Name: A Mouse Model to Evaluate the Long-Term Structural and Functional Outcomes after the Reversal of Prolonged Unilateral Ureteric Obstruction (Video) | JoVE Journal
Uploaded: 2025-07-18T14:30:00.000+00:00
Duration: 5 min 34 s
Description: Learn a reversible unilateral ureteric obstruction protocol in mice to evaluate long-term kidney function and structural recovery after urinary obstruction reversal. This method simplifies surgical procedures for broader research accessibility.

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5 minutes

This research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) under Award Numbers RO1DK128823 and UC2DK126122.

Here, the four-step protocol used for long-term studies is described (Figure 1). As discussed at the end of this manuscript, researchers may choose only to perform three of these (sections 1, 2, and 4) or all four if interested in studying long-term outcomes. All mouse experiments in this manuscript have been approved by the Vanderbilt Institutional Animal Care and Use Committee and adhere to the animal use guidelines.
1. Placement of the ureteric clamp (10 - 15 min/mouse)
NOTE: Rely on the knowledge of mouse kidney anatomy to locate the ureter since it is not possible to see it directly without the surrounding connective tissue. The ureter runs along the lower posterior aspect of the hilar tissue as the kidney is exposed (Figure 2). The choice of vascular clamp is important. These can be expensive but can be cleaned, autoclaved, and reused multiple times. These clamps can be reused at least 100 times without issues.
<ol>
	<li>Autoclave all surgical instruments before surgery at 121 &#176;C for 30 min. If performing surgeries on multiple mice, remove blood and debris from instruments between use and depending on the institutional policy are either sterilized for 10-15 s using the hot bead sterilizer, or autoclaved separately for each mouse. Allow the instruments to cool before using them again.</li>
	<li>Weigh the mouse.</li>
	<li>Anesthetize the mouse using 5-10 mg/kg Xylazine with 90-120 mg/kg Ketamine by intraperitoneal injection (see Table of Materials). This usually takes 3-5 min to take effect.</li>
	<li>Depending on institutional policies, pre-operative analgesia is given for all mice, or only when using a different anesthetic agent, such as isoflurane, since unlike Xylazine, this does not have analgesic effects.</li>
	<li>Shave the surgical site from the hips to the rib cage and around the side, with enough border to keep the hair from contaminating the incision site. Perform in a preparatory area, not where the surgery is performed.</li>
	<li>Cover the heated surgical field with a sterile surgical drape. It will help prevent stray hairs from entering the surgical field and provide an area to lay sterile instruments.</li>
	<li>Apply ophthalmic lubricating ointment to the eyes.</li>
	<li>Apply aseptic prep using betadine-soaked gauze: scrub from the center of the site toward the periphery 3 to 4 times, each time followed by wiping once with an alcohol swab. Remove all betadine from the surgical field prior to incision.</li>
	<li>After confirming surgical plane anesthesia, make a 1.5 cm longitudinal dorsal incision on the midline through the skin and subcutaneous layers using scissors and forceps.</li>
	<li>Make a small incision through the left flank muscle and fascia above the kidney and exteriorize the left kidney.</li>
	<li>Carefully dissect the lower pole fat and some of the connective tissue around where the ureter should be. It is not possible to see the ureter directly without the surrounding connective tissue. Then, separate the ureter with its surrounding connective tissue from the renal pedicle, which contains the renal vein and artery, so the renal pedicle is not included with the ureteric clamp (Figure 2).</li>
	<li>Use the clamp appliers to open it and place it on the ureter directly below the pelvis. Use the markings on the vascular clamp to ensure they are placed with the same pressure on the ureter for each mouse (Figure 3). Do not apply, then remove the clamp to reposition, as this can damage the ureter.</li>
	<li>Once the clamp has been placed, gently push the kidney with the clamp back into the retroperitoneal space using a saline-soaked sterile cotton swab.</li>
	<li>Close the muscle layer using an absorbable suture.</li>
	<li>Close the skin layer using skin clips.</li>
	<li>Administer sustanined release Buprenorphine 3.25 mg/kg analgesia (see&#160;Table of Materials), and administer 0.5 mL of sterile normal saline subcutaneously to hydrate the mice.</li>
	<li>Leave the mouse on the heated hard pad surgical field or transfer it to their home cage with a warmed isothermal pad under half of the cage until the mouse wakes up.</li>
	<li>Return the mouse to the animal room.</li>
