| Background:Kidney is an critical organ of the human body, and it is mainly responsible for the excretion of body and maintaining a normal internal environment. In the modern world, chronic kidney disease has become one of the serious harmful public health diseases. Proteinuria is an independent risk factor for chronic kidney disease. Persistent proteinuria is irreversible, and patients will progress to end-stage renal failure ultimately. Glomerulus is the most important part of kidney filtration and excretion function. In some clinical pathological cases, the glomerular filtration barrier will be damaged and followed by large proteins leakage and proteinuria. The glomerular filtration barrier consists of capillary endothelial cells, basement membrane and podocytes. Podocytes play a key role in the process of glomerular filtration. One of the main pathological changes is proteinuria which caused by glomerular filtration barrier damage. Studies demonstrate that urinary protein can damage proximal renal tubular epithelial cells directly via some inflammatory mediators. These findings suggest that proteinuria can promote the progress of chronic kidney disease by damage proximal tubular epithelial. Previous studies have indicated that the number and density of podocytes and urine protein were significantly negatively correlated, especially in the most massive proteinuria patients, but the mechanism is not clear yet.Integrin a3β1is an transmembrane protein, which can transmit in-outside signals, plays an important role in maintaining sufficient adhesion between podocytes and GBM. On the one hand, integrin a3β1binds ligands on the GBM directly and anchors podocytes on the GBM firmly. On the other hand, podocytes connect to the cytoskeleton indirectly and adjust the dynamic of cytoskeleton for maintaining normal foot process structure. The ILK can connect with integrin a3β1through C terminal and connect with α-actinin-4through the N terminal. ILK can participate integrin signal pathways, and join in α-actinin-4mediated cytoskeleton dynamic process. a-Actinin-4is specifically expressed in podocyte, and connects to the F-actin cytoskeleton to maintain foot process structure. The objective of this study is to observe the change of renal function and total expression of renal ILK, integrin β1and a-actinin-4in albumin overload rats; and investigate the effect of albumin overload on the expression of ILK, integrin β1and a-actinin-4in vitro cultured podocytes. To further provide theoretical basis for in-depth understanding relating mechanisms of podocyte injury and chronic kidney disease.Methods:Effects of albumin overload on kidney function and expression of ILK, integrin β1and a-actinin-4The rat’s24h urine was collected before the experiment start in metabolism cage. The model group rats were treated with BSA (2g per rat) by intraperitoneal injection, and the control group treated with equal volume saline. After albumin injection on the day1st,3th,7th,13th, the24h urine was collected respectively. The rats were sacrificed on14th day, kidneys and blood samples were taken for the follow-up tests.1. Effects of albumin on the renal functionThe body weight and urine volume were recorded, and the total urinary protein and urine creatinine clearance were measured. The left kidney was used for morphology examination (HE staining and transmission electron microscopy), the right kidney was weighted for calculating kidney index first and then frozen in liquid nitrogen for further western blot and RT-PCR analysis. Blood serum creatinine was determined. 2. Effects of albumin on the expression of renal total ILK, integrin β1and α-actinin-4Using western blot and immunohistochemistry staining to observe the effects of albumin on the renal total ILK, integrin β1and a-actinin-4protein level expression; and the mRNA level of those three molecules was detected by RT-PCR.Effects of albumin overload on the expression of ILK, integrin β1and α-actinin-4in podocytesDifferentiated podocytes (MP5) were cultured in1%FCS overnight, and then treated with0,5,10and20mg/mL BSA for12,24and48h. Western blot and immunofluorescence were used for detecting the protein level of ILK, integrin β1and a-actinin-4; Real-time quantitative PCR was used to detect the mRNA level of those three molecules in podocytes.Results:Effects of albumin overload on renal function and expression of ILK, integrin β1and α-actinin-41. Effects of albumin overload on the renal function.The24h urinary total protein sharply rised to peak on day3th after albumin injection, and maintaining a high level from day7th to13th. Mean urine volume decreased apparently compared with control group. In HE; staining, kidney in albumin group revealed glomerular cell fusion, and proximal renal tubular epithelial cell walls damaged. Electron microscopic photographs shown loot process fusion significantly and GBM exposed in albumin group.2. Effects of albumin on the expression of renal total ILK, integrin β1and a-actinin-4Western blot and immunohistochemistry staining showed that the total ILK and a-actinin-4protein were significantly higher than control group, while the integrin β1protein level was much lower than control group. RT-PCR results demonstrated the same change trends of mRNA expression level to the protein expression level above. Effects of albumin overload on the podocytes expression of ILK, integrin β1and a-actinin-4In vitro study, the albumin can up regulate ILK and a-actinin-4protein and mRNA level of cultured podocytes in a concentration-dependent and time-dependent manner. On contrast, the expression of integrin β1protein was down regulated in a concentration-dependent and time-dependent manner in albumin overload treated podocytes. High concentration of albumin can increase the integrin β1mRNA expression first and then decrease the integrin β1mRNA expression. With time extending, the integrin β1mRNA decreased gradually.Conclusion:1. Albumin overload can cause rat renal function disorder. Came up with proteinuria, oliguria, creatinine clearance decreased, kidney index increased and followed by kidney injury.2. The mechanism of renal function disorder may be attributed to abmormal expression of ILK, integrin β1and a-actinin-4in podocytes. |
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