| Background and Objective:Klotho (Kl) is a newly identified antiaging gene, which displays extremely shortened life span with multiple disorders resembling human premature-aging syndromes. There are two forms of klotho protein, the transmembrane form expressed primarily in renal tubular cells and the secreted form circulating in the blood. Although klotho protein is predominantly expressed in distal convoluted tubules in the kidneys and in the choroid plexus in the brain, klotho can exert its effects in tissues or cells that do not express klotho, suggesting that it may function as an endocrine hormone.Atherosclerosis is a generalized and inflammatory vascular disease frequently associated with renal disease. Also, chronic renal failure (CRF) enhances a number of well-established risk factors for atherosclerosis and affects several additional factors that potentially promote atherogenesis, especially the balance between oxidants and antioxidants. Therefore, an anti-oxidative stress strategy may be a promising approach to prevent and treat atherosclerotic disease in CRF condition.Indeed, atherosclerosis is one of the phenotypes observed in Kl-/- mice. Conversely, a previous study had shown that klotho gene delivery could ameliorate vascular endothelial dysfunction which was regarded as the critical factor in the development of artherosclerosis. In our previous study, we found that klotho may play a role in the progression of uremic artherosclerosis. In the meantime, some previous studies revealed that klotho may induce resistance to oxidative stress and protect tissues from oxidative damages.The purpose of this study was to test the hypothesis that klotho gene delivery attenuates the progression of artherosclerosis in the CRF mice.Methods:1. Male apoE–/– mice were allocated to a western-type diet and induced by a two-step surgical procedure. We applied cortical electrocautery to the right kidney through a 2-cm flank incision and performed left total nephrectomy through a similar incision 2 weeks later.2. Serum levels of urea, total cholesterol and triglycerides were measured. The aortic root was sectioned serially in slices and stained with hematoxylin–eosin. The cross-section surface area of the vessel and cross-section surface area of the lesion were measured with computer-assisted image analysis equipment. The mRNA and protein expression of renal klotho were examined by RT-PCR and Western blot methods. The level of serum klotho protein was determined with klotho ELISA kit.3. A recombinant adeno-associated virus vector encoding klotho (pAAV-klotho) was constructed with the technique of molecular cloning. The recombinant expression plasmid or pAAV-LacZ was co-transfected of HEK 293 cells with pHelper and pAAV-RC by lipoplast transfection method. High purified viral particles can be obtained by the combination of gradient centrifugation and virus titer was detected by spot hybridization. Recombinant AAV viral particles were used to infect HEK 293 cells and to estimate the efficiency of transfection as well. rAAV-LacZ in cell culture was harvested for X-gal staining ofβ-galactosidase activity.4. CRF mice with artherosclerosis were established and were randomly divided into four groups which were control, CRF, rAAV-EC-Kl and rAAV-LacZ transplantation group. In rAAV-EC-Kl or rAAV-LacZ transplantation group, AAV mediated-klotho or LacZ were injected into two distinct sites of the anterior tibial muscle of CRF mice. At 2 wk, 4 wk and 6 wk after transfection, blood, aorta and kidneys of mice were collected. Serum TC and TG levels were assessed to analyze the effect of klotho transplantation for uremic artherosclerosis. And the level of NO in serum was detected at the same time after transfection. The concentation of serum klotho was eaqually observed by ELISA. Meantime, the aortic root was sectioned serially in slices and stained with hematoxylin-eosin. The cross-section surface area of the vessel and cross-section surface area of the lesion were measured with computer-assisted image analysis equipment.Results:1. The CRF mouse model was constructed successly. At 6 wk after nephrectomy, CRF mice showed significantly increased aortic plaque area fraction, aortic root plaque area and aortic cholesterol content as compared with non-CRF mice. Serum urea, total cholesterol and triglyceride concentrations were significantly higher in CRF apo-E-/- mice compared with those in non-CRF controls. Moreover, the mRNA and protein expression of renal klotho and the serum levels of klotho protein were markedly decreased in CRF mice as compared with controls.2. After pAAV-LacZ and pAAV-EC-Kl were produced successly, rAAV-EC-klotho and rAAV-LacZ were packed and purified, and their tite were detected. By immunocytochemistry method after transfection, the gene expression efficiency in HEK 293s was about 65%. The highest concentration of klotho protein or LacZ in cell culture was observed after transfection with 1～2×1010IU/ml of titer.3. At 2, 4 and 6 wk after rAAV-LacZ or rAAV-EC-Kl transfection, serum cholesterol and triglyceride concentrations were significantly lower in rAAV-EC-Kl mice as compared with CRF controls or LacZ group. At the same time, the serum NO level and concentration of klotho protein were markly higher in Kl-transfection mice compared with CRF control or LacZ group. And Kl-transfection mice showed significantly decreased in aortic plaque area fraction and aortic root plaque area.Conclusions:1. In the CRF apoE–/– mice with artherosclerosis, the klotho expression was intimatedly correlated with the development of artherosclerosis in CRF.2. pAAV-LacZ and pAAV-EC-Kl were produced successly and viral particle was packed and purified. The titer of rAAV-EC-Kl can be suitable to transfection in vitro.3. After transplantation of rAAV-EC-Kl, the level of blood fat was significantly decreased and NO was increased in Kl-transfection mice compared with CRF mice. And the corruption of aortic artherosclerosis in CRF mice was inhibited obviously in Kl mice. Klotho gene transplantation is beneficial to recover this kind disease.
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