Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation

Abstract

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) 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 Podo^cre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podo^cre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podo^cre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1^-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1^trg/Podo^cre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1^-/- mice or podocytes of Podo^cre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1^trg/Podo^cre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). 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. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

Keywords: NLRP3 inflammasome; acid sphingomyelinase; extracellular vesicle; hyperhomocysteinemia; podocyte.

Graphical abstract

Figure 1.

Characterization of in vitro and in vivo transfection of floxed Smpd1 shRNA into podocytes. A: molecular mechanism of the activation of floxed Smpd1 shRNA by cre recombinase. B: daily imaging confirmation of gene transfection by detection of cotransfected luciferase gene expression by using an in vivo imaging system (IVIS). C: representative images showing that transfection of floxed Smpd1 shRNA led to red fluorescent protein (RFP) expression in podocytes from WT/WT mice and green fluorescent protein (GFP) expression in podocytes from Podo^cre mice. D: representative gel documents showing the expression of acid sphingomyelinase (ASM) in podocytes from Podo^cre mice transfected with floxed scrambled shRNA or floxed Smpd1 shRNA. E: summarized data showing that Smpd1 gene silencing significantly decreased protein level of ASM in podocytes from Podo^cre mice (n = 4). F: summarized data showing that Smpd1 gene silencing significantly decreased mRNA level of ASM in podocytes from Podo^cre mice (n = 3–6). G: representative images showing the immunofluorescent staining of GFP expressed in Cre-positive podocytes and ASM in glomeruli of different groups of mice. H: summarized data showing that transfection of floxed Smpd1 shRNA significantly decreased ASM expression in podocytes of both Podo^cre and Smpd1^trg/Podo^cre mice (n = 4). *P < 0.05 vs. the scrambled shRNA (shScra) group. #P < 0.05 vs. the Podo^cre group. Ctrl, control; shSmpd1, floxed Smpd1 shRNA.

Figure 2.

Inhibition of hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation in glomeruli by podocyte-specific Smpd1 gene silencing. A: representative images showing the glomerular expression of cleaved caspase-1 in different groups of mice. B: summarized data showing the glomerular expression of cleaved caspase-1 in different groups of mice (n = 6). *P < 0.05 vs. the WT/WT-normal diet (ND) group. #P < 0.05 vs. the WT/WT-folate-free (FF) group. §P < 0.05 vs. the Podo^cre-FF-scrambled shRNA (shScra) group. †P < 0.05 vs. the Smpd1^trg/Podo^cre-FF-shScra group. Cle-Casp-1, cleaved caspase-1; shSmpd1, floxed Smpd1 shRNA.

Figure 3.

Attenuation of hyperhomocysteinemia (hHcy)-induced IL-1β production in glomeruli by podocyte-specific Smpd1 gene silencing. A: representative images showing the glomerular expression of IL-1β in different groups of mice. B: summarized data showing the glomerular expression of IL-1β in different groups of mice (n = 6). *P < 0.05 vs. the WT/WT-normal diet (ND) group. #P < 0.05 vs. the WT/WT-folate-free diet (FF) group. §P < 0.05 vs. the Podo^cre-FF-scrambled shRNA (shScra) group. †P < 0.05 vs. the Smpd1^trg/Podo^cre-FF-shScra group. shSmpd1, floxed Smpd1 shRNA.

Figure 4.

Reduction of urinary extracellular vesicle (EV) excretion by Smpd1 gene silencing in podocytes during hyperhomocysteinemia (hHcy). A: representative graphs showing urinary excretion of particles of different groups of mice detected by nanoparticle tracking analysis (NTA). B: summarized data showing urinary excretion of particles sized between 50 and 140 nm of different groups of mice (n = 7–10). *P < 0.05 vs. the WT/WT-normal diet (ND) group. #P < 0.05 vs. the WT/WT-folate-free diet (FF) group. §P < 0.05 vs. the Podo^cre-FF-scrambled shRNA (shScra) group. †P < 0.05 vs. the Smpd1^trg/Podo^cre-FF-shScra group. shSmpd1, floxed Smpd1 shRNA.

Figure 5.

Prevention of immune cell infiltration into glomeruli by podocyte-specific Smpd1 gene silencing during hyperhomocysteinemia (hHcy). A: representative images showing the immunofluorescent staining of CD8 in glomeruli of different groups of mice. B: summarized data showing the levels of CD8 in glomeruli of different groups of mice (n = 5). *P < 0.05 vs. the WT/WT-normal diet (ND) group. #P < 0.05 vs. the WT/WT-folate-free diet (FF) group. §P < 0.05 vs. the Podo^cre-FF-scrambled shRNA (shScra) group. †P < 0.05 vs. the Smpd1^trg/Podo^cre-FF-shScra group. shSmpd1, floxed Smpd1 shRNA.

