LDL Receptor-Related Protein 2 (Megalin) as a Target Antigen in Human Kidney Anti-Brush Border Antibody Disease

Abstract

Primary renal tubulointerstitial disease resulting from proximal tubule antigen–specific antibodies and immune complex formation has not been well characterized in humans. We report a cohort of patients with a distinct, underappreciated kidney disease characterized by kidney antibrush border antibodies and renal failure (ABBA disease). We identified ten patients with ABBA disease who had a combination of proximal tubule damage, IgG-positive immune deposits in the tubular basement membrane, and circulating antibodies reactive with normal human kidney proximal tubular brush border. All but one of the patients also had segmental glomerular deposits on renal biopsy specimen. Patients with ABBA disease were elderly and presented with AKI and subnephrotic proteinuria. Serum from all patients but not controls recognized a high molecular weight protein in renal tubular protein extracts that we identified as LDL receptor-related protein 2 (LRP2), also known as megalin, by immunoprecipitation and mass spectrometry. Immunostaining revealed that LRP2 specifically colocalized with IgG in the tubular immune deposits on the ABBA biopsy specimen but not the control specimen analyzed. Finally, ABBA serum samples but not control samples showed reactivity against recombinantly expressed N-terminal LRP2 fragments on Western blots and immunoprecipitated the recombinantly expressed N-terminal region of LRP2. This case series details the clinicopathologic findings of patients with ABBA disease and shows that the antigenic target of these autoantibodies is LRP2. Future studies are needed to determine the disease prevalence, stimulus for ABBA, and optimal treatment.

AKI is typically caused by nephrotoxic and/or ischemic insult to the renal tubular epithelium and has a broad differential diagnosis that includes other etiologies, such as acute GN, vasculitis, interstitial nephritis, and thrombotic microangiopathy.¹ Autoimmune diseases with antibodies against glomerular antigens are well recognized and routinely evaluated in patients with kidney disease. However, tubular injury as a result of direct immunologic insult is not part of the routine evaluation of patients with AKI.

In 2016, a case was reported describing a patient with a distinctive form of tubulointerstitial nephritis who exhibited immune complexes in the tubular basement membrane (TBM) and rare epimembranous glomerular deposits associated with antibrush border antibodies (ABBAs) reactive with normal proximal tubules.² The disease led to renal failure and recurred in a transplant. Although primary renal tubulointerstitial disease caused by antibodies to proximal tubule antigens with immune complex formation is well described in animal models,^3–7 we found only two other human patient reports with confirmed serum ABBA.^8,9 These findings prompted us to seek other examples of ABBA disease to characterize the clinical and pathologic phenotype and identify the target antigen.

Here, we report the first case series of ABBA disease, including a detailed description of its clinicopathologic characteristics. Sera from these patients were examined by Western blot and immunoprecipitation using human tubulointerstitial extract. Subsequent analysis with mass spectrometry and confirmatory testing with protein-specific reagents enabled identification of the target protein of these circulating autoantibodies.

Results

Clinical and Pathologic Details

We identified ten patients with ABBA disease, including one previously reported (Table 1).² The mean age was 72.9 years old, with a preponderance of men (seven men and three women). All presented with AKI (mean serum creatinine =6.2 mg/dl; median serum creatinine =4.0 mg/dl) and variable non-nephrotic proteinuria. Three patients had positive antinuclear antibodies, but none fulfilled criteria for SLE.

Table 1.

Clinical characteristics at time of presentation and follow-up data of renal disease in patients with anti-LRP2 nephropathy

