Dr. de Caestecker's laboratory is supported by NIH 1RO1 HL093057-01 and 1RC4DK090770-01. Dr. Harris' laboratory is supported by DK38226,DK51265, DK62794 and funding from the Veterans Administration. Support for mouse kidney injury surgeries, serum creatinine, and fibrosis assays also provided by the Vanderbilt O'Brien Kidney Injury Center grant 1P30 DK079341.

<ol>
	<li>Autoclave all surgical instruments before surgery. Note that if one is performing multiple surgeries on different mice, rinse instruments after use and then sterilize using a hot bead sterilizer. It is not sufficient to soak in 70% ethanol.</li>
	<li>Give 0.5 ml S/C sterile normal saline preoperatively and immediately postoperatively to compensate for loss of body fluid during surgery.</li>
	<li>Weigh the mice.</li>
	<li>Anesthetize mouse using IP Xylazine/Ketamine mixture. It usually takes 3-5 min for the mice to reach surgical plane anesthesia.</li>
	<li>Shave the surgical site with enough border area to keep hair from contaminating the incision site. This needs to be performed in a preparatory area, not where the surgery is performed.</li>
	<li>Apply ophthalmic lubricating ointment to the eyes to prevent drying during the procedure.</li>
	<li>Place the mouse prone on the heated surface covered with absorbent bench pad. Tape legs to the surgical surface.</li>
	<li>If using the water bath system, set heat at 38 &deg;C 1 hr prior to surgery.</li>
	<li>Aseptic preparation using Betadine solution swab stick: scrub from the center of the site towards the periphery 3x, each time followed by scrubbing with Nolvasan to remove the Betadine. All Betadine should be removed from the surgical field prior to incision by rinsing with Nolvasan. Before starting aseptic procedure gloves should be changed to be sterile.</li>
	<li>Cover surgical field with a sterile surgical drape. This will prevent stray hair from entering the surgical field and provide an area on which to lay sterile instruments during surgery.</li>
	<li>Palpate kidney location through the skin.</li>
	<li>Cut the dorsal skin along the midline of mouse (approximately 1.5 cm) using scissors and forceps.</li>
	<li>Separate skin and subcutaneous layers over the left and right dorsal sides through this incision by blunt dissection using scissors and forceps.</li>
	<li>Make a small incision through the right flank muscle and fascia above the kidney and exteriorize the right kidney.</li>
	<li>Carefully dissect the upper and lower poles of the kidney free from surrounding tissue. Note that fatty tissue from around the upper pole contains the adrenal gland carrying its own blood supply which can be pushed off the kidney but obviously should not be removed from the mouse (Figure 1). After liberating kidney from surrounding tissue, tie the 4-0 silk suture around the hilum of the right kidney using a double surgical knot. Leave one end of the suture ~2 in long to hold up the kidney as you are cutting distal to the knot so you can visualize the site to be cut. Irrigate the stump with sterile saline.</li>
	<li>Close the muscle layer by using absorbable suture.</li>
	<li>Make a small incision through the left flank muscle and fascia above the kidney and exteriorize the left kidney (Figure 2A).</li>
	<li>Carefully hold kidney using blunt forceps while releasing renal pedicle from surrounding fat tissue using forceps (Figures 2B-2F). Too much pressure on the kidney using the blunt forceps may cause renal injury so one needs to remember this when designing "sham" controls for the studies. If you find that your sham controls are developing renal injury (as evidenced by a rise in serum creatinine), an alternative method you may consider to release the surrounding fat tissue from the renal pedicle is to gently roll the tissues with saline soaked sterile cotton-tipped swabs. In our experience the fat is fairly tightly attached to the renal pedicle and cannot easily be removed with cotton swabs, so this may not be the first approach you would want to use. However, like other aspects of this model, this is something that you will have to establish for yourself with practice and experience.</li>
	<li>Clamp left renal pedicle using non traumatic vascular clamp using holding forceps (to facilitate clamp placement), use timer for different clamp times depending on the mouse strain and injury severity required (see Table 1).</li>
	<li>Cover the left kidney with the skin (Figure 2H).</li>
	<li>Cover skin incision with saline soaked gauze.</li>
	<li>Exteriorize kidney, confirm uniform dusky appearance and release the clamp after the indicated times, and confirm that dusky appearance reverses uniformly throughout the kidney (Figure 2I/G). If the kidney does not rapidly and uniformly re-perfuse (pink up) the mouse should be excluded from further analysis. Gently push kidney back into the retroperitoneal space.</li>
