APOE-Activating Noncoding RNA (AANCR) Regulates APOE in Kidney Cell Types

Background: Apolipoprotein E (APOE) is a lipid carrier protein and polymorphisms of the gene are predictive of the development of chronic kidney disease (CKD).In line with its role in kidney function, APOE knockout mice develop renal disease, indicating that APOE is necessary to maintain normal renal function. However, some studies have suggested it is APOE dysregulation in macrophages or changes in lipid profiles that lead to kidney disease. Hence, the question remains whether APOE function in renal epithelial cells contributes to CKD risk. Recently, we identified a non-coding RNA, APOE-Activating Noncoding RNA (AANCR), that regulates APOE in human cells through a novel mechanism involving R-loop mediated RNA polymerase pausing. In this project, we asked whether AANCR regulates APOE expression in renal cell types. Methods: To quantify AANCR-mediated APOE expression and its effect on renal cell types, we measured gene expression in cultured human proximal tubule cells (HK-2) and murine inner medullary collecting duct cells (IMCD) and performed apoptosis assays in HK-2 cells. Results: First, we measured AANCR and APOE expression in renal cells by RT-qPCR and found that both are expressed in proximal tubule and collecting duct cells. Next, to test if AANCR regulates APOE, we performed siRNA knockdown of AANCR and found a significant reduction in the expression of APOE in renal proximal tubule cells. RNA polymerase pausing is common at stress responsive genes, so we asked if AANCR-APOE are stress responsive in renal cells. We found AANCR is induced in response to both osmotic stress and heavy metal exposure. To explore whether AANCR induction has functional consequences for renal cell types, we asked if APOE has a cytoprotective role in the kidney. APOE is a secreted protein, so we treated cells with osmotic stress to induce APOE expression and then collected the conditioned media. We fractioned the media to have either high or low APOE protein. We found that cells treated with conditioned media with abundant APOE were more resistant to apoptosis induced by osmotic stress, whereas conditioned media without APOE was not protective. Conclusion: We show that the non-coding RNA AANCR regulates APOE expression in renal cell types and plays a cytoprotective role in response to stress. Future work will address whether targeted modulation of APOE expression is protective in CKD. Funding: Other NIH Support - Intramural Research Program of the NIH, National Institute of Environmental Health Sciences ES103361, Private Foundation Support