Glucocorticoid Aristolochic Acid Nephropathy In Clinical And Experimental Research

Part 1. The effect and mechanism of low-dosage steroid therapy in patients with aristolochic acid nephropathyBackground and objective Aristolochic acid(AA)-induced kidney disease, aristolochic acid nephropathy (A AN), is characterized pathologically by chronic interstitial fibrosis with few cell infiltration. It is believed that inflammation or immunization participates in the renal fibrosis. So far, there is no effective treatment for AAN. In recent years, a few reports suggested that glucocorticoid could retard the progression of renal impairment in patients with AAN. In this study, we used low-dosage steroid therapy in AAN patients with mild-to-moderate and severe chronic renal failure to observe the effects of the steroid on the progression of renal failure in chronic AAN.Methods 79 chronic AAN patients in Peking Union Medical College Hospital from November 1998 to April 2010 were included in this study. According to the levels of serum creatinine before treatment, the patients were divided into mild-to-moderate group(n=42, SCr<354μmol/l)and severe group(n=37, Scr>354μmol/1).The mild-to-moderate group was devided into steroid group(SG, n=18, prednisone 0.5mg/kg/d for 1-3 months, tapered off 2.5-5mg every month) and control group(CG, n=24). The severe group was also divided into steroid group(SG, n=23) and control group(CG, n=14). The gender ratio, average age, serum biochemical parameters at basement, hemoglobin and blood pressure in two groups were similar. The mild-to-moderate group patients were followed up for 2 years and the severe group patients were followed up for 1 year. In the observation period, the serum creatinine levels were compared between the SG and CG groups to examine the effects of steroid on renal failure progression of AAN.At the same time, the total efficiency of mild-to-moderate group were compared to that of severe group to determine whether there was any difference of the benefits which were obtained from steroid therapy between different degrees of renal failure.Results In the mild-to-moderate group, the creatinine levels of the patients accepted steroid therapy during the 2 years of follow-up maintained stable. While the renal function of the CG patients kept deteriorating and the creatinine levels were obviously higher than that of the SG patients. The results got from the severe group resembled that of the mild-to-moderate group.Our data also showed that there was no obviously difference of the total efficiency between the mild-to-moderate group and the severe group accepted steroid therapy.The serum biochemical parameters, hemoglobin and blood pressure in all groups were stable during follow-up. The levels of urinary TGF-β1 and MCP-1 decreased obviously for patients accepted steroid therapy. The adverse effects of low-dosage steroid therapy were slightly, only a few patients appeared steroid diabetes and herpesvirus infections. Two older patients appeared femoral head necrosis.Conclusions Low-dosage steroid therapy could reverse or delay the progression of renal failure in AAN patients both for mild-to-moderate group and severe group. The protective effect mainly occured in the first 3 months, and kept at least for 1 to 2 years. The side effects of low-dosage seroid therapy were not severe and could be tolerated to the AAN patients. Steroid therapy can reduce the levels of urinary TGF-β1 and MCP-1, may be one of the mechanisms for chronic AAN. Part 2. Clinical features of Fanconi syndrome in patients with aristolochic acid nephropathyBackground and objective Renal tubular epithelial cells are the target cells of AA. AA can induce cell degeneration, atrophy and renal tubule dysfunction(renal tubular acidosis or Fanconi syndrome). So far, there are many case reports of Fanconi syndrome with AAN patients, but misdiagnosis and missed diagnosis often occur. In this study, we analyze the clinical features of Fanconi syndrome in patients with aristolochic acid nephropathy to raise awareness of Fanconi syndrome with AAN patients.Methods All 38 AAN patients with Fanconi syndrome who were hospitalized in Peking Union Medical College Hospital during 1998 to 2010 were enrolled into this study. The levels of serum creatinine at basement were less than or equal to 354μmol/l. The cohort included 10 males and 28 females. The average age was 55.81±14.14. The patients were divided into control group (n=10) and steroid group (n=28) according to different treatment methods. All patients were followed up for 1 year. In the observation period, clinical manifestation, laboratory parameters and prognosis were observed.Results The clinical manifestations commonly presented with weakness(68.42%), nausea(42.11%), nocturia(15.79%), back pain(21.05%), some patients had obvious polydipsia and polyuria(26.32%), ostalgia, tetania and urinary system infection accounts for 5.26% respectively.The patients were characterized by proximal renal tubular acidosis and multiple renal tubular transport dysfunction, including hypophosphatemia(44.74%), hypouricemia(31.58%), renal glycosuria(100%), aminoaciduria(72.2%) and low-molecular-weight proteinuria(the urine protein of 24 hours was 1.06±0.58g). The levels of urinary (32-MG increased obviously(2855.12±3488.98ng/l). Renal pathohistological studies in 4 patients showed obvious renal tubulointerstitial changes.Most of the patients entered into dialysis and renal glycosuria persisted in control group during follow-up. While in steroid group, the levels of serum creatinine maintained stable, aminoaciduria and renal glycosuria were negative in most of the patients. The levels of urinaryβ2-MG, serum uric acid and serum phosphorus had a certain degree of decline. There were obvious statistical differences when compared to the initial. Conclusions