Name: the mouse isolated perfused kidney technique
Uploaded: 2016-11-17T14:00:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

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
<ol>
	<li>Prepare solutions 1 - 4 and the antidiuretic hormone (ADH) solution (Table 1).</li>
	<li>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.</li>
	<li>Erythrocyte preparation.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Prepare the albumin (bovine serum albumin; BSA) buffer.
	<ol>
		<li>In 200 ml of dialysis buffer, dissolve 44 g of BSA using a stir bar. Filter the solution with filter paper.</li>
	</ol>
	</li>
	<li>Prepare the perfusate.
	<ol>
		<li>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 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Initiating Dialysis and Oxygenation
<ol>
	<li>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 &#176;C and 100% humidity to later hold the kidney) to 37 &#176;C (Figure 2).</li>
	<li>Fill the larger buffer reservoir with the dialysis buffer and the smaller reservoir with the perfusate.</li>
	<li>Turn on the 5% CO2/95% O2 gas inflow to the dialysis buffer.</li>
	<li>Switch on continuous dialysis of the perfusate against the dialysis buffer. Take care to use low-flux dialysis tubing. Proceed to Step 3.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54712/54712fig2.jpg" /> 
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 &#176;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 &#176;C kidney temperature. <a href="http://cloudfront.jove.com/files/ftp_upload/54712/54712fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Surgical Procedure Part 1 (for a diagram of all ligatures, see Figure 3)
Note: Perform all ligatures using 5-0 surgical thread.
<ol>
	<li>Anesthetize a mouse by intraperitoneal injection (10 &#181;l/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.</li>
	<li>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.</li>
	<li>Perform a median laparotomy from the pubic crest to the sternum opening first the skin, then the abdominal muscles, with scissors.</li>
	<li>Remove the intestine and place it on the left side of the mouse lateral from the abdomen.</li>
	<li>Free the bladder from connective tissue and explore both ureters and the urethra.</li>
	<li>Place a ligature around the left ureter (ligature I). Close it.</li>
	<li>Place a ligature around the urethra (ligature II). Close it.</li>
	<li>Place a &#34;lasso&#34; ligature around the whole bladder (ligature III).</li>
	<li>Incise the bladder 1 mm.</li>
	<li>Cannulate the opening with 2 cm PE 50 tubing.</li>
	<li>Close ligature III around the tubing.</li>
	<li>Cut the left ureter and urethra distal from the ligatures. The bladder is now attached to the right ureter only and freely moving.</li>
	<li>Clear the abdominal aorta of connective tissue and fat.</li>
	<li>Place an abdominal mid-aorta ligature (ligature IV).</li>
	<li>Place a ligature around the aorta below the diaphragm between the superior mesenteric artery and the coeliac trunk (ligature V).</li>
	<li>Place a ligature around the superior mesenteric artery (ligature VI).</li>
	<li>Place an aortic ligature directly below the right and above the left renal artery (ligature VII).</li>
	<li>Place a ligature around the caudal vein package (cava) (ligature VIII). Proceed to Step 4.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54712/54712fig3.jpg" /> 
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. <a href="http://cloudfront.jove.com/files/ftp_upload/54712/54712fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Priming of the Perfusion Circuit
<ol>
	<li>Start the rotary pump and fill the tubing with perfusate. Take care to empty all air bubbles from it.</li>
	<li>Fill the windkessel device to approximately mid-level with perfusate.</li>
	<li>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.</li>
	<li>Keep flow at a constant minimal level (0.6 ml/min) and proceed to Step 5.</li>
</ol>
5. Surgical Procedure Part 2
<ol>
	<li>Place a clamp between ligature IV and the branching of the left renal artery.</li>
	<li>Make a small incision in the aorta caudal of ligature IV, taking care to not cut the dorsal wall.</li>
	<li>Dilate the opening in the aorta with a vessel dilator.</li>
	<li>Cannulate the aorta with a needle (2 cm long, pulled PE 50), pushing the tip just to the clamp.</li>
	<li>Open the clamp.</li>
	<li>Push the tip of the needle cranially until it reaches the junction of the right kidney artery and the aorta.</li>
	<li>Close ligature VII.</li>
	<li>Close ligature IV.</li>
	<li>Open the chest with scissors by dissecting the diaphragm. With a single cut, separate the aorta, vena cava, heart and vegetative nerves.&#160;&#160;With this step, the animal is sacrificed via rapid exsanguination under continuous deep anesthesia.</li>
	<li>Start pressure control of the perfusion pump. Maintain the mean pressure between 80 and 100 mmHg.</li>
	<li>Close ligature V.</li>
	<li>Close ligature VI.</li>
	<li>Close ligature VIII.</li>
	<li>Free the right kidney from connective tissue and its embedding into the adipose capsule with scissors.</li>
	<li>Cut the aorta proximally to ligature V.</li>
	<li>Cut the superior mesenteric artery distally to ligature VI.</li>
	<li>Cut the kidney-supporting vessel bundle out, taking care to not cut into the vessels themselves.</li>
	<li>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.</li>
	<li>Take the kidney bundle out of the mouse. Remove the mouse from the moist chamber.</li>
	<li>Place a &#34;lasso&#34; ligature around the connection of liver and kidney (ligature IX).</li>
	<li>Cannulate the vena cava with a venous line (2 cm PE 50).</li>
	<li>Close ligature IX. Venous outflow through the venous line should immediately start.</li>
	<li>Close the moist chamber.</li>
</ol>
6. Downstream Analyses
<ol>
	<li>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.</li>
</ol>

