Defense Date


Document Type


Degree Name

Doctor of Philosophy


Pharmacology & Toxicology

First Advisor

Pin-Lan Li


Cell differentiation and senescence in podocytes are attributed to normal autophagy and associated cellular activities. It is possible that derangement of autophagy under different pathological conditions activates or enhances podocyte dedifferentiation leading to glomerular injury and ultimate glomerular disease. To test this hypothesis, we first tested whether autophagic deficiency due to lysosome dysfunction enhances podocyte dedifferentiation and explored the molecular mechanisms by which this lysosome dysfunction trigger or enhance podocyte dedifferentiation. By Western blot and confocal analysis, lysosome inhibition using an inhibitor or siRNA of V-ATPase inhibitor was found to markedly decrease the epithelial markers (P-cadherin and ZO-1) and increase the mesenchymal markers (FSP-1 and α-SMA). This enhancement of podocyte dedifferentiation (formerly referred to as epithelial-mesenchymal transition, EMT) was accompanied by deficient autophagic flux, as demonstrated by marked increases in LC3B-II and p62/Sequestosome 1. However, inhibition of autophagosome formation using spautin-1 (SP-1) significantly attenuated both enhancement of podocyte dedifferentiation and deficiency of autophagic flux. To explore the mechanisms by which deficient autophagic flux enhances podocyte dedifferentiation, we tested the role of accumulated p62 as a signal hub in this process. Neither the nuclear factor erythroid 2-related factor 2 (Nrf2) nor nuclear factor kappa (NFκ)-light-chain-enhancer pathway regulating p62 function was found to contribute to enhanced dedifferentiation. However, inhibition of cyclin-dependent kinase 1 (CDK1) activity reduced the phosphorylation of p62 and enhanced podocyte dedifferentiation similar to lysosome dysfunction, which indicates that enhanced podocyte dedifferentiation due to lysosome dysfunction may be triggered by accumulation of p62 and associated reduction of p62 phosphorylation. Given the essential role of sphingolipid-ceramide metabolism and transient receptor potential-mucolipin-1 (TRPML1) channel activity in lysosome function, we next sought to test whether altered ceramide metabolism by acid ceramidase (AC) leads to deficient lysosome trafficking and fusion to autophagosome in podocytes and thereby results in autophagic deficiency and podocyte dedifferentiation. Inhibition of AC by a potent and selective inhibitor, carmofur markedly reduced lysosome trafficking and fusion to both autophagosomes and multivesicular bodies (MVBs). Concurrently, enhancement of podocyte AC activity or exposure of podocytes to sphingosine, a product of ceramide metabolism by AC, remarkably increased lysosome trafficking and fusion to autophagosomes and MVBs, indicating that AC activity is critical for lysosome function in podocytes. To further explore the mechanisms by which AC activity contributes to lysosome trafficking, we examined the effects of various sphingolipids related to ceramide metabolism on transient receptor potential-mucolipin-1 (TRPML1) channel, a Ca2+ channel essential for lysosome trafficking and function. It was found that sphingomyelin (SM), a precursor for ceramide production blocked TRPML1 channel activity induced by ML-SA1 (a specific TRPML1 agonist), while ceramide had no effects on TRPML1 channel activity induced by ML-SA1. Interestingly, sphingosine, an AC product of ceramide remarkably enhanced TRPML1 channel activity induced by ML-SA1. These results demonstrate that AC product of ceramide, sphingosine may enhance TRPML1 channel activity, but an upstream sphingolipid, SM may exert inhibitory action on lysosome TRPML1 channel activity. AC may be a key enzyme gating TRPML1 channels by production of sphingosine and changes in upstream substrate SM. These results from in vitro cell studies led us hypothesize that a deficient AC activity may induce podocyte injury through lysosome dysfunction, leading to glomerular damage and proteinuria. To test this hypothesis, we generated a mouse colony with podocyte-specific gene deletion of AC α subunit, namely, Asah1fl/fl/PodoCre mice. In these mice, severe proteinuria and albuminuria were shown compared to their littermates, but they were without global and even focal glomerular sclerosis. These mice also had hypoalbuminemia and edema, and under transmission electron microscopy (TEM) ultrastructural changes of podocytes from their glomeruli exhibited diffuse and flat foot process (podocyte effacement), vacuolation, and microvillus formation, which were not observed in their littermates. Treatment of corticosteroids and specific expression patterns of dystroglycans in glomeruli confirmed that albuminuria in these Asah1fl/fl/PodoCre mice may be resistant to corticosteroids. Together, these results from in vivo animal studies indicate that podocyte-specific gene deletion of AC α subunit may induce a corticosteroid-resistant minimal change disease (MCD). Based on all results from our in vitro and in vivo studies, we conclude that the normal lysosome function is essential for maintenance of autophagic flux and podocyte differentiation, which is regulated by a lysosomal AC-mediated signaling pathway through a TRPML1 channel gating mechanism. AC gene defect or deficiency of its activity induces podocyte injury, which is characterized by a corticosteroid-resistant MCD. These findings indicate an important pathological role of AC deficiency and associated lysosome dysfunction in podocytes injury and corticosteroid-resistant MCD, which may help develop novel therapeutic strategies for prevention or treatment of corticosteroid-resistant MCD.


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