Fluorescence Microangiography for Quantitative Assessment of Peritubular Capillary Changes After AKI in Mice

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Fluorescence Microangiography for Quantitative Assessment of Peritubular Capillary Changes after AKI in Mice

† ‡ Rafael Kramann,* Mari Tanaka,* and Benjamin D. Humphreys*

*Renal Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; †Division of Nephrology and Clinical Immunology, RWTH Aachen University, Aachen, Germany; and ‡Kidney Group, Harvard Stem Cell Institute, Cambridge, Massachussetts

ABSTRACT AKI predicts the future development of CKD, and one proposed mechanism for this (restitutio ad integrum).3,4 After multi- epidemiologic link is loss of peritubular capillaries triggering chronic hypoxia. A ple ischemic events or severe ischemia- precise definition of changes in peritubular perfusion would help test this hypothesis reperfusion injury (IRI), the repair process by more accurately correlating these changes with future loss of kidney function. is often incomplete, resulting in fibrogenesis Here, we have adapted and validated a fluorescence microangiography approach and development of CKD.2,5,6 for use with mice to visualize, analyze, and quantitate peritubular capillary dynamics In contrast to renal tubules, the mi- after AKI. A novel software-based approach enabled rapid and automated quanti- crovasculature lacks substantial regener- tation of capillary number, individual area, and perimeter. After validating perfusion ative capacity, resulting in a persistent in mice with genetically labeled endothelia, we compared peritubular capillary vascular rarefaction after severe or re- number and size after moderate AKI, characterized by complete renal recovery, and current injury.7,8 Indeed, the loss of the after severe AKI, characterized by development of interstitial fibrosis and CKD. renal microvasculature is hypothesized Eight weeks after severe AKI, we measured a 40%67.4% reduction in peritubular to be a key component in fibrogenesis capillary number (P,0.05) and a 36%64% decrease in individual capillary cross- and CKD progression after AKI. As the sectional area (P,0.001) for a 62%62.2% reduction in total peritubular perfusion molecular pathways regulating capillary (P,0.01). Whereas total peritubular perfusion and number of capillaries did not rarefaction begin to be unraveled, pre- change, we detected a significant change of single capillary size following moderate venting this process has been proposed AKI. The loss of peritubular capillary density and caliber at week 8 closely correlated as a therapeutic target.9,10 Capillary rar- with severity of kidney injury at day 1, suggesting irreparable microvascular damage. efaction induces focal hypoxia, activat- These findings emphasize a direct link between severity of acute injury and future ing an injury cascade with inflammation loss of peritubular perfusion, demonstrate that reduced capillary caliber is an un- and ultimately interstitial fibrosis.2,11 It appreciated long-term consequence of AKI, and offer a new quantitative imaging has been described in a variety of rodent tool for understanding how AKI leads to future CKD in mouse models. AKI models,7,12–15 and in human patients with CKD the loss of capillary J Am Soc Nephrol 25: ccc–ccc, 2014. doi: 10.1681/ASN.2013101121 density correlates with the severity of fibrosis.16–18

AKI is a strong risk factor for the future HR, 3.1; 95% CI, 1.9 to 5); and mortality development of CKD and ESRD. In a (pooled HR, 2.0; 95% CI, 1.3 to 3.1).1 Received October 25, 2013. Accepted January 10, 2014. recent meta-analysis, Coca and colleagues Possible mechanisms underlying the compared the risk for CKD, ESRD, and link between AKI to future CKD include Published online ahead of print. Publication date deathinpatientswith or withoutAKIin13 nephron loss, glomerular hypertrophy, available at www.jasn.org. cohort studies that included .1.4 million inflammation, interstitial fibrosis, epithe- Correspondence: Dr. Benjamin D. Humphreys, Re- patients.1 They demonstrated that pa- lial cell-cycle abnormalities, and endothe- nal Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Institutes of Medicine, 2 tients with AKI are at high risk to develop lial injury with capillary rarefaction. Room 536, 77 Avenue Louis Pasteur, Boston, MA CKD, with a pooled adjusted hazard ratio Acute ischemic injury causes epithelial in- 02115. Email: [email protected] fi (HR) of 8.8 (95% con dence interval jury and death, but the tubule can repair Copyright © 2014 by the American Society of [95% CI], 3.1 to 25.5); ESRD (pooled completely after mild-to-moderate injury Nephrology