<ol>
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The authors have no competing financial interests and nothing else to disclose.

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&#39; knowledge, no alternative technique with which to perform this specific task. Biochemical studies on kidney tissue, however, can be performed in a much simpler fashion using kidney slices17. While the technique presented is currently used mostly to study renal physiology, many new applications are being discussed. For example, it could be used for kidney bioengineering during perfusion decellularization and recellularization12. In addition, prolonged normothermic perfusion of kidneys prior to transplantation, with the administration of high doses of anti-rejection or genome-editing drugs into the perfusate, is being studied; even in clinical settings8, 9, 10, 11. The mouse isolated perfused kidney is an excellent tool that could be used for these applications, especially to elucidate the role of individual genes with knockout models.
Three steps are critical for a successful experiment. This paper provides interested labs with the means to reproduce the procedure. First, the buffer needs to be prepared in such a way that the kidney receives most of the nutrients necessary for prolonged viability under reproducible conditions. A balance must be found between fully controlled conditions (synthetic buffer) and a native perfusion condition (whole blood). Therefore, albumin and mammalian erythrocytes to a hematocrit of 10% have to be included. This hematocrit represents, in the authors&#39; experience, the best compromise between sufficient tissue oxygenation and low buffer viscosity (increasing hematocrit decreases flow dramatically during pressure-constant perfusion (compare values presented in this paper to values in kidneys perfused without erythrocytes18)). The quality and freshness of the erythrocytes is critical. During the washing steps, erythrocytes need to be handled with care to prevent hemolysis. The buffer needs to be prepared freshly on the day of the experiment. Second, the surgery needs to be carried out by a trained surgeon within the shortest time possible. The surgeon needs to perform 6 ligatures around the main vessels connecting the kidney to the rest of the body, while keeping the animal&#39;s blood loss to a minimum. For optimal results, the surgical interventions should be accomplished within 30 minutes. If the mean arterial blood pressure of the animal drops, during prolonged surgery or due to anesthesia, below 60 mmHg, renal perfusion will already be minimal and the tissue will be prone to necrosis. To rule this out, the left kidney can be taken after surgery, when perfusion of the right kidney has already begun, and used as an untreated control. Third, during the time of the perfusion the correct functioning of the perfusion apparatus, consisting of a standard small-animal-perfusion circuit, needs to be ensured. While the computer performs the perfusion by itself, human supervision is necessary, for example to check for bubbles in the perfusion circuit.
The representative data provided allows for the assessment of kidney viability after 1 hr of ex vivo perfusion and the comparison of the technical success of any execution of this method to the approach described herein. During at least 1 hr, renal blood flow, vascular resistance, blood gases of the venous outflow and glomerular filtration rate should remain stable.
There are certain limitations to the technique, which one has to keep in mind. Even a kidney issuing from the most successful surgery can only remain viable for a limited time. The authors found 1 hr to be the best time point at which to stop perfusion. Additionally, while perfusion of the isolated kidney allows for the study of the isolated whole organ in depth, it is difficult to differentiate specific effects on one cell type. Kidneys fixed after 1 hr might show defects in S3 segments of proximal tubules and thick ascending limb cells, while glomeruli, S1 segments, DCTs, and CDs should remain intact. The obligatory use of erythrocytes, especially from humans, is also linked to potential risks (e.g., infections) for the experimenter.
In summary, the mouse isolated perfused kidney is a useful technique with which to study the whole organ ex vivo. Like any technique, it has certain advantages and limitations. The authors believe that both the manifold new applications currently arising and this protocol could provide sufficient momentum for other labs to reproduce this effort.

