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 are hallmarks of CKD progression^6,7,8.

Renal denervation (RDNx) is used as a therapeutic option for the treatment of drug-resistant hypertension^9,10,11,12, but the underlying molecular mechanisms are not yet fully understood. Moreover, studies^13,14 defining the role of renal nerve in CKD progression using chemical denervation or antagonists/agonists of sympathetic nerve-derived neurotransmitters (or its receptors) are limited, due to systemic effects that are not specific to the kidney. As reported^15,16,17, RDNx can overcome this limitation and has been successful in defining (in vivo) the roles of the renal nerve, renal nerve-derived factors, and downstream signaling pathways in eliciting renal inflammation and fibrosis.

Provided here is a detailed methodology and representative results for the use of RDNx to study renal tubular injury, inflammation, and fibrosis in a mouse model of CKD.

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.
2. Anesthetize mice using a cocktail containing ketamine (200 mg/kg body weight) and xylazine (16 mg/kg body weight), injected intraperitoneally^16,18.  
3. Check for the lack of response to the toe pinch. Shave the left side of mouse’s back with an electric shaver. Clean the region with an alcohol prep pad, and then ensure asepsis using an iodine-based prep pad.
4. Maintain the body temperature at 36–38 °C on a heating pad with a temperature controller.
   NOTE: Use sterilized surgical instruments for all procedures.
5. Make an incision 1.5 cm long using a surgical blade on the mouse’s back, 5 mm lateral to the spine.
6. Under a surgical microscope, expose the left kidney vessels connected to the kidney through left flank incision. Then, separate the artery and vein carefully from the surrounding connective tissue using micro-dissecting forceps.
7. For stripping the nerves, paint the kidney vessels with filter paper soaked in 95% ethanol for 2 min^15,16.
   NOTE: Ensure that the vessels turn white in color under the microscope.
8. Wash the kidney vessels for 2 min with PBS. Close the muscular layer with absorbable sutures and close the wounded skin using an autoclip.
9. Perform a sham operation in the same manner as above, except strip the kidney nerves.
10. After surgery, inject 0.1 µg/kg body weight (BW) buprenorphine subcutaneously (SC) 1x/day for 3 days to reduce surgical pain.

2. Renal fibrosis models

1. Unilateral ureteral obstruction (UUO) model
   1. Two days after renal denervation, anesthetize two male mice (129S1/SvImJ) aged 8–10 weeks as described in step 1.2.
   2. Check for the lack of response to a toe pinch, then shave the left side of the mouse back.
   3. Clean the region with an alcohol prep pad and ensure that the region is aseptic using an iodine-based prep pad.
   4. Maintain the body temperature at 36–38 °C on a heating pad with a temperature controller.
   5. Expose the left kidney using autoclaved cotton swab through a left flank incision using a surgical blade on the mouse’s back, 1 cm lateral to the spine.
   6. Ligate the left ureter completely near the kidney and pelvis using a 5-0 silk tie^15,18.
   7. Close the muscular layer with absorbable sutures and close the wounded skin using an autoclip.
   8. On the other mouse, perform a sham operation using the same surgical procedure, except for ligation of the ureter.
   9. After surgery, inject 0.1 µg/kg buprenorphine SC 1x/day for 3 days to reduce surgical pain.
2. Ischemic acute kidney injury (AKI) to CKD transition model
   1. Two days after the denervation, anesthetize male 129S1/SvImJ mice aged 8–10 weeks as described in step 1.2.
   2. Check for the lack of response to a toe pinch and shave the left side of the mouse’s back. Clean the region with an alcohol prep pad and ensure that the region is aseptic using an iodine-based prep pad.
   3. Maintain the body temperature at 36–38 °C on a heating pad with a temperature controller.
   4. Expose the left kidney using an autoclaved cotton swab through the left flank incision on the back, 1 cm lateral to the spine, using a surgical blade.
   5. Subject the mice to 30 min of left renal ischemia by clamping the left renal pedicle with both the artery and vein with nontraumatic microaneurysm clamps, as described previously^16,19. Close the incisions temporarily during this ischemic period using clamps.
   6. Once the ischemic period is over, remove the clamps for reperfusion.
      NOTE: Visually confirm reperfusion of the kidneys. The color of the kidneys will change from dark red to bright red upon reperfusion.
   7. Close the muscular layer with absorbable sutures and close the wounded skin using an autoclip.
   8. After the surgery, inject 0.1 µg/kg BW buprenorphine SC 1x/day for 3 days to reduce surgical pain.
   9. Perform a sham operation with the same surgical procedure except clamping the renal pedicle.

