Following the Cellular Itinerary of Renal Aquaporin-2 Shuttling with 4.5x Expansion Microscopy

Aquaporin (AQP) water channels facilitate water diffusion across lipid bilayers following an osmotic gradient. AQPs are especially important in renal water reabsorption. AQP2 in the renal collecting duct mediates fine-tuning of urine volume in response to the antidiuretic hormone arginine vasopressin (AVP). AVP mediates plasma membrane insertion of AQP2 from an intracellular reservoir of AQP2 containing vesicles, which instantly increases water reabsorption and urine concentration. The vesicle population cannot be resolved via conventional fluorescent microscopy therefore, detailed information regarding the AQP2 vesicular population is still lacking. Newly established 4.5x Expansion Microscopy (ExM) can increase resolution to 60-70 nm. Using 4.5x Ex, spinning disk microscopy and image processing, we detected AQP2 vesicles/endosomes. Using different markers of the endosomal system, we obtained detailed information of the cellular AQP2 shuttling itinerary. Thus, our results show that 4.5 ExM is an ideal approach to obtain detailed information regarding the AQP2 vesicle population.