Summary

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

Published: October 18, 2016
doi:

Summary

Questo manoscritto descrive un protocollo dettagliato per fenotipici e analisi quantitativa dei macrofagi residenti da reni di ratto mediante citometria di flusso. Le cellule colorate risultanti possono essere utilizzati anche per altre applicazioni, incluse separazione delle cellule, analisi di espressione genica o studi funzionali, aumentando così le informazioni ottenute nel modello sperimentale.

Abstract

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

Introduction

Renal disease is a global health problem, with increased prevalence, and associated with elevated morbidity and mortality1. One of the most important mechanisms involved in the progression and development of renal injury is inflammation, mainly triggered by macrophages. Macrophages play a pivotal role in many inflammatory diseases, including renal disorders2. Thus, an elevated presence of infiltrating macrophages has been reported in biopsies from patients with acute kidney injury (AKI) or chronic kidney disease (CKD)3,4. Recent studies suggest that the long-term outcome of renal disease could be controlled by macrophages5,6. In response to the local microenvironment, macrophages may differentiate into different phenotypes that play diverse biological functions7. Two well differentiated macrophage phenotypes have been established: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2)8. M1 macrophages promote inflammation, whereas M2 macrophages have an anti-inflammatory role and are involved in tissue repair9. Therefore, a better knowledge of macrophage heterogeneity is necessary to understand their regulation and contribution to renal pathology and develop novel therapeutic approaches.

Both, murine and rats models have been widely used to understand the molecular and cellular mechanism involved in renal injury10. However, there are substantial differences in the diverse markers used to identify macrophages phenotypes between these rodents. Hence, several murine markers, such as F4/80 or Ly6C are not used in rats, thus limiting the extrapolation of findings between these species. Moreover, there is a limited number of markers describing macrophage phenotypes in rats, explaining the scarce studies analyzing macrophage heterogeneity in these animals as compared with mice. Therefore, new strategies for macrophage subset characterization are necessary to understand the role of macrophages in renal disease models in rats.

This manuscript describes a protocol for the phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. This technique can be further followed by several assays, including cell sorting and mRNA or protein expression studies to allow in-depth characterization of the role of macrophages in renal disease.

Protocol

Questo protocollo è stato approvato dalla Institutional Animal Care locali e utilizzare i comitati a seguito della direttiva 2010/63 / UE del Parlamento europeo e con la linea guida nazionale 53/2013. 1. Preparazione dei reagenti e soluzioni Preparare tutti i reagenti e soluzioni in condizioni sterili e utilizzare sotto una cappa a flusso laminare. Mantenere le soluzioni a 4 ° C. Preparare buffer di colorazione (2% siero fetale bovino (FBS) in PBS 1x Dulbecco). Preparare la soluz…

Representative Results

Abbiamo analizzato macrofagi eterogeneità in un modello sperimentale infiammatoria del danno renale associata a maggiore presenza di infiltrazione di macrofagi nel rene. In questo modello, il danno renale è stata indotta mediante somministrazione di aldosterone (1 mg -1 kg -1 die) più sale (NaCl 1%) in acqua potabile per 3 settimane a ratti Wistar, come precedentemente riportato 12. Il progr…

Discussion

I macrofagi sono cellule eterogenee che svolgono un ruolo importante in diverse malattie infiammatorie, incluse le patologie renali. Vi è un crescente interesse nella caratterizzazione dei macrofagi sottoinsiemi nella malattia renale perché ogni sottopopolazione macrofagi contribuisce in modo diverso allo sviluppo di danno renale, come riportato nella glomerulonefrite, nefropatia diabetica e cancro del rene 14-16. Nelle fasi iniziali di danno renale acuta, una predominanza di macrofagi M1 si osserva, promuo…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by grants from FIS/FEDER (Programa Miguel Servet: CP10/00479, PI13/00802 and PI14/00883), Spanish Society of Atherosclerosis, Spanish Society of Nephrology and Fundaciòn Renal Iñigo Alvarez de Toledo (FRIAT) to Juan Antonio Moreno. FIS/FEDER funds PI14/00386 and Instituto Reina Sofìa de Investigaciòn Nefrològica to Jesús Egido. Fundaciòn Conchita Rabago to Melania Guerrero Hue. Fundaciòn Renal Iñigo Alvarez de Toledo (FRIAT) to Alfonso Rubio Navarro.

