Figure 1.
Forskolin attenuated ADR nephrosis.
A: Coomassie blue staining of urine proteins resolved by gel electrophoresis. Creatinine concentration indicated to variable dilutions among urine samples. LMWP: low molecular weight protein; B: Electron microscopy of a capillary loop (×13500). C: Immunohistochemical staining was performed to detect WT-1 positive cells in glomeruli (×200). WT-1 positive cell numbers were counted by one renal pathologist using a blinded method. At least 50 glomeruli per kidney were calculated. Black arrow: WT-1 positive cells. D: A bar graph of the data expressed as average number per glomerulus. E: Immunofluorescence staining of kidney from mice treated with or without forskolin (×200). White arrow: p-CREB positive cells. *: P<0.05 compared with control group, #: P<0.05 compared with ADR group.
Figure 2.
PKA-mediated cAMP protection against podocyte injury.
A: Western blot of MCF7 cells, NRK cells, differentiated and undifferentiated podocytes. B: Immunofluorescence staining of kidney from control mice (×200). C: CCK-8 test was performed using podocytes treated with PAN in the presence or absence of 2Me-cAMP. Data were obtained from five independent studies. D: PKA activity was detected using podocytes treated with pCPT-cAMP for the time indicated. Data were obtained from five independent studies. E: CCK-8 test was performed using podocytes treated with PAN in the presence or absence of pCPT-cAMP. Data were obtained from five independent studies. F: CCK-8 results from podocytes treated with PAN in the presence or absence of pCPT-cAMP and H89. Data were obtained from five independent studies. *: P<0.05 compared with control group, #: P<0.05 compared with PAN group, $: P<0.05 compared with pCPT+PAN group. 2Me: 2Me-cAMP, pCPT: pCPT-cAMP.
Figure 3.
PKA signaling prevented PAN-induced podocyte apoptosis.
A and B: Western blot of podocytes treated with PAN for the indicated time. Bar graph of data obtained from five independent experiments. C and D: Western blot for podocytes treated with PAN in the presence or absence of pCPT-cAMP. Bar graph of data obtained from five independent experiments. E: TUNEL staining results (×100). White arrows indicate the TUNEL positive cells. F and G: JC-1 staining and bar graph of data obtained from four to five independent experiments. *: P<0.05 compared with control group, #: P<0.05 compared with PAN group. 2Me: 2Me-cAMP, pCPT: pCPT-cAMP.
Figure 4.
Mitochondria fission in podocytes was induced by PAN or ADR treatment.
A: Mitochondria staining (×200) and electron microscopy (×17500) of podocytes treated with PAN in the presence or absence of pCPT-cAMP. B: Western blot of podocytes treated with PAN for the indicated time. C: Bar graph of data from five independent experiments. D: Western blot of podocytes treated with PAN for the indicated time. *: P<0.05 compared with control group. pCPT: pCPT-cAMP.
Figure 5.
PKA signaling promoted mitochondria fusion in podocytes.
A and B: Western blot of podocytes treated with pCPT-cAMP for the indicated time. Bar graph of data obtained from five independent experiments. C and D: Western blot results from podocytes treated with pCPT-cAMP for 30 and 60 minutes. Bar graph of data obtained from at least five independent experiments. E: Western blot performed for podocytes treated with pCPT-cAMP for the indicated time. F and G: Western blot performed for podocytes treated with PAN or ADR in the presence or absence of pCPT-cAMP. H: Bar graph of data from five independent experiments. I: Western blot performed for podocytes treated with PAN or ADR in the presence or absence of pCPT-cAMP. J: Immunofluorescence and immunohistochemical staining of kidney from the control, ADR and forskolin+ADR groups (×200). *: P<0.05 compared with control group, #: P<0.05 compared with PAN group. pCPT: pCPT-cAMP.
Figure 6.
Mitochondria fusion/fission was involved in podocyte apoptosis.
A: Mitochondria staining of podocytes treated with ARA or Mdivi. B: CCK-8 results from podocytes treated with ARA for the indicated time. Data were obtained from five independent experiments. C: Western blot of podocytes treated with ARA for the indicated time. D: CCKD-8 was performed using podocytes incubated with the indicated reagents. Data from five independent experiments are shown. E: Western blot of podocytes treated with the indicated reagents. F: Western blot of podocytes treated with PAN in the presence or absence of Mdivi-1. *: P<0.05 compared with control group, #: P<0.05 compared with PAN group, $: P<0.05 compared with pCPT+PAN group. pCPT: pCPT-cAMP.