The Mouse Isolated Perfused Kidney Technique

Podsumowanie

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

Streszczenie

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.

This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney's preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

Wprowadzenie

The isolated perfusion of organs has been the subject of an ongoing effort among physiologists for many decades¹. The technique enables the function of the organ, without systemic influences such as blood pressure, hormones, or nerves, to be studied. Carl Eduard Loebell is considered to be the first to have described the successful perfusion of an isolated kidney, in 1849². Since then, the perfusion apparatus has undergone significant refinement. Frey and Gruber introduced an artificial lung for oxygenation and pulsatile pumps for continuous perfusion². While early researchers mainly studied the kidneys of large mammals-namely, pigs² and dogs³-the first report of the use of rat kidneys, by Weiss et al., was a milestone in the study of small-mammal-organ perfusion⁴. Schurek et al. reported the necessity of adding mammalian erythrocytes to the perfusate if sufficient renal tubular oxygenation was to be achieved⁵. Critical for long-term experiments was the introduction of continuous dialysis of the buffer by the same research group⁶. In 2003, Schweda et al. were the first to report a functional mouse isolated perfused kidney (MIPK)⁷, later refined by Rahgozar et al.¹⁸ and Lindell et al.¹⁴.

While technically more challenging than the rat isolated perfused kidney, the use of the MIPK bears the advantage of enabling the use of a wide array of genetically altered mice. This paper presents the details of the authors' method for perfusing isolated mouse kidneys for 1 hr. The method allows for the continuous assessment of renal flow rate, vascular resistance, hormone release, blood gas analysis, urine analysis, and the application of drugs. Following the procedure, kidneys could be processed for molecular and biochemical analysis, be fixed for microscopy, or transplanted into a recipient mouse (Figure 1).

Figure 1: Overview of Possible Input/Output to the Isolated Perfused Kidney. BGA: Blood gas analysis. Please click here to view a larger version of this figure.

This technique likely will receive increasing attention over coming years, as many innovative applications are being discussed with the dawn of prolonged normothermic kidney perfusion prior to transplantation (with or without the application of anti-rejection or genome-editing drugs)^{8,9, 10, 11}, the bioengineering of whole kidneys from decellularized scaffolds¹², and the application of high doses of fluorescent dyes for multiphoton imaging¹³. It is also an ideal model with which to study the role of specific genes during acute kidney injury¹⁴.

A step-by-step protocol is given to allow other laboratories to perform isolated mouse kidney perfusion successfully. First, the composition and preparation of the buffer is specified. Then, the surgery is described in detail and the critical steps are shown. Third, data is presented that are representative of a successful preparation: renal blood flow, vascular resistance, glomerular filtration rate , and fractional electrolyte excretion-all as functional measurements of viability-and transmission electron micrographs of the morphology of different nephron segments of perfused kidneys fixed after 1 hr of perfusion.

Protokół

All procedures involving animals described in this manuscript were conducted according to Swiss law and approved by the veterinary administration of the Canton of Zurich, Switzerland.

1. Buffer Preparation

1. Prepare solutions 1 - 4 and the antidiuretic hormone (ADH) solution (Table 1).
2. Prepare the dialysis buffer (Table 1).
   NOTE: This is the buffer used as the dialysis buffer during the perfusion. Later, the erythrocytes will be diluted in this buffer to form the final perfusate.
3. Erythrocyte preparation.
   1. Dilute 250 ml of human erythrocyte concentrate (tested material obtained from the local blood bank) to 500 ml with dialysis buffer. Centrifuge at 2,000 x g for 8 min. Remove the buffer, being careful not to remove any erythrocytes. Repeat 3x.
4. Prepare the albumin (bovine serum albumin; BSA) buffer.
   1. In 200 ml of dialysis buffer, dissolve 44 g of BSA using a stir bar. Filter the solution with filter paper.
5. Prepare the perfusate.
   1. Filter the erythrocytes from step 1.3.1 through filter paper into the BSA buffer. Fill up to a total volume of 800 ml with dialysis buffer.
      NOTE: This is the final perfusate. The hematocrit should now be between 8 and 12%. The perfusate can be stored for up to 12 hr at 4 °C.

2. Initiating Dialysis and Oxygenation

1. Turn on the water bath surrounding the larger buffer reservoir, smaller buffer reservoir, and the moist chamber (a small, double-walled chamber brought to 37 °C and 100% humidity to later hold the kidney) to 37 °C (Figure 2).
2. Fill the larger buffer reservoir with the dialysis buffer and the smaller reservoir with the perfusate.
3. Turn on the 5% CO₂/95% O₂ gas inflow to the dialysis buffer.
4. Switch on continuous dialysis of the perfusate against the dialysis buffer. Take care to use low-flux dialysis tubing. Proceed to Step 3.

