Renin-expressing Cell Research Articles

Abstract MP46: Mutagenesis Of PRR Cleavage Site In Mice Attenuates Angiotensin II-induced Hypertension And Suppresses Renin Expression

It is well demonstrated that activation of renal (pro)renin receptor (PRR) contributes to AngII-induced hypertension. Relatively, less is known about involvement of soluble PRR (sPRR), the extracellular domain of PRR, primarily generated by site-1 protease(S1P) and furin. Moreover, the relationship between PRR/sPRR and the renin-angiotensin system (RAS) has been debated. In the present study, we used CRISPR/Cas9 strategy to generate mice with mutagenesis of the overlapping cleavage site for both proteases in PRR (termed as PRR R279V/L282V ) to examine the phenotype during angiotensin II (AngII) infusion with particular emphasis on circulating and intrarenal renin status. PRR R279V/L282V mice exhibited a reduction of sPRR level in plasma by ~53% and in the kidney by ~82%, were fertile, and had no gross developmental abnormalities. At basal condition, PRR R279V/L282V mice had drastically suppressed renin levels from plasma (plasma total prorenin/renin content [ng/ml]: 9.3 ± 0.7 in PRR R279V/L282V mice vs. 12.8 ± 0.9 in WT mice, n = 5, p < 0.05), urine (urinary total prorenin/renin excretion [ng/24h]: 0.54 ± 0.11 in PRR R279V/L282V mice vs. 1.05 ± 0.15 in WT mice, n = 5, p < 0.05), and the kidney as compared to wild-type controls (WT). By telemetry, the hypertensive response of PRR R279V/L282V to AngII infusion (300 ng/min/kg) was blunted (MAP [mmHg] in Day 10: 125.9 ± 4.6 in PRR R279V/L282V mice vs. 138.5 ± 2.8 in WT mice, n = 8 for each group, p < 0.05) in parallel with attenuated response of intrarenal renin and renal medullary α-ENaC expression. We further examined the direct role of sPRR in renin regulation in As4.1 cells, a renin-expressing cell line isolated from mouse renal tumor and M-1 cells, a mouse cell line derived from the collecting duct. The exposure to sPRR-His in both cell types consistently elevated renin activity, and renin expression at both protein and mRNA levels, all of which were sensitive to inhibition by ICG-001, a β-catenin signaling inhibitor. Together, these results represent strong evidence favoring sPRR as a mediator of AngII-induced hypertension and a master regulator of renin expression at systemic and local levels. Therefore, PRR should be considered as an integrative member of the RAS.

Super-enhancers maintain renin-expressing cell identity and memory to preserve multi-system homeostasis.

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.

Fate and plasticity of renin precursors in development and disease

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

The aldo-keto reductase AKR1B7 coexpresses with renin without influencing renin production and secretion

On the basis of evidence that within the adult kidney, the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7, also known as mouse vas deferens protein, MVDP) is selectively expressed in renin-producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wild-type kidneys. Renin cells in AKR1B7-deficient kidneys showed normal morphology, numbers, and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7-deficient mice were normal at standard chow and were lowered by a high-salt diet directly comparable to wild-type mice. Treatment with a low-salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7-deficient and wild-type mice. Under this condition, we also observed a strong retrograde recruitment of renin-expressing cell along the preglomerular vessels, however, without a difference between AKR1B7-deficient and wild-type mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by ANG II and by perfusion pressure. Regarding these parameters, no differences were observed between AKR1B7-deficient and wild-type kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.

CBP and p300 in renin homeostasis: can they drive the fate?

CBP and p300 in renin homeostasis: can they drive the fate?

Interleukin-1β Attenuates Renin Gene Expression Via a Mitogen-Activated Protein Kinase Kinase-Extracellular Signal-Regulated Kinase and Signal Transducer and Activator of Transcription 3-Dependent Mechanism in As4.1 Cells

The precise mechanism by which cytokines such as IL-1beta negatively modulate expression of the renin gene remains incomplete. IL-1beta can repress renin transcription under both baseline and retinoic acid-stimulated conditions in As4.1 cells, a renin-expressing cell line derived from the kidney. This repression does not require a negative regulatory element present in the renin enhancer but is optimal in the presence of the entire renin enhancer. Three tandem copies of the retinoic acid response element is sufficient to attenuate the retinoic acid-response by IL-1beta. The decrease in retinoic acid-induced renin promoter activity in response to IL-1beta was blocked with the general tyrosine kinase inhibitor Genistein. IL-1beta caused an increase in the phosphorylation of ERK, but not p38MAPK or c-Jun N-terminal kinase. PD98059, an Erk kinase inhibitor, significantly decreased IL-1beta-mediated phosphorylation of ERK1/2, and attenuated the repression of baseline renin transcription in response to IL-1beta. PD98059 partially reversed the IL-1beta effect on retinoic acid-mediated transcription. To further investigate this mechanism, we searched the downstream effectors of ERK1/2 pathway. Although there was no effect of IL-1beta on the phosphorylation of ELK, Janus kinase 2, or signal transducers and activators of transcription (STAT) 1, IL-1beta significantly increased tyrosine-phosphorylation of STAT3, an effect attenuated by PD98059. STAT3 overexpression significantly repressed transcription of the renin gene, whereas small interfering RNA-mediated knockdown of STAT3 increased renin at baseline and attenuated the IL-1beta response. We conclude that in As4.1 cells, IL-1beta down-regulates renin gene expression via a mechanism involving the Erk-STAT3 pathway.

