We thank Sarah Williams, PhD, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript.

Animal experiments were approved by the Animal Ethics Committee of Jichi Medical University and comply with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.
1. Obtaining kidney samples from HIGA mice
NOTE: HIGA mice show a stable phenotype of IgA nephropathy after 25 weeks of age8,9,10,11. Balb/c mice should be selected as the control group8,9,10,11. 25-week-old female HIGA mice (n = 10) and 25-week-old female Balb/c mice (n = 10) were obtained. It is necessary to determine the number of mice required for experiments in advance. This step requires about 7&#8211;8 h for a sample size of 10 HIGA mice and 10 Balb/c mice.
<ol>
	<li>Prepare these items: HIGA mice (25-week-old, female), Balb/c mice (25-week-old, female), inhalation anesthesia apparatus, isoflurane, cork sheet, pin, phosphate-buffered saline (PBS), injection syringes, needles, surgical scissors, and forceps.</li>
	<li>Anesthetize a HIGA mouse using 4%&#8211;5% of isoflurane with inhalation anesthesia apparatus and mount it in the dorsal position on the cork sheet using pins. Adjust the concentration of isoflurane to 2%&#8211;3% as the maintenance dose after the induction of anesthesia. 
	NOTE: The depth of anesthesia is maintained at a level at which the pain associated with the invasive procedure is completely eliminated. In particular, isoflurane concentration should be adjusted to 4-5% when excising tissues such as the thorax. Hair removal and eye ointment are not essential. It is not necessary to mount the mouse on a heating pad if the procedure can be performed quickly.</li>
	<li>Make a 3&#8211;4 cm midline incision of the mouse abdominal wall with surgical scissors and forceps and carefully identify both sides of the kidney.</li>
	<li>Incise the ribs and diaphragm with surgical scissors and forceps to expose the heart. After incising the right atrium, inject PBS into the left ventricle until the kidney color changes to pale yellow (this procedure means that the whole body of the mouse is perfused with PBS.)</li>
	<li>Cut the renal artery, renal vein, and ureter, and remove the kidney. Divide the kidney into 30 mg pieces for use in the next step. 
	NOTE: The pathology of IgA nephropathy mainly involves the glomerulus in the renal cortex (outer part of the kidney). Although it is difficult to macroscopically and completely distinguish the cortex and the medulla, it is desirable to mainly collect the outer part of the kidney. These samples can be stored at &#8211;80 &#176;C for several months.</li>
</ol>
2. Purifying total RNA from kidney samples
NOTE: In this step, commercially available miRNA isolation kit is used for the extraction of total RNA. In addition, biopolymer-shredding spin column is used. See Table of Materials for additional details. miRNA isolation kit contains a silica membrane-based spin column, phenol/guanidine-based lysis reagents, guanidine/ethanol wash buffer (wash buffer 1), ethanol wash buffer (wash buffer 2), and nuclease-free water. This step requires about 3 h for a sample size of 10 HIGA mice and 10 control mice.
<ol>
	<li>Prepare the following items: 1.5 or 2.0 mL collection tubes, 100% ethanol, chloroform, silicon homogenizer, micropipettes, pipette tips, centrifuge, biopolymer-shredding spin column, silica membrane-based spin column, phenol/guanidine-based lysis reagents, guanidine/ethanol wash buffer (wash buffer 1), ethanol wash buffer (wash buffer 2), and nuclease-free water.</li>
	<li>Homogenize 30 mg kidney samples using a silicon homogenizer and 700 &#181;L of the phenol/guanidine-based lysis reagent at room temperature. 
	NOTE: The miRNAs in the sample are vulnerable until they have been exposed to phenol/guanidine-based lysis reagent so these steps should be performed promptly.</li>
	<li>Transfer the lysate to the biopolymer-shredding spin column and centrifuge it at 15,000 x g for 2 min at room temperature.</li>
	<li>Add 140 &#181;L of chloroform to the filtrate and mix vigorously for 15 s. Leave for 2&#8211;3 min, then centrifuge at 12,000 x g at 4 &#176;C for 15 min.</li>
	<li>Gently transfer the clear supernatant without touching the middle layer to a new collection tube and add the determined volume (see below) of 100% ethanol. Vortex for 5 s. 
	NOTE: 100% ethanol at 1.5x the volume of the obtained supernatant is required.</li>
