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Jul 2019

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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
双标记PDGFR-β和α-SMA在猪急性肾损伤模型中检测周细胞-肌成纤维细胞转化作为早期纤维化标志物的研究   

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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.

Keywords: PDGFR-β+/α-SMA+ immunofluorescence, (PDGFR-β+/α-SMA+ 荧光免疫检验法), Pericyte-to-Myofibroblast Transition (周细胞-肌成纤维细胞转化), Renal pericytes (肾脏周皮细胞), Swine kidney biopsies (猪肾脏活检), Renal I/R injury (肾脏I / R损伤), Sepsis (败血症), AKI (急性肾损伤), Perivascular cells (血管周细胞), AKI-to-CKD transition (败血症-慢性肾病转变)

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).

Materials and Reagents

  1. Aluminium Foil (Carl Roth GmbH, Rotilabo®, catalog number: 0 954.1 )
  2. StripetteTM Serological Pipets 5 ml, 10 ml (Corning, catalog numbers: 4051 , 4488)
  3. Dish 60 mm (Corning, catalog number: 3261 )
  4. Falcon® centrifuge tube 50 ml,15 ml (Corning, catalog numbers: 352070 , 352096)
  5. Safety safeshield scalpels (Biosigma srl, catalog number: 530050 )
  6. Metal steel base mold (Leica Biosystem, catalog number: 3803081 )
  7. Coverslips for optical microscopy 24 x 40 x 0.16 mm (Bio Optica SpA, catalog number: 09-2040 )
  8. Paper towel
  9. Tissue Embedding Rings (Bio Optica SpA, catalog number: 07-7650 )
  10. Nail varnish
  11. 10% neutral buffered formalin (NBF) (Diapath SpA, catalog number: F0047)
  12. Paraffin Lab O-Wax 56-58 °C (Histo-Line Laboratories srl, catalog number: R0040-20 )
  13. Sterile DPBS (Euroclone SpA, catalog number: ECM4053XL )
  14. Xylene (Bio Optica SpA, catalog number: 06-1304Q )
  15. Ethanol absolute (100%) (Bio Optica SpA, catalog number: 06-10099 )
  16. Deionized water
  17. Microscopy slides Super Frost® Plus (Bio Optica SpA, catalog number: 09-OPLUS )
  18. Normal Goat Serum (Sigma-Aldrich, catalog number: G9023 , stored in aliquots at -20 °C)
  19. Phosphate Buffered Saline Tablets (Sigma-Aldrich, catalog number: P4417 )
  20. α-SMA mouse monoclonal (Santa Cruz Biotechnology, catalog number: sc-32251 )
  21. PDGFR-β monoclonal antibody rabbit anti human (Abcam, catalog number: ab32570 )
  22. AlexaFluor goat anti-rabbit FITC-conjugated (488 nm) antibody (Molecular Probes, catalog number: A11070 )
  23. AlexaFluor goat anti-mouse TRITC conjugated (555 nm) antibody (Molecular Probes, catalog number: A21127 )
  24. TO-PRO3 IODIDE (Invitrogen, catalog number: T3605 )
  25. FluoroMount (Bio-Optica SpA, catalog number: K024 )
  26. Phosphate buffered saline tablet (Sigma, catalog number: P4417 )
  27. Acid citric (Sigma, catalog number: C759 )
  28. Sodium citrate (Sigma, catalog number S4641)
  29. Normal Goat Serum (Sigma-Aldrich, catalog number G9023 , stored in aliquots at -20 °C)
  30. Unmasking Buffer (10 mM Sodium Citrate Buffer) (see Recipes)
  31. Blocking solution (10% Normal Goat Serum) (see Recipes)
  32. Buffer for diluting primary antibody (5% Normal Goat Serum) (see Recipes)
  33. PBS 1x for the IF washing (see Recipes)

Equipment

  1. Autoclave (AHSI S.p.A., model: FVA/A1 )
  2. Tweezers (Thermo Fisher Scientific, catalog number: 10303611 )
  3. Scissors for microscopy (Bio Optica SpA, catalog number: 32-703 )
  4. Gloves and Eye protection
  5. Hot plate with magnetic stirrer
  6. PMP Hellendhal Staining Jar (Kartell S.p.A., LABWARE Division, KartellTM, catalog number: 00 35500 )
  7. Water bath
  8. Fume cupboard (Euroclone SpA)
  9. Pipettes (0.5-10, 10-100 and 100-1,000 μl) (Eppendorf, catalog numbers: 3111000.122 , 3111000.149 , 3111000.165 )
  10. Bard monopty biopsy instrument 16 G x 20 cm length (C.R. Bard.Inc., catalog number: 121620 )
  11. 58 °C paraffin bath Cold plate (Bio Optica SpA, catalog number: PF100 )
  12. Microtome (Leica Biosystem, catalog number: RM2125 RTS )
  13. Histology Bath (Bio Optica SpA, catalog number: WB100 )
  14. Microwave
  15. Pap pen for immunostaining (Bio Optica SpA, catalog number: 11-100 )
  16. Orbital shaker (Thermo Scientific, catalog number: SHKA2000 )
  17. Humidified Chamber
  18. pH meter (Thermo Scientific, catalog number: 3115101 )

