ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: SA-PO082

Quantifying Autophagic Flux in Kidney Tissue with Super-Resolution Structured Illumination Microscopy Imaging

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Elias, Bertha C., Vanderbilt University Medical Center,Division of Nephrology & Hypertension, Nashville, Tennessee, United States
  • Taguchi, Kensei, Vanderbilt University Medical Center,Division of Nephrology & Hypertension, Nashville, Tennessee, United States
  • Brooks, Craig R., Vanderbilt University Medical Center,Division of Nephrology & Hypertension, Nashville, Tennessee, United States
Background

Autophagy, a key homeostatic catabolic pathway in eukaryotic cells, is linked to pathological conditions in most organs as well as cancer and aging. In the kidney, autophagy has been shown to modulate both acute and chronic injury. Despite the importance of autophagy, few methods are available to monitor autophagic flux, i.e. clearance of autophagosomes by the lysosome. Autophagy is usually evaluated by presence of autophagosomes, LC3 II levels or EM but not autophagic flux. We combined the RFP-GFP-LC3 reporter mice with super-resolution structured illumination microscopy (SIM) to measure autophagic flux at an individual autophagosome level in response to kidney ischemia.

Methods

Kidneys of RFP-GFP-LC3 reporter mice were injured by unilateral ischemic reperfusion injury. The GFP of the reporter is sensitive to low pH, quenching the fluorescent signal upon autophagosome/lysosome fusion, leaving only RFP signal. At 48 hours the kidneys were harvested, fixed and paraffin imbedded. The tissue was sectioned at 4-6μm and mounted on silanized coverslips. Immunofluorescence staining was done using antibodies against GFP and RFP and stained with DAPI. The tissue was imaged using an N-SIM microscope to obtain Z stacks. These images were reconstructed using Bitplane's Imaris software. Colocalization of RFP and GFP was quantified using NIS elements.

Results

SIM imaging captured LC3 positive structures ranging in size from <0.3 μm to >4 μm. LC3 positive autophagosomes were present in larger numbers and size in injured kidneys compared to contralateral. Analysis of the colocalization of RFP with GFP, to quantify flux, in the control kidney showed the presence of many small RFP only positive organelles, indicating that these were autolysosomes, i.e. autophagosomes fused with lysosomes. In the injured kidney the organelles were larger and positive for both RFP and GFP, thus indicating more autophagosomes but a lower autophagic flux.

Conclusion

Combining the RFP-GFP-LC3 reporter mouse with SIM imaging allows for the quantification of individual autophagosomes in kidney tissue and measurement of the dynamic flux of the process. In unilateral IRI injury, we found the autophagic flux to be higher in the contralateral kidney than the injured kidney at 48 hours despite the presence of very large autophagosomes. These data suggest autophagic clearance is overwhelmed following ischemic injury.