	<li>Transfer the sample (upper limit 700 &#181;L) to a silica membrane-based spin column and centrifuge the sample at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Add 700 &#181;L of wash buffer 1 to the silica membrane-based spin column and centrifuge the column at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Add 500 &#181;L of wash buffer 2 to the silica membrane-based spin column and centrifuge it at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Add 500 &#181;L of wash buffer 2 to the silica membrane-based spin column and centrifuge it at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Centrifuge the silica membrane-based spin column again without adding anything to remove any additional ethanol, at 15,000 x g for 1 min at room temperature. Throw away the filtrate after centrifugation.</li>
	<li>Change the tube attached to the spin column to a new collection tube.</li>
	<li>Add 30 &#181;L of nuclease-free water to the silica membrane-based spin column and centrifuge it at 8,000 x g for 1 min at room temperature. 
	NOTE: The resulting 30 &#181;L solution contains a high concentration of total RNA. This sample can be stored at &#8211;80 &#176;C for several months.</li>
</ol>
3. Synthesis of cDNA from total RNA
NOTE: In this step, a commercially available reverse transcription kit is used. See Table of Materials for additional details. This kit contains nucleic acid mix, reverse transcriptase mix, and buffer. This procedure must be performed on ice to prevent progress of the reaction. This step requires about 3 h for a sample size of 10 HIGA mice and 10 control mice.
<ol>
	<li>Prepare the following items: 1.5 mL collection tubes, eight-well strip tubes, micropipettes, pipette tips, spectrophotometer, nuclease-free water, ice, nucleic acid mix, reverse transcriptase mix, buffer, and thermal cycler.</li>
	<li>Measure the concentration of total RNA using a spectrophotometer. Then, calculate the required volume of nuclease-free water so that 12 &#181;L contains 1 &#181;g of total RNA.</li>
	<li>Prepare a master mix with the following contents: 2.0 &#181;L of nucleic acid mix, 2.0 &#181;L of reverse transcriptase mix, and 4.0 &#181;L of buffer to a total of 8.0 &#181;L per sample.</li>
	<li>Add 8 &#181;L of the master mix to each well of eight-well strip tubes.</li>
	<li>Dilute total RNA in the volume of nuclease-free water calculated in 3.2. Then, add 12 &#181;L of the total RNA solution (containing 1 &#181;g of total RNA) to each well of eight-well strip tubes.</li>
	<li>Incubate the sample in 8 well strip tubes at 37 &#176;C for 60 min using the thermal cycler. Then, incubate at 95 &#176;C for 5 min using the thermal cycler. 
	NOTE: The solution after incubation contains a high concentration of cDNA.</li>
	<li>Transfer this solution to a 1.5 mL tube and add 200 &#181;L of nuclease-free water. 
	NOTE: The resulting 200 &#181;L of solution can be used for qRT-PCR as a template cDNA. This sample can be stored at &#8211;80 &#176;C for about 1 year.</li>
</ol>
4. qRT-PCR of miRNA
NOTE: In this step, a commercially available PCR kit is used. See Table of Materials for additional details. This kit contains PCR mix, universal primer, and nuclease-free water. Samples should be prepared in duplicate, and the accuracy of the results should be considered in each case. Expression levels of miRNA are quantified by the &#916;&#916;CT method. This step requires about 4 h for a sample size of 10 HIGA mice and 10 control mice.
<ol>
	<li>Prepare the following items: 1.5 mL collection tube, 96-well reaction plate, adhesive film for the 96-well reaction plate, micropipettes, pipette tips, PCR mix, universal primer, nuclease-free water, miRNA-specific primers, and a real-time PCR instrument.</li>
	<li>Prepare the master mix with the following contents: 12.5 &#181;L of PCR mix, 2.5 &#181;L of universal primer, 1.25 &#181;L of 5 &#181;M miRNA-specific primer, and 6.25 &#181;L of nuclease-free water for each well. 
	NOTE: In this assay, primers specific to miRNAs [RNA, U6 Small Nuclear 2 (RNU6-2), miR-155-5p, miR-146a-5p, and miR-21-5p] were used. RNU6-2 was used as an endogenous control14.</li>
	<li>Add 22.5 &#181;L of the master mix to each well of the 96 well reaction plate.</li>
	<li>Add 2.5 &#181;L of cDNA prepared in step 3.7 to individual well of the 96 well plate.</li>
	<li>Cover the 96 well reaction plate with adhesion film, then centrifuge at 1,000 x g for 30 s.</li>
	<li>Run the real-time PCR instrument. Program the PCR instrument as follows: initial activation at 95 &#176;C for 15 min, then 40 cycles of denaturation at 94 &#176;C for 15 s, annealing at 55 &#176;C for 30 s, and extension at 70 &#176;C for 30 s.</li>
	<li>Quantify gene expression using the &#916;&#916;CT method. Relative expression levels are determined as 2&#8211;&#916;&#916;CT. 
