Mechanism Of Roxadustat Improving Renal Interstitial Fibrosis By Reducing Oxidative Stress By Regulating AMPK/PGC-1α Pathway

Background:Renal fibrosis is a common pathological feature of chronic kidney disease(CKD)caused by various causes.Although there have been numerous studies on renal fibrosis,there is no safe and effective strategy to successfully solve renal fibrosis.Increased oxidative stress resulting from overproduction of ROS and insufficient antioxidants is a common cause of various kidney injuries.AMP dependent protein kinase(AMPK)and its downstream target protein peroxisome proliferator activated receptor-γ coactivation factor-1α(PGC-1α)plays an important role in oxidative metabolism.AMPK activation can reduce oxidative stress and inflammation,PGC-1α can fight ROS by regulating mitochondrial biogenesis,respiration and cell defense system.AMPK activation and PGC-1α reduce in renal fibrosis.Therefore,upregulation of AMPK/PGC-1α signaling pathway to reduce ROS production may alleviate the damage of tissue cells caused by oxidative stress.Roxadustat(FG-4592),as a new type of oral hypoxia-inducible factor stabilizer,has been used in clinical treatment of renal anemia in China.At present,it has shown good effectiveness and acceptable safety in basic research and clinical application.In addition to the treatment of renal anemia,studies have found that roxadustat can play an organ protective role by improving oxidative stress in Parkinson’s disease,spinal cord injury and other diseases.In addition,in recent years,there have been a few studies on roxadustat and renal fibrosis,but the relationship between them is still controversial and the specific mechanism of action is not completely clear,which needs further exploration.Therefore,we propose a scientific hypothesis that roxadustat may increase the AMPK/PGC-1α pathway improves oxidative stress and alleviates renal fibrosis.Purpose:1.Build the model of renal interstitial fibrosis in vivo and in vitro,and determine the effect of roxadustat on renal interstitial fibrosis.2.Discuss whether roxadustat can adjust AMPK/PGC-1α to reduce oxidative stress and improve renal interstitial fibrosis.Methods:1.Establish a mouse Unilateral Uretera Obstruction(UUO)model,and intraperitoneal injection of roxadustat for intervention.The experiment was divided into sham operation group(sham group),UUO group,UUO+roxadustat treatment group(10mg/kg/d was administered consecutively on the day of operation,and the last administration was the day before kidney removal).Samples were collected on the 3rd,7th and 14 th day after operation,and renal pathological changes were evaluated by HE and Masson staining.Via TGF-β1 induced HK2 cell fibrosis and established cell fibrosis model.The experiment was divided into normal control group and TGF-β1group,roxadustat,TGF-β1+roxadustat group.The cytotoxicity of roxadustat was detected by Cell Counting Kit-8,RT-q PCR,Western Blot and immunohistochemistry were used to detect and observe the changes of fibrosis indicators.2.Build cell fibrosis model,and divide the experiment into normal control group and TGF-β1 group,roxadustat,TGF-β1+ roxadustat group,NAC group,TGF-β1+NAC group,Compound C group,TGF-β1+roxadustat+Compound C group.The cytotoxicity of antioxidant NAC and AMPK inhibitor Compound C was detected by Cell Counting Kit-8.Detection of fibrosis index by RT-q PCR and Western Blot,and AMPK/PGC-1α Expression of pathway related proteins by Western Blot.DCFH-DA,thiobarbituric acid colorimetry and WST-8 were used to detect the changes of oxidative stress indicators.Results:Part I1.Establishment of mouse UUO model: the kidney of Sham group had no obvious change.After UUO operation,as time goes by,the disease progresses,the ureter on the operation side gradually expands,and the renal cortex gradually becomes thinner due to the urine extrusion,indicating that the modeling is successful.2.Effect of roxadustat on renal histomorphology: renal histopathology staining indicated that with the passage of time after UUO operation,the infiltration of inflammatory cells increased,and the renal damage gradually increased.Roxadustat treatment can reduce the infiltration of inflammatory cells in the kidney after UUO operation,and reduce kidney damage.3.Effect of roxadustat on fibrosis indicators: Masson staining,immunohistochemistry staining,RT-q PCR and Western Blot results of renal tissue showed that renal interstitial fibrosis was gradually aggravated after UUO operation compared with sham group,and roxadustat treatment could reduce the degree of renal interstitial fibrosis after UUO operation,with a statistically significant difference compared with the corresponding UUO group(P<0.05).At the cellular level,TGF-β1 increases the fibrosis indexes Collagen I and α-SMA expression.Roxadustat treatment reduces TGF-β1 induced Collagen I and α-SMA increased(P<0.05).It is suggested that roxadustat can inhibit renal interstitial fibrosis.Part II1.The relationship between oxidative stress and renal interstitial fibrosis and the effect of roxadustat on oxidative stress:After UUO,the level of MDA increased and the level of T-SOD decreased(P<0.05).Compared with the corresponding UUO group,roxadustat treatment reduced the MDA level and increased the T-SOD level after UUO operation,with a statistically significant difference(P<0.05).When HK2 cells were fibrotic,the levels of ROS and MDA,indicators of oxidative stress,increased,and the levels of T-SOD decreased(P<0.05).Antioxidant NAC can significantly reduce ROS and MDA levels and increase T-SOD levels.In addition,the fibrosis indicators Collagen I and α-SMA decreases.The results suggest that oxidative stress increases in renal interstitial fibrosis,while decreasing oxidative stress can inhibit renal fibrosis.Roxadustat treatment can significantly reduce ROS and MDA levels during cell fibrosis,and increase T-SOD levels.To sum up,combined with the first part,the results show that roxadustat can inhibit renal interstitial fibrosis by reducing oxidative stress.2.The relationship between the AMPK/PGC-1α pathway and oxidative stress and renal interstitial fibrosis:After UUO operation,compared with sham group,p-AMPK and PGC-1α in UUO group at lower level,suggesting that the AMPK/PGC-1α pathway is inhibited in renal interstitial fibrosis.At the cellular level,the level of p-AMPK and PGC-1α in HK2 cells induced by TGF-β1 is decreases.AMPK inhibitor Compound C intervention can reduce p-AMPK and PGC-1α horizontal,increase ROS and MDA levels and reduce T-SOD levels,increased fibrosis indicators Collagen I and Expression of α-SMA.It suggests that the AMPK/PGC-1α pathway is inhibited.The inhibition of this pathway,oxidative stress and renal interstitial fibrosis increased.3.The relationship of roxadustat and AMPK/PGC-1α signal pathway:Roxadustat treatment elevated p-AMPK and PGC-1α after UUO,compared with the corresponding UUO group,the difference was statistically significant(P<0.05).Roxadustat increases p-AMPK and PGC-1α level in fibrosis HK2 cells(P<0.05).Compound C intervention can attenuate the increase of roxadustat in p-AMPK and PGC-1α,reduce oxidative stress and inhibit renal interstitial fibrosis.It is suggested that the inhibition effect of roxadustat on renal interstitial fibrosis may be attributed to its effect on AMPK/PGC-1α of pathways regulation.Conclusion:1.Roxadustat can improve renal interstitial fibrosis2.AMPK/PGC-1α pathway and oxidative stress play an important role in renal interstitial fibrosis.3.Roxadustat can up-regulate AMPK/PGC-1α pathway to reduce oxidative stress and thus inhibit renal interstitial fibrosis.