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Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells
Published on: June 20, 2018
[Expression of alpha 1 anti-trypsin in proximal tubular epithelial cell line]
Gong-yao Tang1, Yi-fu Chen, Mei Zhang
1The Center of Nephrology, China-Japan Friendship Hospital, Beijing 100029, China.
Insights
Alpha 1 anti-trypsin (AAT) is expressed in tubular epithelial cells and its expression can be increased by lipopolysaccharide (LPS) stimulation. This study investigated AAT regulation in HKC cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Renal Physiology
Context:
- Alpha 1 anti-trypsin (AAT) plays a crucial role in protecting tissues from inflammatory damage.
- Tubular epithelial cells are key components of the kidney tubules and are involved in various physiological and pathological processes.
- Understanding the regulation of AAT in these cells is important for comprehending kidney physiology and disease.
Purpose:
- To investigate the expression of alpha 1 anti-trypsin (AAT) in tubular epithelial cells (HKC).
- To determine the regulatory effects of lipopolysaccharide (LPS) on AAT expression in HKC.
- To analyze the transcriptional and translational regulation of AAT in response to LPS.
Summary:
- AAT was detected in HKC cells using indirect immunofluorescence and confirmed at the mRNA level via RT-PCR, with sequencing verifying the AAT mRNA sequence.
- Stimulation of HKC cells with 2.0 microg/ml LPS significantly upregulated AAT mRNA expression (4h) and protein synthesis (8h) compared to unstimulated cells.
- Lower concentrations of LPS (0.5, 1.0 microg/ml) did not affect AAT expression in HKC cells.
Impact:
- This study demonstrates that tubular epithelial cells express AAT.
- The findings reveal that LPS can upregulate AAT expression in these cells, suggesting a potential role in inflammatory responses within the kidney.
- This research provides insights into the molecular mechanisms governing AAT regulation in renal tubular cells.
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.
