Ucn Against The Development Of Diabetic Nephropathy And Its Mechanism

Hyperglycemia induces process of overproduction of mitochondrial reactive oxygen stress (ROS) in endothelial cells, which is believed to be the main molecular mechanism of diabetic complications including diabetic nephropathy (DN). Impairment of endothelium-dependent vasodilatation is found in type 2 diabetes. Increased thickness of glomerular basement membrane and augmentation of glomerular extracellular matrix (ECM) are recognized as pathological hallmarks of diabetic nephropathy. ECM accumulation is due to the increased synthesis and decreased degradation of ECM. UCN is a 40-amino-acid peptide related to the corticotrophin-releasing factor (CRF) family, which suppresses production of ROS & expression of VEGF in endothelial cells and sustains endothelium-dependent rat coronary artery relaxation. Furthermore, UCN induces matrix metalloproteinase (MMP-9) expression of cultured cells from human placenta and fetal membranes, which is related to extracelluar matrix (ECM) degradation. These reports highly indicate that UCN may play an important role in the development of DN.We investigated effects of UCN on DN and the possible mechanisms by using cultured rat mesangial cells (MC), db/db mice and DN model rats induced streptozotocin-complete Freund's adjuvant (STZ-CFA). In the first part, advanced glycation end products (AGEs) formation in vitro and cultured MC were used to investigate the effects of UCN on AGEs formation, high glucose/AGEs induced abnormal cell proliferation, overexpression of transforming growth factor (TGF-β1), connective tissue growth factor (CTGF), collagen type IV and TGF-β1 induced overproduction of vascular endothelial growth factor (VEGF). The results showed that UCN did not affect the AGEs formation process, however, it inhibited the over expression of TGF-β1, CTGF, collagen type IV induced by high glucose/AGEs and overproduction of VEGF induced by TGF-β1. Treatment with unselective CRF receptor blocker-astressin reversed such effects.In the second part, db/db mice were used to investigate the amelioration effect of UCN on type 2 DN. UCN was injected intraperitoneally to the mice for 6 weeks. The effects of UCN on blood glucose, body weight & food intake were recorded weekly. At the end of the experiment, plasma insulin, blood glucose, blood urine nitrogen (BUN), creatinine (Crea), AGEs level, sorbitol concentration in RBC and superoxide dismutase (SOD) activity & malonaldehyde (MDA) level in kidney homogenate were examined. The pathology change of kidney was observed by using HE- and PAS- stained paraffin section. The results showed that UCN significantly decreased body weight, plasma AGEs level, BUN level and Crea level. However, food intake, plasma insulin and plasma glucose level remained unaffected. Superoxide dismutase (SOD) activity was markedly increased, whereas malonaldehyde (MDA) level in kidney homogenate and concentration of sorbitol in red blood cells (RBC) were significantly decreased in UCN-treated group. UCN significantly decreased ECM expansion and accumulation in kidney. The beneficial effects of UCN on db/db mice could be abolished by astressin except its inhibition of sorbitol accumulation.In the third part, UCN was injected intraperitoneally to DN rat model induced by STZ-CFA (from the 7th week after the beginning of the experiment) daily for 8 weeks. Body weight and blood glucose were recorded weekly. At the end of the experiment, blood sample was collected to analyze effects of UCN on blood glucose, insulin, BUN, Crea, cholesterol, HDL, LDL, SOD, MDA level and sorbitol concentration in RBC. Urine of 24 h was collected and the effects of UCN on urine volume, Crea, urine nitrogen, microalbuminuria were detected and creatinine clearance rate (Ccr) was calculated. ECM accumulation & mesangial area expansion were observed by using HE- and PAS- stained paraffin section and percent mesangial area was calculated. Overexpression of TGF-β1,CTGF,VEGF and VEGF mRNA in the renal was presented by using immunohistochemistry & RT-PCR. Our results showed that UCN did not influence the serum blood glucose, insulin level and body weight; however, it decreased serum cholesterol, LDL, MDA, BUN, Crea, AGEs level, RBC sorbitol concentration and kidney weight significantly. Likewise, serum HDL level and SOD activity were obviously enhanced by UCN treatment. Total urine volume and microalbumin excretion of 24 h were diminished and Ccr increased significantly by UCN. Furthermore, UCN ameliorated ECM accumulation, mesangial area expansion and collagen type IV expression, which was related to its decreasing effects on overexpression of TGF-β1,CTGF,VEGF,VEGF mRNA in the kidney or glomeruli. Astressin treatment reversed the effects of UCN except its inhibitory effect on polyol pathway activity. RT-PCR and immunohistochemistry were used to compare the expression of UCN & UCN mRNA in kidney between normal rats and STZ-CFA induced DN rats. Although UCN mRNA expression appeared to increase in DN rats, UCN expression was not obviously enhanced. It needs further investigation to do other experiments to quantify the expression of UCN and UCN mRNA due to the time limitation.Taken together, UCN could significantly prevent DN development and this effect was related to CRF receptors. Inhibition of AGE accumulation, polyol pathway activation, ECM expansion & accumulation, TGF-β1, VEGF & CTGF over-expression, ROS over-production and regulation of lipid profiles may all contribute to the amelioration of DN by UCN.