参见作者原研究论文

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Dec 2018

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Immunohistochemistry of Kidney a-SMA, Collagen 1, and Collagen 3, in A Novel Mouse Model of Reno-cardiac Syndrome
心肾综合征新型小鼠模型中肾脏a-SMA,胶原1和胶原3的免疫组化研究   

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Abstract

Cardiorenal syndrome defines a synergistic pathology of the heart and kidneys where failure of one organ causes failure in the other. The incidence of cardiovascular mortality caused by this syndrome, is 20 fold higher in the end stage renal disease (ESRD) population compared to the population as a whole thus necessitating the need for improved therapeutic strategies to combat reno-cardiac pathologies.

Murine in vivo models play a major role in such research permitting precise genetic modification thus reducing miscellany, however presently there is no steadfast model of reno-cardiac syndrome in the most common genetically modified mouse strain, the C57BL/6 mouse. In this study we have modified an established model of chronic renal disease using adenine diet and extended the associated pathology achieving chronic renal failure and consequent reno-cardiac syndrome in the C57BL/6 mouse.

Eight week-old male C57BL/6 mice were acclimatized for 7 days before administration of a 0.15% adenine diet or control diet for 20 weeks after which the experiment was terminated and blood, urine and organs were collected and analyzed biochemically and by immunohistochemistry.

Administration of 0.15% adenine diet caused progressive renal failure resulting in a reno-cardiac syndrome confirmed by a significantly increased heart to body weight ratio (P < 0.0001). Blood biochemistry showed that adenine fed mice had significantly increased serum creatinine, urea (P < 0.0001), and a significantly reduced glomerular filtration rate (P < 0.05), while immunohistochemistry of the kidneys for α-SMA, collagen 1 and collagen 3 showed severe fibrosis.

We present a novel regimen of adenine diet which induces both chronic kidney disease and reno-cardiac syndrome in the C57BL/6 mouse strain. The non-surgical nature of this model makes it highly reproducible compared to other models currently available.

Keywords: Cardio-renal syndrome (心肾综合征), Chronic kidney disease (慢性肾脏疾病), Cardiac hypertrophy (心脏肥大), C57BL/6 mouse (C57BL/6小鼠), Renal fibrosis (肾纤维化), Experimental renal failure (实验性肾功能衰竭), Reno-cardiac syndrome (肾心综合征)

Background

Chronic kidney disease (CKD) results in vascular and cardiac dysfunction, termed cardio renal syndrome, defined more specifically as reno-cardiac syndrome (RCS) by Ronco et al. (2008). RCS is a predominant feature of end stage renal disease (ESRD) resulting in a 20 fold higher incidence of cardiovascular mortality (compared to the normal population as a whole) and cardiovascular events account for approximately 50% of mortality in ESRD patients (de Jager et al., 2009; Steenkamp et al., 2013). As the ESRD population is increasing worldwide research into novel therapies to combat RCS is vital.

Animal models have significantly aided research into many disease pathologies and the development of technology to genetically modify strains has enabled such models to become even more potent through better targeting of pathological genes.

The sub-total nephrectomy model has been up till now the most successful model of RCS and involves the surgical removal or negation of (5/6ths) of the kidney mass (Morrison, 1962). The resulting renal ischaemia/reduced nephron mass initiates hypertension and subsequent left ventricular hypertrophy (LVH) leading to progressive left ventricular dilatation ( Kumar et al., 2014). In parallel, fibroblast activation leads to cardiac hypertrophy and fibrosis ( Hewitson, 2012; Bursac, 2014). The disadvantages of this model is that it requires surgical expertise, expensive surgical facilities and that it is prone to inter and intra operator variability. The preferred rodent used in this model is the rat because in mice, this model is much less effective giving highly variable results (Hewitson et al., 2015). Further to this the C57BL/6 mouse strain, the preferred strain for genetic modification (Seong et al., 2004) has been reported to be resistant to the development of CKD by subtotal nephrectomy (Kren and Hostetter, 1999).

An alternative model of CKD is adenine induced. This non-surgical and therefore highly reproducible method was first described by Yokozawa et al. (1982), who noted that the metabolism of adenine differed from that of other purines and that it was nephrotoxic. However this chemical method although very successful in rats, has not flourished in mice because they do not like the adenine taste resulting in rapid weight loss leading to mortality after 4 weeks. Jia et al. (2013), improved the palatability of the diet by the addition of casein, extending the course of the administration to 8 weeks however this is not long enough to achieve RCS.

We found that we could conceal the adenine taste (without the need for addition of casein), by decreasing the concentration of the diet to 0.15% and by beginning its administration when the mice reached 9 weeks of age, extended the course of the regimen to 20 weeks in order to achieve CKD and subsequently RCS as seen by blood biochemistry and heart/bodyweight ratio data (Table 1), immunoblotting and immunohistochemistry.

Therefore we succeeded in developing a highly reproducible model which does not require surgical expertise or facilities (Kieswich et al., 2018).

Although we did not measure blood pressure (BP) we predict that the main cause of the RCS was haemodynamic due to kidney dysfunction and therefore, if BP was measured consciously, e.g., by a telemetric method, this could also prove to be a successful non-surgical model of hypertension.


Table 1. Results showing evidence of RCS. Control group (n = 6) received standard chow for 20 weeks. Adenine treated group (n = 10) received standard chow with the addition of 0.15% adenine for 20 weeks. Compared with animals receiving normal chow, a 20-week diet of 0.15% adenine caused significant increases in plasma urea (P < 0.0001) and creatinine (P < 0.0001), a significantly reduced glomerular filtration rate (GFR) (P < 0.05), and a significantly greater heart weight-to body weight ratio (P < 0.0001).

