Kidney Week

Abstract: SA-PO584

High Throughput Metabolic Flux Analysis of Intact Kidney Tissue Reveals Abnormal Mitochondrial Respiration During AKI to CKD Transition

Session Information

• AKI: Other Mechanisms and Cell Cultures
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Afzal, Aqeela, Vanderbilt University Medical Center, Nashville,, Tennessee, United States

Aqeela Afzal,

• Urrutia, Andres Amalio, Vanderbilt University Medical Center, Nashville,, Tennessee, United States

Andres Amalio Urrutia,

• Haase, Volker H., Vanderbilt University Medical Center, Nashville,, Tennessee, United States

Volker H. Haase,

Background

Mitochondrial dysfunction has been implicated in the pathogenesis of multiple renal diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD). However, knowledge about mitochondrial function during AKI to CKD transition is limited. The assessment of basal oxygen consumption rates (OCR) and the responses to electron transport chain inhibitors and uncoupling agents represent the first steps in evaluating cellular mitochondrial function. In order to avoid artifacts associated with tissue disruption or primary cell culture, we developed a novel method that permits the measurement of OCR in intact kidney tissue slices and investigated mitochondrial function during AKI to CKD transition.

Methods

9-12 week-old C57/BL6 male mice were subjected to unilateral ischemia reperfusion injury (IRI) induced by 30 minutes of renal pedicle clamping. Kidneys were analyzed immediately after clamp removal (no-reperfusion) and at 24 hours, day 3, day 7 and day 21 post IRI. After removal of the kidney capsule, a portion of the kidney was used for DNA, RNA and histologic analysis. The remainder of the kidney was submerged in oxygenated modified Ringers solution. 1mm punch-biοpsies were obtained from 100 μm vibratome sections of kidney pole tissue and loaded into an islet capture plate. OCR and extracellular acidification rates (ECAR) were analyzed with a Seahorse XF^e24 metabolic flux analyzer.

Results

Basal OCR and ECAR rates were significantly reduced immediately following IRI and recovered incompletely by day 3 post IRI. Day 7 and day 21 were characterized by a further reduction in OCR and ECAR below day 3 levels. The reduction in OCR correlated with significantly diminished mitochondrial DNA and gene expression levels and the development of fibrosis at day 7 an day 21. Similarly reduced ECAR values were associated with reduced glycolytic gene expression.

Conclusion

Here we present a novel approach for high throughput monitoring and analysis of mitochondrial respiration in intact kidney tissue sections. Our data indicate that AKI to CKD transition is associated with reduced mitochondrial respiration and glycolysis rates. A detailed protocol of this method will be presented at the meeting.