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. 2016 Jun;27(6):1714-26.
doi: 10.1681/ASN.2015030321. Epub 2015 Oct 9.

Extrarenal Progenitor Cells Do Not Contribute to Renal Endothelial Repair

Jan Sradnick  1 , Song Rong  2 , Anika Luedemann  1 , Simon P Parmentier  1 , Christoph Bartaun  1 , Vladimir T Todorov  1 , Faikah Gueler  2 , Christian P Hugo  1 , Bernd Hohenstein  3
Affiliations

Extrarenal Progenitor Cells Do Not Contribute to Renal Endothelial Repair

Jan Sradnick et al. J Am Soc Nephrol. 2016 Jun.

Abstract

Endothelial progenitor cells (EPCs) may be relevant contributors to endothelial cell (EC) repair in various organ systems. In this study, we investigated the potential role of EPCs in renal EC repair. We analyzed the major EPC subtypes in murine kidneys, blood, and spleens after induction of selective EC injury using the concanavalin A/anti-concanavalin A model and after ischemia/reperfusion (I/R) injury as well as the potential of extrarenal cells to substitute for injured local EC. Bone marrow transplantation (BMTx), kidney transplantation, or a combination of both were performed before EC injury to allow distinction of extrarenal or BM-derived cells from intrinsic renal cells. During endothelial regeneration, cells expressing markers of endothelial colony-forming cells (ECFCs) were the most abundant EPC subtype in kidneys, but were not detected in blood or spleen. Few cells expressing markers of EC colony-forming units (EC-CFUs) were detected. In BM chimeric mice (C57BL/6 with tandem dimer Tomato-positive [tdT+] BM cells), circulating and splenic EC-CFUs were BM-derived (tdT+), whereas cells positive for ECFC markers in kidneys were not. Indeed, most BM-derived tdT+ cells in injured kidneys were inflammatory cells. Kidneys from C57BL/6 donors transplanted into tdT+ recipients with or without prior BMTx from C57BL/6 mice were negative for BM-derived or extrarenal ECFCs. Overall, extrarenal cells did not substitute for any intrinsic ECs. These results demonstrate that endothelial repair in mouse kidneys with acute endothelial lesions depends exclusively on local mechanisms.

Keywords: endothelial injury; endothelial progenitor cell; endothelial repair; glomerulonephritis; thrombotic.

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Figures

Figure 1.
Figure 1.
Experimental sequence. Three central experiments were used to investigate the role of EPCs for EC repair. In experiment 1, site-selective endothelial injury (sel. EC injury) and I/R injury were used and combined with prior BMTx in experiment 2. For experiment 3, KTx and combined BMTx and KTx with a predefined ischemia time were used.
Figure 2.
Figure 2.
Endothelial injury and regeneration. Representative pictures of zinc fixed sections stained with anti-CD31 in glomeruli (A) and the tubulo-interstitium (B) (large scale=10 μm). Assessment of the glomerular (C) or peritubular endothelium (D) after staining for CD31 positive area measured using densitometry. Reduction of CD31 (E) and CD34 (F) positive cells measured by flow cytometry (fc) following selective EC injury or I/R (G, H).
Figure 3.
Figure 3.
EPCs detected in the kidney mainly express surface markers of ECFC. CD34+/Flk-1 positive cells (measured using flow cytometry) were increased 3 days following endothelial selective injury (A, B). The major part of CD34+/Flk1+ cells express endothelial and lack hematopoietic surface markers (A). ECFCs (C) represented the major and EC-CFUs (D) the minor type of EPC in the murine kidney following selective EC injury.
Figure 4.
Figure 4.
Renal cells expressing EPC surface makers are not bone marrow derived. Most bone marrow (A) and blood cells (B) of chimeric mice (D: tdT+; R: C57BL/6) mice were positive for tdT (donor derived). Significantly more tdT+ cells were recruited to the kidney (C). Cells expressing ECFC surface markers (D, left) were negative for tdT (D, right). Selective EC injury was accompanied by a significant inflammatory response as depicted by influx of neutrophils (E), macrophages (F), dendritic cells (G), CD4+ T cells (H), CD8+ T cells (I), and HSC (J).
Figure 5.
Figure 5.
Histology following EC injury in tdT BM chimeric C57BL/6 mice. Co-staining of tdT+ cells (red) with CD31 (green; A) and CD45 (green; B) using the Alexa flour 488 dye on formalin-fixed frozen sections.
Figure 6.
Figure 6.
Extrarenal progenitor cells do not contribute to EC repair in the mouse kidney. Following renal transplantation in various constellations (A), putative EPCs were increased in all groups and time points (B). ECFCs were negative for tdT (C) (not of extrarenal origin). Inverse transplantation shown in KTx group 3.
Figure 7.
Figure 7.
Histology following experimental kidney transplantation in mice. Co-staining of tdT+ cells (red) with CD31 (green; A, day 2) and CD45 (green; B, day 2) using the Alexa flour 488 dye on formalin-fixed frozen sections. Figure 7C depicts the absence of tdT+/CD31+ EC following combined BMTx with KTx and Figure 7D depicts the BM origin if invading CD45+ extrarenal by their tdT negativity. Virtually no other extrarenal cells can be detected.
Figure 8.
Figure 8.
Limited EC and ECFC proliferation following endothelial injury. Proliferating ECs (A) and ECFCs (B) measured with EdU proliferation assay (flow cytometry) 7 days following endothelial specific injury. Significantly more ECs as well as ECFCs could be detected in injured kidneys.
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