Study On The Diagnosis Strategy Of Early Diabetic Kidney Disease And Intervention Of Dapagliflozin

Research BackgroundDiabetic kidney disease(DKD)is a serious diabetic microvascular complication and the main cause of end-stage renal disease.With the increasing incidence of type 2 diabetes(T2DM),DKD has also become a global disease that seriously endangers human health.According to the American Diabetes Association(ADA),the proportion of T2DM patients who progress to DKD is about 40%.In developed countries,this proportion is even higher,reaching about 50%.The pathogenesis of DKD is not fully understood,but hyperglycemia is always the initiating factor that causes and maintains continuous damage to target tissues and organs.Long-term hyperglycemia can cause increased intra-renal pressure and renal microcirculation disturbance,resulting in damage to the glomerular filtration membrane,accumulation of extracellular matrix of the glomerulus,and tubulointerstitial fibrosis.Under hyperglycemia conditions,it also increases the production of advanced glycation end products(AGEs),reactive oxygen species(ROS)and various inflammatory factors,which induces oxidative stress and inflammation,aggravates kindey damage.For a long time,microalbuminuria has been recognized as a marker of early DKD and its main diagnostic indicator.According to the ADA recommendation,screening and diagnosis of microalbuminuria are performed by measuring the urine albumin/creatinine ratio(UACR)of instant urine specimens.However,many studies have found that microalbuminuria has limited diagnostic value in early DKD.Not only is the occurrence of microalbuminuria inconsistent with the renal pathology of diabetic patients,but it is also manifested that there is no correlation between the progress of microalbuminuria and changes in renal function in diabetic patients.In addition,the measurement of albuminuria will be affected by some special factors,such as exercise,urinary tract infection and heart failure.Clinically,normoalbuminuria diabetic kidney disease(NADKD)patients are not uncommon.All these suggest that microalbuminuria may not be the best marker for early detection of DKD.For these reasons,we urgently need to identify biomarkers that are more sensitive and specific than microalbuminuria to reflect early kidney damage in order to diagnose DKD early.Current standard treatments for DKD include intensive blood glucose control,strict blood pressure control,and medications that inhibit the renin-angiotensin system.In recent years,as the role of sodium-glucose cotransporter-2(SGLT-2)has been gradually recognized,there has been great interest in the role of the kidneys in the formation and maintenance of high glucose levels.The role of the kidney as a target organ for the onset and treatment of diabetes has also received more attention and research,which has led to the development of new drugs that inhibit SGLT-2.SGLT-2 inhibitors prevent blood glucose levels from rising by inhibiting the reabsorption of sodium and glucose by the kidneys and increasing urinary glucose excretion.In addition to lowering blood glucose,SGLT-2 inhibitors have other effects,such as reducing weight,lowering blood pressure,and lowering serum triglyceride(TG)and uric acid(UA)levels.These effects not only help T2DM patients to better control blood glucose,but also may indirectly prevent and improve chronic complications of diabetes,including DKD.In addition,studies have shown that inhibition of SGLT-2 can also produce direct renal protection,including reducing ultrafiltration of glomeruli and renal tubular cell hypertrophy,and alleviating the toxic effect of glucose on renal tubules.Some large clinical studies have shown that SGLT-2 inhibitors delaying the progression of albuminuria and decline in renal function in patients with T2DM,reducing the risk of renal replacement therapy or death from kidney disease,and having renal protection for patients with T2DM.However,the research on SGLT-2 inhibitors for early DKD is still relatively rare.Therefore,this study is mainly divided into two parts.The first part mainly explores the value of multiple serum and urine biomarkers in the diagnosis of early DKD.We have selected 13 biomarkers related to the four aspects of glomerular injury,tubular injury,oxidative stress and inflammation,hoping to find diagnostic indicators of early DKD with higher sensitivity and specificity.We expect to provide clinical data for early intervention and treatment of DKD,and to reduce the risk of renal failure.In the second part,we have selected patients with early DKD of T2DM and used the first SGLT-2 inhibitor marketed in China-dapagliflozin.The changes of urine albumin and some markers of the first part that have diagnostic value in early DKD before and after treatment were observed,and the renal protection effect of dapagliflozin in T2DM with early DKD patients was