	<li>Closely monitor the mouse and give additional doses of Buprenorphine for pain or discomfort for 48 to 72 h. Clinical signs of pain and distress that indicate the mice need additional analgesia include depression or other behavioral changes, abnormal appearance, or postures such as piloerection, hunched posture, lack of grooming and eating, or immobility.</li>
</ol>
2. Removal of the ureteric clamp (10 - 15 min/mouse)
<ol>
	<li>Follow clamp placement procedure steps 1.1-1.10 using the original skin and muscle incisions to begin clamp removal. Remove the wound clips before cleaning the surgical area. Shaving again is usually not necessary since hair has not regrown.</li>
	<li>Due to developed tissue adhesions, use forceps to carefully locate the clamp in the retroperitoneal space without first exteriorizing the kidney. Use the clamp appliers to gently open the clamp while using the forceps to pull the tissue downwards from around the clamp head to remove the clamp.</li>
	<li>Now, exteriorize the kidney so that the renal pelvis can be visualized to determine obstruction. The renal pelvis should be swollen, indicating hydronephrosis.</li>
	<li>Once the kidney is inspected, gently push the kidney back into the retroperitoneal space using a saline-soaked sterile cotton swab.</li>
	<li>Follow steps 1.13-1.18, as outlined above.</li>
</ol>
3. Contralateral nephrectomy (10 - 15 min/mouse)
<ol>
	<li>Shave the area if there is hair growth.</li>
	<li>Use 1-5% isoflurane/air mix for anesthesia to allow an easier recovery due to having prior anesthetic events. Remove wound clips before cleaning the surgical area. 
	NOTE: Since this surgery does not induce kidney injury, which may be mitigated with the use of inhaled anesthesia32.</li>
	<li>Administer Buprenorphine analgesia pre-operatively (see Table of Materials).</li>
	<li>Follow procedure steps 1.1-1.9 using the original skin incision to begin.</li>
	<li>Make a small incision through the right flank muscle and fascia above the kidney and exteriorize the right kidney.</li>
	<li>Hold the kidney gently with smooth, curved forceps and carefully dissect the upper and lower poles of the kidney free from surrounding tissue. 
	NOTE: Tissue from around the upper pole contains the adrenal gland, carrying its own blood supply, which can be pulled off the kidney but obviously should not be removed from the mouse (see Figure 2).</li>
	<li>After liberating the kidney from surrounding tissue, tie the 4-0 silk suture completely around the renal vessels and ureter using a double surgical knot. If left for ~30 s, the kidney goes dark. Hold the kidney with smooth, curved forceps, and remove the kidney by cutting distally to the knot with curved scissors.</li>
	<li>Gently push the remaining renal pedicle back into the retroperitoneal space using a saline-soaked sterile cotton swab.</li>
	<li>Follow steps 1.13-1.15, 1.17-18 as outlined above.</li>
</ol>
4. Tissue collection
<ol>
	<li>Depending on institutional policy, either anesthetize mice by using 5-10 mg/kg Xylazine with 120 mg/kg Ketamine by intraperitoneal injection, or by overdosing with 5% isoflurane/air mix prior to the terminal procedure.</li>
	<li>Open the abdominal cavity to expose the left kidney</li>
	<li>Inspect the kidney to see any swelling of the renal pelvis (see step 2.3).</li>
	<li>If it looks like the renal pelvis is still swollen, insert a small gauge butterfly needle connected to a syringe containing 0.5 mL of a 1 in 5000 dilution of methylene blue in phosphate-buffered saline (PBS). If the ureter is patent, the blue solution will be visible as it travels from the renal pelvis into the bladder. If not, the renal pelvis will swell with blue solution, and it will not appear in the bladder.</li>
	<li>To perfusion fix the kidneys, open the thoracic cavity and expose the heart.
	<ol>
		<li>While the heart is still beating, insert a 23-25 G butterfly needle into the left ventricle, start an infusion of normal saline, and cut the right atrium or ventricle to replace blood volume with saline.</li>
		<li>After about a minute, ensure the kidneys are white. Continue the saline infusion for another minute or swap it with an infusion of 10% formalin solution for another 1-2 min to fix the tissues.</li>
		<li>At this point, the mouse will be dead; harvest the tissues. If the tissues are collected for RNA or protein studies, do not perfusion fix them.</li>
	</ol>
	</li>
	<li>Upon removal of the kidneys from the animal (whether they have been perfusion fixed or not), use a razor blade to cut up to three 2-3 mm thick sagittal slices through the center of the kidney (Figure 4). This gives representative sections that include the renal medulla and cortex and provides enough tissue for histology, protein, or RNA assays.</li>
	<li>Go on to fix/freeze samples according to the study requirements.</li>
</ol>

