Study On The Mechanism Of Nebivolol Improving Renal Interstitial Fibrosis In UUO Rats

Objective:To explore the mechanism of nebivolol in improving renal interstitial fibrosis(RIF)in rats with unilateral ureteral obstruction(UUO)by bioinformatics analysis.Methods:Based on the previous experimental results of our group,bioinformatics analysis was conducted to screen the differential expressed proteins(DEPs)contrary regulated between UUO/Sham and Neb/UUO.Western blot was used to verify the key proteins.Results:Compared with UUO/Sham group,nebivolol reversed 179 DEPs,including 135 upregulated proteins in UUO/sham group but down-regulated proteins in Neb/UUO group;44DEPs down-regulated in UUO/Sham but up-regulated in Neb/UUO.KEGG enrichment analysis showed that the DEPs were mainly enriched in the spliceosome and RNA polymerase pathways.PPI network showed that the DEPs were clustered into five clusters related to spliceosome,cell proliferation and differentiation,actin,endoplasmic reticulum and ribosome assembly.The DEPs contrary altered between the UUO/Sham and Neb/UUO group with degree ≥ 9 were: Cct2,Rbm8 a,Phf5a,Nop58,Actb,Srsf9,Hnrnpr,Pfdn5,Polr2 a,Sart1.Western blot confirmed that Rbm8 a,Srsf9,and Sart1 protein expressions were consistent with the bioinformatics analysis.Conclusion:Our results indicate that the spliceosome may play key role in the amelioration of nebivolol on RIF in UUO rats.Objective:To investigate whether nebivolol(10mg/kg/day,ig,21d)ameliorates RIF in UUO rats by the inhibiting renal endoplasmic reticulum stress(ERS).Methods:(1)Animal experiment:Male SD rats weighted about 220 g were anesthetized by 0.3% sodium pentobarbital(10 ml/kg,ip),and then the abdomen was opened.The left ureter was visualized and slowly separated with a cotton stick dipped in normal saline and ligated twice at 0.5 cm apart to establish UUO rat model.For Sham group,the left ureter was only exposed and separated with normal saline cotton swab without ligation.Rats were divided into Sham group(rats treated with equal volume of distilled water,ig);UUO group(UUO rats treated with equal volume of distilled water,ig);Neb group(UUO rats treated with nebivolol 10 mg/kg/day,ig);At group(UUO rats treated with atenolol 100 mg/kg/day,ig).Based on our preliminary study,the experiment lasted for 21 d.At the end of the experiment,the rats were anesthetized with 0.3% sodium pentobarbital(10 ml/kg,ip),blood samples were collected from the abdominal aorta and centrifuged to obtain serum creatinine(Scr)and blood urea nitrogen(BUN).Then the left kidney was separated,photographed and weighed.The kidney was divided into two along the largest section,and part of the kidney tissue was flash frozen in liquid nitrogen and stored in a refrigerator at-80 ° C: The expressions of α-SMA and ERS-related proteins(ATF6,GRP78,CHOP,IRE1α,PERK)were detected by Western blot.Paraffin blocks were sectioned for HE and Masson staining.(2)Cell experiments:1.Effects of TM(tunicamycin)with different concentrations on the activity of NRK-52E(rat renal tubular epithelial cells).TM(0.01,0.05,0.1,0.2 μmol/L)were used to stimulated NRK-52 E for 48 hours,and the cell survival rate was measured by CCK8 method.2.To establish a ERS model in NRK-52 E induced by TM.NRK-52 E cells were stimulated with TM(0.01,0.05 μmol/L)for 48 h,and ER-associated protein(ATF6)was detected by western blot to determine the appropriate concentration of ERS induced by TM.3.Effects of different concentrations of nebivolol on α-SMA protein expression in ERS model in NRK-52 E cells induced by TM.NRK-52 E cells were divided into the following groups: control group;TM group(TM 0.05 μmol/L);different concentration nebivolol groups(nebivolol 1,3,5,10 μmol/L were cultured with cell 1.5h before the stimulation by TM 0.05 μmol/L).After cultured for 48 h,protein expression of α-SMA was measured by western blot.4.Effects of nebivolol and TUDCA(endoplasmic reticulum stress inhibitor)on protein expressions of α-SMA and ERS related protein expression in ERS model in NRK-52 E cells induced by TM.NRK-52 E cells were divided into the following groups: control group;TM group(TM 0.05 μmol/L);TUDCA group(TUDAC 500 μg/ml was cultured with cell 1.5h before the stimulation by TM 0.05 μmol/L);Neb group(nebivolol 1 μmol/L was cultured with cell 1.5h before the stimulation by TM 0.05 μmol/L).After cultured for48 h,the protein expressions of α-SMA and ERS related proteins(ATF6,GRP78,CHOP,IRE1α,PERK)were detected by Western blot.Results:(一)Results of animal experiments1.Compared with Sham group,the renal cortex of the obstructed kidney in UUO rats was thinner,and the lumen of the renal tubules was larger(HE staining).The renal interstitial fibrosis index of the obstructed kidney from UUO rat was significantly increased(P<0.001)with elevated protein expression of α-SMA(P<0.001,Western blot).In UUO rats,Scr tended to increase,while BUN was significantly increased(P<0.001).Nebivolol 10 mg/kg/day and atenolol 100 mg/kg/day administered for 21 days had no significant effect on Scr and BUN in UUO rats.However,nebivolol significantly improved renal interstitial fibrosis and reduced RIF index and α-SMA protein expression in UUO rats(P<0.05),while atenolol had no significant effect.2.The protein expressions of ATF6,GRP78,CHOP,IRE1α and PERK were significantly increased in UUO rats compared with Sham rats(P<0.05).Nebivolol reduced these ERS related proteins above in UUO rats(P<0.05),while atenolol had no significant effects.(二)Results of cell experiments1.Results of CCK8 indicated that TM 0.01 and 0.05μmol/L had no significant effect on the cell viability of NRK-52 E cells.2.Western blot results showed that NRK-52 E cells stimulated with TM 0.05μmol/L for 48 h significant increased ATF6 protein expression.Therefore,0.05 μmol/L was the optimal concentration of TM to induce ERS in NRK-52 E cells.3.The increased protein expression of α-SMA in NRK-52 E ERS stimulated by TM0.05 μmol/L was reduced by nebivolol(1,3,5,10 μmol/L).Next,nebivolol 1 μmol/L was used for subsequent experiments..4.Nebivolol(1 μmol/L)diminished the protein expressions of α-SMA,ATF6,GRP78,CHOP,IRE1α and PERK in NRK-52 E cells stimulated by TM(P<0.05),which were similar with the results of TUDCA group(P<0.05).Conclusion:Nebivolol(10 mg/kg/day)administration for 21 days significantly ameliorated RIF in UUO rats through inhibiting ERS.