Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis

Background

Renal fibrosis is considered the principal responsible for progression of renal disease and it is associated with a limited capacity of kidney to regenerate after injury. The principal source of interstitial fibrosis in progressive renal diseases (Simone et al., 2014; Fiorentino et al., 2018) is represented by activated fibroblasts, named myofibroblasts, that are recognized by their ability to synthesize de novo α-smooth muscle actin (α-SMA) (Meran and Steadman, 2011; Hewitson, 2012). These cells derived from multiple sources, including not only resident fibroblasts, but also endothelial cells, tubular cells, circulating bone marrow–derived cells and pericytes (Di Carlo and Peduto, 2018). Several studies evaluated the critical role of capillary pericytes in chronic kidney disease (CKD) (Lin et al., 2008; Grgic et al., 2012; Kramann et al., 2015) as well as early phases of acute kidney injury (AKI) (Leaf et al., 2016; Castellano et al., 2018 and 2019).

Recent studies revealed that dysfunctional pericytes are principally involved in sepsis-induced microvascular dysfunction and vascular leakage which are the key hallmarks of end-organ dysfunction and septic shock (Goldenberg et al., 2011; Page and Liles, 2013; Castellano et al., 2014; Stasi et al., 2017; Fani et al., 2018). Consistent with these findings, we found that pigs challenged with LPS led to a dysfunctional response of pericytes in the microvasculature of renal parenchyma (Castellano et al., 2019) (Figure 1).

In recent years, the fate-tracing mapping and the ultrastructural analysis have shed more lights on the pericytes behavior in several mouse model of renal diseases (Lin et al., 2008; Humphreys et al., 2010). In fairness, the identification of pericytes by criteria that requires elaborate techniques as fate-tracing analysis is not practical in large animal model. Since PDGFR-β is defined as the constitutive marker for isolation and characterization of renal pericytes (Chen et al., 2011; Wang et al., 2017) and a-SMA is a marker associated with myofibroblasts (Hewitson, 2012), the double-labeling for PDGFR-β and α-SMA provide a “picture” of pericyte distribution and dysfunctional activation in renal parenchyma during AKI (Guzzi et al., 2019). In accordance with other phenomena of cellular transdifferentation as EndMT, the loss of PDGFR-β and the increased level of α-SMA, Collagen I and MMP proteins is defined as Pericytes to Myofibroblast transition (PMT) (Chang et al., 2012).

We, firstly, performed double immunofluorescent staining of PDGFR-β and α–SMA to study the PMT in swine and mice models of renal I/R injury (Figure 2) (Castellano et al., 2018). Interestingly, in normal swine kidney biopsies we found the colocalization of the PDGFR-β and NG2 in peritubular capillaries, indicating the overall accuracy of PDGFR-β as capillary pericytes markers in pig models whereas glomerular mesangial cells were PDGFRβ⁺ but NG2^−. After 24h of renal I/R we observed the downregulation of the constitute markers PDGFR-β and NG2 and the dramatic upregulation of α-SMA.

These data were also confirmed in our sepsis model of AKI and taken together we demonstrated that PDGFR-β⁺pericytes are able to synthesize pro-fibrotic markers acquiring the typical features of myofibroblasts, leading to extracellular matrix deposition and interstitial fibrosis (Castellano et al., 2019). In accordance, we confirmed our data in vitro and we found that exposition of human placental derived pericytes to I/R injury (C5a) and sepsis stimuli (LPS) led to acquisition of α-SMA contractile stress fibers (Castellano et al., 2018 and 2019).

Therefore, the protocol described here could be useful to characterize pericytes and their dysfunctional activation and will facilitate the research in the early acute kidney injury as well as other fields relating inflammation and fibrosis (Castellano et al., 2018 and 2019).