[Effects of aldosterone on synthesis of fibronectin and expression of transforming growth factor-beta1 mRNA in cultured rat mesangial cells].

AIM: To evaluate the effect of aldosterone (ALD) and spironolactone (SPI, one of the aldosterone receptor antagonists) on synthesis and secretion of fibronectin (FN) and expression of transforming growth factor-beta1 (TGF-beta1) mRNA in cultured rat mesangial cells. METHODS: (1) Mesangial cells were treated with medium containing different concentrations of ALD (10(-11), 10(-9), 10(-7) mol/L) and/or 10(-7) mol/L SPI for 48 h, while control cells were treated with vehicle only. The levels of FN in the supernatants were measured by ELISA method. The expressions of FN mRNA and TGF-beta1 mRNA were detected by semi-quantitative RT-PCR. (2) Mesangial cells were treated with 10(-9) mol/L ALD for 24, 48, 72 h. The levels of FN in the supernatants were measured by ELISA method. The expressions of FN mRNA and TGF-beta1 mRNA were detected by semi-quantitative RT-PCR. RESULTS: ELISA method showed that the level of FN in the supernatant of cultured rat mesangial cells stimulated with ALD increased significantly in a dose- and time-dependent manner. Also the expressions of FN mRNA and TGF-beta1 mRNA were increased significantly by ALD in a dose- and time-dependent manner by semi-quantitative RT-PCR. SPI inhibited the stimulating effect of ALD on synthesis of FN and expressions of FN mRNA and TGF-beta1 mRNA in cultured rat mesangial cells. CONCLUSION: ALD up-regulates the protein synthesis and mRNA expression of FN, and up-regulates the mRNA expression of TGF-beta1 in cultured rat mesangial cells. SPI inhibits the effect of ALD, and thus implication in the treatment of Glomerulosclerosis.

[Expressions of IL-18Ralpha and IL-18Rbeta on rat primary renal tubular epithelial cells].

AIM: To detect the expressions of the IL-18Ralpha mRNA and IL-18Rbeta mRNA in primary rat renal tubular epithelial cells (RTECs). METHODS: The culture of primary RTECs was performed by renal tubular segment sticking mass method. The cellular type was identified by immunocytochemical staining. The IL-18Ralpha mRNA and IL-18Rbeta mRNA expressions in RTECs were detected by RT-PCR. RESULTS: The result of immunocytochemical staining proved that the cultured cells were RTECs, IL-18Ralpha mRNA and IL-18Rbeta mRNA were detected in RTECs. CONCLUSION: IL-18Ralpha mRNA and IL-18Rbeta mRNA are expressed in RTECs, which provide the experimental basis for exploring IL-18R role in renal interstitial fibrosis.

[Development of a method for isolation, culture and proliferation of mouse NK cells].

AIM: To establish a method to isolate, purify, and culture mouse nature killer (NK)-cells in vitro. METHODS: NK-cells were isolated from splenic mononuclear cells (MNC) by a two-step adherence system and magnetic microbeads actived cell sorting (MACS), then these cells were cultivated with feeder cells and IL-2 in RPMI 1640 medium for 5, 10, 15, and 20 days. The enriched cells were counted and stained with anti-mouse CD3-FITC and anti-mouse NK1.1-PE. The purity of the NK-cells was determined by flow cytometry and the cytotoxicity to YAC-1 targets was detected by MTT assay. RESULTS: In the two-step adherence system, the enriched cells cultivated for 5, 10, 15, and 20 days were 0.5 x 10(7), 1.4 x 10(7), 2.6 x 10(7), and 3.0 x 10(7) respectively, and the percent of CD3(-) NK1.1(+) cells was 18.36%, 43.44%, 55.68%, and 60.03% respectively. The cytotoxicity to YAC-1 targets was significantly higher than that of splenic MNC(P<0.01), and increased from 41.93% up to 54.38%, 66.54%, 79.38%, and 83.86% respectively at 25:1 of effector:target ratio. After NK-cells were purified by MACS, the purity reached 93.60%ls, the enriched cells was 1.5 x 10(6), but proliferated to only 1.9 x 10(6) 20 days later. CONCLUSION: NK-cells isolated by using the two step adherence system proliferate abundantly, and the purity reaches 55%-60%. The cytotoxicity to YAC-1 targets is about 80% at 25:1 of effector:target ratio.

