Tissue-Resident Macrophages Promote Renal Cystic Disease

BASIC RESEARCH www.jasn.org

Kurt A. Zimmerman,1 Cheng J. Song,1 Zhang Li,1 Jeremie M. Lever ,2,3 David K. Crossman,4 Addison Rains,1 Ernald J. Aloria,1 Nancy M. Gonzalez,1 John R. Bassler,5 Juling Zhou,2,3 Michael R. Crowley,4 Dustin Z. Revell,1 Zhaoqi Yan,1 Dan Shan,2,3 Etty N. Benveniste,1 James F. George,6,7 Michal Mrug,2,3,7 and Bradley K. Yoder1

Divisions of 2Nephrology and 6Cardiothoracic Surgery, Departments of 1Cell, Developmental, and Integrative Biology, 3Medicine, 4Genetics, 5Biostatistics, and 7Surgery, University of Alabama at Birmingham, Birmingham, Alabama

ABSTRACT Background Mutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12–14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified. Methods We analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation. Results Our data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11clo)to R2a (CD11chi) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane- bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease. Conclusions These data uncover an important pathogenic role for resident macrophages during rapid cyst progression.

JASN 30: 1841–1856, 2019. doi: https://doi.org/10.1681/ASN.2018080810

Cystic kidney disease is a commonly inherited ge- Received August 9, 2018. Accepted May 26, 2019. netic disease resulting from mutations in proteins K.A.Z. and C.J.S. contributed equally to this work. that localize to primary cilia (e.g., polycystin 1 and Published online ahead of print. Publication date available at polycystin 2) or are required for cilia formation www.jasn.org. (e.g.,intraﬂagellar transport 88 [IFT88]).1–4 How- Correspondence: Dr. Bradley K. Yoder, Department of Cell, ever, the mechanism connecting cilia dysfunction Developmental, and Integrative Biology, University of Alabama and renal cyst formation is unknown. In mice, the at Birmingham, 1918 University Boulevard, Birmingham, AL 35294. rate of cystogenesis is inﬂuenced by the age at which Email: [email protected] cilia dysfunction occurs and whether the kidney has Copyright © 2019 by the American Society of Nephrology

JASN 30: 1841–1856, 2019 ISSN : 1046-6673/3010-1841 1841 BASIC RESEARCH www.jasn.org been injured. Disruption of cilia function before postnatal day Significance Statement 12–14 (P12–14, critical switch period) leads to rapid cyst formation throughout the kidney within 2–3 weeks.2,3 In con- Disruption of cilia function before postnatal day 12–14 in mice or trast, induction of cilia dysfunction after P14 (adult induced) renal injury in adult mice with cilia dysfunction results in accelerated 5 renal cyst formation. Macrophages have been implicated in pro- results in slow focal renal cyst formation. The slow rate of cyst fi – moting cyst formation; however, it is unclear whether in ltrating formation in adult-induced Ift88-, polycystin 1 (Pkd1-), bone marrow-derived or kidney resident macrophages are or Pkd2-deficient mice is accelerated by ischemia-reperfusion responsible. The authors show that a specific population of juvenile- injury (IRI) with cysts becoming evident by 14 days post in- like resident macrophages are present during periods of accelera- jury.2,5,6 The mechanism driving the rapid rate of cyst formation ted cyst formation. Inhibition of juvenile-like resident macrophage when cilia function is disrupted in juvenile mice and after injury accumulation using a colony-stimulating factor 1 receptor kinase inhibitor reduced the severity of cystic disease in two different an- in adult-induced mice with cilia dysfunction is unknown, but imal models of cystic disease. These results suggest resident renal raises the possibility that the environmental conditions are sim- macrophages contribute to cystic disease. ilar during both periods of rapid cyst formation.

A complex role of macrophages and other innate immune lo factors in the pathogenesis of renal cystic disease was proposed Our data indicate that CD11c macrophages (referred to as by Mrug et al.7 based on genome-wide expression studies of R2b macrophages) that are present in the kidney during juv- kidneys from the Cys1cpk mouse model. This concept is con- enile periods reappear after renal injury in cilia mutant mice. sistent with the increased number of macrophages observed in Further, we show that resident macrophage accumulation kidneys from patients with autosomal dominant polycystic in cilia mutant mice after injury is independent of periph- kidney disease and orthologous mouse models.8–10 Impor- eral blood monocyte recruitment and that inhibition of – tantly, depletion of phagocytic macrophages in cystic mice CSF1 mediated resident macrophage proliferation mark- using liposomal clodronate reduced cyst severity and im- edly reduces renal cysts in two mouse models of cystic fi proved renal function9,10; however, inhibition of macrophage disease. Importantly, recent ndings from our laboratory recruitment from the bone marrow using bindarit—aphar- indicate that a resident macrophage-like population of 24 macological inhibitor of C-C motif chemokine ligand 2 cells exists in multiple species including humans and (CCL2), CCL7, and CCL8—did not reduce renal cyst forma- may be a viable therapeutic target for patients with cystic tion.11,12 In contrast to this study, recent findings indicate that kidney disease. disruption of Pkd1 or liver kinase B1 (Lkb1) leads to increased accumulation of bone marrow–derived infiltrating macrophages and accelerated cystogenessis.13,14 In these studies, METHODS genetic inhibition of macrophage recruitment using Ccl2- deficient mice reduced the severity of cystic disease. Our Mice f/f previous work also indicated that infiltrating macrophages Eight-week-old CAGG-Cre/Esr1/5Amc/J Ift88 male and fe- promote hepatic fibrosis in Ift88Orpk mice.15 The fact that male mice (referred to as cilia mutant mice) on a C57BL/6J cpk/cpk both inhibition of infiltrating macrophage recruitment and background were bred in-house. The C57BL/6J-Cys1 depletion of all macrophages in the kidney (both infiltrating mice were purchased from the Jackson Laboratory (Bar and resident) reduces cystic severity suggests that multiple Harbor, ME). Animals were maintained in Association for macrophage subtypes are likely important in cystic disease. Assessment and Accreditation of Laboratory Animal Care – In mice, macrophages arise from two distinct origins.16,17 In International accredited facilities in accordance with Institu- contrast to bone marrow–derived infiltrating macrophages tional Animal Care and Use Committee (IACUC) regulations (F4/80lo, CD11bhi), tissue-resident macrophages (F4/80hi, at the University of Alabama at Birmingham (UAB) (approval CD11blo) are derived from embryonic progenitors, migrate numbers: 10130 and 21072). into tissues during organogenesis, are independent of the bone marrow and the transcription factor myb,16 and persist Induction of Cilia Loss within the tissue through local self-renewal.18 Resident Eight- to ten-week-old CAGG-CreERT2 Ift88f/f animals were macrophage self-renewal occurs in part through paracrine given an intraperitoneal injection of tamoxifen at 6 mg/40 g signaling involving colony-stimulating factor 1 (CSF1) as body wt once daily for 3 consecutive days. Deletion of Ift88 was evidenced by an accumulation of CSF1 receptor (CSF1R)– confirmed by PCR, quantitative RT-PCR (qRT-PCR), and West- enhanced green fluorescent protein–tagged macrophages in ern blot (Supplemental Figure 1). The following primers were regions surrounding epithelial-specific, membrane-bound used to detect Ift88 by PCR: 59-GCCTCCTGTTTCTTGAC- CSF1.19–22 Furthermore, inhibition of CSF1R activity reduces AACAGTG, GGTCCTAACAAGTAAGCCCAGTGTT-39 and resident macrophage proliferation and number and alters the 59-CTGCACCAGCCATTTCCTCTAAGTCATGTA-39. Cilia renal injury response.22,23 deletion was confirmed by immunofluorescence microscopy In this study, we tested the hypothesis that resident macro- using the cilia marker acetylated a-tubulin (catalog number phages are involved in the pathogenesis of renal cyst formation. 66200–1; Proteintech) (Supplemental Figure 1).

