DOI: 10.5061/dryad.9zw3r22tn
title: In vivo expression of VCAM1 precedes nephron loss following kidney tubular necrosis

description: Nephron loss is a key event during the onset and progression of chronic
 kidney disease, yet the mechanisms determining whether tubules undergo
 successful repair or progress to atrophy remain poorly understood. While
 fibrosis has been proposed to drive progressive organ damage, antifibrotic
 therapies have failed in clinical trials. Here, we reveal that tubular
 VCAM1-expression precedes nephron loss, fibrosis, and long-term kidney
 dysfunction. Using serial intravital microscopy in transgenic mice, we
 track tubulointerstitial remodeling between injured and intact tissue over
 3 weeks. VCAM1 is rapidly induced in a distinct subset of injured tubules,
 preceding atrophy with sustained fibroblast recruitment. However,
 fibroblasts remain confined to injury sites and do not cause secondary
 damage in uninjured tubules. Finally, in human kidney transplant biopsies,
 tubular VCAM1 expression - but not KIM1 - correlates negatively with early
 and 12-month graft function, underscoring its potential as a biomarker of
 adverse outcomes. These findings position VCAM1 as an early indicator of
 tubular fate and nephron loss.
description: <strong>Study Approval</strong>
 All experimental procedures involving animals were approved by
 the local authorities (Animal Experiments Inspectorate, Denmark, permit
 numbers: 2020-15-0201-00443 and 2024-15-0201-01839) and reported according
 to ARRIVE guidelines. Processing of human samples and data was approved by
 the National Committee on Health Research Ethics (M-20100269). Informed
 and written content was obtained from the participants. The trial was
 conducted according to the Helsinki Declaration.
 <strong>Animals</strong> A total
 of 60 (26 male and 34 female) mice with a mean weight of 22.3 ± 0.5 g and
 age of 12.6 ± 0.6 weeks (mean ± SEM) were included in the study (Tables S1
 and 2). Transgenic mice were obtained from Jax Laboratory and bred in the
 animal facilities at the Department of Biomedicine, Aarhus University:
 PDGFRβCre-ERT2 – Salsa6F reporter mice were generated by crossing
 B6.Cg-Tg(PDGFrβ-cre/ERT2)6096Rha/J (Strain #:030201)
 <sup>27</sup> and
 B6(129S4)-Gt(ROSA)26Sortm1.1(CAG-tdTomato/GCaMP6f)Mdcah/J)] (Strain #:
 031968) <sup>28</sup>. After induction with tamoxifen, this
 strain identifies  PDGFRβcells by Salsa6F, a fusion protein of GCaMP6 and
 tdTomato. Additionally, CycB1-GFP reporter mice
 (Tg(Pgk1-Ccnb1/EGFP)1Aklo/J) (Strain #: 023345) <sup>36</sup>
 were used to detect renal cell proliferation through transient
 nuclear/cytosolic expression of  green fluorescent protein (GFP)
 expression stages S-, G2- and M of the cell cycle. Mice
 were randomly allocated to one of three groups; i) in vivo imaging, ii)
 GFR and urine collection and iii) ex vivo imaging, as summarized in Table
 S2. Group i) was further divided into mice undergoing partial IRI surgery,
 sham surgery or laser injury, respectively. Groups ii) and iii) were
 divided into mice undergoing partial IRI surgery and sham surgery.
 Additionally, some mice from group i) were also used for ex vivo
 correlative microscopy upon sacrificing. Our study design precluded
 blinding of the experimental treatment, as the validation of surgical
 outcomes required close microscopic examination during the procedure.
 Serial intravital microscopy of partial IRI animals was challenged by
 strong tissue remodeling that sometimes caused missing data points from
 late acquisition time points. To compensate for missing data points, we
 included higher n-numbers in the partial IRI group. For statistical
 analysis all available serial imaging data was included. No data was
 excluded.
 <strong>Surgery</strong> Prior to
 surgery mice were accommodated in groups of 2 to 5 within ventilated cages
 (Technoplast model GM500 or GM9000) at 21 ± 2°C and 55 ± 10% relative
 humidity. Littermates were weaned at 3 weeks of age. Mice were maintained
 on a standardized 12-hour light-dark diurnal cycle, with unrestricted
 access to standard diet and water. The bedding material consisted of Aspen
 wood chips measuring 2-3 mm in size (Abedd, bedding midi), complemented by
 compressed cotton tubes for nesting purposes. Cages were enriched with
 Mouse Igloo with spinning wheels, wooden chew sticks, cardboard tubes, and
 Shepherd Shacks. Upon surgery, Mouse Igloo and cardboard tubes were
 replaced by cardbox houses to prevent the abdominal image window from
 getting stuck. Cages were routinely cleaned once a week. To investigate
 whether (myo)fibroblasts engage in progressive and self-perpetuating
 tubulointerstitial fibrosis, two individual surgery procedures were
 performed. First, an unilateral partial ischemia-reperfusion injury (IRI)
 was done by occluding one branch of the left renal artery for 21 min, as
 previously described <sup>7</sup>. Second, a winged abdominal
 image window (wAIW) was placed above the postischemic and non-ischemic
 parts of the partial IRI kidney and implanted into the left flank of the
 mouse as described previously <sup>29, 63</sup>. Thus, it was
 possible to image the border between the ischemic and non-ischemic
 regions. During surgery mice received analgesia through
 intraperitoneal administration of buprenorphine (0.1 mg/ kg BW, Temgesic),
 and anesthetized with isoflurane (3.5% for induction, 1.5–1.75% for
 maintenance, 1.2-1.8 L/min flow rate, 50% oxygen in medical air). Surgery
 was conducted on a heating plate set to 37 ± 0.5°C, while eye ointment
 prevented cornea dehydration. The mouse’s left flank was shaved, and the
 skin was disinfected using chlorhexidine (0.5 % in 70% ethanol). Hereafter
 an incision into the left flank exposed the kidney. The kidney was gently
 repositioned, and the right artery branch was occluded with a clamp for 21
 minutes. During occlusion, kidney temperature, and hydration were
 maintained with a nonwoven swab and occasional flushing with 37°C saline
 <sup>29</sup>. Upon reperfusion, a purse-string suture
 connected the muscle layer and the skin around the incision, and the
 kidney glued to the wAIW using 50 μl cyanoacrylate glue. Lastly, the skin
 was secured around the groove of the wAIW by tightening of the
 purse-string suture <sup>68</sup>. 10 μl/g BW sterile saline
 was injected i.p. for fluid supplementation. Following the image session
 at day 0, meloxicam (1 mg/kg BW meloxicam, Metacam) was administrated s.c.
