SUPPLEMENTAL FIGURE LEGENDS Figure S1 Genetic or pharmacologic COX-2 inhibition led to increased kidney macrophage infiltration. Wild type or COX-2 -/- mice (2 months old, C57/Bl6 background) were treated with a high salt diet (HS) for 4 weeks. (A) Kidney macrophage infiltration was markedly higher in HS plus SC58236-treated mice than in HS-treated mice, as indicated by immunostaining of F4/8, a marker of macrophages/dendritic cells ( P<.1, n = 4 in each group). (B) HS-treated COX-2 -/- mice had more kidney macrophages compared to HStreated wild type mice. Original magnification: x 16 in both A and B. All values are means ± SEM. All P values were calculated by Student s t test. Figure S2 COX-2 inhibition led to increased M1 but decreased M2 markers in vivo and in vitro. (A) Kidney mrna levels of M1/Th1 markers/cytokines including inos, CCL3, TNF-α, IL-1α and IL-1β were markedly higher but that of mannose receptor (MR) were markedly lower in HS plus SC58236-treated mice than in mice with HS alone (P<.1 and P<.1, n = 4 in each group). (B) Freshly isolated peritoneal macrophages treated with 25 µm SC58236 for 24 h had decreased protein levels of MR and arginase 1 (M2 markers) but increased protein levels of TNF- α (M1 marker). All values are means ± SEM. All P values were calculated by Student s t test. Figure S3 Mice with a deficient hematopoietic cell COX-2 pathway had increased heart hypertrophy in response to chronic high salt intake. (A) Heart weight vs. body weight ratios were higher in HS-treated COX-2 -/- -WT BMT mice than in HS-treated WT-WT BMT mice (P<.1, n = 4). (B) Heart weight vs. body weight ratios were also higher in HS-
treated CD11b-Cre; EP 4 mice than in HS-treated EP 4 mice (P<.1, n = 4). All values are means ± SEM. All P values were calculated by Student s t test. Figure S4 Prostaglandin EP 4 receptor tonically suppressed Th1 cytokine expression in cultured macrophages. (A) Murine macrophage RAW264.7 cells expressed COX-2, mpges1 and VEGF-C. EP 4 was the major EP receptors in RAW264.7 cells. COX-1 and EP 1 receptor were undetectable. (B) Treatment of RAW264.7 cells with a selective EP 4 receptor antagonist, L-161,982 (2 µm), led to increased mrna levels of M1/Th1 markers/cytokines, including inos, IL-23α, CCL3, TNF-α, IL-1α and IL-6 (P<.1, n = 3). (C) PGE 2 led to inhibition of inos expression, which was prevented by the selective EP 4 receptor antagonist, L-161,982 (P<.1 vs. PGE 2 alone, n = 4 in each group). All values are means ± SEM. All P values were calculated by Student s t test. Figure S5 Macrophage EP 4 receptor was effectively deleted in CD11b-Cre; EP 4 mice. Peritoneal macrophages were isolated and EP 4 mrna was quantitated by qpcr. Macrophage EP 4 mrna levels were significantly lower in CD11b-Cre; EP 4 mice than in EP 4 mice (P<.1, n = 4). All values are means ± SEM. All P values were calculated by Student s t test. Figure S6 The expression levels of p-ncc were increased in HS-treated mpges-1 -/- - WT BMT mouse than in HS-treated WT-WT BMT control. Figure S7 Increased medium NaCl elevated mrna levels of COX-2 and NFAT5 and VEGF-C in cultured macrophage cells. (A) Addition of 4 mm NaCl to the medium increased RAW264.7 cell COX-2 and NFAT5 mrna levels at 2.5 h and also increased VEGF-C mrna levels at 5 h (P<.5 and P<.1 vs. control, n = 4 in each group). (B)
PGE 2 (1 nm) stimulated RAW264.7 cell NFAT5 mrna expression (P<.1, n = 4). All values are means ± SEM. All P values were calculated by Student s t test. Figure S8 COX-2 -/- -COX-2 -/- BMT mice had increased skin Na and K content and increased water content in response to high salt intake. (A) HS-treated COX-2 -/- -COX-2 -/- BMT mice had higher skin sodium and potassium content, compared to HS-treated WT-WT BMT mice (P<.5, n = 4). (B) HS-treated COX-2 -/- -COX-2 -/- BMT mice had higher skin water content, compared to HS-treated WT-WT BMT mice (P<.5, n = 4). All values are means ± SEM. All P values were calculated by Student s t test. Figure S9 Renal ENaC mrna levels were higher in HS-treated COX-2 -/- -WT BMT and mpges-1 -/- -WT BMT mice than in HS-treated WT-WT BMT mice. Both ENaCβ and ENaCγ mrna levels were significantly higher in COX-2 -/- -WT BMT and mpges-1 -/- -WT BMT mice than in WT-WT BMT mice in response to a high salt diet. (P<.5, n = 5 in each group). All values are means ± SEM. All P values were calculated by Student s t test. Figure S1. Deficiency in hematopoietic cell COX-2 pathway had no effects on water and salt balance. Trained mice were given 1 meq of NaCl via gastric gavage, and urine was collected every 12 h for next 72 h. (A and B) Both urine volume and sodium excretion were comparable between WT-WT and COX-2 -/- -WT BMT mice (n = 4) (A) or between EP 4 mice and CD11b-Cre; EP 4 mice (n = 6) (B). Figure S11 Blood pressure was comparable between control COX-2 -/- -WT BMT and WT-WT BMT Mice measured by tail-cuff microphonic manometer or carotid catheterization (n = 6 in each group).