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Renal IRI Immunofluorescence Microscopy Three weeks after tamoxifen induction, mice were subjected to Following fixation and overnight cryopreservation in 30% unilateral IRI. Mice were weighed and injected with 25 mg/kg sucrose, 8-mm thick, Optimal Cutting Temperature (OCT)– body wt tribromoethanol via intraperitoneal injection and embedded kidney tissue was fixed with 4% PFA for 10 min- maintained under 1% isoflurane during the course of the pro- utes, permeabilized with 1% Triton X-100 for 8 minutes, and cedure. A small incision was made on the back and the kidney was incubated in blocking solution (PBS with 1% BSA, 0.3% Triton removed from the abdominal cavity. Unilateral IRIwas performed X-100, 2% (vol/vol) donkey serum, and 0.02% sodium azide) by clamping the left renal pedicle for 30 minutes followed by for 30 minutes at room temperature. Sections were incubated in clamp release to allow for reperfusion. Mice were injected with primary antibody overnight at 4°C according to the manufac- buprenorphine (0.05 mg/kg) for pain relief and allowed to recover turer’s recommendation, washed with PBS, and incubated with on a 37°C heating pad with free access to food and water. After the appropriate secondary antibodies in blocking solution for the desired number of days, animals were anesthetized with 1 hour at room temperature. Primary antibodies included F4/80 tribromoethanol and perfused with 1% PBS. The kidney receiving (catalog number14–4801, clone BM8; eBioscience), FITC- IRI was harvested for further processing. conjugated Lotus tetragonolobus agglutinin (LTA) (catalog number FL-1321; Vector Laboratories), FITC-conjugated Parabiosis Protocol CD206 (catalog number 141704; Biolegend), CD11c (catalog The parabiosis protocol was modified from the procedure pub- number 14–0114–82; eBioscience) and phycoerythrin-conjugated lished by Kamran et al.25 For this procedure, incisions were Ki67 (catalog number 12–5698–82; eBioscience). Secondary anti- made through the skin and muscle layer, starting from the bodies included the following: Alexa Fluor 647–conjugated elbow joint and extending down the flank to the knee joint. anti-hamster (Armenian) (catalog number 405510; Nonabsorbable 3–0 interrupted sutures were placed around Biolegend) and Alexa Fluor 647–conjugated anti-rat (cat- the knee and elbow joints to prevent strain along the suture alog number 712–606–153; Jackson ImmunoResearch). lines, with care taken to avoid obstructing blood flow to the After the addition of secondary antibody, nuclei were stained distal extremities. Analgesia was maintained on all mice ac- by Hoechst nuclear stain (Sigma-Aldrich) and samples mounted cording to IACUC guidelines. using IMMU-MOUNT (Thermo). All fluorescence images were captured on a PerkinElmer ERS 6FE spinning disk confocal mi- Small Animal Magnetic Resonance Imaging croscope and images were processed and analyzed in Volocity The kidneys of Cys1cpk/cpk mice were visualized using a 9.4T version 6.1.1 software (PerkinElmer). magnetic resonance imaging (MRI) system BioSpec (Bruker BioSpin, Billerica, MA) with surface coil. A low-resolution Flow Cytometry image to confirm the location and orientation of the kidneys After perfusion of the mouse with PBS, kidneys were minced was followed by the acquisition of axial images covering the and digested in 1 ml of RPMI 1640 (Gibco, Grand Island, NY) entire kidney region with a T2-weighted fast spin echo se- containing 1 mg/ml collagenase type I (Sigma-Aldrich) and quence (rapid acquisition with relaxation enhancement) 100 U/ml DNase I (Sigma-Aldrich) for 30 minutes at 37°C with with a slice thickness of 0.5 mm. Kidney volumes were esti- agitation. Kidney fragments were passed through a 70-mm mated based on measurements obtained with ImageJ, version mesh (Falcon; BD Biosciences) yielding single-cell suspen- 1.47 software (National Institutes of Health, Bethesda, MD). sions. Red blood cells were lysed and cells were resuspended in 1 ml PBS containing 1% BSA with Fc blocking solution for CSF-1R Inhibitor Treatment 30 minutes on ice. A total of 23106 cells were stained for GW2580 (catalog number G-5903; LC Laboratories) was 30 minutes at room temperature with primary antibody (Sup- resuspended in 0.5% hydroxypropyl methylcellulose and plemental Table 1). Cells were washed, fixed in 2% PFA for 0.1% Tween 80 using multiple strokes of a Teflon-glass ho- 30 minutes, and resuspended in FACS staining buffer. mogenizer. The resuspended inhibitor was given to mice at a To determine intracellular Ki67 staining, cell suspensions dose of 160 mg/kg through oral gavage once daily throughout were incubated with Foxp3 Fixation/Permeabilization Solu- the RI. For the Cys1cpk/cpk inhibitor studies, mice were random- tion (eBioscience) for 30 minutes at room temperature after ized into vehicle- and drug-treated groups 11 days postbirth addition of primary antibodies. Cells were incubated with anti- based on MRI imaging. mouse Ki67, washed, and resuspended in 13 PBS. After immunostaining, cells were analyzed on a BD LSR II Flow Fixation and Tissue Processing Cytometer. Data analysis was performed using FlowJo version Following harvest, mouse kidneys were immersion fixed in 4% 10 software. (wt/vol) paraformaldehyde (PFA) overnight at 4°C, switched to 70% ethanol overnight, embedded in paraffin, sectioned at RNA Isolation and qRT-PCR 5 mm, and stained using hematoxylin and eosin. Picrosirius RNA was isolated using TRIzol and qRT-PCR was performed red staining was performed by the UAB Comparative Pathol- using TaqMan probes (Supplemental Table 2). To detect ogy Laboratory. membrane-bound and secreted CSF-1, we used SYBR Green

