| BackgroundThe rapidly increasing incidence of hypertensive renal damage has became one of the most important reasons of End Stage Renal Disease (ESRD).In the past,research related to hypertensive renal damage has focused on the renal glomerulus.Recently,it has been considered that the progression and prognosis of chronic renal failure correlates better with tubulointerstitial damage than with glomerular damage.Renal tubular epithelial cell apoptosis plays an important role in this process.Klotho gene,an new anti-aging gene,is predominantly detected in the kidney and cerebullar choroid,significantly in renal tubular epithelial cells.Its deletion in mice results in phenotypes resembling human aging, features resembling clinical manifestation of patients with chronic renal failure.Recently,several studies have confirmed the association between Klotho gene and renal diseases including acute renal failure rat,5/6 nephrectomized rat,ICR-derived glomerulonephritis(ICGN) mice,renal cell carcinoma,chronic renal failure and abnormalities in calcium and phosphorus metabolism.So far,there is no research about the association between Klotho gene and hypertensive renal damage.Cordyceps sinensis(CS) is a type of rare traditional Chinese medicine with excellent reno-protective effects.However,less is known about the mechanisms of its reno-protective effects.In the present study, we used rat proximal renal tubular epithelial cells(NRK-52E) stimulated by AngiotensinⅡ(AngⅡ) and spontaneously hypertensive rat(SHR), and drug intervention including cordyceps sinensis and the AT1 receptor antagonist Losartan(Los) to observe the expression of Klotho and apoptosis-related genes p53,p21,and tubular epithelial cells apoptosis. Then we investigated the role of Klotho in tubular epithelial cells apoptosis in hypertensive renal damage and mechanisms of reno-protective effects of cordyceps sinensis to provide evidence for treatment of hypertensive renal damage.Part 1 Effects of Cordyceps Sinensis Extract on Klotho expression and Apoptosis in NRK-52E Induced by AngiotensinⅡMethods1.NRK-52E cells were incubated with different concentrations of AngⅡ[0(control),10-10,10-9,10-8,10-7,10-6mol/L]for 24 hours.The optimal concentration of AngⅡwas selected to stimulate culture cells for 0(control),6,12,18,24 hours to select the best cultivation time; NRK-52E were incubated with increasing concentrations of cordyceps sinensis extract[0(control),5,10,20,40,80mg/L]with or without AngⅡfor 24,48,72 hours to select the optimal dose and time of cordyceps sinensis extract.Cell viability was determined by MTT assay; reverse transcription-polymerase chain reaction(RT-PCR) were applied to evaluate the Klotho mRNA expression;the caspase-3 activity was analyzed by caspase 3 activity assay kit;the cell apoptosis was assessed by Annexin V-FITC/PI double staining and flow cytometry.2.According to the above results,the optimal concentration of AngⅡ(10-8mol/L) and cordyceps sinensis extract(40mg/L) were selected to incubated NRK-52E for the best cultivate time(24 hours), Losartan(10-5mol/L) as the positive control drug.Cells were divided into five groups,which were control,AngⅡ,AngⅡ+CS,AngⅡ+Los and AngⅡ+CS+Los.The mRNA and protein expression of Klotho,p53 and p21 were measured by RT-PCR and Western Blot;caspase-3 activity was evaluated by caspase-3 activity assay kit;cell apoptosis was determined by Annexin V-FITC/PI double staining and flow cytometry.Results1.AngⅡ(10-9mol/L～10-6mol/L) down-regulated Klotho mRNA expression obviously in NRK-52E compared with control(P<0.01); caspase-3 activity and apoptotic rate in NRK-52E increased gradually as concentrations of AngⅡincreased(10-10mol/L～10-6mol/L) and time prolonged(6h～24h)(P<0.01).2.There was no evidence that cordyceps sinensis extract(5mg/L～80mg/L) alone had any effect on Klotho mRNA expression in NRK-52E (P>0.05);cordyceps sinensis extract(5mg/L～40mg/L) up-regulated Klotho mRNA expression reduced by AngⅡgroup,the effects increased gradually as the concentrations increased(P<0.05 or P<0.01);as compared with AngⅡgroup,caspase-3 activity and apoptotic rate in NRK-52E increased gradually as concentrations of cordyceps sinensis increased(10mg/L～40mg/L).3.AngⅡsignificantly down-regulated mRNA and protein expression of Klotho,enhanced p53 and p21 expression,caspase-3 activity and apoptotic rate of NRK-52E(P<0.01).In the presence of either cordyceps sinensis or Losartan or both,down-regulation of Klotho expression was countered,the increment in p53 and p21 expression were inhibited,caspase-3 activity and apoptotic rate decreased(P<0.05 or P<0.01).No cooperative effects were observed by these two drugs (P>0.05).Conclusions1.AngⅡdown-regulates Klotho expression,up-regulates the expression of p53 and p21,increases caspase-3 activity,and induces apoptosis in NRK-52E,which suggests it maybe one of the mechanisms of AngⅡinvolved in renal tubular epithelial cells apoptosis in hypertensive renal damage.2.Different concentrations of cordyceps sinensis extract alone have no significant