A previously unrecognized tubular–vascular interaction: mTAL-derived extracellular matrix protein EGFL6 maintains DVR integrity and outer medullary homeostasis

Impaired oxygen homeostasis in the kidney outer medulla contributes to the development of acute kidney injury (AKI). In this region, the descending vasa recta (DVR) serve as the main feeding vessels, and both excessive and insufficient blood flow compromise medullary function. Thus, preservation of structural and functional integrity of DVR is essential for maintaining medullary homeostasis. 

The basement membrane confers structural stability and functions as a signaling platform by concentrating growth factors and matrix components that maintain the phenotype of neighboring cells. Here, we focused on epidermal growth factor-like domain 6 (EGFL6), a unique vascular basement membrane protein, and uncovered its essential role in kidney homeostasis.

We utilized Egfl6-H2b-Egfp reporter mice, single-nucleus RNA-seq, immunofluorescence and immunoelectron microscopy to analyze EGFL6-expressing cells and the distribution of EGFL6. We also examined histological and functional changes in the kidneys of Egfl6-KO mice by immunofluorescence and whole kidney RNA-seq. Vascular permeability was assessed by intravenous FITC-dextran injection. As integrin α8 (ITGA8), a receptor for EGFL6, is widely expressed in DVR perivascular fibroblasts, we established ITGA8-overexpressing C3H10T1/2 murine fibroblasts and examined the effects of EGFL6 on cell migration and profibrotic activity. Finally, we analyzed EGFL6 expression in human kidney tissue.

Using Egfl6-H2b-Egfp reporter mice, we confirmed that EGFL6 is produced by the medullary thick ascending limb of Henle’s loop (mTAL). The results of the single-nucleus RNA-seq also supported this finding. Immunofluorescence revealed the deposition of EGFL6 on the DVR basement membrane, and immunoelectron microscopy further demonstrated its localization on the interstitial side of the DVR basement membrane, where adjacent fibroblasts extended protrusions toward it. Egfl6-deficient kidneys showed vascular abnormalities, such as increased permeability and ultrastructural changes including detached pericytes, along with interstitial fibrosis in the outer medulla. Vacuolar degeneration and activation of MAPK pathways, indicative of tubular stress, were observed in some nephron segments, but were most prominent in the mTAL. Furthermore, Egfl6-KO mice showed more severe kidney injury than wild-type controls under LPS-induced AKI. In vitro, EGFL6 suppressed migration and profibrotic gene expression in C3H10T1/2 cells overexpressing ITGA8, indicating its role in maintaining fibroblast quiescence. Finally, analysis of human kidneys showed similar EGFL6 distribution to that of mouse kidneys.

EGFL6 is secreted by mTAL and deposited along DVR basement membrane, where it contributes to the preservation of vascular integrity and, in turn, supports outer medullary homeostasis. Moreover, EGFL6 enhances tolerance to LPS-induced AKI. This previously unrecognized mTAL–DVR interaction may occur through the regulation of perivascular fibroblast behavior mediated by ITGA8. Furthermore, this homeostatic mechanism may be conserved in the human kidney, as supported by histological analyses of human samples.

This work was first presented at JSN 2023, and re-submission is permitted by JSN.