Software

  1. GraphPad Prism® (version 5.0) (GraphPad Software)
  2. Image analysis software (Leica QWin) (Leica)

Procedure

  1. Collection of renal samples
    1. Preparation of work tools for sample processing
      Autoclave clean tweezers and scissors after wrapping them in aluminium foil.
    2. Renal biopsies procedure
      1. A Needle core biopsy was performed at the start of experimental procedure (T0) and at several intermediate time points in healthy and treated pigs. Renal biopsies was performed under direct ultrasound guidance with automated biopsy needles. After the sacrifice, kidneys were explanted and cuneiform biopsies of cortical and medullary regions were obtained with safety safeshield scalpels. 
      2. Each renal biopsy was delivered to the histological laboratory in a 50 ml Falcon containing 20 ml of cold (~4 °C) sterile DPBS and was identified by a unique code for each animal.
      3. All renal biopsies have to be immersed in ice for a half an hour at the most.
    3. Preparation of renal biopsies before fixation
      1. 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.
      2. The specimen size can be reduced to 0.5 x 0.5 cm with a sterile mono-use scalpel.
      3. The renal specimen has to be transferred in another 60 mm dish containing 3 ml of cold (~4 °C) DPBS.

  2. Paraffin slides processing
    1. 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.
    2. Renal specimen dehydration
      1. After fixation, formalin has to be discarded.
      2. The renal specimen has to be dehydrated moving to alcohol grades steps:
        1. 10 ml 50% ethanol for 40 min (max 5 days) at room temperature.
        2. 10 ml 70% ethanol for 40 min at room temperature.
        3. 10 ml of 95% ethanol for 25 min at room temperature.
        4. 10 ml of 95% ethanol for 25 min at room temperature.
        5. 10 ml of 100% ethanol for 25 min at room temperature.
        6. 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.
    3. Renal specimen clarification
      The renal specimen has to be cleared with xylene:
      1. 10 ml of xilene for 15 min.
      2. 10 ml of xilene for 15 min.
    4. 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).
    5. Embedding renal tissues in paraffin blocks
      1. Small amount of molten paraffin has to be put in mold, dispensing from paraffin reservoir.
      2. 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.
      3. 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.
      4. 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.
      5. 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.
      6. 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.
    6. Preparation of paraffin sections: Sectioning protocol:
      1. The paraffin block has to be sectioned at the desired thickness (usually 4-5 µm) on a microtome and float on a 40 °C water bath containing distilled water.
        Notes:
        1. Keep record of the orientation and sequence of the sections. 
        2. The histology bath has to reach a temperature up to 56 °C to allow the paraffin sections to flatten.
      2. The paraffined sections have to be transferred onto Super Frost Plus Microscope slide. 
      3. The slides have to dry overnight and store slides at room temperature until ready for use.