	<li>Close the muscle layer using absorbable suture.</li>
	<li>Close the skin layer using monofilament nylon non-absorbable suture.</li>
	<li>Administer buprenorphine analgesia.</li>
	<li>Transfer mice away from the surgical field onto a heating pad until they wake.</li>
	<li>Return the mice to animal room.</li>
	<li>Closely monitor the mice and give additional doses of buprenorphine as indicated above every 8-12 hr for pain or discomfort. Clinical signs resulting in administration of analgesics include depression or other behavioral changes, abnormal appearance, or posture such as pilo-erection, hunched posture, lack of grooming and eating, or immobility.</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure.+II.+Experimental+models+of+acute+renal+failure:+Imperfect+but+indispensable.">Acute renal failure. II. Experimental models of acute renal failure: Imperfect but indispensable.</a> Am. J. Physiol. Renal Physiol. 278, F1-F12 (2000).</li><li>Dunn, S. R., Qi, Z., Bottinger, E. P., Breyer, M. D., Sharma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+endogenous+creatinine+clearance+as+a+measure+of+renal+function+in+mice.">Utility of endogenous creatinine clearance as a measure of renal function in mice.</a> Kidney Int. 65, 1959-1967 (2004).</li><li>Yuen, P. S., Dunn, S. R., Miyaji, T., Yasuda, H., Sharma, K., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+HPLC+determination+of+creatinine+in+mouse+serum.">A simplified method for HPLC determination of creatinine in mouse serum.</a> Am. J. Physiol. Renal Physiol. 286, F1116-F1119 (2004).</li><li>Hetu, P. O., Gingras, M. E., Vinet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+rapid+liquid+chromatography+isotope+dilution+tandem+mass+spectrometry+(LC-IDMS/MS)+method+for+serum+creatinine.">Development and validation of a rapid liquid chromatography isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for serum creatinine.</a> Clin. Biochem. 43, 1158-1162 (2010).</li><li>Keppler, A., Gretz, N., Schmidt, R., Kloetzer, H. M., Groene, H. J., Lelongt, B., Meyer, M., Sadick, M., Pill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+creatinine+determination+in+mice+and+rats:+An+enzymatic+method+compares+favorably+with+a+high-performance+liquid+chromatography+assay.">Plasma creatinine determination in mice and rats: An enzymatic method compares favorably with a high-performance liquid chromatography assay.</a> Kidney Int. 71, 74-78 (2007).</li><li>Yang, L., Besschetnova, T. Y., Brooks, C. R., Shah, J. V., Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+cycle+arrest+in+G2/M+mediates+kidney+fibrosis+after+injury.">Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.</a> Nat. Med. 16, 535-543 (2010).</li><li>Shanley, P. F., Rosen, M. D., Brezis, M., Silva, P., Epstein, F. H., Rosen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+focal+proximal+tubular+necrosis+after+ischemia+with+reflow+in+the+rat+kidney.">Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.</a> Am. J. Pathol. 122, 462-468 (1986).</li><li>Chawla, L. S., Amdur, R. L., Amodeo, S., Kimmel, P. L., Palant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+severity+of+acute+kidney+injury+predicts+progression+to+chronic+kidney+disease.">The severity of acute kidney injury predicts progression to chronic kidney disease.</a> Kidney Int. 79, 1361-1369 (2012).</li><li>Lo, L. J., Go, A. S., Chertow, G. M., McCulloch, C. E., Fan, D., Ordonez, J. D., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialysis-requiring+acute+renal+failure+increases+the+risk+of+progressive+chronic+kidney+disease.">Dialysis-requiring acute renal failure increases the risk of progressive chronic kidney disease.</a> Kidney Int. 76, 893-899 (2009).</li><li>Burne, M. J., Haq, M., Matsuse, H., Mohapatra, S., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+to+renal+ischemia+reperfusion+injury+revealed+in+a+murine+model.">Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model.</a> Transplantation. 69, 1023-1025 (2000).</li><li>Muller, V., Losonczy, G., Heemann, U., Vannay, A., Fekete, A., Reusz, G., Tulassay, T., Szabo, A. 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Authors have no conflicts of interest to disclose.

We describe two models of IR-AKI to study the effects of moderate and severe renal injury. These models allow us to induce consistent and predictable injury with low mortality. Our protocol outlines many of the difficulties and pitfalls traditionally associated with this model. Moreover, we have shown that depending on the length of renal pedicle clamping, the model induces a largely reversible mild and moderate AKI, or more severe AKI with incomplete recovery and persistent renal fibrosis. Post-injury fibrosis in the se...

