Successful Introduction of Human Renovascular Units into the Mammalian Kidney
Authors: Pleniceanu Oren , Harari-Steinberg Orit , Omer Dorit , Gnatek Yehudit , Lachmi Bat-El , Cohen-Zontag Osnat , Manevitz-Mendelson Eugenia , Barzilai Aviv , Yampolsky Matan , Fuchs Yaron , Rosenzweig Barak , Eisner Alon , Dotan Zohar , Fine Leon G. , Dekel Benjamin , Greenberger Shoshana 
Published: August 2020

Significance Statement
Most cell-based renal regenerative strategies are limited by an inability to generate donor-derived vascular networks upon in vivo transplantation, which is especially problematic in hypoxic CKD kidneys. The authors report that coadministering human renal tubule–forming cells and vessel-forming cells (mesenchymal stromal cells and endothelial colony-forming cells) into mice generates in vivo vascularized grafts comprising renal tubules of different nephron segments and donor-derived vessels connecting to host vasculature. The vessel-forming cells enhanced tubulogenic capacity of renal tubule–forming cells by improving graft perfusion and by inducing a protubulogenic state via paracrine mechanisms. These effects occurred with injection of cells into either the subcapsular renal or intraparenchymatic space. The findings suggest that augmenting the regenerative potential of renal cell–based methods through use of vessel-forming cells hold promise.

Background
Cell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in in vivo formation of vascular networks.

Methods
We administered renal tubule–forming cells derived from human adult and fetal kidneys (previously shown to exert a functional effect in CKD mice) into mice, alongside MSCs and ECFCs. We then assessed whether this would result in generation of “renovascular units” comprising both vessels and tubules with potential interaction.

Results
Directly injecting vessel-forming cells and renal tubule–forming cells into the subcutaneous and subrenal capsular space resulted in self-organization of donor-derived vascular networks that connected to host vasculature, alongside renal tubules comprising tubular epithelia of different nephron segments. Vessels derived from MSCs and ECFCs augmented in vivo tubulogenesis by the renal tubule–forming cells. In vitro coculture experiments showed that MSCs and ECFCs induced self-renewal and genes associated with mesenchymal–epithelial transition in renal tubule–forming cells, indicating paracrine effects. Notably, after renal injury, renal tubule–forming cells and vessel-forming cells infused into the renal artery did not penetrate the renal vascular network to generate vessels; only administering them into the kidney parenchyma resulted in similar generation of human renovascular units in vivo.

Conclusions
Combined cell therapy of vessel-forming cells and renal tubule–forming cells aimed at alleviating renal hypoxia and enhancing tubulogenesis holds promise as the basis for new renal regenerative therapies.