Pt	Age	Sex	Race	Cr, mg/d	Prot, g	Hem	ANA	dsDNA	ANCA	C3	C4	S alb, g/dl	Past medical history	F/U, mo	Treatment	Outcome
1	68	M	H	8.8	NAa	NAa	—	—	—	Nml	Nml	2	Recent B cereus infection	8	Prednisone	Renal replacement
2	78	M	W	3	3.3	+	—	NP	BL+	Nml	Nml	4	DM and HTN	12	Prednisone, cytoxan	Serum Cr: 2 mg/dl; known serologic remission
3	76	M	W	1.7	0.5	+	1:320	—	NP	Nml	Nml	3.6	COPD	4	Rituximab	Renal replacement/deceased
4	69	F	W	2.7	1.5	+	—	—	—	Nml	Nml	3.5	DM	15	Prednisone	Serum Cr: 4.2 mg/dl
5	72	M	W	5	0.8	+	1:40	—	—	Nml	Nml	4.1	DM	3	Prednisone	Renal replacement/deceased
6	70	M	W	2.2	1.1	+	1:640	Weak +	—	Nml	Nml	4	HTN, gout, nephrolithiasis	10	None	Serum Cr: 2.3 mg/dl
7	73	M	W	15.2	1.3	+	—	—	—	Nml	Nml	?	CAD, DM, HTN	48	Prednisone	Kidney transplant with recurrence
8	66	F	W	6.7	0.9	+	—	—	—	Dec	Nml	4	DM, HTN	3	None	Serum Cr: 2.5 mg/d
9	77	F	W	2.3	0.6	+	—	—	—	Nml	Nml	3.7	Sarcoidosis	6	None	Serum Cr: 2.1 mg/dl
10	80	M	W	14.7	NAa	NAa	—	—	—	Nml	Nml	3.3	HTN	1.5	Prednisone	Renal replacement

Pt, patient; Cr, creatinine; Prot, 24-hour proteinuria; Hem, hematuria; ANA, antinuclear antibody; dsDNA, antidouble-stranded DNA; C4, serum complement C4; S alb, serum albumin; F/U, follow-up; M, man; H, Hispanic; NA, not applicable; —, negative; Nml, normal; W, white; NP, not performed; BL, borderline; DM, diabetes mellitus; HTN, hypertension; COPD, chronic obstructive pulmonary disease; F, woman; ?, unknown; CAD, coronary artery disease; Dec, decreased.

^aPatient was anuric on presentation.

Common renal biopsy findings included extensive tubular injury with apical cytoplasmic blebbing, loss of brush border, and regenerative changes (Figure 1, Supplemental Figures 1–3, Supplemental Tables 1 and 2, Table 2). GBMs had segmental “holes” in nine of ten patients, with no glomerular proliferation. Interstitial inflammation was variable, with six patients showing no to mild inflammation and four patients showing moderate to severe inflammation. All had granular IgG and serum complement C3 (C3) staining along TBMs and Bowman’s capsule. The intensity of staining for IgG and C3 was codominant in all patients. Tissue was available from eight patients for C5b9 staining and showed an identical staining pattern in each patient to the pattern of IgG and C3. The apical border of the proximal tubules highlighted with IgG in five of ten patients. Nine patients had rare segmental granular IgG and C3 staining of the GBMs. Glomerular phospholipase A2 receptor staining was negative in all patients. Glomerular thrombospondin type 1 domain–containing 7A staining was negative in the eight available patients. The immune deposits in the TBMs and GBMs were negative for IgA, IgM, or C1q. The IgG4 subclass was positive in all patients and predominant or codominant in nine of the ten (Supplemental Table 3). By electron microscopy, all patients had amorphous electron dense deposits along the TBMs, and nine had rare segmental subepithelial GBM deposits. Two patients had segmental mesangial deposits, whereas none had subendothelial deposits. All ten patients had serum IgG ABBA reactive to the proximal tubular brush border on sections of normal human kidney (Figure 2A).

Figure 1.

Characteristic renal biopsy features of ABBA disease (anti-LRP2 nephropathy). (A and B) Acute tubular injury was present in all biopsies with variable degrees of interstitial inflammation, including some with (A) none and others with (B) a mononuclear interstitial inflammatory infiltrate (hematoxylin and eosin). Original magnification, ×100. (C) Renal cortex with IgG staining of tubular epithelium along the apical surface and basolateral basement membrane (direct immunofluorescence). Original magnification, ×400. (D) Glomerulus with granular staining for IgG along Bowman’s capsule and segmentally within the capillary loops (direct immunofluorescence). Original magnification, ×400. (E and F) Transmission electron photomicrograph showing electron dense deposits (E) within and (F) along the apical aspect of TBM. Original magnification, ×5000.

Table 2.