  3. Immunofluorescence staining
    1. The paraffin sections have to deparaffinated and rehydrated (The entire process must be set up in the fume cupboard)
      1. The slides have to be deparaffinized in pure xylene for 15 min.
        Note: Xylene removes the excess of wax around the tissue and deparaffinates the paraffined sections.
      2. The slides have to rehydrated using graded alcohol series for few minutes and rinse in deionized water:
        1. Ethanol 100% for 6 min.
        2. Ethanol 95% for 1 min.
        3. Ethanol 70% for 1 min.
        4. Ethanol 50% for 1 min.
        5. Deionized water for 5 min (for three times).
    2. Heat-induced Antigen unmasking
      1. 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 .
      2. The slides have to be subjected to three microwave (750 W) cycles of 5 min.
      3. The volume of antigen retrieval buffer has to be top up with distilled water at each cycle in order to avoid evaporation during boiling.
      4. The slides have to be let slowly cool.
        Note: The number of slides must be the same of the positions available in the staining jar at each antigen unmasking process so that the efficiency of unmasking is always the same. If less slides are used, fill up the empty spaces with empty glass slides. This ensures a similar heat distribution to each slide and reduces the differences in antigen retrieval.
    3. Immunofluorescence staining:
      1. The cool slides have to be transferred into glass vertical staining jar with lid filled with deionized water. Wash the slides in deionized water for 5 min on aorbital shaker at room temperature.
      2. The slides have to be rinsed in three changes of PBS 1x for 5 min each with agitations at room temperature.
      3. The slides have to be placed with kidney sections facing up in the humidified chamber.
      4. Each section on the slides has to be limited with a PAP-pen to prevent overflows.
      5. 100-200 μl of blocking buffer (10% Normal Goat Serum diluted in PBS 1x) has to be added to each section and incubated for 1 h, at room temperature, in the humidified box.
      6. 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.
      7. 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.
      8. 100 μl of primary antibodies mix has to be added to each section and incubated in the humidified chamber overnight at +4 °C.
      9. 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.
      10. The slides have to be rinsedin PBS 1x for three times, 5 min each.
      11. 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 (1:200) and Alexa Fluor goat anti-rabbit 488 (1:200) in PBS 1x in the dark.
      12. The secondary antibodies mix (the volume ranges from 50-100 µl depending of the biopsies area) has to be applied on the sections and incubate for 1 h in the humidified box in the dark.
      13. The slides have to be submersed in PBS 1x for 5 min for 3 times.
      14. To counterstain the slides, a solution of TOPRO3 (dilution 1:3,000) in PBS 1x has to be prepared and added on each section. The slides have to be incubated in the humidified box and in the dark for 10 min.
      15. TOPRO3 solution has to be drained off from the slides, and without rising, a drop of fluoromount has to be added on the slides to mount a coverslip. Make sure that no air bubbles are formed.
      16. The slides have to be sealed with a nail polish to preserve staining and stored for a long time in the dark.
      17. 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.



Figure 1. LPS induced PMT in a swine model of LPS-induced AKI. A. Representative pictures of PDGRβ and α-SMA staining performed in swine renal sections of healthy and endotoxemic pigs.Pericytes were double-stained for PDGFRβ (green) and α-SMA (red). In healthy pigs (T9 CTR), α-SMA immunostaining signal was detected only within vascular smooth-muscle cells (dotted white arrow) and PDGFRβ+/αSMA+ perivascular cells were barely detectable. In endotoxemic pigs (T9 LPS) the number of PDGFRβ+/αSMA+ cells significantly increased (white arrows), as shown by the colocalization of the two markers in the interstitiumand at glomerular level. Magnification 630X. B. Quantitative data (n = 5 for each group, P = 0.001). Results are expressed as median ± IQR of five independent pigs for each group. The statistical analysis was performed using the Mann-Whitney test. Modified from Figure 1 in Castellano et al. (2019). License: (http://creativecommons.org/licenses/by/4.0/).



Figure 2. Complement activation induced PMT in a swine model of I/R injury. A. Representative pictures of PDGRβ and α-SMA staining performed on paraffin sections at T0 (before ischemia) and 24 h after reperfusion (T24 CTRL). At T0, PDGFRβ+/αSMA+ cells were rarely detectable (zoomed image) and α-SMA was localized in the arterial wall (white arrow). After 24 h from I/R injury, the number of these cells dramatically increased (T24 CTRL). Magnification 630x. B. Results are expressed as median ± IQR of five independent pigs for each group. The statistical analysis was performed using the Mann-Whitney test. Modified from Figure 4 in Castellano et al. (2018). License: (http://creativecommons.org/licenses/by/4.0/).


   Two independent observers blinded to the origin of the slides counted the number of α-SMA+ and PDGFR-cells in at least 10 consecutive high-power (630x) fields (0.0567 mm2 HPF/section) for each biopsy. The values were then averaged. For each field, positive cells were counted only in the cortical area (peritubular and glomerular capillaries). Subcapsular fibrotic areas, arterial adventitia and medullary areas were excluded from the region of interest. Cell count was determined according to nuclear staining. The final reported count was the mean of the two observer measures. 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.



Figure 3. Statistical analysis by GraphPad Prism Software. A. Open GraphPad Prism and select the parameters indicated by the red arrows. B. Start to insert the values in each group indicated by the red arrow. C-D. Click analyze and choose the test for the analysis. Given the small sample (five independent pigs for each group) we choose non-parametric test, Mann-Whitney test and presented the data as Medians and IQRs.

