Fig 2: The ERK1/2 signaling pathway was activated in vitro and in vivo after GDF11 treatment.(a,b) ERK1/2 phosphorylation and the expression of its downstream effectors c-Myc and cyclin D1 were increased in hPTCs treated with rGDF11, as shown by western blot. (c,d) ERK1/2 phosphorylation and the expression of its downstream effectors c-Myc and cyclin D1 were increased at 72 h after IRI in the kidneys of old mice treated with GDF11. For protein analysis (b), data are means ± SD from three independent experiments. *P < 0.05 vs. NC. For relative protein level determination (d), data are means ± SD; n = 3 in each group. OIC, old mice that underwent IRI for 28 min in the control group; OIG, old mice that underwent IRI for 28 min and were treated with GDF11. *P < 0.05 vs. OIC, **P < 0.01 vs. OIC. ##P < 0.01 vs. Sham.

Fig 3: GDF11 increased proximal tubular cell dedifferentiation, proliferation, and migration in vitro.(a) HPTCs became elongated or stellate in shape, a feature of dedifferentiation, after rGDF11 exposure for 24 h. Magnification, 100×. (b,e) Effect of GDF11 on hPTC dedifferentiation. HPTCs were incubated with rGDF11 for 72 h, and then PCR, immunofluorescence, and western blotting were performed. (b) Relative mRNA levels of the dedifferentiation markers vimentin and Pax2. (c) Photographs of immunofluorescence staining for vimentin and Pax2. Magnification, 600 × for vimentin, and 1800 × for Pax2. (d,e) Relative protein levels of the dedifferentiation markers vimentin and Pax2, the epithelial marker E-cadherin, and the myofibrocyte marker a-SMA in hPTCs after GDF11 treatment for 72 h. (f) The number of hPTCs increased one fold after rGDF11 treatment for 5 days, as assessed by CCK8 tests. (g,j) The percentage of EdU-positive cells increased after rGDF11 incubation for 24 h. Representative images (g) and quantification (j) of the percentage of EdU-positive cells in hPTC cultures. Magnification, 100×. (h,i) Effect of GDF11 on migration following mechanical injury. Confluent monolayers of hPTCs were scraped to create mechanical injury and incubated for 36 h in the presence or absence of rGDF11 (80 ng/mL). (h) Photographs at 40 × magnification. (i) Migration into the wound area was quantified by measuring the migration of cells from the wound edge. For mRNA and protein analyses (b,e), data are means ± SD from three independent experiments. *P < 0.05 vs. NC, **P < 0.01 vs. NC. For the CCK8 assay (f), data are means ± SD from three independent experiments. *P < 0.05 vs. NC, **P < 0.01 vs. NC. For the EdU incorporation test (j), 15 LPFs (magnification,40×) per group from three independent experiments were evaluated. Data are means ± SD. **P < 0.01 vs. NC. For measuring the migration of cells (i), data are means ± SD from three independent experiments (n = 3 in each group). **P < 0.01 vs. NC.

Fig 4: GDF11 increased the survival, renal function, and renal repair of old mice that underwent 28 min of IRI.(a,b) Serum creatinine (a) and BUN levels (b) at 24 h and 72 h for old mice that underwent 28 min of IRI in the control and GDF11 groups. (c) Survival curves at 72 h for old mice that underwent 28 min of IRI in the control and GDF11 groups. (d,e) Renal histology (d) and tubular injury scores (e) at 24 h and 72 h of old mice that underwent 28 min of IRI in the control and GDF11 groups (original magnification, 400×). (f) The percentage of necrotic tubules at 24 h of old mice that underwent 28 min of IRI in the control and GDF11 groups. For serum creatinine (a) and BUN (b), values are means ± SD, n = 6 in each group. OIC, old mice that underwent IRI in the control group; OIG, old mice that underwent IRI in the GDF11 group; **P < 0.01 vs. sham, #P < 0.05 vs. OIG, $P < 0.05 vs. 24 h after IRI, $$P < 0.01 vs. 24 h after IRI. For survival curves (c), OIC, old mice that underwent IRI in the control group; OIG, old mice that underwent IRI in the GDF11 group; P > 0.05 by the log-rank test, n = 20 in OIC, n = 12 in OIG. For tubular injury scores (e) and necrotic tubules (f), values are means ± SD. Approximately 40 HPFs (magnification, 400×) per individual mouse (10 HPFs per slide, four slides per animal) were evaluated. Values are means ± SD, n = 5–6 in each group. OIC, old mice that underwent IRI in the control group; OIG, old mice that underwent IRI in the GDF11 group; #P < 0.05 vs. OIC, $P < 0.05 vs. 24 h after IRI.

Fig 5: GDF11 enhanced tubular cell proliferation and dedifferentiation in old mice that underwent 28 min of IRI.(a,b) Tubular cell proliferation increased in old mice treated with GDF11 that underwent 28 min of IRI. Pictures are shown with 200 × magnification (a). (b) Percentage of EdU-positive tubular cells/HPF in the outer medulla and inner cortex. Approximately 20 HPFs (magnification, 200×) per individual mouse (5 HPFs per slide, four slides per animal) were evaluated. (c) Expression of the proximal tubular cell injury marker Kim1 and the dedifferentiation markers Pax2 and vimentin in the kidneys of sham and old mice treated with or without GDF11 at 72 h after IRI. Pictures are shown with 600 × magnification. (d) PCR for Pax2 and vimentin expression in the whole-kidney homogenates from old mice treated with or without GDF11 that underwent IRI. (e) Western blot for Kim1, Pax2, and vimentin expression in the whole-kidney homogenates from old mice treated with or without GDF11 that underwent IRI. Each lane represents a separate mouse. (f) Relative quantification of Kim1, Pax2, and vimentin expression as in (e), normalized to actin. For the percentage of EdU-positive tubular cells/HPF (b), values are means ± SD, n = 6 in each group. OIC, old mice that underwent IRI in the control group; OIG, old mice that underwent IRI in the GDF11 group. **P < 0.01 vs. OIC. For analysis of Pax2 and vimentin mRNA levels (d), values are means ± SD, n = 6 in each group. OIC, old mice that underwent IRI in the control group; OIG, old mice that underwent IRI in the GDF11 group. ##P < 0.01 vs. Sham, **P < 0.01 vs. OIC. For analysis of Kim1, Pax2, and vimentin protein levels (f), values are means ± SD, n = 3 in each group. OIC, old mice that underwent IRI in the control group; OIG, old mice that underwent IRI in the GDF11 group. #P < 0.05 vs. Sham, ##P < 0.01 vs. Sham, **P < 0.01 vs. OIC.