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Renin is crucial for blood pressure regulation and electrolyte balance, and its expressing cells arise from Forkhead box D1-positive (Foxd1 + ) stromal progenitors. However, factors guiding these progenitors toward renin-secreting cell fate remain unclear. Tcf21, a basic helix-loop-helix (bHLH) transcription factor, is essential in kidney development. Using Foxd1 Cre/+ ;Tcf21 f/f and Ren1 dCre/+ ;Tcf21 f/f mouse models, we investigated the role of Tcf21 in the differentiation of Foxd1 + progenitor cells into juxtaglomerular (JG) cells. Immunostaining and in situ hybridization demonstrated fewer renin-positive areas and altered renal arterial morphology, including the afferent arteriole, in Foxd1 Cre/+ ;Tcf21 f/f kidneys compared with controls, indicating Tcf21's critical role in the emergence of renin-expressing cells. However, Tcf21 inactivation in renin-expressing cells ( Ren1 dCre/+ ;Tcf21 f/f ) did not recapitulate this phenotype, suggesting Tcf21 is dispensable once renin cell identity is established. Using an integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on GFP + cells (stromal lineage) from E12, E18, P5, and P30 Foxd1 Cre/+ ;Rosa26 mTmG control kidneys, we analyzed the temporal dynamics of Tcf21 expression in cells comprising the JG lineage ( n = 2,054). A pseudotime trajectory analysis revealed that Tcf21 expression is highest in metanephric mesenchyme and stromal cells at early developmental stages (E12), with a decline in expression as cells mature into renin-expressing JG cells. Motif enrichment analyses supported Tcf21's significant involvement in early kidney development. These findings underscore the critical role of Tcf21 in Foxd1 + cell differentiation into JG cells during early stages of kidney development, offering insights into the molecular mechanisms governing JG cell differentiation and highlighting Tcf21's pivotal role in kidney development. NEW & NOTEWORTHY This manuscript provides novel insights into the role of Tcf21 in the differentiation of Foxd1 + cells into JG cells. Using integrated scRNA-seq and scATAC-seq, the study reveals that Tcf21 expression is crucial during early embryonic stages, with its peak at embryonic day 12. The findings demonstrate that inactivation of Tcf21 leads to fewer renin-positive areas and altered renal arterial morphology, underscoring the importance of Tcf21 in the specification of renin-expressing JG cells and kidney development.

Background: Juxtaglomerular (JG) cells are sensors that control blood pressure and fluid-electrolyte homeostasis. In response to a decrease in perfusion pressure or changes in the composition and/or volume of the extracellular fluid, JG cells release renin, which initiates an enzymatic cascade that culminates in the production of angiotensin II (Ang II), a potent vasoconstrictor that restores blood pressure and fluid homeostasis. In turn, Ang II exerts a negative feedback on renin release, thus preventing excess circulating renin and the development of hypertension. How Ang II suppresses renin release from JG cells remains elusive. Ang II may inhibit renin release via increased systemic pressure sensed by JG cells, or through a direct effect on Ang II receptors in JG cells, which in turn mediate intracellular calcium (Ca 2+ ) mobilization, a known suppressor of renin release. However, the intricate cellular events mediating Ca 2+ -induced renin inhibition by Ang II are not fully understood. Further, the unique structural organization of the juxtaglomerular apparatus (JGA), with JG cells clustered around afferent arterioles, suggests complex intercellular interactions, potentially facilitating coordinated Ca 2+ activity in response to Ang II. Here, we investigate the cellular processes that control Ca 2+ mobilization and the signaling mechanisms elicited when JG cells are stimulated with Ang II within the intact anatomical context of the JGA. By examining these processes, we aim to elucidate the role of cellular organization in Ca 2+ -mediated signaling and its impact on renin regulation within the JGA., Objective: To define intra- and inter-cellular Ca 2+ dynamics, identify the driving ion channels, and elucidate their functional role in Ang II-stimulated JG cells within the native kidney structure., Methods and Results: We generated mice expressing JG cell-specific GCaMP6f, a genetically encoded Ca 2+ indicator, under the Ren1 c promoter. Ex vivo Ca 2+ imaging in acutely prepared kidney slices revealed that JG cells within clusters exhibit coordinated, robust Ca 2+ oscillations in response to Ang II stimulation, contrary to previous observations in isolated cells. These oscillations showed dose-dependent increases in occurrence and correlated with suppressed renin secretion. Pharmacological inhibition identified key drivers of these oscillations: endoplasmic reticulum Ca 2+ storage and release, extracellular Ca 2+ uptake via ORAI channels, and intercellular communication through gap junctions. Blocking ORAI channels and gap junctions alleviated Ang II inhibition of renin secretion., Conclusion: In intact kidney slices, Ang II elicits synchronized Ca 2+ oscillations in JG cells, driven by endoplasmic reticulum-derived Ca 2+ release, ORAI channels, and gap junctions, leading to suppressed renin secretion., Competing Interests: Competing interests The authors declare no competing interest.