 as postoperative analgesia as well as at days 1 and 2 post surgery.
 Additionally, mice had restricted access to normal drinking water but had
 to drink water containing buprenorphine (1 ml buprenorphine 0.3 mg/ml to
 35 ml water) until 3 days post-surgery.
 <strong>Image acquisition protocol</strong>
 In vivo imaging was conducted using an upright Olympus FVMPE-RS
 2-photon microscope (Olympus, Japan) operated with Fluoview FV31S software
 (Olympus, Japan), and an upright Ultima Investigator Plus multiphoton
 setup (Bruker Corporation, Billerica, MA, USA) with PraireView IV software
 as described previously <sup>7</sup>. The
 Olympus microscope was equipped with a MaiTaiHP DS-OL excitation laser
 (Spectra Physics, United States), a 25X XLPLN25xWMP2 objective, water
 immersion, (Olympus, Japan, NA 1.05; WD 2.00mm), with an IR cut filter of
 690 nm, and the following detection cubes: Ch1: λet = 705/45 nm
 (multialkali PMT), Ch2: λet = 610/35 nm (multialkali PMT), Ch3: λet =
 540/40 (GaAsP), Ch4: λet = 480/40 (GaAsP). The Bruker
 microscope was equipped with a 20X Olympus XLUMPLFLN Objective, water
 immersion (Olympus, Japan, NA 1.00; WD 2.00mm), a 720 sp filter, and the
 following detection cubes: Ch1: λet=525/50 nm (GaAsP), Ch2: λet= 525/50 nm
 (GaAsP), Ch3: λet= 460/50 nm (GaAsP). Prior to imaging,
 1 μl/g body weight of a 2.5 mg/ml conjugated Albumin-Alexa Fluor 594 dye
 solution (Alexa594-albumin, Invitrogen) and 10 μl of propidium iodide (PI,
 0.25 mg/ml, Thermo Fisher) was administered via retro-orbital injection.
 For imaging, the wAIW implant was slotted into a custom 3D-printed frame
 ensuring image stabilization on an upright microscope setup
 <sup>64</sup>. During imaging, anesthesia and temperature were
 maintained with a low-flow (30–50 ml/min, 1.0–1.5%) isoflurane vaporizer
 (SomnoSuite, Kent Scientific), which was equipped with a heating blanket
 placed beneath the animal. We identified ≈ 4 FOV, which
 were imaged using dual-track excitation using 750 nm and 940 nm,
 respectively (512 x 512, dwell time: 4 μs, line averaging x 2, Galvano
 laser). Z-series, with a step size of 1-2 μm and depth of ≈ 60 μm, were
 acquired from each FOV, starting from the capsule. Upon the end of the
 image session, mice recovered from anesthesia and placed back in their
 cage. This process was repeated for each imaging day as detailed in Table
 S2. To induce selective laser-injury in a few nephrons
 within the otherwise healthy PDGFRβ-CreERT2-tdTomato kidneys (n = 4), we
 used the 2-photon laser as a micromanipulator and focused high laser power
 on a kidney region of approximately 60 um<sup>2</sup>. Tissue
 remodeling at the injury site was then monitored via serial in vivo
 imaging for up to 4 weeks as detailed in Tables S1 and 2. At some image
 sessions, a tile scan approach was utilized if the damaged area exceeded
 the normal FOV. These tiles were later stitched using the cell sense
 software (Olympus, Japan). After the last imaging
 session, mice were anesthetized with isoflurane and perfusion-fixated with
 4% PFA. For correlative microscopy the cortical renal area attached to the
 wAIW was separated as a ≈ 2 mm thick tissue section, post-fixated with 4%
 PFA for 1 hour, and then washed in PBS (3 x 5 min). Whereafter they were
 stained ex vivo. <strong>Image
 analysis</strong> During the analysis, all
 tubular segments from each FOV were sequentially numbered, re-identified
 on consecutive imaging time points, and qualitatively assessed for
 categorization into one of the following classifications:
 'undamaged,' 'damaged,' 'recovered,' or
 'atrophic'. Tubular segments were classified as
 'damaged' if they exhibited any of the following criteria:
 positive staining for propidium iodide, dilated or collapsed tubule lumen,
 luminal granular cast accumulation, flattened tubule epithelium, visually
 decreased NADH autofluorescence and/or, in the case of PT-S1, visually
 reduced albumin endocytosis. 'Undamaged' tubule segments were
 defined by the absence of all characteristics mentioned above. Tubule
 segments were classified as 'atrophic' if they adopted a
 strongly collapsed, non-reversible state with decreased NADH
 autofluorescence. ‘Recovered’ tubules were identified with any of the
 damage characteristics outlined above and later adopted an ‘undamaged’
 state. Additionally, atrophic’ tubule segments were further sub-classified
 as 'degraded' if they completely disappeared. The classification
 of individual tubule segments across consecutive imaging time points was
 performed and confirmed by two experimenters. Denoising
 of images was conducted using the BM3D algorithms ^70 ^as previously
 described <sup>69</sup>. In short, a denoising pipeline was
 created utilizing both FIJI <sup>75</sup> and MATLAB. FIJI
 facilitated batch data input/output, handled multichannel images, and
 merged denoised outputs. MATLAB executed BM3D algorithms, processing one
 channel at a time by reading temporary files written on disk by FIJI and
 producing the denoised data in separate temporary files.
