Celluar And Molecular Mechanism Of Aqueous Extract Of Astragali Radix On Renal Water And Sodium Excretion And Its Renal Protective Effects

PARTâ… Effects of aqueous extract of Astragali Radix on renal water and sodium excretion and its molecular mechanism1. Aqueous extract of Astragali Radix induces natriuresis in healthy men through enhanced renal response to atrial natriuretic peptideBackground The diuretic effect of Astragali Radix was described in ancient books of traditional Chinese Medicine, however, this diuretic effect has not been confirmed.Objective To evaluate the diuretic/natriuretic effect of aqueous extract of Astragali Radix (ARE) in healthy men and uncover its possible mechanism.Methods We conducted a double-blind, randomized, crossover study in 12 healthy men. They were randomized to receive either placebo (n=6) or a single oral dosage of 0.3g/kg body weight of aqueous extract of Astragali Radix (ARE) (n=6) before oral administration of 0.9% saline load(10ml/kg). Urinary sodium excretion(U_NaV), urinary cGMP excretion(U_cGMPV), plasma ANP level(pANP) and U_cGMPV/pANP ratio during 4 hours were measured. After two-week washout period, the subjects were crossed over. Results Compared with placebo, ARE produ.ced marked increments in urinary U_NaV which reached a peak at 2h (0.30±0.05 vs. 0.21±0.02 mmol/min, P＜0.05), as well as fractional sodium excretion, urinary excretion of chloride during 4 hours. ARE elevated urinary excretion of U_cGMPV and U_cGMPV/pANP ratio. Further correlation and linear regression analysis between U_cGMPV and plasma ANP showed that ARE could significantly improve the renal reaction to ANP.Conclusion We firstly demonstrate that ARE induces a marked natriuresis in healthy men, which is partly attributed to enhanced renal response to ANE2. Aqueous extract of Astragali Radix can ameliorate the renal resistance to atrial natriuretic peptide in rats with experimental nephrotic syndromeObjective To study the effect of ARE on renal resistance to ANP in nephrotic rats. Methods Forty-eight Male Sprague-Dawley rats were randomly divided into normal control, adriamycin nephropathy (ADR), ADR treated with ARE (2.5 g·kg^-1·d^-1) and ADR treated with benazepril (10mg·kg^-1·d^-1). After 6 weeks, rats received intravenous infusion of 2% body weight isotonic saline over 5 min. Urinary excretion of sodium (U_NaV), urinary excretion of cGMP (UcGMpV) and plasma ANP level(pANP) after volume expansion were measured. Moreover, renal PDE5 activity, PDE5 protein and mRNA expression were detected.Results Compared to ADR rats, ARE increased U_NaV (0.83±0.26 vs.0.37±0.20 mol/min, P＜0.01). ADR rats had a blunted natriuretic response and reduced rate of U_cGMPV after volume expansion despite higher plasma ANP concentration. ARE increased U_cGMPV and restored partly natriuretic response to intravenous infusion of isotonic saline. Renal PDE5 activity, protein and mRNA expression were greater in ADR rats. ARE significantly reduced the PDE5 activity (6.8±0.8. vs. 9.9±1.1pmol/mg/min, P＜0.01), protein abundance (1.01±0.09. vs 1.38±0.14, P＜0.01) and mRNA expression (1.25±0.12.vs.2.05±0.16, P＜0.01).Conclusion ARE may ameliorate the renal resistance to ANP in rats with adriamycin nephropathy. The inhibitory effect of ARE on PDE5 protein and mRNA expression might account for its molecular mechanisms for long term activity.3. Effects of aqueous extract of Astragali Radix on PDE5 activity and expression in cultured mIMCD-3 cellsObjective To study the effects of ARE on PDE5 activity and expression in cultured mIMCD-3 cells.Methods Immortalized mouse mIMCD-3 cells were cultured and incubated with 10^-7M concentration of ANP, different dose of ARE(500,1000,2000μg/ml)and 10^-7M concentration of Sildenafil for 6h. ANP-dependent cGMP accumulation and PDE5 activity in mIMCD-3 cells were measured. The PDE5 protein and mRNA expression were detected by Western blotting and Real-Time PCR.Results 500,1000,2000μg/ml of ARE inhibited ANP-dependent cGMP accumulation and PDE5 activity in mIMCD-3 cells in a dose-dependent manner (P＜0.05). 