Name: analyses of proteinuria, renal infiltration of leukocytes, and renal deposition of proteins in lupus-prone mrl/lpr mice
Uploaded: 2022-06-08T13:00:00
Description: Watch this Scientific Journal Video about Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice at JoVE.com

We thank the Flow Cytometry Core Facility, the Histopathology Laboratory, the Fralin Imaging Center at Virginia Polytechnic Institute, and State University for technical support. This work is supported by various NIH and internal grants.

The present protocol is approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Tech. Since lupus disease has a higher incidence in females, only female MRL/lpr mice were used. The sample collection was started at 4 weeks of age and finished at 15 weeks. The mice were obtained from commercial sources (see Table of Materials) and were bred and maintained in a specific pathogen-free environment following the institutional guidelines.
1. Proteinuria ...

<ol>
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LN is a leading cause of mortality in SLE patients, and factors aggravating the disease remain unclear. The application of this protocol is to characterize renal function using multiple methods, including measurement of proteinuria, FACS analysis of isolated kidney leukocytes, and immunofluorescence staining of frozen kidney sections.
One important point to consider while collecting urine is that one has to be consistent with the time of the day and the location of urine collection. Mice are n...

The kidney&#39;s primary function is the elimination of toxic substances through urine while maintaining the homeostasis of water and salts1. This function is threatened in patients with systemic lupus erythematosus (SLE), leading to so-called lupus nephritis (LN). LN is a consequence of the immune system attacking the kidney, leading to persistent kidney inflammation, therefore losing its ability to clean waste from the blood and regulate salt and fluid concentrations. This will eventually lead to renal failure, which can be fatal. During the nephritic process, circulating B cells, T cells, and monocytes are recruited to the kidney, secreting chemokines, cytokines, and immune complex-forming autoantibodies. This ultimately results in endothelial cell damage, membranous injuries, renal tubular atrophy, and fibrosis2.
MRL/Mp-Faslpr (MRL/lpr) lupus-prone mice is a classical mouse model exhibiting lupus-like clinical signs that resemble human SLE3. This model has been instrumental in understanding one of the leading causes of mortality in SLE patients, lupus nephritis (LN)4. In both human and mouse SLE, LN is characterized by gradual inflammation triggered by renal deposition of immune complexes, followed by complement activation, recruitment of inflammatory cells, and loss of renal function5. The immune complex deposition is the first step to induce chemokine and cytokine production by intrinsic renal cells, which expands the inflammatory response by recruiting immune cells6. The current protocol presents several techniques to follow renal disease progression that analyze cell infiltration and immune complex deposition.
Urine collected every week allows for detection and visualization of the time course of proteinuria before, during, and after lupus onset. Proteinuria as a biomarker can determine the biological progression of LN. Other advantages of this technique are that it is non-invasive, cost-efficient, and easy to implement7. When the kidney is working perfectly, the proteinuria level is consistently low; however, in MRL/lpr mice, after 8-9 weeks of age, a gradual increase of the proteinuria level, that is eventually high enough to cause renal failure8, is observed. Multiple reagent strips and colorimetric reagents are commercially available to monitor the issue. However, the Bradford assay is cheap and very accurate in determining the onset of proteinuria and the course of lupus nephritis. This assay is quick, and the reagent is not affected by the presence of solvents, buffers, reducing agents, and metal-chelating agents that may be in your sample9,10,11.
One important aspect to consider is cell infiltration in the kidney. These infiltrates promote pathogenesis by triggering the secretion of soluble factors such as cytokines to worsen inflammation12. To better understand what cell populations are present in the infiltrates, a useful method is to isolate leukocytes13. Here, the detection of renal infiltration of B cells is used as an example. The procedure begins with a digestion process with deoxyribonuclease (DNase) and collagenase, followed by density gradient separation that removes debris, red blood cells, and dense granulocytes. The reason for isolating B cells (CD19+) and plasma cells (CD138+) is that lupus kidneys can concentrate these cells14. It is suggested that the presence of B cells in small aggregates in the kidney can indicate clonal expansion and, consequently, immunoglobulin (Ig) production. Plasma cells are well-known to be present in these aggregates as well15. Once leukocytes have been isolated, fluorescence-activated cell sorting (FACS) can be used to analyze the cells of interest upon staining with different fluorescence-conjugated antibodies.
