| Summary: | 碩士 === 高雄醫學大學 === 生物化學研究所 === 101 === Diabetes mellitus (DM) is the 5th leading cause in Taiwan in 2012 while diabetic nephropathy (DN) is a common complication of DM. DN is characterized by renal hypertrophy, cell proliferation and extracellular matrices accumulation which results in glomerulosclerosis and renal fibrosis and finally end-stage renal disease. Fibroblast growth factor 23 (FGF23) is phosphaturic because it inhibits renal tubular reabsorption of phosphate. It also inhibits 1??-hydroxylase to inhibit the formation of active 1,25-vitamin D. FGF23 binds to the complex of FGF receptor (FGFR) and klotho to activate signal transduction. Additionally, FGF23 has been associated with chronic kidney disease (CKD) and overexpression of klotho has been found to attenuate animal CKD. Conversely, klotho knockout mice developed features of CKD. However, the roles of FGF23 and klotho in DN remain unknown. Thus, the purpose of this study is to study the roles of FGF23 and klotho in high glucose-cultured normal rat kidney fibroblast (NRK-49F) cells and streptozotocin (STZ)-diabetic rats.
We found that high glucose (30 mM) increased FGF23 and klotho mRNA and protein expression at 3 days while PD98059 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), AG490 (JAK/STAT inhibitor), SB431542 (type 1 TGF-?? receptor inhibitor) and LY294002 (PI3 kinase inhibitor) attenuated high glucose-induced FGF23 and Klotho mRNA and protein expression at 3 days. Exogenous FGF23 and klotho dose-dependently (10 ng to 50 ng and 400 pM to 2 nM, respectively) increased fibronectin, collagen IV, matrix metalloproteinase-9 (MMP-9)and plasminogen activator inhibitor-1 (PAI-1) protein expression at 3 days. Exogeneous FGF23 and klotho also increased TGF-?? transcriptional activity and TGF-???涀ioactivity. FGF23 increased phosphorylations of mammalian target of rapamycin (mTOR), Janus kinase2 (JAK2), signal transducers and activators of transcription3 (STAT3), Akt and Smad2/3. FGF23 increased klotho protein expression whereas klotho (2 nM) attenuated FGF23-induced fibronectin protein expression. Combined treatment of FGF23 (50 ng) and high dose klotho (2 nM) attenuated high glucose-induced extraceuular matrices via attenuating high glucose-induced phosphorylations of mTOR, JAK2, STAT3, Akt, ERK1/2 and Smad2/3. Finally, resveratrol dose-dependently attenuated high glucose-induced FGF23 and klotho protein expression. FGF23 and klotho protein expression increased at 8 weeks in the STZ-diabetic rats whereas resveratrol attenuated fibronectin, FGF23 and klotho protein expression in the STZ-diabetic rats.
We concluded that FGF23 and klotho expression are increased in high glucose-cultured NRK-49F cells and the STZ-diabetic rats. FGF23 or klotho alone were detrimental in high glucose-cultured NRK-49F cells. However, combined treatment of FGF23 and high dose klotho (2 nM) was beneficial. We are going to study the separate and combined effects of resveratrol, FGF23 shRNA and klotho shRNA on high glucose-cultured NRK-49F cells and the STZ-diabetic rats in future studies.
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