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. 2017 Mar;28(3):776-784.
doi: 10.1681/ASN.2016030297. Epub 2016 Sep 13.

Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury

Rafael Kramann  1   2 , Janewit Wongboonsin  3   4 , Monica Chang-Panesso  3 , Flavia G Machado  3 , Benjamin D Humphreys  5
Affiliations

Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury

Rafael Kramann et al. J Am Soc Nephrol. 2017 Mar.

Abstract

Peritubular capillary rarefaction is hypothesized to contribute to the increased risk of future CKD after AKI. Here, we directly tested the role of Gli1+ kidney pericytes in the maintenance of peritubular capillary health, and the consequences of pericyte loss during injury. Using bigenic Gli1-CreERt2; R26tdTomato reporter mice, we observed increased distance between Gli1+ pericytes and endothelial cells after AKI (mean±SEM: 3.3±0.1 µm before injury versus 12.5±0.2 µm after injury; P<0.001). Using a genetic ablation model, we asked whether pericyte loss alone is sufficient for capillary destabilization. Ten days after pericyte ablation, we observed endothelial cell damage by electron microscopy. Furthermore, pericyte loss led to significantly reduced capillary number at later time points (mean±SEM capillaries/high-power field: 67.6±4.7 in control versus 44.1±4.8 at 56 days; P<0.05) and increased cross-sectional area (mean±SEM: 21.9±0.4 µm2 in control versus 24.1±0.6 µm2 at 10 days; P<0.01 and 24.6±0.6 µm2 at 56 days; P<0.001). Pericyte ablation also led to hypoxic focal and subclinical tubular injury, reflected by transient expression of Kim1 and vimentin in scattered proximal tubule segments. This analysis provides direct evidence that AKI causes pericyte detachment from capillaries, and that pericyte loss is sufficient to trigger transient tubular injury and permanent peritubular capillary rarefaction.

Keywords: AKI; CKD; capillary; pericyte.

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Figures

Figure 1.
Figure 1.
Gli1+ pericytes detach from endothelial cells and expand after IRI. (A) Gli1+ cells were genetically tagged using bigenic Gli1-CreERt2; R26tdTomato mice. Tamoxifen was administered and mice underwent severe unilateral IRI 10 days after the last tamoxifen dose and were euthanized 5 days after surgery. (B) Representative images of CD31-stained kidneys from Gli1-CreERt2; R26tdTomato mice at day 5 after IRI. (C) Representative images of distance analysis indicating detachment of pericytes from endothelial cells. Immunofluorescence images of three channels (DAPI, tdTomato, and CD31) were split. Points of maximum intensity of each nucleus were selected and merged with tdTomato+ to mark the nucleus of the Gli1+ pericyte. Selected nuclei and tdTomato+ area were overlaid on the distance map generated from with the CD31 image to measure the distance of nuclei and cytoplasmic edges to the endothelial cells. (D) Scheme of pericyte–endothelial distance measurement. (E) Quantification of tdTomato+ cell number after IRI. (F) Measured distances from tdTomato+ pericytes to the closest endothelial cells indicating detachment after injury. Of note, data represent n=11 mice, six female and five male, in the contralateral kidney group and n=10 mice, five female and five male, in the severe IRI group; mean±SEM in E and F; box and whiskers with 10th–90th percentiles in F; + indicates mean in F; ***P<0.001, by t-test; all scale bars are 50 µm. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 2.
Figure 2.
Cell-specific genetic ablation of Gli1+ cells in healthy mice. (A) Experimental scheme: Gli1-CreERt2; iDTR; R26tdTomato trigenic mice were administered tamoxifen. Heritable expression of DTX receptor was induced in Gli1+ cells. DTX was given to specifically ablate Gli1+ cells as indicated, and the mice were followed and euthanized 10 days or 56 days after the first DTX dose. Control animals were given vehicle instead of DTX. (B) Random images of kidneys after FMA were taken (n=5/kidney section) and analyzed with our MATLAB-based script. (C) Gli1-CreERt2; iDTR; R26tdTomato trigenic mice were administered tamoxifen and received DTX or vehicle (PBS) to validate ablation of tdTomato+ cells. (D) Evidence of endothelial injury after Gli1+ pericyte ablation. Left panel, normal peritubular capillary. Note the very thin endothelial cytoplasm surrounding basement membrane (arrowheads). Center panel, injured endothelial cell with delamination of cell from basement membrane (asterisks) and areas of denuded basement membrane (arrows). Right panel, an injured endothelial cell with swollen cytoplasm and mitochondrial exhibiting disrupted cristae (arrow). Accumulation of electron-lucent material in between the endothelial cell and the capillary basement membrane is evident (arrowheads). Collagen fibers can be seen (asterisk). Scale bars are 50 µm in immunofluorescence images and 2 µm in electron microscopy images. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 3.
Figure 3.
Analysis of cortical microvasculature depicted by FMA reveals capillary rarefaction after Gli1+ pericyte ablation. (A) Ablation of Gli1+ pericytes resulted in reduction of total capillary cross-sectional area (control: 1478±104 µm2, 10 days: 1289±124.6 µm2, and 56 days: 1084±148.6 µm2) and total number of capillaries (control: 67.6±4.7 capillaries/hpf, 10 days: 53.6±4.4 capillaries/hpf, and 56 days: 44.1±4.8 capillaries/hpf; mean±SEM, t-test). (B) Gli1+ pericyte ablation had the highest effect on small capillaries with a significant reduction in capillaries smaller than 15, 25, and 35 µm2. (C) The individual cortical capillary cross-sectional area increased slightly after pericyte ablation (data shown in mean±SEM; box and whiskers with 10th–90th percentiles; + indicates mean; one-way ANOVA with post hoc Bonferroni correction). (D) Representative images of FMA and CD31 staining at 56 days after pericyte ablation versus vehicle (scale bars are 50 µm). Of note, data represent n=7 mice in control, n=4 mice in 10 days group and n=6 in 56 days group; *P<0.05; **P<0.01; ***P<0.001. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 4.
Figure 4.
Ablation of Gli1+ pericytes causes transient proximal tubular injury. (A) Increased Kim1 mRNA and protein expression at 10 days after Gli1+ cell ablation (***P<0.001; one-way ANOVA with post hoc Bonferroni correction). (B, C) Scattered Kim1 expression in proximal tubular (costained with Lotus tetragonolobus lectin [LTL]) brush borders. We observed increased Kim1 expression in areas with reduced FMA perfusion. Scale bars are 50 µm. (C) Injured tubules expressed vimentin indicating dedifferentiation of tubular epithelial cells. Scale bars are 50 µm. (D) Evidence of tubular injury by electron microscopy at 10 days after Gli1+ cell ablation. Asterisks indicate irregular intracytoplasmic vacuolization, arrowheads indicate simplification and thickening of the basal membrane, and arrows indicate enlargement and bulging of mitochondria. Scale bars are 4 µm. (E) Representative images and quantification of pimonidazole staining in kidneys of bigenic Gli1-CreERt2; iDTR mice at 10 days after ablation (n=5 control/vehicle, n=7 DTX). *P<0.05 by t-test, scale bars are 100 µm. (F) Upregulation of hypoxic response genes after Gli1 cell ablation. *P<0.05; **P<0.01 by one-way ANOVA with post hoc Bonferroni. (G) Gli1 ablation did not affect serum BUN or creatinine. (H) Representative images and quantification of collagen 1 and αSMA indicating no significant change after Gli1+ cell ablation. Scale bars are 50 µm. NS, not significant. DAPI, DAPI, 4′,6-diamidino-2-phenylindole.
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