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Despite the established association FMA procedure, and describe how to an- To more precisely define renal peri- between AKI and peritubular capillary alyze FMA pictures automatically using tubular capillary dynamics during pro- loss, the precise nature of microvascular our MATLAB script in the Supplemental gression of AKI to CKD, wild-type mice changes occurring after AKI are poorly Material. We also provide all script files were subjected to severe IRI (28 minutes defined because high-resolution techni- needed for this analysis.) To validate our of clamping), moderate IRI (23 minutes ques have not been used to examine this modified approach, we genetically labeled of clamping), or sham surgery followed question. To date, microvascular density endothelial cells using the VE-Cadherin- by FMA 8 weeks after the surgery (Figure has been measured by immunostaining Cre driver line (Tg[Cdh5-cre]7Mlia) bred 2A). Whereas the BUN levels in the and genetic labeling of the endothelium, against the R26Tomato reporter mouse moderate group showed a complete re- techniques that assess the surface area of (Gt[ROSA]26Sortm9[CAG-tdTomato]Hze/J) covery at 14 days after surgery, BUN endothelial cells or the number of visu- (Figure 1A). Using our modified FMA levels remained significantly elevated ally identified capillaries but not the protocol, which includes solutions pre- at day 14 and week 8 after severe IRI, in- actual capillary lumen.12–15,19 Endothelial warmed to 41°C and intracardiac injec- dicating a progression to CKD (Figure cell antigens used for immunostaining, tion rather than renal artery injection 2B). Immunostaining and quantifica- such as CD31 or genetic labeling (such (see Concise Methods), the fluorescence tion of a-smooth muscle actin–positive as Tie 2), are reported to be expressed microbead (0.02 mm)–low-melting-point surface area demonstrated development on nonendothelial cell types, which may agarose mixture successfully perfuses the of interstitial renal fibrosis only in the introduce error.20–22 Other approaches microvasculature of all solid organs tested severe IRI group, whereas severity of in- have included Microfil infusion7,23 and (Figure 1B). terstitial fibrosis in the moderate IRI radiation microangiography.24 Whereas radiation microangiography does not give information about the capillaries,24 the other techniques allow an estimation of microvascular density; however, they do not provide high-resolution information about perfusion characteristics, such as single capillary cross-sectional area and perimeter. A further limitation to date is the absence of a nonbiased and high-throughput means of quantitating capillary changes. Finally, when the en- dotheliumisusedasasurrogatefor capillary perfusion, overestimation is possible because endothelial cells of clogged or collapsed vessels with micro- embolism will still be stained, even though the capillary is actually nonfunctional. Recently,Advaniandcolleaguesreported use of fluorescence microangiography (FMA) by renal artery injection in the rat 5/6 nephrectomy model.20 They describe changes in glomerular perfusion and a global reduction in peritubular perfusion in this CKD model. Here we modify this technique for use in mice by intracardiac injection and show that it allows visuali- zation of microvasculature in all solid organs tested. We additionally created a MATLAB-based script that allows for pre- Figure 1. FMA provides highly efficient high-resolution perfusion imaging of the kidney, cise and rapid analysis of microvascular heart, and liver. (A) To genetically label endothelial cells in solid organ vascular beds, we characteristics, including single capillary bred the VE-Cadherin-Cre driver mouse against the Rosa26-tdtomato reporter line, re- cross-sectional area and perimeter (Sup- sulting in endothelial cell–specific cre-recombination with removing of the LoxP flanked plemental Figure 1). (We provide a de- STOP codon and expression of the bright red fluorochrome tdtomato. (B) FMA was vali- tailed step-by-step description, including dated in Ve-CadherinCre+,R26Tomato+ mice, showing highly efficient delineation of the notes and a reagent list for the mouse microvasculature in heart, kidney, and liver. DAPI, 49,6-diamidino-2-phenylindole.

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Figure 2. Severe ischemia reperfusion injury leads to CKD with interstitial ﬁbrosis. (A) Wild-type mice were subjected to severe bilateral IRI (clamping for 28 minutes), moderate bilateral IRI (clamping for 23 minutes), or sham surgery and were euthanized following FMA at 8 weeks. (B) BUN measurement revealed signiﬁcantly increased BUN values in both IRI groups at day 1 after surgery. After moderate IRI, mice showed a complete recovery, with BUN levels returned to normal values at day 14 after surgery, whereas severe IRI lead to persistently increased

