Podocyte-Specific Silencing of Acid Sphingomyelinase Gene to Abrogate Hyperhomocysteinemia-Induced NLRP3 Inflammasome Activation and Glomerular Inflammation.

Acid Sphingomyelinase has been reported to increase tissue ceramide and thereby mediate hHcy-induced glomerular NLRP3 inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene attenuates hHcy-induced NLRP3 inflammasome activation and associated exosome release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre mice compared to control littermates. By nanoparticle tracking analysis, floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary exosome excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented Hcy-induced elevation of exosome release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared to WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced exosome secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of exosome release from podocytes was blocked by ASM inhibitor, but not by NLRP3 inflammasome inhibitors. Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and exosome release.

Regulation of NLRP3 Inflammasome Activation and Inflammatory Exosome Release in Podocytes by Acid Sphingomyelinase During Obesity.

The activation of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been reported to importantly contribute to glomerular inflammation and injury under different pathological conditions such as obesity. However, the mechanism mediating NLRP3 inflammasome activation in podocytes and subsequent glomerular injury remains poorly understood. Given that the ceramide signaling pathway has been reported to be implicated in obesity-related glomerulopathy (ORG), the present study was designed to test whether the ceramide-producing enzyme, acid sphingomyelinase (ASM), determines NLRP3 inflammasome activation and inflammatory exosome release in podocytes leading to glomerular inflammation and injury during ORG. In Smpd1trg/Podocre mice, podocyte-specific overexpression of Smpd1 gene which encodes ASM significantly exaggerated high-fat diet (HFD)-induced NLRP3 inflammasome activation in podocytes and immune cell infiltration in glomeruli compared to WT/WT mice. Smpd1 gene deletion, however, blocked these pathological changes induced by HFD in Smpd1-/- mice. Accompanied with NLRP3 inflammasome activation and glomerular inflammation, urinary excretion of exosomes containing podocyte marker and NLRP3 inflammasome products (IL-1ß and IL-18) in Smpd1trg/Podocre mice on the HFD was much higher than that in WT/WT mice. In contrast, Smpd1-/- mice on the HDF had significantly lower urinary exosome excretion than WT/WT mice. Correspondingly, HFD-induced podocyte injury, glomerular sclerosis, and proteinuria were more severe in Smpd1trg/Podocre mice, but milder in Smpd1-/- mice compared to WT/WT mice. Using podocytes isolated from these mice, we demonstrated that visfatin, a prototype pro-inflammatory adipokine, induced NLRP3 inflammasome activation and enrichment of multivesicular bodies (MVBs) containing IL-1ß in podocytes, which was much stronger in podocytes from Smpd1trg/Podocre mice, but weaker in those from Smpd1-/- mice than WT/WT podocytes. By quantitative analysis of exosomes, it was found that upon visfatin stimulation, podocytes from Smpd1trg/Podocre mice released much more exosomes containing NLRP3 inflammasome products, but podocytes from Smpd1-/- mice released much less exosomes compared to WT/WT podocytes. Super-resolution microscopy demonstrated that visfatin inhibited lysosome-MVB interaction in podocytes, indicating impaired MVB degradation by lysosome. The inhibition of lysosome-MVB interaction by visfatin was amplified by Smpd1 gene overexpression but attenuated by Smpd1 gene deletion. Taken together, our results suggest that ASM in podocytes is a crucial regulator of NLRP3 inflammasome activation and inflammatory exosome release that instigate glomerular inflammation and injury during obesity.

Renal Medullary Overexpression of Sphingosine-1-Phosphate Receptor 1 Transgene Attenuates Deoxycorticosterone Acetate (DOCA)-Salt Hypertension.