Biosynthesis of renin in mouse kidney tumor As4.1 cells.

As4.1, a renin-expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42-47 kDa. The predominant intracellular renin was processed into two chains, of 33-34 and 5 kDa. N-glycanase treatment removed N-linked oligosaccharides and yielded products of 41 kDa for prorenin and 31-32 kDa for the heavier chain of two-chain renin. The N-terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N-terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 +/- 1.4% (mean percentage of total precipitable radioactivity +/- SD) of de-novo-synthesized protein secreted into the medium and 0.2 +/- 0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell(-1) h(-1). Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, beta-glucuronidase, by double-fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin-1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.

Tissue-specific expression of novel messenger ribonucleic acids cloned from a renin-expressing kidney tumor cell line (As4.1)

Tissue-specific expression of novel messenger ribonucleic acids cloned from a renin-expressing kidney tumor cell line (As4.1)

Tissue-specific expression of novel messenger ribonucleic acids cloned from a renin-expressing kidney tumor cell line (As4.1).

As4.1 cells are derived from a renin-expressing kidney tumor induced by tissue-specific oncogene-mediated tumorigenesis in transgenic mice. These cells express high levels of renin messenger RNA (mRNA) and synthesize prorenin and renin; they were therefore used as a model to further investigate the molecular biology of renin-producing kidney cells by cloning and characterizing novel mRNAs expressed in these cells. One clone, designated 1.5, was randomly selected from an As4.1 complementary DNA (cDNA) library, and two other cDNA clones, designated 4.9 and 6.9, were obtained by screening the cDNA library using a strategy to identify As4.1 cell-specific mRNAs. Each clone exhibited a highly restricted tissue-specific expression profile, including high level expression in As4.1 cells and low level expression in kidney. No homology was found between the sequence of the partial 1.5 and 4.9 cDNAs and sequences in Genbank. Southern blot analysis revealed that clone 4.9 is encoded by a single copy gene containing at least two separate exons. A homology search of the sequence of clone 6.9 revealed it to encode a cDNA to serum amyloid A protein; consistent with this identification, expression of 6.9 mRNA was highly induced in both kidney and liver after treatment of mice with Escherichia coli lipopolysaccharide.

Isolation and characterization of renin-expressing cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct.

We constructed transgenic mice containing a renin-promoter SV40 T antigen fusion transgene with the intention of inducing neoplasia in renin-expressing cells and isolating renin-expressing cell lines in vitro. We examined six kidney tumors from mice representing three different transgenic lines and found they expressed their endogenous renin gene. Initially, five nonclonal kidney tumor-derived cell lines were established which expressed their endogenous renin gene in addition to the transgene. They retained active renin intracellularly and constitutively secreted an inactive form of renin (prorenin). One of these cell lines was cloned to homogeneity. This line maintained high level expression of renin mRNA throughout 3 months of continuous culture. Although the cells contained an equal proportion of active and inactive renin, the species constitutively secreted into the media was predominantly (95%) prorenin. However, active renin secretion was stimulated 2.3- and 4.6-fold by treatment with 8-bromo-cAMP after 4 and 15 h, respectively. In addition, the presence of multiple secretory granules was confirmed by ultrastructural analysis. These cells, which express renin mRNA and can regulate secretion of active renin, should provide an excellent tool for studying renin gene regulation and secretion. Furthermore, these mice should provide a useful source for the establishment of renin-expressing cell lines from a variety of renin-expressing tissues.

Expression of murine renin genes in subcutaneous connective tissue.

A renin promoter-large tumor antigen (T antigen) fusion gene was constructed to provide a reporter function for renin expression in transgenic mice. These transgenic mice gave rise to tumors in subcutaneous soft tissue, which was attributed to transgene expression at this site. An immunohistochemical analysis of transgenic fetuses from several independent lines revealed scattered T-antigen-containing mesenchymal cells and fibroblasts in the subcutaneous layer of the skin between the panniculus carnosus muscle of the skin and the skeletal muscle of the body wall. This localization is consistent with the location of overt tumorigenesis in adult mice. This pattern was specific for the renin-T antigen fusion gene as no immunohistochemical staining was observed in transgenic fetuses containing a heterologous promoter-T antigen fusion gene. Northern blot analysis of tumor RNA indicated that most of the tumors expressed both T antigen and the endogenous renin gene Ren-1c. In addition, when multiple renin genes were introduced by crossing transgenic mice with nontransgenic DBA/2J mice, which contain another allele of the Ren-1 locus as well as the duplicated locus Ren-2, the resultant tumors expressed the Ren-2 gene. Northern blots were then used to analyze renin expression in the subcutaneous tissue of normal mice. Fully processed renin mRNA was detected in eviscerated 15.5-day postcoitus fetal and newborn carcasses and in newborn skin. Our data indicate that there is a renin-expressing cell population in fetal and newborn subcutaneous tissue.