| Objectives:Diabetic nephropathy(DN), one of the most common complications of diabetes, has become the leading cause of end stage renal disease in china ,which is histologically characterized by hypertrophy of glumeruli and renal tubules, increased thickness of basement membrane, overaccumulation of extracellular matrix, glomerulosclerosis and tubulointerstitial fibrosis .There is no doubt that hyperglycemia is the key factor in the pathogenesis of diabetic nephropathy.Although the glomerulus has been the focus of intensive investigation in the mechanism of diabetic nephropathy in the past decades,in recent years more attentions have been paid on the changes of the tubulointerstitial lesions,because the tubulointerstitial damage is even more important than glomerulosclerosis ,as an important predictor of renal dysfunction and progression of disease course both in diabetic nephropathy and others. AngiotensionⅡand endothelin-1 are two important vasoactive peptide,having powerful vasoconstricting and hemodynamic function,except this,both have nonhemodynamic effects as growth factor to induce proliferation,hypertrophy,to stimulate synthesis of matrix proteins,furthermore,also to inhibit matrix degradation, even to mediate inflammation reaction.Both systems are activated in diabetic nephropathy. There is considerable evidence for an interaction between them.Angiotensin receptor blockade(ARB) ,by binding with AT1 receptor,inhibits AngⅡ-induced proliferation,hypertrophy and extracelluar matrix accumulation. In this study, HKCs cultured in the medium containing high glucose or 10-7 mol/L AngⅡ,experimental diabetic nephropathy and early DN patients were treated with endothelin receptor antagonist and ARB to investigate the interrelation between angiotensinⅡand endothelin-1 in the tubular epithelial cells.Methods:1. Detection of ET-1 and ETA receptor in HKCs cultured in high glucose and AngⅡ(10-7mol/L) mediumHKCs were divided into six groups: low glucose group(5.5mmol/L glucose, LG), low glucose+high mannitol group(5.5mmol/L glucose+24.5mmol/L mannitol, LG+M)(to remove the impact of osmotic pressure) ,high glucose group(30mmol/L glucose , HG), AngⅡgroup(5.5mmol/L glucose + 10-7mol/ L AngⅡ), HG+Los group(30mmol/L glucose+10μmol/L losartan, HG+Los),AngⅡ+ Los group(5.5mmol/L glucose +10-7mol/L AngⅡ+10μmol/L Los,AngⅡ+ Los,Los prior to HG and AngⅡ1 hour in the medium). The cells were cultured in 24-pore plate and 25cm2 plastic culture flask. After treatment for 6, 12, 24, 48 and 72h, the medium was collected and cells were harvested for immunoflurescence, protein extraction and RNA isolation. The protein expressions of ET-1and ETA receptor were observed by immunoflurescence and Western blot. The contents of ET-1 in the supernatants of the cultured HKC were detected by enzyme-linked immunoadsorbent assay(ELISA). ET-1 and ETA receptor mRNA was measured by reverse transcription and polymerase chain reaction ( RT-PCR) .2. Detection of ET-1and ETAR in tubular epithelial cells of diabetic ratsUninephrectomized male Sprague-Dawley rats were firstly divided into two groups: control group and diabetic group. The rats of diabetic group received a single intraperitoneal injection of STZ dissolved in 0.1mol.L-1 sodium citrate (pH 4.5) at a dose of 50mg.kg-1SD. The rats of control group only received an injection of the same volume of 0.1mol.L-1 sodium citrate. The model of diabetes was considered to be successful when the blood glucose was≥13.7mmol.L-1 and the glucose in urine was +++～++++ after 24 hours of the injection. Further 36 diabetic rats were randomly divided into two group: DM group and DM+losartan group. Losartan was administrated to the rats in the losartan-treated group at a dose of 20mg/kg from next day ,equal dose of distilled water was administrated to the rats in control group and DM group.Six rats from each group were respectively sacrificed at weeks 4,8,12. Blood, urine and kidney samples were collected. 24h urine albumin excretion, concentration of serum urea nitrogen (BUN) and creatinine(Cr) were determined, respectively. Partial renal tissure was fixed in 4% formaldehyde and embeded with paraffin for pathological examination. Partial renal cortices were used to isolate proximal tubules and extract total RNA and protein. The levels of expression of ET-1 and ETA receptor were evaluated by immunohistochemistry and Western blot. The expression of preproET-1and ETARmRNA, were measured by reverse transcription and polymerase chain reaction (RT-PCR). Serum AngⅡwas detected with radioimmunoassay and serum ET-1 was detected with ELISA method.3. Detection of serum ET-1 and AngⅡ,urinary ET-1 in early DN patients after treatmen with Losartan and Sodium FerulateFifty–nine early DN patients whose levels of urinary malb were under 300mg/24h were randomly divided into two groups, the Losartan treatment group(50mg oral application per day) and the SF+ Losartan treatment group(50mg oral application per day, normal saline+300mg sodium ferulate vein application per day). Time of therapy was for 4 weeks.The patients'age was at scope of 65±3,and no differrence was found on age and sex between two treatment groups. 