3. Harvesting of kidneys and blood

1. At 1 h, 3 h, 6 h, or 12 h (or 1, 2, 3, or 10 days) post-UUO; or at 0.5, 1, 2, 4, 8, or 16 days post-IRI, anesthetize by placing the mouse’s nose to a 50 mL conical tube with gauze containing isoflurane.
2. Collect either 1) 100 µL blood samples from the retroocular vein plexus using a heparinized capillary tube or 2) 500 µL of blood from the vena cava through a midline abdominal incision using a syringe (25 G needle) containing heparin.
3. For harvesting kidneys, expose the operated kidney by midline abdominal incision. Cut renal vessels and surrounding tissues with surgical blade and then remove the capsules.
   NOTE: Ensure that UUO kidneys have urine filled the pelvis to confirm UUO induction.
4. Collect plasma from the blood by centrifugation at 8,000 x g for 3 min at room temperature (RT) using a heparinized capillary tube. Store at -20 °C until use.
5. For biochemical assays, snap-freeze the kidneys using liquid nitrogen immediately after removal. Store at -80 °C deep freezer until use.

4. Analysis of renal function

1. Anesthetize mice 15 days post-ischemia/reperfusion injury (IRI) as described above.
2. Expose the right kidney using autoclaved cotton swab through a right flank incision on the back, 1 cm lateral to the spine.
3. Remove the kidney (i.e., perform a nephrectomy) from the renal vessels and ureter using scissors, after ligation of the renal vessels and ureter^16,20.
4. Take blood samples from the retroocular vein plexus 6 h prior to placing the mouse in a metabolic cage.
5. To obtain urine samples, place the animals in mouse-specific metabolic cages for 18 h for analysis of glomerular filtration rate (GFR) by creatinine clearance¹⁶.
6. Measure urinary and plasma concentrations of creatinine using a commercially available kit (Table of Materials). In brief, add 30 µL of plasma to a mixture of Reagents A and B (100 µL each). Then, read the optical density both 1) immediately and 2) at 5 min post-incubation at 510 nm and RT using a microplate reader. For urine, use 50 µL of Reagent A, 50 µL of Reagent B, and 100 µL of water per 5 µL of urine.
7. Calculate GFR as follows:
   
8. Alternatively, measure GFR by transcutaneous measurement at 24 h post-Nx, as described previously²¹. In brief, under anesthesia with isoflurane, place a miniaturized fluorescence detector on the mouse’s back, then inject fluorescein-isothiocyanate (FITC)-conjugated sinistrin (an inulin analog, 0.15 mg/g BW) through the retroocular vein plexus. In conscious and freely moving mice, collect data for the half-life of FITC-sinistrin for 1 h and perform the analysis.