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&#39; 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).
<img alt="Figure 1" src="/files/ftp_upload/54712/54712fig1.jpg" /> 
Figure 1: Overview of Possible Input/Output to the Isolated Perfused Kidney. BGA: Blood gas analysis. <a href="http://cloudflare.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.

<table><tbody><tr><td>&lt;strong&gt;Perfusion Circuit:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Moist chamber 834/8</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2901</td><td></td></tr><tr><td>Cannular with basket and side port</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2947</td><td></td></tr><tr><td>Thermostat TC120-ST5&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-4544</td><td></td></tr><tr><td>ISM 827/230V Roller Pump Reglo Analogue</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-0114</td><td></td></tr><tr><td>Reservoir jacketed for buffer solution 1 L</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3438</td><td></td></tr><tr><td>Reservoir jacketed for buffer solution 0.5 L</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3436</td><td></td></tr><tr><td>Pressure Transducer APT300&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3862</td><td></td></tr><tr><td>TAM-D Plugsys Transducer</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-1793</td><td></td></tr><tr><td>SCP Plugsys servo controller</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2806</td><td></td></tr><tr><td>Windkessel&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3717</td><td></td></tr><tr><td>HSE-USB data acquisition</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3330</td><td></td></tr><tr><td>Low-Flux Dialysator Diacap Polysulfone</td><td>B.Braun</td><td>7203525</td><td></td></tr><tr><td>PE-Tubing for aorta cannulation 1.19 mm I.D. x 1.70 mm O.D.</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/8</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Buffer reagents:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aminoplasmal 10%</td><td>B.Braun</td><td>134518064</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256-25G</td><td></td></tr><tr><td>L-Glutamic acid monosodium salt hydrate</td><td>Sigma-Aldrich</td><td>G1626-100G</td><td></td></tr><tr><td>L-(-)-Malic acid sodium salt</td><td>Sigma-Aldrich</td><td>M1125-25G</td><td></td></tr><tr><td>Sodium-L-Lactate</td><td>Sigma-Aldrich</td><td>L7022-10G</td><td></td></tr><tr><td>alpha-Ketoglutaric acid sodium salt</td><td>Sigma-Aldrich</td><td>K1875-25G</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>31434-1KG-R</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761-5KG</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60130-1KG</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378-500G</td><td></td></tr><tr><td>Creatinine</td><td>Sigma-Aldrich</td><td>C4255-10G</td><td></td></tr><tr><td>Ampicillin</td><td>Roche</td><td>10835242001</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; * 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2393-500G</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G8270-1KG</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; * 6H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Riedel-de-Ha&amp;euml;n</td><td>12074</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638-500G</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S0876-500G</td><td></td></tr><tr><td>Antidiuretic Hormone dDAVP</td><td>Sigma-Aldrich</td><td>V2013-1MG</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Filter used for erythrocyte filtration</td><td>Macherey-Nagel</td><td>MN 615</td><td></td></tr><tr><td>BGA Analysis:</td><td></td><td></td><td></td></tr><tr><td>ABL 80 flex</td><td>Radiometer Medical ApS</td><td></td><td></td></tr><tr><td>Electron Microscope:</td><td></td><td></td><td></td></tr><tr><td>Philips CM100 TEM</td><td>FEI</td><td></td><td></td></tr></tbody></table>

the mouse isolated perfused kidney technique

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&#39;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.