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Real-Time PCR Master Mixes with the following primers: RESULTS common forward primer, 59-TCTCCTTGAAAAG- GACTGGAAC-39; secreted reverse primer, 59-TGGT- Renal Resident Macrophages Undergo a Subtype GAGGGGAGGCAGAG-39; and cell surface reverse primer, Transition during Normal Postnatal Renal Maturation 59-GCAAGACTAGGATGATGGCCCG-39. The products To assess whether a specific macrophage subtype is associated from membrane-bound CSF-1 and secreted CSF-1 were verified with the differential rate of cyst formation observed in juvenile by DNA sequencing. versus adult-induced cilia mutant mice, we first analyzed macrophage populations isolated from wild type kidneys at TaqMan Low-Density Array and RNA Sequencing different periods during postnatal development using flow RNAwas isolatedfromFACS-sortedcellsusing the RNeasyPlus cytometry. The gating strategy (Figure 1A) used to distinguish Mini Kit (catalog number 74134; Qiagen) according to bone marrow–derived infiltrating (hereafter referred to as standard procedures. Samples were loaded onto the TaqMan region 1 [R1] macrophages) and embryonic-derived resident low-densityarray (TLDA) card (PN 4342253; Applied Biosystems) macrophages (hereafter referred to as region 2 [R2] macro- and run using a 7900HT Real-Time PCR System. A full list of the phages) was based on differential expression of CD11b and TaqMan probes and associated genes is listed in Supplementary F4/80 as established by Schulz et al.16 R2 macrophages were Table 3. further subgated based on expression of CD11c (R2a CD11chi; For RNA sequencing (RNA-seq), the NEBNext mRNA Li- R2b CD11clo) (Figure 1A). Analysis of R2 cells from kidneys of brary Prep Kit for Illumina with ribosome reduction was used wild-type mice at P4, P19, and P28 reveals a transition from to generate the library which was subsequently sequenced us- R2b (CD11clo) to R2a (CD11chi) during this period (Fig- ing Illumina NextSeq500 with paired-end, 75-bp sequencing ure 1B). This change in R2 macrophage subtype overlaps and aligned to the University of California, Santa Cruz with the period in which there is a switch from a rapid to GRCm38/mm10 reference genome using the STAR software slow rate of cyst formation in response to cilia dysfunction. package.26 At least 25 million reads were obtained for each To broadly characterize the R2 macrophage subtypes that sample. After alignment, HTSeq-Count (version 0.9.1) was are present during these developmental time points, we used to count the number of reads mapping to each gene.27 analyzed gene expression profiles using bulk RNA-seq on Fragments per kilobase of transcript per million mapped reads flow-sorted macrophage subtypes followed by verification of were calculated using the Cufflinks suite (https://github.com/ macrophage-specific genes using TLDAs. There is strong cole-trapnell-lab/cufflinks) and differential expression was correlation between the RNA-seq and TLDA data (0.71), con- then applied to the count files using DESeq2.28 RNA sequenc- firming the reproducibility of these studies. Heat-map analysis ing data have been deposited in the Gene Expression Omnibus generated from RNA-seq of R2a and R2b populations isolated under accession number GSE131896. from 8-week-old C57Bl/6J mice indicates a unique gene expression profile between these populations (Figure 1C). Fur- Statistical Analyses ther analysis comparing TLDA and RNA-seq data shows that Data are presented as the mean6SEM. ANOVA, analysis of R2a cells have increased expression of genes encoding CD11c covariance, two-way ANOVA, and t tests were used for statis- and CCR2, whereas R2b cells have increased expression of tical analysis, and differences were considered significant for genes encoding IL-10, matrix metallopeptidase 9, and P values ,0.05. For studies in which a significant difference CD206—cytokines associated with renal development, injury was observed between male and female mice within a group, a repair, and cyst formation (Supplemental Table 4).29–31 two-way ANOVA was performed with sex as a covariate. Briefly, analyses began by summarizing measures of central Loss of Primary Cilia in Adult Mice Leads to the tendency (sample mean, sample median) and measures of Reappearance of Juvenile-like, CD11clo R2b dispersion (sample variance, sample SD). Factorial ANOVA Macrophages after Renal Injury models, including factors for day of experiment (0, 3, 7, 14, 21, To evaluate the effect of cilia loss on resident macrophage 28, and 35), group (control, cilia mutant), and sex of mouse subtype and number, we compared macrophage profiles in (male, female) were constructed. Specifically, main effects for wild-type and conditional Ift88 mice in which cilia loss was each factor, each two-way interaction between factors, and the induced through tamoxifen injection at P7, followed by har- three-way interaction among factors were examined. Begin- vesting and analysis at P28. The data reveal an increased pro- ning with the highest-order interaction, nonsignificant inter- portion and number of the R2b macrophages as a percentage action terms were removed until a parsimonious model was of total cells in cilia mutant mice compared with control mice achieved. Normal probability plots, histograms, and residual (Figure 2A). In contrast, there were no differences in the plots were used to examine distributional assumptions for the number of R2a macrophages between control and cilia mu- models. Once the parsimonious model was identified, linear tant mice. contrasts were used to test for marginal differences between To determine if the accumulation of R2b macrophages in mouse strains per day of the experiment. All analyses were cilia mutant mice was due to intrinsic defects in resident mac- conducted using SAS 9.4. rophages lacking Ift88,weflow sorted R1 and R2 macrophages

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R2a R2b cell Bgn Sfrp2 Adgrf5 Kdr Ahnak Myom2 Nphs1 Pi16 Emcn Ctgf Serpine2 Dkk2 Sema3g Adamts5 Sparc Cdh5 Heg1 Efnb2 Tns2 Daam2 Pdgfrb Cald1 Exoc3l2 Ptprb Nphs2 Uaca Ehd3 Podxl Synpo Cyp26b1 Plat Epas1 Igfbp5 Tjp1 Gja5 Clic5 Fam129b Shisa3 Tm4sf1 Mapt Angptl2 Hspg2 Myl9 Flt1 Vegfa Hspa12a Col4a1 Nid1 Meis2 Pbx1 Ntn4 Tinagl1 Arhgef15 Cd24a Pecam1 Lama5 Myo1d Cd34 Ptn lgfbp7 Amotl1 Fcrls Mylk Plpp1 Sema5a Lamb2 Plpp3 Tbx3 Gimap4 Cd300lg Slc9a3r2 Enpep Gucy1a3 Itga3 Col4a3 Bcam Palld Rhpn1 Slco2a1 Col1a2 Fat1 F2r Nedd4 Arhgef17 Aebp1 Lmo7 Pcdh12 Ptprg Wt1 Adamts1 Sptbn1 Tspan15 Thsd7a mt-Rnr2 Pcdh1 Fermt2 Ccr2 Itgax Mmp12 Dnase1l3 ﬂ AI RESEARCH BASIC wctmtyplot cytometry ow ﬁ aino R2a of cation -1 -0.5 0 0.5 1 cell R2b R2a 1845 arpae sapretg fttlkde el ssona h mean the as shown is cells kidney total of percentage mean a as as shown macrophages and point time each idtp W)adclamtn ie h ubro 2 n 2 arpae sapretg fttlkde el r quanti are cells kidney total of percentage a as macrophages R2b and R2a of number The mice. mutant cilia and (WT) wild-type Representative 1846 2. Figure n osh bu)sann ncnrladclamtn iehretd3 aspostinjury; days 35 harvested mice mutant cilia and control in staining (blue) Hoescht * and replicates. four to three objective, AI RESEARCH BASIC