5. Confirmation of renal denervation

1. Evaluation of kidney NE level
   1. Homogenize kidneys frozen at -80 °C in a deep freezer with a glass tissue homogenizer. Use homogenizing solution containing 1 mM EDTA and 4 mM sodium metabisulfite in 400 µL of 0.1 N HCl.
   2. Centrifuge the homogenates at 13,600 x g and transfer the supernatant to new tube.
   3. Extract NE from samples by using a cis-diol-specific affinity gel, acylate, then convert enzymatically with a commercially available kit (Table of Materials)^15,16.
      1. Transfer 300 µL of the lysates into the 48 well plate and add 250 µL of distilled water to each well, followed by adding 50 µL of assay buffer and 50 µL of extraction buffer.
      2. Cover the plate with adhesive foil and incubate for 30 min at RT on a shaker (~600 rpm) then remove the foil, empty, and blot dry by tapping the inverted plate on a hand towel.
      3. Wash the plate 2x for 5 min at RT using 1 mL of washing buffer by shaking on a platform shaker, followed by blotting dry as done in step 5.1.3.2.
      4. Add 150 µL of acylation buffer and 25 µL of acylation reagent into the wells, then incubate for 15 min at RT on the shaker.
      5. Empty the plate and blot dry as done in step 5.1.3.2, then wash 1x for 10 min as done in step 5.1.3.3, followed by blot drying as done in step 5.1.3.2. Add 150 µL of 0.025 M hydrochloric acid into the wells, cover the plate with adhesive foil, and incubate for 10 min at RT on the shaker.
   4. Add 25 µL of enzyme solution to the pre-coated NE microtiter strips, followed by 20 µL of supernatant (as done in step 5.1.3.5) and standard solutions of NE (0 ng/mL, 5 ng/mL, 20 ng/mL, 75 ng/mL, 250 ng/mL, and 1,000 ng/mL) into each strip. Add 50 μL of the NE antiserum to the strips. Incubate for 2 h at RT on the shaker.
   5. After discarding the buffer and washing the microtiter strips with 300 μL of washing buffer on the shaker, remove the buffer and blot dry as done in step 5.1.3.2. Incubate for 30 min at RT on the shaker with 100 μL of enzyme conjugate in each well.
   6. After discarding the buffer and washing the strips, add 100 μL of substrate into each well. Incubate for 20–30 min at RT on a shaker, followed by adding 100 μL of stop solution.
   7. Read the absorbance of the solution using a microplate reader at 450 nm and calculate the concentration using standard solution as control.
2. Immunohistochemistry for tyrosine hydroxylase
   1. Fix kidneys in 4% paraformaldehyde and process the paraffin embedding with the central part of the fixed kidney with a cross section of 3 – 4 mm thickness.
   2. Place the paraffin-embedded kidney on a microtome, cut to 3–4 µm thick, and attach the section to the microscope slide.
   3. Deparaffinize the paraffin-embedded kidney sections with xylene. Rehydrate with 100%, 95%, and 90% ethanol (in this sequence) 2x at each step.
   4. Permeabilize the kidney sections in 0.1% SDS for 5 min and wash 3x with PBS at RT.
   5. Autoclave the permeabilized kidney sections in 10 mM sodium citrate at 120 °C for 10 min for antigen retrieval^16,22.
   6. Cool down the autoclaved kidney sections for 30 min at RT and wash 3x with PBS at RT.
   7. Incubate with 3% BSA in PBS for 30 min at RT to prevent non-specific binding.
   8. After removal of BSA by suction, incubate the kidney sections with antibody (1:1000) against tyrosine hydroxylase overnight at 4 °C¹⁵.
   9. Next day, wash the kidney sections 3x with PBS at RT and incubate with HRP-conjugated anti-rabbit antibody for 1 h at RT.
   10. After washing 3x with PBS at RT, apply 3,3 –diaminobenzidine (DAB) to the kidney sections and stain for up to 2 min.
   11. After washing 3x with PBS at RT, dehydrate the kidney sections with 90%, 95%, and 100% ethanol and xylene for 5 min each. Mount the coverslips with a 1:1 mixture of mounting medium and xylene.
3. Western blot analysis for tyrosine hydroxylase
   1. Isolate whole kidney proteins using lysis buffer with phosphatase inhibitor and protease^16,18.
   2. Run 30 µg of kidney protein on PAGE gel in Tris-glycine-SDS buffer at 80 V for 1 h.
   3. Transfer the protein to PVDF membrane from gel in Tris-glycine buffer at 80 V for 100 min at 4°C.
   4. Block the nonspecific proteins on the membrane using 5% skim milk for 30 min at RT.
   5. Incubate membranes with diluted antibody (1:2,000) against tyrosine hydroxylase in 10 mL of a commercially available blocking solution^15,16.
   6. Use anti-β-actin antibody as loading controls on stripped membranes.
   7. Expose membranes to the chemi-luminescence substrate solution. Quantify the bands using analysis software.

