Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells

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.

Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS

“…Obscurin (Obscn; Arhgef30; Cytoskeletal Calmodulin And Titin-Interacting RhoGEF), also upregulated and never associated with FSGS, is a modulator of the cytoskeleton and activator of RhoA [18,19] and might therefore be able to induce FSGS [20]. We also found strongly increased levels of Serine Protease 23 (Prss23) and Sphingolipid Transporter 2 (Spns2), which has been associated with kidney fibrosis before [21]. Strikingly, the transcripts of several circadian genes, including Basic Helix-Loop-Helix Family Member E41 (Bhlhe41, Dec2), the Period Circadian Regulators 2 and 3 (Per2, Per3) and D Site Of Albumin Promoter Binding Protein (Dbp) were massively enhanced.…”

Sphingosine 1‐phosphate/microRNA‐1249‐5p/MCP‐1 axis is involved in macrophage‐associated inflammation in fatty liver injury in mice

“…For instance, the expression of inflammatory cytokines, including TNF‐α, MCP‐1, NOS2 and MIP‐1β is significantly increased in BMMs upon S1P challenge [6]. Downregulation of the S1P Transporter Spns2, which is known to export S1P and thereby reduces intracellular S1P, abolishes the strong induction of MCP‐1 by TNF‐α and IL‐1β in the human renal proximal tubular epithelial cells [37]. S1P mainly acts through five high affinity G protein‐coupled S1P receptors, which are ubiquitously expressed and mediate a complex network of signaling in a cell type dependent manner [38, 39].…”

Morpholino Analogues of Fingolimod as Novel and Selective S1P1 Ligands with In Vivo Efficacy in a Mouse Model of Experimental Antigen-Induced Encephalomyelitis

“…RNA extraction, complementary DNA (cDNA) synthesis, and quantitative PCR analysis were performed as previously described [ 43 ]. Primer sequences of mouse CD3, VCAM-1, and 18S RNA were as previously described [ 15 , 44 ].…”