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aino h ubro 2 n R2b and R2a of number the of cation % of Total Cells 0.00 0.05 0.10 0.15 Days postIR n 3 rgnlmagni Original =3. – * nue ii uatmc.(A) mice. mutant cilia induced ii TCiliaMutant Cilia WT R2b Macrophage ** JASN ** Cilia MutantIR Cilia WTIR 30: ** fi 1841 cation, – ** 86 2019 1856, n fi =8 3 dfor ed 400; – 13, www.jasn.org BASIC RESEARCH as well as ciliated LTA+ proximal tubule epithelium and injured cilia mutant mice. These data indicate that R2b mac- performed qRT-PCR for Ift88. Our data indicate that R1 rophages are located adjacent to cysts and raise the possibility macrophages lack Ift88 gene expression, whereas Ift88 was that they are involved in paracrine/juxtacrine signaling with detected in R2 macrophages and the LTA+ epithelium the cilia mutant epithelium. (Supplemental Figure 2). The low level of Ift88 expression detected in the R2 populationislikelyduetominorcon- Injured Cilia Mutant Mice Express Increased tamination of the R2 cells with CD45-ciliated epithelium Proinflammatory Transcripts Compared with Control (Supplemental Figure 2; note CD45 cells with acetylated Injured Mice tubulin staining showing a cilium). Therefore, the accumu- Toidentify differences in the renal microenvironment between lation of the R2b population in the cilia mutant back- control and cilia mutant mice after injury, we compared ground is unlikely to be due to an intrinsic defect in expression levels of several macrophage chemoattractants, macrophages. proinflammatory cytokines, and extracellular matrix genes Because induction of cilia loss at P7 leads to rapid by qRT-PCR using RNA isolated from whole kidney tissue. cystogenesis with pathology already present at the time of In injured cilia mutant kidneys (day 14 postinjury), there was analysis (P28), we performed flow cytometry on adult- increased gene expression of the monocyte chemoattractant induced (8- to 10-week-old) conditional Ift88 mice at dif- protein (Ccl2) compared with control injured kidneys (Figure ferent time points after undergoing 30 minutes of unilateral 3A). Analysis of gene expression of proinflammatory cytokines IRI. This model allows us to analyze changes in macrophage reveals increased expression of Tumor necrosis factor-a (Tnfa) subtypes and number before cyst initiation and during cyst and Arginase 1 in injured cilia mutant mice compared with in- progression. In this model, the first renal cysts become jured cilia wild-type mice (Figure 3B). Our data also indicate evident around 14 days after injury, followed by rapid pro- increased expression of InhibinBa, Fibronectin 1, and Col3a1 gression (Supplemental Figure 3). Analysis of flow cytom- in injured cilia mutant mice compared with injured wild-type etry data indicates that the number of R2b macrophages was mice although the values were not significant (Figure 3C). Sev- significantly increased in cilia mutant mice beginning 7 days eral of these genes, including those encoding TNFa and Inhibin 32,33 post renal injury and remains significantly increased bA, are known to promote cyst formation. There were no throughout the time course of the analysis (Figure 2B). In overt changes in genes encoding total Csf1,membrane-bound contrast, the number of R2a macrophages was only in- Csf1, IL1b, nitric oxide synthase 2 (Nos2), and Col1a2 (Figure 3). creased in injured cilia mutant mice at 7 and 14 days post We did not observe a difference in expression of inflammatory injury compared with injured wild-type mice (Figure 2B). genes between injured cilia mutant male and female mice at any In these studies, we found that the number of R2b macro- time point analyzed. phages was significantly increased in injured cilia mutant Because iNos (Nos2) gene expression is increased after males compared with injured cilia mutant females at 7 and AKI in mice receiving contralateral nephrectomy34 and our 14 days post injury. Additional analysis of flow cytometry data did not reveal an increase in Nos2 expression between data reveals a significant increase in R1 infiltrating macro- uninjured (day 0) and unilaterally injured mice 1–3days phages in cilia mutant mice at 7 days post injury compared postinjury, we analyzed gene expression of Kidney Injury with control injured mice (Supplemental Figure 4). These Molecule-1 (Kim1)toconfirm injury in our model. Kim1 data indicate that both infiltrating and resident macro- is a commonly used marker of tubular damage after kidney phages are increased in cilia mutant mice before the onset injury.35 Our data indicate that Kim1 expression is signifi- of renal cystogenesis. cantly elevated in both injured control and cilia mutant To determine the spatial relationship between R2b macro- mice 1 day postinjury compared with uninjured controls phages and renal cysts, we conducted immunofluorescence (Supplemental Figure 6). These data indicate that 30 minutes microscopy analysis on kidney sections isolated from mice of unilateral IRI results in significant tubular damage in injured 35 days postinjury using the macrophage markers F4/80 and wild-type and cilia mutant mice. CD11c. To identify R2b macrophages, we used a fluorescently labeled antibody against CD206, a gene (Mrc1) whose expres- Accumulation of Resident Macrophages in Cilia Mutant sion is enriched in R2b macrophages relative to R2a cells (Sup- Kidney Is Independent of Peripheral Blood Input plemental Table 4). To further confirm that this marker is after Injury specific for R2b macrophages at this time point, we analyzed After injury there is an accumulation of R2b macrophages in flow cytometry data collected at 35 days postinjury. Our data cilia mutant mice compared with control injured mice. This indicate that R2b resident macrophages are the predominant accumulation could be driven either through recruitment cell type that express CD206 at this time point (Supplemental of circulating monocytes which differentiate into resident- Figure 5). Analysis of confocal images indicates that most like macrophages or through local paracrine-driven self- macrophages in regions adjacent to cysts coexpress F4/80 proliferation. To address this question, we performed and CD206 (Figure 2C). In contrast, we did not observe parabiosis experiments by joining the circulation of a CD206-positive macrophages around noncystic tubules in CD45.2 control or cilia mutant mouse with a congenic

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A Ccl2 Csf1 Membrane bound Csf1 4 4 1.0 3 3 0.8 * 0.6 2 2 0.4

RQ Value 1 1 0.2 0 0 0.0

(normalized to HPRT) 03714212835 0 3 7 14212835 0 3 7 14212835 Days post IR Days post IR Days post IR B Il1b Tnfa Arg1 Nos2 8 6 * 200 1.5 6 150 * 4 1.0 4 100 2 0.5

RQ Value 2 50 0 0 0 0.0

(normalized to HPRT) 03714212835 03714212835 0 3 7 14212835 0 3 7 14 21 28 35 Days post IR Days post IR Days post IR Days post IR C InhibinβA Fn1 Col1a2 Col3a1 6 20 2.5 20 15 2.0 15 4 1.5 10 10 2 1.0

RQ Value 5 0.5 5 0 0 0.0 0

(normalized to HPRT) 0 3 7 14 21 28 35 03714212835 0 3 7 14212835 0 3 7 14212835 Days post IR Days post IR Days post IR Days post IR

Cilia WT IR Cilia Mutant IR

Figure 3. Cilia mutant kidneys have increased expression of macrophage chemoattractants, proinflammatory, and profibrotic genes after IRI compared with injured wild-type (WT) kidneys. RNA from whole kidney lysates of control and cilia mutant mice was isolated using TRIzol and levels of (A) macrophage chemoattractants and activators (Ccl2,totalCsf-1, and membrane-bound Csf-1), (B) proinflammatory cytokines

(Il1b, Tnfa, Arginase 1 [Arg1], and Nos2), and (C) profibrotic genes (inhibinßA, fibronectin 1 [Fn1], col1a2,andcol3a1) determined by qRT- PCR. Values were normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt). For the qRT-PCR analysis, values represent the mean6SEM. n=2–6 mice, two replicates. Two-way ANOVA *P,0.05. RQ, relative quantification; IR, ischemia reperfusion.