<ol><li>Wu, A. K., Tran, T. C., Sorensen, M. D., Durack, J. C., Stoller, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relative+renal+function+does+not+improve+after+relieving+chronic+renal+obstruction%5BTitle%5D)+AND+%22BJU+Int%22%5BJournal%5D)">Relative renal function does not improve after relieving chronic renal obstruction.</a> BJU Int. 109 (10), 1540-1544 (2012).</li>
<li>Tseng, T. Y., Stoller, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obstructive+uropathy%5BTitle%5D)+AND+%22Clin+Geriatr+Med%22%5BJournal%5D)">Obstructive uropathy.</a> Clin Geriatr Med. 25 (3), 437-443 (2009).</li>
<li>Caddeo, G., Williams, S. T., McIntyre, C. W., Selby, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+kidney+injury+in+urology+patients%3A+incidence%2C+causes+and+outcomes%5BTitle%5D)+AND+%22Nephrourol+Mon%22%5BJournal%5D)">Acute kidney injury in urology patients: incidence, causes and outcomes.</a> Nephrourol Mon. 5 (5), 955-961 (2013).</li>
<li>Sasaki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anemia+and+long-term+renal+prognosis+in+patients+with+post-renal+acute+kidney+injury+of+nonmalignant+cause%5BTitle%5D)+AND+%22Nephron%22%5BJournal%5D)">Anemia and long-term renal prognosis in patients with post-renal acute kidney injury of nonmalignant cause.</a> Nephron. 135 (2), 129-136 (2017).</li>
<li>Berlyne, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distal+tubular+function+in+chronic+hydronephrosis%5BTitle%5D)+AND+%22Q+J+Med%22%5BJournal%5D)">Distal tubular function in chronic hydronephrosis.</a> Q J Med. 30, 339-355 (1961).</li>
<li>Puri, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+kidney+disease+induced+in+mice+by+reversible+unilateral+ureteral+obstruction+is+dependent+on+genetic+background%5BTitle%5D)+AND+%22Am+J+Physiol+Renal+Physiol%22%5BJournal%5D)">Chronic kidney disease induced in mice by reversible unilateral ureteral obstruction is dependent on genetic background.</a> Am J Physiol Renal Physiol. 298 (4), F1024-F1032 (2010).</li>
<li>Buchtler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+origin+and+functional+relevance+of+collagen+I+production+in+the+kidney%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Cellular origin and functional relevance of collagen I production in the kidney.</a> J Am Soc Nephrol. 29 (7), 1859-1873 (2018).</li>
<li>Tapmeier, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reimplantation+of+the+ureter+after+unilateral+ureteral+obstruction+provides+a+model+that+allows+functional+evaluation%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">Reimplantation of the ureter after unilateral ureteral obstruction provides a model that allows functional evaluation.</a> Kidney Int. 73 (7), 885-889 (2008).</li>
<li>Conway, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kidney+single-cell+atlas+reveals+myeloid+heterogeneity+in+progression+and+regression+of+kidney+disease%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease.</a> J Am Soc Nephrol. 31 (12), 2833-2854 (2020).</li>
<li>Connor, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+cell-enriched+miRNAs+in+kidney+injury+and+repair%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Identifying cell-enriched miRNAs in kidney injury and repair.</a> JCI Insight. 5 (24), e140399(2020).</li>
<li>Hesketh, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+murine+model+of+irreversible+and+reversible+unilateral+ureteric+obstruction%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A murine model of irreversible and reversible unilateral ureteric obstruction.</a> J Vis Exp. (94), e51234(2014).</li>
<li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interferon-gamma+improves+renal+interstitial+fibrosis+and+decreases+intrarenal+vascular+resistance+of+hydronephrosis+in+an+animal+model%5BTitle%5D)+AND+%22Urology%22%5BJournal%5D)">Interferon-gamma improves renal interstitial fibrosis and decreases intrarenal vascular resistance of hydronephrosis in an animal model.</a> Urology. 77 (3), 761-768 (2011).</li>
<li>Chaabane, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+functional+decline+and+glomerulotubular+injury+are+arrested+but+not+restored+by+release+of+unilateral+ureteral+obstruction+%28UUO%29%5BTitle%5D)+AND+%22Am+J+Physiol+Renal+Physiol%22%5BJournal%5D)">Renal functional decline and glomerulotubular injury are arrested but not restored by release of unilateral ureteral obstruction (UUO).</a> Am J Physiol Renal Physiol. 304 (4), F432-F439 (2013).</li>
<li>Cochrane, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+structural+and+functional+repair+in+a+mouse+model+of+reversal+of+ureteral+obstruction%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Renal structural and functional repair in a mouse model of reversal of ureteral obstruction.</a> J Am Soc Nephrol. 16 (12), 3623-3630 (2005).</li>
