Lysosomal acid ceramidase (Ac) has been shown to be critical for ceramide hydrolysis and regulation of lysosome function and cellular homeostasis. In the present study, we generated a knockout mouse strain (Asah1 fl/fl /Podo Cre ) with a podocyte-specific deletion of the a subunit (main catalytic subunit) of Ac. Although no significant morphologic changes in glomeruli were observed in these mice under light microscope, severe proteinuria and albuminuria were found in these podocyte-specific knockout mice compared with control genotype littermates. Transmission electron microscopic analysis showed that podocytes of the knockout mice had distinctive foot process effacement and microvillus formation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice bearing the Asah1 podocyte-specific gene deletion. Ceramide accumulation determined by liquid chromatographyetandem mass spectrometry was demonstrated in isolated glomeruli of Asah1 fl/fl / Podo Cre mice compared with their littermates. By crossbreeding Asah1 fl/fl /Podo Cre mice with Smpd1 À/À mice, we also produced a double knockout strain, Smpd1 À/À /Asah1 fl/fl /Podo Cre , that also lacks Smpd1, the acid sphingomyelinase that hydrolyzes sphingomyelin to ceramide. These mice exhibited significantly lower levels of glomerular ceramide with decreased podocyte injury compared with Asah1 fl/fl / Podo Cre mice. These results strongly suggest that lysosomal Ac in podocytes is essential for the maintenance of the structural and functional integrity of podocytes.

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1 −/− mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1 −/− mice as compared to their wild-type littermates. Besides, Mcoln1 −/− mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1 −/− mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1 −/− mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. A. phagocytophilum infected SCID mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner, and A. phagocytophilum is capable of co-opting ASM-independent lipid sources.

Increased secretion of exosomes from arterial smooth muscle cells (SMCs) has been shown to contribute to arterial medial calcification (AMC) associated with accumulation of calcium deposits in the arterial wall and osteoblastic differentiation of SMCs. In this study, we further explored the molecular mechanisms increasing exosome secretion during AMC, focusing on lysosomal‐ceramide/mTOR (mammalian target of rapamycin) signaling in the regulation of lysosome trafficking and exosome release. Smooth muscle‐specific lysosomal acid ceramidase (Ac) gene knock out (Asah1fl/fl/SMcre) mice were treated with a high dose of Vit D (500,000 IU/kg/day) for 4 days. By Alizarin Red S and Von Kossa staining, we observed significantly increased aortic medial calcification with augmented expression of osteogenic markers like osteopontin and RUNX2 in Asah1fl/fl/SMcre mice compared to their wild type littermates, which was markedly attenuated by pretreatment of these mice with Torin‐1, an mTOR inhibitor. Confocal microscopy showed Asah1fl/fl/SMcre mice had markedly increased colocalization of mTORC1 with Lamp‐1(lysosome marker), but decreased colocalization of VPS16 (a multivesicular bodies (MVBs) marker) with Lamp‐1, suggesting mTOR activation and reduced MVBs interaction with lysosomes. However, mTOR inhibition by Torin‐1 significantly reduced the colocalization of mTOR vs Lamp‐1 which inturn increased lysosome‐MVBs interaction. Torin‐1 was also found to reduce accumulation of CD63 and annexin 2 (AnX2) (exosome markers) in the coronary arterial wall of Asah1fl/fl/SMcre mice compared to wild type littermates. Using CASMCs, we observed that Ac gene deletion led to peripheral distribution of lysosomes and increased exosome secretion, which was blocked by Torin‐1. Functionally, Torin‐1 was found to significantly reduce arterial stiffening Asah1fl/fl/SMcre mice as shown by restoration from increased pulse wave velocity (PWV). Altogether, these results suggest that lysosomal ceramide‐mTOR signaling is critical for the control of lysosome trafficking, exosome secretion and arterial stiffening or osteogenesis. Support or Funding Information Supported by NIH grants HL057244, HL075316 and DK120491

Hyperhomocysteinemia (hHcy) has been demonstrated to activate NLRP3 inflammasome leading to podocyte injury and glomerular sclerosis. However, it remains unknown whether exosome‐mediated secretion of NLRP3 inflammasome products is involved in the development of glomerular injury during hHcy. In the present study, we examined the possible role of increased exosome secretion during NLRP3 activation in the inflammatory response and tested whether without robust release of exosomes, inflammatory response and glomerular injury still occur. Exosome biogenesis inhibitor‐GW4869, lysosome trafficking stimulator‐rapamycin or ASM inhibitor‐amitriptyline were used in mice to answer this question. By nanoparticle tracking analysis, it was found that hHcy significantly increased urinary exosome release in WT/WT mice. In Smpd1trg/Podocre (podocyte‐specific ASM transgenic) mice, this hHcy‐induced increase in urinary exosomes was significantly enhanced. Pretreatments of WT/WT mice and Smpd1trg/Podocre mice with amitriptyline (Ami), GW4869 (GW) and rapamycin (Rap) all prevented increases in urinary exosomes during hHcy. In podocytes isolated from WT/WT mice and Smpd1trg/Podocre mice, similar results were obtained when we measured exosomes released from these cells after different treatments. Confocal microscopy demonstrated that Ami not only prevented hHcy‐induced NLRP3 inflammasome formation and activation in glomeruli, but also decreased exosome release in podocytes of WT/WT mice and Smpd1trg/Podocre mice. However, GW and Rap only blocked exosome‐mediated IL‐1β secretion from podocytes, but had no effect on inflammasome formation or activation in glomeruli during hHcy. However, all 3 treatments attenuated the inflammatory response in glomeruli and associated injury as shown by reduced immune cell aggregation, decreased proteinuria and ameliorated glomerular sclerosis. These results suggest that without enhanced exosome release NLRP3 inflammasome activation in podocytes may not produce local glomerular inflammatory response and associated injury during hHcy. Support or Funding Information This study was supported by NIH grants DK54927 and DK102539.