J Am Soc Nephrol 25: ccc–ccc, 2014 Fluorescence Microangiography and Capillary Rarefaction 3 BRIEF COMMUNICATION www.jasn.org group did not differ significantly com- versus sham, capillary number 264% very closely with BUN at day 1, and pared with the sham group (Figure 2, C 63% versus sham) and a similar reduction to a lesser degree at day 14 and week 8 and D). All mice were subjected to the of individual capillary cross-sectional after IRI (Figure 4B, Supplemental Fig- FMA procedure immediately before area and perimeter (Supplemental ure 6), suggesting that severity of initial euthanasia at 8 weeks after surgery. Figure 2). injury is a critical factor for future peri- Confocal microscopy pictures of the The use of genetic lineage tracing is tubular capillary rarefaction. kidneys demonstrate that FMA allows limited by the specificity of the Cre In summary, this method reveals precise delineation of the peritubular drivers used. During this study, we noted novel insight into capillary dynamics in capillary network, including by three- that 15%61% of F4/80+ macrophages response to AKI, including a previously dimensional reconstruction of Z-stack were labeled in VE-Cadherin+;tdTo- unappreciated reduction not only in the images (Figure 3A, Supplemental Vid- mato+ kidneys (Supplemental Figure 3). number but also the caliber of surviving eos 1 and 2). Because macrophage infiltration intensi- peritubular capillaries. Basile et al. pre- Our software-based analysis (script fiesinthepostischemickidney,25 this viously used Microfil injection to dem- available in Supplemental Material) macrophage labeling would induce an onstrate that rats develop a significant demonstrates a significant 62%62% re- error during quantitative measurements reduction of peritubular capillary den- duction of total cortical perfused area after injury. Therefore, we decided to com- sity in the kidney (cortex, 225% to (mm2/high-power field of the inner cor- pare the performance of FMA with stan- 30%; inner stripe of outer medulla, tex) in the severe IRI group compared dard CD31 immunostaining instead. 235% to 40%) at 4–40 weeks after with the sham group and a significant Comparison between FMA and CD31 IRI.7 Horbelt et al. subsequently identi- 52%61% reduction compared with the staining following IRI demonstrated a fied endothelium by immunostaining of moderate IRI group (Figure 3B). This re- larger reduction of the perfused FMA+ postischemic mice and also reported a duction of total perfused cortical area was capillary area compared with the CD31+ 45% decrease of microvascular density due to both a reduction of capillary num- endothelial cell surface area (Supple- at 30 days after IRI.14 Although the ex- ber (Figure 3C) and individual capillary mental Figure 4), suggesting that some perimental protocols differ, our results cross-sectional area and perimeter (Figure capillaries might lack perfusion follow- confirm a substantial reduction in per- 3, B and D), indicating a loss of both cap- ing IRI. Indeed, we observed areas where fusion after acute injury but also report illary number and a reduction of the cal- CD31+ capillaries did not contain FMA+ that surviving capillaries are smaller. iber in remaining capillaries after severe luminal signal following IRI (Supplemen- Thus, an advantage of FMA in assessing IRI. Interestingly, whereas total perfused tal Figure 4). peritubular microvasculature is its abil- cortical area and number of capillaries did For the automated software-based anal- ity to define perfused capillaries and not significantly change after moderate ysis of the FMA sections, we used a lower their precise architecture. IRI compared with the sham group, the cutoff value of 4.9 mm2 because it has been Several future applications can be single cortical capillary surface area and previously reported that the critical diam- envisioned. This mouse FMA approach size did change significantly (Figure 3, eter at which erythrocytes cannot pass should be useful for the study of the B–E). The distribution of cross-sectional through capillaries is 2.5 mm(p3r2=4.9 microvasculature, not just in the kidney area in counted capillaries revealed a bi- mm2)26; to exclude venules, glomerular but in a variety of solid organs, where phasic trend: The number of smallest capillary convolutes, and other larger capillary rarefaction after injury is also capillaries (cross-sectional area ,15 mm2) vessels (arterioles, arteries, veins), we considered to be an important compo- increased slightly after IRI, whereas the used an upper cutoff value of 100 mm2 nent of chronic disease progression.27–29 number of larger capillaries (.15 mm2) (Supplemental Figure 5). Importantly, It should also enable a more precise def- was reduced after IRI (Figure 4A). The the raw data obtained without applying inition of changes within the interstitial most likely explanation for this observa- these cutoffs still showed the same sig- space that accompany injury and repair, tion is a shift in categorization of larger nificant differences after IRI (Supple- such as visualizing pericyte migration capillaries to the smallest group after mental Figure 5). away from endothelium during CKD, IRI. Interestingly, quantification of The loss of total perfused peritubular which has been proposed to underlie fi- medullary perfusion and the number cross-sectional area (mm2/high-power brogenesis.19,30,31 Finally, mouse FMA of medullary capillaries did show a field), peritubular capillary number, may serve as a useful functional readout more dramatic reduction following se- and individual capillary cross-sectional for therapeutics targeting vascular sur- vere IRI (total medullary perfused area area (mm2/capillary) and perimeter vival, such as angiogenic growth factors mm2/high-power field, 278%63% (mm/capillary) at week 8 correlated or drugs.19

BUN levels at 8 weeks after surgery. (C and D) a-Smooth muscle actin staining and quantiﬁcation revealed induction of interstitial ﬁbrosis only after severe IRI. (Graphs show mean6SEM; *P,0.05; **P,0.01; ***P,0.001, one-way ANOVA with post hoc Bonferroni correction). DAPI, 49,6-diamidino-2-phenylindole.

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Figure 3. High-throughput software–based analysis of ﬂuorescence microangiography reveals reduced capillary number and caliber after severe IRI. (A) FMA after sham surgery and moderate and severe IRI, together with CD31 immunostaining, demonstrates capillary rarefaction in response to severity of injury (arrows indicate capillaries with red CD31+endothelial cells surrounding the green FMA solution; all scale bars are 50 mm). (B and C) Severe IRI results in a signiﬁcant reduction of the total cortical cross-sectional capillary area per high-power

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[ROSA]26Sortm9[CAG-tdTomato])reporter mice (The Jackson Laboratories; stock # 006137 and 007909). IRI was performed as previously described.4 Briefly, mice were anesthetized with pentobarbital sodium (60 mg/kg body wt intraperitoneally); buprenorphine (0.1 mg/kg body wt intraperitoneally) was used to achieve analgesia; kidneys were exposed through flank incisions; and mice were subjected to ischemia by clamping therenalpediclewithnontraumaticmicroan- eurysm clamps (Roboz, Rockville, MD) for 28 minutes (severe IRI) or 23 minutes (moderate IRI) or no clamping (sham surgery). Reperfu- sion was visually verified. During the surgery, 1 ml of 0.9% NaCl was subcutaneously adminis- tered. Body temperatures were controlled at 36.5°C–37.5°C throughout the procedure. Mice were bled at 24 hours, 14 days, and 8 weeks after the surgery via the tail vein. BUN was measured using the Infinity Urea assay (Thermo Fisher Scientific) according to the manufac- turer instructions.

