| Background and objective:Diabetic nephropathy(DN)is one of the most common microvascular complications caused by diabetes mellitus(DM),chronic kidney disease(CKD)and end-stage renal disease.The pathogenesis of DN is still not fully understood,and recent studies have found that mitochondrial dysfunction may play a key role in the development and progression of DN.To date,the association between mitochondrial dysfunction,energy metabolism disorders and the risk of developing DN has rarely been reported at home and abroad.Fucoidan is a water-soluble polysaccharide that exerts its biological activities such as antioxidant,antithrombotic,and anti-inflammatory by modulating the gut microbiota structure.However,the mechanism of renoprotective effects of Fucoidans on DN is still unclear.Therefore,this study was intended to include patients with diabetes and diabetic nephropathy,as well as a normal control population,to detect,analyze the serum levels of tricarboxylic acid cycle metabolites in patients and their association with DN,and explore the role of mitochondrial dysfunction and energy metabolism disorders in the development of DN;A 24 week high-fat diet induced early DN mouse model was established,and different doses of fucose were used for intervention to explore the renoprotective effect and mechanism of fucose on DN mice.Research could provide a scientific basis for clarifying the role of mitochondrial dysfunction in the development of diabetic nephropathy and exploring nutritional improvement measures for mechanistic associations.Methods:In this study,a case-control study was conducted,and patients with confirmed diagnosis of DM and DN who visited a city level hospital in Qingdao from January 2017 to December2018 were included,and those who were concluded to be healthy by routine physical examination in this hospital during the same period were included as the normal control population(CON)in this study.A questionnaire survey was used to collect the general demographic characteristics of the study subjects.Blood samples were collected from study subjects,and tricarboxylic acid(TCA)cycle intermediate metabolites were measured in the serum of study subjects by HPLC tandem mass spectrometry.Multivariate logistic regression was used to analyze the relationship between TCA cycle intermediate metabolites and the risk of DN development,and receiver operating characteristic curve(ROC)was used to calculate the area under the curve(AUC)to preliminarily explore the predictive value of TCA cycle intermediate metabolites for the occurrence of DN(ChapterⅠ).Ninety 8-week-old male C57BL/6J mice were selected and randomly divided into six groups according to body weight,with 15 mice in each group:control(NC,0.5%sodium carboxymethylcellulose solution+10%kcal diet,D12450J),model(DM,0.5%sodium carboxymethylcellulose solution+60%kcal high-fat diet,D12492),low-dose fucopolysaccharide intervention(LFC,50 mg/kg fucopolysaccharide+high-fat diet,d12492)The medium dose fucoidan intervention group(MFC,100 mg/kg fucoidan+high-fat diet),high-dose fucoidan intervention group(HFC,200 mg/kg fucoidan+high-fat diet),as well as the positive control(metformin,MTF,150 mg/kg metformin+high-fat diet)groups,mice in each group were gavaged daily with free access to water during the feeding period.The intervention was continuous for 24 weeks,and the glomerular filtration rate(GFR)was determined by applying the fluorescein isothiocyanate(FITC-sinistin)elimination kinetics technique based on percutaneous measurement at 23 weeks.Mice were sacrificed after 12 h of fasting following a single gavage at the end of the 24th week.Intestinal flora was detected using 16srna sequencing,and the levels of short chain fatty acids(SCFAs)in mouse cecal contents were detected using GC-MS.H&E staining and Masson staining were used to observe pathological features of ileum,liver and kidney tissues.We detected zonula occludens protein 1(ZO-1)and occludin in the ileum as well as transforming growth factorβ1,TGF-β1)and collagen I expression levels in the kidney used immunohistochemical.The C-X-C chemokine ligand 1(Cxcl1)content in liver and kidney and lipopolysaccharide(LPS)levels in serum were measured by enzyme-linked immunosorbent assay.The levels of adenosine triphosphate(ATP)in the kidneys were measured by chemiluminescence assay.Quantitative real-time PCR was used to determine mitochondrial DNA copy number in the kidneys,and Western bloting was used to determine the expression levels of key enzymes