# Transcriptome Analysis of Human Reninomas as an Approach to Understanding Juxtaglomerular Cell Biology

Alexandre G Martini, Lucie K Xa, Marie-Josée Lacombe, Alexis

Blanchet-Cohen, Chantal Mercure, Benjamin Haibe-Kains, Edith C H

Friesema, Anton H van den Meiracker, Kenneth W Gross, Michel Azizi, et al.

► To cite this version:

Alexandre G Martini, Lucie K Xa, Marie-Josée Lacombe, Alexis Blanchet-Cohen, Chantal Mercure, et al. Transcriptome Analysis of Human Reninomas as an Approach to Understanding Juxtaglomerular Cell Biology. Hypertension, 2018, 69 (6), pp.1145-1155. 10.1161/hypertensionaha.117.09179. hal-04079203

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Published in final edited form as:

Hypertension. 2017 June ; 69(6): 1145-1155. doi:10.1161/HYPERTENSIONAHA.117.09179.

TRANSCRIPTOME ANALYSIS OF HUMAN RENINOMAS AS AN
APPROACH TO UNDERSTANDING JUXTAGLOMERULAR CELL
BIOLOGY

Alexandre G. Martini<fnref n="1" />,*, Lucie K. Xa<fnref n="2" />,³,*, Marie-Josée Lacombe<fnref n="2" />, Alexis Blanchet-Cohen⁴, Chantal Mercure<fnref n="2" />, Benjamin Haibe-Kains⁴,⁵, Edith C.H. Friesema<fnref n="1" />, Anton H. van den Meiracker<fnref n="1" />, Kenneth W. Gross<fnref n="6" />, Michel Azizi⁷, Pierre Corvol⁸, Genevieve Nguyen⁸,⁹, Timothy L. Reudelhuber<fnref n="2" />,³,⁵,¹⁰, and A.H. Jan Danser<fnref n="1" />

**Abstract**

Renin, a key component in the regulation of blood pressure in mammals, is produced by the rare and highly specialized juxtaglomerular (JG) cells of the kidney. Chronic stimulation of renin release results in a recruitment of new JG cells by the apparent conversion of adjacent smooth muscle cells along the afferent arterioles. Because JG cells rapidly de-differentiate when removed from the kidney, their developmental origin and the mechanism that explains their phenotypic plasticity remain unclear. To overcome this limitation we have performed RNA expression analysis on four human renin-producing tumors. The most highly expressed genes that were common between the reninomas were subsequently used for *in situ* hybridization in kidneys of 5-day old mice, adult mice and mice treated with captopril. From the top 100 genes, 10 encoding for ligands were selected for further analysis. Medium of HEK293 cells transfected with the mouse cDNA encoding these ligands was applied to (pro)renin-synthesizing As4.1 cells. Among the ligands, only platelet-derived growth factor B (PDGFB) reduced the medium and cellular (pro)renin levels, as well as As4.1 renin gene expression. Additionally, PDGFB-exposed As4.1 cells displayed a more elongated and aligned shape with no alteration in viability. This was

**CONFLICT OF INTEREST DISCLOSURE**

None.

<footnote n="1">¹Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands</footnote>

<footnote n="2">²Laboratory of Molecular Biochemistry of Hypertension, Institut de recherches cliniques de Montréal (IRCM), Montréal, Canada ³Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, Canada ⁴Laboratory of Bioinformatics and Computational Genomics, IRCM, Montréal, Canada ⁵Department of Biochemistry, Université de Montréal, Montréal, Canada</footnote>

<footnote n="6">⁶Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA ⁷Hôpital Européen Georges Pompidou, Centre d'Investigations Cliniques 1418, F-75015 Paris, France ⁸Collège de France, Center for Interdisciplinary Research in Biology (CIRB), Paris, 75005, France ⁹INSERM, U1050, Paris, F-75005, France ¹⁰Department of Medicine, Université de Montréal, Montréal, Canada</footnote>

<footnote>Corresponding author: Prof. dr. A.H.J. Danser, PhD; Division of Vascular Medicine and Pharmacology, Department of Internal Medicine, room Ee1418b, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands; telephone: 31-10-7043540, fax: 31-10-7044733, a.danser@erasmusmc.nl.</footnote>

<footnote>contributed equally.</footnote>
accompanied by a downregulated expression of α-smooth muscle actin, and an upregulated expression of interleukin-6, suggesting a phenotypic shift from myo-endocrine to inflammatory. Our results add 36 new genes to the list that characterize renin-producing cells and reveal a novel role for PDGFB as a regulator of renin-synthesizing cells.

Keywords

reninoma; renin; gene expression; hypertension; transcriptome

INTRODUCTION

The aspartyl protease renin catalyzes the rate-limiting step in the renin-angiotensin system (RAS). Renin is produced almost exclusively by so-called juxtaglomerular (JG) cells that line the afferent arterioles at their point of entry into the kidney glomeruli. JG cells convert renin from its inactive precursor (prorenin) by proteolysis and store the resulting active renin in lysosome-like dense cytoplasmic granules whose content can be rapidly released in response to such stimuli as a sudden drop in the perfusion pressure of the afferent arteriole or beta-adrenergic stimulation. During kidney development, renin-producing cells are more broadly distributed along the lumen of the nascent arterioles¹ and gradually become restricted to the glomerular poles after birth. Prolonged exposure to stimuli, such as blood pressure-lowering medication, a low-salt diet or ischemia results in an apparent recruitment of new renin-producing cells along the afferent arterioles,² which has been proposed to represent a partial return to the developmental pattern of JG cell distribution. Altogether, renin-producing JG cells display many unique biological features and because of their central role in modulating blood pressure and fluid balance, there has been long-standing interest in obtaining a better understanding of their origins and biology.