FMA The agarose-fluorescent microbead mixture was made immediately before the procedure by microwaving low-melting-point agarose (Lonza;#50080)1%by massindistilledwater (4.5 ml dH20+0.05 g agarose/mouse), according to the protocol published by Advani et al.20 Following complete dilution of the agarose, 0.02 mm FluoSpheres sulfate (Invi- Figure 4. Severity of initial injury determines shift in capillary size and extent of capillary trogen; #F8845, yellow-green) were added to rarefaction. (A) Distribution of capillary size demonstrates a loss of larger capillaries (.15 the mixture such that they were 10% by vol- mm2) after moderate and severe IRI, with a slight increase in counted small capillaries (,15 m mm2) (data represent three mice per group). (B) FMA assessed total capillary cross-sectional ume (i.e., 500 l FluoSpheres plus 4.5 ml 1% area (total perfused area, mm2/high-power field), individual capillary cross-sectional area agarose/mouse). Mice were anesthetized (mm2), and capillary number (number/high-power field) shows highly significant correlation with pentobarbital (60 mg/kg of body wt in- with day 1 BUN and lower correlation with week 8 BUN. traperitoneally); buprenorphine (0.1 mg/kg body wt intraperitoneally) was given to achieve analgesia; and mice were placed on a surgical CONCISE METHODS were 8- to 10-week-old C57Bl/6J males from heating pad (37°C). The abdomen and thorax Charles River Laboratories (Wilmington, were cut via a midline incision extending from All mouse experiments were performed ac- MA). The Ve-Cadherin-Red Rosa reporter the symphysis pubis to the jugulum. We did cording tothe animal experimental guidelines mice were created by crossing homozygous not achieve satisfactory results when following issued by the Animal Care and Use Commit- VE-Cadherin-Cre (Tg(Cdh5-cre)7Mlia) the Advani protocol in mice until all solutions tee at Harvard University. Wild-type mice with homozygous R26-tdTomato (Gt were prewarmed to 41°C rather than room to

field [3400, inner cortex] (B) and a significant reduction in capillary number (C). (D and E) The cortical individual capillary cross-sectional area (D) (mean6SEM, sham: 30.3160.42 mm2; moderate IRI: 26.2960.28 mm2; severe IRI: 19.3460.38 mm2) and perimeter (E) (sham: 30.1960.31 mm; moderate IRI: 29.1360.22 mm; severe IRI: 24.7760.35 mm) was significantly reduced after both moderate and severe IRI. (Of note, data represent n=3 mice in the sham group and severe IRI group and n=6 mice in the moderate IRI group; mean6SEM in B and C; box and whiskers with 10th–90th percentiles in D and E; + indicates mean in D and E. *P,0.05; **P,0.01; ***P,0.001, one-way ANOVA with post hoc Bonferroni correction). DAPI, 49,6-diamidino-2-phenylindole.

