Name: induction of nephrotic syndrome in mice by retrobulbar injection of doxorubicin and prevention of volume retention by sustained release aprotinin
Uploaded: 2018-05-06T15:00:00
Description: Watch this Scientific Journal Video about Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin at JoVE.com

This study was supported by a grant from the German Research Foundation (DFG, AR 1092/2-1).

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 (Regierungspr&#228;sidium T&#252;bingen).
1. Induction of Experimental Nephrotic Syndrome by Doxorubicin Injection to the Retrobulbar Sinus
<ol>
	<li>Prepare a 0.5 mL syringe with a mounted 30G cannula by marking the stop position of the piston.</li>
	<li>Calculate injection volume...

<ol>
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C., Frokiaer, J., Nielsen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+but+not+targeting+of+ENaC+in+adrenalectomized+rats+with+PAN-induced+nephrotic+syndrome.">Increased expression but not targeting of ENaC in adrenalectomized rats with PAN-induced nephrotic syndrome.</a> Am J Physiol Renal Physiol. 291 (1), F208-F217 (2006).</li><li>Artunc, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-+and+glucocorticoid-inducible+kinase+1+in+doxorubicin-induced+nephrotic+syndrome.">Serum- and glucocorticoid-inducible kinase 1 in doxorubicin-induced nephrotic syndrome.</a> Am J Physiol Renal Physiol. 295 (6), F1624-F1634 (2008).</li><li>Zheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mendelian+locus+on+chromosome+16+determines+susceptibility+to+doxorubicin+nephropathy+in+the+mouse.">A Mendelian locus on chromosome 16 determines susceptibility to doxorubicin nephropathy in the mouse.</a> Proc Natl Acad Sci U S A. 102 (7), 2502-2507 (2005).</li><li>Kimura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstrain+differences+in+murine+daunomycin-induced+nephrosis.">Interstrain differences in murine daunomycin-induced nephrosis.</a> Nephron. 63 (2), 193-198 (1993).</li><li>Wang, Y., Wang, Y. P., Tay, Y. C., Harris, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+adriamycin+nephropathy+in+mice:+sequence+of+histologic+and+immunohistochemical+events.">Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.</a> Kidney Int. 58 (4), 1797-1804 (2000).</li><li>Svenningsen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+in+nephrotic+urine+activates+the+epithelial+sodium+channel.">Plasmin in nephrotic urine activates the epithelial sodium channel.</a> Journal of the American Society of Nephrology : JASN. 20 (2), 299-310 (2009).</li><li>Passero, C. J., Hughey, R. P., Kleyman, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+role+for+plasmin+in+sodium+homeostasis.">New role for plasmin in sodium homeostasis.</a> Curr Opin Nephrol Hypertens. 19 (1), 13-19 (2010).</li><li>Bohnert, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aprotinin+prevents+proteolytic+epithelial+sodium+channel+(ENaC)+activation+and+volume+retention+in+nephrotic+syndrome.">Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome.</a> Kidney Int. 93 (1), 159-172 (2018).</li><li>Pippin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+rodent+models+of+acquired+podocyte+diseases.">Inducible rodent models of acquired podocyte diseases.</a> Am J Physiol Renal Physiol. 296 (2), F213-F229 (2009).</li><li>Bohnert, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+phosphorus+restriction+and+vitamin+D-substitution+on+secondary+hyperparathyroidism+in+a+proteinuric+mouse+model.">Impact of phosphorus restriction and vitamin D-substitution on secondary hyperparathyroidism in a proteinuric mouse model.</a> Kidney Blood Press Res. 40 (2), 153-165 (2015).</li><li>Morton, D. B., Griffiths, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+the+recognition+of+pain,+distress+and+discomfort+in+experimental+animals+and+an+hypothesis+for+assessment.">Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment.</a> Vet Rec. 116 (16), 431-436 (1985).</li><li>Beath, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+aprotinin+concentrations+during+cardiac+surgery:+full-+versus+half-dose+regimens.">Plasma aprotinin concentrations during cardiac surgery: full- versus half-dose regimens.</a> Anesth Analg. 91 (2), 257-264 (2000).</li><li>Kanter, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+toxicology+study+of+liposome+encapsulated+doxorubicin+(TLC+D-99):+comparison+with+doxorubicin+and+empty+liposomes+in+mice+and+dogs.">Preclinical toxicology study of liposome encapsulated doxorubicin (TLC D-99): comparison with doxorubicin and empty liposomes in mice and dogs.</a> In Vivo. 7 (1), 85-95 (1993).</li><li>Ma, L. J., Fogo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+of+robust+induction+of+glomerulosclerosis+in+mice:+importance+of+genetic+background.">Model of robust induction of glomerulosclerosis in mice: importance of genetic background.</a> Kidney Int. 64 (1), 350-355 (2003).</li><li>Kren, S., Hostetter, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+course+of+the+remnant+kidney+model+in+mice.">The course of the remnant kidney model in mice.</a> Kidney Int. 56 (1), 333-337 (1999).</li><li>Mizuno-Horikawa, Y., Mizuno, S., Tamura, S., Kurosawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+glomerulosclerosis+is+reversible+in+nephrotic+mice.">Advanced glomerulosclerosis is reversible in nephrotic mice.</a> Biochem Biophys Res Commun. 284 (3), 707-713 (2001).</li><li>Friberger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromogenic+peptide+substrates.+Their+use+for+the+assay+of+factors+in+the+fibrinolytic+and+the+plasma+kallikrein-kinin+systems.">Chromogenic peptide substrates. Their use for the assay of factors in the fibrinolytic and the plasma kallikrein-kinin systems.</a> Scand J Clin Lab Invest Suppl. 162, 1-298 (1982).</li><li>Haerteis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+kallikrein+activates+the+epithelial+sodium+channel+in+vitro+but+is+not+essential+for+volume+retention+in+nephrotic+mice.">Plasma kallikrein activates the epithelial sodium channel in vitro but is not essential for volume retention in nephrotic mice.</a> Acta Physiologica. , (2018).</li><li>Schork, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Plasminuria+with+Overhydration+in+Patients+with+CKD.">Association of Plasminuria with Overhydration in Patients with CKD.</a> Clin J Am Soc Nephrol. 11 (5), 761-769 (2016).</li></ol>