Efforts to characterize JG cells have historically been hampered by two factors: First, JG cells rapidly de-differentiate when placed in culture, losing their secretory granules and, concomitantly, their ability to proteolytically activate prorenin. As a result, attempts to immortalize both mouse and human renin-producing cells have resulted in the establishment of cell lines that have lost the ability to secrete renin.³, ⁴ Second, JG cells make up only approximately 1/1000 of the cell mass of the kidney, making it difficult to isolate sufficient quantities of pure cells for further characterization. As an alternative approach, Brunskill et al.⁵ targeted yellow fluorescent protein to mouse JG cells and used fluorescence-activated cell sorting (FACS) to enrich tagged cells for expression analysis. The resulting study proposed a 369 gene "signature" that characterizes mouse JG cells, although few of these have been verified in subsequent studies.

In an effort to extend these studies and to determine if the genes identified in the mouse also characterize human renin-producing cells, we have catalogued the gene expression pattern of biopsies derived from 4 human renin-producing tumors, or reninomas. Reninomas are rare, benign tumors arising from a proliferation of JG cells in the kidney cortex⁶, ⁷ and are often detected due to the appearance of fulminant hypertension in relatively young patients (mean age under 30 years) with no obvious explanatory risk factors. RNA sequencing (RNA-Seq)
revealed a set of 54 highly expressed genes that are common to all four tumors and another 30 expressed in 3 of the 4 biopsies tested. *In situ* hybridization of mouse kidney sections subsequently confirmed JG cell expression of 44 of these genes, the vast majority of which have not previously been reported to be expressed in JG cells. Finally, hypothesizing that the highly expressed reninoma genes affect renin-synthesizing capacity, we selected 10 ligands (based on known relevance for blood pressure and kidney disease) and studied their effects on (pro)renin release by As4.1 cells. These cells are derived from a transgene-induced mouse kidney tumor, and do not store renin. They may thus be considered as de-differentiated JG cells which have lost their capacity to secrete lysosomes. Consequently, As4.1 cells might serve as a model to study the effect of reninoma-specific ligands on JG cell plasticity. Results revealed an unexpected suppressant role for platelet-derived growth factor B (PDGFB).

# METHODS

## Human and mouse studies

Total RNA was isolated using Trizol (Invitrogen) from four reninomas surgically obtained from four anonymous patients (Paris1, Paris2, Montreal, Rotterdam), and underwent RNA-sequencing analysis using the HiSeq2000 (Illumina). The top 100 most up-regulated genes presented in all four, and at least in three out of four reninomas were submitted to immunofluorescence and fluorescent *in situ* hybridization (iFISH) in order to analyze their expression in the juxtaglomerular apparatus of kidney mice under different situations (5 day old, 10–12 week old and 10–12 week old treated with captopril for 7 days). For further details, see the Methods section in the online-only Data Supplement.

## Cell culture studies

Human Embryonic Kidney (HEK) 293 cells were transfected with plasmids encoding ligands selected from the transcriptome analysis on four reninomas. The conditioned medium derived from these cells was used to study the effect of these ligands on (pro)renin-synthesizing As4.1 cells. For further details, see the Methods section in the online-only Data Supplement.

## Statistical analysis

Results are expressed as mean±SEM. Data were analyzed for normal distribution using a Shapiro-Wilk's test (P>0.05). Differences were tested using one-way or two-way ANOVA, followed by Holm-Sidak's or Dunnett's multiple comparison test. P<0.05 was considered significant.

# RESULTS

Deep sequencing of RNA (RNA-Seq) was performed on three biopsies of a first reninoma from Paris (Par1B1-B3), one biopsy from a reninoma from Montréal (Mon), two biopsies from a reninoma from Rotterdam (RotB1, B2), and a second reninoma from Paris (Par2) along with a biopsy from adjacent supposedly normal tissue from the same patient (Par2N) (Table S1). We obtained from 45–100 million reads per sample (Table 1) with comparable
overall sample quality (Figure S1). Remarkably, the Fragments Per Kilobase of transcript per Million (FPKM) mapped reads values for renin were quite similar in the four tumor samples and, in each tumor, renin was expressed at 15–41 times the level of the next most abundant transcript, confirming the diagnosis of *bona fide* reninomas (Table 2).

Results obtained from the three independently analyzed biopsies of the Paris1 reninoma demonstrated an inter-sample Spearman correlation of >0.98 (Figure S2, red outlines) and similar results were obtained with the two biopsies of the reninoma from Rotterdam (Figure S2, green outlines) suggesting that the sample preparation and results obtained were reliable and reproducible. In the one case in which we had biopsies of both tumor and adjacent tissue from a single patient (Paris 2), there was a 217-fold increase in the number of renin reads in the reninoma versus the “normal” sample (Table 2) although the samples overall were very highly correlated (0.92 for all genes; see Figure S2, blue outlines). By limiting the gene set analyzed in all the samples to those that were expressed at least 4-fold higher in the reninoma versus the “normal” biopsies for Paris 2, we reduced the gene set to just over 1000 (Table S2) and this resulted in the removal of low reads (which are commonly unreliable) and many “housekeeping” genes from the analysis. Using this limited gene set, we identified the 100 most-expressed genes in each biopsy (Table S3). Re-calculation of the Spearman correlation using these highly expressed genes revealed a higher discrimination between samples (Figure S3). Although the repeat biopsies continued to exhibit a correlation >0.98, the correlation between the reninomas varied between a low of 0.46 (Par1 versus Par2) and a high of 0.74 (RotB1 and Par2). Surprisingly, the highest correlation is between the Par2 and Par2N samples (0.81, Figure S3), raising the possibility that the supposedly normal sample from this patient contains some reninoma. A Venn diagram reveals that 54 of the 100 most expressed genes were common to all 4 reninomas (Table S3 and Figure S4) and another 30 genes were highly expressed in 3 out of 4 of the tumors analyzed. Functional annotation clustering analysis (DAVID) did not reveal any statistically significant correlation for cellular function using the genes most commonly expressed in the reninomas (data not shown).