<li>Souma, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasticity+of+renal+erythropoietin-producing+cells+governs+fibrosis%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Plasticity of renal erythropoietin-producing cells governs fibrosis.</a> J Am Soc Nephrol. 24 (10), 1599-1616 (2013).</li>
<li>Flam, T., Venot, A., Bariety, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversible+hydronephrosis+in+the+rat%3A+a+new+surgical+technique+assessed+by+radioisotopic+measurements%5BTitle%5D)+AND+%22J+Urol%22%5BJournal%5D)">Reversible hydronephrosis in the rat: a new surgical technique assessed by radioisotopic measurements.</a> J Urol. 131 (4), 796-798 (1984).</li>
<li>Hammad, F. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Despite+initial+recovery+of+GFR%2C+long-term+renal+functions+deteriorate+following+short+periods+of+unilateral+ureteral+obstruction%5BTitle%5D)+AND+%22Am+J+Physiol+Renal+Physiol%22%5BJournal%5D)">Despite initial recovery of GFR, long-term renal functions deteriorate following short periods of unilateral ureteral obstruction.</a> Am J Physiol Renal Physiol. 319 (3), F523-F533 (2020).</li>
<li>Hammad, F. T., Lubbad, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+diclofenac+sodium+on+renal+function+in+reversible+unilateral+ureteric+obstruction%5BTitle%5D)+AND+%22Urol+Res%22%5BJournal%5D)">The effect of diclofenac sodium on renal function in reversible unilateral ureteric obstruction.</a> Urol Res. 39 (5), 351-356 (2011).</li>
<li>Ito, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+damage+progresses+despite+improvement+of+renal+function+after+relief+of+unilateral+ureteral+obstruction+in+adult+rats%5BTitle%5D)+AND+%22Am+J+Physiol+Renal+Physiol%22%5BJournal%5D)">Renal damage progresses despite improvement of renal function after relief of unilateral ureteral obstruction in adult rats.</a> Am J Physiol Renal Physiol. 287 (6), F1283-F1293 (2004).</li>
<li>Griffin, M. D., Bergstralhn, E. J., Larson, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+papillary+necrosis-a+sixteen-year+clinical+experience%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Renal papillary necrosis-a sixteen-year clinical experience.</a> J Am Soc Nephrol. 6 (2), 248-256 (1995).</li>
<li>Onen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Grading+of+hydronephrosis%3A+an+ongoing+challenge%5BTitle%5D)+AND+%22Front+Pediatr%22%5BJournal%5D)">Grading of hydronephrosis: an ongoing challenge.</a> Front Pediatr. 8, 458(2020).</li>
<li>Sands, J. M., Layton, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+understanding+the+urine-concentrating+mechanism%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Advances in understanding the urine-concentrating mechanism.</a> Annu Rev Physiol. 76, 387-409 (2014).</li>
<li>Nawata, C. M., Pannabecker, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+urine+concentration%3A+a+review+of+renal+medullary+architecture+and+membrane+transporters%5BTitle%5D)+AND+%22J+Comp+Physiol+B%22%5BJournal%5D)">Mammalian urine concentration: a review of renal medullary architecture and membrane transporters.</a> J Comp Physiol B. 188 (6), 899-918 (2018).</li>
<li>O'Connor, P. M., Cowley, A. W. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+pressure-natriuresis+by+renal+medullary+reactive+oxygen+species+and+nitric+oxide%5BTitle%5D)+AND+%22Curr+Hypertens+Rep%22%5BJournal%5D)">Modulation of pressure-natriuresis by renal medullary reactive oxygen species and nitric oxide.</a> Curr Hypertens Rep. 12 (2), 86-92 (2010).</li>
<li>Vanichapol, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Permanent+defects+in+renal+medullary+structure+and+function+after+reversal+of+urinary+obstruction%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Permanent defects in renal medullary structure and function after reversal of urinary obstruction.</a> JCI Insight. 10 (5), e142017(2025).</li>
<li>Scarfe, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transdermal+measurement+of+glomerular+filtration+rate+in+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Transdermal measurement of glomerular filtration rate in mice.</a> J Vis Exp. (140), e57921(2018).</li>
<li>Li, H., Dixon, E. E., Wu, H., Humphreys, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+single-cell+transcriptional+profiling+defines+shared+and+unique+epithelial+injury+responses+during+kidney+fibrosis%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">Comprehensive single-cell transcriptional profiling defines shared and unique epithelial injury responses during kidney fibrosis.</a> Cell Metab. 34 (12), 1977-1998.e9 (2022).</li>