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 &#181;g/g affect the response of the mice15. When injected with a lower dose such as 14 ...

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 studies3,4,5,6. 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 syndrome9,10. 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 &#947;-subunit4,11,12. 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.

<table>
	<tbody>
		<tr>
			<td>supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro FINE + U-40 (0.30 mm x 8 mm)</td>
			<td>BD Deutschland GmbH, Heidelberg, Germany</td>
			<td>PZN: 07468060</td>
			<td>syring</td>
		</tr>
		<tr>
			<td>ETHILON*II (5/0, 16 mm, 3/8c, 45 cm)</td>
			<td>Johnson&#38;Johnson Medical GmbH, Ethicon Deutschland, Norderstedt, Germany)</td>
			<td>EH7823H</td>
			<td>suture</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>aprotinin (6000 KIU/mg)</td>
			<td>LOXO, Heidelberg, Germany</td>
			<td>CAS 9087-70-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthen, Augen- und Nasensalbe</td>
			<td>Bayer Vital GmbH, Leverkusen, Germany</td>
			<td>PZN: 1578681</td>
			<td>ointment</td>
		</tr>
		<tr>
			<td>Chromogenix S-2251</td>
			<td>HAEMOCHROM DIAGNOSTICA GmbH, Essen, Germany</td>
			<td>82 0332 39</td>
			<td>chromogenic substrate</td>
		</tr>
		<tr>
			<td>doxorubicin (2.0 &#181;g/&#181;L)</td>
			<td>Cell Pharm, Bad Vilbel, Germany</td>
			<td>CAS 25316-40-9</td>
			<td>doxorubicin</td>
		</tr>
		<tr>
			<td>isofluran CP (1 mL/mL)</td>
			<td>CP-Pharma, Burgdorf, Germany</td>
			<td>CAS 26675-46-7</td>
			<td>isofluran</td>
		</tr>
		<tr>
			<td>pellets with matrix-driven sustained release (custom-made)</td>
			<td>Innovative Research of America, Sarasota, FL</td>
			<td>X-999</td>
			<td>pellet</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ELx808 Absorbance Mikroplate Reader</td>
			<td>BioTek, Bad Friedrichshall, Germany</td>
			<td>ELx808</td>
			<td>microplate reader</td>
		</tr>
		<tr>
			<td>MICROSTERIL - 436</td>
			<td>B.M.S., Trescore Balneario, Italy</td>
			<td>GAL/436</td>
			<td>sterilizer</td>
		</tr>
		<tr>
			<td>Hybridization oven/shaker</td>
			<td>GE Healthcare UK Limited, Amersham LIFE SCIENCE, Little Chalfont, UK</td>
			<td>RPN 2511</td>
			<td>heat chamber</td>
		</tr>
		<tr>
			<td>Thermo MAT Pro 10 W, 15x25 cm, Lucky Reptile</td>
			<td>Import Export Peter Hoch GmbH, Waldkirch, Germany</td>
			<td>61201</td>
			<td>mouse warming device</td>
		</tr>
	</tbody>
</table>

induction of nephrotic syndrome in mice by retrobulbar injection of doxorubicin and prevention of volume retention by sustained release aprotinin

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 &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

After the induction of isoflurane narcosis, doxorubicin was rapidly injected via the left retrobulbar sinus. The entire volume of 7.25 &#181;L/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...

Watch this Scientific Journal Video about Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin at JoVE.com

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

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.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...

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.

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