6 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 www.jasn.org BRIEF COMMUNICATION body temperature. One milliliter of heparin- areas smaller than the size of an erythrocyte 5. Venkatachalam MA, Griffin KA, Lan R, Geng ized saline (100 IU/ml heparin [Sagent Phar- (i.e.,4.9mm2)asbackgroundnoise(i.e., prob- H, Saikumar P, Bidani AK: Acute kidney injury: A springboard for progression in chronic maceuticals] in 0.9% NaCl) followed by 1 mm ably no functional capillary) and areas.100 kidney disease. Am J Physiol Renal Physiol m 2 of 3 M KCl (41°C) was injected in the beating m as too large for a capillary (i.e., arterioles, 298: F1078–F1094, 2010 left ventricle using a 27-gauge butterflycathe- arteries, venules, and glomerular convo- 6. Humphreys BD, Xu F, Sabbisetti V, Grgic I, ter (Exel, Corp., #26709). The inferior vena lutes).26 The same analysis was run without Naini SM, Wang N, Chen G, Xiao S, Patel D, cava was then cut and the mouse was per- the array loop to include all data without any Henderson JM, Ichimura T, Mou S, Soeung S, McMahon AP, Kuchroo VK, Bonventre JV: fused with 41°C prewarmed PBS (10 ml), cutoff value (Supplemental Figure 5). Quanti- Chronic epithelial kidney injury molecule-1 fi a – immediately followed by 5 ml of the agarose- cation of -smooth muscle actin positive expression causes murine kidney fibrosis. microbead mixture (41°C). (Note: A rapid surface area was performed by taking random JClinInvest123: 4023–4035, 2013 switch between the different syringes is critical cortical pictures (3200; n=5/kidney) of each 7. Basile DP, Donohoe D, Roethe K, Osborn JL: [i.e., PBS → agarose].) Immediately after the mouse using the number of stained pixels per Renal ischemic injury results in permanent damage to peritubular capillaries and influ- perfusion, kidneys, heart, and liver were ex- total pixels in Adobe Photoshop CS5 (Adobe ences long-term function. Am J Physiol Renal cised and carefully placed in an ice bucket (sur- Systems, Inc., San Jose, CA). Physiol 281: F887–F899, 2001 rounded by ice) for 10 minutes. Thereafter, the 8. Basile DP: Rarefaction of peritubular capil- tissues were fixed in 4% paraformaldehyde on Statistical Analyses laries following ischemic acute renal failure: A ice for 2 hours, then incubated in 30% sucrose Data are presented as mean6SEM. For mul- potential factor predisposing to progressive nephropathy. Curr Opin Nephrol Hypertens in PBS at 4°C overnight and optimum cut- tiple group comparisons, ANOVA with post 13: 1–7, 2004 ting temperature (OCT) embedded (Sakura hoc Bonferroni correction was applied. All 9. Kida Y, Ieronimakis N, Schrimpf C, Reyes M, Finetek). OCT-embedded organs were cryosec- statistical analyses, including linear regres- Duffield JS: EphrinB2 reverse signaling pro- tioned into 7- to 40-mm sections and mounted sion analyses, were performed using Graph- tects against capillary rarefaction and fibrosis JAmSocNephrol on Superfrost slides (Thermo Fisher Scienti- Pad Prism software, version 5.0c (GraphPad after kidney injury. 24: 559–572, 2013 fic) using ProLong Gold Antifade reagent (In- Software Inc., San Diego, CA). A P value,0.05 10. Kida Y, Tchao BN, Yamaguchi I: Peritubular 3 vitrogen). Sections were washed in 1 PBS was considered to indicate a statistically sig- capillary rarefaction: a new therapeutic target fi (335minutes),stainedwith49,6-diamidino- ni cant difference. in chronic kidney disease. Pediatr Nephrol 29: 2-phenylindole and mounted in ProLong 3330–3342, 2014 Gold (Life Technologies). For immunofluores- 11. Tanaka T, Nangaku M: Angiogenesis and hypoxia in the kidney. Nat Rev Nephrol 9: cence staining, sections were blocked in 10% ACKNOWLEDGMENTS 211–222, 2013 normal goat serum (Vector Labs) and incubated 12. O’Riordan E, Mendelev N, Patschan S, with an primary antibody specific for CD31 Patschan D, Eskander J, Cohen-Gould L, This work was supported by National Institutes (1:100; eBioscience; #14–0311), F4/80 (1:200; Chander P, Goligorsky MS: Chronic NOS in- of Health grant DK088923 (to B.D.H.), by an Abcam, Inc.; #ab6640) or a-smooth muscle ac- hibition actuates endothelial-mesenchymal EstablishedInvestigatorAward oftheAmerican transformation. Am J Physiol Heart Circ tin (1:200; Sigma-Aldrich; #A2547) followed Heart Association (to B.D.H.), and by a fel- Physiol 292: H285–H294, 2007 by a Cy5-conjugated secondary antibody lowship from the RWTH Aachen University 13. Yuan HT, Li XZ, Pitera JE, Long DA, Woolf AS: (1:200; Jackson ImmunoResearch Laboratories). 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8 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 Supplementary Material:

Fluorescence Microangiography for Quantitative Assessment of

Peritubular Capillary Changes after Acute Kidney Injury

Rafael Kramann, Mari Tanaka and Benjamin D. Humphreys

Address to correspond to:

Benjamin D Humphreys, MD, PhD; Renal Division, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts; 4 Blackfan Circle 02115, Boston, MA

E-mail: [email protected]

Supplementary figure S1: Our MATLAB script allows automated high-throughput analysis of the microvasculature. A-C: The channels of the original confocal picture of inner cortex (RGB format, A) were splitted in ImageJ (NIH) and the green channel was saved as a grayscale picture (PNG file, B) this picture will be automatically processed by the MATLAB script which generates a binary image of the capillaries (C). D-E: The MATLAB script automatically generates an Excel sheet with number, area (µm2) and perimeter (µm) of the capillaries and also demonstrates the area and perimeter data as a histogramm (D, E). Note, Trough an array loop the script can sort out measurements that do not meet the user defined requirements for a capillary. For example, if the measured area is smaller or larger than a certain value (cut-off values in our case <4.9µm2 and >100µm2) it will be counted as a false and is sorted as a zero.

Supplementary Figure S2: Fluorescence microangiography of the renal medulla. A: Fluorescence microangiography (FMA) of the renal medulla after sham, moderate and severe ischemia-reperfusion injury (IRI) demonstrates a dramatic capillary rarefaction after severe IRI. B: B-C: Severe IRI results in a significant reduction of the total cortical cross- sectional capillary area per high power field [hpf /400x, medulla] (B) and a significant reduction of capillary number (C). D-E: The medullary individual capillary cross sectional area (D: (mean±SEM, sham: 31.67±0.49µm2; moderate IRI: 26.47±0.36µm2; severe IRI: 18.97±0.54µm2) and perimeter (E: sham: 31.08±0.34µm; moderate IRI: 26.41±0.252µm; severe IRI: 22.41±0.41µm) was significantly reduced after both moderate and severe IRI. (of note data represents n=3 mice in sham and severe IRI and n=6 mice in moderate IRI; mean with SEM in B and C; box and whiskers with 10 to 90 percentile in D and E, + indicates mean in D and E; *=p<0.05, **=p<0.01;***=p<0.001 one way ANOVA with posthoc Bonferroni) (all scale bars are 50µm)

Supplementary Figure S3: Cdh5 is expressed in kidney macrophages. A-B: Immunostaining against the macrophage marker F4/80 in non-injured kidneys of Ve- CadherinCre+, R26Tomato+ mice (A) and quantification of positive cells revealed that about 15% of macrophages in the kidney also express VE-Cadherin (B). C: Representative picture of a non-injured kidney from a CadherinCre+, R26Tomato+ mouse stained for the endothelial surface marker CD31.

