BRIEF COMMUNICATION www.jasn.org Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury † ‡ ‡ ‡ Rafael Kramann,* Janewit Wongboonsin, § Monica Chang-Panesso, Flavia G. Machado, and ‡ Benjamin D. Humphreys *Renal Division, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts; †Division of Nephrology and Clinical Immunology, RWTH Aachen University Medical Faculty, RWTH Aachen University, Aachen, Germany; ‡Division of Nephrology, Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, Missouri; and §Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand ABSTRACT Peritubular capillary rarefaction is hypothesized to contribute to the increased risk of after AKI, and whether pericyte loss is future CKD after AKI. Here, we directly tested the role of Gli1+ kidney pericytes in the sufficient to induce peritubular capillary maintenance of peritubular capillary health, and the consequences of pericyte loss loss and altered morphology. during injury. Using bigenic Gli1-CreERt2; R26tdTomato reporter mice, we observed We genetically labeled pericytes using increased distance between Gli1+ pericytes and endothelial cells after AKI (mean6 Gli1-CreERt2 mice crossed against SEM: 3.360.1 mm before injury versus 12.560.2 mm after injury; P,0.001). Using a the R26tdTomato reporter mouse (Gt genetic ablation model, we asked whether pericyte loss alone is sufficient for capillary (ROSA)26Sortm9(CAF-tdTomato)Hze/J; destabilization. Ten days after pericyte ablation, we observed endothelial cell damage Figure 1A). After tamoxifen injection, mice by electron microscopy. Furthermore, pericyte loss led to significantly reduced capil- were subjected to severe unilateral ische- lary number at later time points (mean6SEM capillaries/high-power field: 67.664.7 in mia reperfusion injury (IRI; 28-minute control versus 44.164.8 at 56 days; P,0.05) and increased cross-sectional area (mean6 clamp). The injury was induced 10 days SEM: 21.960.4 mm2 in control versus 24.160.6 mm2 at 10 days; P,0.01 and 24.66 after tamoxifen injection to eliminate the 0.6 mm2 at 56 days; P,0.001). Pericyte ablation also led to hypoxic focal and subclinical possibility of recombination after injury. tubular injury, reflected by transient expression of Kim1 and vimentin in scattered Mice were euthanized 5 days after IRI. proximal tubule segments. This analysis provides direct evidence that AKI causes peri- We performed endothelial cell staining cyte detachment from capillaries, and that pericyte loss is sufficient to trigger transient (with CD31) and quantitatively measured tubular injury and permanent peritubular capillary rarefaction. the distance of tdTomato+ cells to the nearest endothelial cell by generating dis- J Am Soc Nephrol 28: 776–784, 2017. doi: 10.1681/ASN.2016030297 tance maps in ImageJ (Figure 1, B and C, Supplemental Material). Up to 20% of patients that develop AKI becauseoftheabsenceofspecificmark- will go on to develop late stage CKD, and ers for kidney pericytes that would allow Received March 14, 2016. Accepted August 9, these patients are at increased risk of unambiguous fate tracing and genetic 2016. 1,2 ESRD and death. Understanding the ablation in mouse models. R.K. and J.W. contributed equally to this work. mechanisms underlying this epidemio- We recently reported that Gli1 marks a Published online ahead of print. Publication date logic link is a priority, with a strong focus perivascular population of mesenchymal available at www.jasn.org. being on the structural alterations that stem cell-like cells that form an extensive occur in kidney after an episode of network from the arterial adventitia in the Correspondence: Dr. Benjamin D. Humphreys, Di- vision of Renal Disease, Washington University School 3–6 9 AKI. OnehypothesisisthatAKI pericyte niche. Genetic fate tracing ex- of Medicine, 660 South Euclid Avenue, CB 8129, St. causes detachment of pericytes from periments revealed that these perivascular Louis, MO 63110, or Dr. Rafael Kramann, Division of peritubular endothelial cells, triggering Gli1+ cells are a major source of kidney Nephrology and Clinical Immunology, Rheinisch- fi Westfälische Technische Hochschule Aachen Univer- endothelial injury, capillary