Patent Application
20230393147
Described herein are methods and kits for diagnosing and treating minimal change disease, and for identifying subjects for eligibility or treatment before kidney transplant, based on the presence of circulating anti-nephrin autoantibodies.
Diffuse podocytopathy with minimal changes (Minimal Chance Disease (MCD)) is an important and common pathologic diagnosis in adults and children with nephrotic syndrome (NS). MCD is a podocytopathy of unknown etiology, affecting both adults and children, with evidence supporting a role for B-cells10-13. It is characterized by minimal changes by light microscopy, yet extensive injury to glomerular podocytes with diffuse foot process effacement (FPE) and loss of slit diaphragms (SD) by electron microscopy (EM) in the absence of electron dense deposits1. The consequence of these alterations is massive proteinuria secondary to failure of the glomerular filtration barrier (GFB), whose integrity is critically dependent on the specialized junctional SD protein complex linking the interdigitating podocyte foot processes.
Provided herein are methods for diagnosing minimal change disease (MCD) in a subject. The methods include providing a sample from a subject who has, or who is suspected of having, MCD; determining a level of anti-nephrin antibodies in the sample; comparing the level of anti-nephrin antibodies in the sample to a reference level; and diagnosing a subject who has a level of anti-nephrin antibodies in the sample above a reference level as having or at risk of developing MCD.
In some embodiments, the methods include selecting a treatment for MCD to the subject.
Also provided herein are methods for treating minimal change disease (MCD) in a subject. The methods include providing a sample from a subject who has, or who is suspected of having, MCD; determining a level of anti-nephrin antibodies in the sample; comparing the level of anti-nephrin antibodies in the sample to a reference level; identifying a subject who has a level of anti-nephrin antibodies in the sample above a reference level as having or at risk of developing MCD; and administering a treatment for MCD to the subject.
In some embodiments, the treatment for MCD comprises administration of one or more of a glucocorticoid, a treatment that reduces levels of anti-nephrin antibodies, or a treatment that targets B cells, to the subject. In some embodiments, the glucocorticoid is prednisone, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, triamcinolone, prednisolone, or methylprednisolone. In some embodiments, the B-cell targeting therapy is a targeted therapy that depletes B cells or an inhibitor of B lymphocyte stimulation. In some embodiments, the targeted therapy that depletes B cells is an anti-CD20 antibody; anti-CD19 antibody; or an anti-BAFF antibody. In some embodiments, the inhibitor of B lymphocyte stimulation is belimumab, tabalumab, or atacicept. In some embodiments, the treatment that reduces levels of anti-nephrin antibodies is or includes plasmapheresis.
In some embodiments, the methods further include obtaining a subsequent sample from the subject; determining a subsequent level of anti-nephrin antibodies in the sample; comparing the subsequent level of anti-nephrin antibodies to a reference level; and (i) if the level of anti-nephrin antibodies in the subsequent sample is above a reference level, the methods can further include continuing to administer the treatment to the subject or administering a different treatment for MCD to the subject, or (ii) if the level of anti-nephrin antibodies in the subsequent sample is below the reference level, the methods can further include discontinuing the treatment.
Additionally, provided herein are methods that include providing a sample from a subject, optionally a subject who has, or who is suspected of having, MCD; and determining a level of anti-nephrin antibodies in the sample.
In some embodiments, the methods further include comparing the level of anti-nephrin antibodies in the sample to a reference level; and providing the level and the reference level to a healthcare provider and/or the subject, and optionally providing the reference range to the healthcare provider, and an indication of whether the subject is above or below the reference level.
Further, provided herein are methods for determining eligibility of a subject who has end stage renal disease (ESRD) for a kidney transplant. The methods include providing a sample from the subject; determining a level of anti-nephrin antibodies in the sample; comparing the level of anti-nephrin antibodies in the sample to a reference level; and identifying a subject who has a level of anti-nephrin antibodies in the sample above a reference level as ineligible for transplant. In some embodiments, the methods further include selecting and optionally administering a treatment that comprises administration of one or more of a glucocorticoid, a treatment that reduces levels of anti-nephrin antibodies, or a treatment that targets B cells, to the subject to the subject; obtaining a subsequent sample from the subject; determining a subsequent level of anti-nephrin antibodies in the sample; comparing the subsequent level of anti-nephrin antibodies to a reference level; and (i) if the level of anti-nephrin antibodies in the subsequent sample is above a reference level, the methods can further include continuing to administer the treatment to the subject or administering a different treatment to the subject, or (ii) if the level of anti-nephrin antibodies in the subsequent sample is below the reference level, the methods can further include identifying the subject as eligible for transplant.
In some embodiments of the methods described herein, the sample comprises renal biopsy tissue, whole blood, plasma, or serum from the subject.
In some embodiments, determining a level of anti-nephrin antibodies in the sample comprises performing Western blot; enzyme linked immunosorbent assay (ELISA); radio-immunoassay (RIA); immunohistochemistry (IHC); immune-precipitation assay; or fluorescent activated cell sorting (FACS).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Nephrin is an essential structural component of the SD2,3, as illustrated by genetic mutations in nephrin (NPHS1) that cause complete lack of nephrin cell surface localization, underlying Congenital Nephrotic Syndrome of the Finnish Type (CNF)4,5. In contrast to congenital NS with an established genetic basis, the cause of non-congenital NS in both children and adults remains largely unknown. There is strong evidence supporting immune dysregulation with a potential causative circulating factor, however its identity has remained elusive6,7. Glucocorticoids are effective at inducing remission, however relapse, steroid dependence and intolerance are common, often requiring alternative immunosuppressive agents8. In those patients with steroid dependent NS who progress to end stage kidney disease (ESKD) and require kidney transplantation, the disease can promptly recur in the allograft1, a devastating and difficult-to-treat complication.