Normal kidney development requires the coordinated interactions between multiple progenitor cell lineages. Among these, Foxd1+ stromal progenitors are essential for nephrogenesis, giving rise to diverse cell types including the renal stroma, capsule, mesangial cells, renin cells, pericytes, and vascular smooth muscle cells (VSMCs). However, the molecular mechanisms governing their differentiation remain poorly understood. This study investigates the role of Tcf21, a mesoderm-specific bHLH transcription factor, in Foxd1+ cell fate determination. Using single-cell RNA sequencing (scRNA-seq), we analyzed 32,461 GFP+ cells from embryonic day 14.5 (E14.5) Foxd1 Cre/+ ;Rosa26 mTmG ;Tcf21 f/f kidneys ( Tcf21-cKO ) and controls. Clustering identified a predominant stromal population, further divided into six subpopulations associated with healthy kidney development: nephrogenic zone-associated stroma, proliferating stroma, medullary/perivascular stroma, collecting duct-associated stroma, differentiating stroma, and ureteric stroma. Loss of Tcf21 resulted in marked depletion of medullary/perivascular stroma, collecting duct-associated stroma, proliferating stroma, and nephrogenic zone-associated stroma stromal subpopulations, confirmed by immunostaining, which revealed severe constriction of medullary and collecting duct stromal spaces. Additionally, we identified a novel cluster unique to Tcf21-cKO kidneys, characterized by high expression of Endomucin (Emcn), a vascular endothelial marker. These cells spanned across pseudotime trajectories and were distributed broadly across the mutant kidney. The emergence of Emcn-expressing cells in Tcf21-cKO kidneys coincided with a reduction in Acta2-expressing medullary stromal cells, suggesting a population shift. Our findings highlight the critical role of Tcf21 in directing Foxd1+ progenitor differentiation. Loss of Tcf21 disrupts stromal cell fates, leading to aberrant kidney development and providing new insights into the mechanisms underlying congenital kidney anomalies., Translational Statement: This study reveals critical insights into kidney development and congenital anomalies by identifying the developmental origins of stromal heterogeneity and the key role of Tcf21 in stromal progenitor differentiation. These findings enhance our understanding of stromal cell fate decisions and their relevance to congenital disorders. Additionally, this work provides valuable information for improving the recapitulation of the stromal compartment ex vivo, a current challenge in kidney organoid models. The role of Tcf21 in stromal phenotypic modulation underscores its broader significance in tissue repair and fibrotic diseases, suggesting potential avenues for therapeutic intervention.