Effects of specific interleukin-1beta-converting enzyme inhibitor on ischemic acute renal failure in murine models.

AIM: To study the effect of selective interleukin-1beta-converting enzyme (ICE, caspase-1) inhibitor on ischemic acute renal failure (ARF). METHODS: Mouse models of ischemic ARF were treated with the specific ICE inhibitor AC-YVAD-CMK. A renal function assay and renal morphological studies were employed to estimate the renal protective effect of AC-YVAD-CMK. The survival rate of mouse models was also analyzed by a time series test. Furthermore, renal ICE activity, mature interleukin-18 (IL-18) protein expression and interferon-gamma (IFN-gamma) mRNA expression were also detected by fluorescent enzyme-linked immunosorbent assay (ELISA), ELISA, and semi-quantitative reverse transcription-polymerase chain reaction, respectively. RESULTS: The levels of blood urea nitrogen (BUN) and serum creatinine (Scr) increased remarkably in the model controls compared with the sham-operated groups (P<0.01). Typical renal tubular necrosis was found in the model controls. Renal ICE activity, mature IL-18 protein expression, and IFN-gamma mRNA expression were also increased significantly in the model controls compared with the sham-operated groups. The levels of BUN and Scr in the AC-YVAD-CMK therapy group were decreased significantly compared with the untreated model controls (P<0.01). Renal tubulointerstitial lesion was also attenuated significantly (P<0.05). AC-YVAD-CMK therapy alleviated the clinical features of ARF, and increased the survival rate (P<0.01). Furthermore, AC-YVAD-CMK therapy also decreased ICE activity, mature IL-18 protein expression, and IFN-gamma mRNA expression in renal tissue (P<0.05). CONCLUSION: The selective ICE inhibitor AC-YVAD-CMK can effectively protect the kidney from acute ischemic lesions. This protective effect is associated with decreased renal ICE activity and suppressed IL-18 maturation and IFN-gamma mRNA transcription.

[IL-18 expression in whole blood cell cultures from active lupus nephritis and the inhibitory effects of FK506, cyclosporin A and dexamethasone].

AIM: To explore the expression of IL-18 in patients with active lupus nephritis(LN) and the inhibitory effects of immunosupressive agents FK506, cyclosporin A (CsA) and dexamethasone(DEX). METHODS: Peripheral blood samples were collected from 16 LN patients and 10 health volunteers and cultured with or without PHA/LPS, PHA/LPS+FK506, PHA/LPS+CsA and PHA/LPS+DEX for 24 hours. The IL-18 level in cultured supernatants and the IL-18 mRNA expression in cultured whole blood cells were detected by ELISA and semi-quantitative RT-PCR respectively, and the inhibitory effects of FK506, CsA and DEX on IL-18 expression were investigated. RESULTS: The expression levels of IL-18 mRNA and its protein in whole blood cell cultures from LN patients were higher than those in normal control group (P<0.01) either spontaneously or after stimulation with LPS/PHA. FK506, CsA and DEX suppressed significantly the expressions of IL-18 mRNA and its protein (P<0.001) in LPS/PHA-stimulated whole blood cell from LN patients. The inhibitory effects of FK506, CsA and DEX on the IL-18 protein expression in LN patients were stronger than that in normal control group (P<0.01 or P<0.05). CONCLUSION: IL-18 may play an important role in the pathogenesis of LN and inhibition of IL-18 production may be an useful way to treat LN.