Pathology-oriented multiplexing enables integrative disease mapping

Nature, vol. 644, pp. 516–526

Abstract

, the integration of biological layers (that is, cell structure, subcellular domains and signalling activity) remains challenging. This is due to limitations in the compositions of antibody panels and image resolution, which together restrict the scope of image analysis. Here we present pathology-oriented multiplexing (PathoPlex), a scalable, quality-controlled and interpretable framework. It combines highly multiplexed imaging at subcellular resolution with a software package to extract and interpret protein co-expression patterns (clusters) across biological layers. PathoPlex was optimized to map more than 140 commercial antibodies at 80 nm per pixel across 95 iterative imaging cycles and provides pragmatic solutions to enable the simultaneous processing of at least 40 archival biopsy specimens. In a proof-of-concept experiment, we identified epithelial JUN activity as a key switch in immune-mediated kidney disease, thereby demonstrating that clusters can capture relevant pathological features. PathoPlex was then used to analyse human diabetic kidney disease. The framework linked patient-level clusters to organ disfunction and identified disease traits with therapeutic potential (that is, calcium-mediated tubular stress). Finally, PathoPlex was used to reveal renal stress-related clusters in individuals with type 2 diabetes without histological kidney disease. Moreover, tissue-based readouts were generated to assess responses to inhibitors of the glucose cotransporter SGLT2. In summary, PathoPlex paves the way towards democratizing multiplexed imaging and establishing integrative image analysis tools in complex tissues to support the development of next-generation pathology atlases.

Authors 74

1. Malte B. Kuehl corresponding

   Aarhus University · Aarhus University Hospital · University Medical Center Hamburg-Eppendorf

   Affiliation as printed

   Department of Clinical Medicine, Aarhus University, Aarhus, Denmark. malte.kuehl@clin.au.dk

   Department of Pathology, Aarhus University Hospital, Aarhus, Denmark. malte.kuehl@clin.au.dk

   Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany. malte.kuehl@clin.au.dk

   III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. malte.kuehl@clin.au.dk

   Institute of Medical Systems Bioinformatics, Center for Biomedical AI (bAIome), Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany. malte.kuehl@clin.au.dk

2. Yusuke Okabayashi corresponding

   Jikei University School of Medicine · University Medical Center Hamburg-Eppendorf

   Affiliation as printed

   Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan. y_okabayashi@jikei.ac.jp

   Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany. y_okabayashi@jikei.ac.jp

   III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. y_okabayashi@jikei.ac.jp

3. Milagros N. Wong

   Aarhus University · Aarhus University Hospital · University Medical Center Hamburg-Eppendorf

   Affiliation as printed

   Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

   Department of Pathology, Aarhus University Hospital, Aarhus, Denmark

   Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

4. Lukas Gernhold

   University Medical Center Hamburg-Eppendorf

   Affiliation as printed

   Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

5. Gabriele Gut

   University of Zurich · University Hospital Zurich

   Affiliation as printed

   Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland

   Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

6. Nico Kaiser

   Universität Hamburg · Technische Universität Darmstadt · University Medical Center Hamburg-Eppendorf

   Affiliation as printed

   Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   Institute of Medical Systems Bioinformatics, Center for Biomedical AI (bAIome), Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   TU Darmstadt - Technische Universität Darmstadt - Technical University of Darmstadt (Karolinenplatz 5, 64289 Darmstadt - Germany)

7. Maria Schwerk

   University Medical Center Hamburg-Eppendorf

   Affiliation as printed

   Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

8. Stefanie K. Gräfe

   University of Cologne · University Medical Center Hamburg-Eppendorf · University Hospital Cologne

   Affiliation as printed

   Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   UKE - Universitaetsklinikum Hamburg-Eppendorf = University Medical Center Hamburg-Eppendorf [Hamburg] (Martinistraße 52 20251 Hamburg - Germany)

   University Hospital of Cologne [Cologne] (Cologne - Germany)

   Universität zu Köln = University of Cologne (Albertus-Magnus-Platz, 50923 Köln - Germany)

9. Frank Y. Ma

   Monash Medical Centre · Monash University

   Affiliation as printed

   Department of Nephrology, Monash Medical Centre and Department of Medicine, Monash University, Melbourne, Victoria, Australia

10. Jovan Tanevski

    Heidelberg University · University Hospital Heidelberg

    Affiliation as printed

    Institute for Computational Biomedicine, Heidelberg University and Heidelberg University Hospital, Heidelberg, Germany

    Translational Spatial Profiling Center, Heidelberg University Hospital, Heidelberg, Germany

11. Philipp Sven Lars Schäfer

    Heidelberg University · University Hospital Heidelberg

    Affiliation as printed

    Institute for Computational Biomedicine, Heidelberg University and Heidelberg University Hospital, Heidelberg, Germany