CD45.1 wild-type C57BL/6J mouse. The mice were allowed to Resident Macrophages from Injured Cilia Mutant Mice equilibrate for 4 weeks before performing unilateral IRI Have Increased Proliferation Rates Compared with or sham surgery on the CD45.2 mouse (Figure 4A). Our re- Injured Wild-Type Mice sults indicate that R1 infiltrating macrophages from both con- As there is limited peripheral blood monocyte contribution to trol and cilia mutant kidneys have approximately equal levels the R2 population in cilia mutant kidneys after injury, we next of chimerism compared with blood monocytes, indicating a compared the proliferation rate of kidney-resident macro- complete exchange of these cells with circulating precursor phages in control and cilia mutant mice after injury using cells (Figure 4B). The level of chimerism in infiltrating R1 flow cytometry and antibodies against Ki67, a marker of active macrophages in the kidney is similar to previously published cell proliferation.42 Our data indicate that total R2, R2a, and reports.36–38 In contrast to R1 macrophages, both kidney R2a R2b macrophages from cilia mutant mice have significantly and R2b macrophages from control and cilia mutant mice increased proliferation rates at 3 days postinjury compared after injury had 3%–4% chimerism, similar to that observed with injured cilia wild-type mice (Figure 5). Total R2 and in resident microglia, a population that is independent of pe- R2a macrophages from injured cilia mutant mice also have ripheral monocyte recruitment (Figure 4B).39 Thus, our data increased proliferation rates compared with control injured indicate that the observed accumulation of R2b macrophages mice at 14 and 35 days postinjury (Figure 5). Because our data in injured cilia mutant kidneys is largely independent of indicate that infiltrating macrophage numbers are also in- peripheral blood monocyte recruitment. These data are in creased in cilia mutant kidneys compared with control kidneys agreement with previously published data indicating that res- after injury, we analyzed proliferation rates of infiltrating, ident macrophage accumulation after injury is mainly driven bone marrow–derived R1 macrophages via flow cytometry. through self-proliferation.36,40,41 Our data indicate that the proliferation rate of R1 macrophages

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A Cilia WT or Parabiosis IR surgery Cilia Mutant WT CD45.2 CD45.1 Tamoxifen

3 weeks 4 weeks 7 days

B Cilia WT Cilia Mutant **** * 60 *** 60 * ** * * * 40 40

20 20 % Chimerism % Chimerism

0 0 Blood R1 R2a R2b Microglia Blood R1 R2a R2b Microglia monocytes monocytes

Cilia WT IR Cilia Mutant IR

Figure 4. Resident macrophage accumulation in cilia mutant mice is independent of peripheral blood monocytes. (A) Schematic of how wild-type (WT) and cilia mutant CD45.2 mice were parabiotically joined to CD45.1 mice in this experiment. A timeline for key events in the experiment is shown. (B) Percentage chimerism, calculated as percentage nonhost cells (CD45.1 positive cells harvested in CD45.2 mice), is shown for each macrophage population. Values are shown as the mean6SEM. n=5–6 mice, four replicates. One way ANOVA, *P,0.05, **P,0.01, ***P,0.001, ****P,0.0001. IR, ischemia reperfusion. was not different between injured control and cilia mutant alternative RNA splicing.21 Although Csf1 expression from kidneys at any time point tested (Supplemental Figure 7). whole kidney tissue demonstrated minimal changes between There were no differences in proliferation rates between in- injured control and cilia mutant mice, when we analyzed ex- jured male and female cilia mutant mice at any time point pression in flow cytometry–isolated cell types we noted there analyzed. These data indicate that the increased accumulation was a significant increase in mbCsf-1 expression in proximal of R1 cells in injured cilia mutant kidneys is likely due to tubule cells of the injured cilia mutant kidneys compared enhanced Ccl2-dependent peripheral blood monocyte re- with controls (Figure 6A). This increase in mbCsf-1 was not cruitment and that the increased accumulation of R2b mac- observed in the collecting duct (Dolichos biflorus agglutinin rophages in injured cilia mutant kidneys is due to increased positive) or in R1 or R2 macrophage populations. Full-length self-proliferation. CSF-1 did not differ significantly between any of the groups tested (Figure 6B). Both R1 and R2 macrophages express Injured Cilia Mutant Epithelium Expresses Increased CSF-1R (Figure 6C), whereas receptor expression was not de- Levels of Membrane-Bound Csf-1 Compared with tected in either epithelial cell population. These data indicate Control Injured Epithelium excess accumulation and proliferation of R2 macrophages ob- A key factor driving resident macrophage proliferation and served in the cilia mutant mice may be due to local paracrine/ survival is the cytokine CSF-1, which is expressed in a secreted juxtacrine signaling between the injured proximal tubule or membrane-bound (mbCSF-1) form generated through epithelium and R2 macrophages.

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A B Cilia WT IR Cilia Mutant IR Ki67+ R2 29.2 59.4 100 105 105 **** 104 104 80

60 3 3 R2 10 10 ** 0 0 40 * 70.8 -103 -103 40.6 20 3 4 5 3 4 5 0 10 10 10 0 10 10 10 % of R2 macrophage 0 27.8 59.6 0 3 7 14212835 105 105 Ki67+ R2a

4 4 10 10 100 R2a Ki67 103 103 80 ****

0 0 60 ** 72.2 40.4 -103 -103 ** 0 103 104 105 0 103 104 105 40

34.2 56.7 % of R2a macrophage 105 105 0 104 104 0 3 7 14212835 Ki67+ R2b 3 3 R2b 10 10 100 0 0 **** 80 3 65.83 43.3 -10 -10 3 4 5 3 4 5 01010 10 10 0 10 10 60

F4/80 40 Cilia WT IR Cilia Mutant IR 20 % of R2b macrophage 0 0 3 7 14212835 Days post IR

Figure 5. Resident macrophages from cilia mutant mice have increased proliferation compared with injured wild-type (WT) resident macrophages. (A) Representative ﬂow cytometry plots showing Ki67 versus F4/80 staining in resident (R2), R2a (CD11chi), and R2b (CD11clo) macrophages 3 days post injury. (B) Quantiﬁcation of the percentage of each cell population that is positive for Ki67 in R2, R2a, and R2b macrophage populations is shown in the associated graph as the mean6SEM. n=8–13, three to four replicates. Two-way ANOVA, *P,0.05, **P,0.01, ****P,0.001. IR, ischemia reperfusion.

CSF-1R Inhibition Reduces Resident Macrophage vehicle-treated mice (Figure 7A). In contrast, R1 infiltrating Proliferation and Number of R2b Macrophages in Cilia macrophage proliferation was increased after GW2580 treat- Mutant Mice after IRI ment (Supplemental Figure 8). Analysis of resident macro- To test the model that R2b resident macrophage accumulation phage numbers at 3 days postinjury indicates that GW2580 in the injured cilia mutant kidney occurs in part through treatment significantly reduced the accumulation of R2b mac- mbCSF-1–driven local proliferation, we inhibited CSF-1R rophages in injured cilia mutant mice (Figure 7B). The num- activity in vivo using GW2580, a selective small-molecule in- ber of R2a and R1 macrophages in injured cilia mutant mice hibitor of the CSF-1R kinase.43 Injured adult–induced cilia was not affected by GW2580 treatment (Figure 7B, Supple- mutant mice were treated by daily gavage starting 1 day after mental Figure 8). Thus, GW2580 specifically reduced the ac- injury and continuing throughout the 35 day course of the cumulation of R2b macrophages in cilia mutant mice after experiment.43 At 3 days postinjury, GW2580 treatment renal injury. caused a significant reduction in the proliferation rate of To further confirm that GW2580 reduced proliferation and both R2a and R2b resident macrophages compared with accumulation of macrophages at later time points, we stained

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A Membrane bound CSF1 B Full length CSF1 3 * 1.5

