#2090 Dose-dependent NSAID effects on autophagy and fibrosis pathways in renal tubular epithelial cells

Abstract Background and Aims Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used and their chronic administration is associated with reduced renal blood flow, potentially causing papillary necrosis. Autophagy protects renal proximal tubular cells by removing damaged components and promoting cell survival under stress conditions. We have previously demonstrated how hyperosmolarity increases the expression of pro-fibrotic factors in the renal medulla. However, whether different COX-1 and COX-2 inhibitor NSAIDs affect fibrotic pathways or autophagy of kidney tubules remains unknown. Thus, we aimed to study the dose-dependent effects of different NSAIDs on autophagy and fibrosis pathways of human renal tubular cells. Method Human proximal tubular epithelial cells (HK-2, ATCC #CRL2190) were seeded on 24-well plates at 30 000 cells/well density. Cells were then treated with indomethacin (IND; 10, 20, 40 µg/mL), celecoxib (CEL; 0.72, 2.1, 4.3, 8.7 µg/mL), and naproxen (NAP; 15, 30, 60, 110 µg/mL) or DMSO (controls). Cells were harvested after 24 h and markers of fibrosis (e.g. mRNA expression of EGR1, EGR2, TGFB1, ACTA2) and autophagy (LC3-I/II, SQSTM1 /p62/) were investigated with RT-qPCR and immunoblot. Kruskal-Wallis test was performed for statistical analysis. Results As compared to the control group, high doses of NSAIDs induced mRNA expression of ACTA2 (CEL 8.7: 1.6-fold; NAP 110: 2.3-fold, P < 0.001), TGFB1 (CEL 8.7: 2.2-fold; NAP 110: 2.1-fold, P < 0.05), MMP9 (IND 40: 1.6-fold; CEL 8.7: 2.1-fold; NAP 110: 2.4-fold, P < 0.01) and TIMP1 (CEL 8.7: 2.2-fold; NAP 110: 1.9-fold, P < 0.01). The mRNA expression of pro-fibrotic EGR1 (CEL 8.7: 2.2-fold; NAP 110: 2.3-fold, P < 0.05) and EGR2 transcription factors increased as well (IND 40: 1.4-fold, CEL 8.7: 1.8-fold; NAP 110: 2.0-fold, P < 0.01). Interestingly, CEL 8.7 and NAP 110 treatments but not IND 40 elevated SQSTM1 (p62) protein levels significantly (7.1-fold and 7.0-fold, respectively, P < 0.05) although LC3-II/LC3-I ratio was also increased (3.4-fold and 4.2-fold, respectively (P < 0.01), showing a defective autophagy. Conclusion High doses of celecoxib and naproxen activated pro-fibrotic pathways while disrupting autophagic flux in renal proximal tubular cells. Our findings underscore the need for dose optimization of these medications especially in patients with kidney disease to avoid NSAID-induced nephrotoxicity.