	NOTE: Abnormal PCR amplification curves should be considered for exclusion from the results.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+nephropathy.">IgA nephropathy.</a> The New England Journal of Medicine. 368 (25), 2402-2414 (2013).</li><li>Vishnoi, A., Rani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNA+Biogenesis+and+Regulation+of+Diseases:+An+Overview.">MiRNA Biogenesis and Regulation of Diseases: An Overview.</a> Methods in Molecular Biology. 1509, 1-10 (2017).</li><li>Rupaimoole, R., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+therapeutics:+towards+a+new+era+for+the+management+of+cancer+and+other+diseases.">MicroRNA therapeutics: towards a new era for the management of cancer and other diseases.</a> Nature Reviews Drug Discovery. 16 (3), 203-222 (2017).</li><li>Serino, G., Sallustio, F., Cox, S. N., Pesce, F., Schena, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+miR-148b+expression+promotes+aberrant+glycosylation+of+IgA1+in+IgA+nephropathy.">Abnormal miR-148b expression promotes aberrant glycosylation of IgA1 in IgA nephropathy.</a> Journal of the American Society of Nephrology. 23 (5), 814-824 (2012).</li><li>Serino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+a+retrospective+international+study,+circulating+miR-148b+and+let-7b+were+found+to+be+serum+markers+for+detecting+primary+IgA+nephropathy.">In a retrospective international study, circulating miR-148b and let-7b were found to be serum markers for detecting primary IgA nephropathy.</a> Kidney International. 89 (3), 683-692 (2016).</li><li>Muso, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+production+of+glomerular+extracellular+matrix+in+a+new+mouse+strain+of+high+serum+IgA+ddY+mice.">Enhanced production of glomerular extracellular matrix in a new mouse strain of high serum IgA ddY mice.</a> Kidney International. 50 (6), 1946-1957 (1996).</li><li>Kurano, M., Yatomi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+gas+chromatography+mass+spectrometry+to+elucidate+metabolites+predicting+the+phenotypes+of+IgA+nephropathy+in+hyper+IgA+mice.">Use of gas chromatography mass spectrometry to elucidate metabolites predicting the phenotypes of IgA nephropathy in hyper IgA mice.</a> Plos One. 14 (7), 0219403 (2019).</li><li>Hyun, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+improve+renal+function+in+a+mouse+model+of+IgA+nephropathy.">Adipose-derived stem cells improve renal function in a mouse model of IgA nephropathy.</a> Cell Transplantation. 21 (11), 2425-2439 (2012).</li><li>Katsuma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+a+mouse+model+of+immunoglobulin+A+nephropathy+reveals+an+enhanced+PDGF-EDG5+cascade.">Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade.</a> The Pharmacogenomics Journal. 1 (3), 211-217 (2001).</li><li>Morse, S. M., Shaw, G., Larner, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+mRNA+and+protein+extraction+from+the+same+experimental+sample+using+a+commercially+available+column-based+RNA+preparation+kit.">Concurrent mRNA and protein extraction from the same experimental sample using a commercially available column-based RNA preparation kit.</a> BioTechniques. 40 (1), 54-58 (2006).</li><li>Mestdagh, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+quantitative+miRNA+expression+platforms+in+the+microRNA+quality+control+(miRQC)+study.">Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.</a> Nature Methods. 11 (8), 809-815 (2014).</li><li>Sauer, E., Babion, I., Madea, B., Courts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evidence+based+strategy+for+normalization+of+quantitative+PCR+data+from+miRNA+expression+analysis+in+forensic+organ+tissue+identification.">An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensic organ tissue identification.</a> Forensic Science International: Genetics. 13, 217-223 (2014).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+miR-146a+and+miR-155+in+kidney+biopsy+and+urine+from+patients+with+IgA+nephropathy.">Elevated levels of miR-146a and miR-155 in kidney biopsy and urine from patients with IgA nephropathy.</a> Disease Markers. 30 (4), 171-179 (2011).</li><li>Hennino, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-21-5p+renal+expression+is+associated+with+fibrosis+and+renal+survival+in+patients+with+IgA+nephropathy.">miR-21-5p renal expression is associated with fibrosis and renal survival in patients with IgA nephropathy.</a> Scientific Reports. 6, 27209 (2016).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> How to Win the Battle with RNase. 2019 (2), (2019).</li></ol>

