Amlodipine promotes autophagy by inhibiting calcium influx in human podocytes

Acta Universitatis Medicinalis Anhui 2025, 07, v.60 1179-1186     font:big middle small

Fund programs: Academic Funding Program for Top Talents in Colleges and Universities in Anhui Province (No.gxbjZD2021046)

Authors:Zhao Wei; Xu Deping; Liao Kainan; Cai Chunlin; Zang Dandan; Zhou Haisheng

Keywords:human podocytes; amlodipine; angiotensin Ⅱ; autophagy; Ca~(2+); apoptosis;

DOI:10.19405/j.cnki.issn1000-1492.2025.07.003

〔Abstract〕 Objective To explore the effects of the antihypertensive drug Amlodipine on calcium influx and autoph- agy in human podocytes ( HPC) .Methods HPC cells were routinely cultured in vitro. HPC cells were treated with angiotensin Ⅱ ( Ang Ⅱ ) ,the L-type Ca2 + blocker Amlodipine alone or in combination.The Ca2 + imaging system was used to detect the transient changes in the intracellular Ca2 + flux of HPC cells in real time after drug treatment.Western blot was employed to detect the changes in the ratio of autophagy marker proteins LC3B-Ⅱ/ LC3B-Ⅰ , and the expression levels of Beclin-1,P62,as well as apoptosis-related proteins Bcl-2 and Bax.Flow cytometry was used to detect the number of Fluo-4AM positive cells at 488 nm to analyze the level of intracellular Ca2 + influx in HPC cells.Lyso-Tracker Green live cell staining was applied to analyze the fluorescence intensity of lysosomes.Flow cytometry was also used to detect the apoptosis rate of HPC cells.Results Compared with the control group,in the Ang Ⅱ group,the transient Ca2 + flux and the number of Fluo-4AM positive cells increased significantly (P<0. 001) .The ratio of autophagy marker proteins LC3B-Ⅱ/ LC3B-Ⅰ (P<0. 001) and the pro- tein expression of Beclin-1 (P<0. 01) decreased significantly,while the expression of P62 increased (P<0. 01) . The fluorescence intensity of lysosomes weakened (P<0. 05) ,the apoptosis rate increased (P<0. 0001) ,the ex- pression of apoptosis-related protein Bcl-2 decreased (P <0. 01 ) ,and the protein level of Bax increased (P < 0. 001) .Compared with the control group,in the Amlodipine group,the number of Fluo-4AM positive cells de- creased significantly (P<0. 001) ,the ratio of LC3B-Ⅱ/ LC3B-Ⅰ (P<0. 001) and the protein expression of Bec- lin-1 (P<0. 001) increased,the protein expression of P62 decreased (P<0. 05) ,the fluorescence intensity of ly- sosomes enhanced (P<0. 01) ,the apoptosis rate decreased (P<0. 01) ,the protein expression of Bcl-2 increased (P<0. 001) ,and the protein level of Bax decreased (P<0. 001) .Compared with the Ang Ⅱ group,in the Ang Ⅱ + Amlodipine group,the number of Fluo-4AM positive cells decreased significantly (P<0. 001) ,the ratio of LC3B-Ⅱ/ LC3B-Ⅰ (P<0. 01) and the protein expression of Beclin-1 increased (P<0. 05) ,the protein level of P62 decreased (P<0. 01) ,the fluorescence intensity of lysosomes increased (P <0. 05) ,the apoptosis rate de- creased (P<0. 001) ,the protein expression of Bcl-2 increased (P <0. 001 ) ,and the protein level of Bax de- creased (P<0. 001) .Conclusion Amlodipine inhibits calcium influx,promotes autophagy and inhibits apoptosis in human podocytes,which is useful in preventing the development of hypertensive nephropathy.