| Chronic renal failure is one of the major diseases that seriously endanger human health.Its pathological process is chronic and progressive, with the main representation ofglomerular clerosis, tubular analosis and renal interstitial fibrosis in the later period. At last, itmust depend on renal replcement therapy which makes a heavy burden for the family and thesociety. So the treating of slowing its progress has great social and economic benefits.The5/6nephrectomized rat is a common and classic model in the study of chronic renalfailure. After5/6nephrectomy, the nephrons are reduced which cause glomerular highinfusion, high pressure and high filtration. This leads to tubular atrophy, glomerular sclerosisand renal interstitial fibrosis. These changes resemble to the pathological progression ofchronic renal failure in human.There are a lot of mechanisms to participate in its outbreak in the progress of chronicrenal failure. Excessive proliferation of mesangial cells and excessive deposition of extracellular matrix are common manifestations of glomerular clerosis and renal interstitial fibrosis.Excessive proliferation of mesangial cells represents a response to glomerular injury and theactivated mesangial cells are primary sources of extra cellular matrix, which togethercontribute to the pathogenesis of chronic renal failure. Therefore, the process of chronic renaldiseases can be delayed by inhibiting the proliferation of mesangial cells. So it is important tofurther develop the medicines of inhibiting mesangial cells to proliferate.Transforming growth factor-β1(TGF-β1) is an important factor that stimulateshyperplasia of glomerulus mesangial cells, and is also one of the key factors responsible forthe increased fibrotic changes. It can promote the process of chronic renal failure. α-smoothmuscle actin (α-SMA) is a characteristic protein of smooth muscle cells and myofibroblasts,and high expression of α-SMA in the kidney is the marker of tubular epithelial-myofibroblasttransdifferentiation. In addition, α-SMA induced activation of mesangial cells contributes to the deposition of extra cellular matrix and glomerulosclerosis.Hepatocyte growth factor (HGF) was discovered in the serum of rat that the liver waspartly cut off at the beginning, and then was purified from other tissues successively. It is apleiotropic cytokine which has multiple biological functions, such as promoting caryomitosis,accelerating cell locomotion and antiapoptosis. It can also mediate occurrence anddevelopment of fibering diseases and had been verified that it could inhibit hepatic fibrosis.At present, there are many studies showed that it could also inhabit renal interstitial fibrosis.In order to seek a better therapeutic regimen to ameliorate chronic renal failure, we exploredif the gene treating of HGF had potential value for chronic renal failure, and further explorethe mechanisms.Objective:To investigate the efficacy of HGF gene treating in ameliorating chronic renal failure andto elucidate the underlying mechanisms.Methods:1. In vitro study:(1) Rat glomerulus mesangial cells (RGMCs) were brought up outsidebody, With Lipopoly saccharide (LPS) stimulating glomerulus mesangial cells to propagate.To determine the effect of PCI-neo-HGF on RGMCs proliferation, the method of MTT wasused.(2) To determine the effects of PCI-neo-HGF on the mRNA and protein expressions ofTGF-β1and α-SMA, RT-PCR and Western blot were performed.2. The model of chronic renal failure (5/6nephrectomized rat) construction: The ratswere anaesthetized with2%Nembutal by intraperitoneal injection. Cut open the skin at the leftside of the back, and cut off the upper pole and under pole (1/3kidney respectively), stoppedbleeding by axenic string. The second surgery was operated at the7th days after the firstsurgery, and the anaesthetized method was as before. Cut open the skin at the right side of theback, then extirpat the right kidney.3. In vivo study: The animals were randomized into8groups: control (n=12, Ctrl),PCI-neo (n=12, PCI-n), sham-operation (n=12, Sham), model (5/6nephrectomy, n=12,Model), Lotensin (n=12, Loten), low-dose PCI-neo-HGF (n=12, Low), high-dose PCI-neo-HGF (n=12, High), Lotensin+high-dose PCI-neo-HGF (n=12, Lo+H).PCI-neo-HGF was given to Low group (230μg per week), High group (460μg per week) andLo+H group (460μg per week) rats by intramuscular injection(inner muscles of left hind)onthe2nd,3rd and4th week after the2nd surgery. PCI-n group rats were treated with PCI-neo(300μg per week), and Ctrl, Sham, Model and Loten group rats were treated with aluminumhydroxide gel (0.5ml) with the same method and at the same time. Lotensin (0.6mg/100g/day)was given to Loten and Lo+H group rats in drinking water (20ml) from the day after the2ndsurgery, and continued for5weeks. The rats in the other6groups were given the same doseof drinking water at the same time. The animals were executed at the5th and9th after the2ndsurgery respectively. Collected the urine, blood of postcava and took left renal tissue to keepin liquid nitrogen and fastened in10%formalin respectively. The kidney specimens weresubjected to pathological examination by Hematoxylin-Eosin staining and detected oftransforming growth factor-β1and α-smooth muscle actin expressions by RT-PCR, Westernblot and immunohistochemistry.Results:1. PCI-neo-HGF had no effect on the hyperplasia of normal RGMCs. But in the RGMCsthat were stimulated by LPS, it could restrain their proliferation. At the same time, HGF couldinhibit α–SMA and TGF-β1expressions in a dose and time dependent manner in RGMCs thatwere stimulated by LPS, but not in RGMCs that were not stimulated by LPS.2. The model of5/6nephrectomized rat was constructed and showed it was an idealmodel to study chronic renal failure by detecting the renal function and renal pathology.3. PCI-neo-HGF and Lotensin all had renal protection. The reno-protective effect ofPCI-neo-HGF enhanced with the increase of the dose. Lotensin plus high-dose PCI-neo-HGFgroup exhibited the most significant therapeutic effects in protecting renal function,decreasing urine protein and relieving the lesion of renal tubuleinterstitium. Further,PCI-neo-HGF and Lotensin reduced mRNA and protein expressions of α–SMA and TGF-β1.The inhibition for α-SMA and TGF-β1expressions reached to the best level when Lotensinand high-dose PCI-neo-HGF were used together, which were in accordance with the biochemical and pathological changes.Conclusions:In our study, PCI-neo-HGF exhibited significant therapeutic effects in protecting renalfunction, decreasing urine protein and relieving the lesion of renal tubuleinterstitium in themodel of chronic renal failure. This protection may be associated with down-regulation ofα–SMAand TGF-β1expressions in RGMCs and renal tubuleinterstitium. |
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