***P < 0.0001,*P < 0.05

Materials and Reagents

  1. 25 gauge needles (Fisher Scientific, catalog number: 10442204 )
  2. 1 ml syringes (Fisher Scientific, catalog number: 15849152 )
  3. Eppendorf tubes (Fisher Scientific, catalog number: 13094697 )
  4. Embedding cassettes (Fisher Scientific, Simport Acetal Histology Cassettes, catalog number: 10420823 )
  5. Glass slides (Fisher Scientific, catalog number: 12343138 )
  6. Pipettes and tips
    ErgoOne Single-Channel Pipettes 20-200 µl (STARLAB (UK) Ltd, catalog number: S7100-2200 )
    ErgoOne Single-Channel Pipettes 100-1,000 µl (STARLAB (UK) Ltd, catalog number: S7110-1000 )
    Yellow Tips (Sterile), Racked (STARLAB (UK) Ltd, catalog number: S1111-0816-C )
    1,000 µl Blue Graduated Tip (Sterile), Racked (STARLAB (UK) Ltd, catalog number: S1111-6811-C )
  7. Hydrophobic barrier pen (Vector Laboratories, ImmEdge Hydrophobic Barrier Pen, catalog number: H-4001 )
  8. Humidified chamber e.g., a plastic box with airtight lid (prevents tissue from drying out). Place wet tissue paper into the bottom of the box. To keep slides off the wet paper insert a perforated base (can be made by cutting pasteur pipettes into equal size pieces and taping them into a grid formation. Ensure it is level)
  9. Tissue paper
  10. Vessel with slide rack to hold approximately 400-500 ml
  11. Brush (for cleaning microtome blade between samples)
  12. Glass coverslips (Fisher Scientific, catalog number: 12343138 )
  13. 8-week old, male C57BL/6 mice (n = 6 per group) (Charles River, Margate, UK)
  14. 20 Kg Adenine diet (LBS-Biotech, Hookwood, UK, SDS diets, catalog number: 824534 ), RM1 + 0.15% Adenine, storage temperature 4 °C
    Note: Buy 20 Kg 824534 RM1 diet (normal adenine concentration) from SDS diets at same time to use as control). For a detailed composition of the diet see attached file ‘Adenine diet composition’.
  15. Ketamine 100 mg/ml solution (Narketan, MWI Animal Health, Somerset, UK, catalog number: 0 3120257 ), storage temperature–Room Temperature (RTP)
  16. Xylazine hydrochloride 2% w/v solution (Rompun, MWI Animal Health, Somerset, UK, catalog number: 0 2150569 ), storage temperature–RTP
  17. Buprenorphine 0.3 mg/ml solution (Vertegesic, MWI Animal Health, Somerset, UK, catalog number: 0 1300258 ), storage temperature–RTP
  18. Heparin Sodium 5,000 u/ml (MWI Animal Health, Somerset, UK, catalog number: 30394030 ), storage temperature–RTP
  19. Ethanol (Fisher Scientific, catalog number: 10233962 ), storage temperature–RTP
  20. Phosphate Buffered Saline (Fisher Scientific, PBS, catalog number: 10173433 ), storage temperature 4 °C
  21. Formalin (Sigma-Aldrich, catalog number: HT5014 ), storage temperature–RTP
  22. Xylene (Fisher Scientific, catalog number: 10467270 ), storage temperature–RTP
  23. Parrafin (Fisher Scientific, Thermo Scientific Richard-Allan Scientific Histoplast Paraffin, catalog number: 12683026 ), storage temperature–RTP
  24. Hydrogen peroxide (Fisher Scientific, H2O2, catalog number: 10687022 ), storage temperature 4 °C
  25. Triton X-100 (Sigma-Aldrich, catalog number: T8787 ), storage temperature–RTP
  26. Bovine Serum Albumin (Fisher Scientific, BSA, catalog number: 11423164 ), storage temperature 4 °C
  27. Primary antibody, anti α-Smooth muscle actin antibody (Abcam, catalog number: ab5694 ), storage temperature -20 °C
  28. Primary antibody, anti Collagen I antibody (Abcam, catalog number: ab21286 ), storage temperature 4 °C
  29. Primary antibody, anti Collagen III antibody (Abcam, catalog number: ab7778 ), storage temperature 4 °C
  30. Secondary antibody, Goat Anti-Rabbit IgG H&L (HRP) (Abcam, catalog number: ab205718 ), storage temperature 4 °C
  31. Normal Goat serum (Abcam, catalog number: ab7481 ), storage temperature 4 °C
  32. DAB (Fisher Scientific, catalog number: 10006913 ), DAB storage temperature -20 °C. DAB Substrate kit storage temperature 4 °C
  33. Glacial acetic acid (Sigma-Aldrich, catalog number: A6283 ), storage temperature–RTP
  34. TRIS hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: RES3098T-B7 ), storage temperature RTP
  35. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 ), storage temperature–RTP
  36. Ammonium hydroxide (NH4OH) 30% solution (Sigma-Aldrich, catalog number: 221228-1L-A ), storage temperature RTP
  37. Sodium citrate (Sigma-Aldrich, Tri-sodium citrate (dihydrate), catalog number: W302600 ), storage temperature RTP
  38. Tween 20 (Sigma-Aldrich, catalog number: 93773 ), storage temperature RTP
  39. Hematoxylin (Vector Laboratories Ltd, VECTOR®HEMATOXYLINNUCLEAR COUNTERSTAIN, catalog number: H-3401 ), storage temperature RTP
  40. Mounting medium (Vector Laboratories, VectaMount Permanent Mounting Medium, catalog number: H-5000 ), storage temperature RTP
  41. Hydrochloric acid (Sigma-Aldrich, catalog number: 258148 ), storage temperature RTP
  42. Bluing solution (see Recipes)
  43. Acid rinse solution (see Recipes)
  44. Sodium citrate buffer (see Recipes)
  45. TBS buffer (see Recipes)

Equipment

  1. Dissection Forceps (Harvard Apparatus, Jewelers Forceps, No. 7, catalog number: 72-8685 )
  2. Scissors (Harvard Apparatus, Eye Scissors, Straight, Sharp/Sharp, catalog number: 2-8438 )
  3. Ear punch for identifying mice (Harvard Apparatus, Model 4.7.2 Forceps Type Punch, 2.0 mm hole, catalog number: 34-0137 )
  4. Fume hood (Fisher Scientific, Thermo Scientific Hyperclean TruAir Ductless Fume Hood, catalog number: 15613136 )
  5. Orbital shaker (Fisher Scientific, Stuart Orbital Shaker, catalog number: 10759145 )
  6. Vacuum Oven (Fisher Scientific, Technico, catalog number: 13025703 )
  7. Microtome and blade, Fisher Scientific, HM 325 Rotary Microtome, catalog number: 12052999 )
  8. Waterbath (Fisher Scientific, Clifton Unstirred Digital Water Bath, catalog number: 15700619 )
  9. Domestic stainless steel pressure cooker (e.g., Argos, Tower Compact 4 Litre Pressure Cooker, catalog number: 711/5008 )
  10. Hot plate (Fisher Scientific, Stuart Hotplate with Stirrer, catalog number: 11966558 )
  11. Centrifuge (Fisher Scientific, Thermo Scientific Heraeus Megafuge 8, catalog number: 15211026 )
  12. Microscope (3D Histech Ltd, Budapest, Hungary, Panoramic Scanning 250 microscope)
  13. Refrigerator (Fisher Scientific, Loughborough, UK, Liebherr, 12088281)
  14. Staining set consisting of multiple containers (Tissue Tek Slide Staining Dish, Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands, S76316)

Software

  1. Panoramic Viewer software (3D Histech Ltd, Budapest, Hungary)
  2. ImageJ software (https://imagej.nih.gov)
  3. Excel (https://products.office.com/en-gb/excel)
  4. GraphPad Prism (https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Induction of reno-cardiac syndrome in mice and collection of tissue samples
    1. Allow at least 8 week old C57BL/6 strain mice to acclimatize for 7 days in animal facility with free access to food and water, in a room with 12 h light/dark cycle.
    2. On day 8 weigh mice and change cages/diets giving half mice normal chow and the other half chow containing 0.15% adenine.
    3. Continue with diets administration to mice for 20 weeks weighing them every week.

