1 1 2 3 4 Two microRNAs - miR - 330 and miR - 125 b - 5 p - mark the juxtaglomerular cell and balance

28 29 We have shown that microRNAs (miRNAs) are necessary for renin cell specification and kidney 30 vascular development. Here, we used a screening strategy involving microarray and in silico analyses, 31 along with in situ hybridization and in vitro functional assays to identify miRNAs important for renin cell 32 identity. Microarray studies using vascular smooth muscle cells (SMCs) of the renin lineage and kidney 33 cortex under normal conditions and after reacquisition of the renin phenotype revealed that out of 599 34 miRNAs, 192 were expressed in SMCs and 234 in kidney cortex. In silico analysis showed that the 35 highly conserved miR-330 and miR-125b-5p have potential binding sites in smoothelin (Smtn), 36 calbindin 1, smooth muscle myosin heavy chain, α-smooth muscle actin and reningenes important for 37 the myoepithelioid phenotype of the renin cell. RT-PCR studies confirmed miR-330 and mir-125b-5p 38 expression in kidney and SMCs. In situ hybridization revealed that under normal conditions miR-125b39 5p was expressed in arteriolar SMCs and in JG cells. Under conditions that induce reacquisition of the 40 renin phenotype, miR-125b-5p was downregulated in arteriolar SMCs but remained expressed in JG 41 cells. miR-330, normally absent, was expressed exclusively in JG cells of treated mice. In vitro 42 functional studies showed that over-expression of miR-330 inhibited Smtn expression in SMCs. On the 43 other hand, miR-125b-5p increased Smtn expression, whereas its inhibition reduced Smtn expression. 44 Our results demonstrate that miR-330 and miR-125b-5p are markers of juxtaglomerular cells and have 45 opposite effects on renin lineage cells: one inhibiting and the other favoring their smooth muscle 46 phenotype. 47