Supplementary Figure S4: Reduction of perfused vascular area compared to endothelial cell surface area following ischemia reperfusion injury. A: To compare the reduction of the perfused vascular surface area with the reduction of the endothelial cell-surface area following IRI we performed immunostaining for the endothelial- cell surface marker CD31 in kidney sections after application of FMA. The MATLAB based FMA analysis of the perfused vascular area was performed in the same image as the quantification of the CD31 positive surface area (representative picture in A). B: Quantification of the total CD31+ surface area per high power field (hpf n=7 / kidney) demonstrates a reduction of 23±11% after moderate IRI and a reduction of 53±19% following severe IRI whereas analysis of the FMA+ vascular luminal area revealed a slightly higher reduction of 35±14% following moderate IRI and 78±2% following severe IRI. (Of note: no cutt-off values where applied because this would have induced an error in the comparison between CD31+ surface area and FMA+ surface area because application of a cut of value would exclude more FMA+ areas then CD31 as for example glomerula would be automatically excluded regarding their size >100µm2 in the FMA but would be included in the analysis based on their CD31+ surface area .) C: Representative picture of a kidney 8 weeks after moderate IRI demonstrating that some capillaries surounded by CD31+ endothelial cells do not show a luminal FMA signal (arrows) suggesting a that these capillaries lack perfusion.

Supplementary figure S5: High throughput software based analysis of fluorescence microangiography without cut-off values remains significant. Each picture was automatically analyzed using the MATLAB script without the array loop to include all data points without application of cut-off values demonstrating a significant reduction of the average capillary cross sectional area (A) after moderate or severe ischemia reperfusion injury (IRI) (mean±SEM, moderate IRI: 28.19±0.65µm2; severe IRI: 18.01±0.67 µm2) when compared to the sham group (36.39±1.12µm2). Similar results where obtained when the average individual capillary perimeter (B) was determined without cut off values (mean±SEM, sham: 30.03±0.61µm; moderate IRI: 26.14±0.39µm; severe IRI: 20.92±0.47 µm). (box and whiskers with 10 to 90 percentile, + indicates mean ; ***=p<0.001 one way ANOVA with posthoc Bonferroni)

Supplementary figure S6: Correlations between FMA assessed capillary parameters and blood urea nitrogen. FMA assessed total capillary cross sectional area (total perfused area, µm2/ high power field-hpf), average individual capillary cross sectional area (µm2), capillary number (number / hpf), average total capillary perimeter (µm/ hpf) and average individual capillary perimeter (µm/capillary) show highly significant correlation with day 1 blood urea nitrogen (BUN) and to a lower degree with day 14 and week 8 BUN.

Supplementary Figure S7: Labeling strategies of kidney capillaries A: Staining of CD31 in the kidney of a VE-Cadherin+,tdTomato+ mouse shows that the endothelial cell marker CD31 is expressed on the surface of tdTomato+ cells. B: Comparison of capillary demarcation using genetic tagging of endothelial cells via VE- Cadherin (Cdh5) expression and the fluorescence migroangiography (FMA) indicating that some endothelial cells might be missed by the genetic strategy alone (arrows). Of note the genetic Ve-Cadherin labeling strategy seems to miss also some endothelial cells of the arteriole (arrowheads). C: Comparison of capillary demarcation using immunostaining of the endothelial cell-surface antigen CD31 and FMA indicating that some capillaries might be missed by CD31 staining (arrows).

Supplementary Video 1: Confocal Z-Stack (26.4µm) of renal cortex with fluorescence microangiography (FMA) 8 weeks after sham surgery.

Supplementary Video 2: Confocal Z-Stack (26.4µm) of renal cortex with fluorescence microangiography (FMA) 8 weeks after severe ischemia reperfusion injury.

Fluorescence Microangiography (FMA) detailed protocol:

List of reagents needed: ______reagent / tool needed company / Cat No. price ______low melting temperature agarose Lonza / #50081 139$ 0.02µm FluoSpheres sulfate Invitrogen / #F8845 264$ sodium chloride 0.9% Baxter / # 2F7122 4.50$ heparin Sagent Pharmaceuticals NDC25021-400-01 ≈8$/vial 1x PBS (500ml) Life Technologies / #10010-023 17.62$ 3ml syringes various 5ml syringes various 10ml syringes various 20ml Glass Scintillation Vials Fisher Scientific / #03-337-14 ≈0.5$ each 20 gauge needles various 27 gauge butterfly catheter Exel Corp. / #26709 50x / 11.85$ 3M KCL various Pentobarbital (Nembutal™) Oak Pharmaceuticals / NDC #76478-501-20 Buprenorphine (Buprenex™) Reckitt Benckiser /NDC #12496-0757-1 4% paraformaldehyde various / Electron microscopy sciences / #15713 104$ 30% sucrose various / Fisher Scientific #S5500 500g/100$ OCT Sakura Finetek / # 4583 72$ cryo embedding molds Polysciences / #18986 288x/197$ Superfrost Plus Slides Fisher Scientific / #12-550-15 ≈1$/ slide Prolong Gold Antifade reagent Life Technologies / # P36930 10ml/ 139$ water bath (42°C) various microwave various surgical heating pad various scissors / tweezers various MATLAB sofware MathWorks (check for cheaper campus license) ≈500$ Image J NIH download http://rsbweb.nih.gov/ij/download.html free confocal microscope NikonC1 eclipse / various others ______