2 1.0

1 0.5 RQ Value RQ Value (normalized to HPRT) (normalized to HPRT) 0 0.0 R1 R2 LTA+ DBA+ R1 R2 LTA+ DBA+

C CSF1R 3 Cilia WT IR 2 Cilia Mutant IR

1 RQ Value

(normalized to HPRT) 0 R1 R2 LTA+ DBA+

Figure 6. Membrane-bound Csf-1 is increased in LTA+ proximal tubule epithelium from cilia mutant mice. (A–C) mRNA levels of (A) membrane-bound Csf-1, (B) full-length Csf-1, and (C) Csf-1R from FACS-sorted kidney cells 3 days post-IRI as determined by qRT-PCR. Values represent the mean6SEM. n=4–5 mice, two to three replicates. *P,0.05, t test. RQ, relative quantification; HPRT, hypoxanthine- guanine phosphoribosyltransferase; IR, ischemia reperfusion; WT, wild type. kidneysectionsofvehicle-andGW2580-treatedciliamutant the cystic phenotype, there was also a significant reduction mice with LTA, F4/80, and Ki67 35 days post-IRI. In vehicle- in renal fibrosis as indicated by picrosirius red staining in treated kidneys, we detected dual-labeled Ki67+, F4/80+ mice treated with GW2580 compared with vehicle-treated macrophages that are frequently localized to regions sur- mice (Figure 8B). These data indicate that CSF1R-dependent rounding epithelial-derived renal cysts (Figure 7C, white proliferation and accumulation of R2b resident macrophages arrows). In contrast, the number of F4/80+ cells per square is a driving factor in renal cyst formation. millimeter of tissue was markedly reduced in injured cilia To confirm the relevance of CSF-1 signaling during cystic mutant mice receiving GW2580 treatment (Figure 7C). renal disease in the absence of kidney injury and to test whether These data indicate that the number of macrophages in re- inhibition of CSF1R is efficacious in slowing cyst progression gions adjacent to tubule epithelial cells is decreased in in- in mice with prominent renal cysts, we tested the effect jured cilia mutant mice treated with GW2580 at 35 days post of GW2580 in the Cys1cpk/cpk mouse model. Cys1cpk/cpk mice injury. phenocopy aggressive autosomal recessive polycystic kidney disease with rapidly progressive cyst growth.44 In contrast to Inhibition of Resident Macrophage Proliferation with the above-described studies in the Ift88-conditional IRI model GW2580 Reduces Cystic Severity where GW2580 treatment was started at the time of renal in- Next, we assessed whether preventing the accumulation of jury and well before cyst initiation, a 10-day GW2580 gavage R2b macrophages affected cyst number and severity after treatment of Cys1cpk/cpk mice could only be started at P11 due IRI. Kidneys were isolated from male and female cilia mutant to the small size of the mice. At this point, renal cystic disease is mice 35 days post injury. Because our analyses indicate that already moderately severe. Using MRI, we tracked changes in female cilia mutant mice failed to develop severe renal cysts total kidney volume (TKV) as a surrogate for cyst expansion after injury compared with their male counterparts (Supple- in longitudinal studies on the same animals. GW2580 treat- mental Figure 9), we could only analyze the effect of GW2580 ment reduced the severity of renal cystic disease as reflected treatment on injured male cilia mutant mice at 35 days post by the reduced rate of increase (slope) in TKV (TKVreduction injury. Histologic examination of kidneys from male cilia mu- by37%,adjustedmeans2697versus1782mm3, P=0.021) tant mice at 35 days post-IRI revealed a significant reduction and body length–adjusted TKV (TKV/body length, by 34%, in cystic severity in GW2580-treated mice compared with ve- adjusted means 511 versus 358, P=0.033 (Figure 8, C and D). hicle-treated cilia mutant mice (Figure 8A). Importantly, this Body weight–adjusted TKV was also reduced by GW2580 was due to both a reduction in the number and size of cysts treatment although the value was not significant (Supplemen- compared with vehicle controls (Figure 8A). In addition to tal Figure 10). Body weight and body length were

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A Ki67+ R2a Ki67+ R2b

80 ** 80 *** 60 60

40 40 % of parent % of parent 20 20

0 0 Cilia Mutant Cilia Mutant Cilia Mutant Cilia Mutant Vehicle GW2580 Vehicle GW2580

B R2a Macrophage R2b Macrophage

1.5 ** 0.3

1.0 0.2

0.5 0.1 % of total cells % of total cells 0.0 0.0 Cilia Mutant Cilia Mutant Cilia Mutant Cilia Mutant Vehicle GW2580 Vehicle GW2580

C Cilia Mutant Vehicle Cilia Mutant GW2580 LTA F4/80 Ki67 LTA F4/80 Ki67 60 *

20 /Nuclear Area

Cy % Macrophage Area 0 Cilia Mutant Cilia Mutant Vehicle GW2580

Cyst

Figure 7. CSF-1R inhibition reduces resident macrophage proliferation and prevents R2b resident macrophage accumulation. (A) Quantification of Ki67+ cells as a percentage of each cell population is shown for injured cilia mutant mice harvested 3 days after daily treatment with vehicle or GW2580. Values represent the mean6SEM. n=4–5, three replicates. (B) Quantification of the number of R2a and R2b macrophages as a percentage of total kidney cells from cilia mutant mice treated with vehicle or GW2580 for 3 days after injury. Values represent the mean6SEM. n=4–5 replicates. **P,0.01. (C) Confocal images of kidney sections harvested 35 days post injury stained with F4/80 (green), Ki67 (red), and LTA (white). F4/80+ area/nuclear area is quantified in the associated graph as the mean6SEM. n=3, one replicate. *P,0.05, **P,0.01, ***P,0.001, t test. Original magnification, 3400; objective, 340; scale bar, 100 mm. significantly reduced in the GW2580-treated mice compared DISCUSSION with vehicle-treated mice (Supplemental Figure 10). Similar to the Ift88-conditional IRI model, GW2580 treatment decreased The clinical importance of cilia in cystic kidney disease is the number of renal F4/80-positive macrophages compared well established; however, the mechanism by which cilia dys- with vehicle-treated mice (Figures 8E). function leads to cyst formation is still largely unknown.

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A Cilia Mutant IR Cilia Mutant IR C Vehicle Vehicle GW2580 P11 P21 15 **

5 % cystic

0 Vehicle GW2580

** GW2580 8000 P11 P21 6000 4000 2000 No. cysts/area 0 Vehicle GW2580 B Cilia Mutant IR Cilia Mutant IR Vehicle GW2580 * D 15

] p=0.021 p=0.033 3 4000 700 10 3000 600 /cm] 2000 3 500 5 2500 400 2000 0 300 1500

% Picrosirus Red area Vehicle GW2580 1000 200 E Vehicle GW2580 500 100 TKV/BL ratio [mm

F4/80 Hoechst Total kidney volume [mm 0 0 10 15 20 25 10 15 20 25 Cpk Postnatal age [days] Postnatal age [days] 40 Cpk Vehicle 30 Cpk GW2580 20 10 /Nuclear Area % F4/80+ Area 0 Vehicle GW2580

