| Objective:Analyzing and investigating the roles and related factors of protein Klotho and insulin-like growth factor (IGF-1) signal pathway associated factors in diabetic nephropathy (DN), through their levels of them in blood of patientes of DN.Methods:1. Grouping of patientes:there were three groups,42 persons of health examination as normal control were in group NC; second,61 patientes of diabetes without nephropathy were in group DM; 46 patientes of DN in group DN. The patientes in the last two groups were diabetics in affiliated hospital of Binzhou Medical University from 2010-09-01 to 2011-06-30.2.Detection of fasting plasma glucose (FPG), fasting insulin (FINS) and glycosylated hemoglobin (HbAlc):FPG,FINS were measured of peripheral venous blood of patientes with empty stomach early morning, then insulin resistance index (IRI) were got according to the formula (FPG×FINS)/22.5, HbAlc were measured by high-pressure liquid chromatography analytical method.3. Biochemical indicator detection:total cholesterol (TC), Triglyceride (TG), urea nitrogen (BUN) and creatinine (Cr) were measured of peripheral venous blood of patientes with empty stomach early morning; urinary albumin excretion rate was measured of twenty-four-hour urine which was collected a day before blood collection.4. Peripheral blood leucocyte hybridization in situ:expression of iNOSmRNA and eNOSmRNA detected of lmL anticoagulated blood with heparin were compared between groups.5. Enzyme linked immunosorbent assay (ELISA):protein klotho and IGF-1 in blood serum after centrifugation in three groups were detected early morning with empty stomach.6. reverse transcription polymerase chain reaction (RT-PCR):protein klotho and IGF-1 in peripheral blood were detected of lmL anticoagulated blood with heparin.Resultes:1. General condition:there was no statistically significance between groups of different ages(P>0.05); the course of disease in group DN was much longer than group DM (P<0.05); the systolic pressure in group DN was much higher than group NC (P<0.01); the diastolic pressure in group DN and DM were much higher than group NC (P<0.05).2.Detection result of FPG, FINS, HbAlc and UAER:there was not statistically significance between group DN and DM (P>0.05), but the levels of FPG, FINS, HbAlc and UAER in these two groups were higher than group NC (P<0.01); but the level of HbAlc in group DN was higher than group DM (P<0.01).3. Biochemical indicator detection result:there was no statistically significance of TC in three groups; the levels of TG in group DN was higher than group NC (P<0.01); the levels of BUN,Cr, in group DN was higher than group NC (P<0.01); but the level of HbAlc in group DN was higher than group DM (P<0.01).4. Expression of iNOSmRNA and eNOSmRNA detected by hybridization in situ of peripheral blood leucocyte:the levels of expression of iNOSmRNA,eNOSmRNA in groups DN and DM were much higher than the group NC (P<0.01); but the level in group DN is lower than group DM.5. Detection results of protein klotho and IGF-1 in serum:the levels of protein klotho in groups DN and DM were much lower than the group NC (P<0.01); the level of protein klotho in group DN was much lower than the group DM (P<0.05); the level of IGF-1 in group DM was much higher than the group NC (P<0.05); the level of IGF-1 in group DN was much higher than the group NC (P<0.05); there was no statistically significance between the group NC and CM (P>0.05).6. Detection results of klothomRNA and IGF-lmRNA in peripheral blood: the levels of klothomRNA and IGF-lmRNA in groups DN and DM were much lower than the group NC (P<0.01); the level of klothomRNA and IGF-lmRNA in group DN was much lower than the group DM (P<0.05).7. Analytic associativity result:The UAER in group DN had positive correlation with age, course of disease, FPG,1RI, TG and IGF-1 (P<0.05); and had negative correlation with iNOSmRNA, eNOSmRNA and protein klotho in serum (P<0.05). The iNOSmRNA in peripheral blood leucocyte had positive correlation with eNOSmRNA and protein klotho (P<0.01); and had negative correlation with age, course of disease, systol i c pressure, UAER and IGF-1 (P<0.05). The eNOSmRNA in peripheral blood leucocyte had positive correlation with iNOSmRNA and protein klotho (P<0.05); and had negative correlation with age, course of disease, systolic pressure, diastolic pressure, HbAlc, Cr, UAER and IGF-1 (P<0.01).The protein klotho in serum had positive correlation with iNOSmRNA and eNOSmRNA (P<0.01); and had negative correlation with age, course of disease, systolic pressure, HbAlc, TG, TC, Cr and UAER (P<0.05). The IGF-1 in serum had positive correlation with age, course of disease, systolic pressure, FPG, IRI, HbAlc, TG, TC, Cr and UAER (P<0.05); and had negative correlation with protein klotho, iNOSmRNA and eNOSmRNA (P<0.01).Conclusions:1. The expression of klothomRNA in peripheral blood and protein klotho levels in serum of group DM and DN were significantly lower,and to reduce even more in group DN, which suggests the incidence of diabetes and the klotho signal weakens significantly related, and more severe illness klotho signal weakening of the more obvious.2. The IGF-1 levels in serum were significantly increased,the expression levels of IGF-1RmRNA reduced in group DM and DN, and to reduce even more in group DN, which suggests IGF-1 ligand signal enhancement involved in the incidence of DM and DN; While in the pathogenesis of DM and DN, IGF-1 signal enhancement caused by the ligand IGF-1 receptor signaling (IGF-1RmRNA) downward, and the degree of reduction was significantly associated with the degree of exacerbations.The exact mechanism of IGF-1 signaling in the pathogenesis of DM and DN has yet to be explored.3. iNOSmRNA and eNOSmRNA in peripheral blood leucocyte were hardly expressed in group NC, and they were significantly expressed in group DM and DN, but the expression in group DN was much lower than the group DM, which shows two types of nitric oxide synthase transcript levels increased with the occurrence of DM, but progress to DN stage, and both decreased significantly, the results of the mechanism remains to be elucidated. |
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