Figure 1.
Genipin ameliorates body weight loss and urine albumin leakage in diabetic mice.
(a) Blood pressure (BP) of control group, diabetes group treated with vehicle and diabetes group treated with genipin for 12 weeks were measured by a non-invasive blood pressure analysis system (Softron, BP-98A). Values (mmHg) are means ± SE from 6 animals/group. BUN (b) and Scr (c) of control group, diabetes group treated with vehicle and diabetes group treated with genipin were examined in experimental period. Values are means ± SE from 6 animals/group. (d) Fast blood glucose levels of control group, diabetes group treated with vehicle and diabetes group treated with genipin were examined in experimental period. Values (mmol/L) are means ± SE from 6 animals/group at each time point. *P<0.05 vs. sham-control, #P<0.05 genipin vs. vehicle. (e) Body weight of control group, diabetes group treated with vehicle and diabetes group treated with genipin were examined in experimental period. Values (g) are means ± SE from 6 animals/group at each time point. *P<0.05 vs. sham-control, #P<0.05 genipin vs. vehicle. (f) Urine albumin of control group, diabetes group treated with vehicle and diabetes group treated with genipin were examined in experimental period. Values (mg/24h) are means ± SE from 6 animals/group at each time point. *P<0.05 vs. sham-control, #P<0.05 genipin vs. vehicle.
Figure 2.
Genipin attenuates glomerular basement membrane thickening and podocyte injury.
(a–c) Representative electron micrographs show GBM thickening and foot process effacement in the vehicle treated diabetic kidney. These morphologic injuries were attenuated in diabetic mice that received genipin. Scale bar, 1μm. (a) sham-control. (b) Diabetic mice with vehicle. (c) Diabetic mice with genipin. (d) Graphic presentation of the GBM thickness in each group. The data were calculated based on individual values determined on ten fields per mouse, six mice per group (n = 6). Values (nm) are means ± SE. *P<0.05 vs. sham-control, #P<0.05 genipin vs. vehicle.
Figure 3.
Genipin restores the expression of podocin and WT1 in diabetic mice.
(a) Western blot analysis shows the results of podocin and WT1 in the kidneys of each group. The samples were reprobed with actin to confirm equal loading of each lane. Representative pictures show the results of three animals per group. (b) Graphic presentation of relative podocin abundance normalized to actin. *P<0.05 vs. control, #P<0.05, genipin vs. vehicle. (c) Graphic presentation of relative WT1 abundance normalized to actin. *P<0.05 vs. control, #P<0.05, genipin vs. vehicle. (d) Immunofluorescent staining of podocin (green) and WT1 (red) protein in each group, respectively. Collagen IV was stained green to localize WT1 (red). The representative figures after three repetitions were shown. (e) Graphic presentation of relative fluorescent intensity of podocin. *P<0.05 vs. control, #P<0.05, genipin vs. vehicle. (f) Graphic presentation of relative fluorescent intensity of WT1. *P<0.05 vs. control, #P<0.05, genipin vs. vehicle.
Figure 4.
Genipin suppresses the upregulation of UCP2 protein expression in diabetic kidneys.
(a) Western blot analysis shows the results of UCP2 in the kidneys of each group. The membrane was reprobed with actin to confirm equal loading of each lane. Representative pictures show the results of three animals per group. (b) Graphic presentation of relative UCP2 abundance normalized to actin. *P<0.05 vs. control, #P<0.05, genipin vs. vehicle. (c) RT-PCR analysis shows the results of UCP2 mRNA in the kidneys of each group. Representative pictures show the results of three animals per group. (d) Graphic presentation of relative UCP2 mRNA abundance normalized to actin.
Figure 5.
Genipin restores podocin and WT1 expression in cultured podocytes.
(a) Western blot analysis shows the results of podocin and WT1 in podocytes incubated with D-Glucose for different time periods as indicated. The membrane was reprobed with actin to confirm equal loading of each lane. (b) Western blot analysis shows the results of podocin and WT1 in D-Glucose (24 h) incubated podocytes pre-treated (0.5 h) with or without genipin. The membrane was reprobed with actin to confirm equal loading of each lane. (c) Immunofluorescence staining of podocin and WT1 (green) protein, respectively. Cell nuclei were stained with DAPI (blue).
Figure 6.
Genipin reduces UCP2 expression induced by high glucose in podocytes.
(a) Western blot analysis shows the results of UCP2 in podocytes incubated with D-Glucose for different time periods as indicated. The membrane was reprobed with actin to confirm equal loading of each lane. (b) Graphic presentation of relative UCP2 abundance normalized to actin. *P<0.05 vs. control. n = 3. (c) Western blot analysis shows that pre-incubation with genipin inhibited glucose-induced UCP2 expression. The membrane was reprobed with actin to confirm equal loading of each lane. (d) Graphic presentation of relative UCP2 abundance normalized to actin. *P<0.05 vs. control. #P<0.05 vs. glucose incubated cells without genipin pre-treatment. n = 3.
Figure 7.
Downregulation of UCP2 by RNA interference restores podocin and WT1 expression depressed by high glucose in podocytes.
(a) Western blot analysis shows the results of UCP2 in podocytes transfected with different concentrations of UCP2 siRNA as indicated. The membrane was reprobed with actin to confirm equal loading of each lane. (b) Graphic presentation of relative UCP2 abundance normalized to actin. *P<0.05 vs. control. #P<0.05 vs. glucose incubated cells transfected with control siRNA. n = 3. (c) Western blot analysis shows the results of podocin and WT1 in podocytes transfected with different concentrations of UCP2 siRNA as indicated. The membrane was reprobed with actin to confirm equal loading of each lane. (d) Graphic presentation of relative podocin and WT1 abundance normalized to actin. *P<0.05 vs. control. #P<0.05 vs. glucose incubated cells transfected with control siRNA. n = 3.
Figure 8.
Genipin attenuates high glucose induced filtration barrier dysfunction of podocytes.
Graphic presentation shows the albumin flux rate across the differentiated podocyte monolayer in each group after 6 hours' treatment. Values (mg/ml) are means ± SE. *P<0.05 vs. control, #P<0.05 genipin vs. glucose.