Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Summary

Identifying the cell type responsible for secreting cytokines is necessary to understand the pathobiology of kidney disease. Here, we describe a method to quantitatively stain kidney tissue for cytokines produced by kidney epithelial or interstitial cells using brefeldin A, a secretion inhibitor, and cell-type-specific markers.

Abstract

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging.

This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-β, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-β in a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-β-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.

Introduction

It is estimated that >10% of the world's population have some form of kidney disease¹. Defined by its rapid onset, AKI is largely curable; however, an episode of AKI can predispose patients to develop CKD later in life^2,3. Unlike AKI, CKD is marked by progressive fibrosis and worsening kidney function, leading to end-stage renal disease requiring renal replacement therapy. Most injuries to the kidneys target the specialized epithelial cells, such as podocytes or proximal tubule cells, that make up the nephron^4,5. Followi....

Protocol

All animal experiments were performed in accordance with the animal use protocol approved by the Institutional Animal Care and User Committee of Vanderbilt University Medical Center.

1. Animals

1. Use 8-12-week-old BALB/c male mice (body weight: approximately 25 g) for cisplatin- or aristolochic acid-induced nephropathy.
2. Ensure the mice are healthy and have no obvious signs of distress or wounds from fighting.
   NOTE: Wounds, especially to the tail, cou.......

Discussion

Kidney PTCs are known to regulate AKI and CKD through the secretion of TGF-β, TNF-α, CTGF, PDGF, vascular endothelial growth factor, as well as many other proteins^20,21,22,23. Similarly, glomeruli, distal tubules, and other kidney epithelial cells, as well as interstitial cells, secrete these and/or other proteins during injury^24,25.......

Materials

Name	Company	Catalog Number	Comments
1 mL Insulin syringes	BD	329654
10 mL Syringe	BD	302995
2 mL tube	Fisher brand	05-408-138
20 mL Syringe	BD	302830
25 G needles	BD	305125
28 G needles	BD	329424
96-well-plate	Corning	9017
Aristolochic acid-I	Sigma-Aldrich	A9461
α-SMA antibody conjugated with Cy3	Sigma-Aldrich	C6198	RRID:AB_476856
Blade for cryostat	C.L. Sturkey. Inc	DT315R50
Bovine serum albumin (BSA)	Sigma-Aldrich	A7906
Brefeldin A	Sigma-Aldrich	B6542
Cisplatin	Sigma-Aldrich	P4394
Citric acid	Sigma-Aldrich	791725
Confocal microscope	ZEISS	LSM710
Confocal microscopy objectives	ZEISS	40x / 1.10 LD C-Apochromat WATER
Confocal software	ZEISS	ZEN
Coplin jar	Fisher Scientific	19-4
Cover glass	Fisher brand	12545F
Cryostat	Leica	CM1850
CTGF antibody	Genetex	GTX124232	RRID:AB_11169640
Cy3-AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson immunoresearch	711-165-152	RRID: AB_2307443
Cy5-AffiniPure Donkey Anti-Goat IgG (H+L)	Jackson immunoresearch	705-175-147	RRID: AB_2340415
DAPI	Sigma-Aldrich	D9542
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
Disposable base molds	Fisher brand	22-363-553
donkey serum	Jackson immunoresearch	017-000-121
Ethanol	Decon Labs, Inc.	2701
Forceps	VETUS	ESD-13
Glycine	Fisher brand	12007-0050
Heating pads	Kent scientific	DCT-20
Heparin sodium salt	ACROS organics	41121-0010	100mg/15ml of dH2O
Humidified chamber	Invitrogen	44040410	A plastic box covered in foil can be used as an alternative humidified chamber.
Insulin syringes	BD	329461
Inverted microscope	NIKON	Eclipse Ti-E2	immunofluorescence
KIM-1 antibody	R & D	AF1817	RRID: AB_2116446
Lemozole (Histo-clear)	National diagnostics	HS-200
lotus tetragonolobus lectin	Vector	FL-13212
Microscope slide	Fisher scientific	12-550-343
Microtome	Reichert	Jung 820 II
monochrome CMOS camera	NIKON	DS-Qi-2
Mouse surgical kit	Kent scientific	INSMOUSEKIT
NIS Elements	NIKON
Objectives	NIKON	Plan Apo 20x/0.75	image acquisition software linked to Eclipse Ti-E2 (invertd microscope)
OCT compound	Scigen	4586
Pap pen	Vector	H-4000
PBS with calcium and magnesium	Corning	21-030-CV
PBS without calcium and magnesium	Corning	21-031-CV
PDGF-D antibody	Thermo-Fisher scientific	40-2100
PFA	Electron Microscopy Science	15710	RRID: AB_2533455
Plate reader	Promega	GloMax® Discover Microplate Reader	4% PFA is diluted from 16% in PBS.
povidone-iodine (Betadine)	Avrio Health L.P.	NDC 67618-151-17
Pressure cooker	Tristar 8	Qt. Power Cooker Plus
ProLong Gold Antifade Reagent	Invitrogen	P36930
Quantichrom Urea (BUN) assay Kit II	BioAssay Systems	DUR2-100
Single-edge razor blade for kidney dissection (.009", 0.23 mm)	IDL tools	521013
Slide warmer	Lab Scientific Inc.,	XH-2001
Software	NIKON	NIS elements
Sucrose	RPI	S24060
TGF-b1 anitbody	Sigma-Aldrich	SAB4502954
Tris EDTA buffer	Corning	46-009-CM	RRID: AB_10747473
Trisodium citrate dihydrate	Sigma-Aldrich	SLBR6660V
Trisodium citrate dihydrate	Sigma-Aldrich	SLBR6660V
Triton	Sigma-Aldrich	9002-93-1
Tween 20	Sigma-Aldrich	P1379
White Glass Charged Microscope Slide, 25 x 75 mm Size, Ground Edges, Blue Frosted	Globe Scientific	1358D