Purification and analysis of kidney-infiltrating leukocytes in a mouse model of lupus nephritis.

Abstract

Renal injury often occurs as a complication in autoimmune diseases such as systemic lupus erythematosus (SLE). It is estimated that a minimum of 20% SLE patients develop lupus nephritis, a condition that can be fatal when the pathology progresses to end-stage renal disease. Studies in animal models showed that incidence of immune cell infiltrates in the kidney was linked to pathological injury and correlated with severe lupus nephritis. Thus, preventing immune cell infiltration into the kidney is a potential approach to impede the progression to an end-stage disease. A requirement to investigate the role of kidney-infiltrating leukocytes is the development of reproducible and efficient protocols for purification and characterization of immune cells in kidney samples. This chapter describes a detailed methodology that discriminates tissue-resident leukocytes from blood-circulating cells that are found in kidney. Our protocol was designed to maximize cell viability and to reduce variability among samples, with a combination of intravascular staining and magnetic bead separation for leukocyte enrichment. Experiments included as example were performed with FcγRIIb[KO] mice, a well-characterized murine model of SLE. We identified T cells and macrophages as the primary leukocyte subsets infiltrating into the kidney during severe nephritis, and we extensively characterized them phenotypically by flow cytometry.