CXCL16/ERK1/2 pathway regulates human podocytes growth, migration, apoptosis and epithelial mesenchymal transition

Abstract

Primary nephrotic syndrome (PNS) is the commonest glomerular disease affecting children. Previous studies have confirmed that CXC motif chemokine ligand 16 (CXCL16) is involved in the pathogenesis of PNS. However, the exact mechanisms underlying the pathogenesis of PNS remain to be elucidated. Thus, the present study aimed to elucidate the role of CXCL16 in PNS. It was found that the expression of CXCL16 and extracellular signal‑regulated kinases 1 and 2 (ERK1/2) were significantly increased in clinical PNS renal tissues using reverse transcription‑quantitative PCR, western blot analysis and immunohistochemistry. Lentivirus overexpression or short hairpin RNA vector was used to induce the overexpression or knockdown of CXCL16 in podocytes, respectively. Overexpression of CXCL16 in podocytes could decrease the cell proliferation and increase the migration and apoptosis, whereas CXCL16 knockdown increased cell proliferation and decreased cell migration and apoptosis. Results of the present study further demonstrated that ERK2 protein expression was regulated by CXCL16. The knockdown of ERK2 expression reversed the effects of CXCL16 on the proliferation, apoptosis, migration and epithelial mesenchymal transition (EMT) of podocytes. Collectively, the findings of the present study highlighted that the CXCL16/ERK1/2 pathway regulates the growth, migration, apoptosis and EMT of human podocytes.

Keywords: CXC motif chemokine ligand 16; epithelial mesenchymal transition; extracellular signal‑regulated kinases 1 and 2; podocytes injury; primary nephrotic syndrome.

Figure 1.

Expression of CXCL16 in PNS. (A) CXCL16 expression levels were evaluated by reverse transcription-quantitative PCR in PNS patients and normal individuals. (B) Western blotting was used to investigate the CXCL16 protein expression in PNS and normal individuals. (C) Immunohistochemical staining of CXCL16 in normal (left) tissue (original magnification, ×200) and PNS renal tissues (right) statistical results of gray scanning quantization in immunohistochemistry. Arrows represent podocytes. ***P<0.001. CXCL16, CXC motif chemokine ligand 16; PNS, Primary nephrotic syndrome.

Figure 2.

CXCL16 regulates the growth of podocytes. (A and B) The expression of CXCL16 proteins after transduction with lentivirus-CXCL16 or lentivirus-CXCL16 shRNA1 and 2 detected by western blotting. (C and D) CCK8 assay was used to detect the growth of CXCL16 overexpressed and knockdown HPC. (E and F) Apoptosis was detected by flow cytometry. The early-apoptotic cells (Q1), late-apoptotic (Q2), cell debris (Q3) and live cells (Q4) are indicated. The numbers of apoptotic cells within quadrants Q2 and Q3 were counted for statistical analysis. (G and H) Cell migration in immortalized human podocytes was analyzed using Transwell assay (magnification, ×200). *P<0.05, **P<0.01, ***P<0.001. CXCL16, CXC motif chemokine ligand 16; sh, short-hairpin RNA.

Figure 3.

Expression of ERK1/2 in PNS and HPC. (A) ERK1/2 expression levels were evaluated by reverse transcription-quantitative PCR in PNS patients and normal individuals. (B) The expression of ERK1/2 proteins in PNS and normal individuals. (C) IHC staining of ERK1/2 in PNS renal tissues (magnification, ×400). The statistical results of gray scanning quantization in IHC. (D) Correlation analysis of CXCL16 mRNA expression and ERK1/2 mRNA expression in PNS renal tissue of children, correlation coefficient r=0.6310, P<0.05. (E and F) ERK1/2 protein expression in HPC cell lines with CXCL16 overexpression and knockdown detected by western blotting. ***P<0.001. ERK1/2, extracellular signal-regulated kinases 1 and 2; PNS, Primary nephrotic syndrome; HPC, immortalized human podocytes; IHC, immunohistochemistry; CXCL16, CXC motif chemokine ligand 16.

Figure 4.

Knockdown of ERK1/2 expression in podocytes overexpressing CXCL16 could prevent the inhibition of podocyte proliferation, the increase of podocyte apoptosis rate and migration ability. (A) Western blotting detected ERK1/2 expression in CXCL16 overexpressed HPC with ERK1/2 knockdown (referred to as: CXCL16-ERK1/2 shRNA). (B) CCK8 assay was used to detect the growth of CXCL16-ERK1/2 shRNA. (C) Apoptosis was detected by flow cytometry. (D) Cell migration in CXCL16-ERK1/2 shRNA was analyzed using Transwell assay (magnification, ×200). **P<0.01, ***P<0.001. ERK1/2, extracellular signal-regulated kinases 1 and 2; CXCL16, CXC motif chemokine ligand 16; HPC, immortalized human podocytes; sh, short-hairpin RNA.

Figure 5.

Further knockdown of ERK1/2 expression in podocytes could prevent the increase of epithelial mesenchymal transition induced by overexpressing-CXCL16. (A) The expression of N-Cadherin, P-Cadherin, vimentin and ZO-1 protein in CXCL16-ERK1/2 shRNA was detected by western blot assay.(B) Gray scale analysis of western blotting results. *P<0.05, **P<0.01. ERK1/2, extracellular signal-regulated kinases 1 and 2; CXCL16, CXC motif chemokine ligand 16; ZO-1, zonula occludens-1.