Summary of morphologic features on biopsy

Morphologic Finding	Patients Involved
Light microscopy
Acute tubular injury	10/10
Interstitial inflammation
None	3/10
Mild	3/10
Moderate	3/10
Severe	1/10
Immunofluorescence
TBM IgG	10/10
Tubular brush border IgG staining	5/10
Segmental GBM IgG	9/10
Bowman’s capsule IgG	10/10
TBM LRP2	10/10

Figure 2.

Common brush border antigen among ABBA sera but not negative controls. (A) ABBA⁺ serum stains renal tubular brush border (indirect immunofluorescence using normal human kidney tissue, ×100). (B) Western blot (WB) of HTE with individual patient sera (lanes 1–4) or control sera (lanes 5–8); secondary antibody is sheep anti-human IgG1 for lane 1 and sheep anti-human IgG4 for lanes 2–8. (C) HTE was electrophoresed under nonreducing (NR) versus reducing conditions (red); then; it was Western blotted with ABBA⁺ serum (1:100) and detected with sheep anti-human IgG4.

Follow-up was available in all patients (Table 1). Five patients developed ESRD or were deceased at the time of follow-up. One (patient 7) received a renal transplant, and ABBA disease recurred.² Autopsy of patient 3 revealed persistent ABBA disease but no evidence of autoimmune disease in other organs, including the thyroid. Patient 2 was treated with prednisone and cyclophosphamide. At presentation, indirect immunofluorescence of this patient’s serum against normal kidney tubular brush border was positive at a titer of 1:1000. After 4 months, the ABBA titer decreased to 1:100. At 12 months of continuous treatment, the patient’s serum creatinine had stabilized at 2.0 mg/dl, with complete resolution of proteinuria. Indirect immunofluorescence for ABBA was negative at this last follow-up, indicating immunologic remission of disease.

Detection of a Target Antigen in Human Tubular Extract

Because all patients of ABBA-associated disease possessed circulating IgG capable of binding to normal proximal tubular brush border and exhibited TBM immune deposits, we sought to determine if all ABBA⁺ sera identified the same target antigen. Initial Western blots with sera from four patients with ABBA⁺ or controls showed specific binding of the ABBA⁺ sera to a high molecular weight protein in human tubular extract (HTE) (Figure 2B). The band appeared to be a doublet located at or above 300 kD, and reactivity with ABBA was lost under reducing conditions (Figure 2C). The predominant IgG subclass of ABBA was IgG4, with the exception of patient 1. Seventeen control sera, including healthy controls, patients with membranous glomerulopathy, patients with lupus nephritis, and patients with rheumatoid arthritis, failed to recognize the antigenic band.

Identification of LDL Receptor-Related Protein 2 as the Brush Border Antigen

Surface biotinylation of tubulointerstitial cells before extraction showed that the brush border antigen was surface exposed (Supplemental Figure 4) and confirmed that the antigen could be immunoprecipitated by human ABBA⁺ serum. Immunoprecipitates of HTE obtained with ABBA⁺ or control sera were electrophoresed under nonreducing conditions, and the appropriately sized gel region was excised and submitted for mass spectrometric analysis (Supplemental Material). The most promising candidate identified was LDL receptor-related protein 2 (LRP2; megalin), which was enriched by 330-fold in the ABBA⁺ versus the control immunoprecipitate (Supplemental Table 4). Proteomic analysis identified with high mass accuracy four biotinylated LRP2 peptides (Supplemental Figure 5) corresponding to two LDL class B receptor domains and one EGF-like domain. The analysis of the biotinylated sample identified 196 LRP2 peptides achieving 48% protein sequence coverage (Supplemental Figures 6–10). Additional mass spectrometry analysis of immunoprecipitates of partially proteolyzed HTE with ABBA versus control serum also identified LRP2 as the putative antigen with 5% LRP2 sequence coverage (Supplemental Figures 11 and 12). Although full-length LRP2 has a molecular mass of 517 kD, the protein is readily proteolyzed during purification and was initially known as gp330 due to its migration at a similar 330-kD position.¹⁰ The initial immunoprecipitates also contained fibronectin, but it was found to be a coprecipitating protein rather than the primary antigen; this was because it was not identified in our second mass spectrometry analysis with partially proteolyzed starting material, it was not present in the TBM deposits, and ABBA sera were not reactive with human fibronectin by Western blot (Supplemental Figures 13–15).