 Registration of serial imaging data was performed using a
 combination of a manual landmark-based volumetric registration within the
 FIJI plugin BigWarp using rigid rotation transforms
 <sup>76</sup>, and intensity-based medical image registration
 using Elastix <sup>38</sup>. To quantify
 PDGFRβ-cell accumulation, two different approaches were utilized. In the
 first method, PDGFRβ-cells were segmented using Ilastik
 <sup>77</sup> (v5.1.0). Here, tdTomato-labeled PDGFRβ-cells
 were segmented from the registered 940 tracks using pixel classification.
 To train Ilastik, labels were assigned to Z-stack images to identify
 structures/cells as either tdTomato-positive or negative cells. Following
 exportation of the 2D Ilastik predictions, a threshold was applied using
 the Otsu method <sup>78</sup>, whereafter the percentage of
 the of tdTomato cell area was assessed relative to the total FOV area
 (Figures S2B, C). This approach provided an unbiased quantification of
 PDGFRβ-cell abundance on a per FOV basis. In the second
 method, the perimeter of each individual tubule was measured for each day
 equivalating 6903individual measurements. Afterward, it was measured how
 much of the tubule in question was in touch with surrounding PDGFRβ-cells
 and PDGFRβ-cell enclosure was expressed in percent of the total perimeter
 of the tubule (Figure 2E). This approach provided insights into
 PDGFRβ-cell dynamics with individual tubules. Second
 harmonic generation (SHG) was quantified following image segmentation
 using Ilastik. The software was trained to recognize SHG signals from 940
 tracks. Following the export of the 2D Ilastik predictions, a threshold
 was applied using the Otsu method, after which the percentage of the SHG
 area was evaluated relative to the total FOV area. All SHG analysis was
 done on decapsulated, ex vivo samples to avoid bias when separating
 interstitial SHG signal from SHG signal deriving from the kidney
 capsule.  NADH signal quantification was only performed
 on images acquired on the Olympus system to maintain consistent detection
 optics for the quantification. In 750 nm excitation track data, a ROI was
 manually drawn around the epithelium of each tubule, and the average
 intensity of channel 4 was measured.
description: # In vivo expression of VCAM1 precedes nephron loss following kidney
 tubular necrosis Dataset DOI:
 [10.5061/dryad.9zw3r22tn](10.5061/dryad.9zw3r22tn) ## Description of the
 data and file structure `File: Dataset_001_Partial_IRI.zip` **# Data
 Description** Using serial intravital microscopy of transgenic mice, we
 tracked the interactions of renal fibroblasts and pericytes with injured
 and uninjured tubules over 3 weeks and tested whether fibrosis preceded
 secondary tubular damage in uninjured tissue. To achieve this, we used a
 model of partial ischemia-reperfusion injury (partial IRI), which resulted
 in rapid and abundant tubule atrophy and fibrosis in approximately half of
 the kidney while demonstrating uninjured renal tissue in the other half.
 **# Experimental Setup and Protocol Information:** Transgenic
 PDGFRβCre-ERT2–Salsa6F reporter mice were generated by crossing
 B6.Cg-Tg(PDGFrβ-cre/ERT2)6096Rha/J (The Jackson Laboratory, Strain
 #:030201) and B6(129S4)-Gt(ROSA)26Sortm1.1(CAG-tdTomato/GCaMP6f)Mdcah/J)]
 (The Jackson Laboratory , Strain #: 031968). A partial IRI was performed
 in the mice, which was followed by an abdominal imaging window (AIW)
 implementation, and serial intravital imaging under isoflurane anaesthesia
 for up to 21 days post-IRI. **# Experimental Equipment** In vivo 2-photon
 imaging was performed using two setups: 1\) Upright Olympus FVMPE-RS
 2-photon microscope (Olympus, Japan) equipped with MaiTai HP DS-OL
 excitation laser (Spectra Physics, United States), XLPLN25xWMP2 objective
 (Olympus, Japan, NA 1.05; WD 2.00 mm). Excitation wavelengths used were
 750 nm and 940 nm. 2\) Chamelion Ultra II Coherent Laser and an Ultima
 Investigator Plus multiphoton setup (Bruker Corporation, Billerica, MA,
 USA) with PraireView IV software in upright objective configuration (20X
 Olympus XLUMPLFLN Objective, water immersion, 1.00 NA, 2.0 mm WD).
