| Background:Diabetic nephropathy (DN) was the severe complication of Diabetes Mellitus (DM). The treatment of DN was limited to hypertension and hyperglycemia control. The studies on early pathogenic mechanism and the intervention of DN were less. The studies on the pathogenesis of DN were mainly focused on the polyol pathway, protein kinase C pathway activation, glycosylation end products accumulation and inflammatory signaling pathway activation and so on. And these pathways were all related insulin resistance (IR) closely. But the concrete forms of insulin resistance in diabetic nephropathy renal tissue and the intervention on renal insulin resistance still lacked research. AS 160, as a kind of Rab-GTP enzyme activation protein, could regulate the transportation of many transporters including glucose transporters. In kidney, AS 160 was found can regulate sodium and water channel protein, while its role on glucose transporters regulation has not been reported. GLUT4 (glucose transporter 4) was an insulin reactive glucose transporter and an important effector of insulin signaling, which related to insulin resistance closely. In insulin signaling, AS 160 located in the upstream of GLUT4 and phosphorylated AS 160 could promote GLUT4 transport. In the fat and muscle tissue which was conditional gene knockout GLUT4 or AS 160, the mice showed systemic and local insulin resistance. Sodium glucose transporter 1 (SGLT1) and sodium glucose transporter 2(SGLT2) were important to maintain body’s glucose steady state by promoting the proximal renal tubular glucose reabsorption and were the new target of antidiabetic drug. The study on AS 160 and GLUT4, SGLT1/SGLT2 in diabetes renal tissue would help to explore the molecular mechanism of renal insulin resistance and provide possible molecular targets for early intervention of diabetic renal damage. Our previous results showed NF-κB inhibitor Parthenolide (PTN) could improve db/db mice systemic insulin resistance and renal tissues injury. But, the effect of PTN on renal insulin resistance and the possible mechanisms were not reported.Objectives:To study and understand whether abnormal glucose transport occurred in kidney which leading to renal insulin resistance and its possible mechanism in db/db diabetic nephropathy model. To explore whether PTN intervention could improve IR and delay the progress of DN.Methods:Set up the db/db mice model of diabetic nephropathy and PTN intervention group. Three groups of mice were used:1) db/m mice treated with normal saline (NS) as control; 2) db/db mice treated with NS as DN model; 3) db/db mice treated with Parthenolide (PTN) as treatment group. All the mice were enrolled in experiment. The mice were sacrificed at TO (week8), T4 (weekl2), T8 (weekl6), T12 (week20) and the specimens were kept including blood, urine and kidney. GLUT4, AS 160 and p-AS160 (Thr 642) were measured by Real-time PCR, western blotting and immunohistochemistry. To co-localize GLUT4, SGLT1/SGLT2(sodium/glucose transporter 1 and 2) with AS 160/p-AS 160 by immunofluoresent assay and reveal the influence of AS 160 to glucose transporters in the renal IR.Results:(1) The db/db mice diabetic nephropathy model was set up successfully. Since 8 weeks, the body weight of db/db mice was higher than db/m mice. With the increase of weeks, the db/db mice showed increased urine protein excretion, high cholesterol, high blood glucose and insulin, as well as the increased glomerular hypertrophy, mesangial matrix.(2) The immunohistochemistry and immunofluorescence showed kidney expressed AS 160. It distributed mainly in the cytoplasm of renal cortex and medulla renal tubular epithelial cells and presented weaker expression in Bowman’s capsule in glomerular epithelial cells. AS 160 expression in db/m mice didn’t change with the increase of weeks. In db/db mice, the expression of AS 160 protein showed slight increase to db/m mice. After PTN intervention, AS 160 protein showed no obvious change compared with non-PTN db/db mice. AS 160 protein expression of db/db mice as:8w 1.22±0.26,12w 1.27±0.38,16w 1.35±0.25,20w 1.22±0.30 and after PTN intervention, the expression were T0(8w) 1.17±0.22, T4(12w) 1.22 ± 0.31, T8(16w) 1.24+0.39, T12(20w) 1.03+0.35.(3) The immunofluorescence showed p-AS160 (phospho T642) mainly distributed in the cortex renal tubular lumen side, the expression in medulla renal tubular was weak and no positive expression in glomeruli. The expression of p-AS160 in db/db mice was higher than the control group mice in the early weeks, while with the increase of weeks, the expression of p-AS160 decreased. Giving the db/db mice PTN intervention, the expression of p-AS 160 was increased. The expression of p-AS 160 by immunohistochemical as follows:8w 1.55±0.30,12w 1.18±0.22,16w 1.06± 0.20,20w 0.98±0.18. After PTN intervention, the p-AS160 expression as follows: T0(8w) 1.54±0.32, T4(12w) 1.42±0.26,T8(16w) 1.41±0.21,T12(20w) 1.34±0.08.