| ObjectiveDiabetic kidney disease(DKD)is one of the major microvascular complications of diabetes mellitus.With the increase of the incidence of type 2 diabetes,DKD has become the leading cause of chronic kidney disease and end-stage renal disease.Advanced DKD patients need kidney transplantation or dialysis therapy to maintain their renal function.The medical costs of DKD are huge and it becomes a global public health problem.The pathogenesis of DKD remains unclear.It is presently considered that DKD was initiated by a combination of multiple genes and environmental factors.It is closely related to hereditary factor,epigenetic modification,abnormal hemodynamics,inflammatory response,and mitochondrial dysfunction.Accumulating evidence supports that renal mitochondrial dysfunction plays a key role in the occurrence and development of DKD.In early diabetic state,renal intrinsic cells were characterized by different degrees of mitochondrial biosynthesis,energy metabolism and signal transduction abnormalities.Artemisinins,as the representative of antimalarial drugs,has been shown to be effective in inflammatory diseases,autoimmune diseases,tumours and metabolic diseases in recent years.The therapeutic mechanisms of these drugs were also related to the regulation of mitochondrial function.However,the application of these drugs in the field of type 2 DKD has not been reported.Therefore,we explored the distributions of constitution and syndrome,the metabolic characteristics,and the alterations of renal function and morphology in type 2 DKD patients at the early and middle stage.And then to verify whether the artemisinin representative drug artemether could exert renal protection by improving the energy metabolism,renal function,and renal morphological changes in type 2 diabetic mice.Methods1.Clinical researchPatients were selected from the recruited volunteers for clinical observation trial by the department of nephrology,Shenzhen Traditional Chinese Medicine Hospital from December 2016 to December 2018.There were 30 cases in the normal control group(NC group),30 cases in the simple type 2 diabetes group(T2D group),30 cases in the type 2 DKD stage ? and ? group(DKD(1-2)group),and 30 cases in the type 2 DKD stage ? and ? group(DKD(3-4)group).The basic data of gender,age,weight and BMI of each subject were collected.The informations of pulse condition,tongue and facial complexions were collected by the comprehensive diagnostic system of traditional Chinese medicine;the constitution identification questionnaire was filled and then the system automatically identifies various constitutions.Synrome differentiation and classification were carried out according to the collected informations and the standards set by China Association of Chinese Medicine.The blood and urine samples were collected.The volume of kidneys were calculated through measuring the long axis,short axis and thickness of bilateral kidneys by renal ultrasound.Relevant biochemical indicators were detected by using automatic biochemical analyzer.HbAlc was measured by using an Ultra2 HbAlc Analyzer.The serum levels of acetylcarnitine and butylcarnitine were detected by metabolomics.2.Animal experimentsTha 8-week-old mice were allocated randomly to the following groups:lean wild type T2D control(T2D-ctrl)group,db/db group,and db/db+artemether(db/db+Art)group.The T2D-ctrl and db/db group mice were fed a regular diet,and the db/db+Art group mice were fed a regular diet supplemented with 0.67 g/kg artemether.The initial treatment time of artemether was set to 0 week and the treatment lasted for 12 weeks.The 24-hour urine was collected at 0,6 and 12 weeks in metabolic cage,and 24-hour water consumption,urine volume,food intake and feces production were also collected at the same time.The energy expenditure and RER were determined at 0,6 and 12 weeks by using the Comprehensive Lab Animal Monitoring System.Mice were weighed at 0,4,8 and 12 weeks,and fasting blood glucose was measured by blood glucose meter.After 12 weeks intervention,the samples of whole blood,serum,pancreas and kidney tissues were collected.Urinary albumin excretion rate and serum insulin level in each group mice were measured by ELISA kit.Urinary glucose was detected by using an automatic biochemical analyzer.Paraffin-embedded kidney sections were stained with PAS and scanned to measure renal glomerular tuft areas,glomerular mesangial matrix areas,proximal renal tubular areas and tubular lumen areas were measured.The renal glomerular tuft volume and tubular wall area were calculated.Pancreas sections were stained with HE and scanned to measure the mean islet area,islet area/pancreas area,and islet density.The number of islets was counted,and islet density was calculated by islet number/pancreas area.Pancreatic sections were coimmunostained with insulin,glucagon,and somatostatin.The images were visualized and captured on a confocal microscope and were used to calculate the insulin-,glucagon-,and somatostatin-positive area in islets.The transmission electron microscopy photographs were selected to measure foot process width,glomerular basement membrane,and tubular basement membrane.The morphological alterations of cells in islet were also observed under transmission electron microscope.Urinary excretion rate and serum level of H2O2 in each group mice were measured by UltraRed.The distributions of catalase and SOD2 in renal tissue were detected by immunohistochemical staining.The content of PGC-1?,catalase,and SOD2 in renal tissue,and content of MPC1,MPC2,PDK1,PDP1,PDH and VDAC in renal mitochondria were detected by Western blot.Results1.Clinical Research(1)There was no significant difference in gender and age in each group cases and this indicates that the cases included in the study had good consistency.The body weight and BMI of T2D group,DKD(1-2)group and DKD(3-4)group were significantly higher than those of NC group.