia and is implicated in dilated cardiomyopathy. In mice, germline disruption of Dot1L and loss of H3K79me2 disrupt vascular and haematopoietic development. Targeted inactivation of Dot1L in principal cells of the mature collecting duct affects terminal differentiation and cell type patterning. However, the role of H3K79 methylation in mammalian tissue development has been questioned, as it is dispensable in the intestinal epithelium, a rapidly proliferating tissue. Here, we used lineage-specific Cre recombinase to delineate the role of Dot1L methyltransferase activity in the mouse metanephric kidney, an organ that develops via interactions between ureteric epithelial (Hoxb7) and mesenchymal (Six2) cell lineages. The results demonstrate that Dot1L(Hoxb7) is dispensable for ureteric bud branching morphogenesis. In contrast, Dot1L(Six2) is critical for the maintenance and differentiation of Six2+ progenitors into epithelial nephrons. Dot1LSix2 mutant kidneys exhibit congenital nephron deficit and cystic dysplastic kidney disease. Molecular analysis implicates defects in key renal developmental regulators, such as Lhx1, Pax2 and Notch. We conclude that the developmental functions of Dot1L-H3K79 methylation in the kidney are lineage-restricted. The link between H3K79me and renal developmental pathways reaffirms the importance of chromatin-based mechanisms in organogenesis.

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.

Renin cells are crucial for the regulation of blood pressure and fluid electrolyte homeostasis. We have recently shown that renin cells possess unique chromatin features at regulatory regions throughout the genome that may determine the identity and memory of the renin phenotype. The 3-D structure of chromatin may be equally important in the determination of cell identity and fate. CCCTC-binding factor (Ctcf) is a highly conserved chromatin organizer that may regulate the renin phenotype by controlling chromatin structure. We found that Ctcf binds at several conserved DNA sites surrounding and within the renin locus, suggesting that Ctcf may regulate the transcriptional activity of renin cells. In fact, deletion of Ctcf in cells of the renin lineage led to decreased endowment of renin-expressing cells accompanied by decreased circulating renin, hypotension, and severe morphological abnormalities of the kidney, including defects in arteriolar branching, and ultimately renal failure. We conclude that control of chromatin architecture by Ctcf is necessary for the appropriate expression of renin, control of renin cell number and structural integrity of the kidney.

Background: The renin-angiotensin system is highly conserved across vertebrates, including zebrafish, which possess orthologous genes coding for renin-angiotensin system proteins, and specialized mural cells of the kidney arterioles, capable of synthesising and secreting renin. Methods: We generated zebrafish with CRISPR-Cas9-targeted knockout of renin ( ren −/− ) to investigate renin function in a low blood pressure environment. We used single-cell (10×) RNA sequencing analysis to compare the transcriptome profiles of renin lineage cells from mesonephric kidneys of ren −/− with ren +/+ zebrafish and with the metanephric kidneys of Ren1 c −/− and Ren1 c +/+ mice. Results: The ren −/− larvae exhibited delays in larval growth, glomerular fusion and appearance of a swim bladder, but were viable and withstood low salinity during early larval stages. Optogenetic ablation of renin-expressing cells, located at the anterior mesenteric artery of 3-day-old larvae, caused a loss of tone, due to diminished contractility. The ren −/− mesonephric kidney exhibited vacuolated cells in the proximal tubule, which were also observed in Ren1 c −/− mouse kidney. Fluorescent reporters for renin and smooth muscle actin ( Tg(ren:LifeAct-RFP; acta2:EGFP )), revealed a dramatic recruitment of renin lineage cells along the renal vasculature of adult ren −/− fish, suggesting a continued requirement for renin, in the absence of detectable angiotensin metabolites, as seen in the Ren1 YFP Ren1 c −/− mouse. Both phenotypes were rescued by alleles lacking the potential for glycosylation at exon 2, suggesting that glycosylation is not essential for normal physiological function. Conclusions: Phenotypic similarities and transcriptional variations between mouse and zebrafish renin knockouts suggests evolution of renin cell function with terrestrial survival.

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.

The unique spatial arregement of the kidney arterioles is an essential event for its development. However, the mechanisms that govern this process are still poorly understood. During nephrogenesis, a group of stromal cells expressing the Forkhead Box D1 ( FoxD1 ) transcription factor (TF) will give rise to the metanephric progenitors for the mural cells of the kidneys arteries and arterioles. We aim to identify the core TFs involved in the cell fate along the differentiaton pathways of the developing kidney vasculature. Therefore, we generated Foxd1-cre; mTmG mice, whose Foxd1 derivative cells are labeled with green fluorescent protein (GFP). GFP+ cells were isolated from 5 (P5) or 30 (P30) days old mice kidneys, and processed either for single-cell RNA-Seq (scRNA-Seq) or for single-cell Assay for Transposase-Accessible Chromatin (scATAC-Seq ). The top5 highly expressed TFs on scRNA-Seq at P5 are: Tcf21, Zeb2, Meis2, Cebpd and Nme3 (p_adjusted_value(padj)= 0, 3.8E-187, 3.9E-180, 4E-172, 4.1E-172 and 3.2E-154, respectively). They are involved in developmental processes and cell proliferation. At P30, the top5 highly expressed TFs are: Atf3, klf2, Fos, Nr4a2 and Junb (padj= 4.2E-294, 2.1E-200, 3.5E-182, 1.7E-52 and 0.2E-24, respectively). They are implicated with calcium-signaling pathway and inflammation. Additionally, scATAC-Seq identifies regions of accessible chromatin for pontential TFs binding, leading to changes in gene expression content and cell identity. At P30, scATAC-Seq showed differential accessible regions with subsequent putative motif enrichment analysis for the TF N4a2 (padj: 4E-297). This is in accordance with our scRNA-Seq results and might play a role in the Foxd1 progenitors cell fate decisions. Our results tracks the fate of the Foxd1+ cells during the kidney vasculature assembly and suggest a new transcription factors network that might play a role to orchestrate cell fate decisions during kidney vascular development.

