Establishment Of Rat Models Of Type 2 Diabetic Nephropathy And The Research Of Lentiviral Vectors Expressing Small Interfering RNA Targeting CoL I In Rats With Diabetic Nephropathy

Renal fibrosis is the characteristic pathological change of end-stage chronic kidney disease such as diabetic nephropathy. The root cause of renal fibrosis is the excessive accumulation of extracellar matrix, in the process of which, collagen typeâ… plays a critical role. Based on this theory, it could be a new approach to decelerate the process of renal fibrosis by using RNA interference to weaken the over-presence of gene of collagen typeâ… .Part 1: Establishment of rat models of type 2 diabetic nephropathyOBJECTIVE:To optimize a rodent model of type 2 diabetes kidney injury that would replicate the metabolic characteristics of the human syndrome and be suitable for mechanism and therapy. METHODS:Male SD rat at four weeks of age were randomly divide into control group with conventional diet(A group,n=15),model group with high glucose and high fat diet with streptozotocin(STZ) injection(B group,n=15).B group Rats were fed two weeks with the diets enriched w ith sucrose(10%w/w)and lard(12%w/w)and egg(10%w/w)to induce FINS resistance. Hyperglycemia was developed by intraperitoneal injection STZ in these rats with 30mg/kg three times within continuas three days.Observing rats was done for six months after STZ injection. The levels of body weight,urime albumin ,blood glucose .Blood samples were collected for serum creatine ,BUN, serum FINS before experiment,injection and after injection six weeks. RESULTS:There was no significant difference in body weights,blood glucose,serum FINS,serum creatine and BUN before interference among two groups(P>0.05). However,serum FINS in high fat and high glucose diet with STZ group were significantly increased after 2 weeks of diet interference(P<0.01). Blood glucose were significantly increased after 24 hours of injection(P<0.01). Clinic phenomenon showed continuas proteinuia.Pathology study of the kidney showed proliferation of mesangial cells, increase of mesangial matrix . CONCLUSION:Rat model of type 2 diabetic nephropathy can be successfully developed by high fat and high glucose diet with continuas low dose STZ, which preferable.Part 2: Construction of colâ… recombinant lentivirus interference vectors and its transfection to kidney of rat with diabetic nephropathyOBJECTIVE:To construct a recombinant lentivirus vector of RNA interference targeting collagen type I gene and to select the most efficient small interfering RNA(siRNA)to suppress collagen type I in target cells, and to use the selected lentivirus vector to analyze gene transduction of diabetic nephropathy rat kidney in vivo. METHODS: According to Genbank information of collagen type I gene , three siRNA and double strand DNA were designed, synthesized and inserted into the lentivirus vector. The recombinant lentivirus vector system was confirmed by PCR and sequencing, then it was used to transfect the target cells. The change of collagen type I gene expression was determined by RT- PCR . The most efficient lentivirus vector or saline solution (as control) were delivered into kidneys by the left renal artery injection.Animals were killed at 15 days after treatment. The transducted gene expression was determined by GFP observation. RESULTS: The results of enzyme digestion and sequencing showed that the recombinant lentivirus vector was constructed successfully. The result of RT- PCR showed that target 1 had the highest interfering efficiency(about 77%). The presence of GFP in kidney can be observed. CONCLUSION: The recombinant lentivirus vector of RNA interference targeting collagen type I has been successfully constructed. It can effectively suppress collagen type I expression in vitro and can effectively and stably transfer exogenous gene to rat kidney in vivo.Part 3: Inhibitory effect of lentiviral vectors expressing small interfering RNA targeting collagen type I gene in vivi and in rats with diabetic nephropathyOBJECTIVE: To investigate the effect of RNA interference Lentivirus vectors on collagen type I in diabetic nephropathy rats .METHODS: 14 male Sprague-Dawley rats with diabetic nephropathy injected with STZ were randomly divided into RNA interference Lentiviral vectors(n=6) and empty vectors groups(n=6),control group(n=2). Each of the RNA interference Lentivirus vectors and empty vector groups were injected with RNA interference Lentivirus vectors and empty vectors 200ul via renal artery injection respectively. The corresponding blank control group were given doses of saline.Rats were sacrificed at week 2 after a successful modeling. To confirmed the efficacy of RNA interference Lentivirus vectors in rat kidney, the collagen I levels were determined by fluorescence quantitative PCR. The expressions of collagen I protein were detected by immunohistochemical method while examined by Western blot. RESULTS: The collagen type I levels were greatly down-regulated in the RNA interference Lentiviral vectors treated group (48.00+/-3.00)% versus the control group (P < 0.05). The immunohistochemical semi-quantitative method indicated that there was a decrease in the in the RNA interference lentivirus vectors treated group versus the control group: (22.00 +/-8.00)% in collagen I. CONCLUSION: RNA interference Lentivirus on collagen I may be useful in preventing the progression of diabetic nephropathy through influencing the expression of collagen type I .