Abstract
Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate (S1P), which acts as a key regulator of inflammatory and fibrotic reactions, mainly via S1P receptor activation. Here, we show that in the human renal proximal tubular epithelial cell line HK2, the profibrotic mediator transforming growth factor β (TGFβ) induces SK-1 mRNA and protein expression, and in parallel, it also upregulates the expression of the fibrotic markers connective tissue growth factor (CTGF) and fibronectin. Stable downregulation of SK-1 by RNAi resulted in the increased expression of CTGF, suggesting a suppressive effect of SK-1-derived intracellular S1P in the fibrotic process, which is lost when SK-1 is downregulated. In a further approach, the S1P transporter Spns2, which is known to export S1P and thereby reduces intracellular S1P levels, was stably downregulated in HK2 cells by RNAi. This treatment decreased TGFβ-induced CTGF and fibronectin expression, and it abolished the strong induction of the monocyte chemotactic protein 1 (MCP-1) by the pro-inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1β. Moreover, it enhanced the expression of aquaporin 1, which is an important water channel that is expressed in the proximal tubules, and reverted aquaporin 1 downregulation induced by IL-1β/TNFα. On the other hand, overexpression of a Spns2-GFP construct increased S1P secretion and it resulted in enhanced TGFβ-induced CTGF expression. In summary, our data demonstrate that in human renal proximal tubular epithelial cells, SK-1 downregulation accelerates an inflammatory and fibrotic reaction, whereas Spns2 downregulation has an opposite effect. We conclude that Spns2 represents a promising new target for the treatment of tubulointerstitial inflammation and fibrosis.
Highlights
Sphingosine 1-phosphate (S1P) is a central sphingolipid molecule that regulates a variety of important cellular responses, including proliferation and migration, and it modulates inflammatory and fibrotic responses depending on its site of action [1,2,3]
Many studies have shown that under cell culture conditions, proximal tubular epithelial cells are activated by the pro-fibrotic mediator transforming growth factor (TGF)β to undergo epithelial-mesenchymal transition (EMT) which is demonstrated by reduced expression of epithelial markers such as cytokeratin and E-cadherin, and increased the expression of mesenchymal markers, such as α-smooth muscle actin and vimentin
To investigate the contribution of cellular S1P to tubulointerstitial inflammation and fibrosis, we used the human renal proximal tubular epithelial cell line HK2, which represents an important cell type that is involved in tubulointerstitial nephritis that are characterized by both an inflammatory and a fibrotic phase
Summary
Sphingosine 1-phosphate (S1P) is a central sphingolipid molecule that regulates a variety of important cellular responses, including proliferation and migration, and it modulates inflammatory and fibrotic responses depending on its site of action [1,2,3]. Many studies have shown that under cell culture conditions, proximal tubular epithelial cells are activated by the pro-fibrotic mediator transforming growth factor (TGF)β to undergo epithelial-mesenchymal transition (EMT) which is demonstrated by reduced expression of epithelial markers such as cytokeratin and E-cadherin, and increased the expression of mesenchymal markers, such as α-smooth muscle actin and vimentin. This suggests that tubular epithelial cells may transform in vivo to myofibroblasts during fibrosis [22]. These data underline the promise of Spns as an attractive pharmacological target to prevent secretion of iS1P and thereby dampen inflammatory and fibrotic responses, such as those seen in tubulointerstitial nephritis
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