6. Analysis of tubular injury

1. Tubular damage: section paraffin-embedded kidney into 3 µm thick sections using microtome and stain using a periodic acid/Schiff (PAS) stain. First, incubate the sections with 5% PAS stain for 5 min. Then, rinse with distilled water and incubate with Schiff reagent for 15 min.
   1. Wash PAS-stained kidneys with tap water for 5 min, counterstain with hematoxylin for 1 min, and dehydrate after washing with tap water for 5 min. Cover with mounting medium.
   2. Select five fields from each PAS-stained kidney randomly in a blinded manner and photograph using light microscopy.
   3. Score the damaged tubule with flattened lumen according to the loss of brush border, proteinaceous cast, loss of tubular nucleus, and dilation as follows: grade 0 = normal; grade 1 =  >10%; grade 2 = 10%–25%; grade 3 = 26%–50%; grade 4 = 51%–75%; grade 5 = <75%^15,16,18.
2. Apoptotic cell death: analyze apoptotic cell death in paraffin-sectioned kidney samples by performing a TUNEL assay using a commercially available kit (Table of Materials), following the manufacturer’s protocol.

7. Inflammation

1. Analysis of neutrophil
   1. Prepare kidney sections as described in step 5.2 and incubate with antibodies against polymorphonuclear neutrophil overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibody^15,16.
   2. Perform DAB staining and dehydration as described in steps 5.2.10 and 5.2.11.
   3. Take photographic images from cortical or outer medullary regions in a blinded manner.
2. Analysis of macrophage
   1. Prepare kidney sections as described in section 5.2.
   2. Incubate the kidneys with antibodies against F4/80 overnight at 4 °C. Incubate with an HRP-conjugated secondary antibody^15,16.
   3. Take images from cortical or outer medullary regions in a blinded manner.
3. Analysis of inflammatory cytokine level
   1. Following section 5.3, prepare kidney samples.
   2. Incubate the kidney samples with antibodies against ICAM-1, TNF-α, IL-1β, IL-6, and TLR4 overnight at 4 °C. Then, incubate with the corresponding secondary antibodies¹⁵.

8. Evaluation of fibrosis

1. Sirius red stain
   1. Following section 5.2, rehydrate the kidney sections.
   2. Stain the kidney sections with Sirius red solution (0.5 g of Direct Red 80 + 1.3% picric acid in 500 mL of distilled water) for 30 min^15,16,18.
   3. After washing 2x with acidified water (0.5% acetic acid, glacial), physically remove the water from the slides.
   4. After mounting (section 5.2), take images of cortical or outer medullary regions in a blinded manner.
2. Evaluation of fibroblast activation and fibrogenic factors
   1. Following section 5.3., prepare kidney samples.
   2. Incubate the kidney samples with antibodies against alpha-SMA, fibronectin, TGF-β, and phospho-Smad3 overnight at 4 °C. Incubate with the corresponding secondary antibodies¹⁵.
   3. Use anti-β-actin antibody as a loading control on stripped membranes.
   4. Quantify the bands using analysis software.

Results

Removal of renal nerve by renal denervation surgery
Renal denervation (RDNx) was carried out 2 days prior to IRI or UUO to define whether renal nerve contributes to the initiation and development of kidney fibrosis and inflammation. Mice were subjected to either 1) 30 min IRI followed by reperfusion for 1, 2, 4, 8, or 16 days or 2) UUO for 1 h, 3 h, 6 h, or 12 h; 1, 2, 3 or 10 days.

To confirm whether RDNx was successfully applied, tyrosine hydroxylase (TH)-positive symp...

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