Background: Microvasculature loss in the diabetic kidney has been documented but is poorly understood. The proximal tubule has the potential of regulating angiogenesis via secretion of paracrine factors, like VEGF, but the relevance of this in diabetes is unknown. We have shown increased kidney Semaphorins expression (anti-angiogenic) in diabetic mice. However, the role of proximal tubule semaphorin-mediated regulation of angiogenesis in diabetic kidney is an open question. Hypothesis: High glucose stimulates semaphorins in proximal tubule, which inhibit endothelial cell angiogenesis. Methods: The angiogenic properties of conditioned media from polarized RPTEC proximal tubule cells was analyzed in endothelial HUVEC cells in Matrigel. We assessed surface expression of angiogenic receptors in kidney endothelial primary cultures of diabetic Akita mice. Results: The basolateral conditioned media from RPTEC cells stimulated HUVEC angiogenesis (Not conditioned:15.8± 1.8 loops/field vs. conditioned: 20± 2 loops/field, p< 0.05). However, media from RPTEC cells on high glucose (25 mM) did not (8.4± 1.8 loops/field, p< 0.01 vs. conditioned). Apical conditioned media did not have any effect, and media from RPTEC cells conditioned in 20 mM mannitol (osmolality control) was comparable to normal glucose. In a comparable fashion, high glucose-conditioned media failed to stimulate HUVEC migration in wound healing assays (Conditioned: 0.039±0.003 mm2/hr vs. Conditioned high glucose: 0.019±0.002 mm2/hr). To study the role of a semaphorin receptor, we silenced Plexin-D1 via shRNAs and found that migration was stimulated by high glucose-conditioned media (0.059±0.003 mm2/hr). Next, we studied the role of a Plexin ligand: Semaphorin-3D (Sema3D). We silenced Sema3D in RPTEC cells, and observed that high glucose-conditioned media lost their ability to inhibit angiogenesis (12 ± 1.4 vs. 4.4 ± 1 loops/field). Finally, to study whether Sema-Plexin signaling may be enhanced in diabetic kidneys, we measure surface expression of Plexin-D1 in primary cultures of kidney endothelial cells from Akita mice. We found that surface Plexin-D1 expression was 105 ± 62% higher vs. wt (p< 0.01), and stimulated by VEGF only in Akita by 109± 23% (p< 0.05 vs. vehicle). Surface VEGFR2 receptor was not different between wt and Akita, and it was increased by VEGF only in wt (27.6±11%, p< 0.05 vs. vehicle). Conclusion: High glucose-conditioned medium from RPTEC proximal tubule cells inhibits HUVEC angiogenesis via secretion of Sema3D, a ligand for Plexin-D1, a receptor that in turn was increased by VEGF in kidney endothelial cells from diabetic Akita mice. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Kaur et al. (Fri,) studied this question.