Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

The overall goal of this mouse model of experimental nephrotic syndrome is to feature all the facets of the syndrome known from human patients, most importantly, sodium retention and edema formation. This model can help to elucidate the mechanisms of sodium retention in nephrotic syndrome. As will be shown in this article, sodium retention is caused by the excretion of active serum proteases in the urine, which we have termed proteasuria.

The main advantages of this model are that it is easy to induce and that it features all of the facets of human nephrotic syndrome and chronic kidney disease. During the implantation segment of the protocol, Dr.Bohnert, a research fellow, will demonstrate this technique. To begin, warm the doxorubicin in a 37 degrees Celsius chamber.

Next, prepare the injection syringe. Mark the stop position of the piston on the syringe to deliver the required dose of doxorubicin using a two microgram per microliter solution. Then, zero the mass of a prepared syringe and load it into the syringe to the marked point.

For confirmation of the volume, re-weigh the syringe. This injection has to take place approximately 30 seconds after narcosis. After anesthetizing the mouse, position it at right lateral recumbency with its head facing to the operator’s injection hand.

Then, carefully protrude the left eyeball using gentle pressure dorsal and ventral to the eye. Next, insert the cannula into the left retrobulbar sinus from the inner-eye angle, without making contact with the eyeball. Now, slightly tilt the syringe and inject the bolus in one stroke.

There should be no resistance or signs of extravasation, such as exophthalmus or leakage from the injection site. After re-injection, recheck for correct intravenous injection. There should be no signs of extravasation, as mentioned before.

Then, re-weigh the syringe and calculate the actual administered dose of doxorubicin. To begin, prepare the required items for the surgery on a drape. A pair of hair scissors, a pair of skin preparation scissors, a scalpel, surgical tweezers, two pairs of tissue tweezers, a needle holder, and 15 centimeters of non-resorbable MONOFIL suture.

Place a warming device on the surgical stage, cover it with a layer of gauze, and then place an anesthetized mouse, prone, onto the gauze. Now, apply ophthalmic ointment and remove about 0.5 square centimeters of hair centrally from the back, using scissors. Then, disinfect the skin with alcohol.

By minimizing the hair removal, the wound will have natural protection. Now, make an incision in the cranial caudal direction that is about five millimeters long. Then, by bluntly dissecting the subcutaneous connective tissue, make a left lateral pouch about one centimeter deep.

Next, remove a 10-day release pellet from its dry container and place it into the bottom of the pouch in a planar position. Then, close the skin with a few sutures. Make the thread ends very short so it will be difficult for the mouse to open the sutures by gnawing.

Now, have the mice recover from the surgery alone to minimize post-operative distress and suture gnawing. Monitor the mouse until it has regained sternal recumbency. Collect urine from the mice on a daily basis in the morning within about two hours after subjective dawn.

Massage the bladder of the mouse to collect as much urine as possible in a 1.5 milliliter tube. At the same time, look for the development of ascites. Check for an increase of abdominal circumference or for overhanging flanks.

Then, weigh the mouse, the food pellets, and the drinking bottle. Later, measure proteasuria based on the proteolytic activity of urine samples, using S2251 as a chromogenic substrate. Do not use S2251 working solution if it has a strong shade of yellow.

Thaw the urine samples at room temperature and for the assay, prepare a 96-well plate that can be measured by a plate reader. For each sample, add three microliters of urine to two wells. Then, add three microliters of PBS or aprotinin solution to each well.

Then, add 50 microliters of substrate working solution to each well. After all the samples have been set up, cover the plate with a plate sealer and incubate the plate at 37 degrees Celsius for an hour. Then, measure the optical density of each well at 450 nanometers using a microplate reader.

After injecting mice with doxorubicin, food and fluid intake dropped over the first three days due to a general toxic effect of doxorubicin, but recovered thereafter. By five days after injection, the mice started showing signs of marked proteinuria, followed by a massive decline of urinary sodium excretion despite adequate food intake. Three days after the doxorubicin injection, a mouse was implanted with an aprotinin-containing pellet and another was implanted with a placebo pellet.

In these mice, sodium retention and body weight gain were only experienced by the placebo-treated mouse. Next, the effect of aprotinin on urinary serine protease activity was measured with a chromogenic assay using urine samples from the implanted mice. Serine protease activity increased rapidly in the placebo-treated mice, paralleling the onset of proteinuria, whereas urinary serine protease activity were completely inhibited in the aprotinin-treated nephrotic mice.

Once mastered, this model is useful to study the mechanisms of sodium retention in nephrotic syndrome, which center around the excretion of active serine proteases in the urine or proteasuria. This leads to activation of the epithelial sodium channel ENaC in the distal tubule by proteolysis. This model resembles human nephrotic syndrome caused by protocyte loss as observed in focal segmental glomerulosclerosis.

In the course of the model, mice develop progressive renal failure with typical consequences such as secondary hypo-parathyroidism and renal anemia, recapitulating all stages of chronic kidney disease.