Figure 2: Schematic Drawing of the Perfusion Circuit. Scheme shows the main components of the perfusion circuit and the direction of buffer flow. All components surrounded by dark blue are kept at 37 °C with a water bath/thermostat. 1: Dialysis buffer of at least 3 times the volume of the perfusion buffer is continuously bubbled with 95% O₂/5% CO₂. 2: Dialysis buffer and perfusion buffer are continuously dialyzed against each other in a dialysis tube by a roller pump. 3: Due to this dialysis, the perfusion buffer is enriched with 9% O₂/5% CO₂ and electrolyte levels are kept constant throughout perfusion. 4: A roller pump propels the perfusion buffer toward the kidney. 5: A windkessel removes peristaltic waves and acts as a bubble trap. 6: Pressure transducer (connected to 4. (roller pump) to keep continuous pressure while allowing freely alternating flow). 7: Throughout perfusion, the kidney remains in a moist chamber for 100% air humidity and 37 °C kidney temperature. Please click here to view a larger version of this figure.

3. Surgical Procedure Part 1 (for a diagram of all ligatures, see Figure 3)

Note: Perform all ligatures using 5-0 surgical thread.

1. Anesthetize a mouse by intraperitoneal injection (10 µl/g of body weight, 20 mg/ml ketamine and 1 mg/ml xylazine dissolved in 0.9% NaCl). Confirm sufficient depth of anesthesia by testing for absence of rear-foot reflexes.
2. Fix the mouse in a supine position in the moist chamber. Protect the eyes with vet ointment. Place a 1 ml syringe below the spine to elevate the lumbar vessels.
3. Perform a median laparotomy from the pubic crest to the sternum opening first the skin, then the abdominal muscles, with scissors.
4. Remove the intestine and place it on the left side of the mouse lateral from the abdomen.
5. Free the bladder from connective tissue and explore both ureters and the urethra.
6. Place a ligature around the left ureter (ligature I). Close it.
7. Place a ligature around the urethra (ligature II). Close it.
8. Place a "lasso" ligature around the whole bladder (ligature III).
9. Incise the bladder 1 mm.
10. Cannulate the opening with 2 cm PE 50 tubing.
11. Close ligature III around the tubing.
12. Cut the left ureter and urethra distal from the ligatures. The bladder is now attached to the right ureter only and freely moving.
13. Clear the abdominal aorta of connective tissue and fat.
14. Place an abdominal mid-aorta ligature (ligature IV).
15. Place a ligature around the aorta below the diaphragm between the superior mesenteric artery and the coeliac trunk (ligature V).
16. Place a ligature around the superior mesenteric artery (ligature VI).
17. Place an aortic ligature directly below the right and above the left renal artery (ligature VII).
18. Place a ligature around the caudal vein package (cava) (ligature VIII). Proceed to Step 4.

Figure 3: Schematic Drawing of the Ligatures placed during Surgery. View of the open abdomen after the laparotomy. The intestine is moved out to the left. L and R indicate the left and right kidney. The black lines show the area of the respective ligature. Ligatures are first placed and then closed, in the sequence given in the text. X marks the location of the incision for aorta cannulation. Please click here to view a larger version of this figure.

4. Priming of the Perfusion Circuit

1. Start the rotary pump and fill the tubing with perfusate. Take care to empty all air bubbles from it.
2. Fill the windkessel device to approximately mid-level with perfusate.
3. Calibrate the pressure transducer to 0 mm Hg when all tubing is filled and flow is 0. Keep the perfusion needle at kidney level during this time.
4. Keep flow at a constant minimal level (0.6 ml/min) and proceed to Step 5.

