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

Abstract

Renin, a key component in the regulation of blood pressure in mammals, is produced by the rare and highly specialized juxtaglomerular cells of the kidney. Chronic stimulation of renin release results in a recruitment of new juxtaglomerular cells by the apparent conversion of adjacent smooth muscle cells along the afferent arterioles. Because juxtaglomerular cells rapidly dedifferentiate 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 4 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 adult mice treated with captopril. From the top 100 genes, 10 encoding for ligands were selected for further analysis. Medium of human embryonic kidney 293 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 reduced the medium and cellular (pro)renin levels, as well as As4.1 renin gene expression. In addition, platelet-derived growth factor B-exposed As4.1 cells displayed a more elongated and aligned shape with no alteration in viability. This was accompanied by a downregulated expression of α-smooth muscle actin and an upregulated expression of interleukin-6, suggesting a phenotypic shift from myoendocrine to inflammatory. Our results add 36 new genes to the list that characterize renin-producing cells and reveal a novel role for platelet-derived growth factor B as a regulator of renin-synthesizing cells.

Keywords: gene expression; hypertension; platelet-derived growth factor; renin-producing tumor; transcriptome.

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.

Figure 2

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 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.