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IL-13 and IL-4 Receptors Expression Mediate the Injury of Renal Tubular Epithelial Cells

Author: LuZuoZhou
Tutor: LiuZhiHong；LiLeiShi
School: Second Military Medical University
Course: Internal Medicine
Keywords: IL-13 IL-4 Tubular epithelial cells FSGS RANTES Signal transduction and activator of transcription
CLC: R692
Type: Master's thesis
Year: 2009
Downloads: 103
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Abstract

Focal segmental glomerulosclerosis (focal segmental glomerulosclerosis, FSGS) is a common glomerular disease, accounting for 7.0% of the adult primary glomerulonephritis, clinical manifestations of massive proteinuria, early studies have shown that adequate cell damage is closely related to the generation of massive proteinuria, podocyte damage, shedding constitute a key link in the development of glomerulosclerosis occurred. Cellular immune dysfunction plays an important role in FSGS pathogenesis of FSGS patients with T-cell release of cytokines can lead to podocyte injury, which Th2 cytokines, such as IL-13 and IL-4 is considered to be the major virulence factor nephrotic syndrome relapse patients, CD4 and CD8 T cells, IL-13 and IL-4 mRNA expression increased cytoplasm of IL-13 and IL-4 protein expression level also increased. In situ hybridization detected the FSGS kidney tissue of IL-13 and IL-4 mRNA expression increased and tubulointerstitial area of ??IL-13 and IL-4 mRNA expression levels and changes in renal function and 24h urine protein excretion was positively correlated . Studies have shown that podocytes express IL-13 receptor and IL-4 receptor, IL-13 and IL-4 in conjunction with the corresponding receptors can direct injury sufficient cytoskeleton of structure protein connection between the destruction of cells, resulting in filtration destruction of membrane permeability. FSGS patients podocyte injury, often accompanied by renal tubular epithelial cell injury, this tissue morphology and function are clearly reflected, and become an important feature of FSGS diagnosis and differential diagnosis. Previous view massive proteinuria induced tubular epithelial cell injury, glomerular segmental sclerosis caused by a the ischemic injury of the efferent artery stenosis and corresponding nephron glomerular capillary loop and Bauman wall the adhesions to make urine anti-infiltration of the interstitial renal interstitial trigger inflammatory response FSGS tubular interstitial injury reasons, but these mechanisms may cause tissue damage and its outcome is not sufficient to fully explain the phenomenon observed in the clinical. Part of the performance for super heavy proteinuria (gt; 10g/24h) FSGS patients, spectral analysis of the urine protein is often the presence of protein macromolecules and small molecules mixed, this happens only in the proximal tubular absorption function is severely impaired when occur; some of the treatment of sensitive patients, in massive proteinuria ease while, reflect tubular injury indicators also rapidly improved renal biopsy of these patients is often associated with acute proximal tubular injury, we speculated, whether there may be some of the factors leading to podocyte lesions directly attack the tubular epithelial cells and its functional and structural changes, thus triggering the subsequent interstitial lesions and interstitial fibrosis. Since it has been confirmed that the IL-13 and IL-4 by IL-13 receptor and IL-4 receptor on the podocyte lead to podocyte injury, then, we speculate, may also exist in FSGS patients with renal tubular epithelial cell injury same molecular pathogenesis. To investigate the IL-13 and IL-4 on the injury of the proximal tubular epithelial cells, the study with the human proximal tubular epithelial cell line (HK2), studies in human proximal tubular epithelial cell IL-13 receptor and IL 4 receptor expression and IL-13 and IL-4 on renal tubular epithelial cells, and to explore its mechanism. Part I: The purpose of renal tubular epithelial cell IL-13 receptor and IL-4 receptor detection: the detection of human proximal tubular epithelial cell IL-13 receptor and the expression of the IL-4 receptor. Methods: 1. Cells in human proximal tubular epithelial cell line (HK2) were purchased from ATCC (CRL-2910TM). 