investigated.Part 1The role of serum and urinary biomarkers in the diagnosis of early diabetickidney disease in patients with type 2 diabetesPurpose1.To study the changes of 13 biomarkers in different albuminuria periods of T2DM patients,and to explore their correlation with UACR.2.Using receiver operating characteristic curve(ROC)to estimate the diagnostic value of biomarkers in early DKD,and try to establish an effective DKD diagnosis model.Method1.Research subjects and groups287(including 163 males and 124 females)T2DM patients who were admitted to the Department of Endocrinology,Second Affiliated Hospital of Anhui Medical University,were selected as the research subjects.All T2DM patients met the diabetes diagnostic criteria of the World Health Organization(WHO)in 1999.According to the UACR value,it was divided into 144 cases of normoalbuminuria(UACR≤30 mg/g)subgroup,94 cases of microalbuminuria(30 mg/g<UACR<300 m/g)subgroup,and 49 cases of macroalbuminuria(UACR>300mg/g)subgroup.A total of 42 healthy people from our physical examination center were selected as the control group.2.Research indicator detectionThe gender,age,height,weight(W),and blood pressure of all the subjects were recorded,and the body mass index(BMI)was calculated.The duration of diabetes in T2DM patients was recorded and the fundus lesions were collected.All subjects were fasted for more than 10 hours,and venous blood was taken in the morning to measure fasting blood glucose(FBG),hemoglobin Alc(HbAlc),total cholesterol(TC),TG,low-density lipoprotein(LDL),total bilirubin(TBIL),creatinine(Cr),cystatin C(CysC),UA,neutrophil count,lymphocyte count,etc.The estimated glomerular filtration rate(eGFR)and neutrophil to lymphocyte ratio(NLR)were calculated.The first midstream urine was collected in the morning for determination of urinary albumin and Cr,and for the UACR calculation.Simultaneous determination of urinary transferrin(Tf),immunoglobulin G(IgG),podocalyxin(PCX),neutrophil gelatinase-associated lipocalin(NGAL),N-acetyl-beta-glucosaminidase(NAG),alpha-1-microglobulin(α1MG),8-hydroxy-deoxyguanosine(8-OHdG),tumor necrosis factor alpha(TNF-α),and interleukin-18(IL-18).In order to eliminate the influence of urine concentration,all the above urine indexes were corrected by urine Cr value.3.Statistical analysisSPSS 22.0 statistical software was used for statistical description and analysis.The Shapiro-Wilk method was used to test whether the data were normally distributed.Variables with a normal distribution were expressed as mean ± standard deviation.Non-normally distributed variables were expressed as median(interquartile range).The t-test or One-way analysis of variance(ANOVA)was used to analyze the differences between the groups;non-normally distributed data were compared between multiple groups using a rank sum test.Qualitative data was described by composition ratio,and χ2 test was used.Correlation analysis was used to test the correlation with biomarkers and UACR.Multiple linear regression analysis was used to analyze the association between different biomarkers and UACR.Logistic analysis was used to analyze the association between biomarkers and early DKD.ROC analysis was used to calculate parameters such as area under the curve(AUC),sensitivity,and specificity to evaluate the diagnostic value of various biomarkers and combined multiple biomarkers.P<0.05 was statistically significant.Result1.Age,BMI,duration,prevalence of diabetic retinopathy(DR),SBP,DBP,FBG,Cr,eGFR,TG,HbAlc,and 13 biomarkers(urinary Tf;IgG,PCX,NGAL,NAG,α1MG,8-OHdG,TNF-α,IL-18,and serum CysC,TBIL,UA,NLR)in healthy control group,normoalbuminuria group,microalbuminuria group,and macroalbuminuria were statistically different(P<0.05).Among the 13 biomarkers,the levels of urinary Tf,IgG,PCX,NAG,alMG,8-OHdG,IL-18,and serumCysC,UA,NLR levels of patients in the macroalbuminuria group were higher than those in the other three groups(P<0.05).Urinary NGAL,and TNF-α levels in the macroalbuminuria group were higher than those of the healthy control and normoalbuminuria groups(P<0.05).Serum TBIL in the macroalbuminuria group was lower than the normoalbuminuria and microalbuminuria groups(P<0.05).Urinary Tf,IgG,PCX,NGAL,NAG,α1MG,8-OHdG,TNF-α,IL-18,and serum NLR levels of patients in the microalbuminuria group were higher than the healthy controls and normoalbuminuria groups(P<0.05).Urinary Tf,PCX,NGAL,NAG,alMG,IL-18 and serum TBIL levels in patients with normoalbuminuria were higher than the healthy controls(P<0.05).2.Multiple linear regression analysis showed that urinary Tf,IgG,NGAL,and TNF-α in patients with early DKD were significantly correlated with UACR(P<0.001).3.Logistic regression analysis found that urinary Tf,IgG,NGAL,and TNF-α were significantly associated with early DKD(P<0.05).4.After adjusting the relevant variables,the AUC of urinary Tf,IgG,NGAL,and TNF-α for early DKD diagnosis in T2DM