Name: Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection (Video) | JoVE Journal
Uploaded: 2021-11-10T13:00:00.000+00:00
Duration: 10 min 15 s
Description: Learn to identify cytokine-producing cells in injured kidneys using Brefeldin A injection in mice. This method enables visualization of TGF beta and other secreted proteins within specific cell types for kidney disease research.

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5 minutes

American Heart Association (AHA): Kensei Taguchi, 20POST35200221; HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK): Craig Brooks, DK114809-01 DK121101-01.

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<li>Burton, C. J., Combe, C., Walls, J., Harris, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secretion+of+chemokines+and+cytokines+by+human+tubular+epithelial+cells+in+response+to+proteins%5BTitle%5D)+AND+%22Nephrology%2C+Dialysis%2C+Transplantation%22%5BJournal%5D)">Secretion of chemokines and cytokines by human tubular epithelial cells in response to proteins.</a> Nephrology, Dialysis, Transplantation. 14 (11), 2628-2633 (1999).</li>
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<li>Kovacs, S. B., Oh, C., Aachoui, Y., Miao, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+cytokine+production+by+flow+cytometry+using+brefeldin+A+in+mice%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Evaluating cytokine production by flow cytometry using brefeldin A in mice.</a> STAR Protocols. 2 (1), 100244(2021).</li>
<li>Fujiwara, T., Oda, K., Yokota, S., Takatsuki, A., Ikehara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brefeldin+A+causes+disassembly+of+the+Golgi+complex+and+accumulation+of+secretory+proteins+in+the+endoplasmic+reticulum%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum.</a> Journal of Biological Chemistry. 263 (34), 18545-18552 (1988).</li>
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<li>Chung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-%CE%B2+promotes+fibrosis+after+severe+acute+kidney+injury+by+enhancing+renal+macrophage+infiltration%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">TGF-β promotes fibrosis after severe acute kidney injury by enhancing renal macrophage infiltration.</a> JCI Insight. 3 (21), 123563(2018).</li>
<li>Meng, X. M., Nikolic-Paterson, D. J., Lan, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-%CE%B2%3A+the+master+regulator+of+fibrosis%5BTitle%5D)+AND+%22Nature+Reviews.+Nephrology%22%5BJournal%5D)">TGF-β: the master regulator of fibrosis.</a> Nature Reviews. Nephrology. 12 (6), 325-338 (2016).</li>
<li>Kok, H. M., Falke, L. L., Goldschmeding, R., Nguyen, T. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+CTGF%2C+EGF+and+PDGF+pathways+to+prevent+progression+of+kidney+disease%5BTitle%5D)+AND+%22Nature+Reviews.+Nephrology%22%5BJournal%5D)">Targeting CTGF, EGF and PDGF pathways to prevent progression of kidney disease.</a> Nature Reviews. Nephrology. 10 (12), 700-711 (2014).</li>
<li>Engel, J. E., Williams, E., Williams, M. L., Bidwell, G. L., Chade, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+VEGF+%28vascular+endothelial+growth+factor%29+therapy+induces+long-term+renal+recovery+in+chronic+kidney+disease+via+macrophage+polarization%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Targeted VEGF (vascular endothelial growth factor) therapy induces long-term renal recovery in chronic kidney disease via macrophage polarization.</a> Hypertension. 74 (5), 1113-1123 (2019).</li>
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<li>Brooks, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((KIM-1-%2FTIM-1-mediated+phagocytosis+links+ATG5-%2FULK1-dependent+clearance+of+apoptotic+cells+to+antigen+presentation%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation.</a> EMBO Journal. 34 (19), 2441-2464 (2015).</li>
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</ol>

The authors have no conflicts of interest to disclose.

Kidney PTCs are known to regulate AKI and CKD through the secretion of TGF-&#946;, TNF-&#945;, CTGF, PDGF, vascular endothelial growth factor, as well as many other proteins20,21,22,23. Similarly, glomeruli, distal tubules, and other kidney epithelial cells, as well as interstitial cells, secrete these and/or other proteins during injury24,25...

