Abstract:

Objective To investigate the mechanism of iron death induced by TRPC6/NF?κB in glomerular podiocytes mediated by high homocysteine （Hcy）. Methods Mouse glomerulopocytes were cultured in vitro and divided into Control group （0 μmol/L Hcy） and Hcy group （80 μmol/L Hcy）. After 48h of intervention， Western blot was used to detect the expression levels of iron death related proteins GPX4 and SLC7A11 and TRPC6 and NF?κB. Real-time quantitative fluorescence PCR（qRT-PCR） and immunofluorescence were used to detect the expression of TRPC6. The level of podocyte apoptosis was detected by flow cytometry. Malondialdehyde （MDA） assay kit was used to determine intracellular MDA levels. After transfection of TRPC6 interference fragment and TRPC6 negative control （NC）， qRT-PCR was divided into Control， si-NC and si-TRPC6 （Si-TRPC6-1， Si-TRPC6-2， Si-TRPC6-3）. Western Blot was divided into Control， Hcy， si-NC+Hcy， si-TRPC6+Hcy.The expression of TRPC6 mRNA was detected by qRT-PCR. The expression levels of GPX4， SLC7A11， NF?κB and TRPC6 were detected by Western Blot. The level of podocyte apoptosis after interference was detected by flow cytometry. Results （1）Compared with Control group， the expression levels of iron death related proteins GPX4 and SLC7A11 in Hcy group were decreased， and the apoptosis rate was increased（P < 0.05）. （2） Compared with Control group， TRPC6 protein， mRNA levels and immunofluorescence expression were increased in Hcy group.The level of MDA and the expression of NF?κB signaling pathway protein increased in Hcy group， and the comparison between the two groups had statistical significance （P < 0.05）. （3） Compared with the si-NC group， the mRNA expression level of TRPC6 in si-TRPC6 （Si-TRPC6-1， Si-TRPC6-2， Si-TRPC6-3） group was decreased， and the interference effect of Si-TRPC6-3 was the best（P < 0.05）. After transfecting TRPC6 NC and TRPC6 interference fragment and administering Hcy， there was no difference in GPX4， SLC7A11， NF?κB and TRPC6 expression in si-NC+Hcy group compared with Hcy group. Compared with the si-NC+Hcy group， the si-TRPC6+Hcy group had higher expression of iron death related proteins， GPX4 and SLC7A11， lower expression of NF?κB and TRPC6， and decreased apoptosis rate（P < 0.05）. Conclusion This study confirmed that TRPC6/NF?κB can regulate iron death of renal podocytes under the induction of Hcy， which is one of the mechanisms leading to kidney injury.

Key words: iron death, TRPC6, NF?κB, kidney disease, high homocysteine