<li>Gerhardt, L. M. S., Liu, J., Koppitch, K., Cippa, P. E., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-nuclear+transcriptomics+reveals+diversity+of+proximal+tubule+cell+states+in+a+dynamic+response+to+acute+kidney+injury%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Single-nuclear transcriptomics reveals diversity of proximal tubule cell states in a dynamic response to acute kidney injury.</a> Proc Natl Acad Sci U S A. 118 (27), e2101234(2021).</li>
<li>Gerhardt, L. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lineage+tracing+and+single-nucleus+multiomics+reveal+novel+features+of+adaptive+and+maladaptive+repair+after+acute+kidney+injury%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Lineage tracing and single-nucleus multiomics reveal novel features of adaptive and maladaptive repair after acute kidney injury.</a> J Am Soc Nephrol. 34 (4), 554-571 (2023).</li>
<li>Kirita, Y., Wu, H., Uchimura, K., Wilson, P. C., Humphreys, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+profiling+of+mouse+acute+kidney+injury+reveals+conserved+cellular+responses+to+injury%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Cell profiling of mouse acute kidney injury reveals conserved cellular responses to injury.</a> Proc Natl Acad Sci U S A. 117 (27), 15874-15883 (2020).</li>
<li>Canela, V. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+spatially+anchored+transcriptomic+atlas+of+the+human+kidney+papilla+identifies+significant+immune+injury+in+patients+with+stone+disease%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">A spatially anchored transcriptomic atlas of the human kidney papilla identifies significant immune injury in patients with stone disease.</a> Nat Commun. 14 (1), 4140(2023).</li>
<li>Lee, H. T., Ota-Setlik, A., Fu, Y., Nasr, S. H., Emala, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+protective+effects+of+volatile+anesthetics+against+renal+ischemia-reperfusion+injury+in+vivo%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">Differential protective effects of volatile anesthetics against renal ischemia-reperfusion injury in vivo.</a> Anesthesiology. 101 (6), 1313-1324 (2004).</li>
<li>Mohammadi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+the+surgical+ligation+of+the+ureter+in+different+locations+on+the+kidney+over+time+in+the+rat+model%5BTitle%5D)+AND+%22Adv+Urol%22%5BJournal%5D)">Effects of the surgical ligation of the ureter in different locations on the kidney over time in the rat model.</a> Adv Urol. 2024, 6611081(2024).</li>
<li>Imig, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salt-sensitive+hypertension+after+reversal+of+unilateral+ureteral+obstruction%5BTitle%5D)+AND+%22Biochem+Pharmacol%22%5BJournal%5D)">Salt-sensitive hypertension after reversal of unilateral ureteral obstruction.</a> Biochem Pharmacol. 210, 115438(2023).</li>
<li>Provoost, A. P., Molenaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+the+glomerular+filtration+rate+after+unilateral+nephrectomy+in+rats%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">Changes in the glomerular filtration rate after unilateral nephrectomy in rats.</a> Pflugers Arch. 385 (2), 161-165 (1980).</li>
<li>Young, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+regional+renal+blood+flow+after+unilateral+nephrectomy+using+the+techniques+of+autoradiography+and+microautoradiography%5BTitle%5D)+AND+%22J+Urol%22%5BJournal%5D)">Changes in regional renal blood flow after unilateral nephrectomy using the techniques of autoradiography and microautoradiography.</a> J Urol. 160 (3 Pt 1), 926-931 (1998).</li>
<li>Hostetter, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progression+of+renal+disease+and+renal+hypertrophy%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Progression of renal disease and renal hypertrophy.</a> Annu Rev Physiol. 57, 263-278 (1995).</li>
<li>Soranno, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matching+human+unilateral+AKI%2C+a+reverse+translational+approach+to+investigate+kidney+recovery+after+ischemia%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Matching human unilateral AKI, a reverse translational approach to investigate kidney recovery after ischemia.</a> J Am Soc Nephrol. 30 (6), 990-1005 (2019).</li>
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Compared to some of the approaches that have been used to study reversal of prolonged UUO in mice6,7,8,9,10,11,12,13,14, the protocol described here is relatively straightforward and does not require a high level of prior ...

