Background

Diseases of the kidney are rising in incidence and prevalence, placing a significant burden on the healthcare community worldwide and significantly affecting patients’ quality of life (World Kidney Day: Chronic Kidney Disease, 2015). Kidney damage can arise from a wide range of insults including infections, ischemia, toxins, hypertension, genetic and metabolic disorders (Imig and Ryan, 2013). The immediate response to insult functionally is acute kidney injury (AKI), a clinical syndrome characterised physiologically by a rapid (hours to days) decrease in kidney function and histomorphologically by infiltration of inflammatory immune cells into the renal tubulointerstitium, the tissue compartment adjoining the tubules of the kidney (Basile et al., 2012). The subsequent biological responses aim to limit ongoing injury and repair damaged tissue. If unresolved, this inflammation may lead to chronic kidney disease (CKD). CKD is defined as a loss in kidney function that persists for at least 3 months in duration and is characterized histomorphologically, regardless of its cause, by tubulointerstitial fibrosis and chronic inflammation (Kawakami et al., 2014). The presence of inflammatory immune cells in human AKI and CKD is suggestive of functional roles in disease progression. However, the identification and enumeration of these CD45⁺ leukocyte populations in human kidney tissue remain poorly defined.

Until now, the identification of immune cells in human kidney disease has primarily been made using immunohistochemistry (IHC). Numerous IHC-based studies of kidney biopsies report strong associations between the loss of renal function/tubulointerstitial injury and the prominent accumulation of inflammatory immune cell populations of distinct leukocyte lineages, including: T cells (CD3⁺ cells [Hoffmann et al., 2006]), T helper cells (CD4⁺ cells [Liu et al., 2012; Pei et al., 2014]), cytotoxic T cells (CD8⁺ cells [Pei et al., 2014]), B cells (CD19⁺ or CD20⁺ cells [Heller et al., 2007]), natural killer (NK) cells (CD16⁺ or CD56⁺ cells [Furuichi et al., 2001; Hidalgo et al., 2010; Shin et al., 2015]), monocytes/macrophages (CD14⁺ cells [Zhou et al., 2010]), dendritic cells (DC) (HLA-DR⁺ cells [Hart et al., 1981; Markovic-Lipkovski, 1990] or DC-SIGN⁺ cells [Segerer et al., 2008; Pei et al., 2014]) and granulocytes (neutrophils, basophils, eosinophils; CD15⁺ or CD16⁺ cells [Weidner et al., 2004; Segerer et al., 2006]).

However, IHC staining for single antigens is not sufficiently specific to directly identify these immune cell populations, given the broader expression of many of these markers on several leukocyte and non-hematopoietic cell types. For example, single staining for DC-SIGN to identify DC will be non-specific given its co-expression on monocytes/macrophages in kidney tissue (Figel et al., 2011). Similarly, using CD16 or CD56 to identify NK cells will be inadequate, with both markers also expressed on T cells (Markey and MacDonald, 1989; Trinchieri, 1989). Furthermore, IHC-based methods are unable to effectively distinguish expression levels of these antigens, required, for instance, to distinguish subsets of functionally specialized NK cells – CD56^bright CD16^-/low NK cells and CD56^dim CD16⁺ NK cells. Clearly, a progression to multi-parameter staining methodologies is required to more accurately detect and quantify immune cell populations in human kidney disease.

We have developed a novel protocol for obtaining single cell suspensions from fresh kidney biopsies to allow the identification and enumeration of immune cells subsets via multi-color flow cytometry (Kassianos et al., 2013; Law et al., 2017 and 2018; Muczynski et al., 2018). Biopsy tissue (excess to diagnostic purposes) is obtained with informed patient consent, followed by enzymatic digestion into single cells and finally, antibody labeling for acquisition by flow cytometry. This process enables precision in detecting discrete immune cell populations based on the absence/presence and intensity of multiple markers. This approach also allows differentiation of populations based on cell size (forward scatter; FSC), granularity (side scatter; SSC), cell viability (Near-IR) and absolute immune cell counts (fluorescent counting bead standard). This single platform approach is proving versatile in answering basic research questions in kidney disease and is readily translatable to human diagnostics of the future, where immune cell populations may become prognostic markers for the progressive loss of kidney function.