BACKGROUND: Our previous studies showed that renal medullary sphingosine-1-phosphate receptor 1 (S1PR1) mediated sodium excretion, high salt intake increased S1PR1 level, deoxycorticosterone acetate (DOCA) blocked high salt-induced S1PR1 in the renal medulla, and that conditional knockout of S1PR1 in the collecting duct aggravated DOCA-salt hypertension. The present study tested the hypothesis that overexpression of S1PR1 transgene in the renal medulla attenuates the sodium retention and hypertension in DOCA-salt mouse model. METHODS: Male C57BL/6J mice received renal medullary transfection of control or S1PR1-expressing plasmids and then DOCA-salt treatment. Renal sodium excretion and arterial pressure were compared between control and S1PR1-overexpressed mice in response to high salt loading or pressure natriuresis. RESULTS: S1PR1-transfected mice showed significantly enhanced urinary sodium excretion in response to acute sodium loading (0.93 ± 0.27 in control vs. 4.72 ± 1.12 µmol/min/gKW in S1PR1-overexpressed mice, P < 0.05) and the pressure natriuresis (3.58 ± 1.77 vs. 9.52 ± 1.38, P < 0.05), less positive sodium balance in response to chronic high-salt intake (3.05 ± 0.39 vs. 1.65 ± 0.39 mmol/72 hr, P < 0.05), and consequently, the attenuation of DOCA-salt hypertension (134.2 ± 6.79 vs. 109.8 ± 3.54 mm Hg, P < 0.05). The αENaC protein amount in the renal medulla was not changed, however, the ßENaC was significantly decreased and the γENaC was significantly increased in S1PR1-overexpressed mice. The immunostaining showed apical membrane translocation of γENaC, while no change of αENaC and ßENaC in control mice, and that the apical membrane translocation of γENaC was blocked in S1PR1-treasffected mice. CONCLUSIONS: These results suggested that activation of S1PR1 in the renal medulla attenuates DOCA-induced sodium retention and salt-sensitive hypertension associated with inhibition of ENaC.

Contribution of Hepatic Steatosis-Intensified Extracellular Vesicle Release to Aggravated Inflammatory Endothelial Injury in Liver-Specific Asah1 Gene Knockout Mice.

To study the mechanism by which nonalcoholic fatty liver disease (NAFLD) contributes to vascular endothelial Nod-like receptor pyrin domain 3 (NLRP3) inflammasome activation and neointima hyperplasia, NAFLD was established in high-fat diet (HFD)-treated Asah1fl/fl/Albcre (liver-specific deletion of the acid ceramidase gene Asah1) mice. Compared with Asah1 flox [Asah1fl/fl/wild type (WT)] and wild-type (WT/WT) mice, Asah1fl/fl/Albcre mice exhibited significantly enhanced ceramide levels and lipid deposition on HFD in the liver. Moreover, Asah1fl/fl/Albcre mice showed enhanced expression of extracellular vesicle (EV) markers, CD63 and annexin II, but attenuated lysosome-multivesicular body fusion. All these changes were accompanied by significantly increased EV counts in the plasma. In a mouse model of neointima hyperplasia, liver-specific deletion of the Asah1 gene enhanced HFD-induced neointima proliferation, which was associated with increased endothelial NLRP3 inflammasome formation and activation and more severe endothelial damage. The EVs isolated from plasma of Asah1fl/fl/Albcre mice on HFD were found to markedly enhance NLRP3 inflammasome formation and activation in primary cultures of WT/WT endothelial cells compared with those isolated from WT/WT mice or normal diet-treated Asah1fl/fl/Albcre mice. These results suggest that the acid ceramidase/ceramide signaling pathway controls EV release from the liver, and its deficiency aggravates NAFLD and intensifies hepatic EV release into circulation, which promotes endothelial NLRP3 inflammasome activation and consequent neointima hyperplasia in the mouse carotid arteries.

Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel.

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.

Regulation of exosome release by lysosomal acid ceramidase in coronary arterial endothelial cells: Role of TRPML1 channel.

