Active Site And Mechanism Of Rhubarb In The Treatment Of Chronic Kidney Disease And Renal Fibrosis

Chronic renal disease(CKD)is a public problem with high morbidity and mortality,which is caused by myriad etiologies.Kidney fibrosis is the main pathological hallmark of the CKD and represents the common pathway of nearly all progressive nephropathies regardless of cause.Rhubarb,a natural product,has shown potential efficiency in the clinical treatment of CKD,while the primary active ingredients and its precise mechanism of antifibrotic effect remain to be determined.Additionally,metabolomics plays a prominent role in the clinical diagnosis of various diseases as well as the exploration of pathological mechanisms and the identification of valuable biomarkers since it can provide new insight into the dynamic change of metabolites.Therefore,we aim to clarificate the main active site of rhubarb and how it mitigates renal fibrosis of CKD in combination of metabolomics,which might be exploited for potential pharmaceutical intervention.Methods Forty male Sprague-Dawley rats(200±10 g)were randomly divided into control group(CTL),CKD group,CKD + rhubarb petroleum ether(PE)extract group,CKD + rhubarb ethyl acetate(EA)extract group and CKD + rhubarb n-butyl alcohol(BU)extract group.The latter four groups were given 200 mg/kg/d adenine(containing 1% of acacia),and the CTL group was given equal volume saline.In addition to the original dose of adenine,the rats in the treatment group were respectively given a dose of 800 mg/kg/d of PE extract,200 mg/kg/d of EA extract and 600 mg/kg/d of BU extract after three hours of adenine administration.The rat urine of all groups was respectively collected on the third and sixth weekend.At the end of the 6th week,carotid blood and kidney tissue of each group were collected.Serum and urine were used for the studies of biochemical indexes,metabolomics and lipidomics,while renal tissue was utilized for the studies of metabolomics,pathology as well as immunohistochemistry and western blot analysis.Results(1)The analysis of biochemical indexes: Compared with CTL group,creatinine,urea,triglycerides,cholesterol and uric acid were significantly increased in CKD group.After the treatment of rhubarb PE extract,EA extract and BU extract,creatinine,urea,triglycerides,cholesterol and white blood cells were significantly reduced,while creatinine clearance,red blood cells and hematocrit were significantly increased,indicating rhubarb extracts contributed much to the recovery of renal function.(2)The results of pathological study: Hematoxylin-eosin staining(HE)and periodic acid-schiff stain(PAS)showed that expansive renal tubular lumen and increased inflammatory cell infiltration were obviously observed in CKD group;Masson staining demonstrated that there was marked fibrogenesis in renal tubulointerstitial of CKD group.After the treatment of rhubarb PE extract,EA extract and BU extract,the dysregulation of renal tubular lumen,interstitial fibrosis and inflammatory infiltration was mitigated,indicating rhubarb extracts are promising candidates of pharmaceutical exploitation.Of note,rhubarb EA extract showed the best therapeutic effect,and the following was BU extract.(3)The result of immunohistochemical analysis: The expression of ?-SMA,Collagen I,Fibronectin was significantly increased in the CKD group compared to the CTL group.In addition,p-Smad2,Smad2,p-Smad3,Smad3 and Smad4 were also elevated,while Smad7 was significantly decreased in TGF-?/Smad signaling pathway.After the treatment of rhubarb PE extract,EA extract and BU extract,the abnormal expression of above-mentioned proteins was reversed,among which rhubarb EA and BU extract showed the best therapeutic effect.(4)The result of western blot analysis: Compared with CTL group,the expression of TGF-?1,Fibronectin,?-SMA,Collagen I,Collagen III,PAI-1,Vimentin and FSP1 in CKD group was significantly increased,while the expression of E-cadherin was significantly decreased.The expression of E-cadherin was restored after treating with rhubarb PE extract,EA extract and BU extract and the expression of TGF-?1,Fibronectin,?-SMA,Collagen I,Collagen III,PAI-1,Vimentin and FSP1 was dramatically reduced.(5)The result of urine metabolomics: 28 biomarkers were identified from rat urine,which were closely associated with the metabolism of amino acids,creatinine,as well as purine and pyrimidine.After the treatment of rhubarb PE extract,EA extract and BU extract,the expression of N-acetylcysteine,succinyladenosine,creatinine,deoxyadenosine,histamine,hydroxypyruvic acid,methylcytosine was significantly elevated,while the expression of tryptophan,vinylacetylglycine,3-methyldioxyindole,pyridoxine,pyrimidine and trimethylamine was significantly reduced,suggesting rhubarb extracts retarded CKD by alleviating the abnormal metabolism of amino acids,creatinine,purine and pyrimidine.(6)The results of serum metabolomics and plasma lipidomics: 14 biomarkers were identified from serum metabolites by metabolomics,and 10 biomarkers were derived from plasma metabolites through lipidomics,all of which were intimately associated with the metabolism of fatty acid,nicotinic acid,pyrimidine and arachidonic acid.After treating with rhubarb PE extract,EA extract and BU extract,the expression of 5,6-DHET,glucocerebroside,triglyceride(55:1),D-glucose and pipecolic acid was significantly increased,while the expression of triglyceride(53:0),diacylglycerol(38:9),ceramide trihexoside,palmitic acid,histidinyl-glutamine and niacinamide was significantly decreased,hinting rhubarb extracts had a promising effect on the abnormal metabolism of fatty acid,nicotinic acid,pyrimidine and arachidonic acid.(7)The result of renal tissue metabolomics: 21 biomarkers were separated from renal tissue via metabolomics,and metabolic pathway studies revealed that these biomarkers were deeply implicated in the metabolism of amino acid,purine,pyrimidine,fatty acid,tricarboxylic acid cycle and nicotinic acid.After the treatment of rhubarb PE extract,EA extract and BU extract,the expression of microglobulin(18:3),lysophosphatidylcholine(18:1),stearic acid,palmitic acid,linolenic acid was significantly decreased,while the expression of hexadecanedioic acid and kynurenic acid was significantly increased.Among three rhubarb extracts,hypotaurine was significantly reduced in EA extract,and hydroxybutyric acid,2-methylfuran,uracil were significantly elevated in BU extract.In addition,phenylpyruvic acid and niacinamide were dramatically increased in PE extract,while the expression of vaccenic acid and palmitic acid was significantly decreased.These results indicated that rhubarb extracts could mitigate the abnormal metabolism of amino acid,purine,pyrimidine,fatty acid,tricarboxylic acid cycle and nicotinic acid.Conclusion Pharmacology and metabolomics studies uncovered that PE extract,EA extract and BU extract were the main active sites of rhubarb against CKD,which may be closely associated with the inhibition of TGF-?/Smad signaling pathway and the amelioration of amino acid,creatinine,purine,pyrimidine,lipid and nicotinic acid dysregulation.