The recent discovery that anti-CD20 B-cell targeted therapies are effective in children with frequently relapsing or steroid-dependent NS9-11 and in adults12 suggests a potential autoantibody-mediated etiology. However, this possibility is hard to reconcile with the traditional view of MCD lacking IgG deposition on renal biopsy13. Whilst diffuse podocyte-associated IgG is described in MCD, it is minimal compared to that seen in membranous nephropathy (MN), and given the absence of electron-dense deposits by EM, it is generally attributed to non-specific protein resorption of little significance14.
Antibodies targeting the essential SD component nephrin have been shown to cause massive proteinuria when administered in animal models15-17 and when they arise as alloantibodies following kidney transplantation in children with CNF and complete nephrin deficiency18. In both animal models15,16 and cultured podocytes7,19, anti-nephrin antibodies cause a redistribution of nephrin that is identical to that observed in renal biopsies of patients with NS20,21. This redistribution of nephrin away from the SD along with separation of intercellular junctions between adjacent podocytes has long been proposed as a logical concept to explain the proteinuria in these patients; however, the cause of this redistribution remains unknown.
The present inventors hypothesized that autoantibodies against nephrin might underlie non-congenital MCD by interfering with the integrity of the SD complex.
The present invention is based, at least in part, on the discovery of circulating autoantibodies against the extracellular domain of nephrin, the essential constituent of the podocyte SD, in a subset of patients with non-congenital, childhood and adult-onset MCD. These nephrin autoantibodies were specifically present in MCD kidney biopsies, forming distinct clusters together with nephrin. These observations share striking parallels with the autoimmune blistering skin condition pemphigus, in which circulating autoantibodies target the desmosomal cell adhesion molecules desmogleins (dsg), the fundamental structural proteins of the desmosomal cell adhesion complex that links adjacent keratinocytes22, analogous to nephrin in the specialized SD junctional complex between adjacent podocytes. In pemphigus, these dsg autoantibodies directly interfere with cell adhesion through redistribution, clustering and endocytosis of dsg that disrupts the integrity of the desmosome22. Previous reports of experimental anti-nephrin antibody mediated disruption of nephrin homophilic interactions further support this potential mechanism in MCD3. Furthermore, pemphigus exhibits a rapid response to glucocorticoid treatment (within days to weeks) that cannot be explained by reduced IgG synthesis alone and may be due to compensatory desmoglein synthesis in keratinocytesa. Similarly, most cases of MCD respond rapidly to glucocorticoids (within weeks) which have also been shown to upregulate nephrin cell surface expression in cultured human podocytes26. The present findings indicate that the IgG co-localizing with nephrin in MCD kidney biopsies may represent in situ binding of nephrin autoantibodies. Without wishing to be bound by theory, this targeted binding may be sufficient to disrupt nephrin homophilic interactions leading to early loss of SD integrity, and the redistribution of IgG along with its target nephrin may explain this subtle punctate staining pattern, in contrast to the much more intense staining seen with membranous nephropathy labeling large IgG immune complex aggregates that progressively accumulate along the base of the podocyte foot processes.
Fortunately, progression of MCD to ESKD is rare, however in those patients that do or in those with an initial diagnosis of primary FSGS that progresses more commonly, the disease can rapidly recur in the allograft. A role for nephrin autoantibodies in early post-transplant massive proteinuria is illustrated by a patient with steroid dependent MCD that eventually progressed to ESKD with subsequent biopsies showing FSGS. Early post-transplant massive proteinuria occurred in the presence of circulating nephrin autoantibodies identified both prior to transplantation and at the time of disease recurrence, which were successfully treated with plasmapheresis/rituximab, leading to sustained remission associated with their disappearance. These findings are in keeping with previous studies in CNF patients who develop alloantibodies to nephrin in association with disease recurrence in the allograft and respond to plasmapheresis/rituximab.19,36 Importantly, the critical distinction is that in this patient, the nephrin autoantibodies were present both prior to the transplant and at the time of disease recurrence whereas in CNF they arise as a direct consequence of the transplant due to alloimmunization to nephrin.19 FSGS and MCD share some important similarities, such as indistinguishable ultrastructural changes and response to B-cell therapies37, and together with this illustrative case, we would speculate that our findings of nephrin autoantibodies may also extend to a subset of patients with a diagnosis of primary non-genetic FSGS.
Thus, the present results indicate that B-cell targeted therapy can be used in a subset of NS patients, and provide methods to molecularly identify those patients who stand to benefit most from the targeted therapeutic strategies for anti-nephrin antibody positive NS.
Subjects
The present methods can be used in subjects (e.g., mammals, preferably human or non-human veterinary subjects, including human adults and children) who have, or who are suspected to have, nephrotic syndrome (NS), e.g., who have diffuse podocytopathy with minimal changes (Minimal Change Nephrotic Syndrome, MCNS) or Minimal Change Disease (MCD, also known as lipoid nephrosis or nil disease). Methods for identifying subjects are known in the art; see, e.g., Vivarelli et al., Clin J Am Soc Nephrol. 2017; 12(2):332-345. In some embodiments, the subjects have a urine protein-creatinine ratio (UPCR)>3 g/g, profound proteinuria and oval fat bodies, hypovolemia, hypertension, thromboembolism, hypoalbuminemia (less than 2.5 g/dL in children), hyperlipidemia, and/or facial edema. A critical level for diagnosis in children is the presence of proteinuria of more than 40 mg/h/m2, while a threshold of 3.5 g/d/1.73 m2 is useful in adults. See, e.g., Mansur et al., “Minimal-Change Disease,” Mescape, January 2021, available at emedicine.medscape.com/article/243348. The present methods can be used, e.g., in subjects who have not been formally diagnosed, e.g., to aid in diagnosis and/or selection of treatment, or in subjects who have been diagnosed, to aid in selection of treatment.
