CXCL6 Synthesized by Proximal Tubule Cells May Promote Fibrosis in Lupus Nephritis

Philip Carlucci¹, Nalani Sachan², Andrea Fava³, Brooke Cohen², Jasmine Shwetar⁴, Siddarth Gurajala⁵, Qian Xiao⁵, Joseph Mears⁶, Katie Preisinger², Devyn Zaminski⁷, Kristina Deonaraine⁸, Peter Izmirly⁹, Judith James¹⁰, Joel Guthridge¹⁰, Wade DeJager¹¹, David Wofsy¹², Cynthia Loomis², Gyles Ward², Ming Wu¹³, Chaim Putterman¹⁴, Deepak Rao¹⁵, Betty Diamond¹⁶, Derek Fine¹⁷, Jose Monroy-Trujillo¹⁷, H Michael Belmont⁷, William Apruzzese¹⁸, Anne Davidson¹⁹, Richard Furie²⁰, Paul Hoover²¹, Celine Berthier²², Maria Dall'Era²³, Diane Kamen²⁴, Kenneth Kalunian²⁵, Jennifer Anolik²⁶, Jennifer Barnas²⁷, Arnon Arazi²⁸, Soumya Raychaudhuri²⁹, Nir Hacohen³⁰, Robert Clancy³¹, Kelly Ruggles³², Michelle Petri³³ and Jill Buyon², and the Accelerating Medicines Partnership in RA/SLE, ¹New York University School of Medicine, New York, NY, ²NYU Grossman School of Medicine, New York, NY, ³Johns Hopkins University, Baltimore, MD, ⁴New York School of Medicine, Ann Arbor, MI, ⁵Harvard Medical School, Boston, MA, ⁶Michigan University, Ann Arbor, MI, ⁷NYU School of Medicine, New York, NY, ⁸Jacobs School of Medicine, University at Buffalo, Buffalo, NY, ⁹New York University Grossman School of Medicine, New York, NY, ¹⁰Oklahoma Medical Research Foundation, Oklahoma City, OK, ¹¹Oklahoma Medical Research Foundation, Oklahoma City, ¹²University of California San Francisco, SF, CA, ¹³Northwell, New York, NY, ¹⁴Albert Einstein College of Medicine, Safed, Israel, ¹⁵Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ¹⁶The Feinstein Institutes for Medical Research, Manhasset, NY, ¹⁷Johns Hopkins School of Medicine, Baltimore, MD, ¹⁸Brigham and Women's Hospital, Everett, MA, ¹⁹Feinstein Institutes for Medical Research, Manhasset, NY, ²⁰Northwell Health, Manhasset, NY, ²¹Brigham and Women's Hospital, SWAMPSCOTT, MA, ²²University of Michigan, Ann Arbor, MI, ²³UCSF, Corte Madera, CA, ²⁴Medical University of South Carolina, Charleston, SC, ²⁵University of California San Diego, La Jolla, CA, ²⁶University of Rochester Medical Center, Rochester, NY, ²⁷University of Rochester, Rochester, NY, ²⁸Feinstein Institutes for Medical Research, Melrose, MA, ²⁹Brigham and Women's Hospital, Boston, MA, ³⁰Broad Institute of MIT and Harvard, Boston, MA, ³¹Columbia University Medical Center, New York, NY, ³²NYU Grossman School of Medicine, Brooklyn, NY, ³³Johns Hopkins University School of Medicine, Timonium, MD

Meeting: ACR Convergence 2024

Keywords: chemokines, Fibroblasts, Other, Lupus nephritis, Nephritis, Systemic lupus erythematosus (SLE)

Session Information

Date: Monday, November 18, 2024

Title: SLE – Etiology & Pathogenesis Poster

Session Type: Poster Session C

Session Time: 10:30AM-12:30PM

Background/Purpose: Detection of urinary CXCL6, a member of the IL-8 chemokine family, has been linked to CKD and is a proposed marker of chronic damage in IgA nephropathy. While a direct role for lung epithelial derived CXCL6 on fibroblast activation has been described in pulmonary fibrosis, the mechanism by which CXCL6 may promote fibrotic changes in the kidney remains poorly characterized. Accordingly, this study leveraged the Accelerating Medicines Partnership (AMP) and NYU lupus nephritis (LN) cohorts to evaluate whether renal fibrosis associates with CXCL6 production by kidney epithelium, and if CXCL6 can activate renal fibroblasts in vitro.

Methods: LN patients were consecutively enrolled in AMP at the time of a renal biopsy and followed for one year. Dissociated biopsies were passed through a single-cell RNA sequencing (scRNAseq) pipeline and 1200 proteins were quantified in urine collected within 3 weeks of biopsy and at each visit thereafter. Additional biospecimens (urine, plasma, and kidney tissue) were prospectively collected from SLE patients in the NYU lupus cohort to corroborate AMP findings. Primary renal fibroblasts were stimulated in vitro with recombinant CXCL6 for 72 hours and SMAc induction was visualized with immunofluorescence.

