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

Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to transdifferentiate toward alpha-SMA(+) myofibroblasts leading to interstitial fibrosis. Even if precise pericytes identification requires transmission electron microscopy and the co-immunostaining of several markers (i. e., Gli, NG2 chondroitin sulphate proteoglycan, CD146, desmin or CD73) and emerging new markers (CD248 or TEM1, endosialin), previous studies suggested that PDGFR-beta could be used as marker for renal pericytes characterization. Recently, double immunofluorescence staining of PDGFR-beta and alpha-SMA was performed to identify the damage activated pericytes(PDGFR-beta(+)/alpha-SMA(+) cells) in the early phase of fibrosis development. Our data highlighted the crucial role of renal pericytes in the physiopathology of sepsis and I/R associated AKI. In this protocol, we describe the procedure for double immunofluorescence staining of PDGFR-beta and alpha-SMA in swine Formalin-Fixed Paraffin-Embedded (FFPE) kidney biopsies and the method for image analysis and quantification.