To test if these reninoma-enriched genes were also expressed in JG cells, we performed iFISH for 71 of the above 84 common reninoma transcripts (excluding, among others, genes that have no mouse equivalent) in kidney sections from C57bl/6 mice using the equivalent mouse probes (Table 3 and Figure S5). As expected, combined *in situ* hybridization and immunofluorescence for renin reveals perfect co-localization in the JG cells of adult mice (Figure 1A–1D). Furthermore, treatment of the mice with captopril for one week resulted in the expected recruitment of JG cells along the afferent arterioles (Figure 1E), while co-localization was also observed throughout the maturing arterioles of 5 day-old mice (Figure 1F). These results confirm the validity of this approach to assess the co-localization of the transcripts from the reninoma in the mouse JG cells.

In order to obtain a semi-quantitative comparison of gene expression levels in renin-
producing cells, all confocal microscopy images were taken using the same parameters and a
ranking from 0 (for no expression observed) to 4 (highly expressed) was assigned for each
gene under each condition tested (Table 3). Among the 71 genes tested, 44 genes are
expressed in both reninomas and mouse JG cells. Twenty-four of these were detected in all
three conditions (5 day-old, adult and adult treated with captopril kidneys) while the remainder showed preferential expression in one or more of the conditions (Figure S5 and Table 3). Functional annotation clustering analysis using the 44 genes expressed in both the reninomas and mouse JG cells identified “developmental processes” as the most likely gene ontology (data not shown).

From the top 100 highly expressed genes, focusing on blood pressure regulation and renal disease, we selected 10 genes coding for ligands (FSTL1, IGF2, LGALS1, MDK, MFGE8, NPPC, PDGFB, SPARC, SPARCL1, WFDC2) in order to investigate their potential role in JG recruitment. The corresponding mouse cDNAs were cloned with a c-myc epitope and transfected into HEK293 cells to obtain high concentrations of these ligands in the supernatant of the culture medium. WB experiments confirmed the presence of the proteins of each of the transfected genes in the HEK293 cell medium at 48 hours after transfection (Figure S6). Since all ligands were tagged with a c-myc epitope, the same primary antibody could be used.

After adding the 48-hour conditioned HEK293 medium (containing the highest level of the desired protein) to (pro)renin-producing As4.1 cells for 2 days, we studied As4.1 cell morphology and quantified (pro)renin in the medium. Only PDGFB altered As4.1 cell morphology, in that the cells after exposure to this ligand displayed a more elongated densely packed and aligned shape (Figure S7). Moreover, PDGFB also induced parallel decreases in the medium and cellular (pro)renin levels, by 64±5% and 53±10%, respectively (n=11), although only the former was significant (P=0.03; Figure 2A). Under control conditions, the medium contained predominantly (>95%) prorenin. In contrast, the cell lysate contained renin only, at levels corresponding to <1% of the total amount of renin +prorenin in the medium. qPCR experiments revealed that the reduction in (pro)renin also occurred at the expression level, decreasing its level by 84±5% (P=0.005, n=5; Figure 2A). Furthermore, when applying commercially obtained PDGFB directly to As4.1 cells, it lowered (pro)renin concentration-dependently (Figure 2B), and from these data, it could be estimated that the PDGFB level in the conditioned HEK293 medium was >100 ng/mL. MTT assays confirmed that the effect of PDGFB was not due to a reduction in cell viability (Figure 2B). Moreover, PDGFB did not affect total protein content, suggesting that its effect was also not due to a decrease in cell number (data not shown).

Finally, we evaluated the effect of PDGFB-containing HEK293 medium on the expression of markers that illustrate the occurrence of a smooth muscle, endocrine and/or immunological phenotype, i.e., aldo-keto reductase family 1, member B7 (Akr1b7), α-smooth muscle actin (Acta2), interleukin-6 (IL-6), and Recombination signal binding protein for immunoglobulin kappa j (Rbpj). Figure 2C illustrates that PDGFB upregulated IL-6 (P<0.0001, n=4), and downregulated Acta2 (P=0.01, n=6) in As4.1 cells. Non-significant increases in Akr1b7 (P=0.06, n=4) and Rbpj (P=0.06, n=5) expression were also observed.
DISCUSSION

The aim of this study was to gain insight into human JG cell biology. Given their rapid de-differentiation in culture, such knowledge is currently lacking. Instead, information on renin synthesis is largely derived from renin-producing tumor-derived cell lines, like As4.1 (mouse), Calu-6 (human) and human mastocytoma cells (HMC-1). In an attempt to catalogue the genes expressed in JG cells, Brunskill et al.⁵ used a fluorescent reporter mouse model, in which they have used cell sorting to enrich for gene-tagged renin-expressing cells in the mouse kidney. Comparison of gene expression arrays from the sorted cells versus the whole kidney led to the identification of 369 core genes whose expression was proposed to constitute a “gene signature” for the mouse JG cell. In the current study, we have used direct RNA sequencing (RNA-Seq) of human reninomas to independently quantify gene expression levels in each of the biopsies we obtained, and to test for correlation of the gene expression patterns between mouse and human renin-expressing cells. This approach has the additional advantage that a control tissue to which expression can be compared was not required. This was an important consideration since our samples were derived from frozen pathology specimens, and only one of them had a corresponding biopsy of adjacent, presumed non-tumor, tissue. The high reproducibility and sequence “depth” achieved confirm that this approach is valid, robust and applicable to archival samples and the comparison of the reninoma transcriptomes allowed us to derive a gene expression “fingerprint” for the tumors. In addition, the high expression of renin, CD34, and vimentin, all of which have been previously reported as markers of reninomas,⁷,⁸ confirms the diagnosis of bona fide reninoma in all of the tumors.

