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

Summary

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

Abstract

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its γ-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Introduction

Nephrotic syndrome is characterized by heavy proteinuria, hypoalbuminemia, edema and hyperlipidemia, and can be regarded as the most extreme manifestation of proteinuric kidney disease. In rodents, experimental nephrotic syndrome can be induced by single injection of anthracyclines or puromycin which leads to podocyte damage and resembles human minimal change disease and focal segmental glomerulosclerosis (FSGS)¹. After its first description in 1955 by Frenk et al.², puromycin nucleoside nephrosis (PAN) in rats has become a standard model to investigate the pathophysiology of nephrotic syndrome in numerous studies^3,4,5,6. In mice, the corresponding model can be induced by the anthracycline doxorubicin⁷. However, there is a strong strain-dependency that is genetically determined by at least two genetic loci⁸. In addition, there are differences in the proteinuric response and course of the nephrotic syndrome^9,10. Using 129S1/SvImJ mice and rapid intravenous injection of doxorubicin via the retrobulbar sinus, proteinuria responses reach values that are sufficient to induce the typical features of nephrotic syndrome, particularly volume retention characterized by ascites and nearly sodium-free urine⁷. Sodium retention in experimental nephrotic syndrome has been presumed to be the result of activation of the epithelial sodium channel (ENaC) in the distal tubule by aberrantly filtered serine proteases such as plasmin causing proteolysis of its γ-subunit^4,11,12. Recently, this concept was proven in nephrotic mice which were protected from proteolytic ENaC activation and sodium retention by treatment with the serine protease inhibitor aprotinin that was equally effective as the ENaC blocker amiloride¹³. To ensure continuous delivery to the distal tubule, aprotinin was administered via subcutaneously implanted sustained release pellets. Future studies are required to identify the serine proteases that are responsible for proteolytic ENaC activation in nephrotic syndrome which is thought to parallel the human situation. For this purpose, doxorubicin-induced nephrotic syndrome is a valuable model that can be used in wild-type mice or expanded to genetically engineered mice. Its advantages include low cost of the drug, lower complexity of management and good reproducibility¹⁴.

In this article, we demonstrate the induction of experimental nephrotic syndrome by rapid intravenous injection of doxorubicin to the retrobulbar sinus and implantation of sustained-release pellets containing aprotinin to inhibit urinary serine protease activity as measured with a chromogenic assay.

Protocol

All methods were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the German law for the welfare of animals, and they were approved by local authorities (Regierungspräsidium Tübingen).

1. Induction of Experimental Nephrotic Syndrome by Doxorubicin Injection to the Retrobulbar Sinus

1. Prepare a 0.5 mL syringe with a mounted 30G cannula by marking the stop position of the piston.
2. Calculate injection volume of doxorubicin for male (7.25 µL/g body weight (bw) equal to 14.5 µg/g bw doxorubicin) and female mice (6.9 µL/g bw equal to 13.8 µg/g bw doxorubicin) according to the body weight from the morning.
   NOTE: The given dose is appropriate for induction of nephrotic syndrome. Minor changes in dosage lead to a crucial different course of nephropathy reaching from mild chronic kidney disease with only small changes in glomerular filtration rate (12.6 µg/g bw doxorubicin) to acute kidney failure (15.4 µg/g bw doxorubicin)^7,15.
3. Warm the calculated amount of doxorubicin solution (2 µg/µL) in a 37 °C warm chamber.
   Caution: Doxorubicin is harmful if swallowed and can cause cancer. Always wear gloves if working with doxorubicin and avoid any skin contact.
4. Prefill the syringe with doxorubicin solution until the marking point and set the balance to zero. Fill the syringe with the injection volume and check the volume by weighing the syringe.
5. Narcotize the mouse deeply with 5 vol% isoflurane using a vaporizer and oxygen flow of 2 L/min.
   1. Do not use ointment on the eyes prior to the injection. The operator needs a clear view of the orbital cavity for performing a safe and successful retrobulbar injection.
   2. Assess the level of anesthesia by pedal reflex and adjust anesthetic delivery if necessary before starting injection.
6. Place the mouse at a right lateral recumbency with its back facing to the operator's body and its head facing to the operator's injection hand.
   NOTE: The procedure is described for a right-handed person, for left-handed persons performing the injection with the left hand it is easier to do the position and injection reversed right to left.
7. Carefully protrude the mouse's left eyeball from the eye socket by applying gentle pressure to the skin, dorsal and ventral to the eye.
8. Puncture the left retrobulbar sinus from the inner eye angle (medial palpebral commissure). Avoid any contact with the mouse´s eyeball.
9. Slightly tilt the syringe and inject entire volume in one go. Make sure that the volume is injected without any resistance and without any signs of extravasation such as exopthalmus or leakage from the injection site.
10. Since doxorubicin is a highly toxic substance, check well-being at least once a day by using a score-sheet according to Morton and Griffiths et al.¹⁶ and pay special attention to the injection site. If any signs of extravasation like exopthalmus, impaired eyelid closure or any signs of necrosis like swelling or skin lesions occure after injection or in the following days, the mouse should be euthanized.

