Kidney Week

Abstract: FR-PO626

Tryptophan Metabolism as a Link between Cilia and Mitochondria in Renal Tubule Cells

Session Information

• Cystic Kidney Diseases: Mechanisms and Models
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

• Winkler, Brennan, Medical University of South Carolina, Charleston, South Carolina, United States

Brennan Winkler

• Zuo, Xiaofeng, Medical University of South Carolina, Charleston, South Carolina, United States

Xiaofeng Zuo, PhD

• Deng, Peifeng, Medical University of South Carolina, Charleston, South Carolina, United States

Peifeng Deng

• Fitzgibbon, Wayne R., Medical University of South Carolina, Charleston, South Carolina, United States

Wayne R. Fitzgibbon, PhD

• Lipschutz, Joshua H., Medical University of South Carolina, Charleston, South Carolina, United States

Joshua H. Lipschutz, MD, FASN

Background

Primary cilia and mitochondria are centrally involved in ADPKD. We showed that the exocyst trafficking complex is necessary for ciliogenesis, knockdown (KD) of the central exocyst component Exoc5 resulted in absent cilia with increased susceptibility to H₂O₂, overexpression (OE) of Exoc5 increased cilia length with protection from H₂O₂, and renal tubule-specific knockout (KO) of Exoc5 in mice led to PKD. Intraflagellar transport protein 88 (Ift88) is also necessary for ciliogenesis.

Methods

We performed Seahorse assays, staining for reactive oxygen species (ROS), transmission electron microscopy (TEM), and a metabolomics screen in Exoc5 OE, Exoc5 KD, Exoc5 ciliary targeting sequence point mutant (cts-mut), and control Madin-Darby canine kidney tubule (MDCK) cells, and murine Ift88 KO and rescue cells. We also crossed Exoc5^fl/fl and Ift88^fl/fl mice with mice expressing CreERT2 driven by the proximal tubule-specific sodium-dependent inorganic phosphate transporter and performed bilateral ischemia reperfusion (I/R).

Results

There was decreased mitochondrial respiration, increased ROS, and abnormal mitochondria in Exoc5 KD, Exoc5 cts-mut cells, and Ift88 KO cells compared to control MDCK and Ift88 rescue cells, with opposite effects seen in Exoc5 OE cells. In our metabolomics screen tryptophan levels were increased 113- and 58-fold, respectively, in Exoc5 cts-mut and Exoc5 KD compared to control cells. Concomitantly, kynurenine which is directly downstream of tryptophan was decreased in these cells. Similar results were seen in the Ift88 KO compared to rescue cells. In Exoc5 OE cells tryptophan was decreased and kynurenine was increased. Metabolism of tryptophan to kynurenine occurs via indoleamine 2, 3-dioxygenases (IDO1 and IDO2) and changes in mRNA levels by RT-qPCR were consistent with the metabolomics data. Proximal tubule-specific Exoc5 and Ift88 KO mice lacked cilia, had decreased levels of mitochondrial ATP synthase, increased tryptophan, and aggravated renal injury following I/R injury compared to control mice (p=0.005 for Exoc5, p=0.04 for Ift88).

Conclusion

Tryptophan metabolism may be the “cilia-dependent cyst activating” mechanism with cilia loss resulting in a metabolic derangement that prevents cell proliferation and cyst formation. We also show that loss of cilia primes renal tubule cells for injury.

Funding

• Veterans Affairs Support