 Excitation wavelengths used were 750 nm and 940 nm. Emission signal was
 detected after a 720sp filter on 3 GaAsP (Hamamatsu, H7422-40)
 photomultipliers (Ch1: λet = 525/50 nm, Ch2: λet = 525/50 nm, Ch3: λe t=
 460/50 nm). Subject IDs: 008, 009, 010. `File:
 Dataset_002_Laser_Injury.zip` **# Data Description** To explore the
 dynamics of fibroblasts in a locally restricted injury model, we used
 serial *in vivo* imaging via two-photon microscopy and genetic
 identification of PDGFRβ cells in kidneys undergoing laser-induced tubular
 injury. **# Experimental Setup and Protocol Information:** Transgenic
 PDGFRβCre-ERT2–Salsa6F reporter mice were generated by crossing
 B6.Cg-Tg(PDGFrβ-cre/ERT2)6096Rha/J (The Jackson Laboratory, Strain
 #:030201) and B6(129S4)-Gt(ROSA)26Sortm1.1(CAG-tdTomato/GCaMP6f)Mdcah/J)]
 (The Jackson Laboratory , Strain #: 031968). An abdominal imaging window
 (AIW) was implemented in the mice, which was followed by a laser-induced
 tubular injury and serial intravital imaging performed under isoflurane
 anesthesia for up to 28 days. **# Experimental Equipment** *In vivo*
 2-photon imaging was performed using an upright Olympus FVMPE-RS 2-photon
 microscope (Olympus, Japan) equipped with MaiTai HP DS-OL excitation laser
 (Spectra Physics, United States), XLPLN25xWMP2 objective (Olympus, Japan,
 NA 1.05; WD 2.00 mm), and the following detection cubes: Ch1: λet = Empty,
 Ch2: λet = 610 ± 35 nm (multialkali PMT), Ch3: λet = 540 ± 20 nm (GaAsP),
 Ch4: λet = 480 ± 20 nm (GaAsP). `File: Dataset_003_Control.zip` Serial
 intravital imaging via 2-photon microscopy and genetic identification of
 PDGFRβ cells in kidneys undergoing sham ischemia-reperfusion injury (IRI)
 surgery. **# Experimental Setup and Protocol Information:** Transgenic
 PDGFRβCre-ERT2–Salsa6F reporter mice were generated by crossing
 B6.Cg-Tg(PDGFrβ-cre/ERT2)6096Rha/J (The Jackson Laboratory, Strain
 #:030201) and B6(129S4)-Gt(ROSA)26Sortm1.1(CAG-tdTomato/GCaMP6f)Mdcah/J)]
 (The Jackson Laboratory, Strain #: 031968). A sham IRI surgery was
 performed in the mice, which was followed by an abdominal imaging window
 (AIW) implementation, and serial intravital imaging under isoflurane
 anaesthesia for up to 21 days post surgery. **# Experimental Equipment**
 In vivo 2-photon imaging was performed using two setups: 1\) Upright
 Olympus FVMPE-RS 2-photon microscope (Olympus, Japan) equipped with MaiTai
 HP DS-OL excitation laser (Spectra Physics, United States), XLPLN25xWMP2
 objective (Olympus, Japan, NA 1.05; WD 2.00 mm). Excitation wavelengths
 used were 750 nm and 940 nm. 2\) Chamelion Ultra II Coherent Laser and an
 Ultima Investigator Plus multiphoton setup (Bruker Corporation, Billerica,
 MA, USA) with PraireView IV software in upright objective configuration
 (20X Olympus XLUMPLFLN Objective, water immersion, 1.00 NA, 2.0 mm WD).
 Excitation wavelengths used were 750 nm and 940 nm. Emission signal was
 detected after a 720sp filter on 3 GaAsP (Hamamatsu, H7422-40)
 photomultipliers (Ch1: λet = 525/50 nm, Ch2: λet = 525/50 nm, Ch3: λe t=
 460/50 nm). Subject IDs: 008, 009, 010. `File: Dataset_004_Vcam1KIM1.zip`
 Ex vivo imaging of human biopsies from kidney transplantations stained for
 Kim1 and Vcam1. **# Experimental Setup and Protocol Information:**
 Biopsies were obtained from patients undergoing kidney transplantation.
 The biopsy was taken from the transplanted kidney 6 days
 post-transplantation. Human biopsies were stained for Kim1 and Vcam1. The
 primary antibody, Rabbit anti-VCAM1 (Abcam AB0134047, 1:500 dilution) or
 Goat anti KIM1 (Biotechne AF181, 1:500), was incubated overnight at 4°C.