500,1000,2000μg/ml of ARE reduced the PDE5 protein and mRNA expression in mIMCD-3 cells in a dose-dependent manner by 25%,40%,55% and 30%,50%,60% respectively, (P＜0.05). However, Sildenafil exerted no effect on PDE5 protein and mRNA expression (P＞0.05).Conclusion ARE ameliorates the ANP sensitivity in cultured mlMCD-3 cells, its mechanism is involved the inhibition of PDE5 protein and mRNA expression PARTâ…¡Protective effect of aqueous extract of Astragali Radix on podocyte injury in vivo and vitro and its molecular mechanism1. Protective effect of aqueous extract of Astragali Radix on podocyte injury in rats with adriamycin nephrosisBackground Extensive flattening of podocyte foot processes is one of the major pathologic features of minimal change nephrosis (MCN). Adhesion proteins such as a-dystroglycan and a₃ integrin anchor and stabilize podocytes on the glomerular basement membrane (GBM), and play a key role in the podocyte detachment from GBM. Astragali Radix was widely used to treat renal diseases, however, whether it can exert a protective effect on podocyte injury is not identified.Objective To investigate the protective effect of aqueous extract of Astragali Radix (ARE) on podocyte injury in adriamycin nephropathy and its possible mechanism.Methods Forty-eight male Sprague-Dawley rats were randomly divided into normal control, adriamycin nephropathy (ADR), ADR treated with ARE (2.5g·kg^-1·d^-1) and ADR treated with benazepril(10mg·kg^-1. d^-1). After 10 weeks, the urine protein excretion was measured. Podocyte number per square millimeter glomerulus was determined by WT-1 immunohistochemistry staining. The protein and mRNA expression ofα-dystroglycan andα₃ integrin in the renal cortex were detected by western blotting and Real-Time PCR.Results Both ARE and benazepril reduced the urine protein excretion (89.4±4.9 and 70.9±11.8 mg/24h, P＜0.01) compared with ADR group(155.5±18.4 mg/24h). Rats in ADR group exhibited a reduction in the number of podocytes and the protein and mRNA expression ofα-dystroglycan andα₃ integrin compared to the normal control (P＜0.01). The number of podocytes in ARE-treated group was higher than that in ADR group (1220±88 vs 941±80/mm², P＜0.05), as well as the protein and mRNA expression ofα₃-dystroglycan andα3 integrin. Pearson correlation analysis revealed that the protein expression ofα3-dystroglycan andα₃ integrin was positively correlated with the number of podocytes(r=0.50,0.43; P＜0.01) but negatively correlated with urine protein excretion (r=-0.81,-0.87; P＜0.01).Conclusions ARE exerts a protective effect on podocyte loss and ameliorated the proteinuria in adriamycin nephropathy. This protective mechanism may involve its increase in protein and mRNA expression ofα-dystroglycan andα₃ integrin. 2. Protective effect of aqueous extract of Astragali Radix on cultured mouse podocyte injury.Objective To investigate the protective effect of ARE on cultured mouse podocyteMethods Conditionally immortalized mouse podocytes were cultured and treated with 50μg/ml ADR and different dose of ARE (500,1000,2000μg/ml). The cell adhesion ability was measured by centrifugation cell adhesion assay and fluorescence adhesion assay. The protein and mRNA expression ofα-dystroglycan andα₃ integrin in podocytes were detected by western blot and Real-Time PCR.Results Conditionally immortalized mouse podocytes clones grown under nonpermissive conditions display characteristics of differentiated arborized-type. Both centrifugation cell adhesion assay and fluorescence adhesion assay showed that the cell adhesion ability was reduced after ADR treatment, but 2000μg/ml of ARE improved the cell adhesion ability. The protein and mRNA expression ofα-dystroglycan andα₃ integrin were reduced by 60%,50% and 70%,75% (P＜0.05) in ADR-treated podocytes compared to the control(P＜0.05). 2000μg/ml of ARE can up-regulate the protein and mRNA expression ofα-dystroglycan andα₃ integrin by 50%,60% and 60%,200% compared to the ADR-treated podocytes (P＜0.05).Conclusions ARE exerts a protective effect on cultured mouse podocyte injury and improve podocyte adhesion ability which may involve its up-regulation ofα₃-dystroglycan andα₃ integrin protein and mRNA expression.In summary, two conclusions were drawn from above two parts:1.ARE can promote renal water and sodium excretion both in healthy men and nephrotic rats by affecting the ANP-cGMP- PDE5 signal pathway.2. ARE can exert protective effects on the podocyte injury both in vivo and vitro, its mechanism is associated with its upregulation of the adhesion proteinsα-dystroglycan andα₃ integrin expression and alleviation of the podocyte detachment from GBM.