Multilevel Microdissection and Functional-Structural Profiling of Human Renal Arterial Branches

Our research aims to establish standardized methods to isolate and functionally assess human renal arterial branches, uncovering mechanisms of vascular dysfunction and guiding targeted therapies. Previous research relied on animal models or indirect imaging, where our wire myography directly mirrors human renal artery function in vitro. Our method enables precise, human-specific vascular analysis, overcoming species limitations and improving translational drug development.

To begin, ensure the kidney tissue remains fully submerged in liquid during transport to maintain tissue viability and structural integrity. Visually identify the coronal plane by locating the renal hilum and aligning the cut to pass through both the renal pelvis and the lateral convex border. Using a sterile scalpel, bisect the kidney along this coronal plane to create two symmetrical halves, and expose internal structures, such as the renal pyramids and columns.

Under a stereo microscope, use microdissection scissors and forceps to meticulously separate the interlobar, arcuate, and interlobular arteries in a 10-centimeter black-bottomed culture dish. Then, gently remove any surrounding tissue and fat from the dissected arteries in the same 10-centimeter black-bottomed culture dish to clean them thoroughly. Using microdissection scissors, section the cleaned arteries into rings approximately two millimeters in length for vascular function studies.

Carefully insert the first guide wire into an arterial ring in the dish. Bend one side of the guide wire at a 90-degree angle, and transfer the arterial ring with the wire into the chamber. Before fixing the arterial ring on the sample holder, record the vessel length.

Place the ring between the two holders, and read the micrometer scale, where one scale division equals 10 micrometers. Subtract the initial scale value measured when the holders just touch each other to calculate the arterial ring's length and width. Then, fix the arterial ring onto the clam-type sample holder using the instrument provided screws, tightening them in a clockwise direction.

Then, thread a second guide wire through the arterial ring. Wind the wire clockwise around the fixing screws on both ends, securing it tightly to the surface of the sample holder. Select Normalization Settings from the DMT menu, and set the Eyepiece calibration as one millimeter per division, Target pressure as 13.3 kilopascal, IC1/IC100 as 0.9, Online averaging time as three seconds, and Delay time as 60 seconds.

Select the channel corresponding to the target artery, and open the Normalization screen from the DMT menu. In the appropriate fields, enter the Tissue end points as a1 equals zero and a2 equals the measured vessel length in millimeters. Input the wire diameter as 40 micrometers, and enter the Micrometer reading from the scale.

Click Add Point to save the data. Now apply passive stretch and wait for three minutes. Enter the new Micrometer reading as the next point and click Add Point.

Add five milliliters of 60 potassium ion solution to the chamber to induce a potassium-mediated contraction of the vessel. To wash out the 60 potassium ion solution, add five milliliters of Krebs solution three times. To evaluate phenylephrine-induced, concentration-dependent contraction, apply cumulative phenylephrine in half-log increments from 10 to the power of negative nine to 10 to the power of negative four molar.

For additional drug testing, wash the chamber with warm five milliliters of Krebs solution at least five times until tension returns to baseline and remains stable for at least 10 minutes. The interlobar artery was successfully dissected from the renal medulla, showing a thick vascular wall and surrounding adipose tissue that required careful removal to preserve structural integrity. The arcuate artery was isolated at the corticomedullary junction, displaying a thinner vascular wall and an arched trajectory.

The interlobular artery was identified running linearly through the renal cortex with a very thin wall and tightly integrated with cortical tissue. Histological analysis revealed that the interlobar artery had the thickest vascular wall with a distinct adventitia, while arcuate and interlobular arteries exhibited progressively thinner walls and fewer smooth muscle layers. Arterial normalization involved repeated mechanical stretching and potassium-induced stimulation, establishing stable baseline tension conditions across samples.

Cumulative addition of phenylephrine from 10 to the power of negative nine to 10 to the power of negative four molar concentrations induced progressively stronger contractions in arterial rings in a dose-dependent manner. In phenylephrine pre-contracted arteries, increasing concentrations of acetylcholine from 10 to the power of negative eight to three times 10 to the power of negative five molar produced concentration-dependent vasodilation.