 Following primary antibody incubation, sections were washed three times
 for 10 minutes each in a buffer containing 1% BSA, 0.2% gelatin, and 0.05%
 saponin in PBS (pH 7.4). The secondary antibody, Goat anti-Rabbit Cy5
 (Invitrogen A10523, 1:500 dilution) or Donkey anti Goat Alexa 647
 (Invitrogen A2144, 1:1000), was applied for 2 hours at room temperature
 (20°C). Both primary and secondary antibodies were diluted in a solution
 of 0.1% BSA, 0.3% Triton X-100, in PBS (pH 7.4). For nuclear staining,
 Hoechst 33342 (1 µg/mL, Invitrogen H3570) was incubated concurrently with
 the secondary antibody. Sections were mounted with Glysergel mounting
 medium (Dako C0563) containing 0.025 g/mL 1,4-diazabicyclo[2.2.2]octane
 (2.5% w/v, Merck 803456). **# Experimental Equipment** biopsies were
 imaged on an Olympus VS120 upright widefield fluorescence microscope
 (Olympus, Japan) equipped with a 20X UPLSAPO objective (air immersion) and
 a high-sensitivity monochrome camera with 82% quantum efficiency
 (Hamamatsu ORCA-FLASH4.0V2). Images were captured using emission
 wavelengths of 518 nm (exposure time = 500 ms), 455 nm (exposure time =
 100 ms), and 565 nm (exposure time = 800 ms). `File:
 Dataset_005_Ex_vivo.zip` Ex vivo imaging of stained sections following
 partial IRI. **# Experimental Setup and Protocol Information:** On days 4,
 7, and 21 post partial IRI of the kidney, mice were perfusion fixed, and
 the kidney was cut out. Ex vivo samples used for paraffin embedding were
 post-fixated in 4% paraformaldehyde (PFA), whereafter they were
 transferred to 70% ethanol and incubated overnight. The ethanol
 concentration was sequentially increased to 96% for 2 hours, then to 99%
 for an additional 2 hours, followed by overnight incubation in 99% ethanol
 with xylene. Samples were subsequently embedded in paraffin at 60°C and
 stored until staining. Kidney tissue was cut coronally. Upon staining,
 sections were dehydrated until water, using Xylene and a decreasing amount
 of ethanol. Antigen retrieval was performed with HIER pH9. Samples were
 blocked in 50mM NH4Cl for 30 minutes, followed by 1% BSA, 0,2% gelatine,
 0,05% Saponin in PBS pH 7,4. 3x10 minutes. Primary antibodies, Rabbit anti
 VCAM-1 (Abcam AB0134047, 1:500) or Rabbit anti VCAM-1 conjugated to Alexa
 647 (Abcam AB194319-1001, 1:200), Rabbit anti-PDGFRβ (Abcam ab32570,
 1:200), Rabbit anti-CD68 (Abcam 125212, 1:900), Mouse anti αSMA (Dako
 M085, 1:1000), Rabbit anti-PCK1(Abcam 7035, 1:200) were diluted in 0,1%
 BSA, 0,3% Tritin x-100 in PSB pH7,4 and incubated over night at 4°C.
 Samples are washed 3 x 10 min in 0,1% BSA, 0,2% gelatine, 0,05% Saponin in
 PBS pH 7,4, before incubation 1 h with Donkey anti Rabbit Alexa 488
 (Invotrogen A21206, 1:1000), Donkey anti Rabbit Alexa 680 (Invitrogen
 A10043, 1:1000), and  Goat anti Mouse Alexa 488 (Invitrogen A32723,
 1:1000) diluted in 0,1% BSA, 0,3% Tritin x-100 in PSB pH7,4. After
 staining, samples are washed for 100 min. in PBS and mounted with
 Glycergel (Dako C0563) containing 2,5 % w/v 1,4-Diazabicyclo[2.2.2]octane
 (Merck 803456). Samples were imaged on a Zeiss LSM900 Inverted confocal
 laser scanning microscope with Airyscan 2 detection array. For a detailed
 overview, see Table S4. Picrosirius Red staining was performed as
 described before: Upon deparaffinization, sections were consecutively
 incubated in solutions of Celestinblue, Harris Hæmatoxilin, and Sirius
 Red. Collagen was visualized using confocal microscopy (LSM900 Inverted
 confocal laser scanning microscope with Airyscan 2 detection array) using
 an excitation of 561 nm and detection of Sirius Red signal at 635-685 nm.
 **# Experimental Equipment** Samples were imaged on a Zeiss LSM 900 Laser
 Scanning Confocal microscope `File:
 Dataset_006_Correlative_imaging_of_Vcam1.zip` Correlative imaging of
 Partial IRI kidney in vivo and ex vivo stained for Vcam1. **# Experimental
 Setup and Protocol Information:** A partial IRI was performed in the mice,
 which was followed by an abdominal imaging window (AIW) implementation,
 and serial intravital imaging under isoflurane anesthesia for 4 days
 post-IRI. Whereafter, mice were perfusion fixed. Kidney tissue stained
 against VCAM1 was blocked in 1% BSA/2% SEA BLOCK/0,1 % Triton X-100/PBS
 for 1 h and then incubated in Rabbit anti-VCAM-1 (Abcam AB0134047, 1:200)
 for 72 hours at 4 °C. After washing in PBS (3 x 5 min), Donkey anti-Rabbit
 Alexa 405 (Jackson ImmunoResearch 711-175-152, 1:500) secondary antibody
 was incubated overnight at 4°C. For PDGFRβ, the kidney tissue was blocked
 with 1% BSA + 2 % SEA in PBS for 1 hour and incubated in Rabbit
 anti-PDGFrβ (Abcam ab32570, 1:200) overnight at 4°C. After washing in PBS
 (3 x 5 min), the sample was incubated in Donkey anti-Rabbit Alexa 594
 (Invitrogen A21207, 1:500). After incubation in secondary antibody,
 samples were washed in PBS and embedded in PBS between two coverslips
 separated by a 2 mm thick spacer (SunJim Lab). **# Experimental
 Equipment** Upright Olympus FVMPE-RS 2-photon microscope (Olympus, Japan)
 equipped with MaiTai HP DS-OL excitation laser (Spectra Physics, United
 States), XLPLN25xWMP2 objective (Olympus, Japan, NA 1.05; WD 2.00 mm).
