Fig 1.
Diminished renal injury in Sphk2-/- mice after UUO.
(A) Paraffin sections were stained with Hematoxylin & Eosin (H&E) and Periodic Acid Schiff (PAS). Representative photomicrographs taken with Zeiss microscope using 40x objective, WT and Sphk2-/- mice (N = 6) non-obstructed (NO) and obstruced (O) kidneys at day 3 and 5. H&E (A), PAS (B). Infiltration of inflammatory cells, tubular atrophy and widening of interstitial spaces (by H&E stain), and tubular damage (by PAS stain) were considered as the criteria to assess the renal injury. (C) PAS stained slides were blinded and scored for renal injury. A scale of 0–5, zero being the least and 5 being the maximum injury, revealed that Sphk2-/- mice had diminished renal injury after 3 days of obstruction compared WT (**p<0.01). Experiments were repeated at least two times with N = 6 each time. Original magnification; 20x objective. Scale bar; 50μm.
Fig 2.
TGFβ1 expression is diminished in Sphk2-/- mice compared to WT.
TGFβ1 expression significantly increased in obstructed kidneys than in non-obstructed kidneys (N = 6). (A) Representative photomicrographs show immunohistochemical staining for TGFβ1 in WT mice (top panel) and Sphk2-/- (bottom panel) at 3 & 5 days after UUO in non-obstructed (NO) and obstructed (O) kidneys. (B, C) Western blot analysis of TGFβ1 in WT and Sphk2-/- mice at 1, 3 & 5 days and protein bands quantified using Image J and normalized to GAPDH. Results from contralateral non-obstructed kidney were comparable to sham operated kidneys. Experiments were repeated at least two times with N = 6 each time. WT vs Sphk2-/- at 3 day (*p<0.05); WT vs Sphk2-/- at 5 day post ligation (**p<0.01). Original magnification; 20x objective. Scale bar; 50μm.
Fig 3.
αSmooth muscle actin levels are elevated in WT mice when compared to Sphk2-/- mice.
(A) Representative photomicrographs show immunohistochemical staining in non-obstructed (NO) and obstructed (O) kidneys of WT and Sphk2-/- mice. (B, C) Western blot analysis for αSMA in WT and Sphk2-/- mice at 1,3 & 5 days after UUO and quantification of protein bands using image J and normalized to GAPDH. Experiments were repeated at least two times with N = 6 each time. WT vs Sphk2-/- at 3 day (*p<0.05); WT vs Sphk2-/- at 5 day post ligation (*p<0.05). Original magnification; 20x objective. Scale bar; 50μm.
Fig 4.
Differential recruitment of macrophage subtypes to kidneys after UUO: M2 phenotype of macrophages population is predominant in obstructed kidneys of Sphk2-/- mice.
(A) Kidney cells from non-obstructed (NO) and obstructed (O) kidneys of WT and Sphk2-/- mice were isolated by collagenase-1 digestion and stained with other immune cell and macrophage markers for flow cytometry. Total macrophages as identified by CD11b+ F4/80+ (A) increased after obstruction in both WT and Sphk2-/- mice, however the specific population of CD11b+ F4/80+ CD206+ macrophages (B) was significantly increased (p<0.05, WT vs. Sphk2-/- obstructed kidneys) in Sphk2-/- mice compared to WT mice. (C) Representative pseudo color plots in WT and Sphk2-/- mice. (*p<0.05). (Data represents average of 3 experiments, n = 6/experiment). (D) Total RNA was prepared from contralateral non-obstructed (NO) and obstructed (O) kidneys of WT and Sphk2-/- mice. Quantitative real time PCR was performed using Fast SYBR Green Master Mix. The relative fold increase in mRNA expression was normalized to GAPDH and fold increase is represented in the graph. QRT PCR data revealed that Sphk2 mice significantly decreased proinflammatory cytokine mRNA expression levels compared to vehicle treated mice IL1β, TNFα, MCP-1 and CXCL1. (*p<0.05, WT vs. Sphk2-/- obstructed kidneys), n = 6. Experiments were repeated at least two times with n = 6 each time.
Fig 5.
Bone marrow derived macrophages from Sphk2-/- mice preferentially polarize towards the M2 phenotype in the presence of M2 promoting signals.