Materials

Laminar flow hood Faster Or equivalent equipment
Centrifuge Hettich Or equivalent equipment
Flow cytometer (FACSAria) BD Biosciences
Fetal bovine serum BioWest S1820-500
PBS 10x LONZA BE17-515Q
Collagenase Sigma-Aldrich 12/1/9001
ACK Lysing Buffer Thermo Fisher Scientific A10492-01
Flow cytometry strainers BD Biosciences 340626
Falcon cell strainers Thermo Fisher Scientific 352340
Flow cytometry tubes Falcon 352052 5 ml Polystyrene Round-Bottom Tube
Centrifuge tubes Corning centristar 430791
Water bath Memmert GmbH + Co. KG WNE 7 37ºC
Fixation/Permeabilization Solution or Permeabilization/Wash Buffer BD Biosciences 554714
Rompum (Xylazine) Bayer Or equivalent
Ketalar (Ketamine) Pfizer Or equivalent
Hanks’ balanced salt solution Sigma-Aldrich H8264-500ML
Saline solution Braun 622415
Anti-CD45 (clone:OX-1) APC-Cy7 Biolegend 202216 Diluted 1:100
Anti-CD68 (clone: ED1) FITC Bio-RAD MCA341F Diluted 35:1000
anti-CD86 (clone: 24F) PE Biolegend 200307 Diluted 35:1000
anti-CD163 (clone: ED2) Alexa Fluor 647 Bio-RAD MCA342R Diluted 4:100
Live/dead stain  Molecular Probes L34955 Diluted 3:1000

Referências

  1. Gansevoort, R. T., et al. Chronic kidney disease and cardiovascular risk: epidemiology, mechanisms, and prevention. Lancet. 382, 339-352 (2013).
  2. Kon, V., Linton, M. F., Fazio, S. Atherosclerosis in chronic kidney disease: the role of macrophages. Nat. Rev. Nephrol. 7, 45-54 (2011).
  3. Kim, J. H., et al. Macrophage depletion ameliorates glycerol-induced acute kidney injury in mice. Nephron Exp. Nephrol. 128, 21-29 (2014).
  4. Belliere, J., et al. Specific macrophage subtypes influence the progression of rhabdomyolysis-induced kidney injury. J. Am. Soc. Nephrol. 26, 1363-1377 (2015).
  5. Kinsey, G. R. Macrophage dynamics in AKI to CKD progression. J. Am. Soc. Nephrol. 25, 209-211 (2014).
  6. Lech, M., et al. Macrophage phenotype controls long-term AKI outcomes–kidney regeneration versus atrophy. J. Am. Soc. Nephrol. 25, 292-304 (2014).
  7. Murray, P. J., et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 41, 14-20 (2014).
  8. Gordon, S. Alternative activation of macrophages. Nat. Rev. Immunol. 3, 23-35 (2003).
  9. Mosser, D. M., Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958-969 (2008).
  10. Ortiz, A., et al. Translational value of animal models of kidney failure. Eur. J. Pharmacol. 759, 205-220 (2015).
  11. Martina, M. N., Bandapalle, S., Rabb, H., Hamad, A. R. Isolation of double negative alphabeta T cells from the. J. Vis. Exp. , (2014).
  12. Martin-Fernandez, B., et al. Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats. PLoS. One. 11, e0145946 (2016).
  13. Layoun, A., Samba, M., Santos, M. M. Isolation of murine peritoneal macrophages to carry out gene expression analysis upon Toll-like receptors stimulation. J. Vis. Exp. (e52749), (2015).
  14. Komohara, Y., et al. Macrophage infiltration and its prognostic relevance in clear cell renal cell carcinoma. Cancer Sci. 102, 1424-1431 (2011).
  15. Han, Y., Ma, F. Y., Tesch, G. H., Manthey, C. L., Nikolic-Paterson, D. J. Role of macrophages in the fibrotic phase of rat crescentic glomerulonephritis. Am. J. Physiol Renal Physiol. 304, F1043-F1053 (2013).
  16. Ndisang, J. F. Role of the heme oxygenase-adiponectin-atrial natriuretic peptide axis in renal function. Curr. Pharm. Des. 21, 4380-4391 (2015).
  17. Blackbeard, J., et al. Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry. J. Neurosci. Methods. 164, 207-217 (2007).
  18. Strobl, H., Scheinecker, C., Csmarits, B., Majdic, O., Knapp, W. Flow cytometric analysis of intracellular CD68 molecule expression in normal and malignant haemopoiesis. Br. J. Haematol. 90, 774-782 (1995).

Play Video

Citar este artigo
Rubio-Navarro, A., Guerrero-Hue, M., Martín-Fernandez, B., Cortegano, I., Olivares-Alvaro, E., de las Heras, N., Alía, M., de Andrés, B., Gaspar, M. L., Egido, J., Moreno, J. A. Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry. J. Vis. Exp. (116), e54599, doi:10.3791/54599 (2016).

View Video