Delayed Treatment with a Small Pigment Epithelium Derived Factor (PEDF) Peptide Prevents the Progression of Diabetic Renal Injury

Abstract

Our recent publication showed that a small bioactive pigment epithelium derived factor (PEDF) peptide (P78-PEDF) prevents the development of diabetic nephropathy (DN). However, its effects on the progression of established DN were not clear. Therefore, the purpose of this study was to determine the effect of P78-PEDF in the progression of DN and to compare the effects of P78-PEDF and an ACE inhibitor (ACEi), a standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with P78-PEDF or captopril starting at 6 wks of age for 12 wks (early treatment) or starting at 12 wks of age for 6 wks (late treatment). We first established the optimal dose of the P78-PEDF peptide to ameliorate DN in Ins2(Akita) mouse for a 6 wk study period and found that the peptide was effective at 0.1- 0.5 µg/g/day. We next showed that early or late treatment with P78-PEDF resulted in protection from DN as indicated by reduced albuminuria, kidney macrophage recruitment, histological changes, inflammatory cytokines and fibrotic markers (kidney TNF-α, fibronectin, VEGFA and EGFR), and restored nephrin expression compared with vehicle-treated Ins2(Akita) mice. Interestingly, only early but not late treatment with captopril was as effective as P78-PEDF in reducing most DN complications, despite its lack of effect on nephrin, VEGFA and EGFR expression. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in both the development and progression of diabetic renal injury.

Fig 1. P78-PEDF peptide attenuates diabetic renal injury in Ins2 ^Akita mice at 12 wks of age.

Ins2 ^Akita mice were treated with vehicle or P78-PEDF peptide (0.01, 0.05, 0.1, 0.5 μg/g/day) via osmotic minipump for 6 wks. Urine was collected at 12 wks of age for measurement of UAE. Results are means ± SEM. *p<0.001 compared to normal; #p<0.05 compared to vehicle-treated Ins2 ^Akita mice.

Fig 2. P78-PEDF peptide reduces renal histopathological changes in Ins2 ^Akita mice at 12 wks of age.

Sections were stained with PAS and all glomeruli were graded individually at 400x magnification after 12 wks of age in normal (A), vehicle-treated Ins2 ^Akita (B) and P78-PEDF peptide-treated (Dose: μg/g/day; 0.01: C, 0.05: D, 0.1: E, 0.5: F) Ins2 ^Akita mice. Images were taken with 100x (oil) objective with a total magnification of 1000x. Images are representative of 10–13 mice in each group. G: PAS score. Results are means ± SEM. *p<0.001 compared to normal; #p<0.05, ##p<0.001 compared to vehicle-treated Ins2 ^Akita mice.

Fig 3. P78-PEDF peptide reduces macrophage infiltration in Ins2 ^Akita mice at 12 wks of age.

Mac-2-positive macrophages (red arrows) in glomeruli were identified by immunohistochemical staining at 12 wks of age in normal (A), vehicle-treated Ins2 ^Akita (B) and P78-PEDF peptide-treated (Dose: μg/g/day; 0.01: C, 0.05: D, 0.1: E, 0.5: F) Ins2 ^Akita mice. Images are representative of 40 fields from 10–13 mice in each group. G: The number of macrophages/glomerulus. Results are means ± SEM. *p<0.01 compared to normal; #p<0.05, ##p<0.001 compared to vehicle-treated Ins2 ^Akita mice.

Fig 4. Late treatment with P78-PEDF peptide attenuates diabetic renal injury in Ins2 ^Akita mice at 18 wks of age.

Ins2 ^Akita mice were treated with vehicle or P78-PEDF peptide (0.3 μg/g/day) via osmotic minipump or captopril (24 mg/L daily in drinking water; Sigma) starting at 6 wks (early treatment) or 12 wks (late treatment) of age. Urine was collected from each group of mice at 6, 12, and 18 wks of age for measurement of UAE. Results are means ± SEM. *p<0.05, **p<0.001, ***p<0.0001 compared to corresponding normal; #p<0.01 compared to corresponding vehicle-treated Ins2 ^Akita mice.

Fig 5. Late treatment with P78-PEDF peptide reduces renal histopathological changes in Ins2 ^Akita mice at 18 wks of age.

Sections were stained with PAS and all glomeruli were graded individually at 400x magnification after 18 wks of age in normal (A), vehicle-treated Ins2 ^Akita (B), P78-PEDF peptide early treated (C), P78-PEDF peptide late treated (D), captopril early treated (E), or captopril late treated (F) Ins2 ^Akita mice. Images were taken with 100x (oil) objective with a total magnification of 1000x. Images are representative of 7–13 mice in each group. G: PAS score. Results are means ± SEM. *p<0.01, **p<0.001 compared to normal; #p<0.01 compared to vehicle-treated Ins2 ^Akita mice.

Fig 6. Late treatment with P78-PEDF peptide reduces macrophage infiltration in Ins2 ^Akita mice at 18 wks of age.

Mac-2-positive macrophages in glomeruli (red arrows) were identified by immunohistochemical staining at 18 wks of age in normal (A), vehicle-treated Ins2 ^Akita (B), P78-PEDF peptide early treated (C), P78-PEDF peptide late treated (D), captopril early treated (E), or captopril late treated (F) Ins2 ^Akita mice. Images are representative of 40 fields from 7–13 mice in each group. G: The number of macrophages/glomerulus. Results are means ± SEM. *p<0.01 compared to normal; #p<0.05 compared to vehicle-treated Ins2 ^Akita mice.

Fig 7. Late treatment with P78-PEDF peptide reduces inflammatory cytokines and fibrotic markers in Ins2 ^Akita mice at 18 wks of age.

RT-PCR was performed on whole mouse kidney total RNA at 18 wks of age. TNF-α (A), fibronectin (B), VEGFA (C), and EGFR (D) mRNA expression were normalized with GAPDH. Results are means ± SEM. *p<0.05, **p<0.01 compared to normal; #p<0.05, ##p<0.01 compared to Ins2 ^Akita+vehicle.

Fig 8. Late treatment with P78-PEDF peptide restores renal nephrin protein expression in Ins2 ^Akita mice at 18 wks of age.

Kidney nephrin expression was detected using western blot at 18 wks of age. Quantification was performed by densitometry followed by normalization to GAPDH. Results are means ± SEM. *p<0.01, **p<0.01 compared to normal; #p<0.05 compared to vehicle-treated Ins2 ^Akita mice; †p<0.05 compared to Ins2 ^Akita mice treated with captopril early.