Figure 6.

Abolishment of hyperhomocysteinemia (hHcy)-induced glomerular injury by Smpd1 gene silencing in podocytes. A: urinary protein excretion of different groups of mice (n = 7–10). B: urinary albumin excretion of different groups of mice (n = 5–8). C: representative images showing the glomerular morphological changes of different groups of mice. D: summarized data showing the glomerular damage index of different groups of mice (n = 6). *P < 0.05 vs. the WT/WT-normal diet (ND) group. #P < 0.05 vs. the WT/WT-folate-free diet (FF) group. §P < 0.05 vs. the Podo^cre-FF-scrambled shRNA (shScra) group. †P < 0.05 vs. the Smpd1^trg/Podo^cre-FF-shScra group. shSmpd1, floxed Smpd1 shRNA.

Figure 7.

Homocysteine (Hcy)-induced elevation of extracellular vesicle (EV) release from podocytes inhibited by Smpd1 gene silencing. A: summarized data showing EV release from different groups of podocytes (n = 6). *P < 0.05 vs. the WT/WT-vehicle (Vehl) group. #P < 0.05 vs. the WT/WT-Hcy group. §P < 0.05 vs. the Podo^cre-Hcy-scrambled shRNA (shScra) group. †P < 0.05 vs. the Smpd1^trg/Podo^cre-Hcy-shScra group. Ctrl, control; shSmpd1, floxed Smpd1 shRNA. B: summarized data showing EV release from different groups of podocytes (n = 5–12). *P < 0.05 vs. the control (Ctrl) group. #P < 0.05 vs. the Vehl-Hcy group. AMI, amitriptyline; GLY, glycyrrhizin.

Figure 8.

Recovery of lysosome-multivesicular body (MVB) interaction in homocysteine (Hcy)-treated podocytes by acid sphingomyelinase (ASM) inhibitor. A: representative images showing the colocalization of Rab7a and Lamp-1 in different groups of podocytes. B: summarized data showing the colocalization of Rab7a and Lamp-1 in different groups of podocytes (n = 4). *P < 0.05 vs. the control (Ctrl) group. #P < 0.05 vs. the vehicle (Vehl)-Hcy group. AMI, amitriptyline; GLY, glycyrrhizin.

Figure 9.

Homocysteine (Hcy)-induced release of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome products mediated by extracellular vesicles (EVs). A: representative gel documents and summarized data showing the levels of GM130 in podocytes and EVs (n = 4). B: representative gel documents and summarized data showing the levels of CD63 in podocytes and EVs (n = 4). *P < 0.05 vs. the podocyte group. C: summarized data of the measurement of CD63 in EVs isolated from culture medium of podocytes under control condition or treated with Hcy for 24 h by ELISA (n = 8). D: summarized data of the measurement of IL-1β in EVs isolated from culture medium of podocytes under control condition or treated with Hcy for 24 h by ELISA (n = 7). E: summarized data of the measurement of IL-18 in EVs isolated from culture medium of podocytes under control condition or treated with Hcy for 24 h by ELISA (n = 6). *P < 0.05 vs. the control (Ctrl) group.

Figure 10.

Podocyte injury induced by podocyte-derived inflammatory extracellular vesicles (EVs). A: representative gel documents showing the expression of podocin in podocytes of different groups. B: summarized data showing the expression of podocin in podocytes of different groups (n = 6). C: representative gel documents showing the expression of desmin in podocytes of different groups. D: summarized data showing the expression of desmin in podocytes of different groups (n = 6). *P < 0.05 vs. the control (Ctrl) EV group. #P < 0.05 vs. the Ctrl group. Inflam EV, inflammatory EV.

Figure 11.

Glomerular injury and immune cell infiltration induced by podocyte-derived inflammatory extracellular vesicles (EVs). A: representative images showing the immunofluorescent staining of podocin in glomeruli of different groups of mice. B: summarized data showing the expression of podocin in glomeruli of different groups of mice (n = 4). C: representative images showing the immunofluorescent staining of CD8 in glomeruli of different groups of mice. D: summarized data showing the levels of CD8 in glomeruli of different groups of mice (n = 4). *P < 0.05 vs. the control (Ctrl) EV group. Inflam EV, inflammatory EV.