The authors declare that they have no conflicts of interest.

We were able to measure the expression levels of miRNAs in the kidneys of an IgA nephropathy mouse model (HIGA mice) using this new method. IgA nephropathy is an unexplained disease that needs further research to clarify its etiology and therapeutic targets1,3. However, obtaining human kidney samples is highly invasive. This new technique is advantageous in that it allows the study of IgA nephropathy using small animals and should facilitate future research into ...

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA in the renal glomerular mesangial region1,2. It is the most common of the primary glomerulonephritis and leads to the end-stage renal failure in 20%&#8211;40% of patients2. The cause is still unknown but persistent mucosal infection has been implicated1,3. Corticosteroids, immunosuppressants, and renin&#8722;angiotensin system inhibitors have been proposed as therapeutic methods1,3, but have not been completely established3. Therefore, further research is required to clarify the etiology and therapeutic methods of treating IgA nephropathy.
microRNAs (miRNAs) are small, non-coding RNAs that play an important role in regulating gene expression4,5. miRNAs are reported to be involved in the pathogenesis of various diseases, and some have been identified as disease biomarkers and therapeutic agents4,5. In recent years, an association between miRNAs and the pathogenesis of IgA nephropathy has also been reported2,6,7. For example, miR-148b was shown to be involved in structural abnormalities of IgA in patients with IgA nephropathy2,6,7, while miR-148b and let-7b were documented as novel biomarkers for detecting IgA nephropathy7. Although understanding the effects of miRNAs on IgA nephropathy may help further elucidate etiology and treatment2, standard methods for profiling miRNAs in IgA nephropathy using small animal models have not yet been established2.
We herein developed a simple and reliable method for measuring miRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). The HIGA mouse is a characteristic ddY strain showing a particularly high level of serum IgA and the abnormal deposition of IgA in kidney glomeruli8,9,10,11. Therefore, HIGA mice can be used as an IgA nephropathy mouse model8,9,10,11. Our method consists of four major steps: first, surgically obtaining kidney samples from HIGA mice; second, homogenizing samples and purifying total RNA using a silica membrane-based spin column; third, synthesizing complementary DNA (cDNA) from total RNA using reverse transcription; and fourth, detecting the expression levels of miRNA by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The rationale for this method and the reliability of the results are based on previous reports12,13. We show that this is a useful technique to accurately measure miRNA expression levels in an IgA nephropathy mouse model, and that it could be used to facilitate future research into miRNAs in IgA nephropathy.