Urinary tract obstruction (UTO) may be unilateral, which is often asymptomatic, presenting late with chronic obstructive uropathy1, or may present acutely with renal colic and/or hematuria; acute pyelonephritis or post-renal acute kidney injury (AKI)2. Key to the management of UTO is early reversal of obstruction2. However, despite effective reversal and the use of generic therapies to delay the progression of chronic kidney disease (CKD), affected patients remain at increased risk of progressive CKD and hypertension3,4. In addition, long-term urinary concentrating capacity (UCC) is often impaired5, which may predispose susceptible patients to dehydration and recurrent AKI, accelerating the progressive decline in renal function after UTO.
In part, this failure to identify therapeutic approaches to improve long-term outcomes in patients with UTO has resulted from our reliance on the use of irreversible unilateral ureteric obstruction (UUO) models to explore the pathobiology of UTO. However, these models do not replicate the clinical situation in which patients undergo reversal soon after diagnosis and cannot be used to explore the mechanisms and therapeutic approaches that could be used to improve long-term repair after the obstruction is reversed. Reversible UUO (R-UUO) models have been developed to address this, but these are more technically challenging than the irreversible UUO models, more difficult to reproduce by different laboratories, and, as a result, have not been as widely adopted by the scientific community. Mouse models are attractive since they can be used to harness the power of mouse genetics to study disease pathophysiology, but they have been particularly challenging to optimize as the methods often lead to irreversible UUO if the obstruction lasts more than 3 days6. A variety of approaches have been deployed to address this, including multi-surgery, sequential placement of clamps along the ureter every 2 days6,7; UUO followed by bladder reimplantation8,9,10,11; and use non-damaging clamps and tubing to obstruct the ureter12,13,14. Long-term outcome studies in rats and mice have shown improved renal function, reduced fibrosis, and/or glomerular injury after reversal of obstruction, but only partial recovery if UUO lasts longer than 2 days6,7,8,9,12,14,15,16,17,18,19. In addition, while the renal medulla (RM) is particularly susceptible to damage caused by UTO20,21 until recently, there have been no long-term rodent studies in which the effects of prolonged R-UUO on RM structure and function have been studied. This is significant since UCC and the ability to excrete salt loads without increasing blood pressure (the so-called pressure natriuresis response) are dependent on maintaining anatomic integrity of the RM22,23,24, so that incomplete repair of the RM after reversal of UTO may explain why patients have increased susceptibility to recurrent AKI and hypertension after reversal of UTO. However, many of the techniques required to evaluate this require multiple rounds of surgery (five major surgeries for 6 days of obstruction6,7), require surgical expertise that is difficult to teach (e.g., surgical reimplantation of the ureter into the bladder8,9,10,11), and because obstruction is unilateral, make it challenging for investigators to evaluate changes in renal function after reversal of the obstruction.
To address this, we have developed a mouse model of R-UUO that is relatively simple to perform and teach, requires a more limited number of surgeries than other techniques (three&#160;surgeries for 5 to 7 days of obstruction), and allows for the analysis of renal functional recovery without the need for invasive and/or expensive split renal function studies25. For this, mice undergo three major surgeries: 1) placement of a non-traumatic vascular clamp on the proximal left ureter; 2) removal of the vascular clamp 5 to 7 days later, depending on the mouse strain; and 3) removal of the contralateral kidney 10 days later to evaluate renal function. Approximately 20 to 40% of mice die within 2-3 days of the nephrectomy, indicating that the UUO does not reverse in all mice. Long-term follow-up of the surviving mice shows that despite the near complete histological recovery of the renal medulla (RM) 3 months after R-UUO. There is reduced renal function, measured by transdermal glomerular filtration rate (tGFR)26. In addition, there is a marked reduction in UCC, determined by measuring urinary osmolality after water restriction, suggesting there is a permanent defect in RM function. Time course studies show that while there is improvement in tGFR between days 28 and 56 post-RUUO, this stabilizes between days 56 and 84. In contrast, there is no improvement in UCC between days 28 and 84 after R-UUO25. Using scRNA-Seq of isolated RMs, validated by cell lineage and immunohistochemistry studies, we identified strong regenerative responses that restored RM dimensions after R-UUO but that all of the major cellular compartments in the RM showed permanent changes in cell numbers and gene signatures that are likely to impact functional recovery after R-UUO. This included persistent proinflammatory responses in the RM collecting duct and loop of Henle cell populations 84 days after R-UUO, similar to the inflammatory and senescence signatures of failed repair proximal tubular epithelial cells after AKI and UUO27,28,29,30. Similar changes were also seen in RM collecting ducts from patients with recurrent renal stone disease31, suggesting there is a common injury response to RM damage in both humans and mice. In this article, we provide detailed instructions on how we perform these surgeries, how we optimize conditions for its use in different mouse strains, how we determine whether the ureter is still obstructed after reversal, how we evaluate key functional outcomes, and how we harvest renal tissues to assess the renal medullary structures.