(4) GLUT4 was detected in renal cortex and medulla tubular epithelial cells (RTEC) and showed no obvious positive distribution in glomerular by immunohistochemistry and immunofluorescence. The expression of GLUT4 in the kidney of db/m mice showed no change with the increase of weeks. But, in the db/db mice, the expression of GLUT4 in both mRNA level and protein level increased in the early weeks, and then decreased gradually with the increase of weeks. The PTN could increase the expression of GLUT4 in DN kidney. GLUT4 mRNA and protein expression in different weeks of db/db mice as follows:GLUT4 mRNA level 8w 1.79±0.25,12w 1.32±0.33,16w 0.98±0.23,20w 1.00±0.12 and the protein level 8w 1.32±0.28,12w 1.05±0.47,16w 0.85±0.10,20w 0.88±0.25. After PTN intervention for 0 weeks, 4weeks,8weeks and 12weeks, the GLUT4 protein expression of db/db mice was T0(8w) 1.31±0.21, T4(12w) 1.20±0.39, T8(16w) 1.42±0.43, T12(20w) 1.53±0.15.(5) The distribution had overlapped in renal tubules between AS 160/p-AS160 and part of GLUT4. Although SGLT1/SGLT2 mainly expressed in renal cortical tubular lumen side which similar to p-AS160 (Thr642), there was no significant overlap between SGLT1/SGLT2 and p-AS160.Conclusion:(1) The db/db DN mice showed systemic insulin resistance and renal pathological changes. PTN could improve systemic insulin resistance and renal pathological changes of db/db mice.(1) AS 160 and p-AS160 (Thr 642) expressed in kidney and mainly distributed in the in the renal tubules. With the increase of weeks, the expression of p-AS160 in db/db mice kidney tissue decreased gradually.(3) GLUT4 expressed in the renal tubules of db/db mice and decreased gradually with the increase of weeks. It suggested the glucose transportation function were damaged with the progression of DN and that may be the mechanism of renal insulin resistance.(4) The distribution of AS 160 and p-AS160 all showed coexpressed with part of GLUT4 in tubules. It suggested AS 160 in kidney may participate in GLUT4 translocation regulation.(4) PTN increased renal GLUT4 and p-AS160 expression in db/db mice, and it prompted PTN could improve kidney insulin resistance.(5) SGLT1/SGLT2 showed no or little overlap with p-AS160. It suggested AS160 was not the main regulatory protein in SGLT1/SGLT2 transportation.Objectives: Iohexol Plasma Clearance has been validated as a satisfactory reference method for GFR measurement in adult and child. However it has not been applied in China. The early work of the topic had built a method for the quantification of iohexol in serum by high performance liquid chromatography and the method was accurate and stable. And then the study proposed to evaluate the accuracy and practicability for iohexol plasma clearance from the serum and dried bood spots in chronic kidney disease (CKD) children.Methods: Set up the chromatographic conditions of HPLC and validate the linearity of standard curve, precision, trueness. After obtaining informed consents, thirty-three CKD children were included (twelve children had included in the early work and the total number was forty-five) and examined by 99m Technetium-diethylenetriaminepentaacetic acid(99m Tc-DTPA) plasma clearance and iohexol plasma clearance simultaneously. Blood samples were obtained at 2, 4 or 5hours after injection respectively. In the meantime we also evaluated the efficacy of single blood sample method and dried blood spots method in iohexol plasma clearance.Results:(1) The method for determination of the serum iohexol concentration wasestablished by HPLC. The standard curve showed a good linearity(R2 0.9998). The relative standard deviation RSD% of intra-assay and inter-assay were lower than 5% and the recovery rate was more than 96%.(2) Forty-five CKD children completed the iohexol plasma clearance and thirty-six children completed the 99mTc-DTPA plasma clearance at the same time among them. Thirteen children finished the iohexol dried blood spot clearance.(3) The correlation coefficient between 99mTc-DTPA plasma clearance and iohexol plasma clearance was 0.941 and the bias was (6.53±11.6) ml(min. 1.73M),the intraclass correlation coefficient was high (ICC=0.947).(4) The correlation between iohexol single sample plasma clearance and double samples was also strong (r=0.958), the bias was (4.2619.06) ml/ (min.1.73m) , theICC was 0.97.(5) The iohexol clearance by dried blood spots showed a good correlation with theserum iohexol clearance (r=0.950) and the bias was also small [(0.4810.89) ml/(min.1.73m)].Conclusion: Iohexol plasma clearance showed satisfactory agreementwith99mTc-DTPA plasma clearance and can be used as an ideal method tomeasure GFR inCKD children. The single sample method and dried blood spots methodmake iohexolplasma clearance more convenient and practical. |
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