12. Sam Mezher

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

13. Jacobo Sarabia del Castillo

    University of Zurich

    Affiliation as printed

    Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

14. Thiago Goldbeck-Strieder

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

15. Olga I. Zolotareva

    Universität Hamburg · Technical University of Munich

    Affiliation as printed

    Data Science in Systems Biology, TUM School of Life Sciences, Technical University of Munich, Freising, Germany

    Institute for Computational Systems Biology, University of Hamburg, Hamburg, Germany

16. Michael Hartung

    Universität Hamburg

    Affiliation as printed

    Institute for Computational Systems Biology, University of Hamburg, Hamburg, Germany

17. Fernando M. Delgado-Chaves

    Universität Hamburg

    Affiliation as printed

    Institute for Computational Systems Biology, University of Hamburg, Hamburg, Germany

18. Lukas Klinkert

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

19. Ann-Christin Gnirck

    Euroimmun Medizinische Labordiagnostika (Germany) · University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Euroimmun, Lübeck, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

20. Marc Spehr Aachen Department of Chemosensation Institute for Biology II

    RWTH Aachen University

    Affiliation as printed

    Department of Chemosensation, Institute for Biology II, RWTH Aachen University, Aachen, Germany

21. David Fleck Aachen Department of Chemosensation Institute for Biology II

    RWTH Aachen University

    Affiliation as printed

    Department of Chemosensation, Institute for Biology II, RWTH Aachen University, Aachen, Germany

22. Mehdi Joodaki Aachen Institute for Computational Genomics

    RWTH Aachen University

    Affiliation as printed

    Institute for Computational Genomics, RWTH Aachen University, Aachen, Germany

23. Mina Shaigan Aachen Institute for Computational Genomics

    RWTH Aachen University

    Affiliation as printed

    Institute for Computational Genomics, RWTH Aachen University, Aachen, Germany

24. Martin Diebold

    University of Freiburg

    Affiliation as printed

    Faculty of Medicine, Institute of Neuropathology, and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

25. Marco Prinz

    University of Freiburg

    Affiliation as printed

    Faculty of Medicine, Institute of Neuropathology, and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

26. Jennifer Kranz Aachen Department of Urology and Pediatric Urology

    Universitätsklinikum Aachen · Martin Luther University Halle-Wittenberg · RWTH Aachen University

    Affiliation as printed

    Department of Urology and Kidney Transplantation, Martin-Luther-University, Halle, Germany

    Department of Urology and Pediatric Urology, University Hospital RWTH Aachen, Aachen, Germany

27. Johan M. Kux

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Cell Communication and Migration Laboratory, Institute of Biochemistry and Molecular Cell Biology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

28. Fabian Braun

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Martin Zeitz Center for Rare Diseases, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

29. Oliver Kretz

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

30. Hui Min Wu

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

31. Florian Grahammer

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

32. Sven Heins

    Universität Hamburg · University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Institute of Medical Systems Bioinformatics, Center for Biomedical AI (bAIome), Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

33. Marina Zimmermann

    Universität Hamburg · University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Institute of Medical Systems Bioinformatics, Center for Biomedical AI (bAIome), Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

34. Fabian Haas

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

35. Dominik Kylies

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

36. Nicola Wanner

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

37. Jan Czogalla

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

38. Bernhard Dumoulin

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

39. Nikolay Zolotarev

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

40. Maja T. Lindenmeyer

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

41. Pall Karlson

    Aarhus University · Danish Pain Research Center

    Affiliation as printed

    Core Center for Molecular Morphology, Section for Stereology and Microscopy, Aarhus University, Aarhus, Denmark

    Danish Pain Research Center, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

42. Jens Randel Nyengaard

    Aarhus University · Aarhus University Hospital

    Affiliation as printed

    Core Center for Molecular Morphology, Section for Stereology and Microscopy, Aarhus University, Aarhus, Denmark

    Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

    Department of Pathology, Aarhus University Hospital, Aarhus, Denmark

43. Marcial Sebode

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

44. Sören Alexander Weidemann

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Department of Pathology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

45. Thorsten Wiech

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Institute of Pathology, Nephropathology Section, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

46. Hermann-Josef Groene

    Philipps University of Marburg · University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Institute of Pathology, Nephropathology Section, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Institute of Pharmacology, University of Marburg, Marburg, Germany

47. Nicola M. Tomas

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

48. Catherine Meyer‐Schwesinger

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Center for Experimental Medicine, Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

49. Christoph Kuppe Aachen Department of Nephrology

    RWTH Aachen University

    Affiliation as printed

    Department of Nephrology, RWTH Aachen University, Medical Faculty, Aachen, Germany