 Excitation wavelengths used for in vivo were 750 nm and 940 nm, and 800 nm
 for ex vivo. ### Files and variables #### **File:
 Dataset_001_Partial_IRI.zip** Description: Longitudinal imaging of the
 mouse kidney over 3 weeks via in vivo 2-photon microscopy after partial
 ischemia-reperfusion injury (partial IR). **# What each imaging file
 represents** Volumetric image series of renal cortex (mice), approximately
 60 µm depth, step size 1-2 µm. The starting imaging location was selected
 at the depth of the renal capsule (identified via second harmonic
 generation, displayed in blue, in the 940 excitation files). **# Color
 Scheme** Images were acquired using both 750 nm as excitation wavelength,
 and 940 nm as excitation wavelength. For each Field of view, there is a
 file for the 750 excitation and 940 excitation track. For each excitation
 track, light has been collected in either 3 or 4 different channels. 1\)
 for subject ID: 001, 002, 003, 004, 005, 006, 007, the Olympus system with
 the following detection cubes has been used: Ch1: λet = Empty Ch2: λet =
 610 ± 35 nm (multialkali PMT),  Ch3: λet = 540 ± 20 nm (GaAsP), Ch4: λet =
 480 ± 20 nm (GaAsP). What do we see in each channel: •             
 Channel 1. N/A •              Channel 2. 750: Alexa-594 albumin, Propidium
 Iodide (only present in Day 0). Both were injected retroorbitally under
 isoflurane before the imaging session. PDGFRβ–Salsa6F signal (weak). 940:
 PDGFRβ–Salsa6F signal (strong). •              Channel 3. 750: Renal
 epithelial autofluorescence- endogenous signal. PDGFRβ–Salsa6F signal
 (weak). 940: Renal epithelial autofluorescence- endogenous signal.
 PDGFRβ–Salsa6F signal (strong). •              Channel 4. 750: Renal
 epithelial autofluorescence- endogenous signal. 940: Collagen fibers
 images via second harmonic generation (SHG)- endogenous signal. Channel 1,
 which did not convey relevant physiological signals, was occasionally
 disabled during acquisition to optimize data storage and minimize file
 size. In those cases, only 3 channels are present in the file. 2\) for
 subject ID: 008, 009, 010, the Bruker system with the following detection
 cubes has been used: Ch1: λet = Empty Ch2: λet = 595 ± 50 nm (GaAsP), 
 Ch3: λet = 525 ± 50 nm (GaAsP), Ch4: λet = 460 ± 50 nm (GaAsP). What do we
 see in each channel: •              Channel 1. N/A •              Channel
 2. 750: Alexa-594 albumin, Propidium Iodide (only present in Day 0). Both
 were injected retroorbitally under isoflurane before the imaging session.
 PDGFRβ–Salsa6F signal (weak). 940: PDGFRβ–Salsa6F signal (strong).
 •              Channel 3. 750: Renal epithelial autofluorescence-
 endogenous signal. 940: Renal epithelial autofluorescence- endogenous
 signal. •              Channel 4. 750: Renal epithelial autofluorescence-
 endogenous signal. 940: Collagen fibers images via second harmonic
 generation (SHG)- endogenous signal. Channel 1, which did not convey
 relevant physiological signals, was occasionally disabled during
 acquisition to optimize data storage and minimize file size. In those
 cases only 3 channels are present in the file. **# Nomenclature** File
 nomenclature specifies the required information to allocate any imaging
 file to the relevant protocol details, separated by an underscore sign
 (“_“). •                           ZSeries: scan type, volumetric
 acquisition •                           ID0xx: subject experimental ID
 •              FOVx: unique FOV within each experimental subject. Each FOV
 has been serially imaged over different days from partial IRI
 •                           Dxx: days from partial IRI disease model
 •                           WLxx: the wavelength used to excite. Either
 750 nm or 940 nm. •                           Each file name ends with the
 file acquisition date on the format DDMMYYY  **# Subject-specific
 deviations** On the 3-week follow-up protocols, imaging days 14 to 21 from
 partial IRI required imaging deeper to locate the tubular structure, which
 was imaged serially over the initial week from partial IRI. FOVs from
 those days may include volumetric image sequences larger than 60 images.
 Likewise, the laser power used to excite has also been increased. ####
 **File: Dataset_002_Laser_Injury.zip** Description: Serial *in vivo*
 imaging via two-photon microscopy and genetic identification of PDGFRβ
 cells in kidneys undergoing laser-induced tubular injury. **# What each
 imaging file represents** Volumetric image series of renal cortex (mice),
 approximately 60 µm depth, step size 1-2 µm. The starting imaging location
 was selected at the depth of the renal capsule (identified via second
 harmonic generation, displayed in blue, in the 940 excitation files). **#
 Color Scheme** Images were acquired using both 750 nm as excitation
 wavelength, and 940 nm as excitation wavelength. For each Field of view,
 there is a file for the 750 excitation and 940 excitation track. For each
 excitation track, light has been collected in either 3 or 4 different
 channels. Ch1: λet = Empty Ch2: λet = 610 ± 35 nm (multialkali PMT),  Ch3:
 λet = 540 ± 20 nm (GaAsP), Ch4: λet = 480 ± 20 nm (GaAsP). What do we see
 in each channel: •              Channel 1. N/A •              Channel 2.
 750: Alexa-594 albumin, Propidium Iodide. Both were injected
 retroorbitally under isoflurane before the imaging session. PDGFRβ–Salsa6F
 signal (weak). 940: PDGFRβ–Salsa6F signal (strong). •              Channel
 3. 750: Renal epithelial autofluorescence- endogenous signal.
