Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Summary

Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

Abstract

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction defects can be assessed by tracking methylene blue dye flow within the urinary system. Methylene blue dye is injected into the renal pelvis of perinatal embryonic kidneys and dye flow is monitored from the renal pelvis of the kidney through the ureter and into the bladder lumen after applying hydrostatic pressure. Dye accumulation will be evident in the bladder lumen of the normal perinatal urinary tract, but will be constrained between the renal pelvis and the end point of an abnormal ureter, if urinary tract obstructions occur. This method facilitates the confirmation of urinary tract junction obstructions and visualization of hydronephrosis and hydroureter. This manuscript describes a protocol for methylene blue dye injection into the renal pelvis to confirm urinary tract junction obstructions.

Introduction

The urinary system consists of a pair of kidneys and ureters and a common bladder and urethra. The main function of the urinary system is to maintain body homeostasis by managing the water and electrolyte balance of the blood. The kidneys filter the blood to control electrolyte concentrations and acid-base balance,and produce urine to excrete excess water and waste including solutes and metabolites. Urine is then transported through the ureter from the renal pelvis of the kidney to the bladderin a unidirectional manner where it is stored and ultimately eliminated via the urethra¹.

The ureters are straight tubes originating from the nephric duct. After budding from the nephric duct at embryonic day 10.5 (E10.5) in the mouse, the ureter stalk elongates and differentiates into a multilayered structure called urothelium which is impermeable between mouse E12.5 and E16.5. The mesenchymal cells surrounding the ureter stalk are also differentiated into three layers consisting of inner stromal cells, intermediate thick smooth muscle cells, and outer adventitial fibroblasts. Ureteral peristaltic waves initiating in the renal pelvis are propagated through the smooth muscle layer of the ureter wall to the bladder to transport urine^2,3, which is produced starting at E16.5 in the mouse¹.

Congenital anomalies of the kidney and urinary tract (CAKUT) are among the most frequent genetic diseases, present in around 1% of human fetuses^1,4, and composed of a variety of phenotypes including hydronephrosis and hydroureter. The abnormal accumulation of urine in the kidney and ureter results in hydronephrotic kidney and hydroureter formation. One cause of hydronephrosis and/or hydroureter formation is an obstruction of the urinary tract. Ureteropelvic junction obstruction (UPJO) is caused by aberrant urine flow due to a blockage between the proximal ureter and the renal pelvis, resulting in hydronephrosis and proximal hydroureter narrowing with angulation or persistent folding^5,6. In addition, the ectopic insertion of the distal ureter into either the bladder wall or the reproductive tract is called ureterovesical junction obstruction (UVJO). UVJO can also induce hydronephrosis and hydromegaureter formation^7,8. An additional ureterovesical junction (UVJ) defect is vesicoureteric reflux (VUR). VUR is characterized by retrograde urine flow from the bladder toward the kidney at UVJ. Compared to UVJO, perinatal embryos with VUR do not distinctly show a distended hydronephrotic kidney or severe hydroureter phenotype⁹.

In the laboratory mouse, urine flow can be examined by injection of a dye, such as methylene blue, into the renal pelvis⁹. The injectedmethylene blue solution will trace the urine trajectory from the renal pelvis through the ureter and into the bladder. Hydronephrosis can be recognized by an expansion of the dye in the kidney. UPJO can be detected as a blockage of dye flow at the proximal ureter with distended renal pelvis⁵. Any dilation of the ureter indicated by a widened diameter demonstrates an example of hydroureter. Finally, dye accumulation at the bladder wall or at the site of the reproductive tract indicates UVJO with distended hydronephrotic kidney and dilated hydromegaureter^7,10. To detect VUR, the dye solution is injected into the bladder and subsequent retrograde flow is monitored in the kidney⁹.

Here, a protocol for methylene blue dye injection into the renal pelvis of a perinatal embryo is presented. This protocol allows the tracing of urine flow from the renal pelvis through the ureter and into the bladder and verifies potential urinary tract junction obstructions such as UPJO or UVJO.

Protocol

Mice (Wnt5a flox/flox mice (Wnt5a^tm1.1Tpy) and Dll1Cre line, UVJO mouse model)⁷ were managed according to NIH guidelines for the care and use of laboratory animals and studied under a protocol approved by the NCI-Frederick Animal Care and Use Committee.

1. Preparation of Methylene Blue Dye Solution

1. Measure 0.1 g methylene blue powder.
2. Dissolve the methylene blue in 10 mL normal saline or 1x phosphate buffered saline (PBS) completely by vortexing.
3. Filter the 10 mg/mL methylene blue solution with a syringe filter (membrane pore size 0.45µm) to eliminate clogging during injection.
4. Assemble a sterile scalp vein set (27GX3/4″) with a 3 mL disposable syringe and fill the syringe with 3 mL filtered methylene blue solution.
   NOTE: To prevent hydrostatic pressure and subsequent dye flow, place the needle tip above the syringe filled with methylene blue solution.

