Abstract
Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP-LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP-LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.
Highlights
Podocytes are highly specialized epithelial cells that compose the renal filtration barrier, but are considered among the most vulnerable cell types of the kidney
The major goal of the present study is to determine whether CD38mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes
We found that lysosome function inhibitor bafilomycin and inhibition of CD38 by nicotinamide or CD38 gene silencing increased the APs formation, but decreased APLs content in podocytes under basal condition and following induction of autophagy by rapamycin
Summary
Podocytes are highly specialized epithelial cells that compose the renal filtration barrier, but are considered among the most vulnerable cell types of the kidney. Their loss or dysfunction is thought to be critical for the pathogenesis of proteinuria and glomerular disease. As podocytes are terminally differentiated, non-dividing cells with a low capacity for regeneration (37), the mechanisms that contribute to their adaptive responses to stress or injury are likely to play a critical role in their physiology. Autophagy functions in a continuous, reparative way to maintain normal cellular homeostasis, whereas under conditions of stress or pathological stimuli, it may be induced and even develop into autophagic death.
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