Figure 8. GW2580 reduces cystic index and number in two mouse models of cystic disease. (A) Representative hematoxylin and eosin image from male mice treated with vehicle or GW2580. For each group, the number of cysts/total area as well as the cystic index (cystic area/total kidney area) is shown as the mean6SEM. n=7–8, three replicates. **P,0.01, t test. Original magnification, 310; 31ob- jective; scale bar, 1000 mm. (B) Representative image of kidneys that were isolated from cilia mutant mice treated with vehicle only or GW2580 at 35 days postinjury and stained with picrosirius red. Quantification of the percent area that was picrosirius red positive/ kidney area was obtained using ImageJ. Values represent the mean6SEM. n=4–5, two replicates. *P,0.05. Original magnification, 3400; 340 objective; scale bar, 50 mm. (C) Representative pretreatment (P11) and post-treatment (P21) MRI images of Cys1cpk/cpk males treated with GW2580 or vehicle. (D) Graph showing the increase in TKV and TKV/body length (BL) ratio (equivalent to height- adjusted TKV used in humans) in control and Cys1cpk/cpk mice. The effect of GW2580 on body and kidney indices were tested with analyses of covariance with pretreatment scores and gender serving as covariates. (E) Representative confocal images showing Cys1cpk/cpk mice with and without GW2580 treatment that were stained with F4/80 (red) and Hoescht (blue). Quantification of macrophage area/nuclear area is shown. n=2–3. Original magnification, 3400; objective, 340; scale bar, 50 mm.

Additionally, it remains poorly understood why cysts develop Our data suggest that the primary cilium is an important rapidly in juvenile-induced cilia mutant mice (before P12–14), regulatorof the injury and repair process in the kidney. Further, butslowlyinadult-inducedciliamutants(afterP14),and we propose that the primary cilia-dependent repair mecha- why injury in adult-induced cilia mutants reinitiates rapid nism after injury is driven through paracrine/juxtacrine sig- cystogenesis. Our data indicate that R2b resident macrophages naling with nearby tissue-resident macrophages. One of the accumulate in both juvenile-induced and injured adult– candidate pathways involved in this process is mbCSF-1. Our induced cilia mutant mice before the onset of renal cystogenesis. data show that mbCsf-1 is elevated in injured cilia mutant Inhibition of CSF1R-dependent R2b resident macrophage accu- proximal tubule epithelium compared with control injured mulation reduces both the number and severity of renal cysts tubule epithelium. Based on the outcome of the CSF1R inhib- after IRI. These data suggest that proliferation and accumulation itor studies (GW2580), our data indicate that CSF-1 is respon- of R2b macrophages trigger renal cyst formation and that target- sible for local proliferation of R2 macrophages and that this ing this population of cells in human patients may prevent cyst process is increased in cilia mutant mice after injury, resulting formation and expansion. in enhanced and persistent R2b macrophage accumulation.

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This indicates that injured epithelial cells lacking a primary self-proliferation and influx of infiltrating bone marrow– cilium are stuck in a state of persistent injury and that they are derived macrophages.36,38,41 This suggests that renal injury signaling to nearby resident macrophages in an attempt to induces a minor depletion of the resident macrophage niche promotetubulerepair.Inthecaseofinjuredciliamutant immediately after injury and that influx of bone marrow– mice, the continued attempt by resident macrophages to re- derived infiltrating macrophages and resident macrophage pair the epithelium results in enhanced and prolonged epithe- self-proliferation combine to fill that niche. Because the con- lial proliferation and renal cyst formation. tribution of peripheral monocytes to the resident niche is An interesting finding from our studies was that the number minor and the proliferation of resident macrophages 3 days of R2b macrophages was increased in male cilia mutant mice postinjury reaches 70%–80%, it is likely that resident macro- at 7 and 14 days after injury compared with injured female phage self-proliferation is the major source of resident mac- cilia mutant animals. In addition, we found that injured female rophage repopulation. Further, based on the results of our cilia mutant mice failed to develop severe renal cysts 35 days parabiosis studies showing a similar level of chimerism in postinjury compared with their injured male counterparts. R2a/R2b macrophages between injured control and cilia These data provide correlative evidence that the number of mutant mice, it is likely that the increase in R2b resident mac- R2b macrophages that are present at early periods after injury rophages in injured cilia mutant mice is driven by local self- are associated with the severity of renal cysts at later time proliferation and not enhanced recruitment from peripheral points. Interestingly, although the number of R2b macro- blood monocytes. The fact that treatment of injured cilia phages differed between male and female cilia mutant mice mutant mice with a CSF1R inhibitor prevents the accumula- after injury, we did not observe a difference in the proliferation tion of R2b macrophages, but does not affect the number of rate between macrophages that were isolated from these same R1 macrophages in injured cilia mutant mice, provides groups. This suggests that the accumulation of R2b macro- further support of this idea. Additional studies using a lineage phages observed in male cilia mutant mice is due to a reduced tracing approach in which resident macrophages were perma- levels of apoptosis. This hypothesis is further strengthened nently labeled with a tdTomato reporter show that a majority by our data showing that the number of R2 macrophages of resident macrophages that proliferated and accumulated was not different between male and female mice on the day after injury were tdTomato-positive and that only a minor of IRI. Studies that address the effect of sex on apoptosis in number of cells were tdTomato-negative.38 Collectively, these resident macrophages after injury are warranted. data suggest negligible contribution of peripheral monocytes These studies indicate that renal resident macrophages to the resident macrophage pool after injury and that the undergo a phenotypic switch during normal postnatal renal accumulation of R2b macrophages in cilia mutant mice is maturation. Following injury, we observe the reappearance driven through self-proliferation. of juvenile-like macrophages in cilia mutant mice, Our data show that the number of R2b macrophages is indicating a potential reprogramming of these macrophages increased in injured cilia mutant mice compared with injured toward a juvenile-like state. This finding agrees with our pre- control mice and that inhibition of resident macrophage pro- vious data indicating that renal injury induces a transcrip- liferation and accumulation reduced renal cysts in two inde- tional reprogramming in wild-type mice36 and suggests that pendent models of cyst formation. These data suggest that macrophages which are present during juvenile periods and targeting resident macrophages in human patients with cystic after injury in cilia mutant mice likely share some phenotypic disease may be a possibility for therapeutic intervention. and functional overlap that promotes rapid cyst formation. Furthermore, we show that renal resident macrophages self- proliferate independently of peripheral blood input after ACKNOWLEDGMENTS injury to maintain the resident macrophage niche. Interest- ingly, both R2a and R2b macrophages proliferate after injury. We would like to thank members of Dr. Yoder’s, Dr. George’s, Based on our data showing that GW2580 treatment inhibits Dr. Mrug’s, and Dr. Benveniste’s laboratories for suggestions and the proliferation of all R2 macrophages but only prevents technical support on the project. The authors would also like to ac- the accumulation of R2b macrophages, we propose that knowledge Mandy J. Croyle, Ronald Roye, Sean Mullen, David R2a macrophages proliferate to become R2b macrophages. Redden, and Gabriel Rezonzew for their technical assistance. Future fate mapping studies to confirm these findings are Dr. Song and Dr. Zimmerman designed the research studies. warranted. Dr. Song, Dr. Zimmerman, Ms. Gonzalez, Dr. Lever, Dr. Zhou, Our parabiosis data indicate that the majority of resident Dr.Yan,Mr.Li,Ms.Shan,Mr.Aloria,Ms.Rains,andMr.Revell macrophages found in injured cilia mutant kidneys are inde- conductedexperimentsandacquireddata.Dr.Song,Dr. pendent of blood monocyte recruitment. However, there is a Zimmerman, Dr. Lever, Mr. Revell, Dr. Mrug, Dr. Crowley, Mr. small proportion of resident macrophages (3%–4% of total Aloria, Mr. Bassler, Dr. Crossman, and Dr. Yoder analyzed the residents) that are derived from the peripheral blood. Previous data. Dr. Yoder, Dr. George, Dr. Benveniste, and Dr. Mrug pro- work from our group and others has shown that an emptied vided reagents. Dr. Song and Dr. Zimmerman wrote the resident macrophage niche can be repopulated through local manuscript.

