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Renoprotective and Immunomodulatory Effects of GDF15 following AKI Invoked by Ischemia-Reperfusion Injury

Jing Liu,1 Sanjeev Kumar,1,2 Andreas Heinzel ,3 Michael Gao,1 Jinjin Guo,1 Gregory F. Alvarado,1 Roman Reindl-Schwaighofer ,3 A. Michaela Krautzberger,1,4 Pietro E. Cippà,1,5 Jill McMahon,1 Rainer Oberbauer,3 and Andrew P. McMahon1

1Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California; 2Division of Nephrology, Department of Medicine and Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, California; 3Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria; 4Department of In Vivo Pharmacology, Evotec International GmbH, Göttingen, Germany; and 5Division of Nephrology, Regional Hospital of Lugano, Lugano, Switzerland

ABSTRACT Background Gdf15 encodes a TGF-b superfamily member that is rapidly activated in response to stress in multiple organ systems, including the kidney. However, there has been a lack of information about Gdf15 activity and effects in normal kidney and in AKI. Methods We used genome editing to generate a Gdf15nuGFP-CE mouse line, removing Gdf15 at the targeted allele, and enabling direct visualization and genetic modification of Gdf15-expressing cells. We extensively mapped Gdf15 expression in the normal kidney and following bilateral ischemia- reperfusion injury, and quantified and compared renal responses to ischemia-reperfusion injury in the presence and absence of GDF15. In addition, we analyzed single nucleotide polymorphism association data for GDF15 for associations with patient kidney transplant outcomes. Results Gdf15 is normally expressed within aquaporin 1–positive cells of the S3 segment of the proximal tubule, aquaporin 1–negative cells of the thin descending limb of the loop of Henle, and principal cells of the collecting system. Gdf15 is rapidly upregulated within a few hours of bilateral ischemia-reperfusion injury at these sites and new sites of proximal tubule injury. Deficiency of Gdf15 exacerbated acute tubular injury and enhanced inflammatory responses. Analysis of clinical transplantation data linked low circulating levels of GDF15 to an increased incidence of biopsy-proven acute rejection. Conclusions Gdf15 contributes to an early acting, renoprotective injury response, modifying immune cell ac- tions. The data support further investigation in clinical model systems of the potential benefitfromGDF15 administration in situations in which some level of tubular injury is inevitable, such as following a kidney transplant.

JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019090876

AKI is associated with significant morbidity and Received September 4, 2019. Accepted December 23, 2019. – mortality, with mortality rates up to 50% 70% in Published online ahead of print. Publication date available at critically ill patients with AKI.1–3 Even mild AKI is www.jasn.org. associated with a significant increased risk in in- Correspondence: Dr Andrew P. McMahon, Department of Stem hospital mortality.4 Renal ischemia-reperfusion in- Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, 1425 jury (IRI) frequently triggers proximal tubular San Pablo Street, Los Angeles, CA 90089. Email: amcmahon@ damage leading to the clinical syndrome of AKI in med.usc.edu varied settings including kidney transplantation,5 Copyright © 2020 by the American Society of Nephrology