Our previous study on a partial unilateral ureteral obstruction (pUUO) model in neonatal mice showed that the release of obstruction halts the progression of kidney damage and leads to a remarkable repair of the kidney with improvement in renal blood flow. In the current study, we aim to understand the role of mural cells of the renin lineage during kidney damage and repair in the neonatal pUUO model. Our results show a marked increase in renin-positive areas in kidneys obstructed for 3W (Sham-3W: 0.70±0.10%, n=3; Obstructed-3W: 1.82±0.43%, n=3). However, relief of obstruction at 1W restored the renin-positive areas to sham levels (Post-release-2W: 0.70±0.09%; n=3). Lineage tracing using Ren1 d Cre;mTmG mice revealed a significant increase in GFP+ cells in the obstructed kidneys, with a decrease post-release. To understand further the dynamic changes in cells of renin lineage due to obstruction, we ablated the renin cells using DTA (Diphtheria toxin subunit A). We crossed the DTA fl/fl mice with Ren1 d -DTA het ;Ren1 d Cre;mTmG mice and performed pUUO in the resultant pups with DTA in the renin cells (DTA+). DTA+ animals showed thinning of the renal vasculature and a 90% reduction in renin-positive area compared to controls [Control: 0.70±0.10% (n=3); DTA+: 0.06±0.03% (n=3)]. In addition, there was no significant increase in the renin-positive area post-obstruction [Sham-3W: 0.06±0.04% (n=3); Obstructed-3W: 0.12±0.05% (n=4); Post-release-2W: 0.08±0.03% (n=4)]. These results indicate that ablation of renin cells abolished the obstruction-mediated surge in the renin expression. However, measurement of interstitial collagen positive area indicated that despite the absence of renin cells, the fibrotic damage due to obstruction recovered remarkably post-release [Collagen positive area: Sham-3W: 3.38±0.67% (n=3) Obstructed-3W: 62.98±31.50% (n=3); Post-release-2W: 10.93±5.46% (n=4)]. Similarly, vascular damage induced by persistent obstruction and recovery following the relief of obstruction was similar between the DTA+ and non-DTA animals. Our results imply that though the renin and renin lineage cells increase in obstructed kidneys, ablation of renin cells does not affect the regeneration capacity of kidneys following the relief of obstruction.

Inhibitors of the renin-angiotensin system (RAS) are widely used to treat hypertension. Using mice harboring fluorescent cell lineage tracers, single-cell RNA-Seq, and long-term inhibition of RAS in both mice and humans, we found that deletion of renin or inhibition of the RAS leads to concentric thickening of the intrarenal arteries and arterioles. This severe disease was caused by the multiclonal expansion and transformation of renin cells from a classical endocrine phenotype to a matrix-secretory phenotype: the cells surrounded the vessel walls and induced the accumulation of adjacent smooth muscle cells and extracellular matrix, resulting in blood flow obstruction, focal ischemia, and fibrosis. Ablation of the renin cells via conditional deletion of β1 integrin prevented arteriolar hypertrophy, indicating that renin cells are responsible for vascular disease. Given these findings, prospective morphological studies in humans are necessary to determine the extent of renal vascular damage caused by the widespread use of inhibitors of the RAS.

Renin cells are essential for survival perfected throughout evolution to ensure normal development and defend the organism against a variety of homeostatic threats. During embryonic and early postnatal life, they are progenitors that participate in the morphogenesis of the renal arterial tree. In adult life, they are capable of regenerating injured glomeruli, control blood pressure, fluid-electrolyte balance, tissue perfusion, and in turn, the delivery of oxygen and nutrients to cells. Throughout life, renin cell descendants retain the plasticity or memory to regain the renin phenotype when homeostasis is threatened. To perform all of these functions and maintain well-being, renin cells must regulate their identity and fate. Here, we review the major mechanisms that control the differentiation and fate of renin cells, the chromatin events that control the memory of the renin phenotype, and the major pathways that determine their plasticity. We also examine how chronic stimulation of renin cells alters their fate leading to the development of a severe and concentric hypertrophy of the intrarenal arteries and arterioles. Lastly, we provide examples of additional changes in renin cell fate that contribute to equally severe kidney disorders.

The hormone renin plays a crucial role in the regulation of blood pressure and fluid-electrolyte homeostasis. Normally, renin is synthesized by juxtaglomerular (JG) cells, a specialized group of myoepithelial cells located near the entrance to the kidney glomeruli. In response to low blood pressure and/or a decrease in extracellular fluid volume (as it occurs during dehydration, hypotension, or septic shock) JG cells respond by releasing renin to the circulation to reestablish homeostasis. Interestingly, renin-expressing cells also exist outside of the kidney, where their function has remained a mystery. We discovered a unique type of renin-expressing B-1 lymphocyte that may have unrecognized roles in defending the organism against infections. These cells synthesize renin, entrap and phagocyte bacteria and control bacterial growth. The ability of renin-bearing lymphocytes to control infections—which is enhanced by the presence of renin—adds a novel, previously unsuspected dimension to the defense role of renin-expressing cells, linking the endocrine control of circulatory homeostasis with the immune control of infections to ensure survival.