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DISCLOSURES Supplemental Figure 9. Female cilia mutant mice fail to develop severe renal cysts 35 days post injury. Dr.Mrugreportsgrants,personalfees,andnonfinancial support from Supplemental Figure 10. Cys1cpk/cpk mice receiving GW2580 Otsuka; grants, personal fees, and nonfinancial support from Sanofi;personal have reduced body length and body weight compared with vehicle fees from ClearView Healthcare Partners; personal fees from Decision Re- treated mice. sources Group Consulting; personal fees from CLARION Healthcare; personal fees from Chinook Therapeutics, outside of the submitted work; and is the Scientific Advisory Committee Chair of the PKD Foundation. Dr. Yoder reports grants from National Institutes of Health (NIH) and grants from PKD REFERENCES Foundation during the conduct of the study. 1. Yoder BK, Hou X, Guay-Woodford LM: The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized FUNDING in renal cilia. J Am Soc Nephrol 13: 2508–2516, 2002 2. Sharma N, Malarkey EB, Berbari NF, O’Connor AK, Vanden Heuvel GB, Mrug M, et al.: Proximal tubule proliferation is insufficient to induce These studies were supported in part by the following research grants: PKD rapid cyst formation after cilia disruption. JAmSocNephrol24: Foundation grant 214g16a (Dr. Yoder); UAB School of Medicine AMC21 grant 456–464, 2013 (Dr. Yoder, Dr. Mrug, Dr. George); NIH T32 training grant in Basic Immu- 3. Lin F, Hiesberger T, Cordes K, Sinclair AM, Goldstein LS, Somlo S, et al.: nology and Immunologic Disease 2T32AI007051-38 (Zimmerman); NIH R01 Kidney-specific inactivation of the KIF3A subunit of kinesin-II inhibits DK115752 (Dr. Yoder), R01 DK097423 (Dr. Mrug), and R01 NS57563 renal ciliogenesis and produces polycystic kidney disease. Proc Natl (Dr. Benveniste); and Office of Research and Development, Medical Research Acad Sci U S A 100: 5286–5291, 2003 Service, US Department of Veterans Affairs grant 1-I01-BX002298 4. Wilson PD: Polycystic kidney disease. NEnglJMed350: 151–164, (Dr. Mrug). The following NIH-funded cores provided services for this proj- 2004 ect: UAB Hepatorenal Fibrocystic Disease Core Center P30-DK074038, UAB- 5. Davenport JR, Watts AJ, Roper VC, Croyle MJ, van Groen T, Wyss JM, University of California San Diego O’Brien Center for AKI Research et al.: Disruption of intraflagellar transport in adult mice leads to obesity P30-DK079337, and the UAB Comprehensive Flow Cytometry Core P30- and slow-onset cystic kidney disease. Curr Biol 17: 1586–1594, 2007 AR048311 and P30-AI27667. Additional services were provided by the UAB 6. Takakura A, Contrino L, Zhou X, Bonventre JV, Sun Y, Humphreys BD, Comparative Pathology Laboratory and UAB Heflin Genomic Core. et al.: Renal injury is a third hit promoting rapid development of adult polycystic kidney disease. Hum Mol Genet 18: 2523–2531, 2009 7. Mrug M, Zhou J, Woo Y, Cui X, Szalai AJ, Novak J, et al.: Over- SUPPLEMENTAL MATERIAL expression of innate immune response genes in a model of recessive polycystic kidney disease. Kidney Int 73: 63–76, 2008 8. Zeier M, Fehrenbach P, Geberth S, Möhring K, Waldherr R, Ritz E: Renal This article contains the following supplemental material online histology in polycystic kidney disease with incipient and advanced renal at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018080810/-/ failure. Kidney Int 42: 1259–1265, 1992 DCSupplemental. 9. Swenson-Fields KI, Vivian CJ, Salah SM, Peda JD, Davis BM, van Supplemental Table 1. List of relevant information for flow cy- Rooijen N, et al.: Macrophages promote polycystic kidney disease progression. Kidney Int 83: 855–864, 2013 tometry antibodies. 10. Karihaloo A, Koraishy F, Huen SC, Lee Y, Merrick D, Caplan MJ, et al.: Supplemental Table 2. List of Taqman probes used for qRT-PCR Macrophages promote cyst growth in polycystic kidney disease. JAm analysis. Soc Nephrol 22: 1809–1814, 2011 Supplemental Table 3. List of Taqman probes used for microarray. 11. Zoja C, Corna D, Locatelli M, Rottoli D, Pezzotta A, Morigi M, et al.: Supplemental Table 4. Comparison of the relative expression of Effects of MCP-1 inhibition by bindarit therapy in a rat model of poly- – fl cystic kidney disease. Nephron 129: 52 61, 2015 in ammatory genes between RNAsequencing and Taqman arraydata. 12. Mirolo M, Fabbri M, Sironi M, Vecchi A, Guglielmotti A, Mangano G, f/f Supplemental Figure 1. Treatment of CaggcreERT2 Ift88 mice et al.: Impact of the anti-inflammatory agent bindarit on the chemo- with tamoxifen leads to reduction of Ift88 mRNA, IFT88 protein, and kinome: Selective inhibition of the monocyte chemotactic proteins. Eur loss of primary cilia. Cytokine Netw 19: 119–122, 2008 Supplemental Figure 2. Resident macrophages lack primary cilia. 13. Viau A, Bienaimé F, Lukas K, Todkar AP, Knoll M, Yakulov TA, et al.: Cilia-localized LKB1 regulates chemokine signaling, macrophage re- Supplemental Figure 3. Cilia mutant mice begin to form cysts in cruitment, and tissue homeostasis in the kidney. EMBO J 37: e98615, cortical regions 14 days post injury. 2018 Supplemental Figure 4. R1 infiltrating macrophages are increased 14. Cassini MF, Kakade VR, Kurtz E, Sulkowski P, Glazer P, Torres R, et al.: Mcp1 in injured cilia mutant mice compared with injured wild type mice. promotes macrophage-dependent cyst expansion in autosomal dominant – Supplemental Figure 5. Cilia mutant R2b macrophages are the polycystic kidney disease. J Am Soc Nephrol 29: 2471 2481, 2018 15. Zimmerman KA, Song CJ, Gonzalez-Mize N, Li Z, Yoder BK: Primary predominant cell type that express CD206 35 days post injury. cilia disruption differentially affects the infiltrating and resident mac- Supplemental Figure 6. Injured wild type and cilia mutant mice rophage compartment in the liver. Am J Physiol Gastrointest Liver have increased Kim1 gene expression one day following renal injury. Physiol 314: G677–G689, 2018 Supplemental Figure 7. The percentage of proliferating R1 mac- 16. Schulz C, Gomez Perdiguero E, Chorro L, Szabo-Rogers H, Cagnard N, rophages does not differ between injured wild type and cilia mutant Kierdorf K, et al.: A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336: 86–90, 2012 mice. 17. Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, et al.: Fate Supplemental Figure 8. 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