Background: C-X-C chemokine receptor type 4 (CXCR4) has a certain effect on renal fibrosis, and there are few specific studies in cells. We want to investigate the impact of suppressing CXCR4 activity on the expression of renal fibrosis-related genes in primary glomerular endothelial cells, mesangial cells, and podocytes.
Methods: Immunofluorescence assays were used to determine the purity of isolated glomerular endothelial cells, mesangial cells, and podocytes. CXCR4 knockdown cell lines were established by transfecting the short hairpin (sh)RNA against CXCR4. T140 and AMD3100 were used to inhibit the activity of CXCR4. LY294002 was used to inhibit the activity of phosphoinositide 3-kinase (PI3K). The mRNA expression of CXCR4 was determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The protein expression of CXCR4, collagen IV, matrix metallopeptidase (MMP)-9, PI3K, Rac1, and vascular cell adhesion protein 1 (VCAM-1) was evaluated by Western blot analysis.
Results: High purity was observed on isolated primary glomerular endothelial cells and podocytes. However, the purity of isolated mesangial cells was relatively low. The mRNA expression of CXCR4 was significantly suppressed by the transfection of shRNA. Compared to control cells, the expression of CXCR4, collagen IV, MMP-9, PI3K, Rac1, and VCAM-1 were dramatically downregulated in cell lines transfected with shRNA against CXCR4. Furthermore, cell lines treated with T140, AMD3100, or LY294002 also showed downregulated expression of these proteins compared to untreated cells. No significant differences were observed in the protein expression of these proteins between control cells and cells transfected with the shRNA negative control (NC).
Conclusions: Suppressing the activity of CXCR4 downregulated the expression of renal fibrosis-related genes in primary glomerular cells, even under a non-inflammatory state.
Keywords: AMD3100; C-X-C chemokine receptor type 4 (CXCR4); T140; glomerulus; renal fibrosis.
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