 PDGFRβ–Salsa6F signal (weak). 940: Renal epithelial autofluorescence-
 endogenous signal. PDGFRβ–Salsa6F signal (strong). •              Channel
 4. 750: Renal epithelial autofluorescence- endogenous signal. 940:
 Collagen fibers images via second harmonic generation (SHG)- endogenous
 signal. Channel 1, which did not convey relevant physiological signals,
 was occasionally disabled during acquisition to optimize data storage and
 minimize file size. In those cases, only 3 channels are present in the
 file. **# Nomenclature** File nomenclature specifies the required
 information to allocate any imaging file to the relevant protocol details,
 separated by an underscore sign (“_“). •                          
 ZSeries: scan type, volumetric acquisition •                          
 ID0xx: subject experimental ID •              FOVx: unique FOV within each
 experimental subject. Each FOV has been serially imaged over different
 days from partial IRI •                           Dxx: days from partial
 IRI disease model •                           WLxx: the wavelength used to
 excite. Either 750 nm or 940 nm. •                           Each file
 name ends with the file acquisition date in format DDMMYYY **File:
 Dataset_003_Control.zip** Description: Serial intravital imaging via
 2-photon microscopy and genetic identification of PDGFRβ cells in kidneys
 undergoing sham ischemia-reperfusion injury (IRI) surgery. **# What each
 imaging file represents** Volumetric image series of renal cortex (mice),
 approximately 60 µm depth, step size 1-2 µm. The starting imaging location
 was selected at the depth of the renal capsule (identified via second
 harmonic generation, displayed in blue, in the 940 excitation files). **#
 Color Scheme** Images were acquired using both 750 nm as excitation
 wavelength, and 940 nm as excitation wavelength. For each Field of view,
 there is a file for the 750 excitation and 940 excitation track. For each
 excitation track, light has been collected in either 3 or 4 different
 channels. For all subject IDs, the Olympus system with the following
 detection cubes has been used: Ch1: λet = Empty Ch2: λet = 610 ± 35 nm
 (multialkali PMT),  Ch3: λet = 540 ± 20 nm (GaAsP), Ch4: λet = 480 ± 20 nm
 (GaAsP). What do we see in each channel: •              Channel 1. N/A
 •              Channel 2. 750: Alexa-594 albumin, Propidium Iodide (only
 present in Day 0). Both were injected retroorbitally under isoflurane
 before the imaging session. PDGFRβ–Salsa6F signal (weak). 940:
 PDGFRβ–Salsa6F signal (strong). •              Channel 3. 750: Renal
 epithelial autofluorescence- endogenous signal. PDGFRβ–Salsa6F signal
 (weak). 940: Renal epithelial autofluorescence- endogenous signal.
 PDGFRβ–Salsa6F signal (strong). •              Channel 4. 750: Renal
 epithelial autofluorescence- endogenous signal. 940: Collagen fibers
 images via second harmonic generation (SHG)- endogenous signal. Channel 1,
 which did not convey relevant physiological signals, was occasionally
 disabled during acquisition to optimize data storage and minimize file
 size. In those cases, only 3 channels are present in the file. **#
 Nomenclature** File nomenclature specifies the required information to
 allocate any imaging file to the relevant protocol details, separated by
 an underscore sign (“_“). •                           ZSeries: scan type,
 volumetric acquisition •                           ID0xx: subject
 experimental ID •              FOVx: unique FOV within each experimental
 subject. Each FOV has been serially imaged over different days from
 partial IRI •                           Dxx: days from partial IRI disease
 model •                           WLxx: the wavelength used to excite.
 Either 750 nm or 940 nm. •                           Each file name ends
 with the file acquisition date in the format DDMMYYY  **# Subject-specific
 deviations** On the 3-week follow-up protocols, imaging days 14 to 21 from
 partial IRI required imaging deeper to locate the tubular structure, which
 was imaged serially over the initial week from partial IRI. FOVs from
 those days may include volumetric image sequences larger than 60 images.
 Likewise, the laser power used to excite has also been increased. **File:
 Dataset_004_Vcam1Kim1.zip** Description: Ex vivo imaging of human biopsies
 from kidney transplantations stained for Kim1 and Vcam1. **# What each
 imaging file represents** Single image of a human kidney biopsy stained
 for either Vcam1 or Kim1. **# Color Scheme** Biopsies were imaged with an
 emission wavelength of 518 nm, 455 nm, and 565 nm, corresponding to
 channel 1, 2, and 3 in the images of Vcam1 and channel 2, 1, and 3 for
 Kim1. **Vcam1** Ch1: λet = Emission wavelength: 518 nm Ch2: λet = Emission
 wavelength: 455 nm Ch3: λet = Emission wavelength: 565 nm What do we see
 in each channel: •              Channel 1. Autofluorescence from kidney
 tubules (green) •              Channel 2. Hoechst staining (blue)
 •              Channel 3. Vcam1 staining (red) **Kim1** Ch1: λet =
 Emission wavelength: 455 nm Ch2: λet = Emission wavelength: 518 nm Ch3:
 λet = Emission wavelength: 565 nm What do we see in each channel:
 •              Channel 1. Hoechst staining (blue) •              Channel
 2. autofluorescence from kidney tubules (green) •              Channel 3.
 Kim1 staining (red) **# Nomenclature** File nomenclature specifies the
 required information to allocate any imaging file to the relevant protocol
 details, separated by an underscore sign (“_“). •              Vcam1/Kim1:
 staining of either Vcam1 or Kim1 •              IDxxx: randomized ID
 number **File: Dataset_005_Ex vivo.zip** Description: Ex vivo images of
 stained kidney sections 4 days, 7 days, and 21 days after partial IRI.
