In Vivo Disruption of pH Gradients Alters Endo-Lysosomal Dynamics in the Proximal Tubule
- Genetic Diseases: Tubulopathies
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1202 Genetic Diseases of the Kidneys: Non-Cystic
- Sakhi, Imene Bouchra, Universitat Zurich, Zurich, Zurich, Switzerland
- Kaminska, Monika Anna, Universitat Zurich, Zurich, Zurich, Switzerland
- Jankovic, Nevena, Universitat Zurich, Zurich, Zurich, Switzerland
- Hall, Andrew, Universitat Zurich, Zurich, Zurich, Switzerland
Group or Team Name
- Structural and Functional Imaging of the Kidney.
The proximal tubule (PT) displays an apical endo-lysosomal system (ELS) that reabsorbs and degrades filtered plasma proteins. Defects in this process result in proteinuria. Vesicular acidification is integral to endocytotic function and is impaired in some genetic kidney disorders, such as Dent disease; but the nature of pH gradients in the PT ELS and their functional relevance to dynamic processes were not well understood. Data from partially differentiated in vitro models suggest that pH decreases progressively through early endosomes (EEs), late endosomes (LEs) and lysosomes, but whether it is also the case in PTs in vivo was unclear.
We have used small molecules as carriers to target pH dependent and independent fluorescent sensors to the PT ELS in living mice and provide ratiometric readouts of intracellular pH. We have applied intravital multiphoton microscopy to track probes through ELS components in real-time, and have mapped changes in pH to specific structures identified by antibody labeling in fixed tissue. The lipophilic weak base hydroxychloroquine (HCQ) was injected intravenously to explore the effects of rapidly de-acidifying ELS vesicles.
The ratiometric technique was shown to provide a readout of pH across the physiological range in a PT cell line. Clear decreases in pH were identified within PTs in vivo in both EEs and lysosomes, but surprisingly not in LEs. Abolishing these pH gradients with HCQ disrupts recycling of megalin and reroutes it to the degradative pathway, resulting in a severe defect in protein uptake. However, HCQ treatment did not recreate characteristic defects in endosomal maturation described previously in Dent disease models. Meanwhile, de-acidification of lysosomes inhibits dissociation from LEs and prevents their trafficking into the basal, mitochondrial-rich region of the cell.
By using an innovative intravital imaging approach, we have generated the first functional map of ELS pH changes within working PTs, which differs significantly from the previous textbook paradigm. Moreover, we show that vesicular acidification is critical for endocytotic receptor recycling and lysosomal dynamics. However, our results suggest that endosomal alkalinization alone does not fully explain the pathogenesis of Dent disease, meaning that pH-independent mechanisms still need to be considered.
- Government Support – Non-U.S.