5. Surgical Procedure Part 2

1. Place a clamp between ligature IV and the branching of the left renal artery.
2. Make a small incision in the aorta caudal of ligature IV, taking care to not cut the dorsal wall.
3. Dilate the opening in the aorta with a vessel dilator.
4. Cannulate the aorta with a needle (2 cm long, pulled PE 50), pushing the tip just to the clamp.
5. Open the clamp.
6. Push the tip of the needle cranially until it reaches the junction of the right kidney artery and the aorta.
7. Close ligature VII.
8. Close ligature IV.
9. Open the chest with scissors by dissecting the diaphragm. With a single cut, separate the aorta, vena cava, heart and vegetative nerves.  With this step, the animal is sacrificed via rapid exsanguination under continuous deep anesthesia.
10. Start pressure control of the perfusion pump. Maintain the mean pressure between 80 and 100 mmHg.
11. Close ligature V.
12. Close ligature VI.
13. Close ligature VIII.
14. Free the right kidney from connective tissue and its embedding into the adipose capsule with scissors.
15. Cut the aorta proximally to ligature V.
16. Cut the superior mesenteric artery distally to ligature VI.
17. Cut the kidney-supporting vessel bundle out, taking care to not cut into the vessels themselves.
18. Cut the liver at the connection to the kidney. Take care to free the kidney, but leave a small part of the liver adherent to it, so that the vena cava is kept open by it.
19. Take the kidney bundle out of the mouse. Remove the mouse from the moist chamber.
20. Place a "lasso" ligature around the connection of liver and kidney (ligature IX).
21. Cannulate the vena cava with a venous line (2 cm PE 50).
22. Close ligature IX. Venous outflow through the venous line should immediately start.
23. Close the moist chamber.

6. Downstream Analyses

1. During the following hour, continuously monitor blood flow and intravascular pressure¹⁵. Collect venous outflow, which can be used for analysis of, for example, renal renin release⁷. Collect urine for analysis of electrolyte concentration and glomerular filtration rate ¹⁴. After 1 hr of perfusion, kidneys can be snap-frozen for western blotting or be fixed for imaging approaches¹⁶.

Wyniki

With the method described, isolated mouse kidneys can remain viable for at least 1 hr. We tested the tissue viability after 1 hr of continuous perfusion with functional (renal blood flow and vascular resistance, blood gas analysis of venous outflow, glomerular filtration rate, urinary fractional Na⁺ and K⁺ excretion, and urine osmolality) and morphological (transmission electron microscopy, TEM) methods in four kidneys of wildtype C57Bl/6 mice. Additionally, western ...

Dyskusje

The mouse isolated perfused kidney is a tool for studying kidney function in a controlled environment ex vivo for 1 hr, bridging the gap between in vivo experiments in intact animals, which may be flawed by the impact of numerous systemic factors, and in vitro experiments in isolated nephron segments or cultured cells, which necessarily neglect the impact of intact organ structure on function. There is, to the authors' knowledge, no alternative technique with which to perform this specific ...

Materiały

Name	Company	Catalog Number	Comments
Perfusion Circuit:
Moist chamber 834/8	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-2901
Cannular with basket and side port	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-2947
Thermostat TC120-ST5	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-4544
ISM 827/230V Roller Pump Reglo Analogue	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-0114
Reservoir jacketed for buffer solution 1 L	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3438
Reservoir jacketed for buffer solution 0.5 L	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3436
Pressure Transducer APT300	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3862
TAM-D Plugsys Transducer	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-1793
SCP Plugsys servo controller	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-2806
Windkessel	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3717
HSE-USB data acquisition	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3330
Low-Flux Dialysator Diacap Polysulfone	B.Braun	7203525
PE-Tubing for aorta cannulation 1.19 mm I.D. x 1.70 mm O.D.	Scientific Commodities Inc.	BB31695-PE/8
Name	Company	Catalog Number	Comments
Buffer reagents:
Aminoplasmal 10%	B.Braun	134518064
Sodium pyruvate	Sigma-Aldrich	P2256-25G
L-Glutamic acid monosodium salt hydrate	Sigma-Aldrich	G1626-100G
L-(-)-Malic acid sodium salt	Sigma-Aldrich	M1125-25G
Sodium-L-Lactate	Sigma-Aldrich	L7022-10G
alpha-Ketoglutaric acid sodium salt	Sigma-Aldrich	K1875-25G
NaCl	Sigma-Aldrich	31434-1KG-R
NaHCO3	Sigma-Aldrich	S5761-5KG
KCl	Sigma-Aldrich	60130-1KG
Urea	Sigma-Aldrich	U5378-500G
Creatinine	Sigma-Aldrich	C4255-10G
Ampicillin	Roche	10835242001
MgCl2 * 6H2O	Sigma-Aldrich	M2393-500G
D-Glucose	Sigma-Aldrich	G8270-1KG
CaCl2 * 6H2O	Riedel-de-Haën	12074
NaH2PO4	Sigma-Aldrich	S9638-500G
Na2HPO4	Sigma-Aldrich	S0876-500G
Antidiuretic Hormone dDAVP	Sigma-Aldrich	V2013-1MG
FITC-Inulin	Sigma-Aldrich
Filter used for erythrocyte filtration	Macherey-Nagel	MN 615
BGA Analysis:
ABL 80 flex	Radiometer Medical ApS
Electron Microscope:
Philips CM100 TEM	FEI