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Kidney Week

Abstract: PO0699

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

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic


  • Cleveland, Kristan H., The University of Arizona, Tucson, Arizona, United States
  • Schnellmann, Rick G., The University of Arizona, Tucson, Arizona, United States

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, mitochondrial dynamics proteins were altered. Phosphorylation of the mitochondrial fission protein Drp1 increased and the mitochondrial fusion protein Mfn1 decreased. Studies have shown that Drp1 is activated by the RhoA/ROCK1 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 the presence of high glucose (17mM), mannitol (17mM) or no glucose for 96hr and were co-treated with either RhoA (CCG-1423), ROCK1 (Y-27632) or MEK 1/2 (GSK 1120212) inhibitors 24 hr prior harvesting. Cells were subjected to GTPase assays to measure Drp1, RhoA and Mfn1 activity and maximal mitochondrial respiration was measured using Seahorse XF96e analyzer.


RPTC treated with glucose for 96 hr exhibited an increase in RhoA and pDrp1 at 96 hr. This increase corresponded with an increase in GTP-bound RhoA and Drp1. Co-treatment with CCG-1423 or Y-27632 prevented the glucose-induced increase in RhoA and Drp1, respectively. Inhibition of RhoA and ROCK1 restored maximal mitochondrial respiration. Co-treatment with GSK 1120212 prevented the glucose-induced decrease in Mfn1.


Together, these results demonstrate that treatment of RPTC with glucose increases RhoA and Drp1 activity and maximal respiration. Pharmacological inhibition of RhoA and ROCK1 prevented increased activity of RhoA and Drp1 and restored respiration, indicating that the RhoA/ROCK1/Drp1 signaling pathway is responsible for increased mitochondrial fission and respiration in high glucose in RPTC. In contrast, we show that inhibition of the MEK/ERK signaling cascade prevents the decrease in Mfn1 observed in the presence of high glucose. These data indicate that the alteration of mitochondrial dynamics in high glucose in RPTC, are regulated by two independent signaling pathways.


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