The Changes Of Urinary Albumin Fragments In Type 2 Diabetes Mellitus Patients With Different Urinary Albumin Excretion Rate

IntroductionDiabetic nephropathy (DN ) is one of the serious chronic complications of diabetes mellitus ( DM). DN is reversible in trie first stage of the pathological changes; when comes into the clinical albuminuria stage, interfering therapy can only delay the depravation of kidney function but not reverse it, so in these recent years a large number of investigations are focused on early diagnose and interference. The meaning of early diagnose of Urinary albumin fragments ( UAF) for DN also attracted the attention of the scholars. Investigation showed that DM patients with positive urine microalbumin all have positive UAF, and 1/3 normal albumin urines UAF were positive. After two years of regular visit for the latter, 18% -19% turned into microalbuminuria. So detection of UAF had the more detecting rate than urine microalbumin. Anciently, the investigation of UAF were determined. To investigate the meaning of UAF in early diagnose for DN, this experiment did the half quantitative analysis for UAF and discussed the changes of urinary albumin fragments in type 2 diabetes mellitus patients with ? different urinary albumin excretion rate.MaterialDrugs and Reagents:1,Purified anti -human albumin IgG, horseradish peroxidase marked human - albumin, human - albumin standard; purchased from Beijing Institute of Geriatrics, Ministry of Public Health.2 ,Sample disposing liquids, gel -electrophoresis cushion liquids, transfer - eletrophoresis cushion liquids, 3,3 - diaminobenzidine.Samples1 , Experiment group: 60 T2DM patients, divided into three groups according to UAER: normal albuminuria group(UAER <20μg/min) with 22 patients; microalbuminuria group(UAER20 ~200μg/min)with 20 patients; clinical albuminuria group( UAER > 200μg/min) with 18 patients.2, Control group: 20 healthy volunteers.InstrumentUpright electrophoresis instrument, electric transfer instrument, constant temperature box, MetaMorph micro - image analyzing system, automatic biochemical analyzer( HITACHI 7600) , IMMAGE special type albumin analyzer.Method1 , Common clinical determination of subjects: Avoirdupois, stature and blood pressure were determinated and avoirdupois index were counted; age and course of diseases were asked and noted. 10ml elbow vein blood were picked in the morning when they were in limosis, and then HbA1c, blood sugar,creatinine (Cr) , total cholesterol ( TC ) , triglyceride ( TG) , serum albumin were determinated.2, Collection of specimen-. 24h urine of the subjects were collected, mixed, and added 10ml toluol for antisepticising, 2 ml urine were taken and were kept in 4℃ ice -box, UAER and UAF were detected.3 , Determining of UAER: Urine was continuously kept for three times for every subject, concentrations of urine albumin were detected by IMMAGE full automatic special type protein analyzer in velocity scatter method. UAER were counted, averages were taken.4,Determining of UAF: Urine samples, albumin standard liquid and 2 -mercaptoethanol were mixed together, then bathed in boiling water for 3min, 12.5% SDS - polyaerylamide gel electrophoresis( SDS - PAGE) with 20ml sample and the voltage was 200V. The electrophoresis pattern on the gel was trans-ferred to a nitrocellulose sheet for two hours, transferring current was 340mv. Blocked 1h with PBS containing 5% pig blood serum, then anti - human albumin antibody and horseradish peroxidase marked human - albumin, at 37% for 1 h and at 4℃. overnight. Morrow washed by the same PBS, at last visualization was carried out using 3,3- diaminobenzidine, UAF appeared in 45KDa,30KDa and/or 22KDa. Nitrocellilose sheet was scanned by computer, then analyzed by micro- image analyzing system, different fragments relative content were educed and expressed by integrated density value ( IDV ) , relative (?)retion rate was counted by 24h urine quantity.5, Statistical analysis : Statistical analysis were done (?) SPSS 12.0 statistical software. ANOVA, nonparametric test, chi -squ(?), correlation and multivariate regression analysis.Results1 , Measurement of clinical biochemical characters in every groupTable 1 showed that there were no statistical differences among the four groups in aging, sex - distribution and weight - index; courses of disease among the three DM patients had significance(P <0.05) ;systolic pressure, TC, TG of microalbuminuria group and clinical albuminuria were all significantly higher than control group (P <0.05 or P<0.01) ,and clinical group was significantly higher than normal group ( P < 0. 05 ) ; there was significance between clinical and control group in diastolic pressure, low density lipoprotein cholesterol (LDL - C) and high density lipoprotein cholesterol ( HDL -C)(P<0.05); the three experimental groups DM patients' limosis blood sugar were obviously higher than control group(P <0. 05) , but there were no significant difference among these three experimental groups; plasma Cr of clinical group was significant higher than the other groups( P <0.01).2 ,Measurement of UAFTable 2 showed: control group had no UAF; 45KDaUAF of normal albumin group was detected in 7 out of 22; 45KDaUAF was all detected in microalbuminuria group, 30KDaUAF was detected in 8; 45KDa,30KDa UAF were all detec-ted in clinical albumin group, 22 KDaUAF was detected in 10 out of 22. The positive rate of 45 KDaUAF in clinical group and microalbuminuria were significant higher than normal albumin group and control group( P <0.01) , normal albumin group was significant higher than control gr6up(P <0.01) ; the presence of 30 KDaUAF in clinical group was significant higher than microalbuminuria and control group( P <0. 01) ; and the presence of 22KDaUAF in clinical group was significant higher than microalbuminuria group( P <0.01). Table 3 showed that the relative excretion of 45 KDaUAF of three experimental groups had significance between each of them( P < 0. 