High glucose-stimulated secretion of Semaphorin-3G by proximal tubule cells inhibits angiogenesis in peritubular endothelial cells

Hyperglycemia in the diabetic kidney causes tubular and microvascular abnormalities that eventually lead to kidney damage. Given the close association between the proximal tubule and peritubular capillaries, a thorough understanding of their interaction could help identify mechanisms of diabetic kidney disease. Microvascular abnormalities are known to occur in diabetic patients and animal models. Despite known growth factors produced by the proximal tubule that could potentially target neighboring endothelial cells, it is not well understood whether the proximal tubule regulates angiogenesis. Here we propose that the proximal tubule secretes Semaphorin-3G (Sema3G), an anti-angiogenic protein. We hypothesize, that high glucose stimulates Sema3G secretion by proximal tubule cells and inhibits angiogenesis in peritubular endothelial cells.To study the role of secreted Sema3G, we collected the conditioned media from cultured polarized proximal tubule cells (RPTEC cell line) where we silenced Sema3G via lentivirus-delivered shRNAs. To study angiogenesis, we developed primary cultures of mouse kidney peritubular endothelial cells and analyzed angiogenesis via tubulogenesis assays in matrigel.We observed that the basolateral conditioned medium from RPTEC cells stimulated angiogenesis of peritubular endothelial cells in matrigel over 8 hours (p<0.01). However, RPTEC cells pre-grown on high glucose (25 mM) lost this ability. The apical media did not have any effect. To identify a potential proximal tubule anti-angiogenic factor, we measured release of Sema3G from polarized RPTEC cells. We found that RPTEC cells secreted Sema3G basolaterally and high glucose stimulated this by 5.5-fold. (p<0.01). Next, we tested the anti-angiogenic potential of Sema3G in peritubular endothelial cells. While adding recombinant Sema3G (200ng/ml) produced a modest inhibition (28%, p<0.05) of baseline peritubular endothelial cell angiogenesis, instead it completely blocked the stimulatory effect of a dose-response treatment (50–100ng/ml) with the potent angiogenic factor VEGF (p<0.01). Finally, to test the role of endogenous proximal tubule Sema3G, we silenced Sema3G via lentivirus-delivered shRNA in RPTEC cells. We then collected the conditioned medium from cells on normal or high glucose and performed angiogenesis assays. We found that silencing Sema3G in RPTEC cells on high glucose restored the ability of the conditioned medium to stimulate peritubular endothelial angiogenesis.We conclude that RPTEC proximal tubule cells secrete Sema3G, which is stimulated by high glucose, and this anti-angiogenic factor inhibits angiogenesis in mouse peritubular endothelial cells. Henry Ford Hospital, American Heart Association This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.