Özet

Caracterización fenotípica de los macrófagos a partir de riñón de rata por Citometría de Flujo

Published: October 18, 2016
doi:

Özet

Este manuscrito describe un protocolo detallado para fenotípica y análisis cuantitativo de los macrófagos residentes de riñones de rata por citometría de flujo. Las células teñidas resultantes también se pueden utilizar para otras aplicaciones, incluyendo la clasificación de células, el análisis de la expresión génica o estudios funcionales, aumentando así la información obtenida en el modelo experimental.

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

Este protocolo fue aprobado por las autoridades locales Institucional Cuidado de Animales y el empleo Comités siguiendo la Directiva 2010/63 / UE del Parlamento Europeo y la Directriz Nacional 53/2013. 1. Preparación de Reactivos y Soluciones Preparar todos los reactivos y soluciones en condiciones estériles y utilizar bajo una campana de flujo laminar. Mantener las soluciones a 4 ° C. Preparar tampón de tinción (2% de suero bovino fetal (FBS) en PBS de Dulbecco 1x). Preparar…

Representative Results

Se analizó la heterogeneidad de los macrófagos en un modelo experimental de lesión inflamatoria renal asociada a la mayor presencia de la infiltración de macrófagos en el riñón. En este modelo, el daño renal fue inducida por la administración de la aldosterona (1 mg kg -1 -1 día) más de sal (NaCl 1%) en agua de bebida durante 3 semanas en ratas Wistar, como se informó anteriormente 12. <p class="jove_content" fo:keep-together.within-page="…

Discussion

Los macrófagos son células heterogéneas que juegan un papel importante en diferentes enfermedades inflamatorias, incluyendo trastornos renales. Hay un creciente interés en la caracterización de los macrófagos subconjuntos en la enfermedad renal, ya que cada subpoblación de macrófagos contribuye de una manera diferente a la evolución de la lesión renal, como se informa en la glomerulonefritis, la nefropatía diabética y cáncer renal 14-16. En las primeras etapas de la lesión renal aguda, se observ…

Açıklamalar

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

Referanslar

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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).

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