| Part 1 Mechanism and Role of Valsartan and Fosinopril on The Expression of Klotho Gene Regulated by Angâ…¡Charter 1 The Effect of Valsartan on The Expression of Klotho Gene Down-regulated by Angiotensinâ…¡Objective To investigate the mechanism of klotho gene expression regulated by angiotensinâ…¡(Angâ…¡), elucidate the impact of Valsartan (Val) on down-regulation of klotho expression induced by Angâ…¡.Methods Culture cells, rat renal tubular epithelia cells (NRK-52E), were incubated with media either Angâ…¡or Val or both of all. Experimental groups were divided according to different concentration of Angâ…¡[0 (control), 10-9, 10-8, 10-7, 10-6, 10-5mol/L], and Val [0 (control), 10-9, 10-7, 10-5, 10-3 mol/L] for 24 hours. The optimal concentration of AngII(10-7mol/L) was selected to incubated with culture cells for 0 (control), 3, 6, 12, 24 hours. Four groups, which were control, Angâ…¡(10-7mol/L), Angâ…¡(10-7mol/L)+Val (10-5mol/L ) and Val (10-5mol/L), were divided to observe the effect of Val on expression of klotho induced by Angâ…¡. Reverse transcription - polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were applied to evaluate the mRNA and protein expression of klotho gene. 1. In NRK-52E, Angâ…¡down-regulated klotho mRNA expression in a d0se-dependent manner, the stimulatory effect of AnglI (10-7mol/L) was strong in the first 3 hours (P＜0.05), whereas, there was an increasing trend in the latter hours (6-12hours), and reach the control levels (P＞0.05), in the point of 24 hours, klotho mRNA expression was inhibited and decreased by AnglI again as compared to the control levels (P＜0.05)2. No evident was revealed that different concentration of Val had any effect on klotho gene expression (P＞0.05)3. Angâ…¡obviously decreased the levels of kloltho mRNA and protein expression in NRK-52E, this down-regulating effect was reversed by incubating both with Angâ…¡and Val, the expressions of klotho mRNA and protein in Val group were higher than those in Angâ…¡group(P＜0.05), and no statistic significant different was showed as compared to the control (P＞0.05)Conclusions1. Angâ…¡down-regulates klotho gene expression in a dose-dependent manner in NRK-52E;2. Up-regulating klotho gene expression in NRK-52E by Val antagonizing Angâ…¡, suggests that the angiotensinâ…¡type 1 receptor maybe the important factor for Angâ…¡regulating klotho gene expression. Charter 2 The Effect of Fosinopril on The Expression of Klotho Gene Down-regulated by Angâ…¡Objective To investigate the effect of Fosinopril (Fos) regulating klotho gene expression, elucidate the mechanism of Fos regulating the Angâ…¡-induced down-expression of klotho gene.Methods Culture cells, NRK-52E, were incubated with media either Angâ…¡or Fos or both of all. Experimental groups incubated with Fos (10-5mol/L) were divided according to variant points of time for 0 (control), 3, 6, 12, 24 hours. Different concentration of Fos was selected to incubated with culture cells for 0 (control), 10-9, 10-8, 10-7, 10-6,10-5mol/L at the optimal time point (24 hours). Five groups, which were A: control; B: Angâ…¡(10-7mol/L); C: Fos (10-5mol/L); D: Angâ…¡(10-7mol/L) +Fos (10-5mol/L) and E: Cells pretreated with Fos (10-5mol/L) 12hours incubated with Angâ…¡(10-7mol/L) were divided to observe the effect of Fos on expression of klotho induced by Angâ…¡. RT-PCR and IHC were applied to evaluate the klotho mRNA and protein expression, respectively.Results1. Fos up-regulated klotho mRNA in time-dependent manner, and independent of dose-dependent manner;2. Angâ…¡obviously decreased the levels of kloltho mRNA and protein expression in NRK-52E as compared to the control(P＜0.05), the down-regulating effect was reversed by incubating both with Angâ…¡and Fos (P＜0.05), and Fos can inhibit the down-regulated expression of klotho gene induced by Angâ…¡in NRK-52E.Conclusions1. Angâ…¡down-regulates klotho gene expression in NRK-52E;2. Fosinopril up-regulates klotho mRNA in time-dependent manner, and independent of dose-dependent manner in NRK-52E;3. ACEI up-regulates the klotho mRNA and protein expression, and inhibits the down-regulated expression of klotho gene induced by Angâ…¡.Charter 3 Role of Fosinopril and Valsartan on Klotho Gene Expression Induced by Angâ…¡and The Relationship Between Klotho and TGF-β1Objective To investigate role of Fosinopril (Fos) and Valsartan (Val) on klotho gene expression induced by Angâ…¡and the relationship between