05 or P<0.01); the relative excretion of 30KDaUAF had significance between clinical albumin group and microalbuminuria group(P<0.05).3, Correlation analysisThe relative excretion of 45KDaUAF had positive correlation with UREA, course of disease, systolic pressure, TC and TG, the correlation index were 0. 737,0. 513,0. 542,0. 486,0. 528. The relative excretion of 30KDaUAF had positive correlation with UREA, course of disease, systolic pressure, TC, TG and plasma Cr, the correlation index were 0.702,0.520,0.538,0.498,0.551, 0.615.4, Multivariate regression analysisTake age, avoirdupois index, course of disease, UAER, limosis blood sugar, HbA1c, systolic pressure, diastolic pressure, TC, LDL-C, HDL-C, TG, serum albumin and plasma Cr as independent variable; the relative excretion of 45KDa,30KDaUAF as dependent variable. Doing multivariate regression analysis, Affecting factors of the relative excretion of 45KDa,30KDaUAF in DM patients were UAER, systolic pressure and TG, standardation regression index: 45KDa,0.544,0.387,0.268;30 KDa,0.603,0.421,0.302.DiscussionThe amount of urine albumin excretion is a good guide line for judging the state of DN. Albumin in urine may appear abnormal character, representing albumin fragment. Native albumin molecules in human plasma are made up of 585amino acids, molecular weight is 69KDa. There are three subregions, each has 9 peptide rings, peptide rings are connected with 17 pairs of disulfide and hydrogen bridges. Many kinds of proteases exist in human plasma, they can split n-ative albumin molecules forming many incisions or cut of f part of the peptide sequences , this is called modified albumin. There are some differences between n-ative and modified albumin in molecular weight and stereochemical structure, but disulfide bridges still keep the combining state. After treating by the 2 -mercantoethanol and sodium dodecyl sulrate, disulfide bridges are broken to be different molecule fragments. The blots of 45KDa,30KDa,22KDa are UAF by western blot. In 1995, Yagame found it might be a clinical parameter for the early diagnosis of DN. The mechanism of the appearance of UAF is still unknown. Myers proposed that the choosing barrier of the glomerular basement membrane appeared turbulent in DM, since there are some differences in the stereochemical structure and molecular weight between native albumin and modified albumin, but they have almost the same isoelectric point, it may be more easier for modified albumin to get across the glomerular basement membrane into urine.In this experiment, no one in healthy control group appeared UAF. 45KDaUAF of normal albumin group was detected in 7 of 22 ( 31. 8% ) ; 45KDaUAF was also detected in all patients of diabetic early nephropathy and clinical nephropathy, this approximately was same with Yagame's conclusion. Except 45KDaUAF appeared in DM microalbuminuria group, 30KDaUAF was present in 8; both 45KDa and 30KDaUAF were detected in DM clinical albumin group, 22KDaUAF was present in 10. With the UAER increasing of DM patients, relative excretion rate increased, too. There was significance among each group. The relative excretion rate of 45KDaUAF was positive related with UAER, the relative excretion rate of 30KDaUAF was positive related with UAER and plasma Cγ. UAER and plasma Cγ indicate the development and prognosis of DN, it cues that the relative excretion rate of UAF reflect the damage degree of kidney in DM. The relative excretion rate of UAF was also correlated with course of diseases, systolic pressure, TC and TG. The course of diseases is a significantly dangerous factor of the attacking of DN, systolic pressure is an in-dependent warning factor, accelerating the development of DN and the regression of the kidney function; the increase of TC and TG is a dangerous factor for the happening of DN, it is as well as an independent dangerous factor of glomerular filtration rate falling and death rate increasing. It cues that UAF may forecast DN' s happening and reflect its development, and is correlated with DM accompanied by hypertension and turbulence of lipid metabolism.Multivariate regression analysis expressed that the relative excretion rate of UAF was affected by UAER, concentration of TG and systolic pressure. These three factors are correlated and affected with each other, they all have close relation with endothelium - cell function disorder and atherosis. For T2DM patients , albuminuria indicate not only the development of DN but also a dangerous factor of attacking and death for atherosis. Its appearance reflects extensive damage of blood vessel and function disorder in the body. Endothelium - cell function disorder is a link between albuminuria and atherosis. The increase of plasma TG is a major reason to diminish the LDL diameter and rise its density. Small and secret LDL is easy to be oxidized, and has stronger effect on atherosis. High TG blood disease aggravates T2DM patients'atherosis and also directly result in kidney scathe , it is a dangerous factor of cardiovascular disease. Hypertension is a dangerous for the attacking and worsening of kidney disease, further more systolic pressure is a useful forecasting factor for the attacking and death of cardiovascular disease. The rising of systolic pressure and manifold of small and secret LDL is the reason of blood vessel disease of T2DM patients. Turbulence of lipid metabolism and hypertension accelerate each other, and they speed the decline of kidney function. Endodermis - dependent diastolic function damage of vessel is a typical character of DM. Hypertension and high TG blood disease worsen the holdback of DM endothelium function. UAER, TG and systolic pressure affect together on the relative excretion rate of UAF, it presume that UAF may be related with the holdback of endothelium function and big vessel disease of DM.