Lysosomal acid ceramidase (AC) has been reported to determine multivesicular body (MVB) fate and exosome secretion in different mammalian cells including coronary arterial endothelial cells (CAECs). However, this AC-mediated regulation of exosome release from CAECs and associated underlying mechanism remain poorly understood. In the present study, we hypothesized that AC controls lysosomal Ca2+ release through TRPML1 channel to regulate exosome release in murine CAECs. To test this hypothesis, we isolated and cultured CAECs from WT/WT and endothelial cell-specific Asah1 gene (gene encoding AC) knockout mice. Using these CAECs, we first demonstrated a remarkable increase in exosome secretion and significant reduction of lysosome-MVB interaction in CAECs lacking Asah1 gene compared to those cells from WT/WT mice. ML-SA1, a TRPML1 channel agonist, was found to enhance lysosome trafficking and increase lysosome-MVB interaction in WT/WT CAECs, but not in CAECs lacking Asah1 gene. However, sphingosine, an AC-derived sphingolipid, was able to increase lysosome movement and lysosome-MVB interaction in CAECs lacking Asah1 gene, leading to reduced exosome release from these cells. Moreover, Asah1 gene deletion was shown to substantially inhibit lysosomal Ca2+ release through suppression of TRPML1 channel activity in CAECs. Sphingosine as an AC product rescued the function of TRPML1 channel in CAECs lacking Asah1 gene. These results suggest that Asah1 gene defect and associated deficiency of AC activity may inhibit TRPML1 channel activity, thereby reducing MVB degradation by lysosome and increasing exosome release from CAECs. This enhanced exosome release from CAECs may contribute to the development of coronary arterial disease under pathological conditions.

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia.

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1trg/ECcre) and their control littermates (Smpd1trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1trg/ECcre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1trg/ECcre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1trg/ECcre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1trg/ECcre mice than in their control littermates. Using ECs from Smpd1trg/ECcre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1ß levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.

Renomedullary exosomes produce antihypertensive effects in reversible two-kidney one-clip renovascular hypertensive mice.

The rapid fall in blood pressure following unclipping of the stenotic renal artery in the Goldblatt two-kidney one-clip (2K1C) model of renovascular hypertension is proposed to be due to release of renomedullary vasodepressor lipids, but the mechanism has remained unclear. In this study, we hypothesized that the hypotensive response to unclipping is mediated by exosomes released from the renal medulla. In male C57BL6/J mice made hypertensive by the 2K1C surgery, unclipping of the renal artery after 10 days decreased mean arterial pressure (MAP) by 23 mmHg one hr after unclipping. This effect was accompanied by a 556% increase in the concentration of exosomes in plasma as observed by nanoparticle tracking analysis. Immunohistochemical analysis of exosome markers, CD63 and AnnexinII, showed increased staining in interstitial cells of the inner medulla of stenotic but not contralateral control kidney of clipped 2K1C mice. Treatment with rapamycin, an inducer of exosome release, blunted the hypertensive response to clipping, whereas GW-4869, an exosome biosynthesis inhibitor, prevented both the clipping-induced increase in inner medullary exosome marker staining and the unclipping-induced fall in MAP. Plasma exosomes isolated from unclipped 2K1C mice showed elevated neutral lipid content compared to sham mouse exosomes by flow cytometric analysis after Nile red staining. Exosomes from 2K1C but not sham control mice exerted potent MAP-lowering and diuretic-natriuretic effects in both 2K1C and angiotensin II-infused hypertensive mice. These results are consistent with increased renomedullary synthesis and release of exosomes with elevated antihypertensive neutral lipids in response to increased renal perfusion pressure.

Loss of sphingosine kinase 2 protects against cisplatin-induced kidney injury.