JASN 31: ccc–ccc,2020 ISSN : 1046-6673/3104-ccc 1 BASIC RESEARCH www.jasn.org cardiothoracic6 and aortic surgery,7 and sepsis.8 Currently, no Significance Statement therapies exist to prevent or treat established AKI. Identifying potential therapeutic targets is an imperative to enable the Gdf15, which encodes a signaling factor activated by oxidative development of strategies to prevent or ameliorate AKI and stress, DNA damage, and proinflammatory cytokines, is upregu- its sequelae in high-risk patients and those initiating AKI. lated in the human and mouse kidney within a few hours of ischemia-reperfusion injury. Using novel mouse strains, the authors In a previous report, we coupled translating ribosome af- mapped cellular sites of Gdf15 expression in normal and injured finity purification (TRAP) methodology to a bilateral renal IRI kidney and examined Gdf15’s role in ischemia-reperfusion injury. model to characterize early translational profiles in distinct They showed that Gdf15 is expressed within hypoxic regions of the cell lineages of the kidney.9 These studies identified Gdf15, kidney and is predominantly activated within tubular epithelial cells Gdf15 which encodes a divergent member of the TGFb superfamily at injury repair sites; loss of exacerbated injury, enhancing the inflammatory response. In an analysis of clinical data, they 10,11 of factors, as a rapidly induced target of IRI, demonstrated that single nucleotide polymorphisms linked to activated to high levels within a few hours of vascular release. lower circulating GDF15 levels associate with an increased in- Analysis of biopsy specimens from human kidney transplants cidence of biopsy-proven acute rejection. These findings point to also showed the strong induction of Gdf15, within a similar modulating GDF15 levels in patients receiving kidney transplant as time frame, after establishment of the renal vascular circuit,12 a possible therapeutic strategy. and in kidney transplants with delayed graft function.13 Thus, transcriptional activation of Gdf15 is a conserved response injury and inflammation post-IRI. Further, genetic associa- to ischemia-invoked renal stress and tubular damage. Inter- tion studies suggest that lower circulating GDF15 levels link estingly, circulating GDF15 levels have been associated with an to an elevated risk of rejection in patients receiving kidney increased risk of CKD progression14 and elevated Gdf15 ex- transplant. pression is evident in our group’s study of a long-term murine IRI model that replicates the transition from an AKI to the onset of CKD.15 METHODS Gdf15, also known as macrophage inhibitory cytokine 1 (MIC-1),10 placental TGF b (PTGFb),16,17 and nonsteroidal Targeting lacZ anti-inflammatory drug–activated gene 1 (NAG1),17 was first The ES cell clone for Gdf15 was obtained from the Knock- identified as an autocrine regulatory molecule associated with Out Mouse Project (KOMP) Repository (www.komp.org).37 macrophage activation.10 Gdf15 is known to be induced in The vectors for Gdf15 targeting (pL1L2_GT1_LF2A_nEGFPO_ macrophages by proinflammatory cytokines including T2A_CreERT_puro) and dual-recombinase (pDIRE(i- TNFa,IL1b, and IL6, but not by IFNg or LPSs.10 Avariety CRE&Flpo)) were generated in collaboration with scientists in of stressors including inflammation,18–20 oxidative stress,21 the European Conditional Mouse Mutagenesis consortium at DNA damage,19,20,22–24 and integrated stress response25 the Sanger Institute (www.knockoutmouse.org/about/ also activate Gdf15 expression. Further, NF-kB(NFk-light- eucommtools). The ES targeting procedure was performed ac- chain-enhancer of activated B cells)-invoked Gdf15 expression cording to a previously published protocol.38 Correct targeting has been linked to the suppression of macrophage surveillance was confirmed by 59 and 39 PCR with primers: 59 forward: in pancreatic tumors.26 atcggaaatctgacgcaatc; 59 reverse: gttgtgccggatcttgaagt; 39 for- Functional studies in several organ systems have given am- ward: ggcattatttaaagttaggcgcg; 39 reverse: gcttgcacatccattccttc. biguous insights into the actions of Gdf15. GDF15 is reported to exhibit a cardio-protective effect in mouse models of Animal Experiments myocardial IRI.27,28 However, no clear role was observed for Mouse husbandry, handling, and surgical procedures were endogenous Gdf15 after ischemic liver,29,30 cerebral,31 and performed according to the guidelines issued by the Institu- kidney injury.29 The disparate outcomes may reflect actual tional Animal Care and Use Committee (IACUC) at the Uni- differences between organ systems or experimental differences versity of Southern California with protocol number 11911. among these studies in injury-invoked responses. Although Mouse line Gt(ROSA)26Sortm14(CAG-tdTomato)Hze was obtained nuGFP-CE Gdf15 was first identified in 1997,10 a GDF15 receptor was from the Jackson Laboratory (#007914). Gdf15 mouse only reported in 2017.32–36 The receptor termed GFRAL acts line maintained on a C57BL/6 background has been submitted in the brainstem to regulate appetite-associated body to the Jackson Laboratory (#034497). Warm renal IRI was weight.32–36 Whether GFRAL or a different receptor acts to performed on 10–12-week-old (25–28 g) male mice. The mediate GDF15 actions in stress-related responses is not clear. IRI procedure was the same as described in the published pro- Here, we validate new mouse strains that will be of broad tocol15 except that both renal pedicles were clamped for 11.5, utility to the study of Gdf15-andGdf15-transcribing cell types, 15 and 19 minutes. in normal and injured mouse kidneys. The genetic tools en- coded by these strains have facilitated a characterization of Serum and Urine Analysis normal and AKI-associated Gdf15 activity. Functional studies Serum creatinine levels were measured by the UT Southwest- demonstrate that Gdf15 deficiency enhanced renal tubular ern Medical Center O’Brien Center for Kidney Disease