 Sections were stained for Vcam1, CD68, PCK1, αSMA, PDGFRβ, and Picrosirius
 Red. Images have been acquired in the IRI area or in the non-IRI area. Day
 4 data are from 4 different mice (3087, 3088, 3089, 3090) Day 7 data are
 from 3 different mice (2929, 2935, 2937) Day 21 data are from 3 different
 mice (2893, 2895, 2896) Samples were stained for αSMA together with
 PDGFRβ. Samples were stained for CD68 together with Vcam1. Samples were
 stained for PKC1 together with Vcam1. **# What each imaging file
 represents** Single images of renal cortex (mice) stained for Vcam1, CD68,
 PKC1, αSMA, PDGFRβ, and SiriusRed. Each image has three different channels
 as specified: **# Color Scheme** αSMA/PDGFRβ: Ch1: λet = PDGrb staining
 (red) Ch2: λet = aSMA staining (green) Ch3: λet = Tubular autofluorescence
 (blue) CD68/Vcam1: Ch1: λet = Vcam1 staining (red) Ch2: λet = CD68
 staining (green) Ch3: λet = Tubular autofluorescence (blue) PCK1/Vcam1:
 Ch1: λet = Vcam1 staining (red) Ch2: λet = PCK1 staining (green) Ch3: λet
 = Tubular autofluorescence (blue) Picrosirius Red: Ch1: λet = Picrosirius
 Red staining (red) Ch2: λet = Tubular autofluorescence (green) Ch3: λet =
 Tubular autofluorescence (blue) **File: File: Dataset_006\_ Correlative
 imaging of Vcam1.zip** Description: Correlative imaging of Partial IRI
 kidney in vivo and ex vivo stained for Vcam1. Mice were imaged in vivo at
 day 4 after partial IRI, whereafter they were perfusion fixed and the
 kidney taken out, stained for Vcam1, and then the same areas were imaged
 ex vivo. **# What each imaging file represents** Volumetric images of
 renal cortex (mice), approximately 60 µm depth, step size 1-2 µm. The
 starting imaging location was selected at the depth of the renal capsule
 (identified via second harmonic generation, displayed in blue, in the 940
 excitation files). **# Color Scheme** Images were acquired using both 750
 nm as excitation wavelength, and 940 nm as excitation wavelength for in
 vivo and 800 nm for ex vivo. For each Field of view, there is a file for
 the 750, 800, and 940 excitation tracks. For each excitation track, light
 has been collected in either 3 or 4 different channels. 1\) in vivo images
 Ch1: λet = Empty Ch2: λet = 610 ± 35 nm (multialkali PMT),  Ch3: λet = 540
 ± 20 nm (GaAsP), Ch4: λet = 480 ± 20 nm (GaAsP). What do we see in each
 channel: •              Channel 1. N/A •              Channel 2. 750:
 Alexa-594 albumin. PDGFRβ–Salsa6F signal (weak). 940: PDGFRβ–Salsa6F
 signal (strong). •              Channel 3. 750: Renal epithelial
 autofluorescence- endogenous signal. PDGFRβ–Salsa6F signal (weak). 940:
 Renal epithelial autofluorescence- endogenous signal. PDGFRβ–Salsa6F
 signal (strong). •              Channel 4. 750: Renal epithelial
 autofluorescence- endogenous signal. 940: Collagen fibers images via
 second harmonic generation (SHG)- endogenous signal. Channel 1, which did
 not convey relevant physiological signals, was occasionally disabled
 during acquisition to optimize data storage and minimize file size. In
 those cases, only 3 channels are present in the file. 2\) ex vivo images
 Ch1: λet = Empty Ch2: λet = 570 ± 30 nm (multialkali PMT),  Ch3: λet =
 521,5 ± 16,5 nm (GaAsP), Ch4: λet = 452,5 ± 22,5 nm (GaAsP). What do we
 see in each channel: •              Channel 1. N/A •              Channel
 2: PDGFRβ–Salsa6F signal •              Channel 3: Renal epithelial
 autofluorescence- endogenous signal •              Channel 4: Vcam1
 staining Channel 1, which did not convey relevant physiological signals,
 was occasionally disabled during acquisition to optimize data storage and
 minimize file size. In those cases, only 3 channels are present in the
 file. ## Code/software **We recommend opening the images in FIJI** **Image
 Processing** The images have been opened using the free software FIJI Java
 version: 2.14.0/1.54f In case of bending of the field of view over time,
 the serial imaging data were registered to day 0, in order to visualize
 the images in 2D Registration of serial imaging data was performed using a
 combination of a manual landmark-based volumetric registration within the
 FIJI plugin BigWarp using rigid rotation transforms, and intensity-based
 medical image registration using Elastix. For
 Dataset_002_Laser_Injury.zip, a tile scan approach was utilized if the
 damaged area exceeded the normal field of view. These tiles were later
 stitched using the CellSens software (Olympus, Japan). For Dataset_005_Ex
 vivo.zip, the tilescan function has likewise occasionally been used to
 show big overviews. No other processing has been performed on the images
 uploaded in this dataset. ## Access information Other publicly accessible
 locations of the data: * None. The biological interpretation of the data
 is described in detail in the associated manuscript. Data was derived from
 the following sources: * All data presented in this dataset were acquired
 by the authors using the experimental 2-photon microscopy facilities
 provided by the Health Bioimaging Core facility at Aarhus University and
 the Center for Functionally and Integrative Neuroscience (CFIN), Aarhus
 University