Renin-expressing juxtaglomerular (JG) cells possess an intrinsic pressure-sensing mechanism(s) that regulates renin synthesis and release in response to changes in perfusion pressure. Although we recently described the structure of the nuclear mechanotransducer that controls renin transcription, the acute pressure-sensing mechanism that controls the rapid release of renin has not been identified. In JG cells there is an inverse relationship between intracellular calcium and renin release, the 'calcium paradox'. Since the discovery of Piezo2 as the 'touch' receptors, there has been a significant interest in exploring whether they are also involved in other tissues beyond the skin. Given that Piezo receptors are permeable to calcium upon mechanical stimuli, it would be reasonable to hypothesize that Piezo2 controls renin synthesis and/or release in JG cells. To test this hypothesis, we used a variety of novel mouse models and JG cell-specific techniques to define whether Piezo2 controls renin expression and/or release in JG cells. Our in vivo data using constitutive and inducible Cre driver mouse lines and a variety of novel experimental approaches indicate that Piezo2 channels are not necessary for renin synthesis or release in JG cells during normal conditions or when homeostasis is threatened by hypotension, sodium depletion, or inverse changes in blood pressure. Furthermore, Piezo1 channels do not compensate for the lack of Piezo2 in JG cells. Efforts should be devoted to identifying the acute mechanosensory mechanisms controlling renin release., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Renin is crucial for blood pressure regulation and electrolyte balance, and its expressing cells arise from Foxd1+ stromal progenitors. However, factors guiding these progenitors toward renin-secreting cell fate remain unclear. Tcf21, a basic helix-loop-helix (bHLH) transcription factor, is essential in kidney development. Utilizing Foxd1 Cre/+ ;Tcf21 f/f and Ren1 dCre/+ ;Tcf21 f/f mouse models, we investigated the role of Tcf21 in the differentiation of Foxd1+ progenitor cells into juxtaglomerular (JG) cells. Immunostaining and in-situ hybridization demonstrated fewer renin-positive areas and altered renal arterial morphology, including the afferent arteriole, in Foxd1 Cre/+ ;Tcf21 f/f kidneys compared to controls, indicating Tcf21's critical role in the emergence of renin-expressing cells. However, Tcf21 inactivation in renin-expressing cells ( Ren1 dCre/+ ;Tcf21 f/f ) did not recapitulate this phenotype, suggesting Tcf21 is dispensable once renin cell identity is established. Using an integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on GFP+ cells (stromal lineage) from E12, E18, P5, and P30 Foxd1 Cre/+ ;Rosa26 mTmG control kidneys, we analyzed the temporal dynamics of Tcf21 expression in cells comprising the JG lineage ( n =2,054). A pseudotime trajectory analysis revealed that Tcf21 expression is highest in metanephric mesenchyme and stromal cells at early developmental stages (E12), with a decline in expression as cells mature into renin-expressing JG cells. Motif enrichment analyses supported Tcf21's significant involvement in early kidney development. These findings underscore the critical role of Tcf21 in Foxd1+ cell differentiation into JG cells during early stages of kidney development, offering insights into the molecular mechanisms governing JG cell differentiation and highlight Tcf21's pivotal role in kidney development., New & Noteworthy: This manuscript provides novel insights into the role of Tcf21 in the differentiation of Foxd1+ cells into JG cells. Utilizing integrated scRNA-seq and scATAC-seq, the study reveals that Tcf21 expression is crucial during early embryonic stages, with its peak at embryonic day 12. The findings demonstrate that inactivation of Tcf21 leads to fewer renin-positive areas and altered renal arterial morphology, underscoring the importance of Tcf21 in the specification of renin-expressing JG cells and kidney development.

Background: Renin-expressing cells are myoendocrine cells crucial for the maintenance of homeostasis. Renin is regulated by cAMP, p300 (histone acetyltransferase p300)/CBP (CREB-binding protein), and Brd4 (bromodomain-containing protein 4) proteins and associated pathways. However, the specific regulatory changes that occur following inhibition of these pathways are not clear., Methods: We treated As4.1 cells (tumoral cells derived from mouse juxtaglomerular cells that constitutively express renin) with 3 inhibitors that target different factors required for renin transcription: H-89-dihydrochloride, PKA (protein kinase A) inhibitor; JQ1, Brd4 bromodomain inhibitor; and A-485, p300/CBP inhibitor. We performed assay for transposase-accessible chromatin with sequencing (ATAC-seq), single-cell RNA sequencing, cleavage under targets and tagmentation (CUT&Tag), and chromatin immunoprecipitation sequencing for H3K27ac (acetylation of lysine 27 of the histone H3 protein) and p300 binding on biological replicates of treated and control As4.1 cells., Results: In response to each inhibitor, Ren1 expression was significantly reduced and reversible upon washout. Chromatin accessibility at the Ren1 locus did not markedly change but was globally reduced at distal elements. Inhibition of PKA led to significant reductions in H3K27ac and p300 binding specifically within the Ren1 super-enhancer region. Further, we identified enriched TF (transcription factor) motifs shared across each inhibitory treatment. Finally, we identified a set of 9 genes with putative roles across each of the 3 renin regulatory pathways and observed that each displayed differentially accessible chromatin, gene expression, H3K27ac, and p300 binding at their respective loci., Conclusions: Inhibition of renin expression in cells that constitutively synthesize and release renin is regulated by an epigenetic switch from an active to poised state associated with decreased cell-cell communication and an epithelial-mesenchymal transition. This work highlights and helps define the factors necessary for renin cells to alternate between myoendocrine and contractile phenotypes., Competing Interests: None.