rarefaction, myo broblasts and can be targeted thera- sity, Pauwelstrasse 30, 52074 Aachen, Germany. Email: hypoxia and, ultimately, fibrosis with peutically.9,10 In this study, we sought ex- [email protected] or [email protected] 7,8 CKD progression. Direct evidence in perimental evidence for detachment of Copyright © 2017 by the American Society of + support of this hypothesis is lacking Gli1 cells from peritubular capillaries Nephrology 776 ISSN : 1046-6673/2803-776 J Am Soc Nephrol 28: 776–784, 2017 www.jasn.org BRIEF COMMUNICATION 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 tamoxifendoseandwereeuthanized5daysaftersurgery.(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 en- dothelial 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 endo- thelial 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; mean6SEM 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 mm. DAPI, 49,6-diamidino-2-phenylindole. J Am Soc Nephrol 28: 776–784, 2017 Pericyte Loss Triggers Rarefaction 777 BRIEF COMMUNICATION www.jasn.org We detected increased numbers of genetic ablation of Gli1+ cells would swollen endothelial mitochondria with tdTomato+ cells (mean6SEM: 18.462.2 trigger endothelial damage and capillary loss of cristae, thickening of capillary base- versus 77.564.8 cells/high-power field rarefaction. We crossed Gli1-CreERt2; ment membrane, and denudation of en- [hpf]; P,0.001) after IRI, as expected R26tdTomato mice to R26iDTR mice. dothelial cells from the capillary basement (Figure 1, B and E). Importantly the dis- Tamoxifen administration in bigenic membrane (Figure 2D). To quantify peri- tance of tdTomato+ cells to the closest mice leads to heritable expression of tubular capillary changes after pericyte endothelial cell increased significantly the human diphtheria toxin (DTX) recep- ablation, we applied fluorescence micro- (Figure 1, D and F). We measured an in- tor in Gli1+ pericytes and perivascular angiography (FMA) high-resolution im- crease of the minimum as well as maxi- cells. After tamoxifen-induced recombina- aging combined with our automated mum distance from the cytoplasmic edge tion and a 10-day washout period, DTX MATLAB-based analysis of peritubular (minimum distance: 0.460.1–4.26 was administered to specifically ablate capillary size and density (Figure 2B, Sup- 0.2 mm; P,0.001; maximum distance: Gli1+ pericytes (Figure 2A). Mice were eutha- plemental Material).3 As a positive control, 9.860.2–23.960.3 mm; P,0.001) or nized at 10 days or 56 days after the first we used triple transgenic Gli1-CreERt2; the nucleus (3.660.2–13.160.2 mm; DTX dose. We observed evidence of endo- iDTR; R26tdTomato mice to demonstrate P,0.001) of the tdTomato+ pericyte to thelial injury by electron microscopy upon successful depletion of Gli1+ cells after the closest endothelial cell (Figure 1F). pericyte ablation at day 10, consisting of DTX administration (Figure 2C). We next asked whether undifferenti- 2 ated Gli1+ pericytes (tdTomato+/aSMA ) andGli1-derivedmyofibroblasts (tdTomato+/aSMA+) both show increased distance from endothelial cells after in- jury. Indeed, we observed increased dis- 2 tances of both tdTomato+/aSMA cells and tdTomato+/aSMA+ cells to the closest endothelial cells, after IRI (Supplemental Figure 1), suggesting that myofibroblast dif- ferentiation is not a requirement for pericyte detachment from capillaries after injury. In addition to detachment of pericytes, we also detected peritubular capillary rarefaction after IRI (Supplemental Figure 2). Pericyte–endothelial cell crosstalk has been investigated after kidney injury, and experiments by Lin et al. using Col1a1- GFP mice have suggested that pericytes migrate away from capillaries after kidney injury.11 However, the lack of a specific kidney pericyte marker that would allow inducible genetic fate tracing has prevented experimental proof of pericyte migration Figure 2. Cell-specific genetic ablation of Gli1+ cells in healthy mice. (A) Experimental in vivo 11,12 from capillaries . scheme: Gli1-CreERt2; iDTR; R26tdTomato trigenic mice were administered tamoxifen. + Our data indicates that Gli1 myofi- Heritable expression of DTX receptor was