<table><tbody><tr><td>0.9% Sodium Chloride</td><td>VUMC pharmacy</td><td>multiple</td><td></td></tr><tr><td>Absorbable suture Vicryl 6-0</td><td>Med Vet International- ETHICON</td><td>J492G</td><td></td></tr><tr><td>Alcohol Swabs</td><td>BD Brand Isopropyl Alcohol Swabs</td><td>BD 326895</td><td></td></tr><tr><td>Betadine solution</td><td>Hanna Pharmaceutical Supply Co., Inc</td><td>67618015017</td><td></td></tr><tr><td>Clamp applying forceps: S&amp;amp;T CAF-4</td><td>Fine Science Tools</td><td>00071-14</td><td></td></tr><tr><td>Deltaphase Isothermal Pad</td><td>Braintree Scientific</td><td>DPIP</td><td></td></tr><tr><td>Electric trimmer</td><td>Oster</td><td>multiple</td><td></td></tr><tr><td>ENCORE Latex Textured Surgical Gloves</td><td>Ansell</td><td>5785003</td><td></td></tr><tr><td>Ethiqa XR Buprofenor (CIII) VI 1.3 mg/mL</td><td>Patterson Veterinary- Ethiqa</td><td>86084010030</td><td></td></tr><tr><td>EZ Clip 9 mm</td><td>Braintree Scientific</td><td>EZC-CS</td><td>or 7 mm</td></tr><tr><td>EZ Clip applier 9 mm&amp;nbsp;</td><td>Braintree Scientific</td><td>EZC-APL</td><td>or 7 mm</td></tr><tr><td>EZ Clip remover 9 mm&amp;nbsp;</td><td>Braintree Scientific</td><td>EZC-RMV</td><td>or 7 mm</td></tr><tr><td>Gauze sponges 2 &amp;acute; 2</td><td>Fisher Scientific</td><td>22-362178</td><td></td></tr><tr><td>Graefe angled or curved serrated tip</td><td>Fine Science Tools</td><td>11049-10</td><td></td></tr><tr><td>Graefe straight serrated tip</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Heated hard pad 15 &amp;acute; 24</td><td>Braintree Scientific</td><td>HHP 2</td><td></td></tr><tr><td>Heated hard pad pump connectors</td><td>Braintree Scientific</td><td>HHP Gaymar connector</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Insulin Syringe 0.5 mL 29 G</td><td>Exel International</td><td>26028</td><td></td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Patterson Veterinary- Pivetal</td><td>78949580</td><td></td></tr><tr><td>Ketamine 100 mg/mL</td><td>VUMC pharmacy</td><td></td><td></td></tr><tr><td>Non absorable suture Silk 4-0</td><td>Med Vet International- ETHICON</td><td>683G</td><td></td></tr><tr><td>Olsen Hegar needle holder: with suture cutter</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>Optixcare Eye Lube 20 g</td><td>Med Vet International</td><td>PHOPTIXCARE</td><td></td></tr><tr><td>Scissors-Blunt Lexer-Baby</td><td>Fine Science Tools</td><td>14078-10</td><td></td></tr><tr><td>Scissors-Sharp curved</td><td>Fine Science Tools</td><td>14005-12</td><td></td></tr><tr><td>Sterile cotton tipped applicators</td><td>Puritan</td><td>25806 2WC</td><td></td></tr><tr><td>Sterile gauze sponges 4 &amp;acute; 4</td><td>Fisher Scientific</td><td>22-415-469</td><td></td></tr><tr><td>Sterile surgical disposable drape 18 &amp;acute; 26</td><td>Med Vet International</td><td>20001</td><td></td></tr><tr><td>Sterilization Instrument Pouch 5 &amp;acute; 10</td><td>Fisher Scientific</td><td>01-812-54</td><td></td></tr><tr><td>T/pump Heating Cooling Pump</td><td>Braintree Scientific</td><td>TP-700</td><td></td></tr><tr><td>Taylor forceps curved, smooth</td><td>Fine Science Tools</td><td>11017-17</td><td></td></tr><tr><td>Vascular clamp: S&amp;amp;T 5 to 15 g press</td><td>Fine Science Tools</td><td>00396-01</td><td></td></tr><tr><td>Xylazine 100 mg/mL</td><td>Patterson Veterinary- Cronus</td><td>CR04305</td><td></td></tr></tbody></table>