    At week 20:
    1. Prepare anaesthetic Ketamine:Xylazine as 2:1 ratio. Dilute 1:10 with PBS. Weigh mouse and administer at 1.5 ml/kg, intraperitoneally.
      After 3 min when mouse is anaesthetized (check pinch reflex i.e., pinch foot, there should be no response).
    2. Administer buprenorphine (0.03 ml/Kg), intraperitoneally. Wait 3 min.
    3. Remove blood by cardiac puncture, performed by inserting heparinized 25 gauge needle attached to 1 ml syringe into left ventricle (see video link, Stewart and Schroeder, 2020), heparinize needle by briefly drawing up and expelling heparin from a tube, heparin may be reused for multiple needles.
    4. Approximately 0.5 ml of blood should be collected. Perform laparotomy, followed by thoracotomy. Remove heart and weigh. Blood should be centrifuged at 17,000 x g/3 min for separation of plasma and stored in refrigerator.
    5. Remove left and right kidneys. Wash in cold PBS briefly, then cut longitudinally and place into formalin for fixation.
    6. After 24 h make up a solution of 70% ethanol and transfer kidneys from formalin into 70% ethanol.
    7. After 24 h rinse tissue in PBS.
    8. Send blood to a service laboratory (IDEXX, Bioresearch, Ludwigsberg, Germany), for biochemistry measurements (plasma creatinine and urea) to determine degree of renal failure.

  2. Paraffinisation of tissue
    1. Dehydrate tissue
      1. Incubate in 50% ethanol for 10 min, room temperature (RTP).
      2. Then 70% ethanol for 10 min (RTP).
      3. Then 80% ethanol for 10 min (RTP).
      4. Then 95% ethanol for 10 min (RTP).
      5. Then 100% ethanol for 10 min (RTP). Repeat this step 2 more times.
      6. Then 2:1 ratio ethanol:xylene for 10 min (RTP).
      7. Then 1:1 ratio ethanol:xylene for 10 min (RTP).
      8. Then 1:2 ratio ethanol:xylene for 10 min (RTP).
      9. Then 100% xylene for 10 min (RTP). Repeat this step 2 more times.
      10. Transfer samples to a vacuum oven set at 56 °C.
      11. Then 2:1 ratio xylene:paraffin for 30 min, 56 °C.
      12. Then 1:1 ratio xylene:paraffin for 30 min, 56 °C.
      13. Then 1:2 ratio xylene:paraffin for 30 min, 56 °C.
      14. Then 100% paraffin for 2 h, 56 °C.
      15. Then 100% paraffin overnight, 56 °C.
      16. Embed longitudinally cut kidney in fresh paraffin orientated so internal side faces upwards in wax in embedding cassette.
    2. Sectioning
      1. Place embedding cassettes with samples on ice for approximately 2 h or preferably in refrigerator overnight to cool down and facilitate cutting.
      2. Cut 5 μm sections of kidney using microtome.
      3. Place into water bath set at 42 °C.
      4. Collect individual sections onto slides.
      5. Place into slide rack and leave to dry in oven at 37 °C overnight.
    3. Deparaffinize and rehydrate slides
      1. Take slides in rack and incubate in xylene for 3 min, room temperature (RTP). Repeat this step 2 more times.
      2. Then xylene:100% ethanol 1:1 ratio for 3 min (RTP).
      3. Then 100% ethanol for 3 min (RTP). Repeat this step.
      4. Then 95% ethanol for 3 min (RTP).
      5. Then 70% ethanol for 3 min (RTP).
      6. Then 50% ethanol for 3 min (RTP).
      7. Finally rinse off excess ethanol by rinsing under cold tap water. Leave slides rinsing under tap water until ready for antigen retrieval protocol (to prevent drying out).
    4. Antigen retrieval
      1. Pour enough of the sodium citrate pH 6.0 buffer in to the slide container to cover the slides and place into the pressure cooker.
      2. With the pressure cooker on the hotplate turn on to full power. Leave the pressure cooker lid resting on top (unfastened).
      3. Once the cooker is boiling, carefully transfer the slides from the tap water into it using forceps. Fasten the lid according to the manufacturer’s instructions.
      4. Allow the cooker to reach full pressure, and then time 3 min.
      5. After 3 min have elapsed, switch off the hot plate, carefully remove the cooker off the hotplate and place the pressure cooker in a sink.
      6. Activate the pressure release valve and run cold tap water over the cooker. Once de-pressurized, remove the lid and run cold tap water into the cooker for 10 min.
        Slides are now ready for staining.
    5. Staining
      1. Add Triton X-100 to TBS buffer at 0.025% final concentration.
      2. Wash slides 2 x 5 mins in TBS Triton X-100 buffer on gently rotating orbital shaker.
      3. Prepare blocking buffer (TBS + 1% BSA + 10% normal serum).
      4. Block slides in blocking buffer for 2 h on shaker, RTP.
      5. Remove slides and wipe with tissue paper (around sections).
      6. Use hydrophobic barrier pen to draw around sections.
      7. Prepare primary antibody:
        anti SMA antibody (ab5694 diluted 1:250 in TBS buffer/1% BSA)
        or
        anti collagen I (ab21286 diluted 1:200 in TBS buffer/1% BSA)
        or
        anti collagen III (ab7778 diluted 1:400 in TBS buffer/1% BSA).
      8. Prepare humidified chamber (plastic box with tray lined with wet tissue paper, under a raised platform formed of taped together Pasteur pipettes, see Figure 1 below). Place slides flat on top with sections facing up. Apply antibody onto sections on slides. Close lid.


        Figure 1. Humidified chamber. A. Pasteur pipettes taped together. B. Wet tissue paper in box. C. Slides layed on top. D. Lid closed tightly.