At least 3 of the 4 reninomas expressed genes at relatively high levels that were also reported to be highly expressed in mouse renin-expressing cells⁵ including, in addition to renin, *RGS5*, *CRIP1*, and *ATP1B2*. Conversely, a number of the most highly expressed transcripts previously identified in the mouse JG cell preparations were not detected in the 100 most-expressed genes in the reninomas including *Syne2*, *Plac9*, *Myh11*, *Jph2*, *Myo18*, *Akr1b7* and *Mgp*. Interestingly, *Akr1b7*, a member of the alpha-keto reductase gene family was previously found to be highly enriched in mouse JG cell preparations and to serve as a specific marker for JG cells in the mouse kidney.⁵ Even though *Akr1b7* does not have a homologue in humans, its closest human analogues, *AKR1B10* and *AKR1B15*, are not expressed at detectable levels in the reninomas (data not shown). Since more recent results have shown that targeted deletion of *Akr1b7* in mouse JG cells does not affect renin production,⁹ it is possible that the expression of *Akr1b7* in mouse JG cells is unrelated to renin synthesis.

Indeed, of the extended list of 369 core genes previously reported to be enriched in the mouse renin cell preparations,⁵ only 8 were among the 84 genes expressed in at least 3 of the 4 reninomas (Table 3 and Table S3). Although the exact reason for these differences is unclear, they could be due to several factors. First, some of the highly-expressed transcripts in the reninomas might be related to the tumoral transformation of the renin-producing cells. For example, the expression of neither CD34, a commonly reported marker of reninomas and myelo-lymphopoetic precursors, nor insulin-like growth factor 2 (IGF2) was detected in the mouse JG cells. IGF2 also did not affect (pro)renin release by As4.1 cells. Alternatively,
the differences could be due to the fact that in both the current study and that of Brunskill et al.,⁵ the gene expression patterns were obtained from impure populations of renin-producing cells and could reflect expression of a gene in a contaminating non-JG cell. Finally, they could reflect true differences between mouse and humans.

To overcome some of these limitations, we used iFISH to test whether the commonly expressed genes in the reninomas were also expressed in mouse JG cells (Figure S5). Even though the sensitivity of this analysis is limited by the level of expression of the candidate genes, it nevertheless confirmed that 44 of the gene transcripts expressed in the reninomas were also detected in mouse JG cells (Table 3) and thereby significantly extends the list of potential markers for this unique cell type.

Previous studies have reported that JG cells express contractile proteins, are related to pericytes⁵ and, like pericytes, are derived from FoxD1-expressing lineages.¹⁰ Simultaneously, renal CD44(+) mesenchymal stem cell-like cells, whose differentiation is triggered by LXR stimulation,¹¹ have been suggested to differentiate into JG-like renin-expressing cells.¹² The current analysis confirmed the expression of a number of contractile proteins in both reninomas and JG cells, including myosin light chain 9 (MYL9), tropomyosin 2 (TPM2), transgelin (TAGLN), myocyte enhancer factor 2C (MEF2C), caldesmon (CALD1) and very low levels of ACTA2, as well as some pericyte markers including RGS5 and osteonectin (SPARC). We also confirmed the expression of vimentin (VIM), a marker of mesenchyme-derived cells, in both the reninomas and JG cells, but did not detect expression of either LXR receptor or CD44 in the tumors.

Our data also call into question the model that “recruited” JG cells (e.g., after captopril treatment) represent a return to the embryonic stage of renin-producing cells. The *ACTA2* gene codes for α-smooth muscle actin, a marker for smooth muscle cells. Consistent with what has been previously reported¹³ there is a demarcation in *Acta2* expression with the expected higher expression in smooth muscle cells of the afferent arteriole and very low expression in adjacent JG cells of adult mice (Figure 3A). This demarcation persists after recruitment of new JG cells following captopril treatment (Figure 3B). In contrast, there appear to be 2 populations of renin-expressing cells in the developing kidney: one population that exists in clusters similar to the JG apparatus in adult kidney (Figure 3C, arrow) and which express only very low levels of *ACTA2* and others which line the vessels of the developing kidney and exhibit significant overlap of renin staining and *ACTA2* expression (Figure 3C, light blue outline). Other genes whose expression was detected almost exclusively in renin-producing cells of the developing kidney include *A2m*, *Slit3* and *Cxcl12*. Conversely, a number of genes appeared to be preferentially expressed in the JG cells of adult control and captopril-treated mice including *Ptp4a3*, *C4a*, *Tinagl1*, *Lbh*, *Mdk*, *Bgn* and *Crip1*. Taken together, these results suggest that JG recruitment does not simply involve a return to the embryonic renin-producing cell, but may rather reflect differentiation of smooth muscle to either an intermediate or alternative phenotype.

*KISS1*, which codes for a metastasis suppressor and is the second to third most abundant transcript in each of the four of the reninomas (Table 3), also presents a major difference in expression in the reninomas and mouse kidney. The *KISS1* gene is located immediately
adjacent to the renin gene on chromosome 1 in both mice and humans (http://www.ncbi.nlm.nih.gov/gene/3814). When large chromosomal fragments containing both genes are used to generate transgenic mice, coordinate expression of renin and *KISS1* is detected in brain, kidney, lung and placenta.¹⁴ While we detected some expression of *Kiss1* in the JG cells of captopril-treated mice (Figure S5), we did not detect its expression in adult mouse JG cells, raising the possibility that the neighboring *Kiss1* gene is expressed only in conditions of very high renin gene expression. Whether it explains the fact that intra-renal reninomas rarely metastasize is currently unknown.

Also among the most highly expressed genes in all 4 reninomas is *DBNDD2* (Table S3), a paralog of the gene *DTNBP1*, which is a component of the BLOC-1 complex that is required for normal biogenesis of lysosome-related organelles (LRO), such as platelet dense granules and melanosomes. Thus, *DBNDD2* might be an interesting candidate to explain the high content of LROs found in the JG cells.

