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Genipin inhibits mitochondrial uncoupling protein 2 expression and ameliorates podocyte injury in diabetic mice - PubMed

Genipin inhibits mitochondrial uncoupling protein 2 expression and ameliorates podocyte injury in diabetic mice

Wenjing Qiu et al. PLoS One. 2012.

Abstract

Diabetic nephropathy (DN) is one of the most common causes of end stage renal disease (ESRD) in China, which requires renal replacement therapy. Recent investigations have suggested an essential role of podocyte injury in the initial stage of DN. This study investigated the potential therapeutic role of genipin, an active extract from a traditional Chinese medicine, on progression of DN in diabetic mice induced by intraperitoneally injection of streptozocin (STZ). In diabetic mice, orally administration of genipin postponed the progression of DN, as demonstrated by ameliorating body weight loss and urine albumin leakage, attenuating glomerular basement membrane thickness, restoring the podocyte expression of podocin and WT1 in diabetic mice. The protective role of genipin on DN is probably through suppressing the up-regulation of mitochondrial uncoupling protein 2 (UCP2) in diabetic kidneys. Meanwhile, through inhibiting the up-regulation of UCP2, genipin restores podocin and WT1 expression in cultured podocytes and attenuates glucose-induced albumin leakage through podocytes monolayer. Therefore, these results revealed that genipin inhibited UCP2 expression and ameliorated podocyte injury in DN mice.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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
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
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
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
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
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
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
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.

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