ZFYVE21 Sustains Akt-Endothelial Nitric Oxide Synthase (ENOS) Signaling to Promote Vascular Barrier Function in the Kidneys during Aging

Background: We previously identified ZFYVE21 as a Rab5 effector in ECs. ZFYVE21 is a zinc finger-containing protein implicated in cell motility whose functions in vivo are unknown. In cultured human endothelial cells (ECs), we previously observed that ZFYVE21 localized to Rab5+ vesicles where it modulated PI(3,4,5)P3 content to enable Akt activation.As part of these studies, we incidentally observed that ZFYVE21 was highly expressed in glomerular ECs as well as peritubular capillaries. These observations prompted us to examine ZFYVE21 in Akt-mediated ENOS activity, a pathway critical for homeostasis of vascular barriers. Methods: We used gene targeting to generate endothelial cell (EC)-specific ZFYVE21 KO reporter mice where ECs were labeled with GFP while other cells expressed RFP. We performed whole blood chemistries and inulin clearance measurements in mice. We analyzed human and mouse kidney tissues using I.F., in situ hybridization, Western blots, co-immunoprecipitations, and electron microscopy. We performed live cell imaging studies using mutant reporter constructs. Results: We found that ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we analyzed EC-specific ZFVYE21-/- reporter mice. ZFYVE21 EC-/- mice developed accelerated aging phenotypes including reduced ENOS activity, failure to thrive, and renal insufficiency; and kidneys from ZFYVE21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that, in a Rab5- and Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Conclusion: Our work defines a ZFYVE21-mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging. Funding: Veterans Affairs Support, Private Foundation Support