Abstract
The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3pdKO/mVps34pdKO) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes.
Vps34 was originally identified in yeast mutants that exhibited severe defects in vacuolar protein sorting. Subsequent studies revealed that Vps34 is the only phosphatidylinositol 3-kinase (PtdIns3K) in yeast. Mammalian PIK3C3 is classified as a class III PtdIns3K, although it is the most ancient paralog of the three classes of PtdIns3Ks in mammals. Unlike the class I and class II PtdIns3Ks, the class III PtdIns3K can only use phosphatidylinositol as a substrate to generate a single product, phosphatidylinositol-3-phosphate (PtdIns3P), by specifically phosphorylating the D-3 position on the inositol ring of phosphatidylinositol. Previous studies indicate that PIK3C3 plays important roles in intracellular membrane trafficking and autophagy. Autophagy is a tightly regulated intracellular process in which portions of cytoplasm, including proteins and organelles, are sequestered within double-membrane vesicles termed autophagosomes and delivered to lysosomes for degradation and recycling. Mammalian cells are postulated to utilize autophagy as a mechanism for turnover of long-lived proteins, removal of protein aggregates and damaged organelles, and as a survival strategy under metabolic stress conditions such as starvation. Recent studies suggest that autophagy is a key mechanism in maintaining the integrity of podocytes, but the role of PIK3C3 in podocytes is unknown.
Podocytes are highly specialized renal glomerular visceral epithelial cells that intricately wrap around the outer aspect of the glomerular basement membrane and play essential roles in establishing the size- and charge-selective permeability of the glomerular filtration barrier, and in maintaining the structural and functional integrity of the kidney. To begin our understanding of PIK3C3’s physiological role in podocytes, we created a mouse line carrying a conditional allele of the Pik3c3 gene. Specifically, we floxed Pik3c3 exons 20–21 and also rendered all the distal exons out of reading frame so the entire catalytic core, the key aspartic acid-phenylalanine-glycine (DFG) motif, and the ATP binding domain of PIK3C3 can all be deleted upon Cre-mediated recombination. By crossing this Pik3c3-floxed mouse line with a podocin-Cre transgenic mouse, which expresses Cre-recombinase exclusively in podocytes starting from the capillary loop stage during glomerular development, we produced a line of podocyte-specific Pik3c3-knockout (Pik3c3pdKO) mice and compared them with their gender-matched Pik3c3flox/flox;podocin-Cre(-) littermate control (Pik3c3Ctrl) mice.
Homozygous Pik3c3pdKO mice were born at Mendelian ratios and had no apparent phenotype at 2 weeks of age. However, they exhibited growth retardation and were significantly smaller than Pik3c3Ctrl mice by 6 weeks of age, with no difference in kidney-to-body weight ratios. Pik3c3pdKO mice developed significant proteinuria by 3 weeks of age, developed severe kidney lesions by 5–6 weeks of age, with progressive decline of renal function, and died before 9 weeks of age. Renal histology revealed striking podocyte vacuolization and proteinaceous casts, with marked focal segmental and global glomerulosclerosis as well as interstitial inflammation and fibrosis by 6 weeks of age. Immunohistochemistry showed massive fibronectin deposition along the Bowman’s capsule, with moderate increases in the glomeruli and some periglomerular areas.
Electron microscopy revealed numerous enlarged vacuoles and increased autophagosomes in the podocytes, with complete foot process effacement and irregular and thickened glomerular basement membranes, in Pik3c3pdKO mice by 6 weeks of age. Immunoblotting of isolated glomerular lysates revealed markedly increased levels of lysosomal-associated membrane protein 1 and 2 (LAMP1 and LAMP2). Immunofluorescence staining confirmed that the enlarged vacuoles are positive for both LAMP1 and LAMP2. In contrast, podocin levels were decreased drastically. These data suggest that the enlarged vacuoles may have originated from lysosomes. Furthermore, the immunoblotting also revealed that the phosphatidylethanolamine-conjugated form of microtubule-associated protein 1 light chain 3 (LC3-II) and even the cytosolic form of LC3 (LC3-I) are both markedly increased in Pik3c3pdKO mice, consistent with the increases in autophagosomes revealed by electron microscopy.
Additional immunofluorescence staining of younger mouse kidney sections revealed markedly increased LC3 and LAMP1 in Pik3c3pdKO mice at 15 d of age when H&E staining failed to show any abnormality. Of interest, some of the increased LC3-positive granules or puncta were colocalized with the increased LAMP1 (suggesting increases in autolysosomes), whereas others were not colocalized with LAMP1, indicating increases in autophagosomes. Triple immunofluorescence staining confirmed that the increases in both LC3 and LAMP1 occurred only in podocytes. Indeed, electron microscopy further confirmed that the formation of numerous vacuoles and increases of autophagosomes occurred only in the podocytes, with occasional focal foot process effacement, compared with Pik3c3Ctrl littermates. Thus, both marked elevations of LC3 and LAMP1/2 and the earliest ultrastructural alterations (podocyte vacuolization, focal foot process effacement and increased autophagosome formation) had occurred before the onset of proteinuria.
Our results indicate that there is no compensation by other classes of PtdIns3Ks or related genes in the Pik3c3pdKO mice, which all died before 9 weeks of age, highlighting the functional specificity and physiological importance of PIK3C3 in the podocyte. Our results from electron microscopy also showed that many autophagosomes in the podocytes of Pik3c3pdKO mice had irregular inner membranes. This led to our speculation that the enzymatic product of PIK3C3, PtdIns3P, is not only an integral component of autophagosomal membranes but also plays an indispensable role in recruiting critical membrane proteins to maintain the normal membranes of autophagosomes, particularly the inner membrane; a previous study has shown enriched PtdIns3P on the inner membranes of autophagosomes. We further speculate that accumulation of the aberrant autophagosomes and autolysosomes in the podocytes of Pik3c3pdKO mice was caused by loss of PtdIns3P-dependent hydrolase targeting into the lysosome; consequently the engulfed autophagosomal cargos could not be degraded and recycled. Our ongoing studies are directed at the elucidation of the mechanism underlying the disrupted intracellular vesicle trafficking and processing. We are also investigating whether disruption of the endocytic pathway has also contributed to the enlarged vacuoles seen in the podocytes of Pik3c3pdKO mice. Furthermore, it is important to determine the potential interplay between PIK3C3 and the mechanistic target of rapamycin (MTOR) as well as other autophagy-related (ATG) gene products. Moreover, it is also significant to determine whether PIK3C3 inactivation contributes to the pathogenesis of proteinuria and glomerulosclerosis in some cases of focal segmental glomerulosclerosis and diabetic nephropathy.
Acknowledgments
This work was supported by funds from National American Heart Association Scientist Development grant 0630274N, Vanderbilt Diabetes Research and Training Center Pilot and Feasibility grant 2P60DK020593, academic program support funds from the Department of Medicine at Vanderbilt University, startup funds from Georgia Regents University Augusta, and National Institutes of Health R01 grant DK83575 (to J.-K.C.).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.