Recipes

  1. Unmasking Buffer (10 mMSodium Citrate Buffer) (500 ml)
    1. Citric Acid Solution (10.5 g acid citric dissolved in 500 ml distilled water) (41 ml)
    2. Sodium Citrate Solution (14.7g sodium citrate dissolved in 500 ml distilled water) (9 ml)
    3. Distilled water (500 ml)
    Mix to dissolve. Adjust pH to 6.0 with 1 N HCl and mix well. Store this solution at 4 °C for longer storage
  2. Blocking solution (10% Normal Goat Serum)
    1. 5 ml PBS
    2. 500 μl Normal Goat Serum
    3. Store at 4 °C for 1 week
  3. Buffer for diluting primary antibody (5% Normal Goat Serum)
    1. 5 ml PBS 1x
    2. 250 μl Normal Goat Serum
    3. Store at 4 °C for 1 week
  4. PBS 1x for the IF washing
    1. Dissolve one Phosphate buffered saline tablet in 200 ml of deionized water yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4 at 25 °C
    2. Store at 25 °C for 1 week

Acknowledgments

These studies were supported by University of Bari “Aldo Moro”, the Italian Ministry of Health (Ricerca Finalizzata 2009 and Giovani Ricercatori 2011-2012 [GR-2011-02351027] granted to Giuseppe Castellano), a Regional Strategic Grant (Apulia Region (PSR 094) granted to Loreto Gesualdo) and an unrestricted research grant from Pharming Group.

Competing interests

Authors have no conflicts of interest or competing interests to disclose.

Ethics

These studies were approved by the ethical committee of the Ministry of Health, Italy.

References

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简介

[摘要]越来越多的证据表明,肾小管周围的毛细血管周细胞是慢性肾脏病(CKD)以及急性肾损伤(AKI)早期形成疤痕的成纤维细胞的主要来源。在败血症和I / R(缺血再灌注)损伤诱导的AKI的猪模型中,我们证明了肾周细胞能够向α- SMA +肌成纤维细胞转分化,从而导致间质纤维化。即使精确周细胞识别需要透射电子显微镜和几个标志物联合免疫(即。,的Gli ,NG2硫酸软骨素蛋白聚糖,CD146,结蛋白或CD73)和新兴的新的标志物(CD248或TEM1,唾液酸蛋白),以往的研究表明,PDGFR-β可用作肾周细胞表征的标志物。最近,对PDGFR-β和α-SMA进行了双重免疫荧光染色,以鉴定在纤维化发展的早期受损激活的周细胞(PDGFR-β + /α-SMA +细胞)。我们的数据强调了肾周细胞在败血症和I / R相关性AKI的生理病理中的关键作用。在该协议中,我们描述了猪福尔马林固定石蜡包埋(FFPE)肾脏活检中PDGFR-β和α-SMA双重免疫荧光染色的程序以及图像分析和定量方法。

[背景】肾脏纤维化被认为是主要负责肾脏疾病的进展,其与肾的损伤后的容量有限,再生有关。进行性肾脏疾病中间质纤维化的主要来源(Simone等人,2014 ; Fiorentino等人,2018)以活化的成纤维细胞(称为成肌纤维细胞)为代表,这些成纤维细胞的合成能力是从头合成α-平滑肌肌动蛋白( α-SMA)(Meran and Steadman,2011 ; Hewitson,2012)。这些细胞来自多种来源,不仅包括驻留的成纤维细胞,还包括内皮细胞,肾小管细胞,循环的骨髓来源的细胞和周细胞(Di Carlo和Peduto,2018)。一些研究评估了毛细血管周细胞在慢性肾脏疾病(CKD)中的关键作用(Lin等人,2008 ; Grgic等人,2012 ; Kramann等人,2015)以及急性肾损伤的早期阶段(AKI)。(Leaf等人,2016 ; Castellano等人,2018和2019)。

最近的研究表明,功能失调的周细胞主要参与败血症引起的微血管功能障碍和血管渗漏,这是终末器官功能障碍和败血性休克的关键标志(Goldenberg等,2011 ;Page和Liles,2013 ;Castellano等, 2014 ; Stasi等人,201 7 ; Fani等人,2018 )。与这些发现一致的是,我们发现用LPS攻击的猪在肾实质的微脉管系统中导致周细胞功能失调(Castellano等,2019)(图1)。

近年来,命运追踪作图和超微结构分析为几种肾脏疾病小鼠模型的周细胞行为提供了更多的启示(Lin等人,2008 ;Humphreys等人,2010)。公平地说,在大型动物模型中,通过缘分追踪分析需要复杂技术的标准来识别周细胞是不切实际的。由于PDGFR-β被定义为肾周细胞的分离和表征的组成性标志物(Chen等,2011 ; Wang等人,2017),而a-SMA是与成肌纤维细胞相关的标志物(Hewitson,2012)。 PDGFR-β和α-SMA的双标记为AKI期间肾实质中的周细胞分布和功能失调激活提供了“画面” (Guzzi等人,2019)。根据细胞作为EndMT的其他分化现象,PDGFR-β的丧失以及α-SMA,胶原蛋白I和MMP蛋白水平的增加被定义为成肌成纤维细胞转化(PMT)的周细胞(Chang等人,2012)。