Recurrent acute nephrotic syndrome in the allograft, referred to as “recurrent focal segmental glomerulosclerosis” (rFSGS) in patients with a history of acute nephrotic syndrome in the native kidney, is morphologically indistinguishable from MCD. Therefore, a study was also included in a transplant patient with an initial diagnosis of MCD in the native kidney which then progressed to FSGS and eventually ESKD. This patient rapidly developed acute and high proteinuria post-transplant consistent with rFSGS, which was not confirmed by a biopsy; however, her pre-transplant serum showed high levels of anti-nephrin antibodies which decreased after aggressive and successful treatment with rituximab and plasmapheresis; this patient went into lasting remission and retained her transplant. Thus, analysis of anti-nephrin antibodies in this group of patients (with a history of acute nephrotic syndrome, who developed FSGS and then ESKD, and are then considered for a transplant) can be helpful in determining prognosis and identifying subjects who either need to be treated to reduce anti-nephrin antibodies (e.g., using rituximab and plasmapheresis) or who should be excluded from transplant eligibility.
Methods of Diagnosis
Included herein are methods for diagnosing subjects with MCD or antibody-mediated acquired idiopathic nephrotic syndrome (INS), and/or for identifying subjects who would benefit from (and in some embodiments should be treated with) certain therapies as described herein, e.g., subjects who have MCD or an acute acquired nephrotic syndrome or FSGS. The methods rely on detection of anti-nephrin antibodies. The methods include obtaining a sample from a subject, and evaluating the presence and/or level of anti-nephrin antibodies in the sample, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal level of anti-nephrin antibodies, e.g., a level in an unaffected subject, and/or a disease reference that represents a level of anti-nephrin antibodies associated with MCD, e.g., a level in a subject having MCD. Suitable reference values can include those shown in, or determined as described in, Example 1.
As used herein the term “sample”, when referring to the material to be tested for the presence of anti-nephrin antibodies using the methods described herein includes inter alia renal biopsy tissue, whole blood, plasma, or serum; in some embodiments, the methods include testing for the presence of circulating autoantibody in plasma/serum, and then testing in tissue to confirm binding in the biopsy tissue. The type of sample used may vary depending upon the clinical situation in which the method is used. Various methods are well known within the art for the identification of anti-nephrin antibodies in a sample.
The presence and/or level of anti-nephrin antibodies can be evaluated using methods known in the art, e.g., using standard electrophoretic and quantitative immunoassay methods, including but not limited to, Western blot; enzyme linked immunosorbent assay (ELISA); biotin/avidin type assays; protein array detection; radio-immunoassay; immunohistochemistry (IHC); immune-precipitation assay; FACS (fluorescent activated cell sorting); mass spectrometry (Kim (2010) Am J Clin Pathol 134:157-162; Yasun (2012) Anal Chem 84(14):6008-6015; Brody (2010) Expert Rev Mol Diagn 10(8):1013-1022; Philips (2014) PLOS One 9(3):e90226; Pfaffe (2011) Clin Chem 57(5): 675-687). The methods typically include revealing labels such as fluorescent, chemiluminescent, radioactive, and enzymatic or dye molecules that provide a signal either directly or indirectly. As used herein, the term “label” refers to the coupling (i.e. physically linkage) of a detectable substance, such as a radioactive agent or fluorophore (e.g. phycoerythrin (PE) or indocyanine (Cy5), to a probe (e.g., a nephrin protein), as well as indirect labeling of the probe (e.g. horseradish peroxidase, HRP) by reactivity with a detectable substance.
In some embodiments, an ELISA method may be used, wherein a surface such as the wells of a mictrotiter plate are coated with nephrin protein antigen. The sample containing or suspected of containing the anti-nephrin antibodies is then applied to the wells. After a sufficient amount of time, during which antibody-antigen complexes would have formed, the plate is washed to remove any unbound moieties, and a detectably labelled molecule is added. Again, after a sufficient period of incubation, the plate is washed to remove any excess, unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. Variations of the ELISA method, such as the competitive ELISA or competition assay, and sandwich ELISA, may also be used, as these are well-known to those skilled in the art.
In some embodiments, an IHC method may be used. IHC provides a method of detecting a biological marker in situ. The presence and exact cellular location of the biological marker can be detected. Typically a sample is fixed with formalin or paraformaldehyde, embedded in paraffin, and cut into sections for staining and subsequent inspection by confocal microscopy. Current methods of IHC use either direct or indirect labelling. The sample may also be inspected by fluorescent microscopy when immunofluorescence (IF) is performed, as a variation to IHC.
In some embodiments, the presence and/or level of anti-nephrin antibodies is comparable to the presence and/or level of the protein(s) in the disease reference, and the subject has one or more symptoms associated with MCD, then the subject is diagnosed with MCD. In some embodiments, the subject has no overt signs or symptoms of MCD, but the presence and/or level of one or more of the proteins evaluated is comparable to the presence and/or level of the protein(s) in the disease reference, then the subject has an increased risk of developing MCD. In some embodiments, once it has been determined that a person has MCD, or has an increased risk of developing MCD, then a treatment, e.g., as known in the art or as described herein, can be administered.
Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of anti-nephrin antibodies, e.g., a control reference level that represents a normal level of anti-nephrin antibodies, e.g., a level in an unaffected subject or a subject who is not at risk of developing a disease described herein (or a cohort of such subjects), and/or a disease reference that represents a level of anti-nephrin antibodies associated with MCD.
The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n-quantiles being subjects with the highest risk.
In some embodiments, the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.
The predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy population will have a different ‘normal’ range of levels of anti-nephrin antibodies than will a population of subjects which have, are likely to have, or are at greater risk to have, a disorder described herein (e.g., MCD). Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
In characterizing likelihood, or risk, numerous predetermined values can be established.
Methods of Treatment
The methods described herein include methods for the treatment of subjects identified using a method described herein as having a level of anti-nephrin antibodies above a reference level, e.g., having a disorder associated with the presence of anti-nephrin antibodies. In some embodiments, the disorder is MCD. Generally, the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject who has been determined to be in need of such treatment by a method described herein.
As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with anti-nephrin antibodies, e.g., MCD. Often, MCD results in elevated urine protein-creatinine ratio (UPCR) (e.g., over >3 g/g), profound proteinuria and oval fat bodies, hypovolemia, hypertension, thromboembolism, hypoalbuminemia (less than 2.5 g/dL in children), hyperlipidemia, and/or facial edema; thus, a treatment can result in a reduction in any of the above and a return or approach to normal urine protein-creatinine ratio (UPCR) (e.g., less than 3 g/g), normal levels of proteinuria and absence of oval fat bodies, normoolemia, normotension, normoalbuminemia (above 2.5 g/dL in children), normal lipid levels, and/or reduction or absence of facial edema. Administration of a therapeutically effective amount of a treatment described herein for the treatment of a condition associated with anti-nephrin antibodies will result in decreased levels of anti-nephrin antibodies.
An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
Treatments that can be used in the present methods include administration of one or more of glucocorticoids (e.g., prednisone, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, triamcinolone, prednisolone, or methylprednisolone) or B-cell targeting therapies (e.g., targeted therapies that deplete B cells, e.g., anti-CD20 antibodies such as rituximab, ocrelizumab, ofatumumab, veltuzumab, ocratuzamab, ibritumomab, obinutuzumab, tositumomab, ublitiximab, TRU-015, or orublituximab; anti-CD19 antibodies such as blinatumomab, coltuximabravtansine, MOR208, MEDI-551, denintuzumabmafodotin, SAR3419 (huB4-DM4), SGN-CD19A, taplitumomabpaptox, XmAb 5871, MDX-1342, AFM11, inebilizumab, tafasitamab, or antibodies described in U.S. Pat. No. 8,691,952 (see, e.g., Naddafi and Davami, Int J Mol Cell Med. 2015 Summer; 4(3): 143-151); or anti-BAFF antibodies such as belimumab as well as inhibitors of B lymphocyte stimulation, such as belimumab or tabalumab; or Atacicept (a human recombinant fusion protein comprising the binding portion of a receptor for both BLyS (B-Lymphocyte Stimulator) and APRIL (A PRoliferation-Inducing Ligand) (see Hartung et al., Ther Adv Neurol Disord. 2010 July; 3(4): 205-216). In some embodiments, the treatments include plasmapheresis (see, e.g., Kaplan, J Clin Apher 28, 3-10 (2013)).
Treatments that reduce levels of anti-nephrin antibodies can also be used in the methods described herein, including plasmapheresis.
In some embodiments, the present methods include determining a level of anti-nephrin antibodies in a sample from a subject who has, or who is suspected of having, MCD. If the level of anti-nephrin antibodies in the sample is above a reference level, the methods can further include identifying the subject as having or at risk of developing MCD, and optionally administering a treatment as described herein, e.g., a glucocorticoid, a treatment that reduces levels of anti-nephrin antibodies such as plasmapheresis, or a treatment that reduces B cells, to the subject.
In some embodiments, the present methods include determining a level of anti-nephrin antibodies in a sample from a subject who has ESRD and is about to undergo a kidney transplant. If the level of anti-nephrin antibodies in the sample is above a reference level, the methods can further include identifying the subject as at risk of developing severe proteinuria and kidney disease after transplant, and optionally administering a treatment as described herein, e.g., a treatment that reduces levels of anti-nephrin antibodies such as plasmapheresis, or a treatment that reduces B cells, to the subject.
Kits
Also provided herein are kits for use in the present methods. The kits can include a nephrin protein probe, e.g., comprising the extracellular domain of nephrin. An exemplary sequence of the extracellular domain of nephrin (aa1-1059) is as follows:
In some embodiments, the nephrin protein is recombinantly produced and/or is tagged or labelled. The label can include a fluorophore or radiolabel. The tag can include glutathione S-transferase (GST), polyhistidine (e.g., (6×HIS)), c-myc, hemagglutinin, or FLAG™ tag (Kodak, New Haven, Conn.) sequences tags, and can be fused at either the N- or C-terminus of the nephrin protein. The nephrin can be in solution, or can be adsorbed to a surface, e.g., wells of a microtiter plate or on beads, e.g., magnetic or polymeric beads. The kits can include anti-human IgG antibodies to detect anti-nephrin antibodies bound to the nephrin protein probe, and/or anti-nephrin antibodies, e.g., for use as a control or to detect nephrin co-localization with IgG antibodies, e.g., in tissue samples. Optionally the anti-human IgG and/or anti-nephrin antibodies are labelled or are otherwise detectable (e.g., comprises an enzyme or substrate for colorimetric detection). Optionally the kit further includes a secondary antibody that is labelled or otherwise detectable (e.g., comprises an enzyme or substrate for colorimetric detection).
