The overall goal of this procedure is to induce chronic kidney disease in the Lewis rat. This is accomplished by first administering the nitric oxide blocker, NG Nitro, L arginine, or LNNA for a P period of four weeks. Next, the entire right kidney is removed.
Then one week later, two thirds of the left kidney is removed. Finally, while being treated with LNNA and a high salt diet, the development of renal failure in the rat is followed over time. Ultimately, results can be obtained that show established chronic kidney disease through collection of urine for the measurement of protein and hematocrit and UIA in blood, plasma, and or urine and systolic blood pressure.
Renal function is ultimately determined by inulin and para amino hip rate, or PAH clearance. This method can help answer key questions in the field of nephrology, such as a better understanding of the pathology of CKD, and can be used to study the effect of therapeutic interventions Four weeks prior to the initial nephrectomy procedure. Start administering 20 milligrams per liter of the nitric oxide synthase inhibitor LNNA in the rat's drinking water.
According to this timeline. Before beginning the procedure, use 70%alcohol to disinfect the table. To prepare for surgery, begin by sterilizing the following instruments, A blanket one 12 centimeter student tissue forceps with one to two teeth, one student standard pattern forceps, two simkin forceps, one mayo scissors, one student iris scissors, and one Olson heger needle holder with scissors.
Then have the following inventory ready. An operating table with warming pad and lamp oxygen and iso fluorine tissues. A shaver, the sterile surgical instruments five by five and 10 by 10 sterile gauze, 70%alcohol, 0.9%sodium chloride solution, A one milliliter syringe, 25 gauge needles gel foam pads 4.0 and 5.0 Vicryl sutures, 0.03 milligrams per kilogram, buprenorphine, and a clean cage for the rats after surgery.
After inducing anesthesia in an induction chamber with 4%iso fluorine and administering pre analgesia. If necessary, transfer the rat onto the heating pad on its left side on the table and ventilate it using a nose cone with a mixture of 2%isof, fluorine, and one liter of oxygen. Shave the flank of the rat and use alcohol to disinfect the surgical site.
Since surgery takes place in a semi sterile environment, we disinfect the red skin only with alcohol for more sterile preparation. Scrubbing with Betadine can be used, put on a head cap and a mask, then wash and disinfect hands before putting on sterile surgical gloves. Next, using anatomical forceps and blunt scissors, make a one to one and a half centimeter incision parallel to the ribs.
Then with blunt dissection of the back muscles, expose the right kidney. Once the lower pole of the kidney is visible, use the small blunt forceps to carefully grip the perren fat tissue. Then use forceps to gently pull on the perren fat, taking care not to disturb the adrenal gland and externalize the kidney to separate the kidney from the adrenal gland.
Position a forceps at the medial site in the fat tissue and gently move the tissue upwards between the adrenal gland and the kidney. Gently place the kidney on gauze and clearer of any surrounding fat and connective tissue. Identify the renal artery and vein and loosely.
Place a ligature with a single knot around the vessels to prevent the ligature from slipping off. After cutting, move the loose knot carefully along the vessels towards the aorta, approximately 0.5 centimeters to create space between the kidney and the knot. Next, tie two double knots without cutting the ends of the ligature.
If profusion is successfully halted, the color of the kidney will immediately turn brown. Then cut the renal vessels close to the kidney and remove it. Gently pull on the long ends of the ligature to check for bleeding of the renal vessels.
The ligature should stay in place on the vessel. Cut the long ends of the ligature. The remnant renal vessels will retract into the abdominal cavity.
Immediately use 5.0 Vicryl running sutures to close the skeletal muscle into the hind limb. Intramuscularly, inject 0.03 milligrams per kilogram of buprenorphine. Then use 4.0 Vicryl intra cutaneous sutures to close the skin.
Next, blot the excised kidney dry and weigh it. Allow the rat to recover in a clean solitary cage, placed halfway on a heating pad with access to food and water, and monitor it according to the text protocol. Seven days later.
In preparation for a second surgery, calculate the weight of about two thirds of the right kidney and prepare small pieces of gel foam. Repeat the procedure shown to exteriorize the left kidney and detach the adrenal gland from it. Place sharp scissors around the upper pole of the left kidney and resect the upper pole in one stroke cover immediately with a piece of gel foam and exert mild pressure with sterile gauze.
Then resect the lower pole of the kidney and cover it in a similar manner to prevent clotting on the skin. Lift the kidney and use sterile gauze to maintain mild pressure on both gel foam pads until the bleeding stops. If bleeding persists at another foam pad, place the remnant kidney with the adherent gel foam pads inside the abdomen and close the muscle and skin.
Follow the same procedure as described earlier to monitor the recovery and house the rat. After subtotal nephrectomy, approximately one sixth of the total renal mass is left. This figure shows the calculated percentage of removed renal mass of the right kidney with mean and standard deviation.
In our previous experiments in the week after, union nephrectomy hypertrophy of the left kidney occurs resulting in the slight underestimation of the preferred amount of renal tissue that needs to be removed, which was based on the calculated weight of the right kidney. However, since it is not possible to determine the weight of the left kidney, this is the most accurate way to remove about five six of the renal mass. Progression of renal failure is tracked by collection of urine for the measurement of protein and creatinine, blood for plasma urea and creatinine and systolic blood pressure.
Creatinine clearance can be calculated, but tends to underestimate the decline in GFR due to extensive tubular creatinine secretion in rats underlining the importance of the gold standard method to determine renal function by inulin and PAH clearance Conners at all described the complete procedure. Inulin and PIH clearance are calculated from their concentration in the urine sample, urine flow rate, and their plasma concentration. Over time, rats with chronic kidney disease or CKD develop hypertension, EMIA anemia, protein urea, a significant decrease in glomerular filtration rate and effective renal plasma flow.
After six weeks established CKD has developed, which is a suitable time point to test rescue interventions with increased time, strong progression of hypertension and protein urea is observed while hematocrit and renal function show mild deterioration. Other symptoms not focused on in our experiments include derangement and calcium phosphate and lipid metabolism, and many others depending on the strain of rats. Development of CKD can vary markedly with the Lewis rat used here.
A high salt diet and a nitric oxide blocker were added to induce hypertension and endothelial dysfunction. Once small step. This technique can be done in approximately 50 to 20 minutes if it is performed properly.
After watching this video, you should have a good understanding of how to induce chronic kidney disease in the loose reds by performing a stepwise subdues nephrectomy.