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 blots of apical marker proteins of different tubule segments were performed, comparing isolated perfused kidneys to unperfused kidneys of the same animals. In all experiments, constant pressure perfusion was used, keeping the perfusion pressure at ~ 100 mmHg (Figure 4). In those kidneys, perfusate flow and vascular resistance remained stable over a time of 55 min at 15.6 &#177; 0.4 ml/min*g of kidney weight and 35 &#177; 1.0 mmHg*min/ml, respectively (Figure 4 A). A decrease of perfusate flow or an increase in vascular resistance is a sensitive parameter with regards to decreasing tissue viability and should be monitored throughout the perfusion with care. Blood gas and electrolyte analysis in 100 &#181;l samples taken 60 min after the onset of perfusion revealed renal CO2 production and O2 consumption, when the arterial inflow was compared to the venous outflow (Table 2). The other parameters remained unchanged between artery and vein (Table 2). Special emphasis should be placed on unaltered venous potassium release, as an increase may occur during tissue damage. Glomerular filtration rate, assessed via FITC-Inulin clearance14, tended to increase over time (Figure 4 B) but this trend did not reach significance. Fractional excretion of sodium and potassium assessed after 55 minutes of perfusion is increased compared to the in vivo situation (Figure 4 C). Urine osmolality is not elevated above the osmolality of the venous outflow after 55 min of perfusion (Figure 4 D).
For morphological studies, kidneys were fixed after perfusion in 3% paraformaldehyde and 0.1% glutaraldehyde in dialysis buffer as previously described17. Renal ultrastructure was assessed with TEM. Glomeruli and S1 segments of proximal tubules appeared largely unaltered after perfusion (Figures 5 A and B). Some, but not all S2/S3 segments of proximal tubules and the thick ascending limbs that were not close to vascular bundles were showing signs of organ damage, as described previously in rat isolated perfused kidneys5 (Figures 5 C and D). Even upon close inspection, distal convoluted tubules and collecting ducts did not show evident signs of necrosis (Figures 5 E, F, and G). In conclusion, the morphological findings in mouse kidneys mirror the situation found in the rat isolated perfused kidney5.
Western blots of apical marker proteins of the different nephron segments in isolated perfused kidneys (snap-frozen after 1 hr of perfusion) compared to unperfused contralateral kidneys of the same animal (snap-frozen before the onset of perfusion) did not reveal significant alterations. We studied NaPi-IIa (located in proximal tubules), NKCC2 (thick ascending limbs), NCC (distal convoluted tubules), and ENaC (connecting tubules/collecting ducts) (Figures 6 A and B).
<img alt="Figure 4" src="/files/ftp_upload/54712/54712fig4.jpg" /> 
Figure 4: Assessment of Functional Viability of Kidneys Perfused for 55 min. A) During constant pressure perfusion, vascular resistance and perfusate flow remained stable for 55 min. The first 15 min are excluded due to the high variability always seen during this time. B) Glomerular filtration rate (FITC-Inulin) of four kidneys assessed after 25 and 55 min displayed slight but not significant increase over time. C) Fractional excretion of sodium and potassium assessed after 55 min of perfusion. D) Venous plasma and urine osmolality after 55 min of perfusion. n = 4 in all experiments. Data are presented as means &#177; SEM. <a href="http://cloudflare.jove.com/files/ftp_upload/54712/54712fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54712/54712fig5.jpg" /> 
Figure 5: Transmission Electron Microscopy. Representative Morphology of Different Nephron Segments of Perfused Kidneys at an End Point of 1 hr. A) Glomeruli are intact. Detail of a podocyte in a glomerulus. B) S1 segment of a proximal tubule. S1 segments are intact. C) S2/S3 segment of a proximal tubule. Some, but not all S2/S3 segments of proximal tubules are highly necrotic. Cells appear swollen and show large intracellular damage. The tubular outline is maintained only by the basement membrane. D) Thick ascending limb (TAL). Those TALs that are far from the vascular bundles show the first signs of hydropic degeneration. Luminal membranes appear thinned and fragile. E) Distal convoluted tubule (DCT). DCTs are intact. F) Detail of the basolateral infoldings of a DCT cell. Fenestrated endothelium, basement membrane, basolateral infoldings, and mitochondria are intact. G) Collecting duct (CD). CDs are intact. <a href="http://cloudflare.jove.com/files/ftp_upload/54712/54712fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54712/54712fig6.jpg" /> 
Figure 6: Western Blot of Apical Marker Proteins in Isolated Perfused Kidneys Compared to Unperfused Kidneys from the Same Animals. A) Western blots of kidneys perfused for 1 hr and unperfused kidneys. NaPi-IIa is representative for the proximal tubule. NKCC2 is representative for the thick ascending limb. NCC and NCC phosphorylated at threonine 53 (pT53 NCC) are exclusively found in the distal convoluted tubule. Alpha and gamma subunits of ENaC in their full length and their respective proteolytically cleaved products are shown representative for the connecting tubule/collecting duct. B) Quantification of A) did not reveal significant differences between kidneys perfused for 1 hr and unperfused kidneys. n = 3 in all experiments. Data are presented as means &#177; SEM and individual values are shown. <a href="http://cloudflare.jove.com/files/ftp_upload/54712/54712fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54712/54712tbl1.jpg" /> 
Table 1: Composition of Solutions and Dialysis Buffer. <a href="http://cloudflare.jove.com/files/ftp_upload/54712/54712tbl1large.jpg" target="_blank">Please click here to view a larger version of this table.</a>
<img alt="Table 2" src="/files/ftp_upload/54712/54712tbl2.jpg" /> 
Table 2: Blood Gas and Electrolyte Analysis of Arterial Inflow and Venous Outflow of Perfused Mouse Kidneys 60 min after the Onset of Perfusion (n= 4). Data are presented as means &#177; SEM.