Cisplatin is an established chemotherapeutic drug for treatment of solid-organ cancers and is the primary drug used in the treatment of head and neck cancer; however, cisplatin-induced nephrotoxicity largely limits its clinical use. Inhibition of sphingosine kinase 2 (SphK2) has been demonstrated to alleviate various kidney diseases. Therefore, we hypothesized that inhibition of SphK2 could also protect against cisplatin-induced nephrotoxicity. Results from the present study showed that the SphK2 inhibitor ABC294640 or knockdown of SphK2 by siRNA blocked the cisplatin-induced increase of cellular injury markers (neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and cleaved caspase-3) by Western blot analysis in HK-2 cells, a human renal tubular cell line. In addition, SphK2 inhibition blocked cisplatin-induced activation of NF-κB by Western blot analysis and immunostaining analysis. Furthermore, SphK2 inhibition suppressed cisplatin-induced increases of proinflammatory markers (NLR family pyrin domain containing 3, interleukin-1ß, and interleukin-6). Genetic deletion of the SphK2 gene in mice further confirmed that inhibition of SphK2 protected against cisplatin-induced kidney damage in vivo. Compared with wild-type mice, SphK2 knockout mice exhibited less renal dysfunction and reduced promotion of kidney injury markers, inflammatory factors, tubular morphology damage, and fibrotic staining. At the same time, the SphK2 inhibitor ABC294640 failed to interfere with the activity of cisplatin or radiation in two cell culture models of head and neck cancer. It is concluded that inhibition of Sphk2 protects against cisplatin-induced kidney injury. SphK2 may be used as a potential therapeutic target for the prevention or treatment of cisplatin-induced kidney injury.NEW & NOTEWORTHY The present study provides new findings that sphingosine kinase 2 (SphK2) is highly expressed in renal tubules, cisplatin treatment increases the expression of SphK2 in proximal tubular cells and kidneys, and inhibition of SphK2 alleviates cisplatin-induced kidney injury by suppressing the activation of NF-κB, production of inflammatory factors, and apoptosis. SphK2 may serve as a potential therapeutic target for the prevention or treatment of cisplatin-induced nephrotoxicity.

Impairment of Ceramide-Mediated Endothelial Instant Membrane Resealing During Diabetes Mellitus.

Recent studies have indicated that instant cell membrane resealing (ICMR) controls the activation of NOD-like receptor pyrin domain containing 3 (Nlrp3) inflammasomes in endothelial cells, thereby initiating and promoting vascular inflammation. It remains unknown whether this impaired ICMR occurs under diabetic condition or hyperglycemia contributing to endothelial dysfunction leading to vascular inflammation, a hallmark of diabetic vascular injury. The present study aims to examine whether ICMR occurs during in control and diabetic mice and to explore related molecular mechanisms associated with acid sphingomyelinase (ASM)-mediated ceramide production. Using confocal microscopy, we demonstrated that mouse aortic endothelial cells (MAECs) exposed to high glucose levels exhibited much more retarded ICMR after laser-induced membrane injury, compared to that in control cells. The high glucose-induced impairment of membrane resealing in MAECs was prevented when these cells were pretreated with sphingomyelin or C24-ceramide. Mechanistically, high glucose treatment decreased association of membrane ceramide with annexin A5, an essential element of membrane repair machinery. Consistently, the association of ceramide with annexin A5 was significantly reduced in the coronary arterial endothelium of mice with streptozotocin-induced diabetes mellitus compared to that in non-diabetic control mice. Moreover, a marked reduction of the association of ceramide with annexin A5 was observed in coronary arterial endothelium of ASM knockout mice regardless of their diabetic status. Lastly, high glucose treatment or ASM gene deletion substantially impaired ICMR in coronary arterial endothelium of mice receiving membrane puncturing agents. Collectively, our data suggest that ceramide-mediated ICMR in vascular endothelial cells is impaired during diabetes mellitus due to dissociation of ceramide with annexin A5 and ASM play a critical role in this ICMR.

Exosome Biogenesis and Lysosome Function Determine Podocyte Exosome Release and Glomerular Inflammatory Response during Hyperhomocysteinemia.

Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation in podocytes is reportedly associated with enhanced release of exosomes containing NLRP3 inflammasome products from these cells during hyperhomocysteinemia (hHcy). This study examined the possible role of increased exosome secretion during podocyte NLRP3 inflammasome activation in the glomerular inflammatory response. Whether exosome biogenesis and lysosome function are involved in the regulation of exosome release from podocytes during hHcy in mice and upon stimulation of homocysteine (Hcy) in podocytes was tested. By nanoparticle tracking analysis, treatments of mice with amitriptyline (acid sphingomyelinase inhibitor), GW4869 (exosome biogenesis inhibitor), and rapamycin (lysosome function enhancer) were found to inhibit elevated urinary exosomes during hHcy. By examining NLRP3 inflammasome activation in glomeruli during hHcy, amitriptyline (but not GW4869 and rapamycin) was shown to have an inhibitory effect. However, all treatments attenuated glomerular inflammation and injury during hHcy. In cell studies, Hcy treatment stimulated exosome release from podocytes, which was prevented by amitriptyline, GW4869, and rapamycin. Structured illumination microscopy revealed that Hcy inhibited lysosome-multivesicular body interactions in podocytes, which was prevented by amitriptyline or rapamycin but not GW4869. Thus, the data from this study shows that activation of exosome biogenesis and dysregulated lysosome function are critically implicated in the enhancement of exosome release from podocytes leading to glomerular inflammation and injury during hHcy.

