Phenotyping renal leukocyte subsets by four-color flow cytometry: Characterization of chemokine receptor expression

To investigate mechanisms of cell-mediated injury in renal
inflammatory disease it is critical to determine the surface
phenotype of infiltrating renal leukocyte subsets. However, the
cell-specific expression of many leukocyte receptors is difficult
to characterize in vivo. Here, we report a protocol based on flow
cytometry that allows simultaneous characterization of surface
receptor expression on different subsets of infiltrating renal
leukocytes. The described technique combines an adapted method to
prepare single cell suspensions from whole kidneys with subsequent
four-color flow cytometry. We recently applied this technique to
determine the differential expression of murine chemokine receptors
CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this
article, we summarize our current findings on the validity of the
method as compared with immunohistology and in situ hybridization
in two murine models of nonimmune ( obstructive nephropathy) and
immune-mediated ( lupus nephritis) inflammatory renal disease. Flow
cytometry analysis revealed an accumulation of CCR5-, but not
CCR2-positive lymphocytes in inflamed kidneys, compared to the
peripheral blood. Particularly renal CD8(+) cells expressed CCR5
(79% in obstructed kidneys, 90% in lupus nephritis). In both
models, infiltrating renal macrophages were positive for CCR2 and
CCR5. These data corresponded to immunohistological and in situ
hybridization results. They demonstrate that flow cytometric
analysis of single cell suspensions prepared from inflamed kidneys
is a rapid and reliable technique to characterize and quantify
surface receptor expression on infiltrating renal leukocyte
subsets.