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Research (Dallas, TX) using capillary electrophoresis (PA800 mismatch.44,45 The study was approved by the institutional Plus Pharmaceutical Analysis System; Beckman Coulter). review boards of the Medical University of Vienna and the BUN was assayed at the George M. O’Brien Kidney Center Institute for Clinical and Experimental Medicine in Pra- at Yale University using the BUN Procedure No. 0580 for the gue, respectively (EK267/2011, G 05– 04– 03 and A quantitative colorimetric assay with the readout performed on 13–02–01 [83/13]). Hardy–Weinberg equilibrium of vari- a Stanbio Excel analyzer. Urine albumin was assayed using the ants was checked by chi-squared test with continuity Mouse Albumin ELISA Quantitation Set (Cat. No. E90–134; correction. Bethyl Laboratories) on a BIO-RAD microplate reader. The association of rs749451 and rs888663 with the clinical outcome of biopsy-confirmed acute rejection/T cell–mediated qPCR rejection (BCAR/TCR) within the first year after transplanta- Total RNA was prepared from whole kidney tissues using tion was assessed in first-time deceased-donor kidney trans- RNeasy mini kit (Qiagen). cDNA was synthesized with Super- plant recipients who reached primary graft function after script Vilo kit (Thermo Fisher Scientific). qPCR was done transplantation. Odds ratios (ORs) were used to measure the with SYBR green on Applied Biosystems 7500 fast real-time association between specific genotypes and occurrence of PCR system machine according to the manufacturer’sprotocol. BCAR/TCR. Kaplan–Meier analysis and Cox proportional The relative mRNA expression of all was calculated by the hazard modeling were used to assess the association of geno- 2 2 DDCt method using Gapdh as reference. qPCR primers were types with the time to the first BCAR/TCR occurrence. Log- following the online resource: https://pga.mgh.harvard.edu/ rank test was used to test differences between survival curves. primerbank/. The Cox proportional hazard model was adjusted for clinical risk factors (HLA eplet mismatch, recipient and donor sex, Immunofluorescence and donor age). Frozen sections were prepared and stained as described in the published protocol.15 The following antibodies were used in Statistical Analyses this study, recognizing Havcr1 (#AF1817, Kidney injury Quantification values for immunostaining were represented as molecule–1 [KIM-1], goat, 1:1000; R&D Systems), Acta2 histograms and mean6SEM. The significance of difference (a-SMA) (C6198, smooth muscle actin–Cy3, mouse mono- among groups was examined using unpaired two-tailed clonal; Sigma), Cd3 (#ab16669, rabbit; Abcam), Cd4 t test. A P value of ,0.05 was considered significant. All anal- (#550280, rat; BD Pharmingen), Ptprc (#AF114, CD45, goat; yses were on the basis of three independent repeats of exper- R&D Systems), Adgre1 (14–4801, F4/80, rat; eBiosciences), iments with 3–6 biologic replicates. Ptprc (#557390, CD45R (B220), rat; BD Pharmingen), Calb1 (Calbindin-D-28K) (C9848, mouse; Sigma), LTL Study Approval lectin-FITC conjugate (#FL-1321; Vector Laboratories), All surgical procedures and all mouse handling and husbandry Aqp1 (#sc-20810, rabbit; Santa Cruz), Aqp1 (#sc-25287, were performed according to guidelines issued by the IACUC mouse; Santa Cruz), GFP (#AB16901, chicken; Chemicon), at the University of Southern California, and were performed Slc5a2 (#ab85626, rabbit; Abcam), Car4 (#AF2414, goat; on approval of each institution’sIACUC. R&D), Aqp2 (#sc-9882, goat; Santa Cruz), Atp6v1b1 (ab192612; Abcam), and EDU (#900584; Sigma), and were i.p. injected at 40 mg per kg body wt 3 hours before kidney RESULTS collection and analyzed with the Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (#C10640; Thermo Fisher Scientific). Gdf15 Upregulation after Ischemic AKI Fluorescent images were acquired on a Zeiss Axio Scan Z1 In earlier work, we identified cell type–specific responses in slide scanner and Zeiss LSM780 and Leica SP8 confocal acute ischemic AKI by performing TRAP within distinct cell microscopes. lineages of the mammalian kidney 24 hours post-IRI.9 From a more extended TRAP analysis examining the nephron in 1 1 Genetic Polymorphism Association Six2TGC/ ;Gt(ROSA)26Sortm9(EGFP/Rpl10a)Amc/ mice 4 hours The generation and analysis of human sequence datasets has post–vascular release by microarray profiling of the nephron been described previously.39 In short, kidney transplant do- response,9 Gdf15 emerged as a target-of-interest, strongly ac- nors and recipients were genotyped using a transplant-specific tivated as early as 4 hours post-IRI (Figure 1A). A follow-up gene array (Axiom Tx GWAS array; Affymetrix, CA). Haplo- whole-kidney RNA-seq transcriptome analysis15 showed a types were phased using SHAPEIT and imputed with spike of Gdf15 mRNA expression within the first few hours IMPUTE2 using 1000 Genomes Project phase 3 and Genome (2–4 hours) post-IRI, preceding a significant elevation of se- of the Netherlands v5 data as reference panels.40–43 SNP2HLA rum creatinine levels and before the activation of Havcr1 and was used for the imputation of four-digit HLA types on the Lcn2, two genes explored as early diagnostic markers of AKI basis of the Type 1 Diabetes Genetics Consortium reference (Figure 1B).15 Gdf15 levels tracked with the normalization of panel and HLAMatchmaker was used to compute HLA eplet creatinine levels by 14 days post-IRI, but remained elevated

JASN 31: ccc–ccc,2020 Renoprotective Activity of GDF15 3 BASIC RESEARCH www.jasn.org

A Gdf15 B ** 12 1.8 ** 1.6 3000 10 1.4 8 2000 1.2 6 1 1000 0.8 4 0.6

0 (IRI vs Sham) 2 0.4 Nephron specific mRNA

microarray probe intensitiy 0.2

0 Serum Creatinine (mg/dl) RNA seq log2 Fold Change

Sham 0 Sham 2h 4h 24h 48h 72h 7d 14d 28d -2 -0.2 IRI 4 hours No Surgery

IRI 24 hours Havcr1 Lcn2 Gdf15 SCr.