      1. Incubate at 4 °C overnight.
      2. Wash slides 5 min in TBS/0.025% Triton X-100, gently shaking, RTP.
      3. Repeat wash.
      4. Prepare 0.3% H2O2 in TBS
      5. Apply onto sections, incubate 15 min, gently shaking, RTP.
    6. Detection step
      1. Prepare secondary antibody, Goat Anti-Rabbit IgG H&L (HRP) (ab205718) 1:1,000 dilution in TBS/1% BSA.
      2. Apply onto slides and incubate for 1 h, gentle shaking, RTP.
      3. Add DAB chromagen onto slide, 10 min with gentle shaking, RTP.
      4. Rinse slides under running tap water for 5 min.
      5. Counter stain with hematoxylin. Add Vector Hematoxylin onto slide, 5 min with gentle shaking, RTP.
      6. Rinse slides under running tap water until water is colourless.
      7. Dip slides into acid rinse 10 times.
      8. Dip slides into tap water 10 times.
      9. Add Bluing solution onto slide, 1 min with gentle shaking, RTP.
      10. Dip slides into tap water 10 times.
    7. Dehydrate tissue, clear and mount
      1. Incubate slides in 95% ethanol for 10 min, room temperature (RTP).
      2. Then 100% ethanol for 10 min (RTP). Repeat this step 2 more times.
      3. Then 100% xylene for 10 min (RTP). Repeat this step 2 more times.
      4. Mount by draining slides
      5. Apply a drop of mounting medium onto section.
      6. Cover with coverslip ensuring there are no air bubbles.

  3. Imaging acquisition
    Images were recorded using a Panoramic Scanning 250 microscope (3D Histech Ltd, Budapest, Hungary) at 20x magnification (Figure 2).

Data analysis

Statistical analysis was performed using GraphPad Prism 5 Software (GraphPad Software Inc, CA, US). Values are presented as mean ± SEM of n observations. Differences between adenine-treated mice and controls were calculated using the unpaired t-test. P < 0.05 was considered to be statistically significant.

  1. To calculate increase in α-SMA staining between samples open file with a control sample slide in ImageJ. i.e., click ‘File’ then select ‘Open’. Invert the image by clicking ‘Edit’ then select ‘Invert’. Take a background measurement by drawing a rectangle approximately 0.5 cm2 on the dark area surrounding the image. Click ‘Analyze’ then select ‘Measure’. A value will appear in a data file called ‘Results’ which opens automatically. Next drag the same rectangle over the kidney and take 6 random measurements of kidney (by clicking ‘Measure’ each time). Repeat with 2 more control slides and then with 3 Adenine treated kidney slides (ensure that the rectangle is exactly the same size for all slides).
  2. Save all the picture and results files. Finally transfer data to an Excel file by clicking ‘Copy’ in the ‘Results’ files. Subtract background value from all measurements. Calculate average for each slide (Table 2).

    Table 2. Data in Excel spreadsheet


  3. Input results into GraphPad Prism to convert into a column graph (Table 3).

    Table 3. Data in GraphPad Prism


    1. Do an (mean ± SEM) unpaired t-test to calculate statistical significance between the 2 columns (Table 4). Repeat with collagen I and then collagen III.

      Table 4. Statistical analysis of data in GraphPad Prism


    2. Click on ‘Graphs’ icon to obtain graph. Edit graphs (Figure 2).


      Figure 2. Evidence for renal hypertrophy through immunostaining for α-SMA, collagen I, and collagen III. Control group (n = 6) received standard chow for
      20 weeks. Adenine treated group (n = 10) received standard chow with the addition of 0.15% adenine for 20 weeks. Immunostaining of kidneys showed a
      significant increase in α-SMA staining of adenine group (B) compared to control group (A) (P < 0.0001). A significant increase in collagen I staining of
      adenine group (D) compared to control group (C) (P < 0.0001). A significant increase in collagen III staining of adenine group (F) compared to control group
      (E) (P < 0.0001), mean ±SEM unpaired t-test. Representational images (3 mice per group).

Notes

  1. Animal experiments must be performed in accordance with national legal guidelines for animal research and with local ethical committee approval.
  2. Do not administer diet to animals below 9 weeks old.
  3. Do not fix tissue in formalin for more than 24 h as over fixation may inhibit antigen: antibody binding.
  4. Confirm induction of CKD from blood biochemistry results before proceeding with Procedure B. Paraffinisation of tissue.
  5. Steps using xylene should be performed inside a fume hood.

Recipes

  1. Bluing solution
    1.5 ml NH4OH (30% stock) + 98.5 ml of 70% ethanol. Store at RTP for up to 3 months
  2. Acid rinse solution
    2 ml glacial acetic acid + 98 ml of deionized or distilled H2O
    Store at RTP for up to 3 months
  3. Sodium citrate buffer
    10 mM Sodium citrate, 0.05% Tween 20, pH 6.0. Dissolve 2.94 g Tri-sodium citrate (dihydrate) in 1 L distilled water and adjust pH to 6.0 with 1 N HCl
    1. Add 0.5 ml Tween 20 and mix well
    2. Store at RTP for up to 3 months or at 4 °C for longer
  4. TBS buffer
    50 mM Tris-Cl, pH 7.5, 150 mM NaCl
    1. Dissolve 6.05 g Tris and 8.76 g NaCl in 800 ml of H2O
    2. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O
    3. TBS is stable at 4 °C for 3 months

Acknowledgments

We acknowledge the following funding bodies which made this study possible:
Julius Kieswich–Barts and the Royal London Hospital Diabetic Kidney Disease Centre.
Jianmin Chen–China Scholarship Council (grant number 201206240146).
Samira Alliouachene–Barts and the Royal London Hospital Diabetic Kidney Disease Centre.
Paul Caton–Barts and the Royal London Hospital Diabetic Kidney Disease Centre.
Kieran McCafferty–Barts and the Royal London Hospital Diabetic Kidney Disease Centre.
C. Thiemermann–William Harvey Research Foundation.
Muhammad Yaqoob–Barts and the Royal London Hospital Diabetic Kidney Disease Centre.
  The funding body played no role in the design of the study nor in the collection, analysis, or interpretation of data, nor in the writing of the manuscript. A brief version of this protocol appeared in BMC Nephrology (Kieswich et al., 2018).

Competing interests

The authors declare no competing interests.

Ethics

Animal experiments were conducted in accordance with UK Home Office Animals (Scientific Procedures) Act 1986, with local ethical committee approval. Under Home Office Project Licence no: 70/8350 which was granted after review by the Animal Welfare and Ethical Review Body (AWERB) of Queen Mary University, London.

