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

Summary

This article presents a step-by-step protocol for the isolation and functional evaluation of human renal arterial branches, facilitating preclinical studies for pharmaceutical development.

Abstract

Renal vascular dysfunction plays a critical role in the pathogenesis of multiple clinical conditions, including acute kidney injury, renal ischemia, and hypertension, presenting significant challenges in clinical management and adversely affecting patient outcomes. The isolation and functional characterization of intrarenal arteries are crucial for elucidating the mechanisms underlying renal vascular dysfunction, particularly related to kidney injury, and guiding targeted therapeutic development. Despite its clinical importance, standardized approaches for isolating and functionally assessing human intrarenal arteries across different branching levels remain underdeveloped. This protocol provides a comprehensive framework for the systematic isolation and multimodal evaluation of intrarenal arterial branches, incorporating functional and structural assessments under both physiological and pathological conditions. The methodology encompasses three key components: (1) precise anatomical identification and microdissection of intrarenal arteries from donor kidneys, accompanied by Hematoxylin-Eosin (H&E) staining for structural confirmation; (2) rigorous normalization procedures in wire myography to enhance measurement reproducibility and reliability; and (3) quantitative analysis of vasomotor responses using precision wire myography techniques. Normalization is based on the muscle length-tension relationship, where incremental stretching of arterial segments establishes an optimal resting tension to maximize actin-myosin overlap, thereby eliciting peak contractile responses. In wire myography, isolated vessel segments are suspended between two parallel wires, allowing precise measurement of vascular tension. By applying rigorous normalization protocols, this technique enables reproducible and reliable quantification of vascular reactivity across diverse pathophysiological conditions and pharmacological interventions.

Introduction

The kidney is a vital organ responsible for maintaining human homeostasis through clearance of metabolic wastes, electrolyte balance, and fluid regulation - functions that demand exceptionally high blood flow. Under physiological conditions, the kidneys receive approximately 25% of the total blood volume pumped by the heart each minute (cardiac output), underscoring their substantial role in systemic blood flow and the critical importance of renal perfusion in maintaining overall physiological balance¹. The renal arterial system precisely regulates this massive blood delivery to maintain GFR while adapting to systemic hemodynamic changes. ....

Protocol

The protocol and examples described here were reviewed and approved by the Urology Department of Peking University First Hospital (approval No. 2023yan500-002) and conducted in accordance with the Helsinki Declaration. All participants provided written informed consent prior to participation.

1. Solution preparation

1. Prepare Krebs-Ringer solution (Krebs) containing 119.0 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.0 mM MgCl₂, 25.0 mM NaHCO₃, 1.2 mM KH₂PO₄, and 11.0 mM D-glucose at pH 7.4.
2. Prepare high potassium salt solution (60K⁺) containing 64....

Results

In the human kidney, the interlobar artery runs between renal pyramids alongside its corresponding vein. Characterized by a relatively thick vascular wall and significant adipose tissue encasement, this artery requires particularly careful dissection. The surrounding adipose tissue should be removed meticulously to avoid application of excessive force during the isolation procedure (Figure 1C).

The arcuate artery is anatomically positioned at the corticomedullary .......

Discussion

Renal perfusion stability serves as both a critical therapeutic target and a window into hemodynamic pathophysiology^39,40. We present a protocol for hierarchical isolation and functional assessment of human renal arteries from fresh nephrectomy specimens, addressing significant gaps in current methodologies. Unlike existing approaches limited to cadaveric specimens or animal models, our technique enables reliable isolation of viable intrarenal arteries (interloba.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetylcholine chloride	Sigma-Aldrich, Merck, Darmstadt, Germany	A6625
Black bottomed culture dish	Danish Myo Technology, Aarhus, Denmark	300412
CaCl2	Sangon Biotech Co.,Ltd.,Shanghai, China	A501330
D-glucose	Sangon Biotech Co.,Ltd.,Shanghai, China	A610219
Dumont forceps	Danish Myo Technology, Aarhus, Denmark	300413
Geuder dissection scissor	Danish Myo Technology, Aarhus, Denmark	400431
Guide wire	Danish Myo Technology, Aarhus, Denmark	400447	Diameter 40 µm
KCl	Sangon Biotech Co.,Ltd.,Shanghai, China	A100395
KH2PO4	Sangon Biotech Co.,Ltd.,Shanghai, China	A100781
LabChart Professional version 8.3	ADInstruments, Australia	-
MgCl2·6H2O	Sangon Biotech Co.,Ltd.,Shanghai, China	A100288
Multi myograph system	Danish Myo Technology, Aarhus, Denmark	620M
NaCl	Sangon Biotech Co.,Ltd.,Shanghai, China	A100241
NaHCO3	Sangon Biotech Co.,Ltd.,Shanghai, China	A100865
Phenylephrine	Sigma-Aldrich, Merck, Darmstadt, Germany	P6126