Ovarian Hormone-Dependent miR-146b-5p Regulation of Plasminogen Activator Inhibitor-1 in Renal Epithelial Cells.

Chronic kidney disease (CKD) progression differs in males and females and the influence of gonadal hormones on pathology are incompletely understood. We utilized the 5/6 nephrectomy (5/6Nx) rat model, which recapitulates hallmarks of human CKD, to study differences in regulation of CKD pathology between sexes. We previously found that genomic deletion of microRNA 146b-5p (miR-146b) results in a sex-dependent phenotype in 5/6Nx rats. Male miR-146b-/- rats develop renal pathology similar to their WT counterparts. However, female miR-146b-/- rats experience markedly exacerbated renal pathology relative to WT, 5/6Nx, females as well as WT and miR-146b-/-, male, 5/6Nx rats. These sex differences are abolished by ovary removal, suggesting that renoprotective effects of female gonadal hormones are miR-146b dependent. Our objective is to identify a molecular mechanism behind these effects. PAI-1 has previously been shown to be regulated by miR-146b-5p isomiR, miR-146a-5p, which shares a common seed sequence. PAI-1 expression is also known to be ovarian hormone dependent, its renal expression is low in health, but elevated in many disease settings, where it may contribute to remodeling. We hypothesized that PAI-1 expression in rat proximal tubular epithelial cells (NRK-52E) will be dependent upon (1) the presence of miR-146b, (2) ovarian hormones, and (3) transforming growth factor beta (TGFβ), which is elevated in our 5/6Nx rats and known to contribute to renal pathology. To aid in testing our hypothesis we used CRISPR-Cas9 technology to generate miR-146b knockout rat proximal tubule epithelial (NRK-52E) cells. Results. PAI-1 mRNA was highly elevated in untreated miR-146b KO rat NRK-52E cells relative to WT NRK-52E cell controls (11.25 ± 5.3 vs. 1.00 ± 0.68, P<0.05). Conversely, WT NRK-52E cells treated with pre-miR-146b had significantly reduced PAI-1 mRNA expression (0.137 ± 0.14 vs. 1.00 ± 0.45, P<0.05). Treatment of WT NRK-52E cells with β-estradiol did not significantly alter PAI-1 mRNA expression relative to untreated controls, whereas treatment with progesterone (P4) significantly increased it. Treatment of WT NRK-52E cells with TGFβ resulted in a significant increase of PAI-1 mRNA expression. Lastly, treatment of WT NRK-52E cells with P4 and TGFβ combined resulted in a significant increase in PAI-1 mRNA expression, and this expression was significantly reduced by transfection with pre-miR-146b. Collectively, these observations demonstrate that miR-146b, P4, and TGFβ regulate PAI-1 expression in NRK-52E cells and thereby suggest PAI-1 as a mechanistic candidate behind the sex-specific effects of miR-146b in CKD.