Release and Actions of Inflammatory Exosomes in Pulmonary Emphysema: Potential Therapeutic Target of Acupuncture.

BACKGROUND: Exosomes have been reported to mediate activation of the inflammatory response by secretion of inflammasome products such as IL-1ß or IL-18 and that changes in exosomes production or secretion may be a therapeutic target for treatment of a variety of different chronic diseases. The present study tested the hypothesis that exosome-mediated release of NLRP3 inflammasome products instigates the inflammatory response in the lung during emphysema, a type of chronic obstructive pulmonary disease (COPD) and that electroacupuncture (EA) may attenuate emphysema by inhibition of NLRP3 inflammasome activation and consequent inflammation. METHODS: The COPD mice model was developed by injecting porcine pancreatic elastase (PPE) via puncture tracheotomy and instillation. EA (4 Hz/20 Hz, 1 to 3 mA) was applied to the bilateral BL13 and ST36 for 30 min, once every other day for 2 weeks. Micro computed tomography (micro-CT) was performed to measure lung function. Histopathological changes in the lungs were displayed by HE staining. RESULTS: In a mouse model of porcine pancreatic elastase (PPE)-induced emphysema, the lung tissue was found to display several key features of emphysema, including alveolar septal thickening, enlarged alveoli, interstitial edema, and inflammatory cells infiltration. Lungs of mice receiving PPE exhibited substantially increased low attenuation area (LAA) in micro-CT images. The colocalization of NLRP3 vs ASC or caspase-1 detected by confocal microscopy was shown to increase in both bronchial and alveolar walls, indicating the increased formation of NLRP3 inflammasomes. IL-1ß, a prototype NLRP3 inflammasome activating product, was also found to have increased in the lung during emphysema, which was colocalized with CD63 (an exosome marker), an indicative of inflammatory exosome formation. By nanoparticle tracking analysis (NTA), IL-1ß-containing exosomes were shown to significantly increase in the bronchoalveolar lavage (BAL) from mice with emphysema. Therapeutically, IL-1ß production in the lung during emphysema was significantly reduced by EA at the acupoint Feishu (BL13) and Zusanli (ST36), accompanied by decreased colocalization of NLRP3 vs ASC or caspase-1. Increased exosome release into BAL during emphysema was shown to be significantly attenuated in EA-treated mice compared to their controls. However, EA of non-specific BL23 together with ST36 acupoint had no effects on NLRP3 inflammasome activation, exosome release and associated lung pathology during emphysema. CONCLUSION: NLRP3 inflammasome activation in concert with increased release of exosomes containing IL-1ß or other inflammasome products contributes to the development of lung inflammation and injury during PPE-induced emphysema and that EA of lung-specific acupoints attenuates inflammasome activation and exosome release, thereby reducing inflammatory response in the lung of mice with emphysema.

Regulatory role of mammalian target of rapamycin signaling in exosome secretion and osteogenic changes in smooth muscle cells lacking acid ceramidase gene.