References

  1. Bursac, N. (2014). Cardiac fibroblasts in pressure overload hypertrophy: the enemy within? J Clin Invest 124(7): 2850-2853.
  2. de Jager, D. J., Grootendorst, D. C., Jager, K. J., van Dijk, P. C., Tomas, L. M., Ansell, D., Collart, F., Finne, P., Heaf, J. G., De Meester, J., Wetzels, J. F., Rosendaal, F. R. and Dekker, F. W. (2009). Cardiovascular and noncardiovascular mortality among patients starting dialysis. JAMA 302(16): 1782-1789.
  3. Hewitson, T. D. (2012). Fibrosis in the kidney: is a problem shared a problem halved? Fibrogenesis Tissue Repair 5(Suppl 1): S14.
  4. Hewitson, T. D., Holt, S. G. and Smith, E. R. (2015). Animal models to study links between cardiovascular disease and renal failure and their relevance to human pathology. Front Immunol 6: 465.
  5. Jia, T., Olauson, H., Lindberg, K., Amin, R., Edvardsson, K., Lindholm, B., Andersson, G., Wernerson, A., Sabbagh, Y., Schiavi, S. and Larsson, T. E. (2013). A novel model of adenine-induced tubulointerstitial nephropathy in mice. BMC Nephrol 14: 116.
  6. Kieswich, J. E., Chen, J., Alliouachene, S., Caton, P. W., McCafferty, K., Thiemermann, C. and Yaqoob, M. M. (2018). A novel model of reno-cardiac syndrome in the C57BL/ 6 mouse strain. BMC Nephrol 19(1): 346.
  7. Kren, S. and Hostetter, T. H. (1999). The course of the remnant kidney model in mice. Kidney Int 56(1): 333-337.
  8. Kumar, S., Bogle, R. and Banerjee, D. (2014). Why do young people with chronic kidney disease die early? World J Nephrol 3(4): 143-155.
  9. Morrison, A. B. (1962). Experimentally induced chronic renal insufficiency in the rat. Lab Invest 11: 321-332.
  10. Ronco, C., Haapio, M., House, A. A., Anavekar, N. and Bellomo, R. (2008). Cardiorenal syndrome. J Am Coll Cardiol 52(19): 1527-1539.
  11. Seong, E., Saunders, T. L., Stewart, C. L. and Burmeister, M. (2004). To knockout in 129 or in C57BL/6: that is the question. Trends Genet 20(2): 59-62.
  12. Steenkamp, R., Shaw, C. and Feest, T. (2013). UK Renal Registry 15th annual report: Chapter 5 survival and causes of death of UK adult patients on renal replacement therapy in 2011: national and centre-specific analyses. Nephron Clin Pract 123 Suppl 1: 93-123. 
  13. Stewart K. and Schroeder V A. (2020). JoVE Science Education Database. Lab Animal Research. Blood Withdrawal I. JoVE, Cambridge, MA.
  14. Yokozawa, T., Oura, H. and Okada, T. (1982). Metabolic effects of dietary purine in rats. J Nutr Sci Vitaminol (Tokyo) 28(5): 519-526.

简介

[摘要 ] 心肾综合征定义了心脏和肾脏的协同病理,其中一个器官的衰竭导致另一个器官的衰竭。与整个人群相比,该综合征导致的心血管疾病死亡率在终末期肾脏病(ESRD)人群中要高出20倍,因此有必要改善治疗策略以应对肾病。

小鼠体内模型在允许精确基因修饰从而减少杂项的研究中起主要作用,但是目前在最常见的基因修饰小鼠品系C57BL / 6小鼠中还没有稳定的雷诺-心脏综合征模型。在这项研究中,我们使用腺嘌呤饮食修改了已建立的慢性肾脏疾病模型,并扩展了在C57BL / 6小鼠中实现慢性肾功能衰竭和随之而来的肾心脏综合征的相关病理。

使八周大的雄性C57BL / 6小鼠适应7天,然后给予0.15%腺嘌呤饮食或对照饮食20周,此后终止实验,收集血液,尿液和器官并进行生化和免疫组织化学分析。

施用0.15%的腺嘌呤饮食会导致进行性肾功能衰竭,从而导致肾性心脏病综合征,这可通过心体重比显着增加来证实(P <0.0001)。血液生化表明,用腺嘌呤喂养的小鼠血清肌酐,尿素含量显着增加(P <0.0001),肾小球滤过率显着降低(P <0.05),而肾脏的α-SMA,胶原蛋白1和胶原蛋白3免疫组化显示严重的纤维化。

我们提出了一种新型的腺嘌呤饮食方案,该方案在C57BL / 6小鼠品系中诱导了慢性肾脏疾病和肾心综合征。与当前可用的其他模型相比,该模型的非手术性质使其具有很高的可重复性。

[背景 ] 慢性肾脏病(CKD)的结果在血管和Ca rdiac功能障碍,由称为心-肾综合征,更具体地定义为里诺心综合征(RCS)的Ronco 等。(2008)。RCS是终末期肾病(ESRD)的主要特征,导致心血管疾病死亡率(相对于总体正常人群)高20倍,心血管事件约占ESRD患者死亡率的50%(de Jager 等)等人,2009; Steenkamp 等人,2013)。随着ESRD人群的增加,全世界对抗RCS的新疗法的研究至关重要。

动物模型极大地帮助了对许多疾病病理学的研究,并且通过遗传修饰菌株的技术的发展使这种模型能够通过更好地靶向病理基因而变得更加强大。

迄今为止,次全肾切除术模型一直是最成功的RCS模型,涉及手术切除或否定肾脏肿块(5/6)(Morrison,1962年)。所产生的肾脏缺血/肾单位量减少会引发高血压,继而导致左心室肥大(LVH)导致进行性左心室扩张(Kumar 等人,2014)。同时,成纤维细胞活化导致心脏肥大和纤维化(Hewitson ,2012;Bursac ,2014)。该模型的缺点是,它需要外科专业知识,昂贵的外科设施,并且易于在操作者之间和内部发生变化。在该模型中首选的啮齿动物是大鼠,因为在小鼠中,该模型的效果要差得多(产生高度可变的结果)(Hewitson 等人,2015)。此外,据报道,C57BL / 6小鼠品系是用于基因修饰的优选品系(Seong 等,2004),对全肾切除术对CKD的发展具有抗性(Kren and Hostetter ,1999)。

CKD 的替代模型是腺嘌呤诱导的。Yokozawa 等人首先描述了这种非手术方法,因此具有高度可重复性。(1982),他指出腺嘌呤的代谢不同于其他嘌呤,并且具有肾毒性。但是,这种化学方法虽然在大鼠中非常成功,但在小鼠中却没有兴盛,因为它们不喜欢腺嘌呤的味道,导致体重迅速下降,导致4周后死亡。贾等。(2013年),通过添加酪蛋白改善了饮食的适口性,将给药过程延长至8周,但这还不足以达到RCS。

我们发现,通过将饮食中的浓度降低至0.15%,并在小鼠达到9周龄时开始给药,可以掩盖腺嘌呤的味道(无需添加酪蛋白)。如通过血液生化和心脏/体重比数据(表1),免疫印迹和免疫组化所见,为了达到CKD和随后的RCS需要20周。

因此,我们成功开发了不需要手术专业知识或设施的高度可复制的模型(Kieswich 等,2018)。

尽管我们没有测量血压(BP),但我们预测RCS的主要原因是由于肾功能不全引起的血流动力学,因此,如果有意识地测量BP,例如通过遥测方法进行测量,这也可能是成功的。高血压的非手术模型。



