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Kidney Week

Abstract: PO0218

Role of the Exocyst, Cilia, and Mitochondria in AKI

Session Information

  • AKI Mechanisms - 3
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Ilatovskaya, Daria, Medical University of South Carolina, Charleston, South Carolina, United States
  • Zuo, Xiaofeng, Medical University of South Carolina, Charleston, South Carolina, United States
  • Deng, Peifeng, Medical University of South Carolina, Charleston, South Carolina, United States
  • Fitzgibbon, Wayne R., Medical University of South Carolina, Charleston, South Carolina, United States
  • Lipschutz, Joshua H., Medical University of South Carolina, Charleston, South Carolina, United States
Background

AKI has high morbidity and mortality. Management consists of supportive care. Among the critical pathways in AKI are alterations of tubular mitochondrial metabolism, and, as recently suggested, disruption of primary ciliary homeostasis. Mitochondria are also involved in ciliopathies. Here we tested if cilia acting via mitochondria are involved in AKI.

Methods

We previously showed that the exocyst trafficking complex is necessary for ciliogenesis. Overexpression of Exoc5, a central exocyst component, protected renal tubule cells against H2O2-induced injury, whereas Exoc5 knockdown worsened it. In AKI, proximal tubules are most susceptible to injury. To determine the effect of Exoc5 loss, we crossed Exoc5fl/fl mice with mice expressing CreERT2 driven by the proximal tubule sodium-dependent inorganic phosphate transporter (SLC34a-CreERT2). Proximal tubule-specific Exoc5 knockout (KO) mice and littermate controls were subjected to bilateral ischemia reperfusion (I/R) injury by clamping the renal arteries. In order to gain mechanistic insight, we evaluated mitochondrial function in Exoc5 overexpressing (OE), Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence point mutant (cts-mut), and control Madin-Darby canine kidney tubule (MDCK) cells.

Results

Proximal tubule-specific Exoc5 KO mice had worse renal injury, and higher serum creatinine following I/R injury compared to control mice (p=0.005). Seahorse assays revealed diminished spare respiratory capacity in Exoc5 KD and Exoc5 cts-mut cells, which was increased in Exoc5 OE cells, compared to control MDCK cells. Tetramethylrhodamine methyl ester was employed to measure mitochondrial membrane potential and we found mitochondrial uncoupling in Exoc5 KD and Exoc5 cts-mut as compared with control cells. Transmission electron microscopic imaging revealed healthy circular-shaped mitochondria with dense matrix in control and Exoc5 OE cells. Exoc5 KD cells exhibited mitochondrial damage and swelling consistent with the observed reduced respiration. Interestingly, Exoc5 cts-mut cells demonstrated formation of elongated mitochondria with pronounced cristae and large intracristae spaces, which could indicate less intensive bioenergetics, and would explain the reduced respiration.

Conclusion

For the first time we show that the exocyst and cilia are centrally involved in AKI and the effect may be mediated through mitochondria.

Funding

  • Veterans Affairs Support