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Methods
The following materials and methods were used in the Example, below.
Clinical Samples (Kidney Tissue and Serum/Plasma)
Renal biopsies were independently assessed by collaborating renal pathologists across four institutions: Brigham and Women's Hospital (BWH), Massachusetts General Hospital (MGH), Boston Medical Center (BMC) and the Mayo Clinic. Serum/plasma was obtained from patients attending those institutions as either discarded samples originally collected for clinical analysis (BWH/BMC/Mayo clinic) or archival samples from the Kidney Disease Biobank (courtesy of Dr. Sushrut Waikar, Partners Healthcare, in accordance with Partners Healthcare IRB Approval for patients attending BWH or MGH who were consented for serum/plasma collection at the time of renal biopsy). Histological studies were performed on archival kidney tissue that was received for routine clinical evaluation and included diffuse podocytopathies, other nephrotic conditions, and non-neoplastic renal parenchyma from tumor nephrectomies. Medical record review, histological and serological studies were approved by the respective Institutional Review Boards (IRB) for those institutions. Genetic testing was only performed for the patient with post-transplant recurrent disease but not for the other patients enrolled outside the NEPTUNE cohort.
Similarly, sera were obtained from patients with biopsy proven minimal change disease (MCD) from the Nephrotic Syndrome Study Network (NEPTUNE) longitudinal study23 during active disease (urine protein creatinine ratio (UPCR)>3 g/g) and where available, in remission. Complete remission was defined as UPCR<0.3 g/g and partial remission as a>50% reduction in proteinuria. Steroid-dependent nephrotic syndrome (SDNS) was defined as SSNS with 2 or more consecutive relapses during tapering or within 14 days of stopping steroids. No specific exclusion criteria were applied.
Healthy control sera were randomly selected from Partners Healthcare Biobank, specifically excluding those subjects with any renal or autoimmune disease. Sera from nephrotic patients were evaluated for anti-hPLA2R antibodies at the MGH Immunopathology laboratory using a commercial enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence test (IIFT) (Euroimmun). Samples were coded to preserve patient anonymity.
Whole Genome Sequencing of the NEPTUNE Cohort
Whole genome sequencing with a goal median depth of 30×was performed using Illumina Hi-seq. A standard pipeline, Gotcloud, was applied for sequence alignment and variant calling27,28. The variant analysis focused on approximately 70 genes implicated in Mendelian NS. To screen pathogenic variants in the 70 previously implicated Mendelian NS genes, we employed a pipeline similar to one that has been previously reported29. The pathogenicity variants were ultimately classified according to ACMG standards and guidelines30. Analyzed genes: ACTN4, ADCK4, ALGI, ANLN, ARHGAP24, ARHGDIA, AVIL, CD151, CD2AP, CDK20, CFH, COL4A3, COL4A4, COL4A5, COQ2, COQ6, CRB2, DGKE, DLC1, EMP2, FAT1, HNF1B, IL15RA, INF2, ITGA3, ITGB4, ITSN1, ITSN2, JAGI, KANK1, KANK2, KANK4, LAGE3, LAMB2, LMX1B, MAGI2, MTTL1, MYH9, MYO1E, MYO5B, NEIL1, NPHS1, NPHS2, NUP107, NUP205, NUP93, NXF5, OCRL, OSGEP, PAX2, PDSS2, PLCE1, PMM2, PODXL, PTPRO, SCARB2, SGPL1, SMARCAL1, TNS2, TP53RK, TPRKB, TRPC6, TTC21B, UMOD, WDR73, WT1, XPO5, ZMPSTE24.
Human Glomerular Extract
Human glomerular extract (HGE) was prepared as previously described by Beck et al31. Briefly, glomeruli were isolated from human kidneys deemed non-suitable for transplantation (that had been authorized for use in medical research) obtained from New England Donor Services, by graded sieving followed by isolation of glomerular proteins in RIPA buffer (Boston BioProducts). IgG was pre-cleared from tissue lysate by incubation with Protein G Plus agarose beads (Santa Cruz). Only kidneys with less than 20% global glomerulosclerosis, on routine wedge biopsy, were used for glomerular isolation.
Routine Renal Biopsy Processing
After biopsy acquisition, renal cortex was immediately allocated for light (10% neutral-buffered formalin), immunofluorescence (Zeus transport media) and electron microscopy (Karnovsky's fixative) processing. For routine clinical immunofluorescence, 4 μm cryosections were fixed in 95% ethanol for 10 minutes and incubated with FITC-conjugated polyclonal rabbit F(ab)2 anti-human IgG antibody (Dako; F0315) diluted 1:20. FITC-conjugated sheep anti-human IgG1, IgG2, IgG3, IgG4 (Binding Site; AF006, AF007, AF008, AF009, respectively) diluted 1:20 were used for IgG subclass evaluation. Albumin was detected using FITC-conjugated polyclonal rabbit anti-human albumin (Dako; F0117) diluted 1:30. Sections were mounted using Dako fluorescence mounting medium (Dako; S3023) with a #1.5 coverslip. Immunofluorescence images were acquired on an Olympus BX53 microscope with an Olympus DP72 camera at 150 ms exposure.