首先,我们对PDGFR-β和α-SMA进行了双重免疫荧光染色,以研究猪和小鼠肾I / R损伤模型中的PMT (图2)( Castellano等。,2018)。有趣的是,在正常猪的肾脏活检,我们发现PDGFRβ和NG2的肾小管周围毛细血管的共定位,表明PDGFRβ的猪模型的整体精度为血管周细胞标记,而肾小球系膜细胞PDGFRβ + ,但NG2 - 。肾脏I / R 24小时后,我们观察到组成标记PDGFR-β和NG2的下调以及α-SMA的急剧上调。

这些数据在我们的AKI脓毒症模型中也得到了证实,并且综合起来我们证明PDGFR-β +周细胞能够合成获得肌成纤维细胞典型特征的促纤维化标志物,从而导致细胞外基质沉积和间质纤维化(Castellano等。,2019)。因此,我们在体外证实了我们的数据,我们发现人胎盘来源的周细胞暴露于I / R损伤(C5a)和败血症刺激(LPS)导致获得α-SMA收缩应激纤维(Castellano等人,2018和2019)。

因此,本文所述的方案可用于表征周细胞及其功能失调的活化,并将促进早期急性肾损伤以及与炎症和纤维化有关的其他领域的研究(Castellano等人,2018和2019)。

关键字:PDGFR-β+/α-SMA+ 荧光免疫检验法, 周细胞-肌成纤维细胞转化, 肾脏周皮细胞, 猪肾脏活检, 肾脏I / R损伤, 败血症, 急性肾损伤, 血管周细胞, 败血症-慢性肾病转变

材料和试剂

1.铝箔(卡尔罗斯GmbH的,Rotilabo ® ,Ç atalog号:0954.1)        
2. Stripette TM血清移液管5 ml,10 ml(Corning,目录号:4051、4488)        
3.盘子60毫米(Corning,目录号:3261)        
4.猎鹰®离心管50毫升,15毫升(康宁,产品目录号:352070,352096)        
5.安全防护罩手术刀(Biosigma srl ,目录号:530050)        
6.金属钢基础模具(Leica Biosystem ,目录号:3803081)        
7.光学显微镜盖玻片24 x 40 x 0.16 mm(Bio Optica SpA ,目录号:09-2040)        
8.纸巾        
9.组织包埋环(Bio Optica SpA ,目录号:07-7650)        
10.指甲油    
11. 10%的中性缓冲液ED福尔马林(NBF) (Diapath SpA的,Ç atalog号:F0047)    
12.石蜡实验室O型蜡56-58 ℃下(HISTO -Line实验室SRL ,C atalog号:R0040-20)    
13.无菌DPBS(Euroclone SpA,商标号:ECM4053XL)    
14.二甲苯(生物ø ptica的SpA ,Ç atalog号:06-1304Q )                  
15.无水乙醇(100%)(生物ø ptica的SpA ,Ç atalog号:06-10099)    
16.去离子水    
17.显微镜玻片超级冰霜®加(生物。光学SpA公司,Ç atalog号:09-OPLUS)    
18.正常山羊血清(西格玛- Aldrich公司,Ç atalog号码:G9023,在-20以等份试样储存℃)    
19.磷酸盐缓冲盐水片剂(西格玛- Aldrich公司,Ç atalog号:P4417)    
20. α-SMA的小鼠单克隆(Santa Cruz Biotechnology公司,Ç atalog号:SC-32251)    
21. PDGFR-β单克隆抗体兔抗人(Abcam公司,Ç atalog号:ab32570)    
22.的AlexaFluor的山羊抗兔FITC缀合的(488纳米)抗体(分子探针,Ç atalog号:A11070)    
23.的AlexaFluor山羊抗小鼠缀合TRITC(555 nm)的抗体(分子探针,Ç atalog号:A21127)    
24. TO-PRO3碘化物(Invitrogen,目录号:T3605)    
25. FluoroMount (生物。光学的SpA ,Ç atalog号:K024)    
26.磷酸盐缓冲盐水片(西格玛,目录号:P4417)    
27.柠檬酸(Sigma,目录号:C759)    
28.柠檬酸钠(Sigma,目录号S4641)    
29.正常山羊血清(Sigma-Aldrich,目录号G9023,在-20°C下等分储存)    
30.解掩膜缓冲液(10 mM柠檬酸钠缓冲液)(请参阅食谱)    
31.封闭溶液(10%正常山羊血清)(请参阅食谱)    
32.稀释一抗的缓冲液(5%正常山羊血清)(请参阅食谱)    
33.用于中频洗涤的PBS 1x (请参阅食谱)    
 