patients were analyzed with ROC curves,which found to be 0.876(95%CI:0.830-0.923),0.910(95%CI:0.867-0.952),0.896(95%CI:0.856-0.936),and 0.813(95%CI:0.756-0.870),the AUC of urinary Tf,IgG,NGAL,and TNF-α combination is 0.944(95%CI:0.913-0.975),which had the highest AUC when compared with the individual biomarker(P<0.001).Conclusion1.Urinary Tf,IgG,NGAL,and TNF-α in early DKD patients were significantly correlated with UACR and could reflect early kidney damage in T2DM patients.2.Diagnostic value analysis of individual biomarkers,urinary IgG>NGAL>Tf>TNF-α;The combined application of urinary Tf,IgG,NGAL,and TNF-αdemonstrated excellent diagnostic value for early DKD in patients with type 2 diabetes.Part 2The renal protection of dapagliflozin in type 2 diabetes patients with earlydiabetic kidney diseasePurposeTo evaluate the effect of dapagliflozin on UACR and other biomarkers in T2DM patients with early DKD,and to explore the renal protection and mechanism of dapagliflozin on T2DM patients with early DKD.Method1.Research subjects and groupsA total of 185 T2DM patients with early DKD who were admitted to the Department of Endocrinology,Second Affiliated Hospital of Anhui Medical University,from January 2018 to March 2019 were selected.The dapaglifloz:in group used dapagliflozin 10mg once a day,and the control group could receive hypoglycemic drugs other than SGLT2 inhibitors.After stable blood glucose control,the patients were discharged from the hospital.The patients were followed up once every 4 weeks in the endocrine clinic.Hypoglycemia events and other adverse reactions were recorded.The patients were followed up for 24 weeks.2.Indicator detectionPatients’ gender,age,duration of diabetes,comorbidities,medication,blood pressure,height,and W were recorded,and BMI was calculated.Venous blood was collected more than 10 hours overnight,and FBG,HbAlc,TC,TG,LDL,Cr,CysC,UA were measured,eGFR was calculated.Urine was collected at mid-morning,urine albumin,Cr,Tf,IgG,NGAL,TNF-α were measured,and UACR was calculated.After 24 weeks,the patient’s blood pressure and weight were measured again,BMI was calculated,and the above indicators were retested.In order to eliminate the influence of urine concentration,all the above urine indexes were corrected by urine Cr value.3.Statistical methodsStatistical analysis was performed using SPSS22.0 software.The measurement data were used the Shapiro-Wilk test to determine the normality of the sample distribution.If the distribution conformed to the normal distribution,the mean±standard deviation was used to describe it.If the distribution did not conform to the normal distribution,the median(interquartile range)was used for description.For measurement data that meets the normal distribution,independent sample t test was used for comparison between groups,and paired sample t test was used for comparison within the group;Mann-Whitney U test was used for comparison between groups,and Wilcoxon test was used for comparison within the group.Count data were expressed by frequency(%).Comparison between groups were analyzed by Pearson chi-square test and Fisher exact test.Logistic regression analysis assessed association UACR improved with baseline variables and pre-treatment indicators.P<0.05 was considered statistically significant.Result1.There were 89 cases of follow-up in both the dapagliflozin group and the control group.There were no significant difference in gender,age,duration of diabetes,comorbidities,and medications between the two groups(P>0.05).The difference of pre-treatment SBP,DBP,W,BMI,FBG,HbAlc,TC,TG,LDL,UACR,eGFR,and serum Cr,CysC,UA,and urinary Tf,IgG,NGAL,TNF-α between the two groups were not statistically significant(P>0.05).2.The SBP,DBP,W,BMI,FBG,HbA1c,TC,TG,LDL,UACR,serum UA,and urinary Tf,NGAL,TNF-α in the dapagliflozin group after treatment were lower than those before treatment(P<0.05).The SBP,DBP,W,BMI,FBG,HbAlc,TC,TG,LDL,and serum UA after treatment in the control group were lower than those before treatment(P<0.05).3.After 24 weeks treatment,the Wm,BMI,TG,UACR,and urinary Tf,NGAL,TNF-αin the dapagliflozin group were lower than those in the control group(P<0.05).4.Logistic regression analysis showed that the improvement of UACR was significantly correlated with the treatment of dapagliflozin and the absence of hyperlipemia(P<0.05).5.There were no statistically significant differences in incidence of hypoglycemia,reproductive and urinary tract inflammation,gastrointestinal symptoms,and diabetic ketoacidosis(DKA)between the two groups after treatment(P>0.05).ConclusionDapagliflozin can reduce UACR and urinary Tf,NGAL,TNF-α in T2DM patients with early DKD,suggesting that dapagliflozin has significant renoprotective effects on T2DM patients.This effect may be related to dapagliflozin reducing glomerular hyperfiltration,improving tubular function,and alleviating inflammation in the body.