Colocalization of LRP2 and IgG Antibodies in Patients with ABBA

A polyclonal rabbit antibody raised against the C-terminal portion of the human LRP2 protein (amino acids 4447–4655; Abcam, Cambridge, MA) showed strong positive staining along the brush border of proximal tubules in sections from normal kidney, which colocalized with the staining pattern of human sera from patients with ABBA (Figure 3). This particular antibody did not recognize LRP2 within tubular deposits, but a mouse mAb directed against an internal portion of the protein (amino acids 2825–2955) showed positive granular staining in the TBM deposits in all ten patients with ABBA (Supplemental Table 2). There was no LRP2 staining within the segmental GBM deposits by either antibody. As shown by confocal analysis of kidney sections from patient 5 with ABBA⁺ (Figure 3), LRP2 colocalized with IgG in the TBM deposits. An isotype control antibody was negative in TBM deposits. The tubular basement membrane staining was blocked when cell extract expressing the third set of LA repeats (which contains the epitope recognized by the monoclonal antibody) is present (Supplemental Figure 16). The TBM staining for LRP2 was unique to patients with ABBA, because the stain was negative in the TBMs of all 40 controls exhibiting TBM deposits and the ten controls without TBM deposits. Therefore, positive LRP2 staining of TBM deposits had a high degree of sensitivity and specificity for ABBA disease.

Figure 3.

Colocalization of IgG and LRP2. (A–C) Immunofluorescence experiments performed on cryosections of normal human kidney tissue. (A) Staining for LRP2 (red) using a rabbit polyclonal antibody along the apical membrane of the tubular epithelium. (B) Indirect immunofluorescence of serum from a patient with ABBA on the normal human kidney. (C) Strong colocalization of LRP2 and serum antibodies from a patient with ABBA. (D–F) Immunofluorescence experiments performed on a renal biopsy sample from a patient with anti-LRP2 nephropathy shows granular TBM staining in a proximal tubule for LRP2 using (D) a mouse mAb and (E) IgG. (F) There is strong colocalization of LRP2 and IgG in the TBM deposits. (G–I) Immunofluorescence experiments performed on a renal biopsy sample from a patient with lupus nephritis shows staining of the tubular apical membrane for LRP2 using (G) a mouse mAb and (H) granular TBM staining for IgG. (I) There is complete lack of colocalization in a patient without anti-LRP2 nephropathy.

ABBA-Positive Serum Reacts Specifically with a Recombinant N-Terminal Fragment of LRP2

All four sets of LDL receptor class A (LA) repeats in LRP2 (Figure 4A) were individually expressed in HEK293 cells with C-terminal 3XFLAG tags (Figure 4B). When these constructs were probed by Western blot with ABBA and control sera, nine of ten ABBA sera specifically recognized a 45-kD band corresponding to the N-terminal set of seven LA repeats from LRP2. Among them, five patients were also reactive for the fragment LA26–32. One showed additional reactivity with the fragment LA16–25, and another patient (patient 5) was reactive with all four recombinant LRP2 fragments (Figure 4C). None of the control sera recognized any recombinant fragments of LRP2 (Supplemental Figure 17). ABBA sera (but not control sera) were able to immunoprecipitate the N-terminal region of LRP2 (Figure 4D).

Figure 4.

Reactivity of ABBA sera with recombinant (r) LRP2 fragments. (A) The domain structure of LRP2 is composed of four clusters of LA repeats, 17 EGF-like repeats, and eight YWTD (tyrosine-tryptophan-threonine-aspartate)-containing six-bladed β-propeller domains followed by a single transmembrane domain and a cytoplasmic tail. (B) An immunoblot under reducing conditions for the 3xFLAG tag to show the location of all four recombinant fragments. All fragments run slightly above their expected sizes of 35 kD for rLRP2_{(LA 1–7)}, 39 kD for rLRP2_{(LA 8–15)}, 50 kD for rLRP2_{(LA 16–25),} and 32 kD for rLRP2_(LA26–32). (C) ABBA serum from patient 5 is reactive with HTE and the four sets of LA repeats (nonreducing). Full-length LRP2 is close to the origin of the gel; the lower bands are proteolytic fragments that result from protease activity during preparation. (D) Sera from patients with ABBA immunoprecipitate the recombinant 3xFLAG-tagged N-terminal rLRP2_{(LA 1–7)} fragment (lanes 1–4) under reducing conditions, whereas sera from control patients do not (lanes 5–8). The immunoprecipitated product appears distorted on the blot due to the large amount of IgG heavy chain running directly above. IP, immunoprecipitate; MW, molecular weight; WB, Western blot.

