The Role Of Galectin-3 In Diabetic Nephropathy And Interventional Effect Of Astragaloside Ⅳ

Objective:Diabetic nephropathy(DN),one of the most serious microvascular complications associated with diabetes mellitus(DM),has become the second leading cause of end-stage renal disease(ESRD)in china.Currently,the precise pathogenesis of DN has not yet been fully elucidated.Despite aggressive and comprehensive treatment,many patients with DN still develop ESRD.There is a pressing need for the development of novel therapeutics to inhibit the progression of DN.Galectin-3 is a member of the multifunctional lectin family.Galectin-3 plays important regulatory roles in a variety of pathophysiological processes,including fibrogenesis,tissue repair,and inflammation.Galectin-3 has been shown to play a role in the initiation and development of DN.Astragaloside IV(AS-IV)is one of the most important and active compounds of Astragalus membranaceus Bunge.Several studies have shown that AS-IV could alleviate kidney injury both in vivo and in vitro models.The purpose of this study was to examine the clinical relevance of serum galectin-3 levels at various stages of type 2 DN and evaluate the association between galectin-3 levels and loss of renal function.We further explored the possible mechanisms of galectin-3 in DN.In addition,we also investigated whether the protective effect of AS-IV on DN is associated with regulating galectin-3 expression.Methods:The study was made up of two parts:clinical study and animal experiment.The clinical studyThirty type 2 DM and 120 type 2 DN patients were identified between August 2015 and April 2016 from Shenzhen Affiliated Hospital of Guangzhou University of Chinese Medicine.Thirty healthy,age-matched subjects without any medical disease or medical drug treatment were recruited as controls.The stages of DN were categorized using Mogensen DN diagnostic criteria.Patient clinical parameters were obtained,including gender,age,duration of DM,medications administered,body weight,height,body mass index(BMI),systolic blood pressure(SBP)and diastolic blood pressure(DBP).All blood samples were obtained in the morning after an overnight fast,biochemical parameters and galectin-3 were tested.The animal experimental studyHealthy male C57BL/6 mice(5-6 weeks old)weighing 16-18g were purchased from the Laboratory Animal Center of Guangdong Medicine.Experimental diabetic mice were induced,following 4h of fasting,by a single intraperitoneal injection(200mg/kg)of streptozotocin(STZ,Sigma Aldrich,St.Louis,MI,USA)dissolved in 0.1 M citrate buffer(pH 4.2).Normal control mice were intraperitoneally injected with an equal volume of vehicle.The mice were randomly divided into the following three groups:normal control mice(control group),STZ-induced diabetic mice(STZ group),and diabetic mice administered with a diet supplemented with AS-IV(STZ+AS-IV group).Every week,each mouse was weighed and blood samples were obtained by tail vein puncture for blood glucose measurements using a blood glucose meter(Roche,Basel,Switzerland).At the end of 8 weeks of treatment,the mice were sacrificed and blood samples and kidney tissues were harvested immediately.Urine and serum biochemical parameters were measured using a Roche automatic biochemical analyzer.Urine albumin,urine NGAL,urine TGF-β 1 and serum insulin ELISA kits were utilized according to the manufacturer’ s instructions.Periodic acid Schiff stain was used to determine the diameters of the mice glomeruli and glomerular matrix index,while ultrastructural pathological changes of glomerulus and renal tubules were examined by transmission electronic microscopy.Immunoblotting analysis was used to examine the levels of MEK1/2,ERK1/2,RSK2,P-MEK1/2,P-ERK1/2,P-RSK2 and galectin-3 in each group of the mice kidneys.Results:The clinical studyAmong all the basic characteristics,there were no overt differences in gender,weight and BMI.However,it was difficult to match age and duration of DM,because progression of DN is highly time-dependent.Spearman’ s correlation analysis showed a positive correlation between galectin-3 concentration and Scr,β 2—MG,UACR,age,and SBP.There was a negative correlation with HbAlc,FPG and eGFR.Stepwise multivariate linear regression analyses showed galectin-3 was independently associated with eGFR.The level of serum galectin-3 was associated with the primary deficiency syndrome.However,there was no obvious correlation between serum galectin-3 concentration and the secondary excess syndrome.The animal experimental studyThe diabetic model mice exhibited increased levels of blood glucose and decreased body weight compared with the control mice during the entire study period.At 8 weeks,mice in the diabetic model group demonstrated significantly higher urinary glucose levels and lower serum insulin levels compared with the control mice.However,no significant differences were found in these parameters between diabetic model group and AS-Ⅳ treatment group.The SBP,DBP and MAP in diabetic model mice were significant lower than control mice,but the heart rate was significant higher than control mice.AS-IV treatment raised SBP,DBP and MAP,and slowed heart rate overtly.Compared with the normal control group,24 h urinary albumin excretion was significantly increased in diabetic mice at 4 and 8 weeks.Following treatment with AS-IV for 4 weeks,24 h urinary albumin excretion was markedly reduced and this effect continued through to 8 weeks post AS-Ⅳ administration.Compared with the normal control group,there was a significant increase in urinary levels of NAG,NGAL and TGF-β 1 in diabetic mice at 8 weeks.AS-Ⅳ administration resulted in a reduction in urinary NAG,NGAL and TGF-β 1,although no significant differences in NGAL and TGF-β 1 were found after 8 weeks of treatment.The diabetic mice showed significantly higher ALT and AST,and significantly lower ALB and TP compared with the control mice.There were no significant differences in these parameters between the diabetic model and the AS-IV treatment groups.The levels of Scr and BUN in diabetic mice were obviously higher than in the normal control group.AS-Ⅳ administration significantly reduced Scr levels after 8 weeks of treatment.Compared with the control group,the mice in the diabetic model group exhibited significantly higher TG and slightly lower HDL-C.Treatment with AS-IV resulted in an increase in HDL-C,with no concomitant changes to TG levels.In addition,no difference in LDL-C was observed among the three groups at 8 weeks after AS-IV administration.At 8 weeks,the diabetic mice showed larger GTA,greater GTV,thicker GBM,higher mesangial matrix ratios and wider FPW than the control mice.AS-IV treatment significantly ameliorated the mesangial matrix ratio and the FPW.The expression of p-MEK1/2,p-ERK1/2 and p-RSK2 were significantly upregulated in diabetic model mice compared to control mice.Interestingly,after the diabetic mice were treated with AS-IV for 8 weeks,activation of these proteins was significantly inhibited.Conclusion:1.Increased serum galectin-3 levels were independently associated with renal outcome in the present study.Serum galectin-3 could be used as a novel predictor of progressive renal impairment in patients with type 2 DN.2.The level of serum galectin-3 could reflect the dynamic changes of TCM Syndrome Type.Galectin-3 can be used as a objective indicators for TCMsyndrome differentiation.3.AS-IV could attenuate renal injury in STZ-induced diabetic mice.This phenomenon might be partially associated with down-regulated expression of galectin-3 and inhibiting the activation of MEK1/2-ERK1/2-RSK2 signaling.