Impaired endothelial proliferation and mesenchymal transition contribute to vascular rarefaction following acute kidney injury

Abstract

Acute kidney injury induces the loss of renal microvessels, but the fate of endothelial cells and the mechanism of potential vascular endothelial growth factor (VEGF)-mediated protection is unknown. Cumulative cell proliferation was analyzed in the kidney of Sprague-Dawley rats following ischemia-reperfusion (I/R) injury by repetitive administration of BrdU (twice daily) and colocalization in endothelial cells with CD31 or cablin. Proliferating endothelial cells were undetectable for up to 2 days following I/R and accounted for only ∼1% of BrdU-positive cells after 7 days. VEGF-121 preserved vascular loss following I/R but did not affect proliferation of endothelial, perivascular cells or tubular cells. Endothelial mesenchymal transition states were identified by localizing endothelial markers (CD31, cablin, or infused tomato lectin) with the fibroblast marker S100A4. Such structures were prominent within 6 h and sustained for at least 7 days following I/R. A Tie-2-cre transgenic crossed with a yellow fluorescent protein (YFP) reporter mouse was used to trace the fate of endothelial cells and demonstrated interstititial expansion of YFP-positive cells colocalizing with S100A4 and smooth muscle actin following I/R. The interstitial expansion of YFP cells was attenuated by VEGF-121. Multiphoton imaging of transgenic mice revealed the alteration of YFP-positive vascular cells associated with blood vessels characterized by limited perfusion in vivo. Taken together, these data indicate that vascular dropout post-AKI results from endothelial phenotypic transition combined with an impaired regenerative capacity, which may contribute to progressive chronic kidney disease.

Fig. 1.

Cablin antibody-mediated cell sorting in rat kidney. Rat kidney tissue was minced, collagenase digested, and subjected to FACS using an antibody generated against cablin. Cablin +-sorted cells were then plated and cultured for 2 days and endothelial phenotype was evaluated by visualizing acetylated LDL uptake (A), von Willebrand factor staining (B), and expression of CD31/PECAM staining (C). Samples in B and C were counterstained with DAPI.

Fig. 2.

Cablin/PECAM staining of peritubular capillaries of rat kidney. Methanol-fixed rat kidney tissues were subjected to immunofluorescent double labeling using tyramide enhancement as described in methods. Shown are representative confocal images of Cy-3-labeled cablin (A, D shown in red), FITC-labeled PECAM/CD31 (B, E, shown in green). The merged images shown in C and F reveal extensive colocalization in peritubular vessels. Note in images A–C, from renal cortex, that the glomerulus (indicated by g) is largely PECAM+/cablin-, while the arteriole in D and E (indicated by *) is also PECAM+/cablin negative. The experiment was done with dye reversal and yielded similar results (not shown); bar in D = 25 μm.

Fig. 3.

Effect of acute kidney injury (AKI) and vascular endothelial growth factor (VEGF)-121 on renal vascular density following 7 days of recovery from renal ischemia-reperfusion (I/R). Shown are data from morphometric analysis of cablin immunofluorescence-stained microvessel structures intersecting arbitrary grid lines and expressed as a percentage sham-operated control group; n = 5 animals per group. *P < 0.05 vs. sham-operated control. †P < 0.05 VEGF-121-treated vs. vehicle-treated group by ANOVA and Student-Newman-Keuls post hoc test.

Fig. 4.

Identification of proliferating cells in tubular epithelial and vascular endothelial cells following recovery from AKI. Shown are representative confocal images from the kidney of a rat that was given BrdU twice daily for 7 days of recovery from I/R injury. A: BrdU-positive cells in the green channel. B: cablin+ endothelial cells in the red channel. C: merged image. Note the prominent expression of BrdU in tubular epithelial cells. Higher magnification is shown in A′, B′, and C′, which correspond to the inset. Blue arrow indicates a cablin+ BrdU+ cell in the interstitial area, while the yellow arrow indicates a cablin-/BrdU+ in the interstitial area. Magnification is shown in A.

Fig. 5.

Lack of effect of VEGF-121 on proliferation during recovery from AKI. The number of BrdU-positive cells are shown based on their distribution in tubular cells (top), endothelial cells (middle), and perivascular cells (bottom). Data are shown for both cortex and outer medulla at 1, 2, and 7 days of recovery for AKI group (open bar, n = 5 per each time point) and AKI VEGF-121-treated group (striped bar, n = 3). Sham-operated controls (filled bar) was carried out only in the 7-day period. No effect of VEGF-121 was observed on BrdU incorporation in any cellular compartment following I/R injury.

Fig. 6.