klotho and TGF-β1.Motheds Culture cells, NRK-52E, were incubated with medias such as Angâ…¡, Fos and Val. Five groups, control, Angâ…¡(10-7tool/L), Angâ…¡(10-7mol/L)+Fos(10-5mol/L), Angâ…¡(10-7mol/L)+Val (10-5tool/L) and Angâ…¡(10-7mol/L)+Fos(10-5mol/L)+Val(10-5mol/L), were divided to observe the expression of the klotho and TGF-β1 mRNA and protein. The klotho and TGF-β1 mRNA were detected by RT-PCR, and Western Blotting was applied to determine the klotho and TGF-β1 protein expression. The linear relationship between klotho and TGF-β1 protein expression was analyzed by Pearson's.Results1. Angâ…¡induced an up-regulated expression of TGF-β1 mRNA, and associated with down-regulated klotho mRNA in NRK-52E as compared to control (all P＜0.05); in cells incubated with Fos or Val or booth of all, the klotho mRNA expression was enheanced, and the TGF-β1 mRNA expression was diminished as compared to the Angâ…¡group(all P＜0.05); combined Fos and Val could not enhance the expression of klotho gene, no significant different was showed as compared to the Fos group and Val group(all P＞0.05).2. IHC and Western Blotting revealed that ldotho protein was increased, TGF-β1 protein was decreased in Fos group, Val group and combination group as compared to AngII group, but no significantly different among the three groups was showed as compared to the Val group or Fos group(all P＞0.05).3. Negative relationship was showed between klotho and TGF-β1 protein expression(r=-0.717, P＜0.05).Conclusions1. Angâ…¡up-regulates TGF-β1 expression in NRK-52E;2. ACEI and AT1RB up-regulates klotho gene expression, but combination of both the agents can not enhance the expression of klotho gene;3. Negative relationship is showed between klotho and TGF-β1 in NRK-52E.Part 2 Role of Fosinopril and Valsartan on Signal Pathway of Angâ…¡-p53/Spl-klotho in NRK-52EObjective To investigate the role of Fosinopril (Fos) and Valsartan (Val) on signal pathway ofAngâ…¡-p53/Spl-klotho in NRK-52E.Methods Culture cells, NRK-52E, were incubated with medias such as Angâ…¡, Fos and Val. Five groups, control, Angâ…¡(10-7mol/L), Angâ…¡(10-7mol/L)+Fos(10-5mol/L), Angâ…¡(10-7mol/L)+Val(10-5tool/L) and Angâ…¡(10-7mol/L)+Fos(10-5mol/L)+Val (10-5mol/L), were divided to observe the expression of the ldotho and TGF-β1 mRNA and protein. IHC was applied to determine the p-p38, p38, p53, Spl and TGF-β1 protein expression, the klotho mRNA and propein were detected by RT-PCR and Western Blotting, respectively. Corrective data were analyzed by Pearson's analysis.Results1. There was no marked difference showed in p38 expression in NRK-52E among the five groups (P＞0.05), while the expression of p-p38 was obviously higher in Angâ…¡group than in the control(P＜0.05), lower expression of p-p38 in Fos group, Wal group and Fos+Val group than the Angâ…¡group (P＜0.05), and no significant difference was showed among the three groups (P＜0.05).2. The expression of p53 and TGF-β1 protein was increased in Angâ…¡group as compared to those in control (P＜0.05), but in Fos group, Val group and Fos+Val group, p53 and TGF-β1 protein was decreased expression as compared to Angâ…¡group (P＜0.05).3. Positive stains of Sp 1 protein were poorer in Angâ…¡group than the control (P＜0.05), while co-incubated with Fos and/or Val, Spl protein expression was stronger in Fos group, Val group and Fos+Val group than in Angâ…¡group(P＜0.05).4. RT-PCR and Western Blotting revealed that klotho mRNA and protein were increased in Fos group, Wal group and combination group as compared to Angâ…¡group(P＜0.05), but no significant difference among the three groups was showed (all P＞0.05).5. A positive correlation was revealed between TGF-β1 and p53 protein(r=0.684; P＜0.05), and a positive correlation was showed between the expression of Spl and Klotho mRNA and protein (r=0.609 and 0.502 respectively; P＜0.05).Conclusions1. Angâ…¡up-regulates the expression of TGF-β1 and p53, TGF-β1 is positively related with p53, suggesting that p53 may be activated by TGF-β1 and involves in regulating down-stream gene expression;2. Spl is positively related with klotho, and Spl may be involved in the up-regulated expression of klotho gene.Part 3 Expression of Klotho Gene in the renal of Spontaneously Hypertensive Rat and Effects of Fosinopril and Valsartan on Klotho GeneObjective To investigate the expression of klotho gene in the renal of spontaneously hypertensive rats (SHR) and effects of angiotensin