50. Rafael Kramann Aachen Department of Nephrology

    RWTH Aachen University

    Affiliation as printed

    Department of Nephrology, RWTH Aachen University, Medical Faculty, Aachen, Germany

51. Alexandre Karras

    Université Paris Cité · Assistance Publique – Hôpitaux de Paris · Hôpital Européen Georges-Pompidou

    Affiliation as printed

    Department of Nephrology, Georges Pompidou European Hospital, APHP, Université Paris Cité, Paris, France

52. Patrick Bruneval

    Inserm · Université Paris Cité · Paris Cardiovascular Research Center

    Affiliation as printed

    Université Paris Cité, Inserm, Paris Cardiovascular Research Center (PARCC), Paris, France

53. Pierre‐Louis Tharaux

    Inserm · Université Paris Cité · Paris Cardiovascular Research Center

    Affiliation as printed

    Université Paris Cité, Inserm, Paris Cardiovascular Research Center (PARCC), Paris, France

    INSERM - Institut National de la Santé et de la Recherche Médicale (101, rue de Tolbiac, 75013 Paris - France)

    PARCC (UMR_S 970/ U970) - Paris-Centre de Recherche Cardiovasculaire (56 rue Leblanc 75015 PARIS FRANCE - France)

    UFR Médecine UPCité - UFR Médecine [Santé] - Université Paris Cité (15 rue de l’École de Médecine - 75006 Paris - France)

54. Diego O. Pastene

    Heidelberg University · University Medical Centre Mannheim

    Affiliation as printed

    V. Department of Medicine, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany

55. BENITO A. YARD

    Heidelberg University · University Medical Centre Mannheim

    Affiliation as printed

    V. Department of Medicine, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany

56. Jennifer A. Schaub

    University of Michigan · Michigan Medicine

    Affiliation as printed

    Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, MI, USA

57. Phillip J. McCown

    University of Michigan · Michigan Medicine

    Affiliation as printed

    Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, MI, USA

58. Laura Pyle

    University of Washington

    Affiliation as printed

    Department of Pediatrics, Division of Endocrinology and Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA, USA

59. Ye Ji Choi

    University of Washington

    Affiliation as printed

    Department of Pediatrics, Division of Endocrinology and Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA, USA

60. Takashi Yokoo

    Jikei University School of Medicine

    Affiliation as printed

    Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan

61. Jan Baumbach

    Universität Hamburg · University of Southern Denmark

    Affiliation as printed

    Department of Mathematics and Computer Science, University of Southern Denmark, Odense, Denmark

    Institute for Computational Systems Biology, University of Hamburg, Hamburg, Germany

62. Pablo José Sáez

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Cell Communication and Migration Laboratory, Institute of Biochemistry and Molecular Cell Biology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

63. Ivan G. Costa Aachen Institute for Computational Genomics

    RWTH Aachen University

    Affiliation as printed

    Institute for Computational Genomics, RWTH Aachen University, Aachen, Germany

64. Jan‐Eric Turner

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

65. Jeffrey B. Hodgin

    University of Michigan

    Affiliation as printed

    Department of Pathology, University of Michigan, Ann Arbor, MI, USA

66. Julio Sáez-Rodríguez

    Heidelberg University · University Hospital Heidelberg

    Affiliation as printed

    Institute for Computational Biomedicine, Heidelberg University and Heidelberg University Hospital, Heidelberg, Germany

67. Tobias Bruno Huber

    University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

68. Petter Bjornstad

    University of Washington

    Affiliation as printed

    Department of Pediatrics, Division of Endocrinology and Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA, USA

69. Matthias Kretzler

    University of Michigan · Michigan Medicine

    Affiliation as printed

    Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, MI, USA

70. Olivia Lenoir

    Inserm · Université Paris Cité · Paris Cardiovascular Research Center

    Affiliation as printed

    Université Paris Cité, Inserm, Paris Cardiovascular Research Center (PARCC), Paris, France

71. David J. Nikolic‐Paterson

    Monash Medical Centre · Monash University

    Affiliation as printed

    Department of Nephrology, Monash Medical Centre and Department of Medicine, Monash University, Melbourne, Victoria, Australia

72. Lucas Pelkmans

    University of Zurich

    Affiliation as printed

    Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

73. Stefan Bonn

    Universität Hamburg · University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Institute of Medical Systems Bioinformatics, Center for Biomedical AI (bAIome), Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany

74. Victor G. Puelles corresponding

    Aarhus University · Aarhus University Hospital · University Medical Center Hamburg-Eppendorf

    Affiliation as printed

    Department of Clinical Medicine, Aarhus University, Aarhus, Denmark. v.puelles@uke.de

    Department of Pathology, Aarhus University Hospital, Aarhus, Denmark. v.puelles@uke.de

    Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany. v.puelles@uke.de

    III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. v.puelles@uke.de