Colocalization of S100A4/FSP-1 with tomato lectin following renal I/R injury. Biotinylated tomato lectin (TL) binding to vascular endothelial cells is shown in blue, while S100A4/FSP-1 staining is shown in red in kidneys of sham-operated rats (A) and rats following recovery from I/R for 6 h (B) and 7 days (C). Inset: higher magnification of the area in B, which illustrates colocalization of S100A4 staining in peritubulular vascular structures (indicated by the thin black arrows). Vascular structures without corresponding S100A4/FSP-1 staining (white arrows) were also prominent. Note the presence of S100A4/FSP-1+ in the lumen of venular spaces with a macrophage appearance in the early (6-h post-AKI group, thick dark arrow). Magnification is shown in A. Quantification of total number of S100A4/FSP-1+ cells in response to I/R injury is shown in D for both cortex and outer medulla. E: potential endothelial mesenchymal transition states defined as S100A4+/lectin+ structures (filled bars) and potential epithelial mesenchymal states, defined as S100A4+ cells in tubular cells (open bars). ND indicates that TL was not detectable in the outer stripe of outer medulla (OSOM) at 24 h of reperfusion; n = 4 animals per group. *P < 0.05, significant differences as shown based on ANOVA and Student-Newman-Keuls post hoc test.

Fig. 7.

Colocalization of CD31 and S100A4 by immunofluorescence following I/R injury. Representative confocal images are shown from sham-operated rat kidney (A–C) and kidney of rats 6 h following renal I/R injury (D–I). S100A4 staining was achieved by tyramide amplification of fluorescein (A, D), while CD31 staining was visualized directly using Cy3-labeled secondary (B, E; see methods), while merged images are shown in C and F. Magnification is shown in A. D′, E′, and F′ are enlarged from the outlined region and indicate cells expressing both S100A4 and CD31 (white arrow). Negative controls resulting from omission of primary antibodies are shown in green, red, and merged settings are shown in G–I, respectively. Staining representative of 5 animals per group.

Fig. 8.

Colocalization of cablin and S100A4 immunofluorescence following I/R injury. Representative confocal images are shown from kidney of a rat 6 h following renal I/R injury (A, B, and C) and enlarged image of the indicated area are shown in A′, B′, and C′. Cablin staining was achieved by tyramide amplification of fluorescein (A, A′), while S100A4 was visualized directly using Cy3-labeled secondary (B, B′ see methods), while merged images are shown in C and C′. Magnification is shown in A. White arrow indicates double-positive cells, while yellow arrows indicate S100A4-positive/cablin-negative cells. Negative controls (not shown) are similar to Fig. 7. Staining representative of 5 animals per group.

Fig. 9.

Effect of renal I/R and recovery on the yellow fluorescent protein (YFP) expression and colocalization with S100A4 and α-smooth muscle actin (SMA) in kidney of Tie2Cre^+/−YFP^+/− mice. Representative confocal images are shown from transgenic Tie2Cre^+/−YFP^+/− mice 14 days following recovery from sham operation (A, B, C) following renal I/R injury (D, E, and F) and following I/R injury with administration of VEGF-121 during the recovery period (G, H, I). Immunofluorescence of S100A4/FSP-1 is shown by fluorescein (A, D, G), YFP expression is shown by Cy3-enhanced immunofluorescence (B, E, H), and respective merged images are shown in C, F, and I. Colocalization of S100A4 cells with YFP is indicated by the white arrows in D, E, and F. Control immunofluorescence was carried out parallel for S100A4 and YFP in post-AKI tissues from Tie2Cre^−/−YFP^+/− mice (J, K, L), in which no signal was observed for YFP (K). Similar studies were carried out to localize α-SMA and YFP; representative images are shown for I/R vehicle-treated mice (M, N, O). Arrows indicate cells with staining for both α-SMA and YFP. Magnification is shown in A.

Fig. 10.

Effect of AKI and VEGF-121 on the renal interstitial distribution of YFP+ cells from transgenic Tie2Cre^+/−YFP^+/− mice. Data are shown from the renal cortex (open bars) and the renal outer medulla (filled bars) and are expressed as means ± SE. % surface area of YFP+ immunofluorescence was derived from morphometric analysis (see methods). The n for each group is indicated in the parentheses; *P < 0.05 from sham-operated controls; #P < 0.05 VEGF-121-treated vs. vehicle-treated post-AKI, based on ANOVA and Student-Newman-Keuls post hoc test.

Fig. 11.

Effect of renal I/R and recovery on peritubular vascular structure and flow in kidney of Tie2Cre^+/−YFP^+/− mice. Multiphoton images of Tie2Cre^+/−YFP^+/− mouse kidneys were obtained through a lateral flank incision from anesthetized animals 14 days after sham or renal I/R and following the intravenous injection of rhodamine-labeled dextran (MW 500,000). A: patent microvessels containing labeled dextran (red) can be observed and bordered by endothelial cells containing YFP, displayed in pseudocolor as green (thin white arrow). Evidence of flow through these vessels is apparent (see supplemental movie). B: relatively larger cells are observed in the tubular interstitium expressing YFP (green). These cells are found in areas of reduced or absent flow (indicated by thick arrowhead, see supplemental movie). Bar = 25 μm.