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 test

1. Collect urine following the steps below.
   1. Sterilize the hood by spraying 70% ethanol. Place a sterile plastic sheet on the surface. Prepare tips, 1.5 mL tubes, and a pipette to collect about 20 μL of the liquid.
      NOTE: Tips and tubes must be sterile and without any pre-treatment.
   2. Take the cage and spray 70% ethanol before putting it inside the hood.
   3. Hold the mouse with one hand and use the other hand to massage the ventral area from top to bottom gently.
   4. Use a 1.5 mL tube or pipette to collect the urine directly, or if the sample drops, collect it from the plastic sheet. Use fresh gloves, sheets, and tips for each sample.
      NOTE: If this does not work, use a sterile beaker to isolate the mouse, then collect the urine from the beaker after the mouse urinates.
   5. Put the mouse back in the cage. Take out another mouse and repeat steps 1.1.3-1.1.4.
      NOTE: Always clean the plastic sheet and the beaker with 70% ethanol after collecting the sample for each mouse. At least five mice per group are necessary for statistical significance. Urine samples can be stored at -80 °C until use for up to 12 months.
2. Quantify the proteins by the Bradford method²⁴.
   1. Allow urine samples, Albumin standard, and the Bradford reagent to come to room temperature (RT) by keeping them outside the freezer for 10-20 min.
      NOTE: Bradford reagent and Albumin standard are included in a commercially available kit (see Table of Materials). The starting concentration of the Albumin standard is 2 mg/mL. Serial dilutions (1:2 six times) must be done with water.
   2. Add Bradford reagent to a 96-well plate (200 μL/well), undiluted.
   3. Pipette 5 µL of undiluted urine sample per well and use the tip to pipette up and down several times to mix properly.
   4. Add standards (400, 200, 100, 50, 25, 12.5 mg/dL) in duplicates. Leave a couple of wells as blanks.
      ​NOTE: The addition of samples and standards needs to be done quickly.
   5. Let it stand for 5-10 min at RT.
   6. Read the plate at 595 nm in a plate reader as per the manufacturer's protocol (see Table of Materials).

2. Isolation of the kidney cells

1. Euthanize the mouse with CO₂ (flow rate: 30%-70% volume/min, to achieve unconsciousness or death) in a chamber followed by cervical dislocation. Proceed with the dissection to collect both kidneys. Place one-and-a-half-kidneys in an ice-cold C10 medium. Put the other half kidney in OCT (step 3.1.1).
   NOTE: C10 is made with RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM of sodium pyruvate, 1% of 100x MEM nonessential amino acids, 10 mM of HEPES, 55 µM of 2-mercaptoethanol, and 100 U/mL of penicillin-streptomycin (see Table of Materials).
2. Cut the kidneys into grain size (1-2 mm³ pieces) and digest in 5 mL of digestion buffer containing 1 mg/mL of collagenase and 0.2 mg/mL of DNase I (see Table of Materials) in RPMI 1640 medium containing 10 mmol/L of HEPES for 1 h with continuous gentle shaking at 37 °C.
   NOTE: Add 1 mL digestion buffer in a sterile 2 mL tube and use sterile scissors to chop the kidney.
3. Add 10 mL of 1x ice-cold PBS containing 10 mM of ethylenediaminetetraacetic acid (EDTA) and incubate for 10 min on ice. Then vortex the suspension twice.
4. Filter the cell suspension through a 100 µM strainer and wash with 10 mL of HBSS-full.
   NOTE: HBSS-full contains 5 mM of EDTA, 0.1% BSA, and 10 mM of HEPES.
5. Centrifuge at 350 x g for 10 min at RT.
6. Prepare 30%, 37% and 70% Stock Isotonic Percoll (SIP) solution.
   NOTE: Mix 1 part volume of 10x PBS and 9 parts volume of Percoll to prepare SIP (see Table of Materials); use HBSS-full to dilute SIP to 30%, 37%, and 70% SIP.
7. Resuspend cells in 5 mL of 30% SIP and load 37% of 5 mL (top) and 70% of 5 mL (bottom), making SIP gradient, slowly with a paster pipette.
8. Centrifuge with Up9 down0 (or brake 0) at 1000 x g for 30 min at RT.
9. Collect leukocytes from the 37%-70% interface and resuspend them in 5 mL of C10.
10. Centrifuge at 350 x g for 10 min at 4 °C.
11. Resuspend the cell pellet in 3 mL of FACS buffer. Cells are ready for FACS staining.
    NOTE: FACS buffer contains 1x PBS and 0.1% Bovine Serum Albumin (BSA).
12. Centrifuge at 350 x g for 5 min at 4 °C and discard the supernatant (SN), leaving about 50 µL.
    NOTE: To remove the supernatant, carefully pour or pipette the solution away from the solid or use a semiautomatic vacuum to aspirate the supernatant.
13. Add 50 µL of FcR block (see Table of Materials) per tube (prediluted 1/50 in 1x PBS); mix and incubate on ice in the dark for 10 min.
14. Add 2 mL of FACS buffer to wash.
15. Centrifuge at 350 x g for 5 min at 4 °C and discard SN, leaving about 50 µL.
16. Add 20 µL of the appropriate fluorescent dye (dilute 1/100 with 1x PBS, see Table of Materials) and let it stand for 15-30 min on ice.
17. Add 50 µL of antibody¹⁵ mix per tube: CD138-BV711 (1/200 in 1x PBS) and CD45-AF700 (1/200 in 1x PBS) (see Table of Materials).
18. Incubate on ice in the dark for 15-30 min and add 3 mL of FACS buffer.
19. Centrifuge at 350 x g for 5 min at 4 °C and vacuum SN, leaving about 50 µL. Resuspend in 200 µL of 1x PBS. This is now ready to be analyzed by flow cytometry.