in the TCA cycle,mitochondrial respiratory chain complexes I-V(ChapterⅡ).Results:1.A case-control study found that:(1)Malate in the DM group and succinate levels were significantly lower in the con group,fumarate as well asα-Forα-ketoglutarate levels were significantly higher than those in the con group.Patients with DM and DN had more significant TCA disturbances than the general population,in which patients with DN had more severe TCA disturbances than patients with DM,as follows:compared with the DM group,the DN group had significantly higher pyruvate(5.71×10~6ng/L vs 13.69×10~6 ng/L,p<0.05),CIS aconitic acid(0.86×10~5ng/L vs 1.24×10~5 ng/L,p<0.05),isocitrate andα-Ketoglutarate(2.17×10~5ng/L vs19.96×10~5 ng/L,p<0.05)levels were increased compared to the DM group,succinate levels were decreased compared to the DM group(6.60×10~5ng/L vs 3.95×10~5ng/L,p<0.05)。(2)Estimated glomerular filtration rate(e GFR)was estimated in relation to CIS aconitate,isocitrate,pyruvate,andα-Ketoglutarate concentrations were negatively correlated with each other and succinate concentrations were positively correlated with each other.2.Animal experimental results found that after 24 weeks of continuous intervention with high-fat diet,the mice developed increased GFR filtration rate and urinary creatinine levels,and developed pathological changes in glomerulomegaly and tubulostitial fibrosis,judged as an early stage of DN:(1)Fucoidan intervention improved GFR hyperfiltration and decreased urinary creatinine levels in early DN mice;Fucoidan intervention improved glomerular enlargement demonstrated by H&E staining,tubulointerstitial fibrosis demonstrated by Masson staining;downregulated the expression of TGF-β1 and type I collagen.(2)Fucose significantly ameliorated the level of inflammation in the liver of early DN mice through the“gut-liver axis”,and the specific mechanisms were as follows:fucose intervention adjusted the intestinal flora structure of early DN mice,increased the abundance of beneficial bacteria such as Streptococcus,Lactobacillaceae and the concentration of acetic acid in cecal contents;Compared with the DM group,fucoidan intervention significantly upregulated the expression of intestinal tight junction proteins ZO-1 and occludin(p<0.05),decreased the levels of LPS in serum(p<0.05),improved intestinal homeostasis,and in turn decreased the liver proinflammatory cytokines IL-6 and TNF-αAnd levels of Cxcl1 in early DN mice(p<0.05).(3)Fucoidan improved renal mitochondrial function in early DN mice through liver-kidney crosstalk,the specific mechanisms were as follows:compared with the DM group,fucoidan significantly decreased Cxcl1 levels in the kidney,and the trend was consistent with that in the liver(p<0.05);Compared with the DM group,fucoidan intervention decreased the pro-inflammatory cytokines levels of IL-6,IL-1βin the kidneys of early DN mice(p<0.05),increased levels of the anti-inflammatory cytokine IL-10(p<0.05);Compared with the DM group,intervention with fucoidan significantly improved the levels of oxidative stress and TCA cycle key enzymes in the kidney(p<0.05),increased the renal mitochondrial DNA copy number,ATP levels and the expression of complex III and complex V(p<0.05),and significantly improved the quality and quantity of mitochondria in the kidney of early DN mice.(4)Fucoidan ameliorates renal fibrosis in early DN mice by improving mitochondrial function and then subsequently,the specific mechanisms are as follows:compared with the DM group,fucoidan intervention significantly downregulated the expression levels of p-ERK/ERK,p-JNK/JNK in the kidneys of early DN mice(p<0.05),thereby decreasing their downstream renal fibrosis marker TGF-β1and the expression level of typeⅠcollagen,which in turn ameliorates renal fibrosis in early DN mice.Conclusions:A more obvious mitochondrial dysfunction and energy metabolism disturbance were observed in DN patients,and intermediate metabolites of TCA cycle were closely related to e GFR.CIS aconitate was a risk factor for DN development in DM patients,succinate was its protective factor.Fucoidan could improve the homeostasis of intestinal environment and alleviate liver inflammation through the“gut-liver axis”,and then improve the mitochondrial function in the kidney through“liver-kidney crosstalk”,so as to inhibit mitogen activated protein kinase signaling pathway and renal fibrosis and improve the renal function injury in early DN mice induced by long-term high-fat diet. |
PDF Full Text Request