Confocal Microscopy
For confocal microscopy, 4 μm cryosections of human kidney biopsies were fixed in 95% ethanol for 10 minutes and subsequently blocked for one hour at room temperature (RT) with phosphate buffer saline (PBS) supplemented with 0.2% fish gelatin, 2% bovine serum albumin (BSA) and 2% fetal bovine serum (FBS). All antibodies were diluted in this blocking solution and incubated for one hour at RT. Nephrin was detected using 1 μg/ml primary polyclonal sheep anti-human nephrin (R&D systems; AF4269) followed by a secondary AlexaFluor™ 568-conjugated donkey anti-sheep IgG (Invitrogen; A21099). Synaptopodin was detected using anti-synaptopodin (N-terminus) guinea pig polyclonal antiserum (Progen; GP94-N) diluted 1:1000 followed by a secondary AlexaFluor™ 568-conjugated goat anti-guinea pig IgG (Invitrogen; A11075) antibody. Podocin and Wilms Tumor 1 (WT1) were detected using a primary polyclonal rabbit anti-human podocin (Millipore Sigma; P0372) and a primary monoclonal rabbit anti-human WT1 clone SC06-41 (Invitrogen; MA5-32215) diluted 1:500 and 1:300 respectively, followed by a secondary AlexaFluor™ 568-conjugated donkey anti-rabbit IgG (Invitrogen; A10042). IgG immune deposits were detected using a primary monoclonal mouse anti-human IgG antibody (Abcam; ab200699) diluted 1:750 followed by a secondary AlexaFluor™ 488-conjugated donkey anti-mouse IgG (Invitrogen; A21202). All secondary AlexaFluor™-conjugated antibodies were diluted 1:500. Sections were mounted using Vectashield anti-fade mounting medium (Vectashield, H-1000) with a #1.5 coverslip and images were acquired on a Leica TCS SPE microscope.
Structured Illumination Microscopy (SIM)
Structured Illumination Microscopy (SIM) imaging was performed on 4 μm fixed, frozen human kidney biopsy sections processed according to the aforementioned protocol for confocal microscopy. All images were collected using an OMX V4 Blaze (GE Healthcare) microscope equipped with three watercooled PCO.edge sCMOS cameras, 488 nm, 568 nm laser lines, and 528/48 nm, 609/37 nm emission filters (Omega Optical). Images were acquired with a 60×/1.42 Plan-Apochromat objective lens (Olympus) with a final pixel size of 80 nm. Z stacks of 4-8 μm, were acquired with a 0.125 μm z-spacing, and 15 raw images (three rotations with five phases each) were acquired per plane. Spherical aberration was minimized for each sample using immersion oil matching24. Super resolution images were computationally reconstructed from the raw data sets with a channel-specific, measured optical transfer function, and a Wiener filter constant of 0.001 using CUDA-accelerated 3D-SIM reconstruction code32. Axial and lateral chromatic misregistration was determined using a single biological calculation slide, prepared with human kidney tissue stained with a primary mouse anti-human IgG monoclonal antibody (Abcam; ab200699) followed by both secondary AlexaFluor™ 488-conjugated donkey anti-mouse IgG (Invitrogen; A21202) and AlexaFluor™ 568-conjugated goat anti-mouse IgG (Invitrogen; A11031) antibodies on the same tissue cryosection. Experimental data sets were then registered using the imwarp function in MATLAB (MathWorks) 33.
Generation of Recombinant Human Nephrin and Phospholipase A2 Receptor (PLA2R)
Separate plasmids encoding the extracellular subdomains of human nephrin (amino acids 1-1059), comprising the 8 Ig-like C2-type domains and a single fibronectin type III domain, and human phospholipase A2 receptor (hPLA2R), comprising the N-terminal ricin domain, fibronectin type II domain and 8 C-type lectin domains (CTLD), both with C-terminal polyhistidine (6×HIS) tags, were generated by standard cloning techniques. The correct sequences were confirmed by whole plasmid sequencing (MGH DNA core). HEK293-F cells (Thermo Fisher) were transfected with 0.5 μg plasmid per 106 cells using 1.5 μg PEI (polyethylenimine). The plasmid and PEI were pre-incubated for 20 mins in Freestyle media (Thermo Fisher) at one tenth the final volume and then added dropwise to the cells. After 3-5 days, provided the cell viability was >95%, the cell culture media was harvested by centrifugation (300×g for 10 mins). Imidazole was added to a final concentration of 10 mM and the media was filter sterilized (0.2 μm) on ice. Nickel NTA resin (Qiagen) was washed 3× with 10 mM Imidazole in PBS and then incubated with the filtered media overnight at 4° C. on a roller mixer (Thermo Fisher). The Nickel NTA resin was then washed 3× with 10 mM Imidazole in PBS and the recombinant proteins were eluted with 300 mM Imidazole in PBS. The purity of the eluted fractions was confirmed by SDS-PAGE with a 4-12% Bis-Tris gel (Invitrogen), pooled together and concentrated to 1 ml using an Amnicon centrifugation filter with a 10K molecular weight cut off (Millipore). The resultant protein was run over a Sephadex™300 column and 0.5 ml fractions were collected. The purity of the eluted fractions was confirmed by SDS-PAGE on a 4-12% Bis-Tris gel (Invitrogen) and the concentration determined by measuring absorbance at 280 nm using a Nanodrop spectrophotometer (Thermo Fisher). Immunoreactivity of the purified nephrin was confirmed by Western blot analysis, under reducing conditions using a primary sheep anti-human nephrin antibody (R&D) followed by a secondary HRP-conjugated donkey anti-sheep IgG antibody (Jackson immunoresearch), and of the purified hPLA2R under non-reducing conditions using serum from a patient with known anti-PLA2R antibodies (determined by commercial ELISA and IIFT (Euroimmun)) diluted 1:1000 and a secondary HRP-conjugated donkey anti-human IgG antibody (Jackson Immunoresearch).
