| Background Diabetic nephropathy (DN) is one of the leading causes to end stage renal disease(ESRD). Approximately 30-40% of patients with type?and 15% with type?DM develop renal dysfunction. High glycemic level contributes to the development of diabetic nephropathy; however, the mechanisms underlying hyperglycemia induced injury are not fully understood. Recent observations indicate that hyperglycemia triggers the generation of free radicals and oxidant stress in renal tubular epithelial cells, which might be one of the important mechanisms in the development of renal dysfunction in diabetic nephropathy. ROS mediates hyperglycemia-induced activation of signal transduction cascades (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (NF-?B, activated protein-1, and specificity protein-1) leading to upregulation of TGF-?1 and ECM accumulation, and finally result in cellular injury in kidney.Ras superfamily of small molecular weight G-proteins was reported to be as an important factor participating in mitochondrial dynamics, resulting in mitochondrial dysfunction. To be a member of Ras superfamily, Ras-proximate-1b (Rap1b) is a homolog of a well-characterized small GTPase and is known to antagonize the mitogenic and transforming activity of Ras. Recently, Raplb signal has received more and more attention in nephrology because it was found to be involved in progression of renal diseases such as diabetic nephrology and glomerulonephritis. However, very limited literature information is available that documents the effects of Rap1b in HG-induced intracellular ROS generation in the mitochondria of tubular cells in hyperglycemia-induced injury models. Researches have shown that Rap1b is upregulated in the hyperglycemic state and is known to increase B-Raf, an antiapoptotic effector protein. And in addition, our previous studies observed that Raplb ameliorates glucose-induced mitochondrial dysfunction against oxidase stress and thus prevents the apoptosis in renal tubular cells in diabetic nephrology (DN). However, the mechanism is unclear. To this end, experimental research is carried out as follow. Chapter?The expression of Raplb in the kidney of diabetic nephropathy patientsObjective To observe the expression of Rap1b in the kidney in order to investigate the relationship of Rap1b and high gluose ambience in diabetic nephropathy patients.Methods 12 patients were enrolled into the study, they were diagnosed as DN (n=6) or minimal changes disease (primary kidney disease, control group, n=6) based on clinical manifestations and renal pathology. Renal pathological changes were observed by Masson, PASM and PAS staining. Interstitial damage score and collagen deposition score were evaluated. The expression of Rap1b was tested by immunohistochemistry and the relationship of Rap1b and tubular interstitial damage in DN was analyzed.Results Masson, PASM and PAS staining showed that there were many lesions in the kidneys of DN patients such as moderate to severe proliferation of mesangial matrix, nodular sclerosis, diffuse or segmental thickening of the basement membrane, tubular atrophy and compensatory expansion, local interstitial fibrosis. Meanwhile, the lesions in the kidneys of minimal change disease patients were less severe. There were mild mesangial cell and matrix proliferation, basement membrane is not thick, mild granular degeneration and vacuolar degeneration in renal tubular epithelial cells and no significant abnormalities in interstitial and renal vascular. Interstitial damage score and collagen deposition score in the kidneys of DN patients were higher than that in control group. Immunohistochemistry showed that the expression of Raplb was significantly up-regulated in DN patients compared with that in control group.Conclusion The expression of Raplb is significantly up-regulated in the kidney of DN patients and Raplb is negatively related with renal interstitial fibrosis. Chapter?. The expression of Raplb and its role in the process of high glucose induced mitochondrial oxidative injury in human renal tubular epithelial cellsObjective To detect the expression of Raplb in human renal proximal epithelial cell lines (HK-2) and investigate the role of Raplb in the development of high glucose induced induced mitochondrial oxidative injury and apoptosis in HK-2.Methods The cells were exposed respectively in different concentrations of D-glucose(5.5,10,20,30 mM). The expression of Raplb protein was examined by Western Blot. The mitochondrial ROS was detected by Mitosox staining using confocal microscopy. Respiratory Chain Complex Activities assay, ATP assay, Catalase and GSH-Px assay were detected for mitochondrial function. Western blot and Realtime PCR were examined for procaspase-9, procaspase-3 and MnSOD2. Cell apoptosis was measured by Hoechst 33258 staining using fluorescence microscopy. Wild-Type Rap1b plasmid (cDNA3.1/Rap1b) and the Rap1b mutant plasmid (Rap1bS17N and Rap1bT61R) were transfected into HK-2 cells using lipofectamine 2000, respectively. After selection of stable transfectants, cells were maintained in the defined medium.30mM D-glucose was added (5.5mM D-glucose as control).And the detections above were examined in order to investigate the effect of Rap1b on the high glucose-induced mitochondrial injury.Results Stimulation of HK-2 with 30mM D-glucose resulted in a significant decrease in the expression of Raplb protein, compared with medium control group. However, there was no significant difference between the control and D-mannitol group. Decrease of Raplb GTP protein in HK-2 treated by different concentrations of high glucose in a time-dependent manner. In additon, HK-2 treated with 30mM D-glucose resulted in a time-dependent decrease in the expression of Raplb GTP protein, with the peak at 2h. Compared to control (5.5 mM),30 mM D-gluocse (HG) induced mitochondrial dysfunction, including decreased activity of respiratory chain complex?activity, the level of ATP and the decreased capacity of the cellular antioxidant defense system(Catalase, GSH Px and MnSOD2). HG increases overproduction of mitochondrial superoxide. Pro-caspase-3 and pro-caspase-9 were both decreased accompanied by increasing cellular apoptosis. However, over-expression of Rap1b partially reversed such abnormalities.Conclusion Raplb could also protect mitochondria from high glucose-induced injury by reversing the decreased activity of respiratory chain complex?activity, the level of ATP, the decreased capacity of the cellular antioxidant defense system, alteration of expression of Pro-caspase-3 and pro-caspase-9. These data show Rap1b contributes for the inhibition of mitochondria-mediated pathway of apoptosis induced by high glucose. Chapter?Rap1b regulates high glucose induced mitochondrial oxidative injury via modulation of balance of fusion and fission in mitochondria in renal tubular epithelial cellsObjective To investigate the mechanism by which Rap1b modulates the process of high glucose induced mitochondrial oxidative injury in renal tubular epithelial cells (HK-2).Methods Wild-Type Raplb plasmid (cDNA3.1/Rap1b) and the Rap1b mutant plasmid (Rap1bS17N and Rap1bT61R) were transfected into HK-2 cells using lipofectamine 2000, respectively. After selection of stable transfectants, cells were maintained in the defined medium. 30mM D-glucose was added (5.5mM D-glucose as control). Real-time PCR and Western blot were detected for mitochondrial fusion and fission proteins(Drpl,Mfh2) and mitochondria-related transcription factors PGC-1?and NRF-1.ResultsAfter 48h of treatment, high glucose triggers the development of imbalance of fusion and fission in mitochondria, including increased Drp1 and decreased Mfh2 in mRNA levels and protein levels. In addition, mitochondria-related transcription factors PGC-1a and NRF-1 were both decreased. While the effect was significantly reversed in Raplb transfected HK-2 cells, compared with that in Rap1b mutant plasmid.Conclusion Overexpression of Raplb might reverse the effects of oxidative injury induced by 30mM D-glucose in HK-2 via maintaining the PGC-1a/NRF-1-regulated fusion and fission in mitochondria. |
PDF Full Text Request