Abstract
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 α-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-β could be used as marker for renal pericytes characterization. Recently, double immunofluorescence staining of PDGFR-β and α-SMA was performed to identify the damage activated pericytes (PDGFR-β+/α-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-β and α-SMA in swine Formalin-Fixed Paraffin-Embedded (FFPE) kidney biopsies and the method for image analysis and quantification.
Highlights
[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)
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)
Summary
[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. We 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). Tissue Embedding Rings (Bio Optica SpA, catalog number: 07-7650) 10. Xylene (Bio Optica SpA, catalog number: 06-1304Q) 15.
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