Myeloid cells are known mediators of hypertension, but their role in initiating renin-induced hypertension has not been studied. Vitamin D deficiency causes pro-inflammatory macrophage infiltration in metabolic tissues and is linked to renin-mediated hypertension. We tested the hypothesis that impaired vitamin D signaling in macrophages causes hypertension using conditional knockout of the myeloid vitamin D receptor in mice (KODMAC). These mice develop renin-dependent hypertension due to macrophage infiltration of the vasculature and direct activation of renal juxtaglomerular (JG) cell renin production. Induction of endoplasmic reticulum stress in knockout macrophages increases miR-106b-5p secretion, which stimulates JG cell renin production via repression of transcription factors E2f1 and Pde3b. Moreover, in wild-type recipient mice of KODMAC/miR106b−/− bone marrow, knockout of miR-106b-5p prevents the hypertension and JG cell renin production induced by KODMAC macrophages, suggesting myeloid-specific, miR-106b-5p-dependent effects. These findings confirm macrophage miR-106b-5p secretion from impaired vitamin D receptor signaling causes inflammation-induced hypertension., Myeloid cells are involved in hypertension, but their exact role in renin-induced hypertension remains unclear. Here the authors show that impaired vitamin D signaling in myeloid cells causes hypertension via macrophage-specific miR-106b-5p secretion, which activates renin production in the kidney.

Aim Renin cells are essential for regulation of blood pressure and fluid‐electrolyte homeostasis. During homeostatic threat, the number of renin cells in the kidney increases, a process termed as recruitment. It has been proposed that recruitment occurs by proliferation, yet no systematic studies have been performed. We sought to determine the extent to which proliferation contributes to the recruitment process. Methods Mice were subjected to recruitment before analysing the renin cells’ cell cycle. For acute threats, we subjected SV129 and C57Bl6 mice to a low sodium diet plus captopril. Tissue sections from treated mice were co‐stained for proliferation markers (Ki67, PCNA, pH3 and BrdU) and renin. Chronic recruitment was studied in deletion models of aldosterone synthase and angiotensinogen through co‐immunostaining and counting mitotic figures in periodic acid‐Schiff‐stained sections. Finally, RNA‐seq of renin cells isolated from recruited mice was performed to study mitotic signature. Results Mice subjected to low salt and captopril displayed increases in renin cell number (312 ± 40 in controls to 692 ± 85 in recruited animals, P

Congenital obstructive nephropathy is a major cause of chronic kidney disease (CKD) in children. The contribution of changes in the identity of renal cells to the pathology of obstructive nephropathy is poorly understood. Using a partial unilateral ureteral obstruction (pUUO) model in genetically modified neonatal mice, we traced the fate of cells derived from the renal stroma, cap mesenchyme, ureteric bud (UB) epithelium, and podocytes using Foxd1Cre, Six2Cre, HoxB7Cre, and Podocyte.Cre mice respectively, crossed with double fluorescent reporter (membrane-targetted tandem dimer Tomato (mT)/membrane-targetted GFP (mG)) mice. Persistent obstruction leads to a significant loss of tubular epithelium, rarefaction of the renal vasculature, and decreased renal blood flow (RBF). In addition, Forkhead Box D1 (Foxd1)-derived pericytes significantly expanded in the interstitial space, acquiring a myofibroblast phenotype. Degeneration of Sine Oculis Homeobox Homolog 2 (Six2) and HoxB7-derived cells resulted in significant loss of glomeruli, nephron tubules, and collecting ducts. Surgical release of obstruction resulted in striking regeneration of tubules, arterioles, interstitium accompanied by an increase in blood flow to the level of sham animals. Contralateral kidneys with remarkable compensatory response to kidney injury showed an increase in density of arteriolar branches. Deciphering the mechanisms involved in kidney repair and regeneration post relief of obstruction has potential therapeutic implications for infants and children and the growing number of adults suffering from CKD.

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.

An accumulating body of evidence suggests that renin-expressing cells have developed throughout evolution as a mechanism to preserve blood pressure and fluid volume homeostasis as well as to counteract a number of homeostatic and immunological threats. In the developing embryo, renin precursor cells emerge in multiple tissues, where they differentiate into a variety of cell types. The function of those precursors and their progeny is beginning to be unravelled. In the developing kidney, renin-expressing cells control the morphogenesis and branching of the renal arterial tree. The cells do not seem to fully differentiate but instead retain a degree of developmental plasticity or molecular memory, which enables them to regenerate injured glomeruli or to alter their phenotype to control blood pressure and fluid–electrolyte homeostasis. In haematopoietic tissues, renin-expressing cells might regulate bone marrow differentiation and participate in a circulating leukocyte renin–angiotensin system, which acts as a defence mechanism against infections or tissue injury. Furthermore, renin-expressing cells have an intricate lineage and functional relationship with erythropoietin-producing cells and are therefore central to two endocrine systems — the renin–angiotensin and erythropoietin systems — that sustain life by controlling fluid volume and composition, perfusion pressure and oxygen delivery to tissues. However, loss of the homeostatic control of these systems following dysregulation of renin-expressing cells can be detrimental, with serious pathological events.