Discussion

We have identified LRP2/megalin as the target proximal tubular brush border autoantigen in this cohort of patients with ABBA-associated disease. This disorder seems to affect older patients and often presents with AKI and subnephrotic proteinuria. In many patients, the course is progressive, resulting in severe kidney failure and ESRD. Typical renal biopsy findings include severe tubular injury by light microscopy and IgG-positive TBM, GBM, and Bowman’s capsule basement membrane deposits by immunofluorescence.

All individuals in this cohort exhibited circulating antibodies, often of the IgG4 subclass, to the same high molecular weight protein present in human tubulointerstitial extract. Immunoprecipitation and mass spectrometric analyses identified the candidate antigen as LRP2/megalin. In support of this finding, nine of ten of these sera were specifically reactive with the N-terminal recombinant fragment of human LRP2 by Western blot and/or immunoprecipitation (the overall titer of the tenth serum was weak and may not have been sufficient or specific to the truncated recombinant LRP2 protein). LRP2 and IgG colocalized within TBM deposits from ABBA-associated disease but not other tubulointerstitial diseases associated with TBM deposits. This granular TBM staining for LRP2 represents a highly sensitive and specific marker of ABBA-associated disease, which we now propose may be more specifically termed anti-LRP2 nephropathy.

LRP2/megalin is a very large (517-kD) single-pass transmembrane glycoprotein located on the apical membrane of the proximal tubule cells; it is concentrated in clathrin-coated pits at the base of the microvilli,¹¹ where it facilitates the endocytic uptake of albumin and low molecular weight proteins.¹² Its extracellular domain consists of four clusters of LA repeats that enable ligand binding and are separated by one or more tyrosine-tryptophan-threonine-aspartate-containing β-propeller domains,¹³ each flanked by one or two EGF-like domains.¹⁴ The protein was originally characterized as the major target antigen in Heymann nephritis, a rat model of membranous glomerulopathy, and variably called gp330¹⁰ and later, megalin.¹⁵ The mechanism by which antibodies raised against a tubular antigen can induce a glomerular disease (Heymann nephritis) was elucidated when immunohistochemical staining of Lewis rat kidney showed positive staining in both the proximal tubular brush border and the podocytes.¹⁶ In contrast, human LRP2/megalin was shown to be undetectable in podocytes and present only in human proximal tubules by immunohistochemical analysis,¹⁷ leading to the search for the human target antigen in membranous nephropathy that culminated in the identification of phospholipase A2 receptor.¹⁸ Rigorous evidence for anti-LRP2 autoantibodies in humans is limited, although antibodies to the related LRP4 have been identified in myasthenia gravis.¹⁹

Although the tubular damage and TBM deposits are the most dramatic features of anti-LRP2 nephropathy, Bowman’s capsule staining was also present in all patients. Human glomeruli show diffuse low-level LRP2 staining in the parietal and visceral epithelium under normal conditions.²⁰ This likely explains why patients with ABBA frequently show strong circumferential granular IgG staining of Bowman’s capsule. Segmental glomerular subepithelial deposits are also a common feature. These sparse deposits contain human IgG but do not stain for LRP2, making their molecular origins unclear. Their segmental nature might be relevant to disease pathogenesis by allowing filtration of ABBA from the circulation into the tubules. Anti-LRP2 autoantibodies in the tubular lumen would then have direct access to LRP2 on the proximal tubule brush border. The subsequent mechanisms of tubular cell injury are also uncertain as is the pathophysiologic mechanism by which basal deposits might develop due to antibody directed against a predominantly apical protein. A transcytotic mechanism of antigen-antibody complexes is possible, because it has previously been shown that rabbits passively immunized with goat anti-rabbit angiotensin-converting enzyme, an enzyme mainly expressed on the apical proximal tubular cell membrane, also develop immune deposits along the TBMs composed of goat IgG, rabbit angiotensin-converting enzyme, and C3.²¹

