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

Summary

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.

Abstract

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.

Introduction

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA in the renal glomerular mesangial region^1,2. It is the most common of the primary glomerulonephritis and leads to the end-stage renal failure in 20%–40% of patients². The cause is still unknown but persistent mucosal infection has been implicated^1,3. Corticosteroids, immunosuppressants, and renin−angiotensin system inhibitors have been proposed as therapeutic methods^1....

Protocol

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 age^8,9,10,11. Balb/c mice should be selected as the control group^8,9,

Results

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

Discussion

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 targets^1,3. 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 .......

Materials

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