表1 。结果显示了RCS的证据。对照组(n = 6)接受标准食物20周。腺嘌呤治疗组(n = 10)接受标准食物加0.15%腺嘌呤治疗20周。与接受正常食物的动物相比,以0.15%腺嘌呤饮食20周会导致血浆尿素(P <0.0001)和肌酐(P <0.0001)显着增加,肾小球滤过率(GFR)显着降低(P <0.05),心脏重量与体重之比明显更高(P <0.0001)。

参数

控制

腺嘌呤

没有。

6

10

血清尿素,mmol / l

8.867±0.4240

81.57±1.809 ***

血清肌酐,微摩尔/升

16.83±3.554

114.5±5.909 ***

GFR,毫升/分钟

0.1642±0.04598

0.02869±0.003854 *

心/体重比

0.004341±0.0001352

0.006031±0.0001553 ***

*** P < 0.0001,* P <0.05

关键字:心肾综合征, 慢性肾脏疾病, 心脏肥大, C57BL/6小鼠, 肾纤维化, 实验性肾功能衰竭, 肾心综合征

材料和试剂
25号针头(Fisher Scientific,目录号:10442204)
1 ml注射器(Fisher Scientific,目录号:15849152)
Eppendorf管(Fisher Scientific,目录号:13094697 )
包埋盒(Fisher Scientific,Simport 乙缩醛组织学盒,目录号:10420823)
载玻片(Fisher Scientific,目录号:12343138)
移液器和技巧
ErgoOne 单通道移液器20-200 µl (STARLAB(UK)Ltd,目录号:S7100-2200)
ErgoOne 单道移液器100-1 ,000 微升(STARLAB(英国)有限公司,目录编号:S7110-1000)
Yellow Tips(Sterile),Racked(STARLAB(UK)Ltd,目录号:S1111-0816-C )
1 ,000微升蓝毕业提示(无菌),安装机架(STARLAB(英国)有限公司,目录号码:S1111-6811-C )
疏水性隔离笔(Vector Laboratories,ImmEdge 疏水性隔离笔,目录号:H-4001 )
加湿室,例如带密封盖的塑料盒(防止组织变干)。将湿纸巾放入盒子的底部。为了使幻灯片远离湿纸,请插入打孔的底座(可以通过将巴氏移液器切成相同大小的块并将其编成网格形式来制成。请确保其水平。)
卫生纸
带滑架的容器可容纳约400-500毫升
刷子(用于清洁样品之间的切片机刀片)
玻璃盖玻片(Fisher Scientific,目录号:12343138)
8 - 瓦特伊克大,雄性C57BL / 6小鼠(n =每组6只)(查尔斯河,马盖特,UK)
20 Kg 腺嘌呤饮食(LBS-Biotech,Hookwood ,UK,SDS饮食,目录号:824534 ),RM1 + 0.15%腺嘌呤,储存温度4°C
注意:同时从SDS饮食中购买20 Kg 824534 RM1饮食(正常腺嘌呤浓度)以用作对照)。有关饮食的详细组成,请参见附件“腺嘌呤饮食组成” 。
氯胺酮100 mg / ml 溶液(Narketan ,MWI动物卫生,萨默塞特郡,英国,目录号:03120257 ),储存温度–室温(RTP)
盐酸赛拉嗪2%w / v溶液(Rompun ,MWI动物卫生,英国萨默塞特,目录号:02150569 ),储存温度–RTP
丁丙诺啡0.3 mg / ml 溶液(Vertegesic ,MWI动物卫生,萨默塞特郡,英国,目录号:01300258 ),储存温度–RTP
肝素钠5 ,000 U /米升(MWI动物健康,萨默塞特,UK,目录号:30394030 ),存储温度-rtp
乙醇(Fisher Scientific,目录号:10233962),储存温度–RTP
磷酸盐缓冲盐水(Fisher Scientific,PBS,目录号:10173433),储存温度4°C
福尔马林(Sigma - Aldrich,目录号:HT5014),储存温度–RTP
二甲苯(Fisher Scientific,目录号:10467270),储存温度–RTP
Parrafin (Fisher Scientific,Thermo Scientific理查德·艾伦·理查德·艾伦科学组织塑料石蜡,目录号:12683026),储存温度–RTP
过氧化氢(Fisher Scientific,H 2 O 2 ,目录号:10687022),储存温度4°C
Triton X-100(Sigma - Aldrich,目录号:T8787),储存温度–RTP
牛血清白蛋白(Fisher Scientific,BSA,目录号:11423164 ),储存温度4°C
一抗,抗α-平滑肌肌动蛋白抗体(Abcam,目录号:ab5694),储存温度-20°C
一抗,抗I型胶原蛋白抗体(Abcam,目录号:ab21286 ),储存温度4°C
一抗,抗胶原III抗体(Abcam,目录号:ab7778 ),储存温度4°C
二抗,山羊抗兔IgG H&L(HRP)(Abcam,目录号:ab205718),储存温度4°C
正常山羊血清(Abcam,目录号:ab7481),储存温度4°C
DAB(Fisher Scientific,目录号:10006913 ),DAB的耐受温度为-20°C。DAB底物套件的耐受温度为4°C
冰醋酸(Sigma - Aldrich,目录号:A6283),储存温度–RTP
盐酸TRIS(Tris-HCl)(Sigma - Aldrich,目录号:RES3098T-B7 ),储存温度RTP
氯化钠(NaCl )(Sigma - Aldrich,目录号:S9888 ),储存温度–RTP
氢氧化铵(NH 4 OH)30%溶液(Sigma - Aldrich,目录号:221228-1L-A),储存温度RTP
柠檬酸钠(Sigma - Aldrich,柠檬酸三钠(二水合物),目录号:W302600),储存温度RTP
Tween 20(Sigma - Aldrich,目录号:93773 ),储存温度RTP
苏木精(Vector Laboratories公司有限公司,VECTOR ® HEMATOXYLINNUCLEAR染液,目录号:H-3401),存储温度RTP
固定介质(Vector Laboratories,VectaMount 永久固定介质,目录号:H-5000),存储温度RTP
盐酸(Sigma - Aldrich,目录号:258148),储存温度RTP
蓝光解决方案(请参阅食谱)
酸漂洗液(请参阅配方)
柠檬酸钠缓冲液(请参见食谱)
TBS缓冲区(请参阅食谱)
 