(2)The fasting blood glucose and HbAlc in T2D group,DKD(1-2)group and DKD(3-4)group were significantly higher than those in NC group.(3)Compared with NC group,urinary ACR in DKD(1-2)and DKD(3-4)group was significantly higher than that in NC group.Compared with NC group,eGFR increased significantly in DKD(1-2)group and decreased significantly in DKD(3-4)group.Compared with T2D group,urinary ACR in DKD(3-4)increased significantly.And there was no significant difference between T2D and DKD(1-2)group.Compared with T2D group,eGFR in DKD(1-2)group increased significantly,and eGFR in DKD(3-4)group decreased significantly.(4)Compared with NC group,TG in T2D group,DKD(1-2)group and DKD(3-4)group increased significantly.Compared with NC group,HDL in T2D group,DKD(1-2)group and DKD(3-4)group decrea.sed significantly.Compared with NC group,TC and LDL in T2D group,DKD(1-2)group and DKD(3-4)group displayed no obvious changes.(5)Compared with NC group,the volume of left and right kidney in DKD(1-2)group increased significantly.Compared with T2D group,the volume of left and right kidney in DKD(1-2)group also increased significantly.As the disease progresses,the renal volume of DKD(3-4)group reduced,but there was no significant difference when compared with DKD(1-2)group.(6)Compared with NC group,serum metabolites of acetylcarnitine and butylcarnitine in T2D group and DKD(3-4)group increased significantly.Compared with T2D group,acetylcarnitine and butylcarnitine in DKD(3-4)group also increased significantly.The levels of acetylcarnitine and butylcarnitine in DKD(1-2)group was similar to NC group and T2D group.(7)NC group was mainly composed of pinghezhi,qixuzhi,tanshizhi,and shirezhi.There were a few qiyuzhi,yangxuzhi and yinxuzhi.Xueyuzhi cases were not observed in NC group.The T2D group was mainly composed of qixuzhi,tanshizhi,and shirezhi.There were a few qiyuzhi,yangxuzhi,yinxuzhi,and xueyuzhi in the group.No pinghezhi in the group.The DKD(1-2)group was mainly composed of qixuzhi and no pinghezhi in the group.The distribution of other types of constitution was relatively uniform.The DKD(3-4)group was mainly composed of qixuzhi,yangxuzhi,yinxuzhi,and xueyuzhi individuals,and there were no obvious regularity of the constitution distribution.No pinghezhi in the group.(8)The T2D group was mainly composed of Yinxuzaore syndrome and Qiyinliangxu syndrome.Although the DKD(1-2)group was also mainly composed of Yinxuzaore syndrome and Qiyinliangxu Syndrome,but the number of Pishenqixu syndrome and Yinyangliangxu syndrome slightly increased in the group.As the disease progresses,Yinxuzaore syndrome and Qiyinliangxu syndrome gradually reduced.The DKD(3-4)group was mainly consisted of Pishenqixu syndrome and Yinyangliangxu syndrome in this group increased significantly.2.Animal experiments(1)Artemether significantly reduced urinary albumin excretion,decreased kidney weight,alleviated mesangial matrix and ameliorated glomerular and proximal tubular hypertrophy in db/db mice.(2)Artemether attenuated glomerular and tubular basement membrane thickening,and ameliorated foot process effacement in db/db mice.(3)Artemether protected against hyperglycemia and improved diabetic symptoms including polydypsia,polyuria,polyphagia,and increased feces production in db/db mice.(4)Artemether increased serum insulin and recovered the unbalanced ratio of insulin,glucagon and somatostatin content in the islets of db/db mice.It also made the islets densely packed with insulin granules and reduced the chance of being observed of islet ? and ? cells.(5)Artemether increased the RER in db/db mice after 6 weeks and 12 weeks treatment while did not affect energy expenditure.(6)Artemether reduced serum level and and urinary excretion of H2O2 in db/db mice.(7)Artemether increased renal expression of PGC-1? and mitochondrial content of MPC and PDP1 in db/db mice.Conelusion(1)With the progression of DKD,the body constitution also gradually changes from the early qixu,tanshi,and shire to yinxu,yangxu and xueyu.The syndrome type also changed from Yinxuzaore and Qiyinliangxu to Pishenqixu and Yinyangliangxu.This process is also accompanied with metabolic reprogramming which characterized by incomplete glucose oxidation and enhanced protein and fatty acid utilization.(2)This study firstly verified the renal protection of artemether in type 2 diabetic db/db mice.Artemether could reduce the urinary albumin excretion,prevent diabetic kidney hypertrophy,and ameliorate renal pathological injury.(3)This study confirmed for the first time that artemether could increase the RER in db/db mice without affecting the energy expenditure.At the same time,artemether could increase the expression level of PGC-1? in renal tissue,regulate the redox state in kidney,increase the content of MPC and PDP1 in renal mitochondria and then enhance the uptake and oxidation capacity of pyruvate in renal mitochondria. |
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