Roxadustat Regulates the Cell Cycle and Inhibits Proliferation of Mesangial Cells via the Hypoxia-Inducible Factor-1α/P53/P21 Pathway

Yun Cheng ¹ Baijie Feng ^2* Xiuhong Yang ^3* Huimin Min Jin ^1*

• ¹ Division of Nephrology, Pudong Medical Center, Shanghai Pudong Hospital, Fudan University, Shanghai, China
• ² Division of Oncology, Shanghai Pudong Hospital, Fudan University, Pudong Medical Center,, Shanghai, China
• ³ Department of Nephrology, Huadong Hospital, Fudan University, Shanghai, Shanghai Municipality, China

Background: Over-proliferation of mesangial cells (MCs) is one of the main pathological changes in the early stages of diabetic kidney disease (DKD). Roxadustat, an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, is widely studied in different models of kidney disease. Whether roxadustat has beneficial effect on the 2 proliferation of MCs and DKD remains unknown.The optimal concentration of roxadustat for inhibiting MC proliferation was determined using CCK-8 and colony formation assays. Changes in the cell cycle were detected using flow cytometry, and changes in cell cycle-related proteins were screened using quantitative reverse transcription polymerase chain reaction. Reverse experiments were applied using Trp53-/cell line. Co-immunoprecipitation was used to verify the interaction between HIF-1α and p53 and predict sites of interaction. Finally, a corresponding in vivo verification was performed.Results: Optimal concentrations of high glucose (30 mM) and roxadustat (100 μM) were established. Roxadustat showed anti-proliferation effect on MCs through S-phase arrest. HIF-1α/p53/p21 and downstream cyclins (cyclin A1, cyclin A2, and cyclin E1) showed corresponding changes. A reverse experiment confirmed that HIF-1α affected cell cycle and proliferation through p53, and co-immunoprecipitation results showed a interaction between HIF-1α and p53. Molecular docking predicted the possible interaction between Lys328, Pro332, Arg245, and Lys251 of HIF-1α and Ala222, Tyr226, Glu225, and Asp265 of p53, respectively. Finally, animal experiments demonstrated similar effect of roxadustat in db/db mice.Roxadustat regulates and inhibits cell proliferation of MCs via the HIF-1α/p53/p21 pathway. This may be a potential therapeutic target in the early stages of diabetic kidney disease.