effect on Klotho mRNA expression in NRK-52E; cordyceps sinensis extract can increase cell proliferation and Klotho expression,and inhibit apoptosis induced by AngⅡin NRK-52E;The anti-apoptotic effects of cordyceps sinensis extract are related to increased expression of Klotho down-regulated by AngⅡ,inhibition of p53/p21 apoptotic pathway and caspase-3 activition. Part 2 Effects of Cordyceps Sinensis on Klotho Expression in Kidneys and Apoptosis in Renal Tubular Epithelial Cells of Spontaneously Hypertensive RatsObjective To observe renal Klotho expression and tubular epithelial cell apoptosis in spontaneously hypertensive rats and the effects of cordyceps sinensis on them,in order to study the role of Klotho in tubular epithelial cells apoptosis in hypertensive renal damage and mechanisms of reno-protective effects of cordyceps sinensis.Methods Twenty 22 week-old male SHR were randomly divided into four groups for five each:hypertensive group(SHR),CS group(5g/kg/d), Los group(50 mg/kg/d) and CS + Los group(treated with CS 5g/kg/d and Los 50mg/kg/d);six 22 week-old male Wistar-Kyoto rats(WKY) as the control group.After 8 weeks,the body weigh,cuff-tail blood pressure, 24 hour urinary protein(Upro),urinary N-acetyl-β-D-glucosaminidase (NAGase),blood urea nitrogen(BUN) and serum creatinine(Scr) were measured.Renal pathological changes were viewed by HE and Masson staining;in situ detection of DNA fragmentation(TUNEL assay) was applied to evaluate renal tubular epithelial cell apoptosis;the mRNA expression of Klotho,p53 and p21 were detected by RT-PCR;the protein expression of Klotho,p53,p21 and cleaved-caspase-3 were tested by Western Blot.Results 1.At 30 weeks,the blood pressure,Upro,urine NAGase,BUN and Scr of untreated SHR were significantly higher than WKY rats,and typical pathological characteristics of hypertensive renal damage including thickness and hyalinization of glomerular arteriole walls, vacuolar and particulate degeneration of tubular cells,inflammatory cell infiltration in interstitial region,schemic atrophy and partial sclerosis of glomerular capillaries were founded in kidneys of SHR group(P<0.05), cordyceps sinensis or/and Losartan reduced Upro,NAGase,BUN and Scr, and ameliorated renal pathological damage(P<0.05).Cordyceps sinensis had no significant effect on blood pressure(P>0.05).2.Positive stains of Klotho protein were mainly demonstrated in the cytoplasm and nucleus of renal distal convoluted tubule.Compared with WKY rats,Klotho mRNA and protein expression were significantly reduced in kidneys in SHR,concomitant with the increased p53,p21 expression,along with increased cleaved caspase-3 protein expression, and tubular cells apoptotic rate(13.316%±0.645%vs 0.810%±0.115%, P<0.01).Aider treatment with cordyceps sinensis or/and Losartan, Klotho mRNA and protein expression were up-regulated,while the expression of p53,p21 and cleaved-caspase-3 were reduced,tubular cells apoptotic rate were decreased than SHR(decreased to 7.559%±0.520%, 7.928%±0.370%and 7.373%±0.363%separately,P<0.01).No cooperative effects were observed by these two drugs(P>0.05). Conclusions1.The blood pressure,Upro,urine NAGase,BUN and Scr are significantly increased in SHR,cordyceps sinensis or/and Losartan can decrease Upro,urine NAGase,BUN and Scr.cordyceps sinensis does not affect the blood pressure.2.The interstitial damage is appeared in kidneys of SHR, concomitantly the mRNA and protein expression of Klotho are down-regulated obviously,the expression of p53,p21 and cleaved-caspase -3 are up-regulated,tubular epithelial cells apoptotic rate is increased in kidneys.3.Cordyceps sinensis relieves renal interstitial damage of SHR, concomitantly increases renal Klotho expression,inhibits p53/p21 apoptotic pathway and decreases caspase-3 activation,then inhibits tubular epithelial cells apoptosis,its reno-protective effects are independent of blood pressure control.CONCLUSIONS1.AngⅡcan inhibit Klotho gene expression,increase p53,p21 expression and caspase-3 activity,and induce the apoptosis of renal tubular epithelial cells,which suggests it maybe one of the mechanisms of AngⅡinvolved in renal tubular epithelial cell apoptosis in hypertensive renal damage. 2.Cordyceps sinensis extract can reduce apoptosis induced by AngⅡ,its anti-apoptotic effects are related to increased expression of Klotho gene down-regulated by AngⅡ,inhibition of p53/p21 apoptotic pathway and caspase-3 activition.3.The interstitial damage is apparent in kidneys of SHR, concomitantly Klotho gene expression is down-regulated unambiguously, the expression of p53,p21 and cleaved-caspase-3 are up-regulated,while tubular cells apoptotic rate is increased in kidneys.4.Cordyceps sinensis relieves renal interstitial damage of SHR, concomitantly increases Klotho gene expression,inhibits p53/p21 apoptotic pathway and decreases caspase-3 activation,then inhibits tubular cell apoptosis,its reno-protective effects being independent of blood pressure control. |
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