Chronic inflammation and local activation of the renin-angiotensin-aldosterone system (RAAS) play a pivotal role in the pathogenesis and progression of diabetic complications. In patients with type 2 diabetes (T2DM), the prevalence of vitamin D deficiency is almost twice that of non-diabetics, and vitamin d deficiency nearly doubles the risk of developing hypertension and cardiovascular complications compared to diabetics with normal vitamin D levels. Interestingly, mice lacking the vitamin D receptor (VDR) in macrophages (KODMAC) develop renin-dependent hypertension, insulin resistance, and inflammation via up-regulation of macrophage ER stress. Macrophages also express all major components of the RAAS system. However, little is known about the regulation of macrophage-generated renin and its role in modulating the sequelae of VDR signaling in macrophage function and cytokine production. This study found that KODMAC macrophages and vitamin D-deficient macrophages have increased expression and secretion of renin, angiotensin II, ACE, and AT1 receptor and that adhesion, migration, and cytokine release were also increased. Inhibition of ER stress in KODMAC macrophages and vitamin D-deficient macrophages with 4-Phenylbutyric acid (PBA) reduced RAS gene expression and macrophage pro-inflammatory phenotype. Renin 1c gene deletion decreased macrophage adhesion, migration, and cytokine release compared to macrophages with disrupted VDR signaling. Notably, disruption of VDR signaling induced peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) expression in macrophages, and upregulation of renin expression in response to vitamin D deficiency was blunted in PCG1α-deficient macrophages. In conclusion, our findings delineate a mechanism by which impaired VDR signaling induces ER stress to drive PGC1α-dependent expression of renin and RAAS hyperactivation, thereby altering macrophage function and cytokine production. These data implicate RAAS as an essential mediator of VDR-mediated macrophage function and support ongoing investigations of VDR and RAAS modulation as therapeutic approaches in the management of T2DM and its complications.

Renin lineage cells (RLCs) serve as a progenitor cell reservoir during nephrogenesis and after renal injury. The maintenance mechanisms of the RLC pool are still poorly understood. Since RLCs were also identified as a progenitor cell population in bone marrow we first considered that these may be their source in the kidney. However, transplantation experiments in adult mice demonstrated that bone marrow–derived cells do not give rise to RLCs in the kidney indicating their non-hematopoietic origin. Therefore we tested whether RLCs develop in the kidney through neogenesis ( de novo differentiation) from cells that have never expressed renin before. We used a murine model to track neogenesis of RLCs by flow cytometry, histochemistry, and intravital kidney imaging. During nephrogenesis RLCs first appear at e14, form a distinct population at e16, and expand to reach a steady state level of 8-10% of all kidney cells in adulthood. De novo differentiated RLCs persist as a clearly detectable population through embryogenesis until at least eight months after birth. Pharmacologic stimulation of renin production with enalapril or glomerular injury induced the rate of RLC neogenesis in the adult mouse kidney by 14% or more than three-fold, respectively. Thus, the renal RLC niche is constantly filled by local de novo differentiation. This process could be stimulated consequently representing a new potential target to beneficially influence repair and regeneration after kidney injury.

BackgroundWe tested the hypothesis that Foxd1, a transcription factor essential for normal kidney development, is an upstream regulator of the renin-angiotensin system (RAS) during ureteric bud (UB)-branching morphogenesis.MethodsUB branching, RAS gene, and protein expression were studied in embryonic mouse kidneys. RAS mRNA expression was studied in mesenchymal MK4 cells.ResultsThe number of UB tips was reduced in Foxd1-/- compared with that in Foxd1+/+ metanephroi on embryonic day E12.5 (14±2.1 vs. 28±1.3, P

The hormone renin plays a crucial role in the regulation of blood pressure and fluid-electrolyte homeostasis. Normally, renin is synthesized by juxtaglomerular (JG) cells, a specialized group of myoepithelial cells located near the entrance to the kidney glomeruli. In response to low blood pressure and/or a decrease in extracellular fluid volume (as it occurs during dehydration, hypotension, or septic shock) JG cells respond by releasing renin to the circulation to reestablish homeostasis. Interestingly, renin-expressing cells also exist outside of the kidney, where their function has remained a mystery. We discovered a unique type of renin-expressing B-1 lymphocytes that may have unrecognized roles in defending the organism against infections. These cells synthesize and release renin, entrap and phagocyte bacteria and control bacterial growth. The ability of renin-bearing lymphocytes to control infections – which is enhanced by the presence of renin – adds a novel, previously unsuspected dimension to the defense role of renin-expressing cells, linking the endocrine control of circulatory homeostasis with the immune control of infections to ensure survival.

Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear. Pannexin 1 (Panx1) channels localize to both afferent arterioles and juxtaglomerular cells, and provide a transmembrane conduit for ATP release and ion permeability in the kidney and the vasculature. We hypothesized that Panx1 channels in renin-expressing cells regulate renin secretion in vivo. Using a renin cell-specific Panx1 knockout model, we found that male Panx1 deficient mice exhibiting a heightened activation of the renin-angiotensin-aldosterone system have markedly increased plasma renin and aldosterone concentrations, and elevated mean arterial pressure with altered peripheral hemodynamics. Following ovariectomy, female mice mirrored the male phenotype. Furthermore, constitutive Panx1 channel activity was observed in As4.1 renin-secreting cells, whereby Panx1 knockdown reduced extracellular ATP accumulation, lowered basal intracellular calcium concentrations and recapitulated a hyper-secretory renin phenotype. Moreover, in response to stress stimuli that lower blood pressure, Panx1-deficient mice exhibited aberrant “renin recruitment” as evidenced by reactivation of renin expression in pre-glomerular arteriolar smooth muscle cells. Thus, renin-cell Panx1 channels suppress renin secretion and influence adaptive renin responses when blood pressure homeostasis is threatened.

