Insulin directly stimulates VEGF-A production in the glomerular podocyte

Abstract

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.

Keywords: insulin; podocyte; podocyte-specific insulin receptor knockout mice; vascular endothelial growth factor-A.

Fig. 1.

Conditionally immortalized mouse podocytes are insulin sensitive. A: differentiated mouse podocytes responded to insulin predominantly through the phosphatidylinositol 3-kinase (PI3K) pathway. This blot shows control (C) basal cells, insulin-stimulated podocytes, and phosphorylation of PKB [phosphorylated (p)PKB]. Actin loading is also shown. Insulin (10 nM) was applied for 5–20 min. B: mouse podocytes were rapidly insulin sensitive in response to glucose uptake using 2-deoxyglucose (2-DOG) assays. n = 12. ***P = 0.0003, basal vs. insulin (10 nM) for 15 min.

Fig. 2.

Insulin increases VEGF-A mRNA and protein expression in wild-type podocytes. A: human VEGF-A mRNA. Podocytes were exposed to 10 nM insulin or 100 ng/ml IGF-I for 30 or 60 min. n = 3–10 for each group. Each stimulus was compared with matched unstimulated cells and statistically analyzed using t-tests. HPRT, hypoxanthine-guanine phosphoribosyltransferase; C_T, threshold cycle; NS, not significant. **P = 0.0012. B: human VEGF-A secreted protein. Podocytes were exposed to 10 nM insulin (15 min to 24 h) and IGF-I (100 ng/ml for 24 h). Each group was compared with matched controls. n = 3–12 for each group. **P = 0.005. C: mouse VEGF-A mRNA. Mouse podocytes were exposed to 10 nM insulin for up to 4 h. Each group was compared with matched controls. n = 3–8 for each group. *P = 0.018; **P = 0.006. D: mouse VEGF-A protein. Mouse podocytes were exposed to 10 nM insulin for 15 min to 24 h. n = 3–6 for each group. **P = 0.0012. The rest of the conditions were NS compared with basal controls.

Fig. 3.

Rendering the podocyte insulin resistant resulted in reduced VEGF-A production in vitro and in vivo. A: stable podocyte-specific insulin receptor (IR) knockdown (KD) cells produced significantly less VEGF-A compared with wild-type cells and also IGF-IR KD cells. n = 4–10 for each group. **P = 0.026. B: methodology used to produce single cell podocyte suspensions using FACS sorting for insulin-resistant and insulin-sensitive podocytes. CFP, cyan fluorescent protein. C: VEGF-A mRNA production in FACS-sorted insulin-sensitive and insulin-resistant podocytes ex vivo. Four-week-old mice were used. D: in situ VEGF-A production in insulin-sensitive and insulin-resistant podocytes. Representative low (×10)- and high (×40)-power images are shown. podIRKO, podocyte-specific IR knockout (KO) group. E: quantification of the intensity/glomerular area of in situ VEGF-A analysis. n ≥ 15 sections in each group. **P = 0.0011.

Fig. 4.

Prolonged high glucose exposure causes reduced VEGF-A production by the podocyte. Human podocytes were exposed to 25 mM d-glucose for 1–10 days. At 10 days, there was a small but significant decrease in VEGF-A protein secretion into the media compared with cells grown in RPMI (12.5 mmol/l glucose). Each condition was compared with RPMI standard culture conditions. n = 6 for each condition. *P = 0.034.