C Gdf15 **** 30

20

10

0 whole kidney RNA-seq RPKM of kidney transplant Pre Post 1 year 3 months

Figure 1. Expression of Gdf15 transcripts was upregulated post ischemia in mouse and human. (A) Microarray intensity of Gdf15 on 9 the basis of the published nephron-specificmRNAprofile post-IRI. (B) The log2 fold change (IRI versus Sham) of Gdf15 on the basis of the RPKM of the published whole-kidney RNA-seq.15 (C) RPKM value of human kidney transplant on the basis of the published RNA-seq.12 **P,0.01; ****P,0.0001. SCr., serum creatinine. in this severe damage model, where repair is only partially transition which was carried out in the C57BL6 mouse effective (Figure 1B).15 In addition, RNA-seq analysis on pro- strain.15 tocol biopsy specimens obtained from 42 kidney transplant To examine the cellular distribution of Gdf15 expres- nuGFP-CE recipients showed elevated levels of Gdf15 transcripts within sion, Gdf15 mice were crossed to a TDT reporter 1.5 hours post-transplantation (Figure 1C). mouselineGt(ROSA)26Sortm14(CAG-tdTomato)Hze, hereafter referred to as R26TDT. Gdf15 locus–derivedGFPandTDT Localization of Gdf15 Expression in the Mouse Kidney activity faithfully recapitulated Gdf15 expression analysis To examine in detail the cellular distribution and fate of by in situ hybridization46 and RNA-seq analysis of dissected Gdf15-expressing cells, we obtained embryo stem cells nephron segments.47 In the uninjured adult kidney, nu- 1 2 (ESCs) from KOMP (www.komp.org) in which homologous clear GFP localized predominantly in Krt8/18 ,Aqp1 recombination was used to introduce a lacZ gene cassette into cells of the thin descending limb of the loop of Henle lacZ the Gdf15 locus (Gdf15 ). Insertion of the cassette blocks (tDLH) connected to nephrons with cortically positioned production of endogenous GDF15 from the targeted allele and glomeruli (Figure 2, C and D, Supplemental Figure 1, A and 1 is predicted to result in a lacZ expression mirroring the normal B). Most Aqp1 and Lotus Tetragonolobus Lectin activity of Gdf15 (Figure 2A).37 Recombination-mediated (LTL)–bound cells of adjacent S3 segments of the proximal lacZ/1 cassette exchange38 was performed on Gdf15 ES cells, tubule in the outer stripe of outer medulla (OSOM) showed replacing lacZ sequences with a nuclearGFP-F2A-CREERT2- no GFP activity (Figure 2, C and D, Supplemental Figure 1, nuGFP-CE encoding expression cassette resulting in a Gdf15 allele AandB). (Figure 2B). Expression of this cassette is predicted to enable CRE-recombination can be a more sensitive indicator of visualization (nuclear GFP, “nuGFP” in the allele designation) locus activity than locus-driven GFP reporters. To examine and conditional genetic modification (tamoxifen-dependent CRE activity, a single intraperitoneal injection of a high dose CRE recombination; CRE::ERT2, “CE” in the allele designa- of tamoxifen (150 mg/kg body wt) was given to adult 1 1 1 tion) in Gdf15-expressing cell types. Importantly, the inbred Gdf15nuGFP-CE/ ;R26TDT/ mice and TDT cells, indicative C57BL6 background of targeted ESCs enhances phenotypic of Gdf15-driven CE activity, were scored 48 hours later. TDT reproducibility and facilitates comparative analysis with rele- and GFP colabeled cells were observed in the tDLH (Figure 2, vant studies such as a transcriptional analysis of the AKI-CKD C and D, Supplemental Figure 1, A–D)butalsoinseveral

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A ATG TGA B

Gdf15 5’ 3’ Exon1 Exon2

Gdf15lacZ (www.komp.org) 3kb En2 SA IRES lacZ pA neoR pA 2kb 1kb 1kb Exon1 FRT loxP FRTloxP Exon2 loxP 0.5kb

Gdf15nuGFP-CE

En2 SA p2A Nuclear GFP F2ACreERT2 pA puroR pA Exon1 FRT Rox Rox loxP

C TDT GFP Krt8 Merge with Dapi Outer medulla tDLH

D TDT GFPAqp1 Merge with Dapi Outer medulla tDLH

E TDT GFPAqp1 Merge with Dapi OSOM Proximal tubule S3

F 200

150 Proximal tubule S1 Proximal tubule S2 100 Proximal tubule S3 Aqp1- tDLH

per 40X field 50 Collecting duct

# of reporter positive cells 0 GFP TDT TDT 0 12.5 150 Tamoxfien(mg/kg):

Figure 2. Nuclear GFP and TDT reporter activities reflected the endogenous expression of Gdf15 in Gdf15nuGFP-CE mouse line. (A) Schematic diagram of Gdf15 knock-in allele. (B) PCR verification of the targeted allele using primer pairs indicated by black arrows in 1 2 1 (A). Confocal immunofluorescence showed nuclear GFP and TDT in Krt8 ,Aqp1 tDLH (C and D); and TDT but not GFP in Aqp1 proximal tubule S3 segments in the OSOM (E). (F) Quantification of GFP- and TDT-positive cells in different nephron segments per 340 field of confocal microscopy. Scale bar in (C–E), 20 mm.

JASN 31: ccc–ccc,2020 Renoprotective Activity of GDF15 5 BASIC RESEARCH www.jasn.org

2 1 GFP cell types of the nephron and collecting system: (1) (ISOM; Supplemental Figure 1E); (4)Slc5a2 cells of S1 1 1 Aqp1 /LTL S3cellsoftheproximaltubule(Figure2E); region of the proximal tubule (Supplemental Figure 1F); 1 1 (2)Aqp2 principal cells in medullary collecting ducts and (5)Aqp1 tDLH cells in the OSOM (Supplemental 2 1 (Supplemental Figure 1, D and E); (3)Aqp2 /Krt8/18 cells Figure 1G). Thus, CRE activity likely reveals lower levels of of the thin loop of Henle in the inner stripe of outer medulla Gdf15 activity outside of the tDLH. Consistent with this

A 48 hours C GFP Slc5a2 Merged with TDT 2.5 * ns 2.0 ns 1.5 Cortex 1.0

0.5 Proximal tubule S1 0.0 D GFP Car4 Merged with TDT Serum Creatinine (mg/dl) Sham 15min 19min 11.5min Cortex B qPCR at 48 hours Havcr1 Col3a1 Proximal tubule S2 * GFP Havcr1 Merged with TDT 200 * 8 * E * * 150 6 * 100 4 50 2 OSOM 0 0 Proximal tubule S3 Fold change IRI vs Sham Fold change IRI vs Sham Sham Sham 15min 19min 15min 19min F GFP Aqp1 Merged with TDT 11.5min 11.5min

Gdf15 Acta2 * * 4 6

* * tDLH 3 ns ns 4 2 Outer medulla 2 1 G GFP Calb1 Merged with TDT 0 0 Fold change IRI vs Sham Fold change IRI vs Sham Sham Sham 15min 19min 15min 19min 11.5min 11.5min Cortex