A variety of experimental models of acute kidney injury (AKI) have been developed to match the diversity and complexity of the human condition (see reference for a recent, comprehensive review1). Each of these models has its own strengths and weaknesses, and while each mimics the corresponding human conditions with varied efficiencies, none precisely model the pathophysiology of their human counterparts. Ischemia reperfusion (IR)-induced AKI has been developed as a model of acute ischemia-induced renal injury in rodents. While the severity of renal tubular injury seen in this model is rarely observed in patients with renal hypoperfusion injuries2, despite its limitations and largely because of the relatively reproducible nature of this model, its extensive use has provided and, it is expected, will continue to provide important insights into many of the common basic mechanisms of AKI, repair, and therapy3. IR surgery requires familiarity with the mouse kidney anatomy, which we have illustrated in a simplified form in Figure 1. Renal ischemia-reperfusion (IR) injury surgery can be performed via ventral (laparotomy) or dorsal (retroperitoneal) approaches. We use a dorsal approach since it is less traumatic, allowing faster recovery times and improved survival (particularly when first learning the procedure). Renal IR injury can be performed unilaterally or bilaterally. However, differences between effective clamp pressures (which may result from interposition of perihilar fat between the clamp jaws) and/or differences in renal responses to ischemia between sides leads to more variable results. While this is not an insurmountable problem, it can increase variability between experiments, which is a major issue for this model. Unilateral IR can be performed with contra-lateral nephrectomy. This is our method of choice since it reduces variability in clamping between pedicles, and at the same time allows one to evaluate renal function, which is unaffected with unilateral IR alone. There has been discussion about what is the most practical vs. optimal method to assess renal function in mice. Blood urea nitrogen (BUN) provides a measure of renal function and is a useful "first look" marker in some models of AKI, including IR injury. However, BUN levels can be affected by volume status of the mice which may be affected, particularly following ventral approach IR injury, when delayed recovery reduces oral intake of fluids for a number of days post-surgery. Serum creatinine is less influenced by hydration status but is clearly affected by muscle mass. One of the difficulties with serum creatinine measurements has been problems with detection of non-creatinine chromogens in mouse serum using picric acid based techniques. As an alternative, a number of centers have developed an HPLC-based method to quantify mouse creatinine that is not affected by this artifact4. However, unlike BUN and picric acid creatinine assays, which only require 5-10 &mu;l of serum, HPLC-assays require ~ 25 &mu;l serum per assay, which if performed in duplicate will require ~100 &mu;l of whole blood per assay. This can be limiting for mouse studies. Some centers have developed more sensitive HPLC and Mass Spectrometry-based methods that allow analysis of smaller sample volumes5, 6. However, these technologies are not widely available. An alternative, enzymatic cascade assay (which requires only 5-10 &mu;l of serum) has been evaluated in mouse and rat serum samples and shown to closely parallel HPLC measurements of serum creatinine while picric acid assays always over-estimate creatinine values7 . While this assay is not widely used in the AKI literature, the assay is commercially available, simple to use, and we find gives reliable results with this model of IR-induced AKI in mice.