Acid ceramidase (murine gene code: Asah1) (50 kDa) belongs to N-terminal nucleophile hydrolase family. This enzyme is located in the lysosome, which mediates conversion of ceramide (CER) into sphingosine and free fatty acids at acidic pH. CER plays an important role in intracellular sphingolipid metabolism and its increase causes inflammation. The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LEs)/lysosomes may control cargo selection, membrane biogenesis, and exosome secretion, which may be fine controlled by lysosomal sphingolipids such as CER. This lysosomal-CER-mTOR signaling may be a crucial molecular mechanism responsible for development of arterial medial calcification (AMC). Torin-1 (5 mg/kg/day), an mTOR inhibitor, significantly decreased aortic medial calcification accompanied with decreased expression of osteogenic markers like osteopontin (OSP) and runt-related transcription factor 2 (RUNX2) and upregulation of smooth muscle 22α (SM22-α) in mice receiving high dose of Vitamin D (500 000 IU/kg/day). Asah1fl/fl /SMCre mice had markedly increased co-localization of mTORC1 with lysosome-associated membrane protein-1 (Lamp-1) (lysosome marker) and decreased co-localization of vacuolar protein sorting-associated protein 16 (VPS16) (a multivesicular bodies [MVBs] marker) with Lamp-1, suggesting mTOR activation caused reduced MVBs interaction with lysosomes. Torin-1 significantly reduced the co-localization of mTOR vs Lamp-1, increased lysosome-MVB interaction which was associated with reduced accumulation of CD63 and annexin 2 (exosome markers) in the coronary arterial wall of mice. Using coronary artery smooth muscle cells (CASMCs), Pi -stimulation significantly increased p-mTOR expression in Asah1fl/fl /SMCre CASMCs as compared to WT/WT cells associated with increased calcium deposition and mineralization. Torin-1 blocked Pi -induced calcium deposition and mineralization. siRNA mTOR and Torin-1 significantly reduce co-localization of mTORC1 with Lamp-1, increased VPS16 vs Lamp-1 co-localization in Pi -stimulated CASMCs, associated with decreased exosome release. Functionally, Torin-1 significantly reduces arterial stiffening as shown by restoration from increased pulse wave velocity and decreased elastin breaks. These results suggest that lysosomal CER-mTOR signaling may play a critical role for the control of lysosome-MVB interaction, exosome secretion and arterial stiffening during AMC.

Overexpression of MicroRNA-429 Transgene Into the Renal Medulla Attenuated Salt-Sensitive Hypertension in Dahl S Rats.

BACKGROUND: We have previously shown that high salt stimulates the expression of miR-429 in the renal medulla, which induces mRNA decay of HIF prolyl-hydroxylase 2 (PHD2), an enzyme to promote the degradation of hypoxia-inducible factor (HIF)-1α, and increases the HIF-1α-mediated activation of antihypertensive genes in the renal medulla, consequently promoting extra sodium excretion. Our preliminary results showed that high salt-induced increase of miR-429 was not observed in Dahl S rats. This present study determined whether correction of this impairment in miR-429 would reduce PHD2 levels, increase antihypertensive gene expression in the renal medulla and attenuate salt-sensitive hypertension in Dahl S rats. METHODS: Lentiviruses encoding rat miR-429 were transfected into the renal medulla in uninephrectomized Dahl S rats. Sodium excretion and blood pressure were then measured. RESULTS: Transduction of lentiviruses expressing miR-429 into the renal medulla increased miR-429 levels, decreased PHD2 levels, and upregulated HIF-1α target gene NOS-2, which restored the adaptive mechanism to increase the antihypertensive gene after high-salt intake in Dahl S rats. Functionally, overexpression of miR-429 transgene in the renal medulla significantly improved pressure natriuretic response, enhanced urinary sodium excretion, and reduced sodium retention upon extra sodium loading, and consequently, attenuated the salt-sensitive hypertension in Dahl S rats. CONCLUSIONS: Our results suggest that the impaired miR-429-mediated PHD2 inhibition in response to high salt in the renal medulla may represent a novel mechanism for salt-sensitive hypertension in Dahl S rats and that correction of this impairment in miR-429 pathway could be a therapeutic approach for salt-sensitive hypertension.

Regulation of TRPML1 channel activity and inflammatory exosome release by endogenously produced reactive oxygen species in mouse podocytes.