a mouse model to evaluate the long-term structural and functional outcomes after the reversal of prolonged unilateral ureteric obstruction

A Mouse Model to Evaluate the Long-Term Structural and Functional Outcomes after the Reversal of Prolonged Unilateral Ureteric Obstruction

Clinical management of patients with urinary tract obstruction (UTO) requires early intervention to reverse the cause of the obstruction, but despite this, patients with prolonged UTO are at increased risk of chronic kidney disease (CKD) and recurrent acute kidney injury (AKI). However, other than an early reversal of obstruction and generic therapy to delay CKD progression, no therapies have significantly improved long-term renal outcomes after the reversal of UTO. To address this, a number of laboratories have developed models of reversible UUO (R-UUO), but these are technically challenging and have not been widely adopted. In addition, while mouse models of R-UUO are attractive as they can be used to harness the power of mouse genetics to study disease pathophysiology, these have been particularly challenging as methods used often lead to irreversible UUO if the obstruction lasts &#62;3 days. In addition, because of the nature of these models, few studies have evaluated the long-term functional outcomes of reversing ureteric obstruction. To address this, we recently developed a mouse model of R-UUO with delayed contralateral nephrectomy that allows for the analysis of the long-term effects of reversing prolonged UTO on long-term renal structural and functional outcomes. These studies show that despite near complete histological recovery 3 months after reversal, there was a permanent reduction in renal function and a marked and persistent defect in urinary concentrating capacity, indicative of a defect in renal medullary function. The model requires three major survival surgeries but results in a robust and reproducible long-term reduction in renal function that can be reproduced in different mouse strains by adjusting the period of obstruction. In this article, we provide detailed instructions for performing these surgeries, optimizing conditions for use in different mouse strains, evaluating renal functional outcomes, and harvesting renal tissues.

For long-term studies, we generally evaluate renal function by measuring sequential blood urea nitrogen (BUN) and transdermal Glomerular Filtration Rates (tGFR) in conscious mice (refer to a previously published article describing this26), in addition to measuring urinary concentrating capacity (UCC) by evaluating urinary osmolarity from spot urine samples collected after 18 h water deprivation. While we always perform BUN studies, these are most useful to assess the severity of injury at early ti...

This paper provides detailed instructions to enable investigators to perform reversible unilateral ureteric obstruction in mice using a standardized model that allows analysis of the long-term structural and functional outcomes after the reversal of prolonged ureteric obstruction.

Clinical management of patients with urinary tract obstruction (UTO) requires early intervention to reverse the cause of the obstruction, but despite ...

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This video demonstrates the procedure to generate a unilateral ureteral obstruction mouse model. The procedure involves cutting one of the ureters of the mouse to obstruct urine flow.

Unilateral Ureteral Obstruction Murine Model: A Surgical Procedure to Obstruct the Urinary Flow from a Kidney in Mouse Models

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A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

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Learn a reversible unilateral ureteric obstruction protocol in mice to evaluate long-term kidney function and structural recovery after urinary obstruction reversal. This method simplifies surgical procedures for broader research accessibility.

A Mouse Model to Evaluate the Long-Term Structural and Functional Outcomes after the Reversal of Prolonged Unilateral Ureteric Obstruction (Video) | JoVE Journal

A Mouse Model to Evaluate the Long-Term Structural and Functional Outcomes after the Reversal of Prolonged Unilateral Ureteric Obstruction

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