2. Dissection of Prenatal Embryos

1. Clean dissecting scissors and forceps with 70% ethanol.
2. To collect perinatal embryos at E18.5 or E19.5, euthanize pregnant mice first using CO₂ inhalation and then perform cervical dislocation according to NIH guidelines.
   NOTE: The pregnant female mouse usually gives birth starting at E18.5, however, larger kidneys are easier to manipulate. Therefore, kidneys at E19.5 closer to birth are easier to perform this analysis on. However, pups with bilateral urinary tract obstructions die after birth. Depending on the characteristics of experimental mice, an appropriate collection day/time should be empirically determined.
3. Spray 70% ethanol on the ventral abdominal surface and then open the abdominal cavity ventrally using dissecting scissors and forceps.
4. Lift the entire uterus and separate it from the body by cutting with dissecting scissors at the tips of the uterine horns.
5. Rinse the entire uterus with 1x PBS in a petri dish.
6. Cut the uterus segmentally with dissecting scissors and remove placental decidua with dissecting forceps to expose the embryos in yolk sacs.
7. Remove the yolk sac first and then remove the amniotic membrane with dissecting forceps to liberate the perinatal embryos.
8. Decapitate an embryo with dissecting scissors. Wipe off excess blood with sterile gauze pads. Pin the embryo down with its ventral surface up on a dissecting microscope equipped with a digital camera for imaging. Collect its tail for genotyping if necessary.
   NOTE: Perform injections one embryo at a time.
9. Carefully open the abdominal cavity of an embryo with forceps by tearing the skin. Then, carefully remove excess organs and tissues such as liver, stomach, and intestine with forceps by cutting them or pulling them out to expose the kidneys, the ureters, and the bladder that are located dorsally (Figure 1A). Absorb excess blood from the dissected embryo with sterile gauze pads if necessary.
   NOTE: Excess blood interferes with identification of the renal pelvis for the dye injection.

3. Injection of Methylene Blue Dye into the Renal Pelvis and Monitoring Dye Flow

1. Remove bubbles in the needle and tubing by expelling methylene blue solution from the needle tip by hydrostatic pressure. Lift the syringe containing the dye solution above the level of the needle tip to start flow, and then lower the syringe to stop flow.
2. Insert the needle into the renal pelvis near the proximal ureter, taking care not to disturb it once placed. Perform dye injection into a kidney to determine its urinary tract obstruction.
   NOTE: Perform dye injection into any abnormal kidney⁷ first by following the same way to determine its urinary tract obstruction.
3. Lift the syringe up about 20 cm to provide hydrostatic pressure and let 15 µL - 60 µL of the dye solution flow. The flow rate will be about 3 µL/s if the syringe is raised to this height above the embryo.
4. Monitor the blue color of the dye starting first in the renal pelvis, then in the length of the ureter, and finally in the bladder lumen.
   NOTE: It takes about 5 s to see a weak dye color emerge within the bladder lumen if the ureter is properly inserted into the bladder. Allow the dye to accumulate at the blocked site about 15 s if no dye color appears in the bladder lumen.
5. Place the syringe down to stop dye flow and remove the needle from the kidney.
6. Take images of the kidney, the ureter, and the bladder traced with dye solution using the camera and imaging program connected to a stereomicroscope.
7. Make a record of hydronephrosis of the kidney, hydroureter, and the final position of the dye solution in a lab notebook.
8. Perform injection of the contralateral kidney as described in sections 3.1) to 3.5) and allow dye flow about 20 s in total.
   NOTE: The bladder lumen will be filled with dye solution, indicated by a strong blue color, if the ureter is properly inserted into the bladder lumen. After assessment of urinary tract junction obstruction, the ureters and the bladder can be fixed for sectioning.

Results

The kidneys and lower urinary system are located dorsal to most other internal organs such as the liver and intestine. After removing these other internal organs, a pair of kidneys and ureters and a single bladder are visible (Figure 1A). Upon successful dissection, the dye injected into the renal pelvis will flow from the kidney into the bladder via the ureter. The renal pelvis is the funnel-like dilated proximal part of the ureter in the kidney. Therefore, ...

Discussion

Mouse kidneys are functional beginning at E16.5 and a dye injection test is theoretically possible from this time point. However, the kidney is too small to be injected with the dye solution and phenotypes such as hydronephrosis and hydroureter are not clearly observed since these phenotypes are a secondary effect of urine built up due to abnormal urine transport. These phenotypes, from the obstructions such as UPJO or UVJO, are evident at E18.5 by distended kidneys and ureters. Larger kidneys of embryos at E19.5 or neon...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Methylene blue	Sigma-Aldrich	M9140
Quality Biological Inc. NORMAL SALINE - 500ML	Fisherscientific	50-983-204
3 mL disposable syringe with BD Luer-Lok tip	BD	309657
Acrodisc Syringe Filters with Supor Membrane	Pall corporation	4614
Exel Scalp Vein (Butterfly) Sets; 27G x 3/4" 12"	EXELINT	26709
Delicate Operating Scissors	Roboz Surgical Instrument Co.	RS-6702
Micro Dissecting Forceps	Roboz Surgical Instrument Co.	RS-5135
Dumont Tweezers; Pattern #5	Roboz Surgical Instrument Co.	RS-5045
BD PrecisionGlide Needles 30 G x 1/2"	BD	305106