converting enzyme inhibitor (ACEI)—Fosinopril (Fos) and angiotensinâ…¡type 1 receptor block agent (AT1RB)—Valsartan (Val) on klotho gene.Methods Twenty 22 week-old male SHRs were randomly divided into four groups for each of five: hypertensive (SHR) group, Fos group (treated with Fosinopril for 10 mg/kg/d), Val group (treated with Valsartan for 50 mg/kg/d) and Fos+Val group (treated combination both with Fosinopril 10mg/kg/d and Valsartan 50mg/kg/d); five 22 week-old male Wistar-Kyoto rats (WKY) as the control group. After 8 weeks, the body weigh, cuff-taft blood pressure, 24 hours urinary protein (Upro), left renal weigh/body weigh ratio, urinary N-acetyl-β-D-glucosaminidase (NAGase) and serum creatinine (Scr) were measured; the pathological changes were viewed under light microscopy by HE and Masson; klotho and transforming growth factor-β1 (TGF-β1) protein were detected by IHC in renal paraffin section; RT-PCR and Westem Blotting were applied to determine the expression ofklotho, TGF-β1 and collagenâ… mRNA and protein in renal homogenate, respectively. Corrective data were analyzed by Pearson's analysis.Results1. The blood pressure of rats in SHR group was significantly higher than those in the other four groups(P＜0.05), while there was not markedly different in the WKY group, Fos group, Val group and Fos+Val group (P＞0.05); the levels of Scr and urinary NAGase were lower, and the contrast of 24h Upro in pre- and post-experiment was significant in Fos grpup, Val group and Fos+Val group as compared to SHR group (allP＜0.05)2. In morphology, a typical pathological character of hypertensive renal injure, and the increased collagen deposited in renal interstitial area, were showed in the renal of SHR group as compared to the control; but the hypertensive renal injury was ameliorated in Fos group, Val group and Fos+Val group.3. In IHC, positive stains of klotho and TGF-β1 protein were showed in kytoplasm of renal proximal convoluted tubule. Poorer stains of kotho protein and stronger stains of TGF-β1 protein were showed in the renal paraffin section of SHR group (P＜0.05); while in the rats treated with Fos and/or Val, the increased expression of klotho protein and the decreased expression of TGF-β1 protein were obviously viewed in renal (P＜0.05).4. RT-PCR analysis revealed that the decreased expression of klotho mRNA, the increased expression of TGF-β1 and collagenâ… mRNA were showed in SHR group, but a higher klotho mRNA expression, concomitant with a lower TGF-β1 and collagenâ… mRNA expression, was evident in Val group, Fos group and Fos+Val group(P＜0.05).5. The levels of klotho protein were down-regulated, and the levels of TGF-β1 and collagenâ… protein were up-regulated in SHR group; however, the ldottho protein showed an up-regulated expression, while the TGF-β1 and collagenâ… protein were showed a down-regulated expression in renal of the rats treated with Fos and/or Val. Furthermore, the expression of klotho protein was higher in Val group than in Fos group and Fos+Val group (P＜0.05); but there was not significant different for TGF-β1 and collagenâ… protein expression among the Fos group, Val group and Fos+Val group(P＞0.05).6. Linear correlation analysis revealed that there was a negative relationship between klotho protein and TGF-β1 and collagenâ… protein (r=-0.566 and -0.721, respectively. P＜0.05); and a positive correction between TGF-β1 protein and collagenâ… protein (r=0.893, P＜0.05).Conclusions1. The expression of klotho gene is up-regulated, and the expression of TGF-β1 and collagen I are down-regulated in the renal of SHR;2. Negative relationship between klotho and TGF-β1 and collagen I in renal, suggests that the down-regulated expression of klotho may be associated with the renal interstitial fibrosis closely;3. Fosinopril and Valsartan up-regulate the klotho gene expression and down-regulate the TGF-β1 and collagen I in the renal of SHR;4. ACEI and AT1RB ameliorate the renal interstitial fibrosis probably by up-regulating klotho expression.CONCLUSIONS1. Klotho gene is anomai-expression in the renal of SHR and NRK-52E treated with Angâ…¡;2. TGF-β1—p53/Sp1—klotho signal pathway may be involved in the pathologic process of renal interstitial fibrosis;3. The reno-protection of ABEI and AT1RB is realized probably by the up-regulation of klotho gene, and independent of decreasing the blood pressure. |
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