Mitochondrial Fission and Fusion Dynamics are Regulated by Multiple Pathways in Renal Proximal Tubule Cells Treated with High Glucose

Abstract

In type 2 diabetes, hyperglycemia leads to proximal tubular dysfunction, which is accompanied by altered mitochondrial homeostasis. We previously demonstrated that in renal proximal tubule cells (RPTC) grown in high glucose, as well as in diabetic db/db mice, proteins involved in mitochondrial dynamics were altered. Phosphorylation of the mitochondrial fission protein Drp1 increased and expression of the mitochondrial fusion protein Mfn1 decreased. One potential activation pathway of Drp1 is the RhoA/ROCK1/Drp1 signaling cascade in the presence of high glucose, leading to increased mitochondrial fission. Conversely, Mfn1 can be activated by MEK/ERK signaling. However, these pathways have not been investigated in the proximal tubule. Therefore, we determined the signaling pathways responsible for altered Drp1 phosphorylation and Mfn1 expression in RPTC. Primary cultures of RPTC were grown in high glucose (17mM), mannitol (17mM) or no glucose for 96 hr and were co‐treated with either RhoA (CCG‐1423), ROCK1 (Y‐27632) or MEK 1/2 (GSK 1120212) inhibitors 24 hr prior to harvesting. Cells were subjected to GTPase pulldown assays to measure Drp1, RhoA and Mfn1 activity and maximal mitochondrial respiration was measured using Seahorse XF96 analyzer. RPTC treated with glucose for 96 hr exhibited an increase in both RhoA and pDrp1 and an increase in GTP‐bound RhoA and Drp1. Co‐treatment with CCG‐1423 or Y‐27632 prevented glucose‐induced increases in RhoA and Drp1, respectively. Inhibition of RhoA and ROCK1 restored maximal mitochondrial respiration. Co‐treatment with GSK 1120212 prevented glucose‐induced decreases in Mfn1. Together, these results demonstrate that treatment of RPTC with high glucose increases RhoA and Drp1 activity and maximal respiration through the RhoA/ROCK1/Drp1 signaling pathway and is responsible for increased mitochondrial fission and respiration. In contrast, the MEK/ERK signaling cascade prevents the decrease in Mfn1 observed in the presence of high glucose. These data indicate that mitochondrial dynamics in high glucose in RPTC, are regulated by two independent signaling pathways.