Sphingosine Kinase 2 Deficiency Attenuates Kidney Fibrosis via IFN- γ

Abstract

Maladaptive repair after AKI may lead to progressive fibrosis and decline in kidney function. Sphingosine 1-phosphate has an important role in kidney injury and pleiotropic effects in fibrosis. We investigated the involvement of sphingosine kinase 1 and 2 (SphK1 and SphK2), which phosphorylate sphingosine to produce sphingosine 1-phosphate, in kidney fibrosis induced by folic acid (FA) or unilateral ischemia-reperfusion injury. Analysis of Masson trichrome staining and fibrotic marker protein and mRNA expression 14 days after AKI revealed that wild-type (WT) and Sphk1^-/- mice exhibited more kidney fibrosis than Sphk2^-/- mice. Furthermore, kidneys of FA-treated WT and Sphk1^-/- mice had greater immune cell infiltration and expression of fibrotic and inflammatory markers than kidneys of FA-treated Sphk2^-/- mice. In contrast, kidneys of Sphk2^-/- mice exhibited greater expression of Ifng and IFN-γ-responsive genes (Cxcl9 and Cxcl10) than kidneys of WT or Sphk1^-/- mice did at this time point. Splenic T cells from untreated Sphk2^-/- mice were hyperproliferative and produced more IFN-γ than did those of WT or Sphk1^-/- mice. IFN-γ blocking antibody administered to Sphk2^-/- mice or deletion of Ifng (Sphk2^-/-Ifng^-/- mice) blocked the protective effect of SphK2 deficiency in fibrosis. Moreover, adoptive transfer of Sphk2^-/- (but not Sphk2^-/-Ifng^-/- ) CD4 T cells into WT mice blocked FA-induced fibrosis. Finally, a selective SphK2 inhibitor blocked FA-induced kidney fibrosis in WT mice. These studies demonstrate that SphK2 inhibition may serve as a novel therapeutic approach for attenuating kidney fibrosis.

Keywords: T cells; folic acid; ischemia-reperfusion; renal fibrosis; sphingolipid.

Figure 1.

FA treatment induces fibrosis in kidneys of WT and Sphk1^−/− mice but Sphk2^−/− mice are protected. (A) Plasma creatinine in WT, Sphk1^−/−, or Sphk2^-/ mice measured 3 and 14 days after vehicle (0.3 M sodium bicarbonate) or FA (250 mg/ml, intraperitoneal) treatment (see Supplemental Figure 3 for timeline). (B) Deposition of extracellular collagen indicated by Masson trichrome staining (blue) in kidney sections of WT and Sphk1^−/− is much greater than in Sphk2^−/− mice euthanized 14 days after FA treatment. Scale bar, 1 mm. (C) Extent of fibrosis determined by quantitative stereological analysis of Masson trichrome-stained sections (expressed as percentage of total surface area of kidney section occupied by interstitial fibrosis). (D) Higher magnification of cortex in Masson trichrome-stained sections from WT mouse kidney showing interstitial cell infiltration (arrows). Scale bar, 100 μm. n=3–4. *P<0.05; **P<0.01; ***P<0.001.

Figure 2.

FA induces increased expression of fibrotic markers in kidneys of WT and Sphk1^−/− but not Sphk2^−/− mice. Tissue samples from same mice as Figure 1 (euthanized on day 14). Expression of (A) α-SMA (Acta2), (B) fibronectin (Fn1), and (C) vimentin (Vim) measured by real-time quantitative RT-PCR (relative to glyceraldehyde 3-phosphate dehydrogenase; n=3–4) and immunofluorescence labeling (representative photomicrographs from the same groups of mice). Scale bar, 100 μm. *P<0.05.

Figure 3.

FA induces infiltration of immune cells in kidneys of WT and Sphk1^−/− but not Sphk2^−/− mice. (A) Macrophage (CD11b⁺F4/80^low), neutrophil (CD11b⁺GR-1^high [Ly6G]), and T cell (CD3⁺) number in kidneys determined by flow cytometry 14 days after treatment with vehicle or FA. Tissue samples from same mice as Figure 1 (euthanized on day 14). n=3–4. *P<0.05; **P<0.01; ***P<0.001 relative to corresponding vehicle control. (B) Immunofluorescent labeling of neutrophils (7/4), monocytes (F4/80), and T cells (CD3) in kidney sections from mice 14 days after vehicle or FA. Blue 4′,6-diamidino-2-phenylindole–labeled nuclei. Green autofluorescence reveals architecture of tubules. Scale bar, 100 μm.

Figure 4.

FA induces increased expression of Tgfb in kidneys of WT and Sphk1^−/− but not Sphk2^−/− mice, and increased Ifng in Sphk2^−/− but not WT and Sphk1^−/− mice. RNA was extracted from kidneys harvested 14 days after administration of vehicle (sodium bicarbonate buffer) or FA (treatments as in Figure 1). mRNA levels were measured by quantitative RT-PCR and expressed relative to glyceraldehyde 3-phosphate dehydrogenase. n=3–4. *P<0.05; **P<0.01.

Figure 5.