Enzyme Linked Immunosorbent Assay (ELISA)
Nunc MaxiSorp™ ELISA plates (Thermo Fisher) were coated with either 1 μg/ml recombinant extracellular domain of human nephrin or hPLA2R diluted in coating buffer (Biolegend) and incubated O/N at 4° C. Uncoated control wells were used to determine non-specific binding (in the absence of antigen) for each patient sample and this allowed for background subtraction. The plates were washed 3× with 300 μl PBS+0.05% Tween 20 (PBST). Plates were blocked with 300 μl of Superblock (Thermo Fisher) for 1 hr at RT and then incubated O/N at 4° C. with 100 μl of patient samples diluted 1:100 in SuperBlock containing 0.1% Tween 20 (SuperT). Samples with an initial high titer were subsequently diluted to 1:200 or 1:400. Plates were washed a further 5× with 300 μl of PBST, followed by incubation with 100 μl of biotin-conjugated goat anti-human IgG Fc, highly x-absorbed antibody (Thermo Fisher) diluted to 0.75 μg/ml in SuperT, shaking at 500 r.p.m for 1 hr at RT. Plates were washed 5× with PBST followed by incubation with 100 μl of HRP-conjugated avidin (Biolegend) diluted to 1:2000 in SuperT, shaking at 500 r.p.m for 30 min at RT. Following 5 final washes with PBST, 100 μl of tetramethyl benzidine (TMB) substrate (Biolegend) was added and the plates incubated for 10 min at RT. 100 μl of stop solution (Biolegend) was added and the absorbance at 450 nm was measured. Background subtraction was performed by subtracting the average OD of duplicate uncoated wells from the average OD of duplicate antigen coated wells for each individual patient sample34. Anti-nephrin antibody titers were then determined using a standard curve derived from a serial two-fold dilution series of a positive patient sample (MCD15+) in which a 1:100 dilution was arbitrarily defined as containing 1,000 units/ml.
Immunoprecipitation and Western Blot
1 volume of patient serum or plasma was mixed with 5 volumes of RIPA buffer containing HGE, or 100 ng recombinant extracellular domain of human nephrin, and incubated overnight (O/N) at 4° C. IgG-antigen complexes were precipitated with Protein G plus agarose beads (Santa Cruz) for 2 hours at 4° C. The beads were collected by centrifugation and washed 3 times with Tris buffered saline supplemented with 0.2% Tween-20 (TBST) and a final wash with distilled water. Proteins were eluted from the beads and denatured under reducing conditions by heating at 95° C. for 5 mins in 1×Laemmli buffer (Biorad) containing 2.5% beta-mercaptoethanol. Samples were loaded into precast 7.5% Mini Protean Tris-glycine gels (Biorad) and electrophoresed at 100V for 90 minutes in the presence of Novex Tris-glycine-SDS running buffer (Thermo Fisher). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore) using the Pierce Power Blotter system (Thermo Fisher) for 10 mins at 25V, 1.3 A. Membranes were blocked for one hour at RT in TBST containing 5% skimmed milk (w/v) followed by incubation O/N at 4° C. with 1 μg/ml polyclonal sheep anti-human nephrin antibody (R&D systems; AF4269) diluted in TBST with 2% skimmed milk (w/v). All other antibodies were diluted in TBST containing 5% skimmed milk and incubated for one hour at RT. Membranes were washed with TBST 3 times for 5 minutes each, followed by a secondary horseradish peroxidase (HRP)-conjugated donkey anti-sheep IgG antibody (Jackson Immunoresearch; 713-035-147) diluted 1:20,000. Human IgG heavy chain was detected using HRP-conjugated donkey anti-human IgG antibody (Jackson Immunoresearch; 709-035-149) diluted 1:10,000. Finally, membranes were washed 3 times with TBST and incubated with Supersignal™ West PICO PLUS or FEMTO chemiluminescent substrate (Thermo Fisher) for 3 minutes and images were acquired on a Universal Hood III gel dock system (Biorad).
Clinical Case Details
To illustrate a potential role of pre-transplant, nephrin autoantibodies in early massive post-transplant proteinuria recurrence, we present the case of a 27-year-old woman with an initial diagnosis of steroid-responsive MCD at age 2 who became steroid dependent (SDNS), and eventually progressed with subsequent biopsies showing FSGS. Importantly, clinical whole exome sequencing (Prevention Genetics) found no known NS disease causing variants. She eventually developed ESKD and initially underwent hemodialysis for 5 years and then received a pediatric deceased donor kidney (cold ischemia time 19 hours) with immediate graft function (
To first determine whether circulating autoantibodies against nephrin are detectable in the serum of patients with biopsy proven MCD and no known genetic basis (lacking known pathogenic variants in established Mendelian NS genes), we evaluated serum obtained from the Nephrotic Syndrome Study Network (NEPTUNE) longitudinal cohort study23 consisting of 41 (66%) children and 21 (34%) adults (Table 2). We developed an indirect enzyme-linked immunosorbent assay (ELISA) using a recombinant, affinity purified extracellular domain of human nephrin (hNephrinG1059) and established a threshold for anti-nephrin antibody (ab) positivity, based on the maximum titer in a healthy control population (n=30) (
The patients' clinical characteristics (Table 1) and the median time from enrollment to complete remission (CR) were similar between the anti-nephrin ab positive and negative groups (4.4 months vs 5.4 months respectively; p=0.7288) (FIG. S2). However, the relapse-free period was shorter for the anti-nephrin ab positive group compared with the ab negative group, although this finding did not reach conventional levels of statistical significance (median time to relapse 6.0 months vs 21.57 months respectively; p=0.0945).
