ROCK1 Mediates The Pathogenesis Of Protein-energy Wasting Caused By Chronic Kidney Disease

Objective:1.To investigate the mechanism of ROCK1 stimulating chronic kidney disease-induced muscle atrophy.2.To study whether ROCK1 inhibitor could turnover chronic kidney disease-induced muscle atrophy.Methods:1.30 male C57BL/6 mice,8w,20-25 g,divided into 4 groups:?1?normal control group?CTL?,?2?fasudil group?Fasudil?,?3?chronic renal failure group?CKD?,?4?chronic renal failure+fasudil group?CKD+F?;24 pair-feeded wild-type and knockout mice,male,8-week-old,divided into 4 groups:?1?ROCK1^+/+ sham group,5 mice,?2?ROCK1^-/-sham group,5 mice,?3?ROCK1^+/+ CKD group,7 mice,?4?ROCK1^-/-CKD group,7 mice.After 2 weeks of adaptive feeding normal diet,5/6 of kidney of the last 2 groups was removed.After 8 weeks later,serum creatinine,urea nitrogen of all the mice was measured.Fasudil group and CKD+F group were given a intraperitoneal injection of fasudil 50mg/?kg·d?lasting for 6 weeks.Venous blood and muscles were collected when mice were executed.2.C2C12 cells were cultured with DMEM contained with10% fetal bovine serum?FBS?and 1% P-S.When the cells covered about 80% of the six-well plate area,medium was changed to DMEM contained with 2% horse serum?HS?.M-Fv-Casp3,AP20187 and Fasudil?100?g / ml?were added into the cells after they were differentiated to myotubes and the treatment were lasting for 48,24 and 24 hours respectively.Then the cells were scraped and extracted for protein,mRNA or other kinds of samples.3.Morphology: anti-laminin immunofluorescence staining to measure the cross sectional area of TA muscle and the expression of ROCK1;Western blot,co-immunoprecipitation and ELISA: examine the activity of PTEN,ROCK1,Rho A,PI3 K,Akt and the protein levels of p-MYPT,p-GSK3?,GAPDH;RT-PCR: detect the mRNA levels of Atrogin-1 and MuRF-1.Protein degradation using isotope: measure the degradation rate of extensor digitorum longus and soleus.4.Statistics of all the results were analyzed by SPSS 19.0.Results:1.CKD reduces PI3 K activity but the level of activated Akt is more lower accompanied with PTEN activation increasing: In CKD muscle,the activity of PI3 K and Akt were all supressed?P<0.05,P<0.01?,but decreasing of Akt was more notable?P<0.01?.And PTEN activation was increased 2.3 fold contrasted with control group?P<0.05?.2.ROCK1 is activated in the muscle of CKD mice: cleaved fragment of ROCK1 was increased in CKD mice.ROCK1 activity increased 2.6 fold?P<0.01?.Activity of Rho A GTPase was not different with control group?P>0.05?.3.ROCK1 knockout prevents CKD-induced muscle atrophy in mice: there was no difference of myofiber distribution in ROCK1^+/+ sham group and ROCK1^-/-group.But there was a right shift of myofiber distribution in ROCK1^-/-CKD group compared with ROCK1^+/+ CKD group.Difference of protein degradation rate in ROCK1^+/+ sham group and ROCK1^-/-sham group was not significant?P>0.05?.In contrast to ROCK1^+/+ CKD group,the protein degradation rate of ROCK1^-/-CKD group was significantly decreased?P<0.05?.Compared with ROCK1^+/+ CKD group,mRNA levels of Atrogin-1 and MuRF-1 in ROCK1^-/-CKD group were decreased?P<0.01?,while Akt activity increased,but PTEN suppressed?P<0.01?.4.Activated ROCK1 diminishes Akt through PTEN: In C2C12 cells,after active ROCK1 with caspase-3,activity of PTEN was increased?P<0.01?and while the activation of Akt was decreased.Fasudil could diminish the activation of PTEN?P<0.05?and actived Akt.5.Fasudil,a ROCK1 inhibitor prevents CKD-induced muscle wasting: grip strength of CKD group was decreased compared with control group?P<0.05?.We discovered that fasudil attenuates CKD-induced muscle loss through weighing the muscle mass of tibialis anterior muscle,extensor digitorum longus and soleus?P<0.05?as well as the body weight?P<0.05?.Fasudil can also increased the cross-sectional area of myofiber?P<0.05?.What is more,fasudil can stimulate the phosphorylation level of Akt?P<0.05?and suppress the mRNA level of Atrogin-1 and MuRF-1?P<0.01?.Conclusions:1.ROCK1 activation mediates CKD-induced muscle wasting through inducing PTEN activation and then deducing Akt activity.2.Fasudil can attenuate CKD-induced muscle atrophy which provides a basis for clinical therapy.