Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

Abstract

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34-deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cellspecific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux.

Active vacuolar H+ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P2 loss under PIKfyve or Vps34 deficiency

“…For example, terminally differentiated 3T3L1 adipocytes in culture, as opposed to the rapidly dividing precursor 3T3L1 fibroblastic cells, do not form cytoplasmic vacuoles under pharmacological inhibition or expression of the dominantinterfering kinase-deficient mutant of PIKfyve (27,50). Likewise, fat cells, muscle cells, or glomeruli podocytes in the context of the respective adipose-specific, muscle-specific or podocytes-specific mouse model with Pikfyve inactivation through Cre recombinase expression driven by the corresponding promoters, do not exhibit cytoplasmic vacuoles in situ (22,23,28,64). Whereas in these mouse models incomplete Pikfyve inactivation might be an admissible cause for lack of vacuoles, it is remarkable that mitotic podocytes that outgrow in a culture from the very same nonvacuolating terminally differentiated glomerular podocytes exhibit the vacuolation phenomenon (64).…”

“…Likewise, fat cells, muscle cells, or glomeruli podocytes in the context of the respective adipose-specific, muscle-specific or podocytes-specific mouse model with Pikfyve inactivation through Cre recombinase expression driven by the corresponding promoters, do not exhibit cytoplasmic vacuoles in situ (22,23,28,64). Whereas in these mouse models incomplete Pikfyve inactivation might be an admissible cause for lack of vacuoles, it is remarkable that mitotic podocytes that outgrow in a culture from the very same nonvacuolating terminally differentiated glomerular podocytes exhibit the vacuolation phenomenon (64). Concordantly, Pikfyve inactivation in cells that are under robust and constant turnover, such as proximal tubular and intestinal epithelial cells, readily trigger cytoplasmic vacuoles in situ in the respective tissue-specific Pikfyve KO mouse model (61,64).…”

Role of FGF and Hyaluronan in Choroidal Neovascularization in Sorsby Fundus Dystrophy

“…Fluorescence intensity of HABP staining was quantified using integrated density analysis as previously described [37,38]. For all the RPE cell culture confocal microscopy images, fluorescence was quantitated using a standard measure of integrated density, which is the product of area and mean gray value.…”

PIKfyve Deficiency in Myeloid Cells Impairs Lysosomal Homeostasis in Macrophages and Promotes Systemic Inflammation in Mice

“…A hypomorphic PIKfyve mouse model is viable, but it dies early due to defects in multiple organs and tissues, including neural tissues, heart, lung, kidney, thymus, and the hematopoietic system (20). Subsequently, conditional PIKfyve knockout mice were developed using the Cre-Lox system and demonstrated the essential roles of PIKfyve in specific tissues (19,(21)(22)(23)(24)(25)(26). In particular, we previously showed that mice with platelet-specific deletion of PIKfyve have impaired lysosomal homeostasis and develop aberrant inflammatory and prothrombotic responses (22).…”