AAN may be associated with Fanconi syndrome. Further examination is needed to make a definite diagnosis. Steroid therapy has a certain curative effect for Fanconi syndrome in aristolochic acid nephropathy. Part 3. The influence of Dexamethasone on AA induced epithelial-mesenchymal transition in HKC cellsBackground and objective It is well known that epithelial-mesenchymal transition (EMT) is an important event in renal interstitial fibrosis. Aristolochic acid can induce EMT of renal tubular epithelial cells into myofibroblasts, and can also act on the interstitial fibroblasts possibly through the cell "cross talk", such as the release of cytokines, which can synthesize and secrete a large number of extracellular matrix and result in renal interstitial fibrosis. In recent years, a few reports suggest that glucocorticoid could be used for AAN patients. This study is to explore the influence of Dexamethasone on AA induced epithelial-mesenchymal transition in HKC cells.Methods Cultured HKC cells were divided into five groups:negative control, incubated with AA(10μg/ml) alone, with Dex (100μmol/l) alone, with AA(10μg/ml) and absolute ethanol(100μmol/l), or with AA(10μg/ml) and Dex at different concentrations (100,10,1, 0.1,0.01μmol/l) for 48 hours. Confocal microscope was used to detect expressions of a-SMA and E-cadherin. The mRNA and protein expressions of a-SMA, E-cadherin were assessed with real-time PCR and Western Blot respectively. The culture supernatants were collected to examine the expressions of fibronectin (FN) and collagenⅠ(COLⅠ) using enzyme linked immunosorbent assay (ELISA) methods. In the other experiment, HKC cells were incubated with AA(10μg/ml) in the absence or presence of Dex (100μmol/l) respectively. The mRNA and protein expressions of a-SMA, E-cadherin were assessed with real-time PCR and Western Blot at different time points respectively. The culture supernatants were collected to examine the expressions of FN and COL I using ELISA methods.Results 1 As revealed by confocal microscope, compared to the control group, AA inhibited E-cadherin expression and enhanced a-SMA expression in HKC cells. There were no obvious differences of these two markers between the group of Dex alone and the control group. For AA+Dex group, Dex dramatically abrogated AA induced a-SMA expression and restored the E-cadherin expression in HKC cells in a dose-dependent manner.2 Real-time PCR, Western Blot and ELISA showed that mRNA and protein expressions of a-SMA significantly decreased, E-cadherin significantly increased, the expressions of FN and COL I in the supernatant were downregulated concomitantly in cells incubated with AA and Dex(100-1μmol/l), compared with that treated with AA alone (P<0.05). There were no significant differences between the group of Dex alone and the control group for mRNA and protein expressions ofα-SMA and E-cadherin in HKC cells.3 Both mRNA and protein expressions ofα-SMA in HKC cells incubated with AA alone increased, E-cadherin decreased and the expressions of FN and COL I in the supernatant increased steadily within 48 hours, there were obvious statistical differences in 24 and 48 hours when compared to control group. While mRNA and protein expressions of a-SMA decreased, E-cadherin increased significantly in group AA+Dex group at 48h when compared to AA group (P<0.05). The expressions of FN and COL I in the supernatant were downregulated concomitantly in group AA+Dex at 48h (P<0.05).Conclusions The results documented that AA may induce EMT in HKC, and Dex may partially inhibit AA-induced EMT in HKC cells in vitro, mainly appeared at 24 and 48 hour after intervention. Part 4. Smad transduction pathway involved in supression of Dex on AA induced EMT of HKCBackground and objective It is reported that Smad3 plays an important role in EMT and renal interstitial fibrosis in chronic AAN and there is no obvious EMT when Smad3 konckout. It is also noted that the main pathway of Smad3 activation is TGF-β1/Smads. Lately, there have been a few reports of glucocorticoid used for AAN patients, which can relieve renal interstitial fibrosis. Another report implied that Dexamethasone can suppress Smad3 activation in osteoblasts. This study is to explore the mechanism of Dexamethasone on AA induced EMT in HKC cells.Methods Cultured HKC cells were divided into five groups:negative control, incubated with AA(10μg/ml) alone, with Dex (100μmol/l) alone, with AA(10μg/ml) and absolute ethanol(100μmol/l), or with AA(10μg/ml) and Dex at different concentrations (100,10,1, 0.1,0.01μmol/l) for 48 hours. In the other experiment, HKC cells were incubated with AA(10μg/ml) in the absence or presence of Dex (100μmol/l) respectively. The mRNA expressions of TGF-β1 and Smad7 were assessed by real-time PCR. The protein expressions of TGF-β1, p-Smad3, Smad3 and Smad7 were assessed by Western Blot.Results 1 Real-time PCR and Western Blot showed that mRNA and protein expressions of p-Smad3 and TGF-β1 significantly decreased, Smad7 significantly increased in cells incubated with AA and Dex(100-1μmol/l), compared with that treated with AA alone (P<0.05). There were no significant differences between the group of Dex alone and the control group for mRNA and protein expressions of p-Smad3 and TGF-β1 in HKC cells.2 In group AA, mRNA and protein expressions of p-Smad3 and TGF-β1 increased, Smad7 decreased obviously within 48h when compared with control group. While in group AA+Dex, mRNA and protein expressions of p-Smad3 and TGF-β1 decreased, Smad7 increased. There were obvious statistical differences in 24 and 48 hours when compared to AA group.Conclusions The results documented that Dex may partially inhibit AA-induced EMT in HKC cells in vitro, which is probably contributed to the repression of TGF-β1 and p-Smad3, upregulation of Smad7. Further study is still needed.