[Effect of spironolactone on the expression of Toll-like receptor 4 in renal tubular epithelia cells exposed to high glucose].
OBJECTIVE
To study the expression of Toll-like receptor 4 (TLR4) in renal tubular epithelial cells exposed to high glucose and the effect of spironolactone on the TLR4 expression.


METHODS
In vitro renal tubular epithelial cells (NRK-52E) were randomly exposed to DMEM culture solution with low glucose (5 mmol /L), high glucose (25 mmol/L) or 10(-7) mol/L spironolactone plus 25 mmol/L glucose. Immunohistochemistry, RT-PCR and Western blot were used to determine TLR4 protein and mRNA expression. The levels of IL-6 and TNF-alpha in the cell culture supernatant were determined using ELISA.


RESULTS
The expression of TLR4 mRNA in the high glucose group began to increase 6 hrs and remained at a higher level up to 24 hrs after exposure as compared with the low glucose group. The TLR4 mRNA expression in the spironolactone treatment group was significantly lower than that in the high glucose group, although it was higher than that in the low glucose group between 6 and 24 hrs after exposure. TLR4 protein expression increased significantly in the high glucose group 24 and 48 hrs after exposure compared with that in the low glucose group. The TLR4 protein expression in the spironolactone treatment group was lower than that in the high glucose group, but higher than that in the low glucose group. IL-6 and TNF-alpha expression in the supernatant from the NRK-52E cells in the high glucose groups increased significantly as compared with the low glucose group. The spironolactone treatment group had significantly reduced IL-6 and TNF-alpha expression compared with the high glucose group.


CONCLUSIONS
High glucose triggers an increase in the expression of TLR4 and inflammatory factors in NRK-52E cells. TLR4 may participate in the progress of diabetic nephropathy. Spironolactone can reduce expression of TLR4 and inflammatory factors, which might be attributed to one of the mechanisms of protection by spironolactone against diabetic nephropathy.
Liu
Kang-Han
KH
Kidney Disease and Blood Purification Research Center/Department of Nephropathy, Xiangya Hospital of Central South University, Changsha 410008, China.
Zhou
Qiao-Ling
QL
Ao
Xiang
X
Tang
Tian-Feng
TF
Hong
Xue-Min
XM
Bao
Rui-Lan
RL
D002478:Cells, Cultured; D003928:Diabetic Nephropathies / Q000209:etiology*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism*; D007150:Immunohistochemistry; D015850:Interleukin-6 / Q000032:analysis; D007684:Kidney Tubules / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D013148:Spironolactone / Q000494:pharmacology*; D051197:Toll-Like Receptor 4 / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D015850:Interleukin-6; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D013148:Spironolactone