<table><tbody><tr><td>BALB/cCrSlc (25-week-old, female)</td><td>Japan SLC, Inc.</td><td>none</td><td>Mouse for control</td></tr><tr><td>HIGA/NscSlc (25-week-old, female)</td><td>Japan SLC, Inc.</td><td>none</td><td>IgA nephropathy mouse model</td></tr><tr><td>MicroAmp Optical 96 well reaction plate for qRT-PCR</td><td>Thermo Fisher Scientific</td><td>4316813</td><td>96-well reaction plate</td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4311971</td><td>adhesive film for 96-well reaction plate</td></tr><tr><td>miScript II RT kit</td><td>Qiagen</td><td>218161</td><td>Experimental kit for synthesis of cDNA</td></tr><tr><td>miRNeasy Mini kit</td><td>Qiagen</td><td>217004</td><td>Experimental kit fot extraction of total RNA</td></tr><tr><td>miScript Primer Assay (RNU6-2)</td><td>Qiagen</td><td>MS00033740</td><td>miRNA-specific primer</td></tr><tr><td>miScript Primer Assay (miR-155-5p)</td><td>Qiagen</td><td>MS00001701</td><td>miRNA-specific primer</td></tr><tr><td>miScript Primer Assay (miR-146a-5p)</td><td>Qiagen</td><td>MS00001638</td><td>miRNA-specific primer</td></tr><tr><td>miScript Primer Assay (miR-21-5p)</td><td>Qiagen</td><td>MS00009079</td><td>miRNA-specific primer</td></tr><tr><td>miScript SYBR Green PCR kit</td><td>Qiagen</td><td>218073</td><td>Experimental kit for real-time PCR</td></tr><tr><td>QIA shredder</td><td>Qiagen</td><td>79654</td><td>biopolymer-shredding spin column</td></tr><tr><td>QuantStudio 12K Flex Flex Real-Time PCR system</td><td>Thermo Fisher Scientific</td><td>4472380</td><td>real-time PCR instrument</td></tr><tr><td>QuantStudio 12K Flex Software version 1.2.1.</td><td>Thermo Fisher Scientific</td><td>4472380</td><td>real-time PCR instrument software</td></tr><tr><td>takara biomasher standard</td><td>Takara Bio</td><td>9790B</td><td>silicon homogenizer</td></tr></tbody></table>

detection of microrna expression in the kidneys of immunoglobulin a nephropathic mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

We investigated the expression levels of miRNAs in the kidneys of HIGA mice (n=10). This result was obtained completely based on the described protocol. The kidneys of Balb/c mice were selected as the control (n=10). In both groups, aged 25 weeks were selected. Only female HIGA mice were available from the supplier. The expression levels of three miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p; Figure 1) were detected, which were previously reported to be associated with IgA nephropathy<sup c...

Watch this Scientific Journal Video about Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice at JoVE.com

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

detection-microrna-expression-kidneys-immunoglobulin-nephropathic

Research

JoVE Journal

Immunology and Infection

1.8K Views.  Jichi Medical University. microRNA's zijn betrokken bij de pathogenese van IgA-nefropathie. We hebben een betrouwbare methode ontwikkeld voor het detecteren van microRNA-expressieniveaus in de nieren van een IgA-nefropathiemuismodel (HIGA-muizen). Deze nieuwe methode zal het gemakkelijker maken om te controleren op betrokkenheid van miRNA's bij IgA-nefropathie.

Video: Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

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

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

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

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.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Biochemistry

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Genetics

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of their target messenger RNAs (mRNAs) by destabilizing them and disrupting their translation. Although the miRNA expression profiles in various mouse organs and tissues have been investigated, there have been no standard methods for purifying and quantifying mouse kidney and serum miRNAs. We have established an effective and reliable method for extracting and evaluating the miRNA expression in the serum and kidney of mice with age-dependent renal impairment. 
The method uses quantitative reverse-transcription-polymerase chain reaction (qRT-PCR), and the protocol requires six steps: (1) preparing senescence-accelerated mouse resistance 1 (SAMR1) mice and senescence-accelerated mouse prone (SAMP1) mice; (2) extracting serum samples from these mice; (3) extracting a kidney sample from each mouse; (4) extracting total RNA (including miRNA) from kidney and serum samples from each mouse; (5) the synthesis of complementary DNA (cDNA) with reverse transcription from the miRNA; (6) conducting a qRT-PCR using the cDNA obtained. 
This protocol was used to confirm that, compared to the controls, the expression of miRNA-7218-5p and miRNA-7219-5p was significantly changed in the kidney and serum of a mouse model of age-dependent renal impairment. This protocol also clarified the relationship between the kidney and serum of the mouse model of age-dependent renal impairment. This protocol can be used to determine miRNA expression in the kidney and serum of mice with age-dependent renal impairment.

We present a method for evaluating microRNA expression in the kidney and serum of mice with age-dependent renal impairment by quantitative reverse-transcription polymerase chain reaction.