Results: The AMP scRNAseq revealed expression of CXCL6 by proximal tubule (PT) cells and PT CXCL6 protein expression was confirmed by immunofluorescence (Fig 1A-C). The proportion of PT cells expressing CXCL6 significantly associated with biopsy chronicity (Fig 1D). Fibroblasts from biopsies with greater than 1% of PT cells expressing CXCL6 had higher average ACTA2 expression suggesting myofibroblast transdifferentiation (Fig 1E). The proportion of CXCL6 expressing PT cells significantly associated with matched urinary CXCL6 levels, but there was no correlation between CXCL6 quantified in matched plasma and urine samples from LN collected at NYU, suggesting intrarenal production as the source of urinary CXCL6 (Fig 1F-G). Urinary CXCL6 correlated with higher chronicity and decreased eGFR in both the AMP and NYU cohorts (Fig 2A-D). In the AMP study, patients classified as nonresponders to standard of care at 52 weeks had higher urinary CXCL6 at baseline, 12, 26, and 52 weeks compared to complete responders (Fig 2E). In the NYU cohort, urinary but not plasma CXCL6 was significantly higher in patients with current LN (UPCR >0.5) compared to LN patients in remission (UPCR < 0.25/normal creatinine, median time from biopsy = 9 years) and SLE nonrenal patients (Fig 2F-G). The association with current LN vs LN in remission/SLE nonrenal (as a combined variable) persisted after adjusting for GFR in a logistic regression (p=0.001). Stimulation of primary renal fibroblasts in vitro with recombinant CXCL6 resulted in the expression of SMAc protein suggesting fibroblast activation.

Conclusion: CXCL6 secretion by PT cells associates with LN biopsy chronicity and is higher in SLE patients with persistent proteinuria. Further, CXCL6 treatment results in renal myofibroblast transdifferentiation in vitro. These data suggest that PT to fibroblast crosstalk through a CXCL6 axis contributes to renal fibroblast activation and irreversible scar formation in LN.

Figure 1: (A) UMAP of the clusters identified in the AMP scRNAseq. (B) Feature plot showing the expression of CXCL6 in defined clusters. (C) Representative immunofluorescence staining of CXCL6 and LRP2 (a proximal tubule marker) in a lupus kidney biopsy, DAPI (blue), LRP2 (red), CXCL6 (green). (D) Spearman correlation between the proportion of CXCL6 positive proximal tubule PT cells by scRNAseq and biopsy chronicity, n=140 biopsies. (E) Average fibroblast ACTA2 expression in biopsies with less than 1% PT cells expressing CXCL6 (n=28 biopsies) compared to biopsies with greater than 1% PT cells expressing CXCL6 (n=56 biopsies). P-value generated using Wilcoxon rank-sum test; biopsies with less than or equal to five total fibroblasts were excluded. (F) Spearman correlation between the proportion of CXCL6 positive PT cells by scRNAseq and urinary CXCL6 protein abundance by multiplex proteomics, n=137 AMP patients. (G) Spearman correlation between plasma CXCL6 concentration and urinary CXCL6 abundance by ELISA in the NYU lupus cohort, n=22 NYU patients. TNK: T and natural killer, PT: proximal tubule, INTL: interstitial, GLOM: glomerular, LOH: loop of henle, DN: distal nephron

Figure 2: (A) Spearman correlation between biopsy chronicity and urine CXCL6 abundance in the AMP cohort, n=168 AMP patients. (B) Spearman correlation between biopsy chronicity and CXCL6 abundance in the NYU cohort, n=38 NYU patients. (C) Spearman correlation between estimated GFR and urine CXCL6 abundance in the AMP cohort, n=194 AMP patients. (D) Spearman correlation between estimated GFR and urine CXCL6 abundance in the NYU cohort, n=38 NYU patients. (E) Urine CXCL6 abundance at baseline, 12, 26, and 52 weeks in complete responders (red), partial responders (green), and nonresponders (blue) to standard of care in the AMP cohort. P-values generated using Wilcoxon rank-sum test compare complete vs nonresponders, *<0.05, **<0.01. (F) Plasma CXCL6 concentration in the NYU cohort comparing patients with current LN (n=22), LN in remission (n=8), and SLE nonrenal (n=9). P-values generated using Wilcoxon rank-sum test. (G) Urine CXCL6 abundance in the NYU cohort comparing patients with current LN (n=38), LN in remission (n=8), and SLE nonrenal (n=9). P-values generated using Wilcoxon rank-sum test.

Figure 3: Immunofluorescence of primary renal fibroblasts incubated for 72 hours in the following conditions: (A) unstimulated, (B) recombinant CXCL6 treatment, (C) recombinant TGF1 treatment. Cells were stained with SMAc (smooth muscle actin) according to an established protocol. Images are representative of one experiment performed in duplicate.

