Vascular Endothelial Growth Factor and Podocyte Protection in Chronic Hypoxia: Effects of Endothelin-A Receptor Antagonism

Abstract

Background: Podocytes are major components of the filtration barrier and a renal source of vascular endothelial growth factor (VEGF). Chronic renovascular disease (RVD) progressively degrades the renal function, accompanied by podocyte damage and a progressive reduction in VEGF. We showed that the endothelin (ET) pathway contributes to this pathological process and ET-A (but not ET-B) receptor antagonism protects the kidney in RVD. We hypothesize that ET-A-induced renoprotection is largely driven by the protection of podocyte integrity and function.

Methods: To mimic the renal environment of chronic RVD, human podocytes were incubated under chronic hypoxia for 96 h and divided in untreated or treated with an ET-A or ET-B receptor antagonist. Cells were quantified after 96 h. Cell homogenates and media were obtained after 1, 24 and 96 h to quantify production of VEGF, anti-VEGF soluble receptor s-Flt1, and the expression of apoptotic mediators. A separate set of similar experiments was performed after addition of a VEGF-neutralizing antibody (VEGF-NA).

Results: Hypoxia decreased podocyte number, which was exacerbated by ET-B but improved after ET-A antagonism. Production of VEGF was preserved by ET-A antagonism, whereas s-Flt1 increased in hypoxic cells after ET-B antagonism only, accompanied by a greater expression of pro-apoptotic mediators. On the other hand, treatment with VEGF-NA diminished ET-A-induced protection of podocytes.

Conclusion: ET-A antagonism preserves podocyte viability and integrity under chronic hypoxia, whereas ET-B antagonism exacerbates podocyte dysfunction and death. Enhanced bioavailability of VEGF after ET-A antagonism could be a pivotal mechanism of podocyte protection that significantly contributes to ET-A receptor blockade-induced renal recovery in chronic RVD.

Figure 1. Wt-1 expression is preserved in podocytes after 7–12 passages, indicating preserved podocyte characteristics

Cell homogenates from normoxic podocytes were used and expression determined in duplicates by western blotting.

Figure 2. Chronic hypoxia reduces podocyte activity

Production of VEGF (A) and s-Flt1 (B) after 1, 24, and 96 hours of hypoxia. Only ET-A antagonism significantly improved VEGF availability (expressed in pg/protein of interest/μg of total protein), whereas ET-B antagonism progressively increased production of s-Flt1. * p<0.05 vs. Normoxic; † p<0.05 vs. Hypoxic; ‡ p<0.05 vs. Hypoxic+ET-A

Figure 3. ET-A blockade restore the expression of podocin, nephrin, and VEGF

ET-A antagonism distinctly preserved the expression of VEGF and the slit-associated proteins podocin and nephrin (determined by western blot at the 96 hours time-point and quantified related to β-actins, A and B, respectively), suggesting a reduction in podocyte damage. * p<0.05 vs. Normoxic; † p<0.05 vs. Hypoxic; ‡ p<0.05 vs. Hypoxic+ET-A.

Figure 4. ET-A blockade improved expression of apoptotic and survival factors

4a) Podocyte expression (determined by western blot at the 96 hours time-point, A) and quantification (B) of pro-apoptotic BAX, AIF, HSP 60, p53 and Smac were all increased by hypoxia and ET-B antagonism but restored by ET-A blockade, accompanied by improved expression of pro-survival p-akt. 4b) Representative pictures (96 hours) from normoxic (A), hypoxic (B), and hypoxic podocytes treated with ET-A (C) or ET-B (D) antagonist. Cell counts and morphological changes suggestive of apoptosis (e.g.: nuclear condensation and fragmentation) were improved after ET-A but not ET-B antagonism. These results suggest a decrease in pro-apoptotic activity in hypoxic podocytes after ET-A antagonism. * p<0.05 vs. Normoxic; † p<0.05 vs. Hypoxic; ‡ p<0.05 vs. Hypoxic+ET-A

Figure 4. ET-A blockade improved expression of apoptotic and survival factors

4a) Podocyte expression (determined by western blot at the 96 hours time-point, A) and quantification (B) of pro-apoptotic BAX, AIF, HSP 60, p53 and Smac were all increased by hypoxia and ET-B antagonism but restored by ET-A blockade, accompanied by improved expression of pro-survival p-akt. 4b) Representative pictures (96 hours) from normoxic (A), hypoxic (B), and hypoxic podocytes treated with ET-A (C) or ET-B (D) antagonist. Cell counts and morphological changes suggestive of apoptosis (e.g.: nuclear condensation and fragmentation) were improved after ET-A but not ET-B antagonism. These results suggest a decrease in pro-apoptotic activity in hypoxic podocytes after ET-A antagonism. * p<0.05 vs. Normoxic; † p<0.05 vs. Hypoxic; ‡ p<0.05 vs. Hypoxic+ET-A

Figure 5. VEGF-NA decreased the beneficial effects of ET-A blockade on podocyte counts and pro-apoptotic signaling

Incubation for 96 hours with VEGF-NA further decreased cell count (A) and diminished the protective actions of ET-A on cell survival and apoptotic signaling (B), suggesting the ET-A antagonism-induced protection is largely mediated by VEGF. * p<0.05 vs. Normoxic; † p<0.05 vs. Hypoxic; ‡ p<0.05 vs. Hypoxic+ET-A; ‡‡ p<0.05 vs. untreated.