2. Implantation of Sustained-Release Pellets Containing Aprotinin

1. Order pellets with the desired dose per day and release duration in advance. In case of aprotinin, choose a dose of 1 mg per day to be released over 10 days.
   NOTE: In this study, the aprotinin pellets contain a dose of 10 mg.
   1. Store the pellets under dry conditions and avoid any exposure to humidity.
2. Prepare required items for the surgery, including 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 cm monofile suture non resorbable.
   1. Sterilize the instruments using a heat sterilizer for 5 min at 240 °C. Before using the instruments, wait for 5 min until they reach room temperature.
      1. Avoid any contact of sterilized instrument tips to nonsterile surfaces.
      2. Avoid skin burn by hot instruments.
3. Narcotize mouse with isoflurane 5 vol% followed by 1.5 vol% using a vaporizer and oxygen flow of 2 L/min.
4. Place the mouse in a prone position on a warming device covered with a layer of gauze to avoid thermal injuries to the mouse. Surface temperature is about 37 °C.
5. Protect the eyes with ointment.
6. Remove the hair on the middle back in an area of about 0.5 cm² using scissors or a hair trimmer and disinfect the hairless skin with a disinfectant suitable for skin disinfection. Remove as less hair as possible so it stays more difficult for the mouse to reach the wound and thread ends later and to keep body temperature.
7. Incise the hairless skin with a scalpel in cranio-caudal direction in a length of about 5 mm. Prepare a left lateral pouch of about 1 cm depth in the subcutaneous connective tissue using a blunt preparation.
   1. Assess the level of anesthesia by pedal reflex and adjust anesthetic delivery if necessary before starting surgery.
8. Insert one 10-day release pellet into the prepared left lateral pouch using sterile tissue tweezers. Leave the pellet at the bottom of the pouch in a planar position.
   1. Avoid any contact of the pellet to fluid and humidity till placed in the prepared pouch.
9. Close the skin with 2-3 sutures. Only leave very short thread ends to make it more difficult for the mouse to open the sutures by gnawing.
10. Place the mice individually to reduce postoperative distress and to prevent opening of the sutures by gnawing. Keep the mouse in view until it has regained sufficient consciousness after narcosis.
    NOTE: There is no need for postoperative pain management since this intervention is well tolerated without any signs of discomfort or pain. Alternatively we recommend topical analgesics, e.g. a drop of bupivacaine on the incision prior to closure which would provide up to 8 h of analgesia.

3. Assessment for Model Induction, Signs of Nephrotic Syndrome and well-being

1. Collect morning urine (08:00 am) into a reaction cup (1.5 mL) every day from the injection day by massaging the bladder.
   1. Measure proteinuria by using Bradford protein assay and normalize to creatinine.
      NOTE: For induction of nephrotic syndrome, proteinuria should reach a threshold of 120 mg/mg creatinine between day 7 and 10 after doxorubicin injection.
2. Look for the development of ascites. Check for increase of abdominal circumference or overhanging flanks.
3. Weigh the mouse, the food pellets and the drinking bottle in the morning (08:00 am) every day.
4. Check well-being at least once a day by using a score-sheet according to Morton and Griffiths et al.¹⁶