A subsequent serum sample was available during either complete (UPCR<0.3 g/g) or partial remission (>50% reduction in proteinuria) from 12 of the 18 anti-nephrin ab positive patients, in whom we observed a complete absence or a significant reduction of nephrin autoantibodies respectively (
To further investigate a potential pathogenic role of these nephrin autoantibodies, we next sought to establish whether they are present within kidneys of patients with MCD. One limitation of the NEPTUNE cohort is that biopsy material from these patients was not available for further evaluation and so we turned to our own institution and collaborators for biopsy and serum samples.
For many years, we have observed a delicate punctate staining for IgG in a subset of patients with MCD (MCD+) by routine immunofluorescence staining that is distinct from the background (
We utilized confocal microscopy to further evaluate this punctate IgG in renal biopsies that were received and processed by us over the last 3 years. We observed two predominant patterns of IgG distribution: GBM-associated fine punctate or curvilinear structures and more apically located punctate and vaguely vesicular clusters, with the latter being more common. These disparate staining patterns may reflect different stages of antibody binding and/or redistribution. In all the MCD+ biopsies evaluated, we observed specific co-localization of nephrin with the punctate IgG and not the background (
To confirm that MCD+ patients with punctate IgG on renal biopsy do indeed have circulating autoantibodies against nephrin, we evaluated serum or plasma that was available specifically during active disease for 9 of them. As expected, all 9 patients were serologically positive for anti-nephrin antibodies by ELISA, in contrast to 12 control patients lacking punctate IgG on renal biopsy, who were all serologically negative (
Finally, to highlight a potential role of pre-transplant nephrin autoantibodies in post-transplant disease recurrence, which generally shows morphologic features indistinguishable from MCD, we identified a 27-year-old patient with childhood onset, steroid dependent MCD and no underlying genetic basis (as determined by clinical whole exome sequencing) who progressed to ESKD requiring kidney transplantation (detailed clinical history given above). In keeping with a pathogenic role for anti-nephrin autoantibodies, she developed massive proteinuria early post-transplant, that in contrast to CNF18,19 was associated with high pre-transplant levels of nephrin autoantibodies (
The threshold for a positive anti-nephrin antibody (α-nephrin Ab) level was based on a randomly selected, healthy control population with no renal disease. Complete remission was defined as a UPCR<0.3 g/g. Partial remission was defined as a>50% reduction in proteinuria that did not fall below 0.3 g/g. The continuous variables are presented as median (interquartile range). Statistical analysis was performed using the Mann-Whitney test for continuous variables and Fisher's exact test for categorical variables (*p<0.05).
Table 2 provides relevant clinical information for the patients or controls. All patients in the NEPTUNE cohort had biopsy proven minimal change disease (MCD); however, the renal biopsy IgG deposition status was not reported and neither immunofluorescence images nor biopsy material were available for further assessment. Proteinuria values (Urine Protein Creatinine ratio (UPCR)) are from the same day (or within one day) that the serum sample was collected for anti-nephrin antibody (α-Nephrin Ab) testing during active disease. #For patient N13, the UPCR was calculated to be 323 g/g on the day of serum collection and so the value for the next available UPCR (assessed 20 days later) is given. Peak sCr (serum creatinine) was the highest serum creatinine reached during the follow-up period. Partial remission was defined as >50% reduction in the UPCR and complete remission (CR) as UPCR<0.3 g/g. A patient was deemed to have relapsed with a UPCR>3 g/g after first reaching CR. In those patients not reaching CR, the relapse status is not applicable (N/A). Serum was obtained from a randomly selected healthy control cohort from Partners Healthcare Biobank. The threshold for a positive anti-nephrin antibody titer was based on the maximum value for the healthy cohort of 187 U/ml. Antibody titer is given as the mean±S.D. of replicate samples (n≥3) for each patient. Serum from patients who tested positive for anti-human PLA2R antibodies (hPLA2R+) by two clinically validated assays, ELISA and IIFT (Euroimmun), were obtained from MGH Immunopathology Laboratory. Designations: P, prednisolone; CNI, calcineurin inhibitor; MMF, mycophenolate mofetil; CTX, cyclophosphamide; RIT, rituximab; FLU, flucytosine; PRTL, partial; CMPLT, complete; * indicates Hispanic or Latino ethnicity.
The BWH/MGH/BMC/Mayo Clinic cohort consists of patients whose renal biopsy was evaluated for IgG by immunofluorescence staining (IF) and a concurrent serum sample, where available, was evaluated for anti-nephrin antibodies. For the control patients that had a tumor nephrectomy for RCC (N×1, N×2) an area of non-neoplastic renal parenchyma was evaluated by IF. Proteinuria values are given as either UPCR (g/g) or urine dipstick (negative, 3+, 4+) unless otherwise stated (#For patient MCD17−, proteinuria is given as urine albumin creatinine ratio (UACR) (g/g)). Serum Creatinine (Serum Cr) and proteinuria values are those closest to the time of serum sampling for patients evaluated for anti-nephrin antibodies and closest to the biopsy for those who were not. The predominant IgG subclass is given in parenthesis where known (ND indicates that the IgG subclass was not determined due to lack of additional biopsy material). FSGS, focal segmental glomerulosclerosis; TL, tip lesion; MN, membranous nephropathy; RCC, renal cell cancer; MCD, Minimal change disease. * indicates Hispanic or Latino ethnicity.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/104,306, filed on Oct. 22, 2020. The entire contents of the foregoing are incorporated herein by reference.
This invention was made with Government support under Grant Nos. DK007053 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/056157 | 10/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63104306 | Oct 2020 | US |
