A Renal Denervation Approach to Prevent Inflammation and Fibrogenesis in Chronic Kidney Disease

Summary

Described here is a protocol for renal denervation that is used to define the role of renal nerve-derived signaling in persistent renal tubular injury, inflammation, and fibrogenesis. It is focused on sympathetic nerve-mediated signaling.

Abstract

Chronic kidney disease (CKD) is affecting increased numbers of people across the world, and there remains no effective treatment strategy. Sympathetic nerve activation has been recognized as an important factor in the development and progression of cardiovascular disease, hypertension, and CKD. Catheter-guided renal denervation is useful to control blood pressure (BP) in patients with resistant hypertension and CKD. Sympathetic nerve-derived norepinephrine (NE) has been implicated in tissue homeostasis and the progression of various diseases, including CKD. The molecular mechanisms and signaling pathways triggered by sympathetic nerve activation, which drive renal inflammation and fibrogenesis in CKD progression, remain undefined. Presented here is the detailed methodology for renal denervation (RDNx) in experimental models of CKD. The results show that this method effectively ablates the renal nerve, as evidenced by the loss of tyrosine hydroxylase immunoreactivity and levels of kidney NE. This results in the suppression of renal tubular injury, inflammation, and fibrogenesis in CKD models. Competence of surgeons performing surgical procedures to denervate the kidney is a requirement to achieve consistent results. RDNx can be utilized to study the roles of renal nerve, nerve-derived neurotransmitters, and factors, as well as unveil their downstream signaling pathways. Defining the molecular mechanisms and underlying functions will lead to the design of novel therapeutic interventions for CKD, regardless of its etiology (e.g., diabetes, hypertension, and cardiovascular complications).

Introduction

CKD, characterized by tubular injury, persistent renal inflammation, and fibrosis, ultimately leads to end stage kidney disease (ESKD)^1,2,3. Sympathetic nervous system governs both normal and pathological functions of diverse organ systems, including those in the kidney⁴. One type of catecholamine, norepinephrine (NE or noradrenaline), originates from sympathetic neurons and is an effector of the sympathetic nervous system⁵. In both patients and experimental models, increased sympathetic nerve activity and tissue levels of NE ar....

Protocol

Mice were cared for prior to and during the experiment in accordance with the policies of the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska Medical Center (UNMC), and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All portions of the protocol received prior approval from the UNMC-IACUC.

1. Renal denervation

1. Use male (129S1/SvImJ) mice (8–10 weeks old) from the Jackson Laboratories.......

Discussion

This protocol describes the detailed methods for renal nerve ablation in a mouse model.  Further, the pivotal role of the renal nerve in triggering inflammatory and fibrotic responses to injury in CKD models is demonstrated. Complete separation of renal artery from connective tissue and veins is a critical step for successful RDNx that allows full exposure of renal nerves and complete nerve ablation. Since there is an overlap of the renal artery over the vein, the portion overlapped is not well-exposed to the alcoho.......

Materials

Name	Company	Catalog Number	Comments
129S1/SvlmJ	Jax Lab	Stock #000090
0.1% SDS	BioRad	1610416
0.5% acetic acid (glacial)	Fisher Chemical	BP1185	Sirius Red Stain
1mM EDTA	Sigma	E6758
1.3% Picric acid	Sigma	P6744	Sirius Red Stain
10 mM sodium citrate, pH6.0	Sigma	C9999
3% BSA	Sigma	A7906
3,3-diaminobenzidine (DAB)	Vector Lab	SK-4100
4% Paraformaldehyde	Electron Microscopy Sciences	15710-S
4mM Sodium metabisulfite	Sigma	S9000
5% skim milk	BioRad	1706404
5-0 Silk	Oasis	MV-682-V	Ureteral obstruction
70% Isopropyl alcohol	Fisher Chemical	A459
95% Ethanol	Decon Labs	2701	Removal of renal nerve
Anti-α-SMA antibody	Sigma	A5228
Anti-β-actin antibody	Sigma	A-5316
Anti-F4/80 antibody	Proteintech	18705-1-AP
Anti-Fibronectin antibody	Cedarlane	CL5495AP
Anti-ICAM-1 antibody	Santa Cruz	SC-1511-R
Anti-IL-1β antibody	Abcam	ab9722
Anti-IL6 antibody	Abcam	ab83339
Anti-Phospho-Smad3 antibody	Abcam	ab51451
Anti-PMN antibody	Accurate	AIAD51140
Anti-TGF-β antibody	Santa Cruz	SC-7892
Anti-TLR4 antibody	IMGENEX	IMG-579A
Anti-TNF-α antibody	Abcam	ab9739
Anti-Tyrosine Hydroxylase antibody	Abcam	ab112
Autoclave	Tuttnauer	EZ9PLUS
Autoclip	MikRon	205016
Bouin’s Fixative	Polysciences	16045-1
Coplin Jar	Grainger	3WEF1
Cotton tip	Midline	MDS202055
Creatinine Assay Kit	BioAssay Systems	DICT-500
DC Temperature Controller	FHC	40-90-8D
Direct Red 80	Sigma	365548	Sirius Red Stain
Filter paper	Whatman	3030917	Removal of renal nerve
FITC-conjugated sinistrin	MediBeacon	N/A	GFR analysis
Heparinized capillary tube	Fisher Scientific	22-260-950
Heparinized tube	Terumo Medical Corp.	Capiject
HRP-conjugated anti-rabbit antibody	Vector Lab	PI-1000
Insulin syringe	Becton Dickinson	305500
Ketamine	Par Pharmaceutical	Ketalar	Anesthetic agent
Lab Works analysis software	Ultra-Violet Products	N/A	Analysis of Western blot band density
Light microscope	Leica	Leica DMR
Metabolic cage	Tecniplast	3600M021	GFR analysis
Microaneurysm clamp	Roboz	RS-5422	Ischemia/reperfusion
Microdissecting forcep	Roboz	RS-5069
Microplate reader	Tecan	Infinite 200 PRO
Mounting medium	Fisher Scientific	SP15-100
Noepinephrine ELISA kit	ALPCO Diagnostics	17-NORHU-E01.1
PAGE gel of Any KD	BioRad	456-9034
Phosphatase inhibitor	Sigma	P5726
Povidon-Iodine Prep Pad	Professional Disposables International	C12400
Protease	Calbiochem	539134
Protein lysis buffer	Thermo Scientific	78510
PVDF membrane	BioRad	162-0176
Scalpel Handle	Roboz	RS-9843
Scissors	Roboz	RS-5882
Surgical blade	Bard-Parker	371110
Surgical microscope	Nikon	SMZ-745
Superblock	Thermo Scientific	37535
Transcutaneous Measurement System	MediBeacon	N/A	GFR analysis
Tris-Glycine buffer	BioRad	1610771
Tris-Glycine-SDS buffer	BioRad	1610744
TUNEL assay kit	Roche	11684795910
Tweezers	Roboz	RS-5137
Western Lightning Chemiluminescence Substrate solution	PerkinElmer	NEL10400
Xylazine	Akorn Animal health	139-236	Anesthetic agent
Xylene	HistoPrep	HC700