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome in podocytes has been implicated in the initiation of glomerular inflammation during hyperhomocysteinemia (hHcy). However, the mechanism by which NLRP3 inflammasome products are released from podocytes remains unknown. The present study tested whether exosome secretion from podocytes is enhanced by NADPH oxidase-produced reactive oxygen species (ROS), which may serve as a pathogenic mechanism mediating the release of inflammatory cytokines produced by the NLRP3 inflammasome in podocytes after Hcy stimulation. We first demonstrated the remarkable elevation of endogenously produced ROS in podocytes treated with Hcy compared with control podocytes, which was abolished by pre-treatment with the NADPH oxidase inhibitors, gp91 ds-tat peptide and diphenyleneiodonium (DPI). In addition, Hcy induced activation in podocytes of NLRP3 inflammasomes and the formation of multivesicular bodies (MVBs) containing inflammatory cytokines, which were prevented by treatment with gp91 ds-tat or the ROS scavenger, catalase. Given the importance of the transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited by endogenously produced ROS in podocytes after Hcy stimulation. By GCaMP3 Ca2+ imaging, we confirmed the inhibition of TRPML1 channel activity by Hcy which was remarkably ameliorated by catalase and gp91 ds-tat peptide. By structured illumination microscopy (SIM) and nanoparticle tracking analysis (NTA), we found that ML-SA1, a TRPML1 channel agonist, significantly enhanced lysosome-MVB interaction and reduced exosome release in podocytes, which were attenuated by Hcy. Pre-treatment of podocytes with catalase or gp91 ds-tat peptide restored ML-SA1-induced changes in lysosome-MVB interaction and exosome secretion. Moreover, we found that hydrogen peroxide (H2O2) mimicked the effect of Hcy on TRPML1 channel activity, lysosome-MVB interaction, and exosome secretion in podocytes. Based on these results, we conclude that endogenously produced ROS importantly contributes to inflammatory exosome secretion from podocytes through inhibition of TRPML1 channel activity, which may contribute to the initiation of glomerular inflammation during hHcy.

Lysosome Function in Cardiovascular Diseases.

The lysosome is a single ubiquitous membrane-enclosed intracellular organelle with an acidic pH present in all eukaryotic cells, which contains large numbers of hydrolytic enzymes with their maximal enzymatic activity at a low pH (pH ≤ 5) such as proteases, nucleases, and phosphatases that are able to degrade extracellular and intracellular components. It is well known that lysosomes act as a center for degradation and recycling of large numbers of macromolecules delivered by endocytosis, phagocytosis, and autophagy. Lysosomes are recognized as key organelles for cellular clearance and are involved in many cellular processes and maintain cellular homeostasis. Recently, it has been shown that lysosome function and its related pathways are of particular importance in vascular regulation and related diseases. In this review, we highlighted studies that have improved our understanding of the connection between lysosome function and vascular physiological and pathophysiological activities in arterial smooth muscle cells (SMCs) and endothelial cells (ECs). Sphingolipids-metabolizingenzymes in lysosomes play critical roles in intracellular signaling events that influence cellular behavior and function in SMCs and ECs. The focus of this review will be to define the mechanism by which the lysosome contributes to cardiovascular regulation and diseases. It is believed that exploring the role of lysosomal function and its sphingolipid metabolism in the initiation and progression of vascular disease and regulation may provide novel insights into the understanding of vascular pathobiology and helps develop more effective therapeutic strategies for vascular diseases.

Podocyte Sphingolipid Signaling in Nephrotic Syndrome.

Podocytes play a vital role in the pathogenesis of nephrotic syndrome (NS), which is clinically characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, and peripheral edema. The pathogenesis of NS has evolved through several hypotheses ranging from immune dysregulation theory and increased glomerular permeability theory to the current concept of podocytopathy. Podocytopathy is characterized by dysfunction or depletion of podocytes, which may be caused by unknown permeability factor, genetic disorders, drugs, infections, systemic disorders, and hyperfiltration. Over the last two decades, numerous studies have been done to explore the molecular mechanisms of podocyte injuries or NS and to develop the novel therapeutic strategies targeting podocytopathy for treatment of NS. Recent studies have shown that normal sphingolipid metabolism is essential for structural and functional integrity of podocytes. As a basic component of the plasma membrane, sphingolipids not only support the assembly of signaling molecules and interaction of receptors and effectors, but also mediate various cellular activities, such as apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation. This review briefly summarizes current evidence demonstrating the regulation of sphingolipid metabolism in podocytes and the canonical or noncanonical roles of podocyte sphingolipid signaling in the pathogenesis of NS and associated therapeutic strategies.