Fate mapping and genetic manipulation of renin cells have relied on either noninducible Cre lines that can introduce the developmental effects of gene deletion or bacterial artificial chromosome transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. To circumvent these problems, we generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells that is expressed in a few extrarenal tissues. We confirmed the proper expression of Cre using Akr1b7 CreERT2/+ ; R26R mTmG/+ mice in which Akr1b7 + /renin + cells become green fluorescent protein (GFP) + upon tamoxifen administration. In embryos and neonates, GFP was found in juxtaglomerular cells, along the arterioles, and in the mesangium, and in adults, GFP was present mainly in juxtaglomerular cells. In mice treated with captopril and a low-salt diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. We generated Akr1b7 CreERT2/+ ;Ren1 cFl/- ;R26R mTmG/+ mice to conditionally delete renin in adult mice and found a marked reduction in kidney renin mRNA and protein and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin + cells. Most importantly, these mice developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. We conclude that Akr1b7 CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells. NEW & NOTEWORTHY Fate mapping and genetic manipulation are important tools to study the identity of renin cells. Here, we report on a novel Cre mouse model, Akr1b7 CreERT2 , for the spatial and temporal regulation of gene expression in renin cells. Cre is properly expressed in renin cells during development and in the adult under basal conditions and under physiological stress. Moreover, renin can be efficiently deleted in the adult, leading to the development of concentric vascular hypertrophy.

Competing Interests: Disclosures None.

Competing Interests: Disclosures None.

Aim: Ureteral obstruction leads to significant changes in kidney renin expression. It is unclear whether those changes are responsible for the progression of kidney damage, repair, or regeneration. In the current study, we aimed to elucidate the contribution of renin-producing cells (RPCs) and the cells of the renin lineage (CoRL) towards kidney damage and regeneration using a model of partial and reversible unilateral ureteral obstruction (pUUO) in neonatal mice., Methods: Renin cells are progenitors for other renal cell types collectively called CoRL. We labeled the CoRL with green fluorescent protein (GFP) using genetic approaches. We performed lineage tracing to analyze the changes in the distribution of CoRL during and after the release of obstruction. We also ablated the RPCs and CoRL by cell-specific expression of Diphtheria Toxin Sub-unit A (DTA). Finally, we evaluated the kidney damage and regeneration during and after the release of obstruction in the absence of CoRL., Results: In the obstructed kidneys, there was a 163% increase in the renin-positive area and a remarkable increase in the distribution of GFP + CoRL. Relief of obstruction abrogated these changes. In addition, DTA-expressing animals did not respond to pUUO with increased RPCs and CoRL. Moreover, reduction in CoRL significantly compromised the kidney's ability to recover from the damage after the release of obstruction., Conclusions: CoRL play a role in the regeneration of the kidneys post-relief of obstruction., (© 2023 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

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: Gata3 fl/fl ; Ren1 dCre/+ ( Gata3-cKO ) and control Gata3 fl/fl ; Ren1 d+/+ 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-α 2 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.

During embryonic and neonatal life, renin cells contribute to the assembly and branching of the intrarenal arterial tree. During kidney arteriolar development renin cells are widely distributed throughout the renal vasculature. As the arterioles mature, renin cells differentiate into smooth muscle cells, pericytes, and mesangial cells. In adult life, renin cells are confined to the tips of the renal arterioles, thus their name juxtaglomerular cells. Juxtaglomerular cells are sensors that release renin to control blood pressure and fluid-electrolyte homeostasis. Three major mechanisms control renin release: (1) β-adrenergic stimulation, (2) macula densa signaling, and (3) the renin baroreceptor, whereby a decrease in arterial pressure leads to increased renin release whereas an increase in pressure results in decrease renin release. Cells from the renin lineage exhibit plasticity in response to hypotension or hypovolemia, whereas relentless, chronic stimulation induces concentric arterial and arteriolar hypertrophy, leading to focal renal ischemia. The renin cell baroreceptor is a nuclear mechanotransducer within the renin cell that transmits external forces to the chromatin to regulate Ren1 gene expression. In addition to mechanotransduction, the pressure sensor of the renin cell may enlist additional molecules and structures including soluble signals and membrane proteins such as gap junctions and ion channels. How these various components integrate their actions to deliver the exact amounts of renin to meet the organism needs is unknown. This review describes the nature and origins of renin cells, their role in kidney vascular development and arteriolar diseases, and the current understanding of the blood pressure sensing mechanism., Competing Interests: Disclosures None.