FMA Protocol (in our experience the best results are achieved with 2 persons performing this procedure)

1. prepare the following solutions (for 1 mouse):

A) 1ml 0.9 % NaCL + 100 IU heparin

B) 1ml 3M KCl

C) 10ml 1x PBS

D) FMA solution: Prepare the solution in a 20ml glass scintillation vial 4.5ml

dH2O + 0.05g low melting temperature agarose ( Lonza #50080), microwave

until the agarose is dissolved add 500µl 0.02µm FluoSpheres sulfate

(Invitrogen #F8845, yellow-green)

è transfer the solutions into a syringe using a 20g needle (2x3ml

syringe for solution A+B and 1x 10ml syringe for solution C and 1x

5ml syringe for solution D, place the syringes in a water bath at 41°C

until immediately before injection into the mouse (set up the water

bath next to the surgical heating pad where you are planning to

perform the procedure with the mouse).

2. Anesthesize mouse with pentobarbital (60mg / kg bodyweight) via intraperitoneal

injection and additional analgesia with 0.1mg/kg bodyweight of buprenorphine i.p.

3. Place the mouse on a surgical heating pad to remain the bodytemperature at 37°C (to

prevent an early gelation of the injected agarose).

4. Cut abdomen and thorax via a midline incision extending from the symphysis pubis to

the jugulum.

5. Inject solution A (100 IU/ml heparin, 0.9% NaCl, 41°C) using a 27 gauge butterfly

catheter (Exel Corp. #26709) directly into the beating left ventricle of the mouse, keep

the needle in the left ventricle and switch the syringe to solution B (3M KCl, 41°C),

inject solution B slowly (the perfusion works best with 2 persons, person A can make sure that the butterfly catheter remains in the left ventricle and person B injects the

different solutions)

6. Cut the vena cava inferior directly proximal of the bifurcation (be careful not to injure

the aorta and to remain the butterfly catheter needle in the left ventricle during this

procedure)

7. Inject solution C (10ml 1x PBS, 41°C) slowly via the butterfly catheter

8. Inject solution D (FMA solution), monitor the perfusion i.e. the green solution should

exit the circulation trough the vena cava incision.

9. Remove the kidney (and / or other organs of interest) carefully using tweezers and

scissors and place them directly on ice (make a little hole in the ice bucket for each

organ to make sure that it´s completely surroutweended by ice). After 10 min, cut the

kidney carefully into half using a razor blade and transfer it in 4% paraformaldehyde

at 4°C for 2 hours, then 30% sucrose over night.

10. Embed the tissue in OCT (Sakura Finetek) and store at -80°C, you can now make

cryosections mount them on Superfrost Plus slides (we did 7- 40µm) and store them at

-80°C.

11. You can perform a DAPI counterstaining or a immunostaining of your antigens

(optimaly using a Cy5 secondary antibody to prevent bleed trough). For this you

should carefully wash the slides using 1x PBS (3x5min) and then incubate with your

antibodies (follow a standard immunofluorescence protocol) or DAPI (1mg/ml 1:1000

for 5 min). Wash again for 2x5 min (1xPBS) after the DAPI or 3x10min (1xPBS) after

the secondary antibody.

12. Add one drop of the Prolong Gold Antifade reagent directly onto the tissue and add a

coverslip (remove the air bubbles using weak pressure of a pipette tip). Let the

coverslips dry over night in the dark at roomtemperature. 13. Seal the coverslips with nail polish and proceed to confocal microscopy (for later

quantification using the MATLAB script (below) please make sure that you are using

the same laser intensity and gain for all pictures.

MATLAB script for the automated high-throughput analysis of the microvasculature:

1.) This script generates all raw data of capillary area (in µm2) and perimeter (in µm) without any cut off value (cut off values can be choosen within the excel sheet or by analyzing the pictures with script #2: folder_name = uigetdir; %Prompts user to select folder filename = uigetfile; %Prompts user to select file to be analyzed uiimport = (filename); %Imports selected file name

I = imread(filename); %Reads imported file background = imopen(I,strel('disk', 15)); %Standardizes background and threshold figure, surf(double(background(1:8:end,1:8:end))),zlim([0 255]); set(gca,'ydir','reverse');

I2 = I - background; %Removes excess noise imshow(I2); level = graythresh(I2); bw = im2bw(I2, level); bw = bwareaopen(bw,50); %States capillary area cc = bwconncomp(bw,4); cc.NumObjects; labeled = labelmatrix(cc); whos labeled;

RGB_label = label2rgb(labeled, @spring, 'c', 'shuffle'); %colors individual capillaries figure, imshow(RGB_label); capillarydata = regionprops(cc,'all'); %reads all perimeter data of the capillaries capillary_peri = [capillarydata.Perimeter]; capillary_area = [capillarydata.Area];