Insight into the pathogenesis of tubular cell injury can be derived from studies of Heymann nephritis, in which the severity of proximal tubular lesions correlates with ABBA.⁶ In passive Heymann nephritis, fixation of Ig to the proximal tubule brush border and tubular injury occur within 48 hours of infusion. The injury could not be attributed to proteinuria, because in rats with serum sickness, similar amounts of proteinuria did not cause proximal tubule injury.²² Heymann nephritis–induced proximal tubular injury is independent of complement.²³ Consistent with this, IgG4, which does not fix complement, is usually the predominant IgG subclass in human anti-LRP2 nephropathy. The tubulointerstitial morphologic phenotype in animals with ABBA is similar to those in humans with anti-LRP2 antibodies, including proximal tubular degeneration with interstitial fibrosis accompanied by sparse mononuclear interstitial inflammation by light microscopy and granular TBM deposits by immunofluorescence.^3,4,7 Thus, there is strong experimental data to support the hypothesis that antibodies to the brush border are cytotoxic to proximal tubule epithelium.

In our cohort, most of the patients (nine of ten) exhibited reactivity with the N-terminal set of LA repeats. Interestingly, the major epitope in Heymann nephritis has also been identified in the N-terminal part of megalin.²⁴ In addition to the N-terminal fragment, five patients showed reactivity against a fragment of the most C-terminal set of LA repeats, two patients showed reactivity against the third set of LA repeats, and one patient was reactive against all four sets of LA repeats. Interestingly, in Heymann nephritis, Shah et al.²⁵ showed a similar pattern of epitope spreading, with higher reactivity against the N-terminal set of LA repeats followed by the fourth set, then the third set, and at least, the second set. Of note, the patient who had reactivity to all four sets of LA repeats had severe clinical manifestations and died within 3 months. This correlation between clinical disease severity and intramolecular epitope spreading is well known and has been shown in Heymann nephritis²⁵ as well as primary membranous nephropathy.²⁶

Anti-LRP2 nephropathy is probably underdiagnosed, because elderly patients with AKI are infrequently biopsied. As early as 1981, Douglas et al.⁹ described a patient with segmental membranous nephropathy with granular deposits in Bowman’s capsule and proximal TBM. This patient’s serum was found to be reactive against the brush border of normal human kidney, but the antigen was not identified. The recent case report detailing the clinical and pathologic features of one of the patients in this current series has rekindled interest in this disorder.² Whereas double staining of the patient serum and LRP2 did not show colocalization in that report, in retrospect, this was probably due to the specificity of the antibody to the C terminus of megalin. An alternative anti-LRP2 antibody used in this study disclosed LRP2 and IgG colocalization both in the TBM deposits and by indirect immunofluorescence (Figure 3).

The presence of any IgG-containing TBM immune deposits should alert the clinician and pathologist to the possibility of this disorder. Similar tubular deposits can be seen in the setting of lupus nephritis and IgG4-related disease. Tubular deposits in the setting of lupus generally correlate with the presence of proliferative glomerular lesions,²⁷ which were not seen in any of our patients with anti-LRP2 nephropathy. IgG4-related tubulointerstitial nephritis is the entity with the most morphologic overlap, because both it and anti-LRP2 nephropathy can exhibit interstitial inflammation and glomerular deposits in addition to the tubular deposits. However, most patients with IgG4-related disease show intense interstitial inflammation, whereas inflammation in anti-LRP2 nephropathy was absent to mild in most patients. Additionally, the characteristic sclerosing pattern of IgG4-related disease²⁸ is not present in anti-LRP2 nephropathy. When doubt exists, tissue staining for LRP2 and serum testing for ABBA should resolve the diagnostic dilemma. Other common etiologies of tubulointerstitial nephropathy, such as drug-induced hypersensitivity reactions and sarcoidosis, do not show TBM IgG deposits in the vast majority of patients.