设备
解剖钳(哈佛仪器,珠宝商钳,第7号,目录号:72-8685)
剪刀(哈佛仪器,眼用剪刀,笔直,锋利/锋利的产品,目录号:2-8438)
用于识别小鼠的耳模(哈佛仪器,型号4.7.2镊子式打孔器,2.0毫米孔,货号:34-0137)
通风柜(Fisher Scientific,Thermo Scientific Hyperclean TruAir 无管通风柜,目录号:15613136)
轨道振动器(Fisher Scientific,Stuart轨道振动器,目录号:10759145)
真空烤箱(Fisher Scientific,Technico ,目录号:13025703)
切片机和刀片,Fisher Scientific,HM 325旋转切片机,目录号:12052999)
水浴(Fisher Scientific,克利夫顿无搅拌数字水浴,目录号:15700619)
家用不锈钢压力锅(例如Argos,Tower Compact 4 升压力锅,目录号:711/5008)
热板(Fisher Scientific,带搅拌器的Stuart热板,目录号:11966558)
离心机(Fisher Scientific,Thermo Scientific Heraeus Megafuge 8,目录号:15211026)
显微镜(匈牙利布达佩斯3D Histech Ltd,全景扫描250显微镜)
冰箱(Fisher Scientific,英国拉夫堡,利勃海尔,12088281)
包含多个容器的染色套件(Tissue Tek Slide染色皿,Sakura Finetek Europe,Alphen aan den Rijn,荷兰,S76316)
 
软件
Panoramic Viewer软件(匈牙利布达佩斯的3D Histech Ltd)
ImageJ软件(https://imagej.nih.gov)
Excel(https://products.office.com/en-gb/excel)
GraphPad Prism(https://www.graphpad.com/scientific-software/prism/)
 
程序
小鼠肾性心脏病综合征的诱导和组织样品的收集
允许至少8周大的C57BL / 6 品系小鼠在具有12小时光照/黑暗周期的房间内的动物设施中适应7天,可以自由获取食物和水。
在第8天,给小鼠称重并更换笼子/饮食,使一半的小鼠正常食物,另一半的食物包含0.15%腺嘌呤。
继续对小鼠进行20 周的饮食管理,每周称重一次。
在第20周:
制备anaesthe 抽动氯胺酮:甲苯噻嗪为2:1倍的比例。用PBS稀释1:10。称重小鼠,并以1.5 ml / kg的剂量腹膜内给药。
当小鼠为3分钟后anaestheti ž ED (检查捏反射即,捏脚,应该没有响应)。
腹膜内给予丁丙诺啡(0.03 ml / Kg)。等待3分钟
通过心脏穿刺去除血液,方法是将附着在1 ml注射器上的25针肝素化针插入左心室(参见视频链接,Stewart和Schroeder ,2020年),通过短暂地吸出并从管中排出肝素来使肝素化针,可以重复使用肝素用于多针。
大约应收集0.5 毫升血液。进行剖腹手术,然后进行开胸手术。移开心脏并称重。血液应在17离心,000 X 克/ 3分钟为分离血浆并储存在冰箱中。
取出左右肾脏。短暂地在冷PBS中洗涤,然后纵向切割并放入福尔马林进行固定。
24小时后,制成70%乙醇的溶液,将肾脏从福尔马林转移到70%乙醇中。
24小时后,用PBS冲洗组织。
将血液送至服务实验室(IDEXX,Bioresearch,德国路德维希堡),以进行生化测定(血浆肌酐和尿素)以确定肾功能衰竭的程度。
组织石蜡化
脱水组织
在室温(RTP)中于50%乙醇中孵育10分钟。
然后加入70%乙醇10分钟(RTP)。
然后在80%的乙醇中放置10分钟(RTP)。
然后在95%乙醇中放置10分钟(RTP)。
然后100%乙醇浸泡10分钟(RTP)。重复此步骤2次。
然后以2:1的比例混合乙醇:二甲苯10分钟(RTP)。
然后用1:1的比例Ë THANOL:二甲苯10分钟(RTP)。
然后以1:2的比例混合乙醇:二甲苯10分钟(RTP)。
然后100%二甲苯10分钟(RTP)。重复此步骤2次。
将样品转移到设置为56的真空烘箱中 ℃。
然后在56 °C下以2:1的比例混合二甲苯:石蜡30分钟。
然后在56 °C下以1:1的比例混合二甲苯:石蜡30分钟。
然后在56 °C下以1:2的比例混合二甲苯:石蜡30分钟。
然后在56 °C下100%石蜡干燥2 h 。
然后100%石蜡在56 °C 过夜。
将纵向切开的肾脏以新的石蜡方向包埋,使包埋盒的蜡内侧朝上。
Sectio 宁
将带有样品的包埋盒在冰上放置约2小时,或最好在冰箱中放置过夜,以冷却并便于切割。
使用切片机切开5μm 的肾脏切片。
放入设定为42 °C的水浴中。
将各个部分收集到幻灯片上。
放入滑架,并在37 °C的烤箱中干燥过夜。
Deparaffini ž Ë 和幻灯片补充水分
将玻片放在架子上,并在室温(RTP)中于二甲苯中孵育3分钟。重复此步骤2次。
然后以二甲苯:100%乙醇1:1的比例混合3分钟(RTP)。
然后加入100%乙醇3分钟(RTP)。重复此步骤。
然后在95%乙醇中放置3分钟(RTP)。
然后加入70%的乙醇3分钟(RTP)。
然后加入50%乙醇3分钟(RTP)。
最后用冷自来水冲洗掉多余的乙醇。让载玻片在自来水下冲洗,直到准备好抗原回收方案(防止变干)。
抗原回收
将足够的柠檬酸钠pH 6.0缓冲液倒入玻片容器中,以覆盖玻片并放入高压锅中。
将压力锅放在电炉上,将其打开至全功率。将压力锅盖放在顶部(未拧紧)。
锅煮沸后,用镊子将载玻片从自来水中小心地转移到锅中。按照制造商的说明拧紧盖子。
让炊具达到最大压力,然后等待3分钟。
3分钟后,关闭电热板,小心地从电热板上取下电磁炉,然后将压力锅放入水槽中。
启动压力释放阀,并在电磁炉上注入冷自来水。减压后,取下盖子,将冷自来水倒入锅中10分钟。
载玻片现在可以染色了。
染色
将Triton X-100以0.025%的最终浓度添加到TBS缓冲液中。
在TBS Triton X-100缓冲液中轻轻旋转的轨道振荡器上洗涤载玻片2 x 5分钟。
准备封闭缓冲液(TBS + 1%BSA + 10%正常血清)。
在RTP振荡器上,在封闭缓冲液中将载玻片封闭2小时。
取出幻灯片并用纸巾擦拭(在各个部分周围)。
使用疏水性隔离笔在各部分周围绘制。
准备一抗:
抗SMA抗体(在TBS缓冲液/ 1%BSA中以1:250稀释的ab5694)
要么
抗胶原蛋白I(在TBS缓冲液/ 1%BSA中以1:200稀释的ab21286)
要么
抗胶原蛋白III(ab7778在TBS缓冲液/ 1%BSA中以1:400稀释)。
准备加湿室中(PLAS 抽动盒托盘用湿纸巾内衬,粘贴在一起而形成的凸起的平台下巴斯德移液管,参见图URE 低于1)。将幻灯片平放在顶部,部分朝上。将抗体涂在幻灯片上的切片上。关上盖子。
 
