Agonism of Prostaglandin E2 Receptor 4 Ameliorates Tubulointerstitial Injury in Nephrotoxic Serum Nephritis in Mice

Figure 1. EP4 receptor agonism improved the phenotype of nephrotoxic serum nephritis (NTS). Fourteen days after NTS induction, kidneys of mice treated with vehicle, high-dose EP4 receptor agonist, or low-dose EP4 receptor agonist were harvested, and PAS-staining was performed (A). Representative pictures are shown. Stained kidney sections were quantified for glomerular and kidney damage (B–D). Urine samples collected on day 7 (E) and 14 (F) were analyzed for albumin and creatinine. In order to quantify for tubular damage acute tubular injury score (G) and tubular cast formation, six high-power fields (HPF) (H) were evaluated. Venous dilatation score (I) was quantified. Serum neutrophil gelatinase-associated lipocalin (NGAL) levels (J) and serum blood urea nitrogen (BUN) levels (K) were evaluated on day 14. (* p ≤ 0.05, *** p ≤ 0.001).

Figure 2. Hypotensive episodes occurred after EP4 receptor agonist administration. Blood pressure was assessed in vehicle- (n = 4), EP4 receptor high-dose agonist- (n = 4), and EP4 receptor low-dose agonist- (n = 4) treated mice after NTS induction. Blood pressure was measured via the tail cuff method immediately (A) and 30 min after EP4 agonist injection (B) in all three groups. Mean arterial pressure (MAP) is represented as mean ± SEM. Statistical significances are provided compared to vehicle-treated mice (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Figure 3. Effects of EP4 agonism on kidney-infiltrating immune cells. Fourteen days after induction of NTS, renal infiltration of CD4+ (A), CD8+ T cells (B,) and CD68+ macrophages (C) were evaluated in mice treated with high- and low-dose EP4 receptor agonist compared to vehicle. Ly6G+ (Gr1) cells were evaluated in glomeruli (D) and the interstitium (E) on day 14 after NTS induction. Fourteen days after NTS induction, quantitative PCR was performed to evaluate Gata3, FoxP3, and Il17a expression in the kidney and lymph node (F–H) in mice treated with vehicle (n = 6), high-dose EP4 receptor agonist (n = 3–4), and low-dose EP4 receptor agonist (n = 7–8). Results were compared to the kidneys of healthy mice (n = 3). Data are provided (mean ± SEM) as fold increase compared to healthy kidneys. Means are indicated by a horizontal line. Statistical significances are provided compared to vehicle-treated mice (* p ≤ 0.05).

Figure 4. EP4 receptor agonism increases tubular proliferation in vitro. To evaluate proliferative capacity of DCT cells, the MTS test was performed. DCT cells were treated with vehicle, EP4 receptor agonist (A), PGE2 (B), EP4 receptor antagonist (C), and the EP4 receptor antagonist together with PGE2 (D), as well as EP4 receptor antagonist together with EP4 receptor agonist (E) under starving conditions for 72 h. Data are provided as mean ± SEM from at least five independent experiments. Statistical significances are provided compared to vehicle-treated cells (** p ≤ 0.1, *** p ≤ 0.001).

Figure 5. EP4 agonist treatment significantly decreased cells in the sub-G1 phase and increased cells in the G1 phase of DCTs. To evaluate cell viability, a cell-cycle analysis was performed. DCT cells were treated with vehicle (n = 7), EP4 receptor agonist (n = 6–7) (A) PGE2 (n = 5–6) (B), EP4 receptor antagonist (n = 5–6) (C), and the EP4 receptor antagonist together with PGE2 (n = 6–7) (D) or together with the EP4 receptor agonist (n = 4) (E) under starving conditions for 72 h. Data are shown as mean ± SEM. Statistical significances are provided compared to vehicle (* p ≤ 0.05, ** p ≤ 0.01).

Figure 6. EP4 agonist-treated mice showed increased proliferation of tubular cells in vivo. Fourteen days after NTS induction, mice treated with vehicle, high-dose EP4 receptor agonist or low-dose EP4 receptor agonist were evaluated for PCNA+ tubular cells per six high power fields (HPF). Means are indicated by horizontal lines. Statistical significances are provided compared to vehicle-treated mice (* p ≤ 0.05).

Figure 7. EP4 stimulation of DCTs with ONO AE1-329 (30 nmol/L or 300 nmol/L) significantly increased cAMP production DCTs. Cells were treated with either PGE2 30 nmol/L (A) or 300 nmol/L (B), EP4 antagonist ONO AE3-208 (1000 nmol/L), or EP4 agonist ONO AE1-329 30 nmol/L (C) or 300 nmol/L (D). As a control, cells were either not treated (veh) or as a positive control with Forskoline (FSK). Cells were lysed, followed by a 5-, 10- or 30-min incubation before assay. Experiments were performed in duplicates. In total, at least five different experiments were performed. Statistical significances are provided compared to vehicle (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001)).