Insulin restores renal neprilysin (NEP) and attenuates the shedding of urinary NEP and KIM-1 in diabetic Akita mice

Figures

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  FIGURE 1. Treatment with insulin implants maintain euglycemia in diabetic Akita mice. Blood glucose levels were measured weekly over a 15-week period in wild-type nondiabetic control mice (WT); sham-operated diabetic Akita mice (Akita), and diabetic Akita mice treated with subcutaneous insulin implants (0.1 U/day) (Akita + insulin). Values are means ± SE of the group size (n = 10). Two-way ANOVA analysis followed by the Bonferroni post hoc test showed that insulin treatment caused a significant decrease in blood glucose levels in diabetic Akita mice (*p < 0.0001 vs. WT mice, # p < 0.0001 vs. diabetic Akita mice).

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  FIGURE 2. Treatment with insulin implants ameliorate diabetic nephropathy in diabetic Akita mice: (A) Glomerular mesangial matrix expansion: Representative photomicrographs of glomerular Periodic acid-Schiff (PAS) staining (left) and quantitative analysis of mesangial matrix expansion (right) in wild-type (WT), diabetic Akita, and insulin-treated Akita mice (n = 2 mice/group). Insulin treatment (0.1 U/day for 16 weeks) significantly attenuated the diabetes-induced increase in PAS-positive area. Values are mean ± SE (n = 15 glomeruli/kidney section). *p < 0.05 vs. WT; # p < 0.05 vs. untreated Akita. (B) Renal cortical tubulointerstitial fibrosis: Representative photomicrographs of Masson’s trichrome staining for renal cortical tubulointerstitial fibrosis (left) and corresponding quantitative analysis (right). Insulin treatment significantly reduced the diabetes-induced fibrosis. Values are mean ± SE (n = 10 non-overlapping random fields/kidney section). *p < 0.05 vs. WT; # p < 0.05 vs. untreated Akita.

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  FIGURE 3. Western blot analysis reveals altered urinary NEP expression and fragmentation in diabetic Akita mice: (A) Western blot analysis of urinary NEP in younger (9 weeks) wild-type non-diabetic control mice (WT) and diabetic Akita mice (Akita). WT kidney lysate was used as a positive control. Immunoreactive NEP band was observed at 95 kDa for kidney lysate (first lane) and urinary samples from three WT and diabetic Akita mice. The two-sample t-test showed a significant decrease in urinary NEP expression in diabetic Akita mice compared to age-matched WT mice (p < 0.05). Values are means ± SE of the group size (n = 7-8). (B) Western blot analysis of urinary NEP in older (27 weeks) wild-type nondiabetic control mice (WT); sham operated diabetic Akita mice (Akita) and diabetic Akita mice treated with subcutaneous insulin implants (0.1 U/day) (Akita + insulin). The first line represents mouse kidney lysate (positive control). In diabetic Akita mice, smaller immunoreactive NEP fragments were observed at 70 kDa, 50 kDa, and 37 kDa. One-way ANOVA analysis of the full-length NEP band at 95 kDa revealed a significant reduction in urinary NEP expression in Akita mice compared to WT controls.…

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  FIGURE 4. Renal NEP expression is reduced in diabetic Akita mice and restored with insulin implants treatment. Western blot analysis of renal NEP expression in kidney lysate from 27 weeks old wild-type non-diabetic control mice (WT); sham-operated diabetic Akita mice (Akita) and diabetic Akita mice treated with subcutaneous insulin implants (0.1 U/day) (Akita + insulin). An immunoreactive NEP band was observed at 95 kDa in all groups. One-way ANOVA showed a significant decrease in renal NEP expression in Akita mice compared to age-matched WT controls. Insulin treatment significantly increased renal NEP protein expression in diabetic Akita mice compared to untreated diabetic Akita mice. Values are means ± SE of the size of the group (n = 7-8). *p < 0.05 vs. WT. # p < 0.05 vs. untreated diabetic Akita mice.