设备
 
高压釜(AHSI SpA ,型号:FVA / A1)
镊子(Thermo Fisher Scientific,目录号10303611 )
显微镜用剪刀(Bio Optica SpA ,目录号:32-703)
手套和护目镜
带电磁搅拌器的热板
PMP Hellendhal染色缸(卡特尔SpA的,实验室器皿司卡特尔TM ,Ç atalog号:0035500)
水浴
通风柜(Euroclone SpA )
移液器(0.5-10,10-100和100-1 ,000微升)仪(Eppendorf,产品目录号:3111000.122,3111000.149,3111000.165)
巴德单片穿刺活检仪长16 G x 20厘米(CR Bard.Inc 。,货号:121620)
58 °C石蜡浴冷却板(Bio Optica SpA ,目录号:PF100)
切片机(Leica Biosystem,目录号:RM2125 RTS)
组织学浴(Bio Optica SpA ,目录号:WB100)
微波
用于免疫染色的木棒笔(Bio Optica SpA ,目录号:11-100 )
轨道振动筛(Thermo Scientific,目录号:SHKA2000)
加湿室
pH值米ETER(Thermo Scientific的,Ç atalog号:3115101)
 
软件
 
的GraphPad Prism ® (版本5.0)(格拉夫派得软件)
图像分析软件(Leica QWin)(Leica)
 
程序
 
收集肾脏样本
准备用于样品处理的作业工具
在包装后其中高压灭菌干净镊子和剪刀铝箔。
肾脏活检程序
在实验过程开始时(T0),在健康和治疗猪的几个中间时间点进行针头活检。肾活检是与自动活检针直接超声引导下进行。处死后,将肾脏移出,并用安全的安全防护手术刀对骨皮质和髓质区域进行楔形活检。
每次肾脏活检均在装有20 ml冷(〜4°C)无菌DPBS的50 ml F alcon中递送至组织学实验室,并通过每只动物的唯一代码进行识别。
所有肾活检必须在最浸入冰半小时。
固定前准备肾脏活检
每次肾脏活检都必须转移到一个装有3 ml冷(约5°C)DPBS的60 mm皿中,并用无菌镊子将其清除掉任何残留的结缔组织或脂肪组织。
用无菌一次性手术刀可以将样本尺寸减小到0.5 x 0.5厘米。
肾标本必须在含有3毫升冷的另外60 mm皿转移(〜4℃)DPBS。
 
石蜡切片处理
肾脏标本固定
肾脏标本必须在室温下转移到装有10%中性福尔马林(NBF)的15 ml锥形管中8 h,但不得超过24 h。确保您有足够的固定剂覆盖组织。固定体积应为组织体积的5-10倍。
注:牛逼,他的肾标本不石蜡盒子托起。手动进行组织处理以模仿自动化处理的各个累积步骤。
肾标本脱水
固定后,福尔马林必须被丢弃。
肾标本有脱水移动酒精等级的步骤:
在室温下10 ml 50%乙醇放置40分钟(最多5天)。
10 ml 70%乙醇在室温下放置40分钟。
10 ml 95%乙醇在室温下放置25分钟。
10 ml 95%乙醇在室温下放置25分钟。
10 ml 100%乙醇在室温下放置25分钟。
10 ml 100%乙醇在室温下放置25分钟。
注意:这些步骤应完全完成(为这些步骤留出足够的时间),因为脱水不足会导致组织降解。
肾脏标本澄清
肾脏标本必须用二甲苯清除:
10 ml的二甲苯15分钟。
10 ml的二甲苯15分钟。
石蜡浸润肾标本
肾标本必须被转移到贝克尔在含有液体石蜡58 ℃的石蜡浴:10毫升液体石蜡2 h中的烘箱中在56℃-58 ℃(石蜡的熔融温度)。
将肾组织包埋在石蜡块中
熔融石蜡的少量具有被置于模具中,从石蜡储分配。
使用热镊子,肾样品必须被转移到阱中健康块金属钢基体的模具,将切割的一面朝下。
嵌入环必须被放入钢基础模具(在56 ℃)。石蜡色带漂浮在温水浴的表面上,在那里展开并完美展平。
模具具有可迅速地转移到冷却板(-20 ℃),并轻轻压平组织。石蜡将固化成薄薄的一层,将组织固定在适当的位置。
热石蜡已被添加到从石蜡分配器模具。确保有足够的石蜡覆盖组织。石蜡块应在-20°C下30分钟内固化。
当蜡完全冷却并硬化后,必须很容易地从模具中弹出石蜡块。蜡块不应粘住。如果蜡破裂或薄纸排列不正确,只需再次将其融化并重新开始。
制备石蜡切片:切片方案:
石蜡块具有在对一个40切片机和浮子期望厚度(通常为4-5微米)被分段含有蒸馏水℃水浴中。
笔记:
记录各部分的方向和顺序。
组织学浴必须达到最高56°C的温度,以使石蜡切片变平。
石蜡切片必须转移到Super Frost Plus显微镜载玻片上。
载玻片必须干燥过夜,并在室温下保存,直到可以使用为止。
 
