BASIC RESEARCH www.jasn.org Expression Profiling of Fibroblasts in Chronic and Acute Disease Models Reveals Novel Pathways in Kidney Fibrosis Atsuko Y. Higashi,1 Bruce J. Aronow,2 and Gregory R. Dressler1 1Department of Pathology, University of Michigan, Ann Arbor, Michigan; and 2Department of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio ABSTRACT Background Renal interstitial fibrosis results from activation and proliferation of fibroblasts to myofibro- blasts, secretion and accumulation of extracellular matrix, and displacement of normal renal tubules. In contrast to chronic renal disease, acute injury may be repaired, a process that includes a decrease in the number of myofibroblasts in the interstitium and degradation of the accumulated extracellular matrix, leaving little evidence of prior injury. Methods To investigate whether activated fibroblasts demonstrate changes in gene expression that cor- respond with regression after acute injury but are not observed in chronic models of fibrosis, we used microarrays to analyze gene expression patterns among fibroblast populations at different stages of injury or repair. We then mined the data for signaling pathways in fibroblasts corresponding to the acute pro- liferative, regression, and chronic phases of renal injury. Results We identified multiple gene clusters with changes that correlate with the three phases of renal injury, including changes in levels of receptors for the antifibrotic factor PGE2. In adult renal fibroblast cultures, PGE2 was able to upregulate many genes that are suppressed by the profibrotic cytokine TGF-b, whereas many PGE2-downregulated genes were activated by TGF-b. High levels of TGF-b suppressed expression of a subset of PG receptors in fibroblast cultures, making these cells resistant to any effects of PGE2. Conclusions Inherent gene expression changes in activated fibroblasts accompany the transition from AKI to repair and regeneration. In chronic models, however, activated fibroblasts are resistant to the antifi- brotic effects of PGE2 due to suppression of a subset of PGE receptors. J Am Soc Nephrol 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018060644 Fibrosis is a common pathology due to the activa- cytokines.4,5 Using definitive genetic cell lineage– tion of interstitial fibroblasts to myofibroblasts, the tracing methods, the origins of renal interstitial increased secretion and accumulation of extracel- fibroblasts and myofibroblasts have clearly been lular matrix (ECM), and the loss or perturbation of established in animal models of renal fibrosis.6 The tissue-specific epithelial, endothelial, and other cell lineages.1,2 In CKD, this expansion and deposition of ECM compromises renal function by displacing Received June 22, 2018. Accepted November 7, 2018. proximal and distal tubules, by increasing glomer- Published online ahead of print. Publication date available at ular mesangium to affect the filtration barrier, and www.jasn.org. by affecting the hemodynamics of the renal arteri- Correspondence: Prof. Gregory R. Dressler, Department of Pa- oles.3 Myofibroblast induction is also accompanied thology, University of Michigan, BSRB 2049, 109 Zina Pitcher by innate immune pathways, partly through the Drive, Ann Arbor, MI 48109. Email: [email protected] expression and secretion of immune-mediating Copyright © 2019 by the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc, 2019 ISSN : 1046-6673/3001-ccc 1 BASIC RESEARCH www.jasn.org best available data point to pericytes and perivascular fibroblastic Significance Statement cells, in addition to preexisting, resident interstitial fibroblasts, as the major sources of myofibroblasts in renal fibrosis models. Activation of fibroblasts to myofibroblasts is a key factor that drives Thus, to identify optimal therapeutic targets for CKD, it is es- renal interstitial fibrosis; regression of activated fibroblasts in the sential to understand both the intrinsic genomic programming interstitium is a key feature of repair, which can occur after acute fi injury but not in chronic renal disease. Using models of AKI and CKD of pericytes and interstitial, activated broblasts as well as their to study genes and signaling pathways that may mediate repair and responses to injury and genetic abnormalities. fibrosis regression, the authors found that activated fibroblasts Expansion of interstitial fibroblasts has been studied in AKI display changing gene expression patterns, including for PGE2 in human biopsy samples7 and in rodent models.8 AKI is the result receptors. These inherent changes accompany the transition from of renal ischemia or nephrotoxic injury and results in the death acute injury to repair, but differ from patterns observed after chronic injury. The authors also found that high levels of the profibrotic of proximal tubule epithelial cells primarily from the S2 and S3 cytokine TGF-b in cultured adult fibroblasts may inhibit PGE2’s segment of the nephron. However, these segments retain the abil- antifibrotic effects by suppressing a subset of PGE receptors, a ity to regenerate and