Bone marrow cells isolated from WT and Sphk2-/- mice were differentiated into macrophages in medium containing Macrophage Colony stimulating growth factor (MCSF) and stimulated either with IL4 or IL13 for 24 hours. Flow cytometry analysis shows that Sphk2-/- mice were able to polarize significantly (p<0.05) towards M2 macrophages positive for CD11b+ F4/80+ CD206+. Pseudo color plot (A) and percent over control increase (B) for CD206+ cells is shown. QRT PCR analysis on total RNA prepared from IL4 stimulated macrophages from WT and Sphk2-/- macrophages revealed that mRNA expression levels for M2 markers Arg-1 and Ym-1 (C) were increased in BMDMs from Sphk2-/- mice compared to WT cells. (Data represents average of 3 experiments, n = 3/experiment). (D) Glycolytic rate in BMDM as assessed by the extracellular acidification rate (ECAR) using the Seahorse XF analyzer showed a significant increase in the glycolytic rate which was further enhanced upon addition of the glycolytic driver Oligomycin in WT but not Sphk2-/-/ cells, indicating that the glycolytic pathway is largely inactive in Sphk2-/- cells. Suppression of glycolysis with 2-DG has no effect on Sphk2-/- cells. **p<0.01; *p<0.05. Data represents average of 2 experiments, experiments repeated at least two times with N = 6 each time.
Fig 6.
Sphk2 inhibitor treatment diminishes renal injury.
6–8 week old WT mice were treated with the Sphk2 inhibitor (SK2i) at 3mg/kg. Renal injury was assessed in H&E stain (A), PAS stain (B) and scored (C). Myofibroblast infiltration indicated by protein levels of αSMA is significantly lower in SK2i treated mice (D), protein bands quantified using Image J and normalized to GAPDH (E). **p<0.01; *p<0.05. N = 6. Experiments repeated at least two times with N = 6 each time. Original magnification; 20x objective. Scale bar; 50μm.
Fig 7.
Reduced EMT transition in absence of Sphk2 following renal injury.
6–8 week old WT, Sphk2-/- or WT mice were treated with Sphk2 inhibitor (SK2i) at 3mg/kg or vehicle control, daily by i.p three days prior to and following the UUO surgery (6 days total) and expression of EMT markers were assessed in whole kidney lysates. Western Blot analysis indicated reduced expression of EMT markers Vimentin and Fibronectin in Sphk2-/- (A, B) and SK2i treated mice (C, D), protein bands were quantified using Image J and normalized to GAPDH. **p<0.01; *p<0.05. N = 3.
Fig 8.
Inflammatory cytokines are diminished in mice treated with SK2i.
Mice subjected to UUO were treated with either vehicle (2%cyclodextrin) or SK2i (3mg/kg) and kidney tissue was harvested for qRT PCR analysis for inflammatory cytokine mRNA shows that expression levels of IL1β, TNFα, MCP-1 and CXCL1 were significantly lower in SK2i compared to vehicle treated group, N = 6. (B) Flow Cytometry analysis of isolated kidney cells show that the percentage CD11b+F4/80+ cells did not change in vehicle and SK2i treated mice whereas the proportion of anti-inflammatory M2 cells (F4/80+ CD11b+ CD206+) is significantly higher (C) in the SK2i group. Fig 7C shows pseudo color plots for CD206+ cells of vehicle and SK2i treated mice and isotype control. (**p<0.01; *p<0.05). (Data represents average of 3 experiments, n = 3/experiment).
Fig 9.
Diminished renal fibrosis in Sphk2-/- and SK2i treated mice.
Sphk2-/- mice and WT treated with SK2i were subjected to UUO for 7 days with appropriate control groups. Masson’s Trichrome staining indicates reduced renal fibrosis after 7 days of obstruction in Sphk2-/- (A&B) and SK2i treated mice (C&D) compared non-obstructed controls and quantitated by Image J. N = 6. (Data represents average of 3 experiments, n = 3/experiment). **p<0.01). Original magnification; 20x objective. Scale bar; 50μm.
Fig 10.
Sphk2 in both immune cells and kidney microenvironment contribute to renal fibrosis.
(A). Recipient wild-type and Sphk2-/- mice were irradiated and injected with 1 × 106 bone marrow cells obtained from WT or Sphk2-/- mice by tail vein. 8 weeks after engraftment mice were subjected to UUO for 7 days and trichrome staining performed on kidney sections. Image J quantification (B) indicates that WT mice receiving WT cells have the highest degree of renal fibrosis, followed by Sphk2-/- recipients receiving WT cells and WT mice receiving Sphk2-/- bone marrow. Sphk2-/- mice receiving Sphk2-/- transplanted cells had the least fibrosis. N = 4/genotype. **p<0.01; *p<0.05. Original magnification; 20x objective. Scale bar; 50μm.