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  FIGURE 5. Treatment with insulin implants attenuates elevated renal Arginase-2 (Arg-2) expression in diabetic Akita mice: Western blot analysis of renal Arg-2 expression in kidney lysate from 27-week-old wild-type non-diabetic control mice (WT); sham-operated diabetic Akita mice (Akita), and diabetic Akita mice treated with subcutaneous insulin implants (0.1 U/day) (Akita + insulin). An immunoreactive Arg-2 band was detected at ∼39 kDa in all groups. One-way ANOVA showed a significant increase in renal Arg-2 expression in diabetic Akita mice compared to WT controls (p < 0.05). Treatment of diabetic Akita mice with insulin for 16 weeks significantly decreased renal Arg-2 expression in Akita mice compared to untreated diabetic Akita mice (p < 0.05). Values are means ± SE of the size of the group (n = 7-8). *p < 0.05 vs. WT. # p < 0.05 vs. untreated diabetic Akita mice.

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  FIGURE 6. Treatment with insulin implants restores renal NEP activity in diabetic Akita mice. NEP activity was measured in kidney lysate (1 µg of protein) from 27-week-old wild-type non-diabetic control mice (WT); sham operated diabetic Akita mice (Akita) and diabetic Akita mice treated with subcutaneous insulin implants (0.1 U/day) (Akita + insulin). One-way ANOVA revealed a significant decrease in renal NEP activity in diabetic Akita mice compared to WT controls (p < 0.05). Insulin treatment significantly increased renal NEP activity in diabetic Akita mice compared to untreated diabetic Akita mice (p < 0.05). Values are means ± SE of group size (n = 7-8). *p < 0.05 vs. WT. # p < 0.05 vs. untreated diabetic Akita mice.

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  FIGURE 7. Insulin treatment enhances urinary NEP activity in diabetic Akita mice: NEP activity was determined in urine samples (normalized to 1 µg creatinine) collected from 27-week-old wild type non-diabetic control mice (WT); sham-operated diabetic Akita mice (Akita), and diabetic Akita mice treated with subcuntaneous insulin implants (0.1 U/day) (Akita + insulin). One-way ANOVA revealed no significant difference in urinary NEP activity between diabetic Akita mice and WT control mice. However, insulin treatment significantly increased urinary NEP activity in diabetic Akita mice compared to untreated diabetic Akita mice (p < 0.05). Values are means ± SE of group size (n = 7-8). *p < 0.05 vs. untreated Akita mice.

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  FIGURE 8. Effect of insulin treatment on ELISA quantification of urinary NEP concentration. NEP concentration in urine samples was measured using ELISA. Urine samples (20 μL, diluted 1:5) were collected from 27-week-old wild type non-diabetic control mice (WT); sham-operated diabetic Akita mice (Akita), and diabetic Akita mice treated with subcuntaneous insulin implants (0.1 U/day) (Akita + insulin). Urinary NEP concentration is presented as ng/day (A) and normalized to creatinine as ng/μg creatinine (B). Values are means ± SE of the size of the group (n = 7-8). *p < 0.05 vs. WT. # p < 0.05 vs. untreated diabetic Akita mice.

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  FIGURE 9. Treatment with insulin attenuates diabetes-induced urinary KIM-1 elevation in diabetic Akita mice. Western blot analysis of KIM-1 expression in urine samples (normalized to 1 µg creatinine) collected from 27 weeks old non-diabetic control mice (WT); sham-operated diabetic Akita mice (Akita), and diabetic Akita mice treated with subcutaneous insulin implants (0.1 U/day) (Akita + insulin). Lanes 1-3 represent urine samples from WT mice, lanes 4-6 represent samples from diabetic Akita mice, and lane 7-9 represent samples from Akita mice treated with insulin. An immunoreactive band for KIM-1 was observed at ∼70 kDa in urine samples from Akita mice (lanes 4–6). One-way ANOVA revealed a significant increase in urinary KIM-1 expression in diabetic Akita mice compared to WT controls (*p < 0.05). Insulin treatment significantly reduced urinary KIM-1 expression compared to untreated diabetic Akita mice (# p < 0.05). Values are means ± SE of the size of the group (n = 7-8).