图URE 1.加湿室中。一。巴斯德移液器用胶带粘在一起。乙。W¯¯ 箱等生活用纸。Ç 。小号大环内酯类奠定之上。d 。我的身子紧闭。

在4 °C下孵育过夜。
在TBS / 0.025%Triton X-100中将载玻片洗涤5分钟,轻轻摇动,RTP。
重复洗涤。
在TBS中准备0.3%H 2 O 2 。
涂在切片上,孵育15分钟,轻轻摇动,RTP。
检测步骤
准备在TBS / 1%BSA中以1:1,000稀释的二抗山羊抗兔IgG H&L(HRP)(ab205718)。
涂在玻片上,温育1小时,轻轻摇动,RTP。
将DAB 色原加到玻片上,轻摇10分钟,RTP。
在流动的自来水下冲洗载玻片5分钟。
用苏木精染色。将Vector苏木精加到玻片上,轻轻摇动5分钟,RTP。
用自来水冲洗幻灯片,直到水无色。
将玻片浸入酸洗液中10次。
浸入自来水中10次。
将Bluing解决方案添加到幻灯片上,轻轻摇动1分钟,RTP。
浸入自来水中10次。
使组织脱水,清除并固定
在室温(RTP)下于95%乙醇中孵育玻片10分钟。
然后100%乙醇浸泡10分钟(RTP)。重复此步骤2次。
然后100%二甲苯10分钟(RTP)。重复此步骤2次。
通过排水滑轨安装
在部分上滴一滴安装介质。
盖上盖玻片以确保没有气泡。
 
影像采集
使用Panoramic Scanning 250显微镜(匈牙利布达佩斯3D Histech Ltd)以20倍的放大倍率记录图像(图2 )。

数据分析
使用GraphPad Prism 5软件(GraphPad Software Inc,CA,美国)进行统计分析。值表示为n个观测值的平均值±SEM。使用未配对的t 检验计算经腺嘌呤治疗的小鼠和对照组之间的差异。P <0.05被认为具有统计学意义。
要计算样品之间的α-SMA染色增加,请使用ImageJ中的对照样品玻片打开文件。我。e。,单击“文件”,然后选择“打开”。通过单击“编辑”反转图像,然后选择“反转”。通过在图像周围的黑暗区域绘制一个约0.5 cm 2 的矩形进行背景测量。单击“分析”,然后选择“测量”。值将出现在名为“结果” 的数据文件中,该文件将自动打开。接下来,将相同的矩形拖到肾脏上,对肾脏进行6次随机测量(每次单击“测量”)。重复2个对照玻片,然后再用3个经腺嘌呤治疗的肾脏玻片(确保矩形与所有玻片的大小完全相同)。
保存所有图片和结果文件。最后,单击“结果”文件中的“复制”,将数据传输到Excel文件。从所有测量值中减去背景值。计算每个滑块e的平均值(表2 )。

表2 。Excel电子表格中的数据


将结果输入到Gra phPad Prism中,以转换为柱形图(表3 )。
 
表3 。GraphPad Prism中的数据

单击“图形”图标以获取图形。编辑图(图2 )。
 
图2 。通过α- SMA,胶原蛋白I和胶原蛋白III的免疫染色来证明肾脏肥大。对照组(n = 6)接受标准食物20周。腺嘌呤治疗组(n = 10)接受标准食物加0.15%腺嘌呤治疗20周。与对照组(A)相比,肾脏的免疫染色显示腺嘌呤组(B)的α- SMA染色显着增加(P <0.0001)。与对照组(C)相比,腺嘌呤组(D)的胶原蛋白I染色显着增加(P <0.0001)。与对照组(E)相比,腺嘌呤组(F)的胶原蛋白III染色显着增加(P <0.0001),平均值±SEM未配对t 检验。代表性图像(每组3只小鼠)。


笔记

必须根据国家动物研究法律准则并获得当地伦理委员会的批准进行动物实验。
请勿对9周以下的动物进行饮食控制。
不要在福尔马林中固定组织超过24 小时,因为过度固定可能会抑制抗原:抗体结合。
CKD的确认感应从血液生化结果与继续之前P ROC edure B. Paraffinisation 组织。
使用二甲苯的步骤应在通风橱内进行。
 
菜谱
蓝光解决方案
1.5 ml NH 4 OH(储备液为30%)+ 98.5 ml的70%乙醇。在RTP上存储长达3个月
酸洗液
2毫升冰醋酸+ 98毫升去离子或蒸馏水H 2 O
在RTP上存储长达3个月
柠檬酸钠缓冲液
10 mM柠檬酸钠,0.05%Tween 20,pH 6.0。将2.94 g 柠檬酸三钠(二水合物)溶于1 L蒸馏水中,并用1 N HCl将pH调节至6.0
加入0.5 ml吐温20并混合均匀
在RTP中储存长达3个月,或在4 °C下储存更长时间
TBS缓冲区
50 mM Tris-Cl,pH 7.5,150 mM NaCl
将6.05 g Tris和8.76 g NaCl溶解在800 ml H 2 O中
用1 M HCl将pH调节至7.5,并用H 2 O 调节至1 L
TBS在4°C下稳定3个月
 
致谢
我们感谢以下资助机构使这项研究成为可能:
Julius Kieswich – 巴特斯和伦敦皇家医院糖尿病肾病中心。
建民陈- 中国国家留学基金委(授权号201206240146)。
Samira Alliouachene – 巴茨和伦敦皇家医院糖尿病肾病中心。
保罗·卡顿(Paul Caton)– 巴茨和伦敦皇家医院糖尿病肾病中心。
基兰·麦卡菲蒂(Kieran McCafferty)– 巴特斯和伦敦皇家医院糖尿病肾病中心。
C. Thiemermann – 威廉·哈维研究基金会。
Muhammad Yaqoob – 巴特斯和伦敦皇家医院糖尿病肾病中心。
  资助机构在研究的设计,数据的收集,分析或解释以及手稿的撰写中均不起作用。该协议的简短版本出现在BMC Nephrology(Kieswich et al。,2018)中。

利益争夺

作者宣称没有利益冲突。

伦理
根据英国内政部动物(科学程序)法1986进行动物实验,并得到当地伦理委员会的批准。根据内政部项目许可证编号:70/8350,该许可证是由伦敦玛丽皇后大学动物福利和道德审查机构(AWERB)审查后授予的。

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引用:Kieswich, J. E., Chen, J., Alliouachene, S., Caton, P. W., McCafferty, K., Thiemermann, C. and Yaqoob, M. M. (2020). Immunohistochemistry of Kidney a-SMA, Collagen 1, and Collagen 3, in A Novel Mouse Model of Reno-cardiac Syndrome. Bio-protocol 10(18): e3751. DOI: 10.21769/BioProtoc.3751.
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