In patients with diabetic kidney disease (DKD), plasma renin activity is usually decreased, but there is limited information on urinary renin and its origin. Urinary renin was evaluated in samples from patients with longstanding type I diabetes mellitus and mice with streptozotocin-induced diabetes mellitus. Renin-reporter mouse model (Ren1d-Cre;mT/ mG) was made diabetic with streptozotocin to examine whether the distribution of cells of the renin lineage was altered in a chronic diabetic environment. Active renin was increased in urine samples from patients with DKD (n=36), compared with those without DKD (n=38; 3.2 versus 1.3 pg/mg creatinine; P

Formation of the metanephric kidney requires coordinated interaction among the stroma, ureteric bud, and cap mesenchyme. The transcription factor Foxd1, a specific marker of renal stromal cells, is critical for normal kidney development. The prorenin receptor (PRR), a receptor for renin and prorenin, is also an accessory subunit of the vacuolar proton pump V-ATPase. Global loss of PRR is embryonically lethal in mice, indicating an essential role of the PRR in embryonic development. Here, we report that conditional deletion of the PRR in Foxd1+stromal progenitors in mice ( cKO) results in neonatal mortality. The kidneys of surviving mice show reduced expression of stromal markers Foxd1 and Meis1 and a marked decrease in arterial and arteriolar development with the subsequent decreased number of glomeruli, expansion of Six2+nephron progenitors, and delay in nephron differentiation. Intrarenal arteries and arterioles in cKO mice were fewer and thinner and showed a marked decrease in the expression of renin, suggesting a central role for the PRR in the development of renin-expressing cells, which in turn are essential for the proper formation of the renal arterial tree. We conclude that stromal PRR is crucial for the appropriate differentiation of the renal arterial tree, which in turn may restrict excessive expansion of nephron progenitors to promote a coordinated and proper morphogenesis of the nephrovascular structures of the mammalian kidney.

The renin-angiotensin system (RAS) evolved early amongst vertebrates and remains functioning throughout the vertebrate phylogeny and has adapted to various environments. The RAS is crucial for the regulation of blood pressure, fluid-electrolyte balance and tissue homeostasis. The RAS is also expressed during early ontogeny in renal and extra-renal tissues, and exerts unique vascular growth and differentiation functions. In this brief review, we describe advances from molecular-genetic and whole animal approaches and discuss similarities and unique aspects of the RAS in the context of embryonic development and vertebrates’ phylogeny.

Renin progenitors appear early and are found in multiple tissues throughout the embryo. Besides their well known role in blood pressure and fluid homeostasis, renin progenitors participate in tissue morphogenesis, repair, and regeneration, and may integrate immune and endocrine responses. In the bone marrow, renin cells offer clues to understand normal and neoplastic hematopoiesis.

Renin or a renin-like enzyme evolved in ancestral vertebrates and is conserved along the vertebrate phylogeny. The ontogenic development of renin, however, is not well understood in nonmammalian vertebrates. We aimed to determine the expression patterns and relative abundance of renin mRNA in pre- and postnatal chickens (Gallus gallus, White Leghorn breed). Embryonic day 13 (E13) embryos show renal tubules, undifferentiated mesenchymal structures, and a small number of developing glomeruli. Maturing glomeruli are seen in post-hatch day 4 (D4) and day 30 (D30) kidneys, indicating that nephrogenic activity still exists in kidneys of 4-week-old chickens. In E13 embryos, renin mRNA measured by quantitative polymerase chain reaction in the adrenal glands is equivalent to the expression in the kidneys, whereas in post-hatch D4 and D30 maturing chicks, renal renin expressions increased 2-fold and 11-fold, respectively. In contrast, relative renin expression in the adrenals became lower than in the kidneys. Furthermore, renin expression is clearly visible by in situ hybridization in the juxtaglomerular (JG) area in D4 and D30 chicks, but not in E13 embryos. The results suggest that in chickens, renin evolved in both renal and extrarenal organs at an early stage of ontogeny and, with maturation, became localized to the JG area. Clear JG structures are not morphologically detectable in E13 embryos, but are visible in 30-day-old chicks, supporting this concept.

Conditional deletion of RBP-J, the major transcriptional effector of Notch signaling, specifically within renin-expressing cells leads to the development of B-cell leukemia. However, the influence of contributing factors such as mouse strain, cell of origin and Cre recombinase copy number are unknown. In this study, we compared RBP-J deletion efficiency using one versus two copies of Cre recombinase. Further, we compared the incidence and timing of leukemia development in two unique strains of mice, C57BL/6 and 129/SV, as well as at different B-cell developmental stages. We found that animals expressing two copies of Cre recombinase developed B-cell leukemia at an earlier age and with more fulminant disease, compared with control animals and animals expressing one copy of Cre recombinase. In addition, we found a difference in leukemia incidence between C57BL/6 and 129/SV mouse strains. Whereas deletion of RBP-J in renin-expressing cells of C57BL/6 mice leads to the development of B-cell leukemia, 129/SV mice develop dermatitis with a reactive, myeloproliferative phenotype. The difference in phenotypes is explained, in part, by the differential expression of extra-renal renin; C57BL/6 mice have more renin-expressing cells within hematopoietic tissues. Finally, we found that deletion of RBP-J in Mb1- or CD19-expressing B lymphocytes does not result in leukemia development. Together, these studies establish that renin progenitors are vulnerable cells for neoplastic transformation and emphasize the importance of genetic background on the development of inflammatory and malignant conditions. This article has an associated First Person interview with the first author of the paper.