3. Immunofluorescent staining

1. Perform the sample collection.
   1. Embed the half kidney in the small plastic cryomold with the Optimal Cutting temperature (OCT) compound (see Table of Materials).
   2. Add dry ice into a polystyrene foam box (see Table of Materials), and carefully put the cryomold on top of the dry ice. Ensure that the cryomolds are placed flat.
      NOTE: It takes 3-4 min for the OCT compound to solidify and turn white.
   3. Take the cryomold out and wrap it in aluminum foil. Store it at -80 °C until further use.
      NOTE: This can be stored at -80 °C for at least 1 year.
2. Perform sectioning and fixing of the sample.
   1. Cut the samples into 4 µm sections, wait for at least 2-3 h to dry, and then fix with cold acetone (at 4 °C) for 10 min.
      NOTE: Be careful when using acetone, which requires a specialized chemical hood.
   2. After fixing the slides, air dry for 0.5-1 h and store them for a short time at -20 °C, or for a long time at -80 °C.
3. Stain the samples.
   1. Refix the slides in cold acetone for 5-10 min.
   2. Allow the sections to warm up to RT. Use a PAP pen (see Table of Materials) around the section and allow it to dry.
      NOTE: Nail enamel can be used as well. This step prevents antibody dilution from floating all over the slide.
   3. Place slides in 1x Tris-buffered saline (TBS) in the Coplin jar (see Table of Materials) for 20 min, put the jar on the shaker, and shake gently.
   4. Pour off 1x TBS and fill the jar with 1x TBS, 5% BSA, and 0.1% Tween 20. Incubate for 20 min on the shaker, shake gently.
   5. Take the slides out and wipe the bottom of the slides dry. If necessary, shake the slides dry. Add 100 µL of conjugated antibodies²⁵ C3-FITC (1/100) and IgG2a-PE (1/100) to the section (see Table of Materials). Incubate at RT in a humidified chamber for 1 h.
   6. Wash the section 3 times in 1x TBS, 5% BSA, and 0.1 % Tween 20 for 10 min on the shaker.
   7. Shake dry the slides and mount the section with an antifade reagent (see Table of Materials) then a coverslip.
   8. Store the slides at 4 °C until viewing under the microscope (e.g., confocal microscope or imaging system microscope). Quantify images using software and subtract background signals when comparing the fluorescence intensity between regions.
   9. For image analysis using the ImageJ software (see Table of Materials), outline the desired region.
   10. Set standard parameters by clicking on Analyze, then Set Measurements and Measure Area to get the results.
   11. Transfer the data obtained from the measure windows to a spreadsheet.
       NOTE: A small area can be selected from the image as a background; it shouldn't have any fluorescence.
   12. Once done, click on Analyze to measure the region and transfer data again to the previous spreadsheet.
   13. Repeat steps 3.3.11-3.3.12 for several regions.
   14. Calculate the mean fluorescence as Corrected Cell Fluorescence (CCF) = Integrated density - (Selected cell region x Mean background fluorescence).
   15. Calculate for each region and analyze data using the graphing and statistics software (see Table of Materials).

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

Name	Company	Catalog Number	Comments
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