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>
	<li>Lee, V. W., 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=Adriamycin+nephropathy:+a+model+of+focal+segmental+glomerulosclerosis.">Adriamycin nephropathy: a model of focal segmental glomerulosclerosis.</a> Nephrology (Carlton). 16 (1), 30-38 (2011).</li><li>Frenk, S., Antonowicz, I., Craig, J. M., Metcoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+nephrotic+syndrome+induced+in+rats+by+aminonucleoside;+renal+lesions+and+body+electrolyte+composition.">Experimental nephrotic syndrome induced in rats by aminonucleoside; renal lesions and body electrolyte composition.</a> Proc Soc Exp Biol Med. 89 (3), 424-427 (1955).</li><li>Ichikawa, I., 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=Role+for+intrarenal+mechanisms+in+the+impaired+salt+excretion+of+experimental+nephrotic+syndrome.">Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.</a> J Clin Invest. 71, (1983).</li><li>Deschenes, G., Wittner, M., Stefano, A., Jounier, S., Doucet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+duct+is+a+site+of+sodium+retention+in+PAN+nephrosis:+a+rationale+for+amiloride+therapy.">Collecting duct is a site of sodium retention in PAN nephrosis: a rationale for amiloride therapy.</a> J Am Soc Nephrol. 12 (3), 598-601 (2001).</li><li>Lourdel, 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=Hyperaldosteronemia+and+activation+of+the+epithelial+sodium+channel+are+not+required+for+sodium+retention+in+puromycin-induced+nephrosis.">Hyperaldosteronemia and activation of the epithelial sodium channel are not required for sodium retention in puromycin-induced nephrosis.</a> J Am Soc Nephrol. 16 (12), 3642-3650 (2005).</li><li>Seigneux, S., Kim, S. W., Hemmingsen, S. 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 ...

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