[Expression and regulation of adiponectin in human renal glomerular endothelial cells].

OBJECTIVE: To investigate the expression and regulation of adiponectin in human renal glomerular endothelial cells (HRGEC). METHOD: The adiponectin expression in human renal tissue was examined with immunohistochemistry assay, and the adiponectin mRNA and protein expression of HRGEC was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. PCR product was sequenced. To investigate the regulation action of TNF-alpha, the adiponectin produced by HRGEC was semi-quantitatively determined with real-time PCR in mRNA level and quantitatively measured with dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) in protein level, respectively. RESULTS: (1) Positive adiponectin staining was observed on human glomerular capillary wall with immunohistochemistry assay. (2) Adiponectin mRNA expression in HRGEC was detected by RT-PCR, and the DNA sequence of this PCR product is consistent with adiponectin DNA sequence in Gene Bank. Adiponectin protein was also found in the supernatant of cultured HRGEC by Western blot. (3) Compared with control groups, the adiponectin mRNA expression in HRGEC determined by real-time PCR was significantly up-regulated after 250 IU/ml and 500 IU/ml TNF-alpha stimulation for 24 h (P < 0.05 and P < 0.01), and the adiponectin protein levels in culture supernatant measured by DELFIA were also significantly increased in these two groups (P < 0.05 and P < 0.01). (4) Compared with control groups, the adiponectin mRNA expression in HRGEC was significantly up-regulated after 500 IU/ml TNF-alpha stimulation for 6 h, 12 h and 24 h (P < 0.05, P < 0.05 and P < 0.01), and the adiponectin protein levels in culture supernatant were also significantly increased after stimulation for 24 h and 48 h (P < 0.01). CONCLUSIONS: HRGEC is able to synthesize adiponectin, and TNF-alpha can up-regulate its mRNA and protein expression.

[Expression of alpha 1 anti-trypsin in proximal tubular epithelial cell line].

OBJECTIVE: To investigate the expression and regulation of alpha 1 anti-trypsin (AAT) in tubular epithelial cells. METHODS: The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction (PCR) in transcription level respectively, and PCR product was sequenced. In order to observe the response of HCK to LPS, the different concentrations of LPS (0, 0.5, 1.0, 2.0 microg/ml) were used in the research and the regulation of AAT in HKC was semi-quantitatively detected with Western Blot and Real-Time PCR respectively. RESULTS: Indirect immunofluorescence staining showed that AAT was positive in HKC, but negative in renal interstitial fibroblast. By PCR, a significant messenger RNA band of AAT was found in HKC, but not in renal interstitial fibroblast. DNA sequencing indicated that the sequence of PCR product is consistent with AAT mRNA sequence in Gene Bank. Real-time PCR showed that the expression of AAT mRNA was up-regulated (fluorescence intensity ratio is 3.43 +/- 0.88 versus 1.22 +/- 0.20; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 4 h, and Western Blot showed that the synthesis of AAT significantly increased (band density ratio is 0.88 +/- 0.12 versus 0.59 +/- 0.05; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 8 h, as compared with unstimulated HKC. 0.5, 1.0 g/ml of LPS has no effect on the expression of AAT mRNA and AAT protein in HKC. CONCLUSIONS: HKC express AAT and the expression can be up-regulated by LPS.