A number of ligands and receptors were also found to be highly expressed in the reninomas and in mouse renin-producing cells, some of which might play a role in either the maintenance of the JG cell phenotype or in JG cell recruitment. Of particular interest is the high level of expression of the Notch3 receptor and PDGF receptor β (*PDGFRβ*) in the reninomas and JG cells. Indeed, RBPJ, the main effector involved in Notch receptor signaling, activates the myo-endocrine program of the JG cell, and conditional deletion of *Rbpj* in renin-producing cells results in a dramatic decrease in the number of JG cells seen in adult mice.¹⁵ Yet, in humans mutations in *NOTCH3* have been identified as the underlying cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), with no known effects on renin. In the current study we observed that PDGFB suppressed (pro)renin synthesis in As4.1 cells, and altered their morphology. The concomitant suppression of Acta2 and upregulation of IL-6 suggests an As4.1 phenotype change from myo-endocrine to inflammatory. IL-6 upregulation by PDGFB is well-known, and occurs in a RBPJ-independent manner.¹⁶ No significant alterations were observed in Akr1b7 and Rbpj, nor did PDGFB affect cell viability. Classically, endothelium-derived PDGFB recruits pericytes and promotes VSMC proliferation.¹⁷ Importantly, pericytes in the human kidney were recently reported to produce renin.¹⁸ Hypoxia transforms renin-producing cells into erythropoietin-producing cells,¹⁹ and erythropoietin-expressing cells are assumed to be derived from capillary pericytes.²⁰ Finally, following severe mesangial injury, renin-positive precursor cells moved into intraglomerular sites and differentiated into (renin-negative), PDGFRβ-expressing mesangial cells.²¹ As shown in Figure 3 (panels D–F), *PDGFRβ* modestly overlapped with renin in kidneys of adult, young, and captopril-treated adult mice, in addition to its well-known expression in interstitial cells. Moreover, Rider et al. observed that renin cells in the zebrafish kidney express both Acta2 and Pdgfrβ.²² Given the well-known role of endothelial PDGFB as a facilitator of mesangial cell proliferation and migration, an additional effect of PDGFB might now be suppression of renin expression. Here it should be mentioned that neither conditional deletion of the PDGFRβ in renin-producing cells nor deletion of endothelial PDGFB production affected the normal development of renal renin-expressing cells.²³ This may not be too surprising, given the fact that PDGFB actually is a negative regulator of renin expression, possibly coming into play only under pathological conditions.
Besides the high level of *PDGFRβ* expression in the reninomas and the JG cells, both also express the genes for SPARC, SPARCL1 (SPARC-like 1 or hevin) and MFGE8 (lactadherin) at relatively high levels. It has been reported that SPARC can both prevent the binding of PDGF to its receptor²⁴ and potentiate its profibrotic effects,²³ while lactadherin potentiates PDGFRβ signaling.²⁵ This raises the possibility that a more complex interplay between SPARC, SPARCL1, MFGE8 and PDGF regulates either the recruitment or other physiological responses of JG cells in their natural setting. In agreement with this concept, the current study revealed no effect of either SPARC, SPARCL1 or MGE8 alone on (pro)renin synthesis by As4.1 cells. Additionally, ligands that are known to be involved in blood pressure regulation (NPPC, or natriuretic peptide precursor C), diabetic nephropathy (LGALS1 or galectin-1)²⁶, nephrogenesis (MDK or midkine)²⁷, renal fibrosis (WFDC2, or WAP four-disulfide core domain protein 2)²⁸, and renal injury prevention (FSTL1, or follistatin-related protein 1)²⁹ also did not directly affect (pro)renin synthesis by As4.1 cells.

Somewhat surprisingly, no proteases were included in the 100 most expressed genes in the reninomas (Table S3) where we might have expected to find one or more candidate prorenin processing enzymes (PPE) necessary for proteolytic activation of the renin secreted by these tumors. However, examination of the gene lists before subtraction of the genes expressed in the Par2N “normal” biopsy (Table S1) reveals that the rank of Cathepsin B (*CTSB*) expression varies from the 8th (Par1B1) to the 78th (Par2) position in the reninomas, although these expression levels did not appear to correlate with the apparent renin expression levels in each sample (not shown). However, because *CTSB* was only expressed 1.1-fold more in the Par2 reninoma than in the Par2N control kidney biopsy, it was eliminated from our gene list. Nevertheless, because *CTSB* has been co-localized with renin in JG cells secretory granules³⁰ and shown to accurately process human prorenin in vitro,³¹ it is quite possible that it fulfills the role of the PPE in the reninomas, even though it is a ubiquitously distributed protease. This analysis also reveals one of the potential shortcomings of our approach since by focusing on genes whose expression was consistently enriched in tumor biopsies we may have eliminated consideration of genes that play an important functional role in the biology of the JG cell, but whose expression is more widely distributed.

The present study is the first to use a reninoma transcriptome analysis as an approach to understand human JG cell biology. It has yielded multiple new candidates that might play a role in the developmental plasticity of these cells and, among them, raises the PDGFB-PDGFRβ signaling pathway as a promising novel pathway that controls renin-expressing cells.

## PERSPECTIVES

By comparing the transcriptomes of 4 reninomas and subsequently mapping the expression patterns of the most highly expressed genes in mouse kidney during development, adulthood and following renin-producing cell recruitment in response ACE inhibition, we have confirmed the expression of 36 new genes (Table 3) in this unique cell type. As a result, we propose the PDGFB-PDGFRβ signaling pathway as a promising novel pathway that controls the developmental plasticity of JG cells.
# Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

# Acknowledgments

The authors gratefully acknowledge the technical advice of Dr. Eric Lécuyer for the iFISH analysis, and the analytical help of Frank P.J. Leijten, Ingrid M. Garrelds and Gardi J. Voortman.

