Down-regulation Of Alpha-2u Globulin In Renal Mitochondria Of STZ-induced Diabetic Rats Observed By A Proteomic Method

Objective: To identify the changes of mitochondrial protein expressionin diabetic renal parenchyma and to characterize their molecular functions andbiological processes in diabetes.Method: Male Sprague-Dawley rats were randomly divided into adiabetic group and control group. Diabetic group rats received anintraperitoneal injection of sterile STZ (65mg/kg body weight) to establish ananimal model of diabetes. Control group rats received an equal volume ofsodium citrate buffer. After twelve weeks of diabetes, the renal damagein diabetic rats was assessed by hematoxylin-eosin staining (HE)staining and electron microscopy. Renal mitochondria of diabetic group ratsand control group rats were extracted by differential centrifugation.Crude mitochondria were purified with density gradient centrifugation.Mitochondrial purity was evaluated by determination of 5’-nucleotidase(5’-NT) activity and electron microscopy. Mitochondrial proteins extractedfrom renal parenchyma mitochondria of diabetic group rats and control grouprats were separated by two-dimensional polyacrylamide gel electrophoresis(2-DE) , the significantly differently expressed proteins were identified bymatrix-assisted laser desorption/ionization tandem time-of-flight massspectrometry (MALDI-TOF-TOF MS). Differences in protein expressionwere validated by Western blotting, the bioinformatics analysis of thevalidated proteins were performed at the end.Result: Urine glucose in control group rats were negative (-), while diabetic group rats were strongly positive (++++). Compared with controlgroup, diabetic group showed significantly hyperglycemia after STZinjections. Some pathological changes in the glomerular of diabetic rats wereevident, including proximal tubule epithelial cell edema and hypertrophy,thickening of the glomerular basement membrane, mesangial regionsproliferative and expansion, and foot process effacement or fusion. The resultof 5’-nucleotidas analysis demonstrated that the 5′-nucleotidas activity inhomogenate was extremely high, which was decreased significantly insuspension of purified mitochondria and mitochondrial proteins, it was almostzero in mitochondrial proteins. 2-DE maps of renal parenchyma mitochondriawere obtained; eleven proteins from 533 visualized protein spots expresseddifference significantly in diabetic kidneys. Among these altered proteins, twoproteins with the greatest changes in protein expression were identified asalpha-2u globulin (mature protein, named A2) and its proteolytically modifiedform (named A2-fragment) respectively. They were found in mitochondria ofmale rat kidney and were proved to be down-regulated in diabetic rats byproteomic analysis and Western blotting. Bioinformatics analysis showed thatA2 is a secreted protein and is synthesized in the liver of adult male rats. Aftersecreted into the blood, a portion of A2 excreted in the urine, and another partof A2 undergoes endocytosis by the proximal tubule epithelial cells, in whichA2 was converted to A2-fragment by hydrolytic enzymes within lysosomes.A2-fragment can bind long-chain fatty acids and is most likely to assist themitochondrialβ-oxidation of long-chain fatty acids.Conclusion: Compared with normal male rats, the expression of A2 andA2-fragment were significantly reduced in the mitochondria of renalparenchyma of STZ-induced diabetic rats, down-regulation of these two proteins may be associated with an abnormalβ-oxidation of long-chain fattyacids during diabetes.