I connecting tubue Proximal tubule S1 H GFP Aqp2 Merged with TDT Proximal tubule S2 Proximal tubule S3 Aqp1+ tDLH Collecting duct Connecting tubule Medulla collecting duct

Figure 3. Gdf15 was upregulated in renal epithelial tubules at 48 hours postischemia. (A) Serum creatinine level at 48 hours post sham and 11.5-, 15-, and 19-minute ischemia. *P,0.05. NS, not significantly changed. (B) Quantitative PCR showing fold changes of Havcr1, Gdf15, Col3a1,andActa2 after IRI compared with sham. Confocal immunofluorescence showed nuclear GFP and tamoxifen- 1 1 1 induced TDT in (C) Slc5a2 cortical proximal tubule S1; (D) Car4 cortical proximal tubule S2; (E) Havcr1 proximal tubule S3 in the 1 1 1 OSOM; (F) Aqp1 tDLH in the outer medulla; (G) Calb1 cortical connecting tubule; and (H) Aqp2 collecting duct. (I) Pie chart 1 displays the distribution of number of GFP cells per 340 field of confocal microscopy among different nephron segments. Scale bar in (A–F), 20 mm.

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A C Sham Proximal tubule S3 in the OSOM

TDTKrt8 Aqp1 Merge with Dapi Sham

48 hours 28 days 28

Proximal tubule S3 in the OSOM the in S3 tubule Proximal 28 days D tDLH in the OSOM

TDT Krt8 Aqp1 Merge with Dapi Sham

B Sham 28 days 28

48 hours E tDLH in the OSOM

Acta2 (αSMA) Ptprc (CD45) Krt8 Merge with Dapi

Sham tDLH in the OSOM the in tDLH

28 days 28 days 28

Figure 4. Proximal tubule S3 and tDLH in the OSOM marked by Gdf15 reporter showed distinct cell fates after IRI. (A and B) H&E staining on kidneys collected at 48 hours and 28 days after sham and 15-minute ischemia. (A) Proximal tubule S3; (B) tDLH. (C–E) Confocal immunofluorescence on kidneys collected at 28 days post sham and IRI. Tamoxifen was injected at 48 hours after IRI. 1 Gdf15-expressing cells represented by tamoxifen-dependent TDT in the proximal tubule S3 (C); and Aqp1 tDLH cells costained with 1 1 Krt8 (green) (D) that were surrounded by Acta2 (aSMA) and Ptprc (CD45) cells (E). Scale bar in (A and B), 10 mm; in (C–E), 20 mm.

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ns ns A ns ns B 0.6 ns ns 80 ** **

60 0.4 40 0.2 20 Serum Creatinine (mg/dl)

0 Blood Urea Nitrogen(mg/dl) 0 Wt Wt Wt Wt KO KO KO KO Het Het Het Het Sham Sham Sham Sham 11.5min 15min 11.5min 15min

C D E Havcr1 ns ** * ns ns ns 250 40 *** ** 80 *** ** **** ** ** ** 200 30 60 150 20 40 100

10 20 50

0 Number of EDU+ cells 0 0 Number of Havcr1+ tubules qPCR Fold Change IRI vs Sham Wt Wt Wt Wt Wt Wt Wt Wt Wt KO KO KO KO KO KO KO KO KO Het Het Het Het Het Het Het Het Het Sham Sham Sham Sham Sham Sham Sham 11.5min 15min Cortex OSOM ISOM Cortex OSOM ISOM 11.5min 11.5min

F Col1a1 G Col3a1 H Acta2 * ns * ns ns ns 30 40 4 ** ns ** * 30 3 * * 20 20 2 10 10 1

0 0 0 Wt Wt Wt Wt Wt Wt qPCR Fold Change IRI vs Sham qPCR Fold Change IRI vs Sham qPCR Fold Change IRI vs Sham KO KO KO KO KO KO Het Het Het Het Het Het Sham Sham Sham Sham Sham Sham 48 hours 28 days 48 hours 28 days 48 hours 28 days 15min 15min 15min

Figure 5. GDF15 deficiency aggravated acute tubular injury. IRI in wild-type and GDF15-deficient mice. Creatinine (A) and BUN (B) 1 were measured with serum samples collected at 48 hours post 11.5- and 15-minute ischemia. Quantification of number of Havcr1 (C) 1 tubules and EDU cells (D) per 340 field of confocal microscopy at 48 hours post 11.5-minute ischemia. (E) qPCR for Havcr1 with whole-kidney samples at 48 hours post 11.5- and 15-minute ischemia. (F–H) qPCR for Col1a1, Col3a1,andActa2 at 48 hours and 28 days post 15-minute ischemia. *P,0.05; **P,0.01; ***P,0.001. NS, not significantly changed. Het, heterozygous; KO, knockout; Wt, wild type. view, when the tamoxifen dosage was markedly decreased to segments, quantifying the distribution of each cell type in 12.5 mg/kg body wt, TDT activity was predominantly re- particular target cell populations. stricted to the S3 region of the proximal tubule and the tDLH, more similar to the nuGFP reporter (Supplemental Gdf15 Upregulation in Defined Renal Compartments Figure 2, A–D). Without tamoxifen, around 0.2% of proxi- after IRI 1 nuGFP-CE mal tubular cells in S3 segments were TDT only in the Before examining the postischemia response with Gdf15 Gdf15nuGFP-CE/nuGFP-CE homozygous kidneys, suggesting mice, we first calibrated IRI responses in 10–12-week old minimal leakiness (data not shown). Figure 2F summarizes male C57BL/6J mice. Three cohorts were subjected to differ- the GFP and TDT reporter activity in multiple nephron ent periods of ischemia (11.5, 15, or 19 minutes) and serum