BACKGROUND: Recent guidelines recommend a tighter control of systolic blood pressure (SBP) in type 2 diabetes mellitus (T2DM). However, it is unclear whether intensive SBP lowering increases the incidence of chronic kidney disease (CKD) in T2DM. METHODS: Systolic Blood Pressure Intervention Trial (SPRINT) tested the effects of SBP goal < 120 vs. < 140 mm Hg in persons without diabetes. Action to Control Cardiovascular Risk in Diabetes (ACCORD) BP trial tested a similar SBP intervention in T2DM. In participants without CKD at baseline (N = 4305 in ACCORD BP; N = 6677 in SPRINT), we related SBP interventions to incident CKD (defined as a >30% decrease in eGFR to

The close relationship between endothelial and hematopoietic precursors during early development of the vascular system suggested the possibility of a common yet elusive precursor for both cell types. Whether similar or related progenitors for endothelial and hematopoietic cells are present during organogenesis is unclear. Using inducible transgenic mice that specifically label endothelial and hematopoietic precursors, we performed fate-tracing studies combined with colony-forming assays and crosstransplantation studies. We identified a progenitor, marked by the expression of helix-loop-helix transcription factor stem cell leukemia (SCL/Tal1). During organogenesis of the kidney, SCL/Tal1(+) progenitors gave rise to endothelium and blood precursors with multipotential colony-forming capacity. Furthermore, appropriate morphogenesis of the kidney vasculature, including glomerular capillary development, arterial mural cell coating, and lymphatic vessel development, required sphingosine 1-phosphate (S1P) signaling via the G protein-coupled S1P receptor 1 in these progenitors. Overall, these results show that SCL/Tal1(+) progenitors with hemogenic capacity originate and differentiate within the early embryonic kidney by hemovasculogenesis (the concomitant formation of blood and vessels) and underscore the importance of the S1P pathway in vascular development.

We previously identified aldo-keto reductase 1b7 (AKR1B7) as a marker for juxtaglomerular renin cells in the adult mouse kidney. However, the distribution of renin cells varies dynamically, and it was unknown whether AKR1B7 maintains coexpression with renin in response to different developmental, physiological, and pathological situations, and furthermore, whether similar factor(s) simultaneously regulate both proteins. We show here that throughout kidney development, AKR1B7 expression—together with renin—is progressively restricted in the kidney arteries toward the glomerulus. Subsequently, when formerly renin-expressing cells reacquire renin expression, AKR1B7 is reexpressed as well. This pattern of coexpression persists in extreme pathological situations, such as deletion of the genes for aldosterone synthase or Dicer. However, the two proteins do not colocalize within the same organelles: renin is found in the secretory granules, whereas AKR1B7 localizes to the endoplasmic reticulum. Interestingly, upon deletion of the renin gene, AKR1B7 expression is maintained in a pattern mimicking the embryonic expression of renin, while ablation of renin cells resulted in complete abolition of AKR1B7 expression. Finally, we demonstrate that AKR1B7 transcription is controlled by cAMP. Cultured cells of the renin lineage reacquire the ability to express both renin and AKR1B7 upon elevation of intracellular cAMP. In vivo, deleting elements of the cAMP-response pathway (CBP/P300) results in a stark decrease in AKR1B7- and renin-positive cells. In summary, AKR1B7 is expressed within the renin cell throughout development and perturbations to homeostasis, and AKR1B7 is regulated by cAMP levels within the renin cell.

The development of the kidney arterioles is poorly understood. Mature arterioles contain several functionally and morphologically distinct cell types, including smooth muscle, endothelial, and juxtaglomerular cells, and they are surrounded by interconnected pericytes, fibroblasts, and other interstitial cells. We have shown that the embryonic kidney possesses all of the necessary precursors for the development of the renal arterial tree, and those precursors assemble in situ to form the kidney arterioles. However, the identity of those precursors was unclear. Within the embryonic kidney, several putative progenitors marked by the expression of either the winged-forkhead transcription factor 1 (Foxd1+ progenitor), the aspartyl-protease renin (Ren+ progenitor), and/or hemangioblasts (Scl+ progenitor) are likely to differentiate and endow most of the cells of the renal arterial tree. However, the lineage relationships and the role of these distinct progenitors in renal vascular morphogenesis have not been delineated. We, therefore, designed a series of experiments to ascertain the hierarchical lineage relationships between Foxd1+ and Ren+ progenitors and the role of these two precursors in the morphogenesis and patterning of the renal arterial tree. Results show that 1) Foxd1+ cells are the precursors for all the mural cells (renin cells, smooth muscle cells, perivascular fibroblasts, and pericytes) of the renal arterial tree and glomerular mesangium, and 2) Foxd1 per se directs the origin, number, orientation, and cellular composition of the renal vessels.

The activity of the renin–angiotensin–aldosterone system is triggered by the release of the protease renin from the kidneys, which in turn is controlled in the sense of negative feedback loops. It is widely assumed that Ang II (angiotensin II) directly inhibits renin expression and secretion via a short-loop feedback by an effect on renin-producing cells (RPCs) mediated by AT 1 (Ang II type 1) receptors. Because the concept of such a direct short-loop negative feedback control, which originates mostly from in vitro experiments, has not yet been systematically proven in vivo, we aimed to test the validity of this concept by studying the regulation of renin synthesis and secretion in mice lacking Ang II-AT 1 receptors on RPCs. We found that RPCs of the kidney express Ang II-AT 1 receptors. Mice with conditional deletion of Ang II-AT 1 receptors in RPCs were normal with regard to the number of renin cells, renal renin mRNA, and plasma renin concentrations. Renin expression and secretion of these mice responded to Ang I (angiotensin I)–converting enzyme inhibition and to Ang II infusion like in wild-type (WT) controls. In summary, we did not obtain evidence that Ang II-AT 1 receptors on RPCs are of major relevance for the normal regulation of renin expression and secretion in mice. Therefore, we doubt the existence of a direct negative feedback function of Ang II on RPCs.