T cells from Sphk2^−/− mice proliferate faster and make more IFN-γ. (A–C) T cell proliferation measured in Cell Trace Violet dilution studies. T cells were isolated from spleens of untreated mice (control conditions), stimulated in culture with anti-CD3- and anti-CD28-conjugated beads, incubated with Cell Trace Violet for 10 minutes and allowed to proliferate for 72 hours. (A) Cell Trace Violet measured by flow cytometry. Numbers in each panel are percentage of the total population of Cell Trace Violet-labeled cells that has divided over the course of 3 days. Results are from a representative experiment; similar results were achieved in a replicate experiment by labeling cells with CFSE. (B) Overlay of proliferation histograms for splenic T cells from the four types of mice. (C) Proliferation measured in triplicate wells of cells (representative experiment from two mice per group) and expressed as percentage of the population of cells that divided at least once during the 3-day incubation period (mean±SEM). (D) FACS analysis of intracellular IFN-γ in proliferating T cells. (E) Percentage of proliferating, stimulated T cells that are IFN-γ–positive; from triplicate wells analyzed by FACS as shown in panel D. (F) IFN-γ concentration (measured by ELISA) in supernatant of triplicate wells of T cell cultures (representative experiment from three mice per group, repeated at least three times) 4 and 7 days after stimulation with vehicle or anti-CD3- and anti-CD28-conjugated beads. **P<0.01 compared with values on day 7 for WT and Sphk1^−/−. n.d., not determined in mice lacking IFN-γ.

Figure 6.

Protection from unilateral kidney IRI-induced fibrosis in Sphk2^−/− mice is dependent on expression of IFN-γ. (A–D) Mouse kidneys were exposed to sham surgery or 26 minutes unilateral ischemia followed by reperfusion. To reveal kidney function, the contralateral unclamped kidney was removed (and saved as the control) on day 13. (A) Plasma creatinine was measured 1 day after nephrectomy on day 14 after unilateral IRI. (B) Masson trichrome staining (blue) in kidney sections 13 (contralateral control kidney) or 14 days (IRI kidney) after IRI. Scale bar, 1 mm. (C) Extent of fibrosis determined by quantitative stereological analysis of Masson trichrome-stained sections. n=4–5. (D) mRNA levels of fibrotic markers, chemokines, and cytokines in IRI and contralateral control kidneys (Cntrl) measured by quantitative RT-PCR and expressed relative to glyceraldehyde 3-phosphate dehydrogenase. Two-way ANOVA with post hoc analysis revealed statistical significance between Sphk2^−/− and WT or Sphk1^−/− mice in level of expression of various markers that was reversed in Sphk2^−/− Ifng^−/− mice, but comparisons have been omitted from the figure for simplicity. For CXCL9 and CXCL10, ***P<0.001 relative to all other groups. (E) Mice were subjected to unilateral IRI and contralateral nephrectomy as in A–D, and were treated with IgG1 isotype control antibody or anti–IFN-γ blocking antibody (150 μg per mouse, administered intraperitoneally) on days 2, 5, 8, 11, and 13 after IRI (see timeline in Supplemental Figure 3). Plasma creatinine was measured on day 14. n=4–5. *P<0.05; **P<0.01; ***P<0.001.

Figure 7.

Adoptive transfer of splenic CD4⁺ T cells from Sphk2^−/− mice 7 days before FA protects kidneys of recipient WT mice from injury. CD4⁺ T cells were isolated from spleens of WT, Sphk1^−/− Sphk2^−/−, or Sphk2^−/−Ifng^−/− mice, and 1.5×10⁶ cells were intravenously injected via the tail vein into WT mice 7 days before treating with vehicle or FA (as in Figure 1). (A) Masson trichrome staining (blue) in kidney sections 14 days after vehicle or FA. Scale bar, 1 mm. (B) Extent of fibrosis determined by quantitative stereological analysis of Masson trichrome-stained sections. (C) Relative expression of fibrotic markers in kidneys measured by quantitative RT-PCR (relative to glyceraldehyde 3-phosphate dehydrogenase). n=3 for vehicle, 4–5 for FA groups. *P<0.05. NC, no cells.

Figure 8.

SphK2 inhibitor reduces FA-induced fibrosis in kidneys. (A) Relative expression of fibrotic markers measured by quantitative RT-PCR (relative to glyceraldehyde 3-phosphate dehydrogenase) 14 days after vehicle (sodium bicarbonate) or FA in kidneys of WT mice that were treated once daily with the SphK2 inhibitor SLP120701 (10 mg/kg, administered intraperitoneally) beginning 1 day after FA. n=3 for vehicle, 4–5 for FA or FA+SLP120701. *P<0.05; **P<0.01; ***P<0.001 relative to vehicle or comparisons as shown. (B) Masson trichrome staining (blue) in kidney sections. Scale bar, 1 mm. Acta2, actin α2 smooth muscle aorta; Col3a1, collagen type III α1; Ctgf, connective tissue growth factor; Fn1, fibronectin; Tgfb, transforming growth factor β1; Vim, vimentin.

Figure 9.

Mechanisms of antifibrotic effects of SphK2 deficiency. In WT mice or SphK1-deficient mice, FA or IRI leads to early epithelial injury followed by progressive fibrosis. Fibroblasts and pericytes transform into myofibroblasts leading to extracellular matrix deposition. Deletion of Sphk2 leads to enhanced early injury to an extent greater than WT or Sphk1^−/− mice following IRI (Supplemental Figure 1A), an effect that is dependent on the proinflammatory effects of IFN-γ (Supplemental Figure 1B). Despite the increase in initial injury in Sphk2^−/− mice, there is reduced fibrosis at 14 days in both unilateral IRI and FA-induced injury. T cells from SphK2-deficient mice are hyperproliferative and produce more IFN-γ in vitro. Although T cells may be one source of IFN-γ, other hematopoietic and nonhematopoietic cells likely to contribute during the period of time following initial injury to the kidney IFN-γ levels and IFN-γ response genes that lead to reduced fibrosis. Developing drugs that block SphK2 may lead to therapeutic agents that attenuate kidney fibrosis.