Background
Methods
Cells culture and treatment
Western blotting
RNA isolation and qPCR
Gene | Forward primer | Reverse primer |
---|---|---|
hIL-1β | 5’-CTGAGCTCGCCAGTGAAATG-3’ | 5’-TGTCCATGGCCACAACAACT-3’ |
rIL-1β | 5’-CAGCAGCATCTCGACAAGAG-3’ | 5’-CATCATCCCACGAGTCACAG-3’ |
hNLRP3 | 5’-AAGGCCGACACCTTGATATG-3’ | 5’-CCGAATGTTACAGCCAGGAT-3’ |
rNLRP3 | 5’-GTAGGTGTGGAAGCAGGACT-3’ | 5’-CTTGCTGACTGAGGACCTGA-3’ |
hcaspase 1 | 5’-CTCAGGCTCAGAAGGGAATG-3’ | 5’-CGCTGTACCCCAGATTTTGT-3’ |
rcaspase 1 | 5’-CCGTGGAGAGAAACAAGGAG-3’ | 5’-GGACAGGATGTCTCCAGGAC-3’ |
rASC | 5’-TGGCTACTGCAACCAGTGTC-3’ | 5’-GGCTGGAGCAAAGCTAAAGA-3’ |
hGAPDH | 5’-CCAGGTGGTCTCCTCTGA-3’ | 5’-GCTGTAGCCAAATCGTTGT-3’ |
rGAPDH | 5’-GCAAGTTCAACGGCACAG-3’ | 5’-GCCAGTAGACTCCACGACAT-3’ |
Establishment of early-stage CKD animal model
Kidney histopathological examination
Detection of serum creatinine and UA
Influence of UA on the production of ROS
Measurements of IL-1β, TNF-α, and ICAM-1 by ELISA
Statistical analysis
Results
UA induced the expression of IL-1β and ICAM-1 in HUVECs
UA induced the expression of NLRP3 complexes and activation of NLRP3 inflammasome in HUVECs
NLRP3 siRNA or inhibitor of caspase 1 affected the components of NLRP3 inflammasome and the expression of downstream cytokines
UA regulated the activation of NLRP3 inflammasome by activation of ROS and K+ efflux
UA induced the vascular endothelial injury of early stage CKD by activating NLRP3/ IL-1β pathway
Group | n | Creatinine (μM) | UA (μM) |
---|---|---|---|
Sham | 5 | 30.4 ± 7.2 | 54.8 ± 9.4 |
CKD | 5 | 31.9 ± 4.8 | 55.9 ± 6.1 |
CKD + OXO | 5 | 31.2 ± 5.7 | 178.5 ± 8.4* |
CKD + OXO + ALLO | 5 | 30.7 ± 7.7 | 74.9 ± 6.1# |
Cytokines | Sham | CKD | CKD + OXO | CKD + OXO + ALLO |
---|---|---|---|---|
IL-1β (pg/mL) | 42.38 ± 7.96 | 45.72 ± 8.13 | 88.73 ± 5.64* | 47.25 ± 6.14# |
TNF-a (ng/mL) | 163.70 ± 10.83 | 174.20 ± 11.24 | 293.10 ± 12.31* | 183.60 ± 14.28# |
ICAM-1 (ng/mL) | 284.10 ± 22.64 | 311.50 ± 27.83 | 592.18 ± 98.20* | 328.70 ± 76.12# |