repopulate the sites of injury if the degree of finding with potential implications for therapeutic strategies. injury is limited.9,10 Coincident with AKI is widespread activation and proliferation of interstitial fibroblasts. As AKI is repaired, the Michigan. Mice were housed in a specificpathogen–free fa- activation of interstitial myofibroblasts subsides and expansion cility with a 12-hour light/12-hour dark cycle and given free regresses, often leaving little evidence of residual fibrosis. How- access to food and water. ever, repeated bouts of AKI may alter the ability of activated in- For the UUO model, mice were anesthetized by isoflurane terstitial myofibroblast to regress, leading to irreversible inhalation.Throughamidlineabdominalincision,theleftureter interstitial fibrosis and a chronic renal disease state.11,12 Neverthe- was tied off at the level of the lower pole and themidureteral level less, after an initial bout of AKI the number of activated myofi- with fine suture materials (5–0 silk), then cut between the two broblasts decreases as injury is repaired, suggesting that these cells ligated points to induce a complete obstruction. Mice were al- have the ability to reversibly deactivate or undergo apoptosis and lowed to recover from anesthesia and were supplied food and removal by mechanisms that remain to be determined.13 water ad libitum until they were euthanized at 14 days after the In this report, we have begun to address inherent differences in surgery. For the acute tubular necrosis model, mice were in- renal interstitial fibroblasts after AKI and in chronic disease models. jected intraperitoneally with a single dose of 250 mg/kg FA in Using a folic acid (FA) model of AKI and the unilateral ureteral 0.15 M NaHCO . Mice were euthanized at 3, 7, and 14 days after obstruction model (UUO) of renal fibrosis in mice, we isolated 3 injury. Mice were put under constant observation for the first interstitial fibroblasts by fluorescence-activated cell sorting (FACS) 48 hours after injection. and determined gene expression changes with Affymetrix micro- arrays. We compared the transcriptome of fibroblasts from unin- juredkidneysandUUO-derivedfibroblasts, as well as fibroblasts Cell Sorting from different times after AKI. We hypothesized that there may be In brief, kidneys were minced and digested with 0.5 mg/ml Lib- specific genes and pathways that mediate the regression of erase DH (#05401054001; Roche) and 100 U/ml DNaseI fibroblasts after AKI that are not active in chronic models of fibro- (11284932001; Roche) in PBS solution supplemented with sis, such as the UUO model. The data show dynamic changes in the 10% FBS at 37°C for 30 minutes with triturate by P1000 tip every patterns of fibroblast gene expression in the acute and recovery 5 minutes, followed by centrifugation at 3003g for 5 minutes at phases that include WNT and TGF signaling pathways, fibroblast 4°C. The resulting pellet was resuspended in ice-cold PBS with growth factors, and genes involved in ECM deposition or turnover. 2 mM EDTA and was passed through a 70-mm cell strainer Furthermore, we show that suppression of the PG receptor Ptger3 (Falcon), then centrifuged at 3003g for 5 minutes at 4°C. The correlates with acutely injured fibroblasts but increases with re- pellet was incubated with 0.53 red blood cell lysis buffer covery. Strikingly, TGF-b–cultured kidney fibroblasts suppress (0.15 M NH4Cl, 14 mM NaHCO3, and 0.1 mM Na2 EDTA) expression of multiple PG receptors making cells recalcitrant to for 5 minutes on ice, to remove red blood cells, and centrifuged PGE2 signaling. Given the proposed antifibrotic effects of at 3003g for 5 minutes at 4°C. The single isolated kidney cells PGE2,14–16 these data suggest a novel mechanism for the regres- were stained with APC-conjugated anti-PDGFRa Antibodies sion of interstitial fibroblasts that is deregulated in a high–TGF-b (Ab) (#A18382; Life Technologies) and PE-Cyanine7–conju- environment. gated anti-CD11b Ab (#25–0112–81; eBioscience) and subjected to cell sorting using FACS Aria II cell sorter (BD Biosciences). FACS Aria II was also used for surface pattern analysis. The METHODS antibodies used for surface pattern are anti-PDGFRa,APC-Cy- anine7–conjugated anti-CD45 Ab (#103115; Biolegend), PE- Animals Cyanine7–conjugated anti-CD105 Ab (#120409; Biolegend), Mice were kept according to National Institutes of Health PE-conjugated anti-CD44 Ab (#103007; Biolegend), PE-con- guidelines and all procedures were approved by the University jugated anti-PDGFrb Ab (#136005; Biolegend), and PE-con- Committee on Use and Care of Animals at the University of jugated anti-CD31 Ab (#102507; Biolegend). 2 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH No injury FA3 FA7 FA14 U14 HE staining Nuclei / PDGFR Nuclei / SMA Nuclei / PDGFR Nuclei / Collagen1 Nuclei / Fibronectin Figure 1. Interstitial fibroblasts expand and regress after acute kidney injury (FA3–FA14) but not in the chronic obstruction model (U14). Representative sections of kidneys taken at the indicated times post-FA or -UUO or from uninjured controls are shown.