Endothelial Cell‐Secreted Semaphorin 3F Promotes Proximal Tubule Cell Maturation and Polarization

Abstract

Endothelial cells secrete trophic factors that contribute to the maturation of surrounding tissues. The notion of the microvasculature as niche for local differentiation and homeostasis has implication in diseases like diabetic kidney disease, in which the microvasculature is lost at early stages. Endothelial cells are known to produce semaphorins (Sema) as signal molecules that target cells in proximity. The isoform Sema3F is secreted by endothelial cells throughout the body, and it is known to regulate angiogenesis in an autocrine fashion. However, Sema3F can potentially reach other neighboring cell types, in particular kidney proximal tubule, since this epithelium is closely associated with peritubular capillaries. It is not known whether Sema3F influences proximal tubule homeostasis. We hypothesize that endothelial cells secrete Sema3F, which in turn promotes proximal tubule epithelial maturation and polarization. We utilized the human proximal tubule cell line RPTEC/hTERT since these cells polarize in trans‐well permeable support by 2 weeks, and they retain expression and apical‐basolateral polarization of proximal tubule proteins. To study the role of endothelial cells in RPTEC/hTERT maturation, we co‐culture them with the human endothelial cell line HUVEC. First, we measured Sema3F release from HUVEC endothelial cells by ELISA and observed that they secrete 206 ± 3 pg Sema3F/cm2/day. Next, to determine whether HUVEC endothelial cells promote proximal tubule maturation, we monitored the progressive acquisition of trans‐epithelial resistance as a measure of tight junction maturation. We observed that in co‐culture with HUVEC endothelial cells, RPTEC/hTERT cells developed a trans‐epithelial resistance that was 11±3% higher at 1 week, and 27±5% higher at 2 weeks compared to monoculture (p<0.05). To test whether Sema3F stimulates RPTEC/hTERT trans‐epithelial resistance, we added 200ng/mL of recombinant human Sema3F to cell cultures. We observed that Sema3F increased RPTEC/hTERT trans‐epithelial resistance at 1 week (vehicle= 152±5 Ω/cm2 vs. Sema3F= 171±3 Ω/cm2; p<0.05) and 2 weeks (vehicle= 150±2 Ω/cm2 vs. Sema3F= 182±4 Ω/cm2; p<0.05). Also, Sema3F accelerated polarization of the basolateral protein E‐cadherin measured by surface biotinylation over 2 weeks. Finally, to determine whether Sema3F produced by endothelial cells stimulate RPTEC/hTERT maturation, we silenced Sema3F in HUVEC endothelial cells via lentivirus‐transduction of silencing shRNAs. We observed that silencing Sema3F in HUVEC cells prevented the stimulation of RPTEC/hTERT trans‐epithelial resistance, while control shRNA transduction showed the expected stimulation at 7 days (monoculture= 222±4 Ω/cm2 vs. coculture‐shSema3F= 218±7 Ω/cm2; p=NS vs. coculture‐shControl= 231±8 Ω/cm2; p<0.05). We conclude that HUVEC endothelial cells secrete Sema3F, which reaches RPTEC/hTERT proximal tubule cells in co‐culture and stimulates tight junction maturation and polarization. These findings have relevance as a possible mechanism of disease in pathologies like diabetic kidney disease where the microvasculature is compromised.