Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase‐1 expression and activity

Abstract

Background and purpose: Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved. Experimental approach: MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate. Key results: Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration. Conclusions and implications: These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.

Purinergic signalling in the kidney in health and disease

“…ATP and UTP induce migration of mesangial cells by upregulating sphingosine kinase-1 expression and activity [193].…”

Interleukin-11 protects against renal ischemia and reperfusion injury

“…SK activity was measured, as described previously (23), using a modified protocol according to Vessey et al (57). To preferentially measure SK1 activity, we supplemented the assay buffer with 250 mM KCl plus 0.5% Triton X-100 (27,42).…”

Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts

“…Primary cultures of mouse renal mesangial cells were isolated from C57BL/6 mice (Wt) or SK-2 knockout (ko) mice exactly as previously described (Klawitter et al, 2007;Hofmann et al, 2008). Outgrown mesangial cells were subcultured and further used up to passage 20.…”