免疫荧光染色
石蜡切片必须脱蜡并重新水化(整个过程必须在通风柜中进行设置)
载玻片必须在纯二甲苯中脱蜡15分钟。
注意:二甲苯去除了组织周围多余的蜡,并去除了石蜡切片。
载玻片必须使用梯度酒精系列再水化几分钟,然后用去离子水冲洗:
100%乙醇浸泡6分钟。
乙醇95%1分钟。
乙醇70%1分钟。
乙醇50%1分钟。
去离子水5分钟(连续3次)。
热诱导抗原暴露
载玻片必须转移到塑料垂直染色罐(适用于微波)中,其中装有100 ml pH = 6的柠檬酸钠缓冲液10 mM(装满帽沿),并放入水浴中。
注意:获得的水棒是将滑动罐放入一杯水(适用于微波)中,然后将整个东西放入微件中。这减少了罐内的沸腾。
载玻片必须经受5分钟的三个微波(750 W)循环。
抗原修复缓冲液的体积必须沸腾期间在每一周期为顶起来用蒸馏水,以避免蒸发。
幻灯片必须是让慢慢冷却。
注意:在每个抗原揭露过程中,载玻片的数量必须与染色罐中可用的位置相同,以便揭露的效率始终相同。如果使用的载玻片较少,请用空的玻璃载玻片填充空白处。这样可确保每个载玻片具有相似的热量分布,并减少了抗原回收的差异。
免疫荧光染色:
冷却的载玻片必须转移到装有去离子水的玻璃垂直染色罐中。在室温下,用去离子水在定轨摇床上清洗载玻片5分钟。
载玻片必须在室温下搅拌下,用三份PBS 1x漂洗5分钟,每次5分钟。
幻灯片必须放置与肾脏切片朝上在加湿室。
幻灯片上的每个部分都必须用PAP笔限制,以防止溢出。
100-200微升封闭缓冲液(10%正常山羊血清的PBS中稀释的1×)必须被添加到每个部分,并温育1个小时,在室温下,在加湿箱中。
染色组合必须制备组合并适当稀释的两种不同的抗体(封闭混合物(山羊血清的5%)中的α - SMA 1:100,PDGFR-β1:100),请注意之前单独测试的稀释度。
封闭缓冲液必须被轻轻地除去(例如,通过倾斜滑动侧身在纸巾上),而不添加第一抗体之前漂洗。
100微升的初级抗体混合物必须被添加到每个部分,并在加湿室中温育过夜,在4 ℃。
中频反应的阴性对照具有获得孵育同种型对照抗体代替初级抗体上的良好分隔部混合在幻灯片中的一个。
将载玻片必须被rinsedin PBS 1 X三次,每次5分钟。
必须分别混合两种二抗以结合两种一抗的混合物,并进行适当稀释:Alexa Fluor山羊抗小鼠555(1:200)和Alexa Fluor山羊抗兔488(1:200)在黑暗中PBS 1x。
二次抗体(50-100体积范围微升取决于混合的活检区域)具有在黑暗中的加湿箱上的部分和孵化要施加1个小时。
载玻片必须浸入PBS 1x中3分钟5分钟。
为了对幻灯片,TOPRO3的溶液(稀释1:3 ,000)的PBS 1X必须制备并添加于各部分编 载玻片必须在加湿箱中在黑暗中孵育10分钟。
TOPRO3溶液必须从载玻片上排出,并且在不上升的情况下,必须在载玻片上添加一滴氟胶以安装盖玻片。确保没有气泡形成。
载玻片必须用指甲油密封以防止弄脏,并在黑暗中长时间保存。
载玻片必须通过共聚焦显微镜Leica TCS SP2(Leica)获取。
 
数据分析
 
使用配备有氩k气(488 nm),绿氖气(543 nm)和氦氖气(633 nm)激光器的Leica TCS SP2(Leica)进行共聚焦显微镜检查。通过该截面的z轴以500 nm的间隔拍摄共焦图像,总共深度为7 µm。分析来自单个光学平面和多个串行光学部分的图像,并在所有三个激光通道中依次扫描图像。这些影像是使用63X物镜拍摄并导出为TIFF文件,如图表明小号1和2。
 


