Regulation of KLF2 Expression in Glomerular Endothelial Cells by Glomerular Hyperfiltration

Background: Krüppel-like factor 2 (KLF2) is known to mediate flow-dependent phenotype in endothelial cells and confers endoprotective effects by inhibition of pro-inflammatory pathways, thrombotic activation, and uncontrolled angiogenesis. Over the last several years, we have generated interesting data to support the protecting role of KLF2 against glomerular endothelial cell (GEC) injury in diabetic kidney disease (DKD) mice by using knockout and overexpression approaches. Here, we further explored how KLF2 expression is regulated in GECs in vitro and in mice with unilateral nephrectomy (uNx) and DKD. Methods: We analyzed the public datasets of kidney bulk and single cell RNA-sequence. We made two reporter constructs that express human KLF2 promoter-driven luciferase or GFP. We generated mouse GEC cell lines. We also developed an in vitro microperfusing system of GEC culture to mimic high and low shear stress (SS). We also generated transgenic KLF2-GFP reporter mice that allow the in vivo monitoring of KLF2 expression. Results: scRNA-seq data of mouse and human kidneys show that KLF2 expression is predominantly expressed in endothelial cell subsets, i.e., GECs, which was confirmed in human kidneys by analysis of KPMP datasets. The Nephroseq datasets confirmed the reduced glomerular KLF2 expression in patients with DKD, which was negatively correlated with serum creatinine levels. Interestingly, KLF2 expression is lower in male than female, and is significantly increased in endothelial cells of patients treated with SGLT2 inhibitors. Using the reporter assay, qPCR, and western blot, we confirmed that KLF2 expression is downregulated by TNF-α and IL-1β, but upregulated by estradiol (E2) and SGLT2 inhibitors in cultured GECs. In the microperfusing system, we found that high SS increased KLF2 expression in GECs. In KLF2-reporter mice, we confirmed an increase of KLF2 expression in mice at day 3, 14, 28 post-uNx, which was parallel to GFR. In contrast, we did not observe any significant increase of KLF2 expression in GECs of DKD mice regardless of increase in GFR. However, treatment of DKD mice with empagliflozin increased KLF2 expression in GECs. Conclusion: KLF2, an endo-protective factor, is highly regulated by shear stress, cytokines, and SGLT2i. KLF2 is upregulated in vivo by glomerular hyperfiltration in uNx mice but not in DKD mice. Funding: NIDDK Support