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 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.

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) . 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 . 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   c. All renal biopsies have to be immersed in ice for a half an hour at the most.
3. Preparation of renal biopsies before fixation a. Each renal biopsy has to be transferred in a 60 mm dish containing 3 ml cold (~5 °C) DPBS and cleaned from any residual connective or adipose tissue by sterile tweezers. www.bio-protocol.org/e3779 b. The specimen size can be reduced to 0.5 x 0.5 cm with a sterile mono-use scalpel.
c. The renal specimen has to be transferred in another 60 mm dish containing 3 ml of cold (~4 °C) DPBS.
B. Paraffin slides processing

Renal specimen fixation
The renal specimen has to be transferred into 15 ml conical tube with 10% neutral buffered formalin (NBF) at room temperature for 8 h but no longer than 24 h. Make sure you have enough fixative to cover tissues. Fixative volume should be 5-10 times of tissue volume.
Note: The renal specimen is not hold up in paraffin cassettes. Tissue processing was carried out manually to mimic the individual cumulative steps of automated processing.

Renal specimen dehydration
a. After fixation, formalin has to be discarded.
b. The renal specimen has to be dehydrated moving to alcohol grades steps: i. 10 ml 50% ethanol for 40 min (max 5 days) at room temperature.
ii. 10 ml 70% ethanol for 40 min at room temperature.
iii. 10 ml of 95% ethanol for 25 min at room temperature.
iv. 10 ml of 95% ethanol for 25 min at room temperature.
v. 10 ml of 100% ethanol for 25 min at room temperature.
vi. 10 ml of 100% ethanol for 25 min at room temperature.
Note: These steps should be completed fully (with sufficient time set aside for these steps) because insufficient dehydration can lead to tissue degradation.

Renal specimen clarification
The renal specimen has to be cleared with xylene: a. 10 ml of xilene for 15 min.

Renal specimen infiltration by paraffin
The renal specimen has to be transferred into a becker containing liquid Paraffin in 58 °C paraffin bath: 10 ml of liquid Paraffin for 2 h in an oven at 56 °C-58 °C (the melting temperature of paraffin).

Embedding renal tissues in paraffin blocks
a. Small amount of molten paraffin has to be put in mold, dispensing from paraffin reservoir.
b. Using warm forceps, the renal specimen has to be transferred into the well of the metal steel base mold in health block, placing cut side down.
c. The embedding ring has to be put into the steel base mold (at 56 °C). The paraffin ribbon is floated onto the surface of a warm water bath, where it spreads out and flattens perfectly.
d. The mold has to be transferred quickly to cold plate (-20 °C), and gently press tissue flat.
Paraffin will solidify in a thin layer that holds the tissue in position. www.bio-protocol.org/e3779 e. Hot paraffin has to be added to the mold from the paraffin dispenser. Be sure there is enough paraffin to cover the tissue. The paraffin block should solidify in 30 min at -20 °C.
f. When the wax is completely cooled and hardened the paraffin block has to be easily popped out of the mold; the wax blocks should not stick. If the wax cracks or the tissues are not aligned well, simply melt them again and start over. b. The slides have to rehydrated using graded alcohol series for few minutes and rinse in deionized water: i. Ethanol 100% for 6 min.
v. Deionized water for 5 min (for three times).

Heat-induced Antigen unmasking
a. The slides have to be transferred into a plastic vertical staining jar (suitable for microwave), containing 100 ml of sodium citrate buffer 10 mM at pH = 6 (filled to the brim) and put in a water bath.

Note: The water bat is obtained put slide jar into a glass of water (suitable for microwave)
and put the entire thing into the microware. This reduces the boiling inside the jar .
b. The slides have to be subjected to three microwave (750 W) cycles of 5 min.
c. The volume of antigen retrieval buffer has to be top up with distilled water at each cycle in order to avoid evaporation during boiling. f. The staining mix has to be prepared combined and diluted appropriately the two different antibodies (α-SMA 1:100, PDGFR-β 1:100) in the blocking mix (5% of Goat Serum), respecting the dilution tested individually before.
g. The blocking buffer has to be removed gently (e.g., by tipping the slide sideways onto a paper towel), without rinsing before adding primary antibody.
h. 100 μl of primary antibodies mix has to be added to each section and incubated in the humidified chamber overnight at +4 °C.
i. Negative control of the IF reaction has to be obtained incubating the isotype control antibody instead of the primary antibodies mix on a well delimited section in one of the slides.
j. The slides have to be rinsedin PBS 1x for three times, 5 min each. k. A mix of the two secondary antibodies, respectively to bind the two primary antibodies, has to be prepared combined and diluted appropriately: Alexa Fluor goat anti-mouse 555 q. The slides has to be acquired by confocal microscope Leica TCS SP2 (Leica).

Data analysis
Confocal microscopy was performed using the Leica TCS SP2 (Leica), equipped with argon-krypton (488 nm), green neon (543 nm), and helium-neon (633 nm) lasers. Confocal images were taken at 500-nm intervals through the z-axis of the section, encompassing a total of 7 µm in depth. Images from individual optical planes and multiple serial optical sections were analyzed, and the images were sequentially scanned in all three laser channels. The images were captured using a 63x objective lens and exported as TIFF files as showed in Figures 1 and 2. In no case was the interobserver variability greater than 20%.
Results are expressed as median ± interquartile range (IQR) Statistically significant differences were assessed by the Mann-Whitney test. A P value < 0.05 was significant. Statistical analysis was performed using GraphPad Prism Software 5 as indicated in Figure 3.