IGF-1 protects tubular epithelial cells during injury via activation of ERK/MAPK signaling pathway

Abstract

Injury of renal tubular epithelial cells can induce acute renal failure and obstructive nephropathy. Previous studies have shown that administration of insulin-like growth factor-1 (IGF-1) ameliorates the renal injury in a mouse unilateral ureteral obstruction (UUO) model, whereas the underlying mechanisms are not completely understood. Here, we addressed this question. We found that the administration of IGF-1 significantly reduced the severity of the renal fibrosis in UUO. By analyzing purified renal epithelial cells, we found that IGF-1 significantly reduced the apoptotic cell death of renal epithelial cells, seemingly through upregulation of anti-apoptotic protein Bcl-2, at protein but not mRNA level. Bioinformatics analyses and luciferase-reporter assay showed that miR-429 targeted the 3'-UTR of Bcl-2 mRNA to inhibit its protein translation in renal epithelial cells. Moreover, IGF-1 suppressed miR-429 to increase Bcl-2 in renal epithelial cells to improve survival after UUO. Furthermore, inhibition of ERK/MAPK signaling pathway in renal epithelial cells abolished the suppressive effects of IGF-1 on miR-429 activation, and then the enhanced effects on Bcl-2 in UUO. Thus, our data suggest that IGF-1 may protect renal tubular epithelial cells via activation of ERK/MAPK signaling pathway during renal injury.

Figure 1. IGF-1 administration significantly ameliorates UUO-induced kidney injury.

(A) Experimental design: We performed UUO on mice, while some of which received daily i.p injection of IGF-1 (UUO+IGF-1), while the other received daily i.p. injection of saline as a control (UUO). Another group of mice that received sham operation were also daily i.p. injected with saline as another control (Sham). At 3 days after UUO, we digested the kidney from some of the mice for sorting of renal epithelial cells by fluorescence-activated cell sorting (FACS). These purified renal epithelial cells were further analyzed. At 14 days after UUO, the rest of the mice were sacrificed for histology analysis to confirm the model. (B,C) Masson trichrome staining was done to analyze and score for the degree of renal fibrosis regions in these mice, shown by representative images (B) and by quantification (C). N = 10. *p < 0.05.

Figure 2. IGF-1 administration significantly decreases UUO-induced renal epithelial cell apoptosis.

(A) At 3 days after UUO, we digested the kidney from some of the mice for sorting of renal epithelial cells by FACS, based on expression of E-cadherin (E-cad). These purified renal epithelial cells were further analyzed for apoptosis. (B,C) Renal epithelial cell apoptosis was analyzed using fluorescence-based apoptosis assay, shown by quantification (B) and by representative flow charts (C). N = 10. *p < 0.05.

Figure 3. IGF-1 increases anti-apoptosis protein Bcl-2 in renal epithelial cells.

(A,B) Bcl-2 was significantly increased by IGF-1 treatment, at protein level by Western blot (A), but not at mRNA level by RT-qPCR (B). N = 10. NS: non-significant. *p < 0.05.

Figure 4. IGF-1 suppresses miR-429 that targets 3′-UTR of Bcl-2 mRNA to inhibit its translation in renal epithelial cells.

(A) Bioinformatics analyses for Bcl-2 targeting miRNAs showed that miR-429 had a binding site at the 3′-UTR of Bcl-2 mRNA ranged from 440^th to 447^th base site. (B) The levels of miR-429 in renal epithelial cells were significantly decreased In IGF-1-treated mice that received UUO. (C) We either overexpressed miR-429, or inhibited miR-429 in the purified renal epithelial cells, by transfecting the cells with plasmids carrying either a miR-429-mimic (miR-429), or a miR-429 antisense (as-miR-429). The renal epithelial cells were also transfected with a plasmid carrying null sequence as a control (null). The overexpression or inhibition of miR-429 in renal epithelial cells was confirmed by RT-qPCR. (D) MiR-429-modified renal epithelial cells were then transfected with 1 μg of Bcl-2 3′-UTR luciferase-reporter plasmid or with 1 μg of Bcl-2 3′-UTR luciferase-reporter plasmid with a site mutation between 440^th to 447^th base site. The luciferase activities were quantified in these cells. (E,F) Bcl-2 mRNA (E) and protein (F) in miR-429-modified renal epithelial cells. N = 10. *p < 0.05.

Figure 5. IGF-1 suppresses miR-429 through ERK1/2 signaling pathway.

Renal epithelial cells received 100 ng/ml IGF-1 (and) 10 μmol/l ERK/MAPK-p42/p44 inhibitor PD98059. (A) Western blot for phosphorylation of ERK1/2. (B) RT-qPCR for miR-429. (C) RT-qPCR for Bcl-2. (D) Western blot for Bcl-2. N = 5. *p < 0.05.

Figure 6. Schematic of the model.

IGF-1 may protect tubular epithelial cells against renal injury via ERK/MAPK signaling pathway-dependent suppression of miR-429, and miR-429 inhibits Bcl-2-dependent anti-apoptosis.