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

Summary

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.

Abstract

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 >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.

Introduction

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 hypertension^3,4. In addition, long-....

Protocol

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....

Results

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.......

Discussion

Compared to some of the approaches that have been used to study reversal of prolonged UUO in mice^{6,7,8,9,10,11,12,13,14}, the protocol described here is relatively straightforward and does not require a high level of prior .......

Materials

List of materials used in this article

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