FC023: Human Adult Renal Progenitor Cells Secrete in the Kidney Very High Levels of the Anti-Ageing Protein Klotho Sustained by the Long No-Coding RNA Hotair

Abstract BACKGROUND AND AIMS The complex system of human adult renal stem/progenitor cells (ARPCs) and their crucial role in the renewal of epithelial renal cells and regenerative processes in the adult kidneys have been recently discovered. In the human kidney, CD133+/CD24+ ARPCs are present within different segments of the nephron as a population with epithelial expansion, self-renewal and differentiation capabilities. Understanding the factors that regulate the ARPCs behaviour is fundamental to completely take advantage of their potential and to develop treatments for renal injury. Recent studies showed that long no-coding RNA (lncRNAs) are essential to establish developmental patterning and maintain the stem cell pluripotency network, further underscoring their important role in stem cell biology/technology and cellular reprogramming. lncRNAs can be dysregulated in different types of diseases and can also modulate the cellular senescence processes. For the first time, we studied their function in ARPCs showing that they express high levels of a particular lncRNA, HOTAIR, influencing cell senescence. METHOD Whole-genome lncRNA expression was performed by Agilent microarray. lncRNA expression was validated by real-time PCR. CRISPR/Cas9 system has been used to knock-down HOTAIR lncRNA. SA-β-Gal experiments were used to evaluate cellular senescence in normal ARPCs and ARPCs knock-out for HOTAIR. By ELISA, it was evaluated the expression of secreted anti-ageing protein Klotho. Fluorescence-activated cell sorting (FACS) was applied to measure CD133 and protein p15 expression in normal and transfected cells. Chromatin immunoprecipitation assay (ChIP) was used to evaluate H3K27me3 in the promoter of p15. RESULTS We studied the lncRNA profile of renal proximal tubular cells (RPTEC) and tubular ARPCs. We found 611 lncRNAs specifically expressed in ARPCs compared with RPTECs (Fold Change > 2; FDR < 0.05). Among the most significantly modulated lncRNAs, we demonstrated that the lncRNA HOX Transcript Antisense RNA (HOTAIR) is highly expressed in ARPCs (Fold change = 15; P < 0.001). To study the functions related to the ARPCs properties, knock-out cell lines for HOTAIR were created by the CRISPR/Cas9 technique. The silenced lines for HOTAIR immediately assumed a senescent phenotype confirmed by the beta-galactosidase assay and decreased proliferation (proliferation rate of ˂60%, P < 0.001). Moreover, we found that the constitutional, functional and inverse-senescence marker CD133+ was downregulated in knock-out cells (Fold change = 15; P < 0.01). We then studied the secreted levels of the anti-ageing protein α-Klotho which is essential to the maintenance of normal renal function and has potential clinical applications in the diagnosis of acute renal failure (AKI) and chronic kidney disease (CKD). We discovered that ARPCs produce α-Klotho at much higher levels compared to RPTEC, at a mean concentration of 200 pg/mL versus 33 pg/mL (Fold increase = 6.6; P < 0.001). Interestingly, following the HOTAIR knock-out, the levels of α-Klotho secreted by the ARPCs dropped sharply, with a 4-fold decrease (P < .001). In addition, we found that HOTAIR was responsible for the ARPC self-renewal through the epigenetic induction of the cell cycle inhibitor p15 through the trimethylation of histone H3K27 associated with the promoter region of p15. In ARPCs knock-out for HOTAIR p15 increased significantly (Fold increase = 3; P < 0.005) and the H3K27me3 in the promoter of p15 was less enriched (Fold decrease = 1.5; P < 0.05), leading to the increase of the cell cycle inhibitor and cell senescence. CONCLUSION These data demonstrated that HOTAIR regulates the self-renewal capacity of ARPCs and prevents them from becoming senescent in the short term. Moreover, HOTAIR influences ARPC ability to secrete high levels of α-Klotho, and those renal progenitors, through α-Klotho secretion, are able to influence its levels in surrounding tissues and to modulate, therefore, kidney ageing.