图1.在LPS诱导的AKI的猪模型中,LPS诱导的PMT。A.在健康和内毒素猪的猪肾脏切片中进行PDGRβ和α-SMA染色的代表性图片。对于PDGFRβ(绿色)和α-SMA (红色),周细胞被双重染色。在健康猪(T9 CTR),α-SMA免疫染色信号才被内血管平滑肌细胞(虚线白箭头)和检测到的PD GFR β + / α SMA +血管周围细胞中几乎检测不到。在内毒素血症的猪(T9 LPS)PDGFRβ的数量+ /αSMA +细胞显著增加(白色箭头),如由在两个标记的共定位interstitiumand在肾小球水平。放大率630X。B.定量数据(每组n = 5 ,P = 0.001)。结果表示为每组五只独立猪的中值±IQR 。使用Mann - Whitney检验进行统计分析。根据Castellano等人的图1进行修改。(2019 )。许可证:(http://creativecommons.org/licenses/by/4.0 /)。
 


图2.在I / R损伤的模型中补体激活诱导的PMT 。A.在T0(缺血前)和再灌注后24 h(T24 CTRL)对石蜡切片进行PDGRβ和α-SMA染色的代表性图片。在T0,PDGFR β + / α SMA +细胞是很少可检测的(缩小图像)和α-SMA在动脉壁(白色箭头)本地化。I / R损伤24小时后,这些细胞的数量急剧增加(T24 CTRL)。放大率630x。B.结果表示为每组五只独立猪的中值±IQR。使用Mann - Whitney检验进行统计分析。根据Castellano等人的图4进行修改。(2018 )。许可证:(http://creativecommons.org/licenses/by/4.0/)。
 
  两名对切片的来源不了解的独立观察员对每次活检至少连续10次高倍(630x)视野(0.0567 mm 2 HPF /切片)中的α-SMA +和PDGFR细胞计数。然后将这些值平均。对于每个视野,仅在皮质区域(周围和肾小球毛细血管)中计数阳性细胞。囊下纤维化区域,动脉外膜和髓质区域被排除在目标区域之外。根据核染色确定细胞计数。最终报告的计数是两项观察员措施的平均值。观察者之间的差异绝不会大于20%。
  结果以中位数±四分位数间距(IQR)表示。通过Mann - Whitney检验评估统计学上的显着差异。甲P值< 0.05是显著。小号使用GraphPad Prism软件5进行tatistical分析如图3所示。
 


图3.通过GraphPad Prism软件进行的统计分析。A.打开GraphPad Prism并选择红色箭头指示的参数。B.开始在红色箭头指示的每个组中插入值。光盘。单击分析,然后选择要进行分析的测试。给定小样本(每组五只独立的猪),我们选择非参数检验,Mann - Whitney检验,并将数据表示为中位数和IQR。
 
菜谱
 
Unmasking Buffer(10毫升柠檬酸钠缓冲液)(500 ml)
柠檬酸溶液(10.5 g柠檬酸溶于500 ml蒸馏水)(41 ml )
柠檬酸钠溶液(将14.7g柠檬酸钠溶于500 ml蒸馏水)(9 ml )
蒸馏水(500毫升)
混合溶解。用1 N HCl将pH调节至6.0,并充分混合。将此溶液存储在4 °C以便更长的存储时间
封闭溶液(10%正常山羊血清)
5毫升PBS
500 μ升正常山羊血清
在4°C下保存1周
稀释一抗的缓冲液(5%正常山羊血清)
5毫升P BS 1 x
250 μ升正常山羊血清
在4°C下保存1周
PBS 1x用于中频洗涤
在200 ml去离子水中溶解一块磷酸盐缓冲盐水片,在25 °C时产生0.01 M磷酸盐缓冲液,0.0027 M氯化钾和0.137 M氯化钠,pH 7.4
在25°C下保存1周
 
致谢
 
这些研究得到了意大利卫生部的Bari“ Aldo Moro”大学(授予Giuseppe Castellano的Ricerca Finalizzata 2009和Giovani Ricercatori 2011-2012 [GR-2011-02351027])的支持,这是一项区域战略拨款(普利亚地区(PSR) 094)授予Loreto Gesualdo),并获得Pharming Group的无限制研究资助。
 
利益争夺
 
作者没有利益冲突或竞争利益要披露。
 
伦理
 
这些研究得到了意大利卫生部伦理委员会的批准。
 
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引用:Stasi, A., Franzin, R., Divella, C., Gesualdo, L., Stallone, G. and Castellano, G. (2020). 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. Bio-protocol 10(19): e3779. DOI: 10.21769/BioProtoc.3779.
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