In summary, we have identified a unique and likely under-reported cause of severe, acute, and progressive renal tubular injury, which is associated with circulating autoantibodies to the tubular brush border protein LRP2/megalin and characterized by IgG- and LRP2-containing immune complexes in the TBM. Future studies to detect the disease by measuring circulating anti-LRP2 antibodies may help reveal the true prevalence of the disease and enable earlier detection and treatment to avoid the grave outcome seen in several of the patients reported here.

Concise Methods

Patient Population

The diagnosis of ABBA disease was made using renal biopsy and serologic testing on samples submitted for diagnosis to Arkana Laboratories or Massachusetts General Hospital. Biopsies were processed by light, immunofluorescence, and electron microscopy using routine techniques (Supplemental Material).²⁹ Serum samples from patients whose renal biopsy showed tubular injury by light microscopy and IgG-positive granular TBM deposits by immunofluorescence were tested for the presence of ABBA by indirect immunofluorescence on sections of normal human kidney. The study protocol was approved by the Schulman Institutional Review Board and conformed to the Declaration of Helsinki principles.

Immunostaining for LRP2/Megalin

LRP2 staining was performed on all renal biopsies from patients with ABBA as well as 50 controls, including 40 biopsies with granular IgG TBM deposits (lupus nephritis, 27; IgG4-related disease, seven; BK nephritis, six) and ten biopsies with kidney injury negative for TBM deposits (toxic acute tubular injury, five; interstitial nephritis, five). Formalin-fixed paraffin-embedded sections, cut at 3 μm, were deparaffinized, and antigen retrieval was performed at 99°C. The sections were reacted with mouse monoclonal anti-human LRP2 (1:1000; EMD Millipore, Billerica, MA), then polyclonal (Rhodamine Red-X) goat anti-mouse IgG Fcγ subclass 1 (Jackson ImmunoResearch, West Grove, PA), and polyclonal (FITC-conjugated) goat anti-human IgG (1:100; Kent Laboratories, Bellingham, WA). Colocalization of IgG and LRP2 in the TBM was examined by confocal microscopy.

Human Renal Tubular Protein Extract

Cortical tissue retrieved from kidneys deemed unsuitable for transplantation by the New England Organ Bank (with consent for research) was minced and mechanically sieved as described.¹⁸ Glomeruli were retained on the final sieve, and the cellular material passing through was collected in PBS, centrifuged to obtain a tubulointerstitial cellular pellet, and detergent extracted on ice, yielding HTE. The extract was gel electrophoresed in the presence or absence of reducing agents and transferred to nitrocellulose membranes for immunoblotting using routine protocols.

Proteomic Analyses

Immunoprecipitates of HTE and patient serum were electrophoresed, and gel regions corresponding to bands visible by Western blot analysis were excised and subjected to in-gel digestion as described previously with some modifications.³⁰ Digested peptides were separated using chromatographic techniques, and high resolution mass spectrometry data were collected using high-accuracy mass spectrometry as described in Supplemental Material.³⁰

Expression of Human LRP2/Megalin

RNA isolated from fresh human cortical tissue was used to generate 4–5 kB cDNA fragments from human LRP2 using specific primers (Supplemental Material). Using these templates, domain-limited regions of LRP2 were subcloned into a mammalian cell expression vector that directed expression of these constructs with a C-terminal 3XFLAG tag (Sigma-Aldrich). For these experiments, we focused on the sets of LA repeats. After expression, whole-cell lysate or isolated constructs purified via their C-terminal 3XFLAG tag were assayed by immunoblot and immunoprecipitation for reactivity with sera from patients who were ABBA⁺ or controls.

Supplementary Information

The reader is directed to Supplemental Material for additional experimental details and results.

Data files for acquired liquid chromatography mass spectrometry data (.RAW) and peak lists (.mzXML) and compressed search results (.mzIdentML) files were deposited in the MassIVE (http://massive.ucsd.edu) data repository (MassIVE ID no. MSV000080885) with the Center for Computational Mass Spectrometry at the University of California, San Diego and shared with the ProteomeXchange (ID no. PXD006258; www.proteomexchange.org).

Disclosures

None.