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A Sham WT D

* 500 * 400

300

HET KO 200

100

0 number of Cd45 (Ptprc) positive cells per 40X field in around cortical vessels Sham WT HET KO N=6 N=9 N=6 N=16

B Cd3 Ptprc(CD45) Pecam1 Sham WT HET KO

C HET KO Cd3 Ptprc(CD45) Cd3 Ptprc(CD45)

Ptprc(CD45R(B220)) Merge Ptprc(CD45R(B220)) Merge

Figure 6. GDF15 deficiency enhanced adaptive immunity. Kidneys from wild-type, heterozygous, and homozygous mutant mice were collected 28 days after sham and 15-minute ischemia. (A) H&E staining. (B–C) Confocal immunofluorescence showed merged (B) and 1 separate channels (C) in the aggregates near the cortical vessels. (D) Quantification of number of Ptprc (CD45) cells per 340 field of confocal microscopy in the aggregates. *P,0.05. Scale bar in (A–D), 20 mm. HET, heterozygous; KO, knockout; WT, wild type.

JASN 31: ccc–ccc,2020 Renoprotective Activity of GDF15 9 BASIC RESEARCH www.jasn.org

Table 1. Clinical covariables by genotypes of rs749451 rs749451 Characteristic CC CT TT Number of recipients 169 283 100 Recipient age, yr 54.25 (46.08–62.12) 57 (46.00–64.15) 56.76 (41.37–63.86) Recipient (female) 62 (37%) 99 (35%) 42 (42%) Donor age, yr 51 (41.00–61.00) 53 (43.00–61.00) 52 (37.00–60.00) Donor (female) 64 (38%) 102 (36%) 42 (42%) Follow up, yr 5.67 (2.99–7.25) 5.17 (2.67–7.25) 5.67 (2.81–7.92) HLA serotype mismatch 3 (2–4) 3 (2–4) 3 (2–4) HLA eplet mismatcha 41 (31.75–56.00) 42 (32.00–54.00) 38 (30.00–53.25) Highest PRA% 4 (0.00–20.00) 4 (0.00–21.00) 4 (0.00–21.75) TCR within first yr 21 (12%) 53 (19%) 24 (24%) Patient characteristics are described by median and first and third quartile for continuous variables or by frequency and percentage for binary variables. PRA%, panel-reactive antibodies. aAvailable for 477 donor-recipient pairs. creatinine levels were measured 48 hours after vascular asignificant elevation of Gdf15 and Acta2 expression that release (Figure 3A). Whereas all time points led to signif- was not observed in 11.5-minute IRI kidney analysis, and icant elevation of Havcr1 and Col3a1 mRNA levels 48 hours which was more marked in the 19-minute IRI sample postreperfusion, consistent with renal injury and an early (Figure 3B). Hereafter, we refer to the characterized injury fibrotic response, only the 19-minute IRI resulted in a ro- evoked by different periods of ischemia as mild (11.5 bust and significant elevation in serum creatinine levels minutes), moderate (15.0 minutes), and severe (19.0 (Figure 3B). The 15- and 19-minute IRI also resulted in minutes).

Strata rs749451=CC rs749451=CT rs749451=TT 1.00

0.75

0.50 Rejection free survival 0.25 p = 0.048

0.00

0 100 200 300 400 Time (days) Number at risk rs749451=CC 169 149 147 145 0 rs749451=CT 283 231 222 218 0

Strata rs749451=TT 100 80 74 73 0 0100 200 300 400 Time (days)

Figure 7. GDF15-related single nucleotide polymorphisms are associated with acute rejection episodes after kidney trans- plantation. Kaplan–Meier plot of BCAR stratified for rs749451 genotypes. The P value was derived from the log-rank test.