Contribution of podocyte inflammatory exosome release to glomerular inflammation and sclerosis during hyperhomocysteinemia.

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis in response to hyperhomocysteinemia (hHcy). However, it remains unknown how the products of NLRP3 inflammasome in cytoplasm are secreted out of podocytes. In the present study, we tested whether exosome release serves as a critical mechanism to mediate the action of NLRP3 inflammasome activation in hHcy-induced glomerular injury. By various approaches, we found that hHcy induced NLRP3 inflammasome activation and neutrophil infiltration in glomeruli of WT/WT mice. Lysosome-MVB interaction in glomeruli remarkably decreased in WT/WT mice fed with FF diet, leading to elevation of urinary exosome excretion of these mice. Podocyte-derived exosomes containing pro-inflammatory cytokines increased in urine of WT/WT mice in response to hHcy. The release of inflammatory exosomes from podocytes was prevented by Smpd1 gene deletion but enhanced by podocyte-specific Smpd1 gene overexpression (Smpd1 encodes Asm in mice). Pathologically, hHcy-induced podocyte injury and glomerular sclerosis were blocked by Smpd1 gene knockout but amplified by podocyte-specific Smpd1 gene overexpression. Taken together, our results suggest that Asm-ceramide signaling pathway contributes to NLRP3 inflammasome activation and robust release of inflammatory exosomes in podocytes during hHcy, which together trigger local glomerular inflammation and sclerosis.

Role of phosphodiesterase 1 in the pathophysiology of diseases and potential therapeutic opportunities.

Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca2+/calmodulin (CaM), resulting in the integration of Ca2+ and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.

Collecting duct-specific knockout of sphingosine-1-phosphate receptor 1 aggravates DOCA-salt hypertension in mice.

OBJECTIVE: We have previously reported that renal medullary sphingosine-1-phosphate (S1P) regulates sodium excretion via the S1P type-1 receptor (S1PR1). As S1PR1 is predominantly expressed in collecting ducts (CD), the present study tested the hypothesis that the CD-S1PR1 pathway plays a critical role in sodium excretion and contributes to salt-sensitive hypertension. METHODS: CD-specific S1PR1 knockout mice were generated by crossing aquaporin-2-Cre mice with S1PR1-floxed mice. Renal sodium excretion and arterial pressure were compared between wild type and KO mice in response to high-salt challenges and treatment of deoxycorticosterone acetate (DOCA) salt. RESULTS: Protein levels of renal medullary S1PR1 were increased by 100% after high-salt intake, whereas DOCA treatment with high-salt intake blocked the increase of S1PR1 levels. Urinary sodium excretions in knockout mice were decreased by 60% compared with wild type mice after acute intravenous sodium loading (0.84 ±â€Š0.16 vs. 2.22 ±â€Š0.62 µmole/min per g kwt). The pressure natriuresis was impaired in knockout mice compared with wild type mice (4.32 ±â€Š1.04 vs. 8.73 ±â€Š0.19 µmole/min per g kwt). The chronic high-salt intake-induced positive sodium balance was enhanced in knockout mice compared with wild type mice (5.27 ±â€Š0.39 vs. 2.38 ±â€Š1.04 mmol/100 g BW per 24 h). After 10-day DOCA-salt treatment, knockout mice developed more severe hypertension than wild type mice (SBP 142 ±â€Š8 vs. 115 ±â€Š4 mmHg). CONCLUSION: The deletion of CD-S1PR1 reduced sodium excretion, promoted sodium retention, and accelerated DOCA-salt-induced salt-sensitive hypertension, suggesting that the CD-S1PR1 signaling is an important antihypertensive pathway by promoting sodium excretion and that impairment of renal medullary S1PR1 may represent a novel mechanism for salt-sensitive hypertension.