# SOURCES OF FUNDING

This work was supported by a grant from the Canadian Institutes for Health Research to TLR, a grant from Naturali and Biologia to GN, two grants (HL48459 and/RPCI Core Grant P30CA016056) to KWG, and a CAPES Foundation grant (99999.006209/2015-07) to AGM.

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NOVELTY AND SIGNIFICANCE

What is new?

This is the first report of a gene expression catalogue for human reninomas.

What is relevant?

This study is significant because it advances our knowledge of the unique biology of human renin-producing cells and may provide a foundation for understanding renin cell recruitment in the context of anti-hypertensive therapy. Among others, it has identified the PDGFB-PDGFRβ signaling pathway as a promising novel pathway that controls renin-expressing cells

Summary

Deep sequencing of 4 human renin-producing tumors with transcript mapping in the kidneys of mice under different conditions yielded a list of genes specifically expressed in renin-producing cells. When evaluating the effect of 10 of these genes on (pro)renin-producing As4.1 cells, it was observed that platelet-derived growth factor B (PDGFB) suppressed (pro)renin levels and renin gene expression. Moreover, PDGFB-exposed cells displayed a phenotypic shift from myo-endocrine to inflammatory. Taken together, our results add 36 new genes to the list that characterize renin-producing cells and reveal a novel role for PDGFB as a regulator of renin-synthesizing cells.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** iFISH for renin in mouse kidney. Where possible, glomeruli are outlined in red and vessels in light blue. (A) Immunofluorescence for renin in adult mouse kidney is shown in blue; (B) In situ hybridization for renin in the same section is shown in yellow; (C) merged image from panels a and b, including phase contrast microscopy showing that renin is expressed at the base of the glomeruli as expected; (D) iFISH overlay for renin in kidneys from adult; (E) adult mouse treated for one week with captopril, and (F) 5 day-old mouse. Original magnification X 20. Scale bar=50µm.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2.</figcaption></figure>

A) Total renin (i.e., renin + prorenin) levels in the medium, and renin levels in the cell lysate obtained from As4.1 cells incubated for 48 hours with medium obtained from HEK293 cells transfected with 10 genes expressing ligands that were upregulated in the 4 reninomas. Renin gene expression is also provided. Data are mean ±SEM of 5–11 experiments and have been expressed as a percentage of the levels in cells exposed to medium of control-transfected cells, i.e., cells treated with the transfection mix, but without cDNA. *P<0.01. B) Cell viability and total renin levels in the medium of As4.1 cells exposed to PDGFB for 24 or 48 hours. Data are mean ±SEM of 3 experiments and have been expressed as a percentage of the levels in cells not exposed to PDGFB ('vehicle'=0). *P<0.01, #P<0.05 vs. 100%. C) Aldo-keto reductase family 1, member B7 (Akr1b7), α-smooth muscle actin (Acta2), interleukin-6 (IL-6), and recombination signal binding protein for immunoglobulin kappa j (Rbpj) expression in As4.1 cells incubated for 48 hours with medium obtained from HEK293 cells transfected with 10 genes expressing ligands that were upregulated in the 4 reninomas. Data are mean ±SEM of 4–6 experiments and have been expressed as fold change versus the levels in cells exposed to medium of control-transfected cells, i.e., cells treated with the transfection mix, but without cDNA ('vehicle'). *P<0.01, #P<0.05 vs. 1.0.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** iFISH showing renin immunofluorescence in blue and in situ hybridization for Acta2 or Pdgfrβ in yellow in kidneys from adult (A, D), captopril-treated (B, E), 5 day-old (C, F) mice. Note the existence of 2 types of renin-producing cells in the 5 day-old mouse: those that co-express Acta2 (outlined in the vessel in light blue) and those that don't (arrow). Original magnification X 40. Scale bar=50μm.</figcaption></figure>
<caption>**Table 1**</caption>

Quality control of sequenced reads.

<table><thead><tr><td>Sample</td><td>Number of reads before trimming</td><td>Number of reads after trimming</td><td>% reads post-filtering</td><td>Exonic alignment rate</td></tr></thead><tbody><tr><td>Par1B1</td><td>88058080</td><td>83611860</td><td>94.95</td><td>0.811</td></tr><tr><td>Par1B2</td><td>88713782</td><td>84187709</td><td>94.90</td><td>0.803</td></tr><tr><td>Par1B3</td><td>74683086</td><td>71782557</td><td>96.12</td><td>0.813</td></tr><tr><td>Mon</td><td>76131363</td><td>73212125</td><td>96.17</td><td>0.760</td></tr><tr><td>Rot1B1</td><td>117331030</td><td>109192033</td><td>93.06</td><td>0.753</td></tr><tr><td>Rot1B2</td><td>95468148</td><td>88616034</td><td>92.82</td><td>0.766</td></tr><tr><td>Par2</td><td>55053372</td><td>47536436</td><td>86.35</td><td>0.766</td></tr><tr><td>Par2N</td><td>117480551</td><td>101148769</td><td>86.10</td><td>0.647</td></tr></tbody></table>
<caption>Table 2</caption>

Ratio of the number of renin reads (FPKM) obtained in the various biopsies versus those for the next most abundant transcript. Also shown is the ratio of renin reads obtained between the putative tumor and normal adjacent tissue (Par2N) biopsies for the Par2 samples. Data are drawn from Table S1.