There is increasing interest in the long-term cardiovascular health of women with complicated pregnancies and their affected offspring. Emerging antenatal risk factors such as preeclampsia appear to increase the risk of hypertension and cardiovascular disease across the life course in both the offspring and women after pregnancy. However, the antenatal programming mechanisms responsible are complex and incompletely understood, with roots in alterations in the development, structure, and function of the kidney, heart, vasculature, and brain. The renin-angiotensin-aldosterone system is a major regulator of maternal-fetal health through the placental interface, as well as kidney and cardiovascular tissue development and function. Renin-angiotensin-aldosterone system dysregulation plays a critical role in the development of pregnancy complications such as preeclampsia and programming of long-term adverse cardiovascular health in both the mother and the offspring. An improved understanding of antenatal renin-angiotensin-aldosterone system programming is crucial to identify at-risk individuals and to facilitate development of novel therapies to prevent and treat disease across the life course. Given the inherent complexities of the renin-angiotensin-aldosterone system, it is imperative that preclinical and translational research studies adhere to best practices to accurately and rigorously measure components of the renin-angiotensin-aldosterone system. This comprehensive synthesis of preclinical and translational scientific evidence of the mechanistic role of the renin-angiotensin-aldosterone system in antenatal programming of hypertension and cardiovascular disease will help (1) to ensure that future research uses best research practices, (2) to identify pressing needs, and (3) to guide future investigations to maximize potential outcomes. This will facilitate more rapid and efficient translation to clinical care and improve health outcomes.

Rationale: Renin 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 from FoxD1 + 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., Objective: We sought to comprehensively evaluate the chromatin states and transcription factors (TFs) that drive the differentiation of FoxD1 + progenitor cells into those that compose the kidney vasculature with a focus on renin cells., Methods and Results: We isolated single nuclei of FoxD1 + progenitor cells and their descendants from FoxD1 cre/+ ; R26R-mTmG mice at embryonic day 12 (E12) (n cells =1234), embryonic day 18 (E18) (n cells =3696), postnatal day 5 (P5) (n cells =1986), and postnatal day 30 (P30) (n cells =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 of Nfix in JG cell development and renin expression, and identified the myocyte enhancer factor-2 (MEF2) family of TFs as putative drivers of JG cell differentiation., Conclusions: We 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.

Polycystic kidney disease (PKD) is an inherited disorder that results in large kidneys, numerous fluid-filled cysts, and ultimately end-stage kidney disease. PKD is either autosomal dominant caused by mutations in PKD1 or PKD2 genes or autosomal recessive caused by mutations in the PKHD1 or DZIP1L genes. While the genetic basis of PKD is known, the downstream molecular mechanisms and signaling pathways that lead to deregulation of proliferation, apoptosis, and differentiation are not completely understood. The Notch pathway plays critical roles during kidney development including directing differentiation of various progenitor cells, and aberrant Notch signaling results in gross alternations in cell fate. In the present study, we generated and studied transgenic mice that have overexpression of an intracellular fragment of mouse Notch1 ('NotchIC') in renin-expressing cells. Mice with overexpression of NotchIC in renin-expressing cells developed numerous fluid-filled cysts, enlarged kidneys, anemia, renal insufficiency, and early death. Cysts developed in both glomeruli and proximal tubules, had increased proliferation marks, and had increased levels of Myc. The present work implicates the Notch signaling pathway as a central player in PKD pathogenesis and suggests that the Notch-Myc axis may be an important target for therapeutic intervention., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.

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.

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.