<table border="1">
	<tbody>
		<tr>
			<td>Drug or solution</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ketamine (anesthetic)</td>
			<td>Webster Veterinary</td>
			<td>07-881-9413 (100 mg/ml)</td>
			<td>Final concentration 30 mg/ml Ketamine with 3 mg/ml Xylazine administered at 3-4 ml/kg (100 mg/kg Ketamine and 10 mg/kg Xylazine)</td>
		</tr>
		<tr>
			<td>Xylazine (analgesic)</td>
			<td>Webster Veterinary</td>
			<td>07-808-1939 (100 mg/ml)</td>
			<td>As above</td>
		</tr>
		<tr>
			<td>Antisedan (anti-Xylazine)</td>
			<td>Webster Veterinary</td>
			<td>07-867-7097 (5 mg/ml)</td>
			<td>Optional: to reverse effects of Xylazine in order to increase the rate of recovery after surgery Use 0.5-0.7 mg/kg</td>
		</tr>
		<tr>
			<td>Buprenorphine (analgesic)</td>
			<td>Bedford Laboratories</td>
			<td>NDC 55390-100-10 (0.3 mg/ml)</td>
			<td>Dose 0.05-0.1 &mu;g/g every 8-12 hr. Make up a 1:5 dilution in sterile normal saline preoperatively and give 2.5-5 &mu;l/g of the diluted solution.</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra</td>
			<td>NDC 17033-211-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine swab sticks</td>
			<td>Purdue Products L.P.</td>
			<td>NDC 67618-153-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nolvasan Surgical Scrub</td>
			<td>Pfizer Animal Care</td>
			<td>300253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Drugs and solutions for I/R surgery.</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Integra Miltex</td>
			<td>VWR 21909-404</td>
			<td>For skin and muscle dissection</td>
		</tr>
		<tr>
			<td>Curved iris forceps</td>
			<td>FST</td>
			<td>11065-07</td>
			<td>To hold skin and muscle, to remove peri-nephric fat</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>ROBOZ</td>
			<td>RS-5228</td>
			<td>To manipulate kidneys</td>
		</tr>
		<tr>
			<td>Vascular clamp (795 g pressure)</td>
			<td>ROBOZ</td>
			<td>RS-5459</td>
			<td>Good for C57BL/6 and BALB/c mice strains</td>
		</tr>
		<tr>
			<td>Schwartz clip applying forceps</td>
			<td>ROBOZ</td>
			<td>RS-5450</td>
			<td>For RS-5459 clamps</td>
		</tr>
		<tr>
			<td>Vascular clamp (75-85 g pressure)</td>
			<td>FST</td>
			<td>18055-02</td>
			<td>Optimal for CD1 mice strain, but also can be used for C57BL/6 and BALB/c</td>
		</tr>
		<tr>
			<td>Micro-Serrefine Clamp Applicator-14 cm with Lock</td>
			<td>FST</td>
			<td>18056-14</td>
			<td>For FST 18055-02 clamps</td>
		</tr>
		<tr>
			<td>Gayman T/Pump (water-bath heated platform)</td>
			<td>Braintree Scientific</td>
			<td>TP-650</td>
			<td>The water heater is set at 38 &deg;C 1 hr prior surgery</td>
		</tr>
		<tr>
			<td>15" W x 24" Heated platform</td>
			<td>Braintree Scientific</td>
			<td>HHP-2</td>
			<td>Connect to Gayman heated water pump</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Braintree Scientific</td>
			<td>Model 39DP</td>
			<td>Uses microwave for heating. This is useful for warming during recovery</td>
		</tr>
		<tr>
			<td>Absorbable suture (Vicryl 5-0)</td>
			<td>ETHICON</td>
			<td>VCP834G</td>
			<td>For fascia and muscle</td>
		</tr>
		<tr>
			<td>Non-absorbable suture: monofilament nylon MONOMID 6/0 660B)</td>
			<td>CP Medical</td>
			<td>CP-B660B-03</td>
			<td>For skin</td>
		</tr>
		<tr>
			<td>Halsey needle holder: Tungsten Carbide/13 cm</td>
			<td>FST</td>
			<td>12501-13</td>
			<td>To hold suture needles</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>FST</td>
			<td>06-662-3</td>
			<td>To quantify ischemia time</td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>FST</td>
			<td>18000-45</td>
			<td>To sterilize instruments between mice if you are performing multiple surgeries (it is not sufficient to soak instruments in ethanol)</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Braintree Scientific</td>
			<td>CLP-22965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Durapore</td>
			<td>1538-0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td>EXELint</td>
			<td>26044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile cotton tipped applicators</td>
			<td>Kendall</td>
			<td>8884541300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbent BenchPad</td>
			<td>VWR</td>
			<td>56617-014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical drapes</td>
			<td>VWR</td>
			<td>21902-985</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr>
			<td>2'' x 2'' Gauze Pads</td>
			<td>Medi-First</td>
			<td>60673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Cardinal Health</td>
			<td>2D7251</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Equipment and instruments for I/R surgery.</td>
		</tr>
	</tbody>
</table>

ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

Moderate IR-induced AKI. Unilateral IR with simultaneous contralateral nephrectomy substantially reduces variability in results, but with shorter clamp times necessary for the mice to survive this procedure, we still found that only 50-60% of mice developed the expected renal insufficiency 24 hr after injury (Figure 3A). In practical terms this creates difficulties evaluating data unless the studies involve treatment regimens that can be initiated at least 24 hr after injury, allowing ti...

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Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

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39.2K Views.  Vanderbilt University Medical Center. We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.