<table><thead><tr><th>Sample</th><th>Par1 (ave.)</th><th>Mon</th><th>Rot (ave.)</th><th>Par2</th><th>Par2N</th><th>Par2/Par2N</th></tr></thead><tbody><tr><td>Ren FPKM</td><td>74372</td><td>110034</td><td>110062</td><td>117805</td><td>541</td><td>217</td></tr><tr><td>2nd transcript (FPKM)</td><td>GNAS (2218)</td><td>GNAS (2652)</td><td>KISS1 (6830)</td><td>KISS1 (8110)</td><td></td><td></td></tr><tr><td>Ratio</td><td>33.53</td><td>41.49</td><td>16.11</td><td>14.53</td><td></td><td></td></tr></tbody></table>
<caption>Table 3</caption>

Summary of the iFISH analysis on mouse kidney for the genes most highly expressed in the reninomas. The genes found to express in renin-producing cells (RPC) are listed in their order of expression in the reninomas from highest to lowest (see Table S1) and their relative expression in each sample is estimated on a scale of 0 (no detectable expression) to 4 (highest expression), based upon the data in Figure S5.

<table><thead><tr><th rowspan="2">Gene symbol</th><th rowspan="2">Gene ID</th><th colspan="4">Expression in mouse renin-producing cells (iFISH)</th><th rowspan="2">Notes</th></tr><tr><th>Developing Kidney</th><th>Adult Kidney</th><th>Adult Kidney + Captopril</th></tr></thead><tbody><tr><td colspan="7">PRESENT IN ALL 4 RENINOMAS</td></tr><tr><td>REN*#</td><td>Renin</td><td>4</td><td>2</td><td>4</td><td></td><td></td></tr><tr><td>KISS1</td><td>KiSS-1 metastasis-suppressor</td><td>1</td><td>0</td><td>2</td><td></td><td></td></tr><tr><td>GNAS</td><td>GNAS complex locus</td><td>4</td><td>1</td><td>4</td><td></td><td></td></tr><tr><td>A2M</td><td>alpha-2-macroglobulin</td><td>3</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>PTP4A3*</td><td>protein tyrosine phosphatase type IVA, member 3</td><td>1</td><td>0</td><td>4</td><td></td><td></td></tr><tr><td>SPARCL1</td><td>SPARC-like 1 (hevin)</td><td>4</td><td>4</td><td>4</td><td></td><td></td></tr><tr><td>C4A</td><td>complement component 4A (Rodgers blood group)</td><td>0</td><td>1</td><td>2</td><td></td><td></td></tr><tr><td>VIM#</td><td>vimentin</td><td>4</td><td>2</td><td>4</td><td>also in mesangial cells and arterioles</td><td></td></tr><tr><td>MYL9</td><td>myosin, light chain 9, regulatory</td><td>4</td><td>3</td><td>3</td><td>strongest in developing arterioles</td><td></td></tr><tr><td>NOTCH3</td><td>Notch homolog 3 (Drosophila)</td><td>1</td><td>1</td><td>2</td><td></td><td></td></tr><tr><td>MFGE8</td><td>milk fat globule-EGF factor 8 protein</td><td>2</td><td>2</td><td>4</td><td></td><td></td></tr><tr><td>PDGFRβ</td><td>platelet-derived growth factor receptor, beta polypeptide</td><td>3</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>SPARC</td><td>secreted protein, acidic, cysteine-rich (osteonectin)</td><td>2</td><td>2</td><td>0</td><td>mostly in cells adjacent to JG</td><td></td></tr><tr><td>JAG1</td><td>jagged 1 (Alagille syndrome)</td><td>0</td><td>1</td><td>1</td><td>mostly in arterioles adjacent to JG</td><td></td></tr><tr><td>SERPINI1*</td><td>serpin peptidase inhibitor, clade 1 (neuroserpin), member 1</td><td>0</td><td>0</td><td>1</td><td></td><td></td></tr><tr><td>TBC1D1</td><td>TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1</td><td>0</td><td>0</td><td>1</td><td></td><td></td></tr><tr><td>CXCL12</td><td>chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)</td><td>3</td><td>0</td><td>1</td><td>mostly in cells adjacent to RPC in developing arterioles</td><td></td></tr><tr><td>TPM2</td><td>tropomyosin 2 (beta)</td><td>1</td><td>0</td><td>1</td><td></td><td></td></tr><tr><td>SLIT3</td><td>slit homolog 3 (Drosophila)</td><td>3</td><td>0</td><td>1</td><td>only in some RPC 5 day and recruited JG capto (faint)?</td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="2">Gene symbol</th><th rowspan="2">Gene ID</th><th colspan="3">Expression in mouse renin-producing cells (iFISH)</th><th rowspan="2">Notes</th></tr><tr><th>Developing Kidney</th><th>Adult Kidney</th><th>Adult Kidney + Captopril</th></tr></thead><tbody><tr><td>ITGA7</td><td>integrin, alpha 7</td><td>0</td><td>0</td><td>2</td><td></td></tr><tr><td>GLA4</td><td>gap junction protein, alpha 4, 37kDa</td><td>1</td><td>0</td><td>2</td><td></td></tr><tr><td>DBNDD2</td><td>dystobrevin binding protein 1) domain containing 2</td><td>3</td><td>2</td><td>2</td><td></td></tr><tr><td>FLNA</td><td>filamin A, alpha (actin binding protein 280)</td><td>3</td><td>2</td><td>4</td><td></td></tr><tr><td>ISYNA1</td><td>inositol-3-phosphate synthase 1</td><td>1</td><td>1</td><td>2</td><td></td></tr><tr><td>CALDI</td><td>caldesmon 1</td><td>4</td><td>2</td><td>4</td><td>also mesangial cells?