Supplemental Table 1. Renal and Hematologic Parameters After BMT Parameters Na (mm) K (mm) Cl (mm) TCO 2 (mm) BUN (mg/dl) HCT (%) Hgb (g/dl) Control 148 ± 1 5.5 ±.7 115 ± 2 21 ± 1 21 ± 4 52 ± 2 17 ± 1 WT-WT 148 ± 1 5.6 ±.6 117 ± 2 22 ± 2 2 ± 1 5 ± 1 17 ± 1 COX-2 -/- -WT 148 ± 2 5.6 ± 1.2 116 ± 2 25 ± 3 17 ± 3 48 ± 3 16 ± 1 Six weeks after BMT, renal and hematologic parameters were measured. Data were presented as mean ± s.e.m (n = 3 in each group).
A C57/Bl6 control C57/Bl6 SC58236 Renal macrophage density (cells/field) 5 4 3 2 1 Vehicle SC-58236 B C57/Bl6 wild type C57/Bl6 COX-2 -/- Supplemental Figure 1
A mrna levels 4. 3.5 3. 2.5 2. 1.5 1..5 HS HS + SC58236 inos CCL3 TNF-α IL-1α IL-1β MR mrna levels.5.4.3.2.1 HS HS + SC58236 B Vehicle SC-58236 MR Arginase-1 TNF-α α-tubulin Supplemental Figure 2
A.55 Heart weight/body weight.5.45.4.35.3 WT-WT COX-2 -/- -WT B Heart weight/body weight Supplemental Figure 3.65.6.55.5.45.4.35.3 EP 4 CD11b-Cre; EP 4
A Mouse macrophage cell line RAW264.7 COX1 COX-2 mpges VEGF-C EP1 EP2 EP3 EP4 B mrna levels 3 25 2 15 1 5 Control L-161,982 inos mrna levels Control L-161,982 3. 2.5 2. 1.5 1..5 TNF-α CCL3 IL-23α IL-1α IL-6 C inos mrna levels 14 12 1 8 6 4 2 Vehicle PGE 2 PGE 2 L161,982 Supplemental Figure 4
Macrophage EP 4 mrna levels 2 15 1 5 EP 4 CD11b-Cre; EP 4 Supplemental Figure 5
WT-WT mpges-1 -/- -WT p-ncc immunostaining: original magnification: x 25. Supplemental Figure 6
A B mrna levels 6 5 4 3 2 1 COX-2 NFAT5 h 2.5 h 5. h NFAT5 mrna levels 15 12 9 6 3 VEGF-C mrna levels 3.5 3. 2.5 2. 1.5 1..5 h 2.5 h 5. h Basal PGE 2 (12h) Supplemental Figure 7
A Skin Na content (%).8.75.7.65.6.55.5 Skin K content (%).5.45.4.35.3.25 WT-WT COX-2 -/- -COX-2 -/- WT-WT COX-2 -/- -COX-2 -/- B Skin water content (%) 76 74 72 7 68 66 64 62 6 WT-WT COX-2 -/- -COX-2 -/- Supplemental Figure 8
COX-2 COX- 2- /- -/- -WT BMT mpges- 1- /- mpges-1 -/- BMT -WT Renal mrna levels (fold of WT-WT) 2. 1.6 1.2.8.4 ENaCβ ENaCγ Supplemental Figure 9
A B Urine volume (ml/12 h) 3. 2.5 2. 1.5 1..5 WT to WT COX- 2- /- to WT 12 24 36 48 6 72 Urine volume (ml/12 h) 3. 2.5 2. 1.5 1..5 EP4 CD11b-Cre;EP4 12 24 36 48 6 72 Time after acute salt loading (h) Time after acute salt loading (h) Na excretion (meq/12 h) 1..8.6.4.2 WT to WT C OX - 2- /- to WT 12 24 36 48 6 72 Na excretion (meq/12 h) 1..8.6.4.2 EP4 CD11b-Cre;EP4 12 24 36 48 6 72 Time after acute salt loading (h) Time after acute salt loading (h) Supplemental Figure 1
Tail cuff Carotid catheterization 175 175 15 15 SBP (mmhg) 125 1 75 5 SBP (mmhg) 125 1 75 5 25 25 WT-WT COX-2 -/- -WT WT-WT COX-2 -/- -WT Supplemental Figure 11