2 from the mRNA and protein levels by RT-PCR and immunofluorescence method to detect the HK2 cells on the expression of IL-13 receptor and IL-4 receptor. Results: 1.RT-PCR results showed that the mRNA expression of IL-13 receptor type I and IL-4 II receptor composed of sub-chain-IL-13Rα1 and IL-4Rα the HK2 cells have normal training. 2 immunofluorescence showed normal cultured HK2 cells with the protein expression of the IL-13 in the composition of the receptor type I and type II receptor of the IL-4 alkylene chain-of IL-13Rα1 and the IL-4Rα, granular uniformly distributed in HK2 extracellular pulp. Conclusion: human proximal tubular epithelial cells express IL-13 receptor type I receptor and IL-4 II. The second part: IL-13 receptor and IL-4 receptor purpose tubular injury: by observing the IL-13 and IL-4 on proximal tubular epithelial cell injury markers RANTES, and signal transduction pathways JAK -STAT6 impact analysis of the role of IL-13 and IL-4 on renal tubular epithelial cell injury, and explore its mechanism. Methods: 1.IL-13 and IL-4 effects HK2 cells by immunofluorescence observed with different concentrations (10ng/ml, 50ng/ml and 100ng/ml) IL-13 and IL-4 in intervention HK2 12-48 hours after the cell the HK2 injury markers of RANTES changes. 2. IL-13 and IL-4 on HK2 cell injury mechanisms to detect different times) and Western blot - concentrations of IL-13 and IL-4 were intervention HK2 cells, JAK-STAT6 signaling pathway. 2) and Western blot detection of specific inhibitors of JAK-STAT6 signaling pathway - leflunomide on IL-13 and IL-4 role HK2 cells JAK-STAT6 signaling pathway activation. 3) observed with immunofluorescence method of leflunomide on IL-13 and IL-4 role HK2 cells after RANTES change. Results: 1.IL-13 and IL-4 was the time - the dose-dependent effects of proximal tubular epithelial cells RANTES expression. 50ng/ml IL-13, 100ng/ml IL-4 in the role of HK2 cells 48h after injury is the most obvious. HK2 cells cultured normal RANTES negative, 50ng/ml IL-13 and 100ng/ml IL-4 intervention after 48 hours, the positive cells were significantly increased. JAK-STAT6 phosphorylation level of 2. IL-13 treatment group began to increase the 50ng/ml role 10min (phosphorylation level of 222.4% ± 81.9% of the control group), and reached the peak at 50ng/ml 20min (phosphate the level of the control group, 307.8% ± 40.7%); (phosphorylation level of the control group, 294.0% ± 59.7% of IL-4-treated JAK-STAT6 phosphorylation levels also began to increase when 50ng/ml role 10min) and reached a peak 100ng/ml 20min (phosphorylation level of the control group 1618.0 ± 229.9%). Leflunomide intervention no significant change in IL-13 treated and leflunomide pre-intervention in the IL-4 treatment group compared with the negative control, JAK-STAT6 phosphorylation levels, phosphorylation levels compared with the negative control group were: 43.6% ± 0.4% vs 41.2% ± 2.4%, P gt; 0.05,20.6% ± 0.8% vs 14.9% ± 6.7%, P gt; 0.05, no statistical difference; STAT6 phosphorylation level compared to the positive control. significantly lower compared were: 43.6% ± 0.4% vs 89.3% ± 8.3%, P lt; 0.01,20.6% ± 1.4% vs 44.8% ± 7.5%, P lt; 0.05, were statistically significant. Leflunomide intervention IL-13 treatment group compared with IL-13 treatment group, leflunomide intervention IL-4 treatment group compared with IL-4 treatment group the HK2 cells RANTES expression were significantly weakened, with the negative control had no significant difference. Conclusion: 1. IL-13 and IL-4 by human proximal tubular corresponding receptors on epithelial cells cause cell damage, the performance increase RANTES expression. 2, IL-13 is closely related to the above effects of IL-4 and intracellular JAK-STAT6 signaling pathway.

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