Integrins are the largest family of cell adhesion molecules. β1-Integrin (Itgb1) is the most abundantly expressed β subunit and is present in almost every cell type. Previous studies showed that Itgb1 is required for normal development of the ureteric bud and podocytes and for the function of the proximal tubule. However, its role in the kidney vasculature has not been explored. Renin cells are crucial for blood pressure homeostasis and for normal nephrovascular development. The mechanisms involved in their morphogenetic functions are not well understood. We found that Itgb1 is highly expressed in renin expressing cells throughout development. Therefore, to study the role of Itgb1 in renin cells we generated a conditional deletion (cKO) of Itgb1 in cells of the renin lineage by crossing floxed Itgb1 mice with mice expressing cre recombinase driven by the renin locus. Itgb1 cKO mice were smaller in size (20.32 ± 4.35 g vs 29.55 ± 7.39 g, p=0.016 ), had smaller kidney to body weight ratio (0.92 ± 0.30 vs 1.31 ± 0.30, p=0.017 ), hypotension (MABP 82.33 ± 4.00 mmHg vs 92.55 ± 7.72 mmHg, p=0.02 ), anemia (hemoglobin 11.26 ± 1.48 g/dL vs 15.07 ± 1.31 d/dL, p=0.003 ; hematocrit 40.39 ± 5.48% vs 53.8 ± 4.21%, p=0.002 ), renal failure (BUN 71.44 ± 32.81 mg/dL vs 29.4 ± 7.67mg/dL, p=0.004), and lower plasma renin levels (6664.58 ± 3251.92 vs 43357.09 ± 17032.63 pg/ml, p =0.001 ). Mutants also developed hyposthenuria (urine osmolality 452 ± 138.98 mOsm/kg vs 1460.5 ± 482.09 mOsm/kg, p ). Histological analysis revealed excessive collagen deposition in the interstitium and peri-glomerular areas; fibrocystic glomeruli; tubular dilatation and protein casts in the tubules. Immunostaining for renin and α-smooth muscle actin showed a marked decrease in renin protein expression and abundant α-smooth muscle actin expression in the interstitium. Microdissection of the renal arterial tree combined with renin immunostaining confirmed the marked decrease in renin and evidenced the overall vascular abnormalities including fewer and shorter arterial and arteriolar branches. Overall, this study shows that β1-Integrin in cells of the renin lineage is crucial for renin expression, morphogenesis of the renal vasculature and maintenance of the normal kidney architecture and function.

The cardiac endothelium plays a crucial role in the development of a functional heart. However, the precise identification of the endocardial precursors and the mechanisms they require for their role in heart morphogenesis are not well understood. Using in vivo and in vitro cell fate tracing concomitant with specific cell ablation and embryonic heart transplantation studies, we identified a unique set of precursors which possess hemogenic functions and express the stem cell leukemia (SCL) gene driven by its 5′ enhancer. These hemo-vascular precursors give rise to the endocardium, atrioventricular cushions and coronary vascular endothelium. Furthermore, deletion of the sphingosine-1-phosphate receptor 1 (S1P1) in these precursors leads to ventricular non-compaction cardiomyopathy, a poorly understood condition leading to heart failure and early mortality. Thus, we identified a distinctive population of hemo-vascular precursors which require S1P1 to exert their functions and are essential for cardiac morphogenesis.

During development, renin cells are precursors for arteriolar smooth muscle, mesangial cells, and interstitial pericytes. Those seemingly differentiated descendants retain the memory to re-express renin when there is a threat to homeostasis. Understanding how such molecular memory is constructed and regulated would be crucial to comprehend cell identity which is, in turn, intimately linked to homeostasis.

Obstructive nephropathy, the leading cause of kidney failure in children, can be anatomic or functional. The underlying causes of functional hydronephrosis are not well understood. miRNAs, which are small noncoding RNAs, regulate gene expression at the post-transcriptional level. We found that miR-145-5p, a member of the miR-143/145 cluster that is highly expressed in smooth muscle cells of the renal vasculature, was present in the pelvicalyceal system and the ureter. To evaluate whether the miR-143/145 cluster is involved in urinary tract function we performed morphologic, functional, and gene expression studies in mice carrying a whole-body deletion of miR-143/145. miR-143/145–deficient mice developed hydronephrosis, characterized by severe papillary atrophy and dilatation of the pelvicalyceal system without obvious physical obstruction. Moreover, mutant mice showed abnormal ureteral peristalsis. The number of ureter contractions was significantly higher in miR-143/145–deficient mice. Peristalsis was replaced by incomplete, short, and more frequent contractions that failed to completely propagate in a proximal-distal direction. Microarray analysis showed 108 differentially expressed genes in ureters of miR-143/145–deficient mice. Ninety genes were up-regulated and 18 genes were down-regulated, including genes with potential regulatory roles in smooth muscle contraction and extracellular matrix-receptor interaction. We show that miR-143/145 are important for the normal peristalsis of the ureter and report an association between the expression of these miRNAs and hydronephrosis.

The mechanisms underlying the establishment, development, and maintenance of the renal vasculature are poorly understood. Here, we propose that the transcription factor “recombination signal binding protein for immunoglobulin kappa J region” (RBP-J) plays a key role in the differentiation of the mural cells of the kidney arteries and arterioles, as well as the mesangial cells of the glomerulus. Deletion of RBP-J in renal stromal cells of the forkhead box D1 (FOXD1) lineage, which differentiate into all the mural cells of the kidney arterioles along with mesangial cells and pericytes, resulted in significant kidney abnormalities and mortality by day 30 postpartum ( P30). In newborn mutant animals, we observed a decrease in the total number of arteries and arterioles, along with thinner vessel walls, and depletion of renin cells. Glomeruli displayed striking abnormalities, including a failure of FOXD1-descendent cells to populate the glomerulus, an absence of mesangial cells, and in some cases complete loss of glomerular interior structure and the development of aneurysms. By P30, the kidney malformations were accentuated by extensive secondary fibrosis and glomerulosclerosis. We conclude that RBP-J is essential for proper formation and maintenance of the kidney vasculature and glomeruli.