10 JASN JASN 31: ccc–ccc,2020 www.jasn.org BASIC RESEARCH

1 1 Compound heterozygous Gdf15nuGFP-CE/ ; R26TDT/ male Gdf15nuGFP-CE/nuGFP-CE (KO) kidneys 48 hours post-IRI. mice were subjected to severe ischemia (19 minutes) and a BUN but not serum creatinine levels were significantly ele- single low dose of tamoxifen (12.5 mg per kg body wt) admin- vated in wild-type mice 48 hours after initiation of both mild istered shortly after surgery. Analysis 48 hours post injury of and moderate IRI (Figure 5, A and B). However, quantitative kidneys showed nuclear GFP and TDT reporter activity in PCR confirmed an enhanced injury in Gdf15 KO kidneys multiple cell types within renal tubular segments identified after mild IRI and immunostaining suggested increased in- by coanalysis with antibodies recognizing key cell markers in- jury within cortical PTregions (Figure 5, C–E, Supplemental 1 cluding: (1)Slc5a2 in the S1 segment of the proximal tubule; Figure 4). However, examining mild IRI–invoked cell pro- 1 (2) Car4 cells in the S2 segment of the proximal tubule; (3) liferation by EDU labeling of S-phase nuclei 45 and 48 hours 1 1 Havcr1 cells in the S3 segment of the proximal tubule in the post-IRI showed a striking increase in EDU cells in the 1 OSOM; (4)Aqp1 cells in the outer medullary tDLH; (5) medullary region of KO kidneys (Figure 5D, Supplemental 1 1 1 Calb1 (Calbindin D28K) cells in the connecting tubules Figure 4). DNA replication was observed in Aqp1 ,Havcr1 1 1 (CNT) in the cortex; and (6)Aqp2 principal cells in the proximal tubule cells; Pdgfrb interstitial cells; Ptprc 1 1 medullary collecting ducts (Figure 3, C–H). Taken together, (CD45) immune cells (data not shown); and Aqp2 prin- these results showed a marked upregulation of Gdf15 expres- cipal cells in the epithelium of the medullary collecting duct sion in proximal tubule segments, tDLH, and distal domains (Supplemental Figure 5). Thus, loss of Gdf15 activity results of the nephron, and principal cells of the collecting duct. in distinct cellular responses within several different regions and cell populations within the kidney 2 days after an initi- Fate of Gdf15-Expressing Cells within Ischemic ating mild ischemic episode. Stressed Segments of the Nephron Moderate IRI (15 minutes) in Gdf15 KO kidneys signifi- To examine the fate of cells activating Gdf15, we performed a cantly increased Havcr1, Col1a1, Col3a1,andActa2 levels at 1 1 15-minute bilateral IRI on adult Gdf15nuGFP-CE/ ; R26TDT/ 48 hours although no long-lasting difference could be scored mice to invoke a moderate injury then injected tamoxifen at by qPCR analysis 4 weeks post-IRI (Figure 5, E–H). However, 48 hours (12.5 mg/kg) post vascular release, then visualized a marked increase in lymphocyte aggregation was observed 1 TDT cells in kidneys 28 days post-IRI. around blood vessels in five of 16 (31.3%) KO mutant mice Histologic analysis showed extensive sloughing off of cells (Figure 6, Supplemental Figure 6). These aggregates contained 1 1 in the S3 region of the PT and tDLH cells 2 days after IRI Cd3 T (Figure 6, B–D) and Ptprc (CD45R) B cells sugges- (Figure 4, A and B, Supplemental Figure 3A). However, by tive of tertiary lymphoid organs arising in kidney transplants 28 days post-IRI, much of the epithelium of the S3 region (Figure 6, B and C)48 that are also observed in the transition was histologically normal, although some cells contained from acute injury to chronic disease in mouse studies.15,49,50 two nuclei (yellow arrows in Figure 4A), and others exhibited Together these data suggest that Gdf15 has an early renal pro- an abnormally light staining of nuclei and cytoplasm (black tective response which in the longer term suppresses arrows in Figure 4A). Compared with the sparse contribution inflammation. 1 1 of TDT cells to the Aqp1 S3 segment of the proximal tubule after sham-surgery, there was a marked increase in the percentage Association between GDF15-Related Single 1 of TDT cell clusters in S3 segments 28 days post-IRI (Figure 4C). Nucleotide Polymorphisms and Acute Rejection 1 1 2 Further, many of these TDT cells were Aqp1 /Krt8 in- Episodes after Kidney Transplantation dicative of repair and recovery of the S3 segment. In contrast, To investigate the clinical relevance of our experimental find- 1 in focal regions at the proximal end of the Apq1 tDLH in ings, we examined genetic polymorphisms in a cohort of 522 the outer medulla, the lumen of the nephron was markedly recipients who received a first deceased-donor kidney trans- enlarged (Figure 4, B–E, Supplemental Figure 3). Here, plant from two transplant centers in Vienna and Prague. 1 1 TDT cyst-like tubular structures showed ectopic Krt8 ,a Among the single nucleotide polymorphisms previously asso- hallmark of maladaptive repair.15 These regions were sur- ciated with the circulating concentration of GDF15,51 we se- 1 rounded by clusters of Acta2 myofibroblasts and exhibited lected rs749451 and rs888663. Considering the low linkage amarkedinfiltration of Ptprc (CD45)-positive immune cells disequilibrium between the two single nucleotide polymor- (Figure 4E). Collectively, these findings indicate that activa- phisms (0.43), we performed two independent analyses in tion of Gdf15 occurred within cells that participated sub- which we stratified the study population on the basis of their stantially in normal repair in some nephron segments as well genotype into GDF15 high and low expressers and asserted the as in segments where compromised epithelial repair was as- differences in BCAR/TCR between the groups. Because of the sociated with inflammation and renal fibrosis. low minor allele frequency (20%) of rs888663, and because only 23 recipients with the GG genotype were available for GDF15 Deficiency Aggravated Acute Tubular Injury analysis, we thus focused on rs749451. The clinical character- and Enhanced Inflammation Postischemia istics of the groups were similar, except for the number of To explore the role of Gdf15, we examined the effects of recipients who had a BCAR/TCR within the first year, and mild (11.5 minutes) and moderate (15 minutes) IRI on are presented in Table 1. The genotypes in the study

