title: Inhibition of hyperhomocysteinemia-induced inflammasome activation and glomerular sclerosis by NLRP3 gene deletion.
abstract: OBJECTIVE
  Hyperhomocysteinemia (hHcys) has been reported to initiate Nod-like receptor protein 3 (NLRP3) inflammasome formation and activation in podocytes, leading to glomerular dysfunction and sclerosis. However, it remains unknown whether Nlrp3 gene is critical for the formation and activation of inflammasomes in glomeruli of hHcys mice.
  
  
  METHODS
  Plasma homocysteine concentration was estimated utilizing HPLC, inflammasome formation and immunofluorescence expression from confocal microscopy, IL-1β production from ELISA.
  
  
  RESULTS
  Uninephrectomized Nlrp3 knockout (Nlrp3(-/-)) and wild type (Nlrp3(+/+)) and intra renal Nlrp3 shRNA-transfected wild type mice (Nlrp3 shRNA) were fed a folate free (FF) diet or normal chow (ND) for 4 weeks to produce hHcys. The plasma Hcys levels were significantly elevated in both Nlrp3(-/-) and Nlrp3(+/+) mice fed a FF diet compared to ND fed mice. The FF diet significantly increased the colocalization of Nlrp3 with apoptosis-associated speck-like protein (ASC) or caspase-1, caspase-1 activity and IL-1β production in glomeruli of Nlrp3(+/+), but not in Nlrp3(-/-) mice and local Nlrp3 shRNA transfected mice. Correspondingly, the glomerular damage index (GDI) and urinary protein excretion were significantly higher in Nlrp3(+/+) mice compared to ND fed mice. However, the hHcys-induced increase in GDI and proteinuria were significantly lower in Nlrp3(-/-) and local Nlrp3 shRNA transfected mice than in Nlrp3(+/+) mice. Immunocytochemical analysis showed that hHcys decreased expression of podocin and nephrin, but increased desmin expression in glomeruli of Nlrp3(+/+) mice compared to Nlrp3(-/-) mice.
  
  
  CONCLUSIONS
  Nlrp3 gene is an essential component of Nlrp3 inflammasomes and that targeting Nlrp3 may be important therapeutic strategy to prevent inflammasome activation and thereby protect podocytes and glomeruli from hHcys-induced injury.
authors.affiliation: Department of Pharmacology and Toxicology, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Xia
authors.forename: Sabena M
authors.initials: SM
authors.lastname: Conley
authors.forename: Guangbi
authors.initials: G
authors.lastname: Li
authors.forename: Pin-Lan
authors.initials: PL
authors.lastname: Li
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Boini
mesh_terms: D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D017931:DNA Primers; D017353:Gene Deletion; D020868:Gene Silencing; D005923:Glomerulosclerosis, Focal Segmental / Q000209:etiology* / Q000235:genetics / Q000473:pathology; D020138:Hyperhomocysteinemia / Q000150:complications / Q000235:genetics* / Q000473:pathology; D058847:Inflammasomes / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; D034741:RNA, Small Interfering