10 normal individuals were as control group .Indexs,including serum endothelin(ET-1),angiotensinⅡ(AngII),urinary ET-1,and malb were detecteded before and after treatment.Results:1. Expression of ET-1 and ETA receptor in HKCs cultured in high glucose and AngⅡ(10-7mol/L) medium①Immunofluorescence cytochemical staining showed that ET-1 protein was expressed in cytoplasm of HKCs. Compared with low glucose groups and LG+M group, the expression of ET-1 was increased in high glucose group and AngⅡgroup from 6h point ,the expression intensity enhanced with the time , arrived peak level at 24h, then decreased slightly.However,there was no evident difference of ET-1 protein expression between high glucose group and AngⅡgroup. Compared with high glucose group and AngⅡgroup, expression of ET-1 protein decreased in high glucose +losartan group and AngⅡ+ losartan group,respectively, but there was no significant difference between high glucose +losartan group and AngⅡ+ losartan group.②Western blot showed that, compared with low glucose group and LG+M group , the expressions of ET-1 protein(from 6h)and ETA receptor protein(from 12h) were increased in high glucose group (p<0.05) in time dependent manner; the expressions of ET-1 protein, and ETA receptor protein were increased in AngⅡgroup from 6h; Compared with high glucose group and AngⅡgroup, the expressions of ET-1 and ETA receptor protein were decreased correspondingly in high glucose +Losartan group,from 12h and AngⅡ+ Losartan group, from 6h.③RT-PCR showed that the relative expression level of ET-1 and ETA receptor mRNA was increased in high glucose group and AngⅡgroup (P<0.01) in a time dependent manner,but there was no difference for expression of ET-1mRNA beween LG group and LG+M group .The expression of ET-1mRNA arrived peak level at 24h , the expression of ETA receptor mRNA arrived peak level at the 48h in high glucose group and AngⅡgroup.In high glucose +Losartan group and AngⅡ+ Losartan group, the expression of ET-1mRNA and ETA receptormRNA were decreased correspondingly(p<0.05).④The concentration of ET-1 in supernatants of the HKCs in high glucose group and AngⅡgroup was higher than those in low glucose group at different stimulated times from 6h to 72h(p<0.01), roughly being coincident with the results of Immunofluorescence cytochemical staining.The secretion of ET-1 in HKCs cultured in high glucose and 10-7mmol/LAngⅡmedium could be inhibited by losartan(compared with high glucose group and AngⅡgroup, P <0.01).2. Renal morphological changes and expression of ET-1and ETA receptor in tubular epithelial cell of diabetic rats①Light microscopically ,the glomeruli were enlarged and proximal tubular epithelial cells were swelling in the residual kidneys in diabetic rats at week 8. Glomerular sclerotic lesion, dilation and atrophy of the tubules, interstitial edema and inflammatory changes were found significantly at week 12. The scope of glomerular damage and the tubular lesions was increased with the course of diabetes.②The renal function indexes such as serum urea nitrogen, creatinine and 24h urine protein were increased in diabetic group with time ( compared with control group, P<0.05).③Immunhistochemistry ,RT-PCR and western blot analysis indicated that the exprssions of ET-1and ETA receptor ,in protein and mRNA levels,in diabetic group were markedly increased than those in control group(P<0.01).④Compared with control group, the level of serum ET-1 and AngⅡis increased in diabetic group with time (p<0.01).3. Effects of losartan on ET-1 and ETA receptor expressions in tubular epithelial cells of diabetic rats①The pathological changes of the residual kidney, including interstitial cellular infiltration, tubular atrophy, dilation or microcysts, mesangial proliferation and glomerular sclerosis, were markedly lighten in different extent with time .After treatment of Losartan, renal function indexs, including serum creatinine, BUN, and urinary protein excretion ,were lower in losartan-treated rats than that in untreated diabetic rats(p<0.01).②Immunhistochemistry ,RT-PCR and western blot analysis indicated that the exprssions of ET-1and ETA receptor , in protein and mRNA levels, were down-regulated in losartan-treated group(p<0.01).③Compared with control group,the level of serum ET-1and AngⅡwas also decreased in losartan-treated group.4.Comparision of serum AngⅡ,ET-1 and urinary ET-1 and 24hmalb in different therapy group before and after treatmentThe level of AngⅡ,ET-1 and 24hmalb in early DN patients before treatment was higher than that in normal control group(p<0.01) , The level of ET-1 and 24hmalb were decreased significantly in both groups after treated with losartan and SF for 4 weeks(p<0.05,p<0.01),the level of AngII in the SF+ Losartan treatment group decreased also(p<0.05) ,but AngII in the Losartan treatment group had no change(p>0.05).Conclusions:①The expressions of ET-1 and ETA receptor were up-regulated and the content of ET-1 in the supernatants was also increased in the HKCs cultured in high glucose and AngⅡmedium, suggesting that high glucose or AngⅡwhen their concentration in medium exceed to physiological condition, could induce excessive expression of ET-1 and ETA receptor in HKCs. Losartan could inhibit and down-regulate the expression of ET-1 and ETA receptor,probably by the common receptor of endothelin-1 and angiotensinⅡin HKCs.②Treatment with Losartan could down-regulate the overexpression of ET-1 and ETA receptor in tubular epithelial cells of dibetic rats, attenuate tubulointerstitial fibrosis in remnant kidney and postpone the progression of chronic renal insufficiency in diabetic rats. These suggest that the protective effects of ARB for renal function might be partly through blocking ETA receptor .③The renin angiotensin system and endothelin system were activated and the levels of serum AngⅡand ET-1 were increased in early stage DN,which could be inhibited by endothelin receptor antagonist and angiotensinⅡreceptor antagonist ,and combined use of both can take better effects. |
PDF Full Text Request