JASN 31: ccc–ccc,2020 Renoprotective Activity of GDF15 11 BASIC RESEARCH www.jasn.org population did not violate the Hardy–Weinberg equilibrium Analysis of IRI in mouse kidneys in Gdf15 KOssupportsa (rs749451: P50.36; rs888663: P50.86). In both analyses, ho- renal-protective function for GDF15 attenuating fibrotic and mozygous carriers of the allele previously linked to a lower inflammatory responses. The appearance of multiple foci of GDF15 concentration in plasma (rs749451: T; rs888663: G) lymphocyte aggregates in kidneys several weeks after a mod- displayed a significantly higher incidence of biopsy-proven erate IRI episode resembles aggregates observed in human acute rejection episodes in the first year after transplantation. kidneys from patients with lupus nephritis62 and tertiary lym- Recipients with the rs888663 G/G genotype exhibited 35% phoid organs in human kidney allografts and mouse kidneys and those with the T/T genotype exhibited 16% of BCAR/ transitioning to CKD.48,50 Interestingly, the mouse studies TCR (OR, 2.85; 95% confidence interval [95% CI], 1.15 to may have clinical relevance. Kidney transplantation begins 7.03; P50.023). Likewise, recipients with the rs749451 T/T with IRI. The initial response to injury is important in mod- genotype exhibited 24% and with the C/C genotype 12% ulating the subsequent immune response, although how is of BCAR/TCR (OR, 2.23; 95% CI, 1.16 to 4.25; P50.016). not clear.49 An association between GDF15-linked genetic Kaplan–Meier analysis revealed that this effect was dose- polymorphisms influencing GDF15 serum levels in acute dependent (P50.048; Figure 7). A Cox model adjusted for transplant rejection supports further study of the potential HLA eplet mismatch, recipient and donor sex, and donor immunomodulatory action of GDF15.63–68 age found an 80% (hazard ratio 95% CI, 1.06 to 3.08; We attempted to directly test whether systemic delivery of P50.030) increased risk for BCAR/TCR in recipients carrying recombinant GDF15 might suppress IRI-invoked injury re- one or two copies of the Tallele. A higher immune reactivity in sponses. However, mice showed a dramatic weight loss before association with lower GDF15 levels is consistent with the IRI precluding further study. Recent studies have demon- more pronounced lymphocytic infiltrates observed after kid- strated that GDF15 binding to a hypothalamic localized ney injury in the mouse model. GFRAL triggers a receptor pathway leading to a rapid and marked reduction in food intake and body weight.32–36 This raises the possibility that an upregulation of GDF15 after kid- DISCUSSION ney injury might contribute to weight loss and cachexia after AKI and in the progression to CKD given the link between The normal kidney is constantly under metabolite-, toxin-, circulating GDF15 and CKD.14,69,70 and hypoxia-invoked stresses.52–55 Our genetic studies high- In terms of GDF15’s mechanism of action, we have failed to light Gdf15 as a stress response gene. Using a newly generated, detect mRNA for Gfral according to published renal micro- sensitive recombination-mediated reporter, we identified sev- array9 or whole-organ15 or single-cell60 RNA-seq datasets, eral normal domains of Gdf15 expression. The outer medul- raising the question as to whether IRI-induced GDF15 func- lary region is relatively hypoxic52,56 and the kidney region tions similarly as a hormone or through alternative receptors most sensitive to IRI-invoked cell death.57 Reporter activity outside of the brain. GDF15 has been reported to inhibit in- 2 indicated the strongest Gdf15 activity in Apq1 tDLH cells in tegrin activation and mouse neutrophil recruitment through this region associated with short loop cortical nephrons the TGF b receptor I (TGF-bRI) (–like kinase emerging later in kidney development58,59 and in the S3 seg- 5 [ALK-5]) and TGF-b receptor II (TGF-bRII) heterodimer,71 ment of the proximal tubule in the OSOM.52,56 These although a later in vitro receptor screen indicated only a low- findings are in good agreement with predicted expression affinity interaction with GDF15.32–36 in “KidneyCellExplorer” (https://cello.shinyapps.io/ Although the exact target of GDF15’s signaling function in kidneycellexplorer/), a single-cell RNA-seq–based molecu- modulating renal response to IRI remains to be determined, lar anatomic atlas of cell types in the adult mouse kidney the observed changes in the immune cell compartment sup- (Supplemental Figure 7).60 Anaerobic glycolysis may result port an immunomodulatory action consistent with studies in in the decrease of cellular pH in the hypoxic medullary re- other organ systems. In the mouse heart, GDF15 deficiency gion52 and Gdf15 expression was detected in principal cells resulted in an enhanced inflammatory cell infiltration in both in this region. transient27 and prolonged28 ischemia models, although only Surprisingly, even a moderate ischemic episode, which the former showed an enlarged infarct in the absence of does not significantly affect serum creatinine levels 48 hours GDF15. In the liver, GDF15 deficiency had no reported effect 30 postinjury, results in substantial induction of Gdf15 in multi- on acute injury invoked by a single high dose of CCl4 but ple segments of renal tubular epithelium. Gene expression exhibited an enhanced inflammatory response in chronic liver analysis of kidneys with delayed graft function confirmed a fibrosis resulting from repeated, low-dose administration of 12,13 72 similar induction in the human kidney. Acidosis had been CCl4 over 3 weeks. Furthermore, the recent report of a car- 1 shown to induce Gdf15 in the Aqp2 collecting duct epithe- dioprotectiveroleforGdf15 in sepsis supports a role for lium leading to the proliferation and expansion of the acid- GDF15 in promoting survival during acute inflammation secreting cellular pool.61 This is in line with the increased rather than direct pathogen control.73 Gdf15 reporter activity in the principal cells of the collecting The nuclear GFP and CRE-ERT2 reporter mouse line ducts and cortical connecting tubules postischemia. driven by Gdf15 provides a valuable sensitive genetic tool

12 JASN JASN 31: ccc–ccc,2020 www.jasn.org BASIC RESEARCH that will aid in further elucidation of Gdf15 pathway actions in Supplemental Figure 5. Prominent proliferation in the medulla in the kidney. Further, the strain will facilitate broader analysis of the GDF15-deficient kidneys 48 hours post 11.5-minute ischemia. stress-related events in the kidney and potentially other organs Supplemental Figure 6. Inflammatory cell infiltration in the kid- where Gdf15 activation is invoked early in the response to neys 28 days post 15-minute IRI. ischemic injury,28,29,31 idiopathic pulmonary fibrosis,74 and Supplemental Figure 7. Expression of Gdf15 in adult mouse septic AKI.75 scRNA-seq datasets.

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