Immunofluorescence is one of the immunohistochemistry (IHC) detection methods that allows for fluorescent visualization of proteins in 4 &#956;m thick kidney tissue samples. Other IHC detection methods depend on the nature of analytes, binding chemistry, and other factors16. Immunofluorescence is a rapid identification method that exposes the antigen to its counterpart antibody labeled with a specific fluorochrome (or a fluorescent dye). When excited, it produces light that a fluorescence microscope can detect. This technique can be used to observe the deposition of complement C3 and IgG2a17. Excessive complement cascade activation could be associated with an uncontrolled immune response and loss-of-function18. Immune deposition of anti-double-stranded DNA (anti-dsDNA) autoantibodies in the kidney is a major concern19, where those with IgG2a isotype have been associated with LN20. Specifically, anti-dsDNA antibodies exhibit more pathogenicity and affinity to nuclear materials, forming immune complexes21. When IgG2a is present, the complement cascade, including C3, is activated to clear the immune complexes22. The C3 and IgG2a markers can be quantified individually or overlaid to establish their correlation.
Notably, serum creatinine measurement is another reliable technique that can be used together with microscopic hematuria and kidney biopsies to diagnose LN23. However, the presence of proteinuria is a strong indicator of glomerular damage. In that sense, monitoring the proteinuria level during lupus can detect disease onset and complement other methods for diagnosing lupus. In addition, immune complexes deposited in glomeruli can induce an inflammatory response, activate the complement system, and recruit more inflammatory cells. Another noteworthy point of this protocol is B cell infiltration in the kidney. This, together with the infiltrated T cells, amplifies local immune responses that trigger organ damage. Importantly, the classification of LN is not only based on glomerular morphologic changes seen in microscopy but also immune deposits observed with immunofluorescence. Therefore, in this protocol, accurate and cost-effective methods for the analysis of renal function are offered in laboratory settings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris-Buffered Saline (TBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>J60764.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human/Mouse C3</td>
			<td>Cedarlane</td>
			<td>CL7632F</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD138 BV711</td>
			<td>Biolegend</td>
			<td>142519</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 AF700</td>
			<td>Biolegend</td>
			<td>103127</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Sigma-Aldrich</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope LSM 880</td>
			<td>Zeiss</td>
			<td>LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Fisher Scientific</td>
			<td>50-212-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold</td>
			<td>Fisher Scientific</td>
			<td>NC9511236</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient medium</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Percoll</td>
		</tr>
		<tr>
			<td>DEPC-Treated water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9906</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>dsDNA-EC</td>
			<td>InvivoGen</td>
			<td>tlrl-ecdna</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic Acid</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>EVOS M5000 Microscope imaging system</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMF5000</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Fusion Cell sorter</td>
			<td>BD Biosciences</td>
			<td>FACS Fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium, Heat Inactivated</td>
			<td>R&#38;D systems</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand 96-Well Polystyrene Plates</td>
			<td>Fisher Scientific</td>
			<td>12-565-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad prism</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175-079</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus reuteri Kandler et al.</td>
			<td>ATCC</td>
			<td>23272</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>MRL/MpJ-Fas lpr /J Mice (MRL/lpr)</td>
			<td>Jackson Lab</td>
			<td>485</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail enamel</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any conventional store</td>
		</tr>
		<tr>
			<td>O.C.T compound</td>
			<td>Tisse-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way Disposable Embedding Molds</td>
			<td>Polysciences</td>
			<td>R-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 20x TBS Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>28358</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Coomassie Plus (Bradford) Assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23236</td>
			<td>Albumin standard included</td>
		</tr>
		<tr>
			<td>ProLon Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>FcR block</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>SoftMax Pro 6.1 software</td>
		</tr>
		<tr>
			<td>Sterile cell Strainers 100 &#181;M</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin Hydrochloride</td>
			<td>Goldbio</td>
			<td>V-200-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua</td>
			<td>Biolegend</td>
			<td>423102</td>
			<td>fluorescent dye for flow cytometry analysis</td>
		</tr>
	</tbody>
</table>

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 protocol uses multiple methods to assess MRL/lpr mice for lupus nephritis. First, a procedure is described to study increased proteinuria levels due to kidney dysfunction over time. As shown in Figure 1, female mice were treated with oral gavage of 200 &#181;L of phosphate-buffered saline (1x PBS) as the control group and probiotic Lactobacillus reuteri as the treatment group, at a concentration of 109 cfu/mL, twice a week. Treatment started at 3 weeks old and finishe...

Watch this Scientific Journal Video about Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice at JoVE.com

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in 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 ...

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