Disclosures: P. Carlucci: None; N. Sachan: None; A. Fava: Annexionbio, 2, Arctiva, 2, AstraZeneca, 2, Exagen, 5, Novartis, 6, UCB, 2; B. Cohen: None; J. Shwetar: None; S. Gurajala: None; Q. Xiao: None; J. Mears: None; K. Preisinger: None; D. Zaminski: None; K. Deonaraine: None; P. Izmirly: Hansoh Bio, 2; J. James: GlaxoSmithKlein(GSK), 1, Progentec Diagnostics, Inc., 5, 10; J. Guthridge: AstraZeneca, 5, Bristol-Myers Squibb(BMS), 5; W. DeJager: None; D. Wofsy: Amgen, 2, Novartis, 2; C. Loomis: None; G. Ward: None; M. Wu: None; C. Putterman: Equillium, 2, Progentec, 2; D. Rao: Amgen, 6, AnaptysBio, 2, AstraZeneca, 1, Bristol-Myers Squibb, 2, 5, GlaxoSmithKline, 2, HiFiBio, 2, Janssen, 5, Merck, 5, Scipher Medicine, 2; B. Diamond: adicet, 2, alpine, 12, dsmb, atara, 2, DBV, 2, icell, 2, sail, 2; D. Fine: None; J. Monroy-Trujillo: None; H. Belmont: Alexion, 1, Aurinia, 6; W. Apruzzese: None; A. Davidson: EMD Serono, 5; R. Furie: AstraZeneca, 1, 2, 6; P. Hoover: None; C. Berthier: None; M. Dall'Era: AstraZeneca, 2, Aurinia, 2, Genentech, 2, GlaxoSmithKlein(GSK), 2, Janssen, 2; D. Kamen: Alpine Immune Sciences, 1, Bristol Myers Squibb (BMS), 1; K. Kalunian: None; J. Anolik: None; J. Barnas: None; A. Arazi: None; S. Raychaudhuri: Janssen, 1, Mestag, 8, Nimbus, 2, Pfizer, 1, Sonoma, 8, Third Rock Ventures, 2; N. Hacohen: None; R. Clancy: None; K. Ruggles: None; M. Petri: Amgen, 2, AnaptysBio, 2, Annexon Bio, 2, Arthros-FocusMedEd, 6, AstraZeneca, 2, 5, Atara Biosciences, 2, Aurinia, 5, 6, Autolus, 2, Avoro Ventures, 2, Biocryst, 2, Boxer Capital, 2, Cabaletto Bio, 2, Caribou Biosciences, 2, CTI, 1, CVS Health, 1, Eli Lilly, 2, 5, Emergent Biosolutions, 1, Ermiium, 2, Escient Pharmaceuticals, 2, Exagen, 5, Exo Therapeutics, 2, Gentibio, 2, GlaxoSmithKlein(GSK), 2, 5, iCell Gene Therapeutics, 2, Innovaderm Research, 2, IQVIA, 1, Janssen, 5, Kira Pharmaceuticals, 2, Merck/EMD Serono, 1, Nexstone Immunology, 2, Nimbus Lakshmi, 2, Novartis, 2, PPD Development, 2, Precision Biosciences, 2, Proviant, 2, Regeneron Pharmaceuticals, 2, Sanofi, 2, Seismic Therapeutic, 2, Senti Bioscienes, 2, Sinomab Biosciences, 2, Takeda, 2, Tenet Medicines Inc, 2, TG Therapeutics, 2, UCB, 2, Vertex Pharmaceuticals, 2, Worldwide Clinical Trials, 1, Zydus, 2; J. Buyon: Artiva Biotherapeutics, 1, Bristol-Myers Squibb(BMS), 1, 2, Equillium, 1, GlaxoSmithKlein(GSK), 1, 2, Otsuka Pharmaceuticals, 1, Related Sciences, 1, 2.

To cite this abstract in AMA style:

Carlucci P, Sachan N, Fava A, Cohen B, Shwetar J, Gurajala S, Xiao Q, Mears J, Preisinger K, Zaminski D, Deonaraine K, Izmirly P, James J, Guthridge J, DeJager W, Wofsy D, Loomis C, Ward G, Wu M, Putterman C, Rao D, Diamond B, Fine D, Monroy-Trujillo J, Belmont H, Apruzzese W, Davidson A, Furie R, Hoover P, Berthier C, Dall'Era M, Kamen D, Kalunian K, Anolik J, Barnas J, Arazi A, Raychaudhuri S, Hacohen N, Clancy R, Ruggles K, Petri M, Buyon J. CXCL6 Synthesized by Proximal Tubule Cells May Promote Fibrosis in Lupus Nephritis [abstract]. Arthritis Rheumatol. 2024; 76 (suppl 9). https://acrabstracts.org/abstract/cxcl6-synthesized-by-proximal-tubule-cells-may-promote-fibrosis-in-lupus-nephritis/. Accessed .

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