[min_perim, idx] = min(capillary_peri); capillary = false(size(bw)); capillary(cc.PixelIdxList{idx}) = true;

%Converts perimeter data to micrometers

PDataInMicrons =capillary_peri*0.30120'; %Insert conversion factor here in microns per pixel

%Converts Area data to Micrometers

ADataInMicrons =capillary_area*0.0907'; %Insert conversion factor here in microns-squared per pixel-squared

nbins = 50; figure, hist(ADataInMicrons, nbins) %Generates capillary Area histogram title('Histogram of Capillary Area Data') figure, hist(PDataInMicrons, nbins) %Generates capillary Perimeter histogram title('Histogram of Capillary Perimeter Data')

SA = ADataInMicrons';

SP = PDataInMicrons'; csvwrite('AreaQuant1.csv', SA) %Writes data to area excel sheet csvwrite('PerimQuant1.csv', SP) %Writes data to perimeter excel sheet

B) This script generates data of capillary area (in µm2) and perimeter (in µm) with a cut off value of individual capillary area (>4.9µm2 and < 100µm2) to automatically exclude arterioles, arteries, veins, venules and glomerular capillary convolutes:

folder_name = uigetdir; %Prompts user to select folder filename = uigetfile; %Prompts user to select file to be analyzed uiimport = (filename); %Imports selected file name

RGB_label = label2rgb(labeled, @spring, 'c', 'shuffle');

%colors individual capillaries with pretty colors figure, imshow(RGB_label); capillarydata = regionprops(cc,'all'); %reads all perimeter data of the capillaries capillary_peri = [capillarydata.Perimeter]; capillary_area = [capillarydata.Area];

[min_perim, idx] = min(capillary_peri); capillary = false(size(bw)); capillary(cc.PixelIdxList{idx}) = true;

%Converts perimeter data to micrometers

PDataInMicrons =capillary_peri*0.30120';

%Insert conversion factor here in microns per pixel %Converts Area data to Micrometers

ADataInMicrons =capillary_area*0.0907';

%Insert conversion factor here in microns-squared per pixel-squared

%counter variable n = 1;

%number of entries in the data set you are looking for arraysz = length(ADataInMicrons);

%while loop: look at each point, it it is greater than 10, put in into new vector while n < (arraysz+1)

if ADataInMicrons(n) > 4.9

if ADataInMicrons(n) < 100

Data1Sorted(n) = ADataInMicrons(n);

end

n = n + 1; end

%reset your counter n = 1; nbins = 50; figure, hist(Data1Sorted, nbins) %Generates capillary Area histogram title ('Histogram of Capillary Area Data') figure, hist(PDataInMicrons, nbins) %Generates capillary Perimeter histogram title ('Histogram of Capillary Perimeter Data')

SA = Data1Sorted';

SP = Data2Sorted'; csvwrite('AreaQuant1.csv', SA) %Writes data to area excel sheet csvwrite('PerimQuant1.csv', SP) %Writes data to perimeter excel sheet

Analysis of the confocal microscopy picture using the MATLAB script (above):

1. The first thing to do is to open each picture in Image J and split the channels, save the green channel (FMA) in grayscale mods a PNG file. 2. Make a central folder (call it MATLAB) to import into MATLAB with the following files: the script called “QuantUnsort”, area.csv file, perimeter.csv file, and all the pictures you are going to analyze (all these files are available as online supplements). [the “QuantUnsort“script will give you data for all measured vessels i.e. including large arteries and veins to get only capillary data use the “QuantFinalR“ script (online supplements) instead of the “QuantUnsort” script, this will exclude all areas smaller than 4.9µm2 and larger than 100µm2. 3. Open MATLAB 4. Import the folder into MATLAB. You might be prompted to set this folder as the MATLAB “path”. Say yes to this. 5. Type “QuantUnsort” into the command window. 6. A window will pop up and ask you which folder you want to open. Click on the MATLAB folder and press enter. The MATLAB folder may also be pre-selected. If this is the case, just press “open”. 7. Another window will pop up and ask you to choose a file. This will be asking you which image you want to analyze. If you can’t see your file in the window, make sure the file type is set to “All Files”. A common problem is that by default MATLAB will only look for matlab files and will not “see” any .png files. Chose your image and press enter. The script will run and analyze it. 8. A series of 4 images will pop up. The first is the original image, the second is the same image but with different colors assigned to different capillaries. These are there to make sure that the right image was analyzed and to track progress. The next two images are histograms of the area data and perimeter data for the analyzed image. By this point, the data has been exported to the respective AreaQuant1.csv or PerimeterQuant1.csv files. 9. Open up Excel and open the AreaQuant1.csv and/or PerimeterQuant1.csv file. All of the measurements should be in one column. The area is in µm2 and the perimeter is in µm. Copy and paste this into another excel file where you can compile your data. After you do that, make sure you close the area.csv or perimter.csv files or else when you analyze the next image, MATLAB won’t be able to export the new data to these files. 10. Return to matlab and type “clc” then press enter into the command window. This resets the program and allows you to analyze another image. Make sure you do this after each picture! 11. Re-do steps 1-9 for the rest of the images.