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

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.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Medicine

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Biology

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

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including the kidneys. Under such circumstances, the kidney loses its ability to clean waste from the blood and regulate salt and fluid concentrations, eventually leading to renal failure. Women, particularly those of childbearing age, are diagnosed nine times more often than men. Kidney disease is the leading cause of mortality in SLE patients. The present protocol describes a quick and simple method to measure excreted protein levels in collected urine, tracking lupus progression over time. In addition, an approach to isolate kidney mononuclear cells is provided based on size and density selection to investigate renal infiltration of leukocytes. Furthermore, an immunohistochemical method has been developed to characterize protein deposition in the glomeruli and leukocyte infiltration in the tubulointerstitial space. Together, these methods can help investigate the progression of chronic inflammation associated with the kidneys of lupus-prone MRL/lpr mice.

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.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including ...

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

Treating Immunoglobulin A Nephropathy (IgAN) with Dioscin in a Murine Model

Dioscin Mediated IgA Nephropathy Alleviation by Inhibiting B Cell Activation In Vivo and Decreasing Galactose-Deficient IgA1 Production In Vitro

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal immune responses, which is a critical link in the pathogenesis of IgA nephropathy (IgAN). Peyer&#39;s patch, the prominent place where B lymphocytes are transformed into IgA-secreting plasma cells, is the primary source of IgA. In addition, the lower expression of core 1&#946;-1,3-galactosyltransferase (C1GalT1) and its molecular chaperone, C1GalT1-specific molecular chaperone (Cosmc), is related to abnormal glycosylation of IgA1 in IgAN patients. Our clinical experience shows that Dioscoreae Nipponicae Rhizoma&#39;s (DNR) herbal medicine can relieve proteinuria and hematuria and improve renal function in IgAN patients. Dioscin (DIO) is one of the main active ingredients of DNR, which has various pharmacological activities. This study explores DIO&#39;s possible mechanism in treating IgAN.The IgAN model mouse was established by mucosal immune induction. The mice were divided into the control, model, and DIO gavage groups. The glomerular IgA deposition in mice, renal pathological changes, and B cell markers CD20 and CXCR5 expression in Peyer&#39;s patch were detected by immunofluorescence and immunohistochemistry. After lipopolysaccharide (LPS) stimulation, DIO&#39;s effects on DAKIKI cells proliferation, IgA and Gd-IgA1 secretion, C1GalT1, and Cosmc expression were studied by cell counting kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA) test, quantitative real-time polymerase chain reaction (QRT-PCR), and western blotting (WB). In in vivo studies, IgA deposition accompanied by glomerular mesangial hyperplasia and increased expression of CD20 and CXCR5 in Peyer&#39;s patch in the IgAN model mouse was alleviated by DIO. In vitro studies showed 0.25 &#181;g/mL to 1.0 &#181;g/mL DIO inhibited LPS-induced DAKIKI cell proliferation, IgA and Gd-IgA1 secretion, and up-regulated the mRNA and protein expression of C1GalT1 and Cosmc. This study demonstrates that DIO may reduce Gd-IgA1 production by inhibiting excessive activation of IgA-secreting cells and up-regulating C1GALT1/Cosmc expression.

Author Spotlight: Investigating the Potential of Chinese Herbal Medicinal Active Dioscin in Treating IgA Nephropathy

This study provides experimental data for treating immunoglobulin A nephropathy (IgAN) with Dioscin (DIO), the active ingredient of Dioscoreae Nipponicae Rhizoma (DNR), and a paradigm for studying herbal medicine's effects and underlying mechanisms in vivo and in vitro.

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal ...

Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely used to elucidate the molecular and cellular mechanisms underlying kidney interstitial fibrosis and to identify potential therapeutic targets. This model is established in mice through surgical ligation of a unilateral ureter, a procedure that is relatively straightforward and easy to perform. However, the UUO mouse model is known to exhibit significant variability and inconsistency, influenced by factors such as mouse strain, age, sex, anesthesia type, duration of surgery, body temperature during the procedure, the operator's surgical skills, feeding conditions, and the overall health status of the mice. Variations in surgical techniques, suture placement, and the duration of obstruction contribute to the variability in outcomes. Additionally, inconsistent sampling of obstructed kidneys further increases variability in the assessment of kidney fibrosis. This study outlines the process of developing the UUO mouse model and evaluating interstitial fibrosis, discusses the technical challenges contributing to the model's unpredictability, and proposes potential solutions. These insights aim to establish a more standardized and universally applicable approach for investigating kidney fibrosis.

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.

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely ...

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

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