It is estimated that &#62;10% of the world&#39;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 life2,3. 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 nephron4,5. Following injury, the surviving epithelial cells help coordinate the repair response through the secretion of cytokines and other proteins. In this way, the surviving cells can modulate the immune response, direct extracellular matrix remodeling, and aid organ recovery.
Cytokines are small, secreted proteins essential for modulating the maturation, growth, and responsiveness of multicellular organisms6,7. They function as signal messengers among various cell types, including immune and epithelial cells8. Although cytokines are thought to be secreted mainly by immune cells, long-standing research has demonstrated that kidney epithelial and interstitial cells also secrete cytokines as signals for other resident kidney cells, such as tubule cells, interstitial cells, and immune cells9,10. PTCs, in particular, play an important role in the initiation and recovery phase after AKI11. However, maladaptively repaired PTCs are known to secrete profibrotic cytokines such as transforming growth factor-&#946; (TGF-&#946;), platelet-derived growth factor-D (PDGF-D), and connective tissue growth factor (CTGF), contributing to CKD progression12. Thus, kidney epithelial cells use secreted cytokines to modulate kidney injury.
While it is known that kidney epithelial cells secrete cytokines, the exact source and relative contribution of each cell type have been difficult to determine due to the technical challenges of studying secreted proteins13. Flow cytometry, a common approach used to measure cytokines, is challenging to perform on injured kidneys, especially in highly fibrotic ones. With Cre recombinase driven by a cytokine promoter, cytokine reporter mice are often used to identify the cell type that expresses a given cytokine. However, the use of reporter mice is limited because of the requirement to cross reporter mice into various knockout backgrounds, the lack of suitable reporters, and the fact that only one cytokine can be analyzed at a time. Thus, it is necessary to develop a simple, versatile, and affordable technique for detecting cytokine-releasing kidney cells.
We hypothesized that injection of BFA, a secretion inhibitor that blocks endoplasmic reticulum-Golgi transport in vivo would allow the staining of secreted proteins in kidney tissue (Figure 1A,B), as shown with flow cytometry-based assays14,15. Along with cell-type-specific makers, this technique could be used to identify the source and relative contribution of cytokine-producing cells in injured kidneys. Unlike samples for flow cytometry, fixed tissues can be kept long-term with preservation of proteins and cellular structures, allowing for a more thorough investigation of the secretory cells. To test this hypothesis, mouse kidneys were injured with a model of AKI (cisplatin) and a model of CKD (aristolochic acid nephropathy (AAN)), injected with BFA, and stained using standard immunofluorescent techniques.

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

identification of the source of secreted proteins in the kidney by brefeldin a injection

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

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-&#946;), 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-&#946;, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-&#946; in&#160;a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-&#946;-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.

To examine the role of tubular epithelial cells in cytokine production following cisplatin-induced AKI, cisplatin was injected at a concentration of 20 mg/kg followed by an intravenous injection of 0.25 mg of BFA on day 3 after the cisplatin injection. The kidneys were harvested 6 h later. Paraffin-embedded kidneys were sectioned and stained with TGF-β, PDGF-D, and CTGF, representative cytokines responsible for tissue repair in AKI. As shown in Figure 2A, TGF-β+ vesicles are obser...

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.

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 ...

identification-source-secreted-proteins-kidney-brefeldin

Research

JoVE Journal

Biology

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Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Assessment of Kidney Function in Mouse Models of Glomerular Disease

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

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Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys.

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice

The present protocol describes the establishment of a membranous nephropathy (MN) animal model, and how Kemeng Fang's inhibition reduces MN rat podocyte apoptosis by activating the PI3K/AKT signaling pathway.

Mechanism of Kemeng Fang's Inhibition of Podocyte Apoptosis in Rats with Membranous Nephropathy through the PI3K/AKT Signaling Pathway

This protocol describes a method for inducing unilateral ureteral obstruction (UUO) in mice to study the progression of tissue fibrosis in obstructive nephropathy. It includes surgical procedures, post-operative care, and methods for fibrosis assessment.

Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Learn to identify cytokine-producing cells in injured kidneys using Brefeldin A injection in mice. This method enables visualization of TGF beta and other secreted proteins within specific cell types for kidney disease research.

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

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