4. Measurement of Urinary Serine Protease with a Chromogenic Assay

1. Prepare substrate working solution by adding 15 mL of sterile phosphate buffered saline (PBS) to a bottle of 25 mg of lyophilised chromogenic substrate S-2251, yielding a concentration of 2 mM. Dissolve aprotinin in PBS to obtain an aprotinin solution with a concentration of 2 mg/mL.
   1. Store prepared solutions at -20 °C. This significantly slows the auto-degradation of S-2251. Do not use old solutions with a strong shade of yellow.
2. Use freshly prepared or thawed substrate working solution and warm it in a 37 °C heat chamber.
3. Thaw urine samples at room temperature.
   1. Prevent protease degradation by avoiding repeated thawing and freezing. Store urine samples at -20 °C.
4. Use a 96 microwell plate suitable for photometric measurement. Add 3 µL of undiluted urine in each of two wells and add 50 µL of substrate working solution with 3 µL of either PBS or aprotinin solution to the wells. Cover the plate with a plate sealer.
5. Incubate the covered microwell plate for 60 min in a 37 °C heat chamber.
6. Measure the optical density of each well at 450 nm using a microplate reader.
7. Take the difference between the OD without aprotinin and with aprotinin as urinary aprotinin-sensitive serine protease activity.
   NOTE: Measurement of a paired sample with and without aprotinin increases specificity of the assay since the substrate could also be cleaved by other proteases that do not play a pathophysiological role.

Results

After the induction of isoflurane narcosis, doxorubicin was rapidly injected via the left retrobulbar sinus. The entire volume of 7.25 µL/g bw was injected without any resistance and extravasation proving correct intravenous location of the cannula. The mouse recovered from narcosis rapidly and had no impairment on that day and thereafter. Particularly, there was no sign of damage at the left eye. After doxorubicin injection, food and fluid intake dropped over the first 3 days due to...

Discussion

Here, we demonstrate that doxorubicin injection via retrobulbar sinus injection induces experimental nephrotic syndrome in 129S1/SvImJ mice with proteinuria, sodium retention, hypoalbumenia and hyperlipidemia. However, there are two critical issues that have to be taken into account when using this model. Firstly, the model induction is strictly dose dependent and deviations of doxorubicin dose as small as 0.3 µg/g affect the response of the mice¹⁵. When injected with a lower dose such as 14 ...

Materials

Name	Company	Catalog Number	Comments
supplies
BD Micro FINE + U-40 (0.30 mm x 8 mm)	BD Deutschland GmbH, Heidelberg, Germany	PZN: 07468060	syring
ETHILON*II (5/0, 16 mm, 3/8c, 45 cm)	Johnson&Johnson Medical GmbH, Ethicon Deutschland, Norderstedt, Germany)	EH7823H	suture
Name	Company	Catalog Number	Comments
reagents
aprotinin (6000 KIU/mg)	LOXO, Heidelberg, Germany	CAS 9087-70-1
Bepanthen, Augen- und Nasensalbe	Bayer Vital GmbH, Leverkusen, Germany	PZN: 1578681	ointment
Chromogenix S-2251	HAEMOCHROM DIAGNOSTICA GmbH, Essen, Germany	82 0332 39	chromogenic substrate
doxorubicin (2.0 µg/µL)	Cell Pharm, Bad Vilbel, Germany	CAS 25316-40-9	doxorubicin
isofluran CP (1 mL/mL)	CP-Pharma, Burgdorf, Germany	CAS 26675-46-7	isofluran
pellets with matrix-driven sustained release (custom-made)	Innovative Research of America, Sarasota, FL	X-999	pellet
Name	Company	Catalog Number	Comments
equipment
ELx808 Absorbance Mikroplate Reader	BioTek, Bad Friedrichshall, Germany	ELx808	microplate reader
MICROSTERIL - 436	B.M.S., Trescore Balneario, Italy	GAL/436	sterilizer
Hybridization oven/shaker	GE Healthcare UK Limited, Amersham LIFE SCIENCE, Little Chalfont, UK	RPN 2511	heat chamber
Thermo MAT Pro 10 W, 15x25 cm, Lucky Reptile	Import Export Peter Hoch GmbH, Waldkirch, Germany	61201	mouse warming device