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

Summary

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.

Abstract

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.

Introduction

The kidney'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-Fas^lpr (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 sample^9,10,11.

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 μm 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.

Protocol

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 …

Representative Results

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 µL 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…

Discussion

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…

Materials

10x Tris-Buffered Saline (TBS)	Thermo Fisher Scientific	J60764.K2
2-mercaptoethanol	Thermo Fisher Scientific	21985-023
Anti-Human/Mouse C3	Cedarlane	CL7632F
Anti-Mouse CD138 BV711	Biolegend	142519
Anti-Mouse CD45 AF700	Biolegend	103127
Bovine Serum Albumin	Sigma-Aldrich	A9418-100G
Collagenase D	Sigma-Aldrich	11088882001
Confocal Microscope LSM 880	Zeiss	LSM 880
Coplin jar	Fisher Scientific	50-212-281
Cryomold	Fisher Scientific	NC9511236
Density gradient medium	GE Healthcare	17-1440-02	Percoll
DEPC-Treated water	Thermo Fisher Scientific	AM9906
DNase I	Sigma-Aldrich	D4527
dsDNA-EC	InvivoGen	tlrl-ecdna
Ethylenediaminetetraacetic Acid	Fisher Scientific	S311-500	EDTA
EVOS M5000 Microscope imaging system	Thermo Fisher Scientific	AMF5000
FACS Fusion Cell sorter	BD Biosciences	FACS Fusion
Fetal Bovine Serum – Premium, Heat Inactivated	R&D systems	S11150H
Fisherbrand 96-Well Polystyrene Plates	Fisher Scientific	12-565-501
Graphpad prism	GraphPad	N/A
Hank’s Balanced Salt Solution	Thermo Fisher Scientific	14175-079
HEPES	Thermo Fisher Scientific	15630-080
ImageJ software	National Institutes of Health	N/A
Lactobacillus reuteri Kandler et al.	ATCC	23272
MEM non-essential amino acids	Thermo Fisher Scientific	11140-050
MRL/MpJ-Fas lpr /J Mice (MRL/lpr)	Jackson Lab	485
Nail enamel	N/A	N/A	Any conventional store
O.C.T compound	Tisse-Tek	4583
PAP pen	Sigma-Aldrich	Z377821
Peel-A-Way Disposable Embedding Molds	Polysciences	R-30
Penicillin-Streptomycin	Thermo Fisher Scientific	15140-122
Phosphate-buffered saline (PBS)	Thermo Fisher Scientific	70011069
Pierce 20x TBS Buffer	Thermo Fisher Scientific	28358
Pierce Coomassie Plus (Bradford) Assay kit	Thermo Fisher Scientific	23236	Albumin standard included
ProLon Gold Antifade Mountant	ThermoFisher	P36934
Purified Rat Anti-Mouse CD16/CD32	BD Biosciences	553141	FcR block
RPMI 1640	Thermo Fisher Scientific	11875-093
Sodium pyruvate	Thermo Fisher Scientific	11360-070
SpectraMax M5	Molecular Devices	N/A	SoftMax Pro 6.1 software
Sterile cell Strainers 100 µM	Fisher Scientific	22363549
Tween 20	Fisher Scientific	BP337-500
Vancomycin Hydrochloride	Goldbio	V-200-1
Zombie Aqua	Biolegend	423102	fluorescent dye for flow cytometry analysis