| Aim: The tssiue kallikrein–kinin system plays an importment role in regulation of cardiovascular and renal function. It is one of the major regulators for renal circulation. Potential protective roles of the tissue kallikrein-kinin system to improve renal function have been manifested in our recent studies in renal injury were used. The therapeutic effect of tissue kallikrein gene on chronic renal failure was not involved in recent studies. To investigate the effects and mechanism of tissue Kallikrein gene therapy on chronic renal failure ,we based on previous studies and the first injected intravenously recombinant adeno-associated virus-mediated (rAAV) human tissue kallikrein gene (HK) to chronic renal failur rats by 5/6 nephrectomized and observed changes of renal function and morphology, as well as receptors mRNA expression,which express in kidney and regulate renal function. We also studied in vitro the regulation of bradykinin on bradykinin-B2 receptor mRNA, and then the possible involved signal transduction pathways were explored. Methods and results一,Human kallikrein cDNA was packed in a rAAV-based plasmid vector.The rAAV particles were produced by transfection in 293 cells, and its titer was measured by dot blot methods. Male wistar 24 were divided into two groups .A control group (sham Op) of 6 rats underwent sham operation consisting of laparotomy and manipulation of the renal pedides. Surgery group of 18 rats underwent 5/6 nephrectomization published by Hinojosa-Labord, beginning with resection of approximately two thirds of left kidney, one week after , right kidney was removed .One month after surgery, 18 rats with 5/6 nephrectomized were randomly divided into three groups: Only operation group of 6 rats (op only) without receiving virus injection ; GFP group of 6 rats (GFP) injected single dose rAAV-GFP via the tail vein with 1×1011p.f.u.; HK group of 6 rats (HK) injected single dose rAAV-HK via the tail vein with 1×1011p.f.u. At before surgery and every month after surgery until rats were executed, the caudal arterial pressure was measured using tail cuff blood pressure system; blood and 24h urine samples were collected to measure the serum creatinine, 24h urinary microalbumin and urinary infiltrator pressure. The liver, heart, aorta and kidney of rats were obtained for histological analysis, reverse transcription-polymerase chain reaction (RT-PCR), Real-time RT-PCR and western Blot. The expression of HK gene in experimental rats was assessed by enzyme-linked immunosorbent assay (ELISA), RT-PCR and western Blot. The expression of receptors mRNA, including bradykinin B2 (B2R),Dopamine D1-like(D1R), angiotensinⅡAT1(AT1R), endothelin ETA(ETAR) and vasopressin V2 receptor (V2R), were assessed by real-time RT-PCR. Results showed: 1.rAAV-HK injected via the tail vein to achieve a long-term and stable expression of tissue kallikrein in chronic renal failure rats with 5/6 renal mass reduction. 2. Systolic blood pressure of 5/6 nephrectomized rats was significant higher than that of sham -operation rats (182±12mmHg,n=18, vs 103±9mmHg,n=6, p<0.001), prior to gene delivery. One-month postgene delivery, blood pressure significantly reduced in rAAV-HK treated rats, as compared with rAAV-GFP treated rats. Three months postgene delivery, rAAV-HK treated rats maintained a significant reduction in blood pressure compared with only operation and rAAV-GFP treated rats ( 163±13mmHg vs217±16mmHg vs220±13mmHg , n=6, p<0.001). 3. Serum creatinin level of 5/6 nephrectomized rats was significant higher than that of sham -operation rats (45.3±7.2umol/L,N=18,vs 6.5±19umol/L,N=6, P<0.001). One month postgene delivery ,there were no significant difference in serum creatinine between rAAV-HK treated rats and rAAV-GFP treated rats .Two months postgene delivery, there were a increase in serum creatinin levels of rAAV-GFP treated and only operation rats, as compared with rAAV-HK treated rats,no significant difference. Three months after gene delivery , rAAV-GFP treated and only operation rats maintained increase in serum creatinine levels , rAAV-GFP treated rats had no significant difference in serum creatinine levels compared with one month postgene delivery , but there were significant difference in serum creatinine levels between rAAV-HK treated rats and rAAV-GFP treated rats(58.9±12.3umol/L vs 127.8±20.33 umol/L , n=6 , p<0.001). 4. Three months postgene delivery, there were markedly decreased in 24h urinary excretion of microalbumin of rAAV-HK treated rat(6.95±0.83mg/24h vs 12.85±1.54 mg/24h vs 13.35±1.12 mg/24h,n=5,P<0.05,), and increased in urinary infiltrator pressure(589.3±74.1 Osmd/Kg vs 413.5±68.2 Osmd/Kg vs 433.3±81.1 Osmd/Kg, n=5 ,P<0.05, )as compared to only operation and rAAV-GFP treated rats. 5. Kidney sections stained with PAS and HE to illustrate beneficial effects of kallikrein gene delivery on nephrectomized rats, it reduced tubular dilation, brush border disruption, glomerular sclerosis, PAS positive area, glomerular cellular number. 6. The expression of B2R and D1R mRNA in kidney were up-regulated, the other way round, the expression of AT1R, ETAR and V2R mRNA were down-regulated in HK group rats as compared with only op group and GFP group rats. 7. Western Blot indicated the expression of B2R protein in HK group rats was up-regulated.二,In vitro, we incubated 293 cell with bradykinin(BK) , BK +HOE-140, BK +MAPK inhibitor(apigenin),BK +PI3K inhibitor(LY294002)and BK +PKC inhibitor(H7)inhibitor to assess the expression of B2R mRNA by real-time RT-PCR and evaluate the expression and phosphorylation of signalling molecules MAPK as well as PI3K by western Blot. Results found: 1.BK markedly up-regulated the expression of B2R mRNA in 293 cell. 2.Apigenin and LY294002 inhibited up-regulation of B2R mRNA induced by BK, H7 didn't inhibit. 3. Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2R by BK-mediated.Conclusions: 1.rAAV-HK injected via the tail vein in chronic renal failure rats with 5/6 renal mass reduction offers long-term and stable expression. 2.rAAV-mediated HK cDNA delivery attenuates hypertension, improve renal function and reduce glomerular sclerosis injury in chronic renal failure rats with 5/6 renal mass reduction. 3. rAAV-mediated HK cDNA delivery reduced blood pressure and improved renal function, and attenuated glomerular sclerosis in 5/6 nephrectomized rats through up-regulating expression of B2R and D1R mRNA, which improved renal function, and down-regulating expression of AT1R, ETAR and V2R mRNA in kidney. 4. In vitro, our findings indicated that BK up-regulates the expression of B2R mRNA in 293 cell. 5. Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2 receptor by BK-mediated.
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