</td></tr><tr><td>ADCY3</td><td>adenylate cyclase 3</td><td>1</td><td>1</td><td>0</td><td></td></tr><tr><td>MXRA8</td><td>matrix-remodelling associated 8</td><td>1</td><td>1</td><td>2</td><td></td></tr><tr><td>OAZ2</td><td>ornithine decarboxylase antizyme 2</td><td>2</td><td>2</td><td>3</td><td></td></tr><tr><td>TIMP1</td><td>TIMP metallopeptidase inhibitor 1</td><td>0</td><td>0</td><td>1</td><td></td></tr><tr><td>TINAGL1</td><td>tubulointerstitial nephritis antigen-like 1</td><td>1</td><td>2</td><td>4</td><td></td></tr><tr><td>LBH</td><td>limb bud and heart development homolog (mouse)</td><td>1</td><td>1</td><td>4</td><td></td></tr><tr><td>NRARP</td><td>NOTCH-regulated ankyrin repeat protein</td><td>0</td><td>1</td><td>1</td><td></td></tr><tr><td>IGF2</td><td>insulin-like growth factor 2 (somatomedin A)</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>IFITM3</td><td>interferon induced transmembrane protein 3 (1-8U)</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>SFRP4</td><td>secreted frizzled-related protein 4</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>CSRP2</td><td>cysteine and glycine-rich protein 2</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>IFITM1</td><td>interferon induced transmembrane protein 1 (9-27)</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>COL6A2</td><td>collagen, type VI, alpha 2</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>CPE</td><td>carboxypeptidase E</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>LGALS1</td><td>lectin, galactoside-binding, soluble, 1</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>TGM2</td><td>transglutaminase 2 (C polypeptide, protein-glutamine-gamma-glutamyltransferase)</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>CBLN4</td><td>cerebellin 4 precursor</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>SRGN</td><td>serglycin</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>TBX2</td><td>T-box 2</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>COL6A1</td><td>collagen, type VI, alpha 1</td><td>0</td><td>0</td><td>0</td><td>in extraglomerular cells surrounding JG</td></tr></tbody></table>
<table><thead><tr><th rowspan="2">Gene symbol</th><th rowspan="2">Gene ID</th><th colspan="4">Expression in mouse renin-producing cells (iFISH)</th><th rowspan="2">Notes</th></tr><tr><th>Developing Kidney</th><th>Adult Kidney</th><th>Adult Kidney + Captopril</th></tr></thead><tbody><tr><td>N75DC2</td><td>5'-nucleotidase domain containing 2</td><td>0</td><td>0</td><td>0</td><td>weak expression in cells adjacent to JG</td><td></td></tr><tr><td>TPM1</td><td>troponomyosin 1 (alpha)</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>GPR124</td><td>G protein-coupled receptor 124</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>HSPB6</td><td>heat shock protein, alpha-crystallin-related, B6</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>CD3#</td><td>CD34 molecule</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>HCFC1R1</td><td>host cell factor C1 regulator 1 (XPO1 dependent)</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>PLEKHH3</td><td>pleckstrin homology domain containing, family H (with MyTH4 domain) member 3</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td colspan="7" style="text-align: center;">PRESENT IN 3 OUT OF 4 RENINOMAS</td></tr><tr><td>MDK</td><td>midkine (neurite growth-promoting factor 2)</td><td>1</td><td>1</td><td>4</td><td></td><td></td></tr><tr><td>CPA3</td><td>carboxypeptidase A3 (mast cell)</td><td>1</td><td>2</td><td>0</td><td></td><td></td></tr><tr><td>RGS5*</td><td>regulator of G-protein signaling 5</td><td>4</td><td>4</td><td>2</td><td></td><td></td></tr><tr><td>ACTA2</td><td>actin, alpha 2, smooth muscle, aorta</td><td>4</td><td>0</td><td>2</td><td>mostly in VSMC in adult</td><td></td></tr><tr><td>CD248</td><td>CD248 molecule, endosialin</td><td>3</td><td>2</td><td>4</td><td></td><td></td></tr><tr><td>IFITM2*</td><td>interferon induced transmembrane protein 2 (1-8D)</td><td>0</td><td>0</td><td>1</td><td></td><td></td></tr><tr><td>TUBA1A</td><td>tubulin, alpha 1a</td><td>2</td><td>3</td><td>4</td><td></td><td></td></tr><tr><td>BGN</td><td>biglycan</td><td>1</td><td>2</td><td>4</td><td></td><td></td></tr><tr><td>ATP1B2*</td><td>ATPase, Na+/K+ transporting, beta 2 polypeptide</td><td>2</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>MCAM</td><td>melanoma cell adhesion molecule</td><td>2</td><td>2</td><td>3</td><td></td><td></td></tr><tr><td>TAGLN</td><td>transgelin</td><td>3</td><td>2</td><td>3</td><td>also in adjacent arterioles</td><td></td></tr><tr><td>CRIP1*</td><td>cysteine-rich protein 1 (intestinal)</td><td>0</td><td>1</td><td>4</td><td></td><td></td></tr><tr><td>MEF2C</td><td>myocyte enhancer factor 2C</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>TGFBI</td><td>transforming growth factor, beta-induced, 68kDa</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>TPSB2</td><td>tryptase alpha/beta 1; tryptase beta 2</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>NPPC</td><td>natriuretic peptide precursor C</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>GPC1</td><td>glypican 1</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr><tr><td>FKBP10</td><td>FK506 binding protein 10, 65 kDa</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="2">Gene symbol</th><th rowspan="2">Gene ID</th><th colspan="4">Expression in mouse renin-producing cells (iFISH)</th><th rowspan="2">Notes</th></tr><tr><th>Developing Kidney</th><th>Adult Kidney</th><th>Adult Kidney + Captopril</th></tr></thead><tbody><tr><td>FXYDI*</td><td>FXYD domain containing ion transport regulator 1</td><td>0</td><td>0</td><td>0</td><td></td><td></td></tr></tbody></table>

* Described as part of a 369 gene list that makes up a renin-producing cell signature, according to Brunskill et al.⁵.

# Reported as reninoma marker. Of the genes confirmed by iFISH, 36 have not been reported before in relationship to renin-producing cells.