We examined the antihypertensive effects of valsartan, aliskiren, or both drugs combined on circulating, cardiac, and renal components of the renin–angiotensin system in congenic mRen2.Lewis hypertensive rats assigned to: vehicle (n=9), valsartan (via drinking water, 30 mg/kg per day; n=10), aliskiren (SC by osmotic mini-pumps, 50 mg/kg per day; n=10), or valsartan (30 mg/kg per day) combined with aliskiren (50 mg/kg per day; n=10). Arterial pressure and heart rate were measured by telemetry before and during 2 weeks of treatment; trunk blood, heart, urine, and kidneys were collected for measures of renin–angiotensin system components. Arterial pressure and left-ventricular weight/tibia length ratio were reduced by monotherapy of valsartan, aliskiren, and further reduced by the combination therapy. Urinary protein excretion was reduced by valsartan and further reduced by the combination. The increases in plasma angiotensin (Ang) II induced by valsartan were reversed by the treatment of aliskiren and partially suppressed by the combination. The decreases in plasma Ang-(1–7) induced by aliskiren recovered in the combination group. Kidney Ang-(1–12) was increased by the combination therapy whereas the increases in urinary creatinine mediated by valsartan were reversed by addition of aliskiren. The antihypertensive and antiproteinuric actions of the combined therapy were associated with marked worsening of renal parenchymal disease and increased peritubular fibrosis. The data show that despite improvements in the surrogate end points of blood pressure, ventricular mass, and proteinuria, dual blockade of Ang II receptors and renin activity is accompanied by worsening of renal parenchymal disease reflecting a renal homeostatic stress response attributable to loss of tubuloglomerular feedback by Ang II.

The renin–angiotensin system (RAS) in the brain is a critical determinant of blood pressure, but the mechanisms regulating RAS activity in the brain remain unclear. Expression of brain renin (renin-b) occurs from an alternative promoter-first exon. The predicted translation product is a nonsecreted enzymatically active renin whose function is unknown. We generated a unique mouse model by selectively ablating the brain-specific isoform of renin (renin-b) while preserving the expression and function of the classical isoform expressed in the kidney (renin-a). Preservation of renal renin was confirmed by measurements of renin gene expression and immunohistochemistry. Surprisingly, renin-b–deficient mice exhibited hypertension, increased sympathetic nerve activity to the kidney and heart, and impaired baroreflex sensitivity. Whereas these mice displayed decreased circulating RAS activity, there was a paradoxical increase in brain RAS activity. Physiologically, renin-b–deficient mice exhibited an exaggerated depressor response to intracerebroventricular administration of losartan, captopril, or aliskiren. At the molecular level, renin-b–deficient mice exhibited increased expression of angiotensin-II type 1 receptor in the paraventricular nucleus, which correlated with an increased renal sympathetic nerve response to leptin, which was dependent on angiotensin-II type 1 receptor activity. Interestingly, despite an ablation of renin-b expression, expression of renin-a was significantly increased in rostral ventrolateral medulla. These data support a new paradigm for the genetic control of RAS activity in the brain by a coordinated regulation of the renin isoforms, with expression of renin-b tonically inhibiting expression of renin-a under baseline conditions. Impairment of this control mechanism causes neurogenic hypertension.

In response to a homeostatic stress the number of cells that make renin increases dramatically along the renal arteriolar tree resembling the embryonic pattern. We have shown that this “recruitment” occurs by re-expression of renin in smooth muscle cells that differentiated from embryonic renin cells. A recent study proposed that during recruitment, renal CD44+ mesenchymal stem-like cells can differentiate into juxtaglomerular (JG)-like renin-producing cells. To test such hypothesis, we assessed the distribution and role of CD44+ cells in renin cell recruitment. Mice with homozygous (KO) and heterozygous (het) deletion of CD44 (knockin for LacZ) were treated with low-sodium diet (0.05%) plus captopril (0.5 g/l) for 10 days (n: 9 treated, 7 controls). Body and kidney weights and BP were not different between KO and het mice. BUN and creatinine were significantly increased in both KO and Het treated mice. The number of renin expressing cells in the kidney and circulating renin increased similarly in treated mice (ELISA, untreated: het 131,503 ± 19,319 pg/mL vs KO 84,714 ± 29,065 pg/mL p=0.2517; treated: het 367,850 ± 38,189 pg/mL vs KO 495,120 ± 80,311 pg/mL p=0.2311). Interestingly, immunostaining for CD44 was negative in kidneys of untreated and treated wild type mice. We occasionally observed in CD44-LacZ het or KO mice isolated LacZ positive cells inside the glomeruli (1 or less per sagittal kidney section) and none in the JG area. On the other hand, immunostaining for CD44 on kidney sections of Ren1cKO mice revealed positive cells within perivascular infiltrates. To confirm these results we performed qRT-PCR for CD44 on kidney samples from CD44 het and KO treated, untreated, control, and Ren1cKO mice. CD44 mRNA expression confirmed the histological findings. In summary: 1) CD44 is dispensable for renin expression and recruitment, and 2) CD44+ cells do not contribute to the pool of renin expressing cells in the kidney during basal conditions or in response to a homeostatic stress as previously suggested. However, they do participate in the inflammatory process observed surrounding the vessels in mice with deletion of the renin gene, suggesting that they derived from the circulation and not from the kidney.

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.