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 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 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-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 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.
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
2. Isolation of the kidney cells
3. Immunofluorescent staining
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 finished at 15 weeks old. The mice group treated with L. reuteri had a more aggressive proteinuria progression than the control group.
Figure 1: Detection of protein levels in the urine of MRL/lpr mice over time. n = 5 mice per group. The statistics were performed with the simple linear regression test. *P < 0.05, **P < 0.01. Please click here to view a larger version of this figure.
Figure 2 shows the FACS analysis of the isolated kidney leukocytes. The mice were treated with vancomycin (2 g/L) or vancomycin plus E. coli dsDNA (80 µg) in this experiment. Vancomycin was given along with the drinking water during the indicated period of time. Oral gavage of bacterial DNA was administered to the vancomycin-treated mice once a week for four consecutive weeks at 4, 5, 6, and 7 weeks of age. E. coli dsDNA was expected to trigger the renal infiltration of plasma cells leading to compromised kidney function, but no significant difference was found.
Figure 2: Analysis of plasma cells in isolated kidney leukocytes. (A) Sequential gating strategy: total cells, single cells, live cells, CD45+ cells, and finally CD138+ cells. (B) Percentage of plasma cells after treatment with van (Vancomycin) or van + DNA (Vancomycin + E. coli dsDNA) for 3 weeks (n ≥ 5 mice per group). No statistical significance ('ns') was observed. Please click here to view a larger version of this figure.
Figure 3 shows immunofluorescence staining of complement C3 and IgG2a on female MRL/lpr kidney sections. In this experiment, the effect of environmental factors was compared concerning the renal deposition of C3 and IgG2a. In-house mice were bred in the animal facility for several generations, and they were compared to recently purchased mice from a vendor. The immunohistochemical analysis did not show any difference between different facilities.
Figure 3: Detection of complement C3 and IgG2a in kidney sections via immunofluorescence staining. (A) Representative pictures of kidney sections stained with anti-C3-FITC (Green) and anti-IgG2a-PE (Red). (B) Comparison of corrected cell fluorescence (CCF) between the two groups (n ≥ 5 mice per group). Scale bar = 100 µM. No statistical significance ('ns') was observed. Please click here to view a larger version of this figure.
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 nocturnal animals, so the most accurate samples are collected in the late afternoon before mice start being active. Another reason for choosing this time is that MRL/lpr mice tend to drink a lot of water at night, leading to diluted urine. Thus, collection at the wrong time may increase the urine's concentration and/or volume variabilities. It is also important to collect as much urine as possible to get accurate results.
The Bradford assay gives us an estimate of how much protein is present. It is not specific for any SLE marker proteins but good enough to give an idea of disease progression26. In addition, this method is cost-effective compared to others. Notably, the Bradford assay is time-sensitive; therefore, it must be completed within half an hour. Samples tend to have increased values or high false results if one waits too long. Another benefit compared to the commercial reagent strips for urinalysis is that it gives accurate numbers rather than a range within the urine sample. Scoring strips can give ambiguous results, particularly in the higher range9,10,24.
For the isolation of kidney leukocytes, the first key step is to perform the dissection as fast as possible. The viability of kidney cells greatly impacts the success of subsequent FACS analysis. When kidneys are being processed, each step is time- and temperature-sensitive. It needs to be noted that DNase I and collagenase concentrations and temperature are important for maximum efficiency, as they eliminate DNA and allow the disintegration of the organ, respectively27. The second key step is isolating cells, divided into two important processes. The first one is using the strainer that allows the removal of tissue debris; the second process involving Percoll is the most crucial step and requires focus and patience. When doing the density gradient with Percoll, it is important to maintain clear interfaces between different concentrations of SIP. It is recommended to use a Pasteur pipette since the flux and pressure can be controlled manually. Additional phases can be better built with the slow dispense of the SIP solution or even drop by drop. Once phases are established, the tube needs to be handled gently; otherwise, the phases will mix immediately, and samples will be lost, as it is impossible to separate the phases again. In addition, it is important to centrifuge the tube without a break because breaking could be too aggressive, leading to the loss of separate phases. The leukocytes are then recovered from interphase between 30% and 37% SIP.We analyzed one cell population in this example, plasma cells (CD45+ and CD138+). However, this technique is not limited to B cells; it can be used for other cell types and intracellular staining28.
The third method is a powerful tool for visualizing intracellular localizations of given protein depositions. Once tissues are fixed with acetone, it is important to perform adequate washing, so antibodies only attach to the target. Next, blocking with BSA reduces nonspecific binding and false positives. Direct immunofluorescence is usually recommended, as it is faster than indirect immunofluorescence with a secondary antibody, which is time-consuming, especially considering the extra washing and incubation steps. However, direct immunofluorescence restricts the flexibility of antibody selection compared to indirect immunofluorescence29. Therefore, both can be valuable methods depending on the purpose. Furthermore, it needs to be mentioned that dilution factors are key while staining for IHC. If the dilution is not well done, increased background (not enough antibody dilution) or weak staining (too much dilatation) will show on the microscopy images. It is important to start with serial dilutions of the antibody to optimize the antibody concentration for a better target-to-background ratio. Another step to consider is the amount of incubation time with the antibodies. The longer the antibody is in contact with the sample, the more nonspecific binding can be created.
The limitations of the present methods are as follows. Certain clinical parameters for LN, such as the albumin/creatinine ratio, cannot be revealed using these methods. In addition, some samples may have poor acid solubility leading to a high concentration reading above the standard curve. This requires further dilutions of samples or the addition of surfactants to precipitate the dye30. Moreover, this protocol may not be suitable if many cells are needed after kidney leukocyte isolation. The current kidney cell isolation protocol involves several washing steps, digestion, and isolation steps to maximize the removal of other cells, so even though the cell purity is high at the end, the cell number is usually low. Furthermore, it is a long procedure, so cell viability can be compromised. Other ready-to-use assays are more suitable for clinical use; however, the present methods may provide accurate results with a smaller budget for research laboratories.
In summary, three different efficient and accurate techniques are presented in this protocol to characterize the LN progression. Combining the Bradford method for the proteinuria level, FACS analysis for renal infiltration of the leukocytes, and IHC analysis for the renal deposition of IgG2a and C3, a clear picture of renal dysfunction in female MRL/lpr mice as a model of human LN is successfully established.
The authors have nothing to disclose.
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.
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 |