A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

Altered expression of microRNA in AKI may be useful for biomarker and therapy. This protocol is simple and achieves constant results between laboratories. IRI of the kidney represents one of the major risk factor for AKI. Quantifying microRNAs that are altered by IRI leads to a diagnosis. MicroRNAs purified from kidney sample is applied to other system. Carefully monitor for hypothermia, intestinal moisturization, and depth of anesthesia throughout the procedure. This is because there are many manual techniques to get used to.

[Narrator] After confirming a lack of response to toe pinch, place the anesthetized mouse on a heated surgical pad in the supine position. After making a midline abdominal incision, use a cotton swab moistened with PBS to gently push the bowel to the left side of the extra abdominal cavity to expose the right kidney and ureter and use angled forceps to lift the right ureter. Ligate the ureter in two places with 4-0 silk braided sutures, leaving longer ties for the suture that is closer to the kidney, and cut between the sutures. Carefully holding the remaining suture, bluntly dissect the connective tissue and fat along the kidney and identify a blood vessel that is not the renal arteries or veins that supplies blood to the right kidney. Ligate the vessel and, when the right kidney has been sufficiently detached, ligate the renal vein and artery. Using a cotton swab moistened with PBS, gently push the intestine toward the right side of the extra abdominal cavity to expose the left kidney and cover the intestine with a moistened drape. After preparing the left kidney as demonstrated, clamp the renal artery and vein one centimeter from the kidney to induce left kidney ischemia. A successful ischemia can be visually confirmed by a gradual uniform darkening of the kidney tissue. When ischemia is confirmed, return the intestinal tissue to the abdominal cavity, taking care to avoid intestinal twisting, and temporarily close and drape the abdominal skin. After 25 to 45 minutes, remove the clamp and confirm that the blood flow reperfusion and kidney color improve. After confirming that the bowel is not twisted, instill intraperitoneal PBS and close the abdominal muscle and skin in two layers using 4-0 nylon sutures. 24 hours after ischemic injury induction, reopen the midline incision and use a 27-gauge needle to collect the blood from the inferior vena cava. Make a midline incision in the chest wall and insert a 23-gauge needle into the left ventricle. After confirming backflow into the needle, make an incision in the right lobe of the liver and inject 20 milliliters of PBS. The liver will change red to pink, confirming that sufficient blood has been drained. Use forceps and scissors to remove the left kidney and wash the excised tissue in a dish of PBS. Then use the forceps to remove the renal fascia without drying out the kidney and cut the kidney in half along the vertical midline. For microRNA purification, place 30 milligrams of the frozen kidney sample into a glass homogenizer containing 700 microliters of phenylguanidine-based lysis reagent on ice and slowly and repeatedly twist the pestle to completely homogenize the sample. When the tissue has been completely dissociated, transfer the homogenized lysate to the biopolymer-shredding system in a micro centrifuge spin column in a two-milliliter collection tube and collect the sample to the bottom of the tube by centrifugation. Transfer the lysate to a new micro centrifuge tube containing 140 microliters of chloroform and mix the tube by inversion for 15 seconds with the cap securely tightened. After a two- to three-minute incubation at room temperature, sediment the sample by centrifugation and transfer the supernatant to a new micro centrifuge tube containing 1.5 times the volume of 100% ethanol without disturbing the pellet. Mix the sample for five seconds by vortexing before transferring 700 microliters of the sample to a silica-membrane based spin column in a two-milliliter collection tube. After a brief centrifugation, discard the flow-through and wash the sample two times with 500 microliters of wash buffer 2 in a new silica-membrane based spin column per wash to remove any traces of salt. After the second wash, centrifuge the silica-membrane based spin column again and, after discarding the flow-through, transfer the column to a new 1.5-milliliter collection tube. Add 25 microliters of RNase-free water to the column and incubate the sample for five minutes at room temperature before centrifuging the sample again. Then transfer the 25-microliter microRNA-containing eluite to a new micro centrifuge tube. In this study, seven microRNAs that expressed in humans that were upregulated in the kidneys of acute kidney injury mice were investigated. The levels of six of the microRNAs were found to be significantly increased in the kidneys of ischemia reperfusion injury mice compared to those in mock mice using quantitative real-time PCR as demonstrated. Serum blood urea nitrogen and creatinine levels and acute tubular necrosis were also significantly upregulated.

The biomarkers for IRI diagnosis and specific treatment of AKI remains unclear. I hope that microRNA research will advance and contribute to the diagnosis and treatment of AKI.