The Transcription Factor TCF21 is necessary for adoption of cell fates by Foxd1+ stromal progenitors during kidney development.

Normal kidney development requires coordinated interactions between multiple progenitor cell lineages. The Foxd1+ stromal progenitors are critical for normal nephrogenesis and their heterogeneity is increasingly appreciated. However, the molecular mechanisms and trajectories that drive the differentiation of Foxd1+ cells toward the renal stroma, capsule, mesangial cells, renin cells, pericytes, and vascular smooth muscle cells (VSMCs) are poorly understood. Recent work has implicated Tcf21, a mesoderm-specific bHLH transcription factor critical for embryogenesis, in the development of the kidney stroma and perivascular cells. To investigate the role of Tcf21 in Foxd1+ cells, we performed single-cell RNA sequencing (scRNA-seq) on GFP+ cells from E14.5 Foxd1 Cre ;Rosa26 mTmG ;Tcf21 f/f kidneys ( Tcf21 -cKO) and Foxd1 Cre controls. Clustering of the entire dataset identified a large stromal population and a smaller representation of non-stromal lineages. Subclustering of stromal cells identified six populations associated with healthy kidney development: medullary/perivascular, proliferating, differentiating nephron, nephrogenic zone-associated, collecting duct-associated, and ureteric. Loss of Tcf21 resulted in a dramatic reduction in the medullary/perivascular, proliferating, nephrogenic zone-associated, and collecting duct-associated stromal subpopulations. Immunostaining confirmed that Tcf21 -cKO has a severe constriction of the medullary and collecting duct-associated stromal space. We identified and validated a cluster unique to Tcf21 -cKO kidneys exhibiting mosaic expression of genes from nephrogenic, proliferating, medullary, and perivascular stromal cells spanning across all pseudotime, suggesting cells halted in the midst of differentiation. These findings underscore a critical role for Tcf21 in the emergence of Foxd1+ derivatives, with loss of Tcf21 leading to a shift in stromal cell fates that results in abnormal kidney development. NEW & NOTEWORTHY: The mechanisms responsible for the emergence of renal stromal heterogeneity has been unknown. Using scRNA-seq on Foxd1+ enriched cells from E14.5 kidneys, we identified seven molecularly distinct stromal populations and their regional association. The data suggest that the transcription factor Tcf21 regulates the adoption of fates by Foxd1+ cells that is required to form the normal milieu of stromal derivatives for the development of a kidney of normal size and function.

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

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.

An efficient inducible model for the control of gene expression in renin cells.

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 Akr1b7CreERT2/+;R26RmTmG/+ 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 Akr1b7CreERT2/+;Ren1cFl/-;R26RmTmG/+ 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 Akr1b7CreERT2 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, Akr1b7CreERT2, 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.

Tcf21 as a Founder Transcription Factor in Specifying Foxd1 Cells to the Juxtaglomerular Cell Lineage.

Renin is crucial for blood pressure regulation and electrolyte balance, and its expressing cells arise from Foxd1+ stromal progenitors. However, the factors guiding these progenitors toward the renin-secreting cell fate are not fully understood. 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 significantly fewer renin-positive areas and altered renal arterial morphology in Foxd1 Cre/+ ;Tcf21 f/f kidneys compared with controls, indicating Tcf21's necessity for renin cell emergence. 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. 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 revealed that Tcf21 expression peaks at embryonic day 12, crucial for early JG cell specification. Subsequent analyses confirmed Tcf21's critical role in early kidney development, with expression declining as development progresses. Our results highlight the temporal and spatial dynamics of Tcf21, showing its importance in the early specification of Foxd1+ cells into JG cells. These findings provide new insights into the molecular mechanisms governing JG cell differentiation and underscore Tcf21's pivotal role in kidney development. The data suggest that Tcf21 expression in Foxd1+ progenitors is essential for the specification of renin-expressing JG cells, but once renin cell identity is assumed, Tcf21 becomes redundant. 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.

Cells of the renin lineage promote kidney regeneration post-release of ureteral obstruction in neonatal mice.

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.

Renin Cells, From Vascular Development to Blood Pressure Sensing.

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) ß-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.

The role of Gata3 in renin cell identity.

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

Determinants of renin cell differentiation: a single cell epi-transcriptomics approach.

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.

Comparative Studies of Renin-Null Zebrafish and Mice Provide New Functional Insights.

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 Ren1c-/- and Ren1c+/+ 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 Ren1c-/- 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 Ren1YFP Ren1c-/- 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.

Inhibition of the renin-angiotensin system causes concentric hypertrophy of renal arterioles in mice and humans.

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 ß1 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.

Patterns of differentiation of renin lineage cells during nephrogenesis.

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.

Renin Cell Baroreceptor, a Nuclear Mechanotransducer Central for Homeostasis.

[Figure: see text].

A primitive type of renin-expressing lymphocyte protects the organism against infections.

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.

Renin Cells, the Kidney, and Hypertension.

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.

Deciphering the Identity of Renin Cells in Health and Disease.

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.

Expression of Acsm2, a kidney-specific gene, parallels the function and maturation of proximal tubular cells.

The acyl-CoA synthetase medium-chain family member 2 (Acsm2) gene was first identified and cloned by our group as a kidney-specific "KS" gene. However, its expression pattern and function remain to be clarified. In the present study, we found that the Acsm2 gene was expressed specifically and at a high level in normal adult kidneys. Expression of Acsm2 in kidneys followed a maturational pattern: it was low in newborn mice and increased with kidney development and maturation. In situ hybridization and immunohistochemistry revealed that Acsm2 was expressed specifically in proximal tubular cells of adult kidneys. Data from the Encyclopedia of DNA Elements database revealed that the Acsm2 gene locus in the mouse has specific histone modifications related to the active transcription of the gene exclusively in kidney cells. Following acute kidney injury, partial unilateral ureteral obstruction, and chronic kidney diseases, expression of Acsm2 in the proximal tubules was significantly decreased. In human samples, the expression pattern of ACSM2A, a homolog of mouse Acsm2, was similar to that in mice, and its expression decreased with several types of renal injuries. These results indicate that the expression of Acsm2 parallels the structural and functional maturation of proximal tubular cells. Downregulation of its expression in several models of kidney disease suggests that Acms2 may serve as a novel marker of proximal tubular injury and/or dysfunction.