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The Mouse Isolated Perfused Kidney Technique

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.

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 ...

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Nanyang Technological University

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JoVE Journal

Biology

21.6K Views.  University of Zürich. 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.

Video: The Mouse Isolated Perfused Kidney Technique

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Medicine

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Biochemistry

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Immunology and Infection

Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a significant approach to understanding the fundamental physiological and molecular processes of renal pathophysiology. The methods and protocols we present here will allow for the molecular dissection of the kidney in each specific region of interest related to disease sequelae. To determine the effects of disease on specific kidney regions and structures with unique functions, the goals of this protocol are to demonstrate simplified mouse kidney compartmentalization and renal cortical tubule isolation techniques in tandem with streamlined label-free quantitative proteomic workflows. Combining these methods will assist in the identification of perturbed molecular patterns in the whole kidney, medullary compartments, and cortical tubule structures of kidneys, with the ultimate and eventual goal of single-cell proteomics in pathological contexts. Applying these methods in virtually any disease model will be helpful in delineating mechanisms of pathology related to kidney dysfunction.

Here, we present a detailed protocol for proteomic analysis of the whole kidney, isolated cortical tubule, and medullary proteomes. The study also compares regional proteomes in a diabetic mouse model and non-diabetic mice.

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Glomeruli Isolation: A Technique to Obtain Intact Glomeruli from Murine Kidney

Source: Eymael, J. et al. <a href="https://www.jove.com/video/60324" target="_blank">Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation.</a> J. Vis. Exp. (2020)
This video describes the method to isolate intact glomeruli from mouse kidneys. The encapsulated and decapsulated glomeruli thus obtained can further be used for culturing parietal epithelial cell outgrowths from the murine kidney.

Izolacja kłębuszków nerkowych: technika uzyskiwania nienaruszonych kłębuszków nerkowych z mysiej nerki

Source: Eymael, J. et al. Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation. J. Vis. Exp. ...

The Mouse Isolated Perfused Kidney Technique

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Use of a Hanging-weight System for Isolated Renal Artery Occlusion

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Mouse Kidney Transplantation: Models of Allograft Rejection

Reliable and High Efficiency Extraction of Kidney Immune Cells

Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice

Isolation of Mouse Kidney-Resident CD8⁺ T cells for Flow Cytometry Analysis

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Izolacja kłębuszków nerkowych: technika uzyskiwania nienaruszonych kłębuszków nerkowych z mysiej nerki