Developmentally heterogeneous renin-expressing cells serve as progenitors for mural, glomerular, and tubular cells during nephrogenesis and are collectively termed renin lineage cells (RLCs). In this study, we quantified different renal vascular and tubular cell types based on specific markers and assessed proliferation and de novo differentiation in the RLC population. We used kidney sections of mRenCre-mT/mG mice throughout nephrogenesis. Marker positivity was evaluated in whole digitalized sections. At embryonic day 16 , RLCs appeared in the developing kidney, and the expression of all stained markers in RLCs was observed. The proliferation rate of RLCs did not differ from the proliferation rate of non-RLCs. RLCs expanded mainly by de novo differentiation (neogenesis). Fractions of RLCs originating from the stromal progenitors of the metanephric mesenchyme (renin-producing cells, vascular smooth muscle cells, and mesangial cells) decreased during nephrogenesis. In contrast, aquaporin-2-positive RLCs in the collecting duct system, which embryonically emerges almost exclusively from the ureteric bud, expanded postpartum. The cubilin-positive RLC fraction in the proximal tubule, deriving from the cap mesenchyme, remained constant. In summary, RLCs were continuously detectable in the vascular and tubular compartments of the kidney during nephrogenesis. Therein, various patterns of RLC differentiation that depend on the embryonic origin of the cells were identified. NEW & NOTEWORTHY The unifying feature of the renal renin lineage cells (RLCs) is their origin from renin-expressing progenitors. RLCs evolve to an embryologically heterogeneous large population in structures with different ancestry. RLCs are also targets for the widely used renin-angiotensin-system blockers, which modulate their phenotype. Unveiling the different differentiation patterns of RLCs in the developing kidney contributes to understanding changes in their cell fate in response to homeostatic challenges and the use of antihypertensive drugs.

[Figure: see text].

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 renin-angiotensin system (RAS) evolved early among 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., (Copyright © 2020. Published by Elsevier Inc.)

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.

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that no conflict of interest exists., (Copyright © 2020. Published by Elsevier Inc.)

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., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS) is, in combination with diuretics, the first-choice treatment for hypertension, although 10-20% of patients do not respond adequately. Next to the RAAS, the nitric oxide/cGMP/protein kinase G (PKG) system is the second fundamental blood pressure regulator. Whether both systems influence each other is not well-studied. It has been shown that nitric oxide (NO) supports renin recruitment via activation of soluble guanylate cyclase (sGC) and subsequent generation of cGMP. Whether this leads to an ensuing activation of PKGs in this context is not known. PKGIα, as well as PKGII, is expressed in renin-producing cells. Hence, we analyzed whether these enzymes play a role regarding renin synthesis, secretion, or recruitment. We generated renin-cell-specific PKGI-knockout mice and either stimulated or inhibited the renin system in these mice by salt diets. To exclude the possibility that one kinase isoform can compensate the lack of the other, we also studied double-knockout animals with a conditional knockout of PKGI in juxtaglomerular cells (JG cells) and a ubiquitous knockout of PKGII. We analyzed blood pressure, renin mRNA and renal renin protein content as well as plasma renin concentration. Furthermore, we stimulated the cGMP system in these mice using BAY 41-8543, an sGC stimulator, and examined renin regulation either after acute administration or after 7 days (application once daily). We did not reveal any striking differences regarding long-term renin regulation in the studied mouse models. Yet, when we studied the acute effect of BAY 41-8543 on renin secretion in isolated perfused kidneys as well as in living animals, we found that the administration of the substance led to a significant increase in plasma renin concentration in control animals. This effect was completely abolished in double-knockout animals. However, after 7 days of once daily application, we did not detect a persistent increase in renin mRNA or protein in any studied genotype. Therefore, we conclude that in mice, cGMP and PKG are involved in the acute regulation of renin release but have no influence on long-term renin adjustment.

The kidney vasculature has a unique and complex architecture that is central for the kidney to exert its multiple and essential physiological functions with the ultimate goal of maintaining homeostasis. An appropriate development and coordinated assembly of the different vascular cell types and their association with the corresponding nephrons is crucial for the generation of a functioning kidney. In this review we provide an overview of the renal vascular anatomy, histology, and current knowledge of the embryological origin and molecular pathways involved in its development. Understanding the cellular and molecular mechanisms involved in renal vascular development is the first step to advance the field of regenerative medicine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.

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., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The kidney vasculature is extremely complex, yet, despite recent progress, our understanding of how the renal vascular system develops is limited. By using advanced tissue engineering techniques and in vivo and in vitro depletion of specific populations of endothelial cell precursors, Halt et al. have identified a CD146-expressing precursor as an important player in the development of the renal vasculature., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)