Patent Application
20230400465
This document relates to methods and materials involved in identifying and/or treating mammals having membranous nephropathy (e.g., lupus membranous nephritis) likely or unlikely to progress to end stage kidney disease. For example, this document provides methods and materials for determining if a mammal having membranous nephropathy (e.g., lupus membranous nephritis) is likely or unlikely to progress to end stage kidney disease. This document also provides methods and materials for administering one or more immunosuppressive agents (e.g., corticosteroids, cyclosporine, mycophenolate mofetil, or a B-cell reduction or depletion agent such as Rituximab) to a mammal (e.g., a human) having membranous nephropathy that was identified as being likely to progress to end stage kidney disease.
Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in white adults (Couser, Clin. J. Am. Soc. Nephrol., 12:983-997 (2017); Makker et al., Semin. Nephrol., 31:333-340 (2011); and Ronco et al., The Lancet, 385:1983-1992 (2015)). Primary MN accounts for approximately 70-80% of cases, and the remaining 20-30% cases are secondary MN. In the primary MN, M-Type Phospholipase A2 Receptor antigen (PLA2R), Thrombospondin Type-1 Domain-Containing 7A antigen (THSD7A), Neural epidermal growth factor-like 1 protein (NELL1), and Semaphorin 3B (Sema3B) have been identified as target antigens (Beck et al., N. Engl. J. Med., 361:11-21 (2009); Tomas et al., N. Engl. J. Med., 371:2277-2287 (2014); Sethi et al., Kidney International, 97:163-174 (2020); and Sethi et al., Kidney International, 98:5-1253-1264 (2020)). Secondary MN is associated with autoimmune diseases, malignancies, infection, and drugs (Couser, Clin. J. Am. Soc. Nephrol., 12:983-997 (2017)). Recently, two novel proteins, Exostosin 1 (EXT1) and Exostosin 2 (EXT2), were recently discovered in the glomerular basement membrane (GBM) in a small cohort of PLA2R-negative MN (Anders et al., Nature Rev. Nephrol., 15:595-596 (2019); and Sethi et al., J. Am. Soc. Nephrol., 30:1123-1136 (2019)). More than 80% of EXT1/EXT2-positive patients had an autoimmune finding, confirmed by serology, clinical presentation, or kidney biopsy findings of an underlying autoimmune disease (Sethi et al., J. Am. Soc. Nephrol., 30:1123-1136 (2019)).
This document provides methods and materials involved in identifying and/or treating mammals (e.g., humans) having membranous nephropathy (e.g., lupus membranous nephritis) likely or unlikely to progress to end stage kidney disease. For example, this document provides methods and materials for identifying a mammal (e.g., a human) having membranous nephropathy (e.g., lupus membranous nephritis) as being likely or unlikely to progress to end stage kidney disease. As described herein, mammals (e.g., humans) having membranous nephropathy (e.g., lupus membranous nephritis) that contain kidney tissue (e.g., GBM) that does not express EXT1 or EXT2 polypeptides (e.g., kidney tissue that is EXT1-negative and EXT2-negative) can be identified as being likely to progress to end stage kidney disease. In such cases, the mammal can be classified as being likely to progress to end stage kidney disease. As also described herein, mammals (e.g., humans) having membranous nephropathy (e.g., lupus membranous nephritis) that contain kidney tissue (e.g., GBM) that expresses EXT1 or EXT2 polypeptides (e.g., kidney tissue that is EXT1-positive and EXT2-positive) can be identified as being unlikely to progress to end stage kidney disease. In such cases, the mammal can be classified as being unlikely to progress to end stage kidney disease. Identifying mammals (e.g., humans) having membranous nephropathy as being likely or unlikely to progress to end stage kidney disease can allow clinicians and patients to proceed with appropriate membranous nephropathy treatment options.
This document also provides methods and materials for treating membranous nephropathy (e.g., lupus membranous nephritis). For example, a mammal (e.g., a human) having membranous nephropathy (e.g., lupus membranous nephritis) that was identified as being likely to progress to end stage kidney disease as described herein can be administered one or more immunosuppressive agents (e.g., corticosteroids, cyclosporine, mycophenolate mofetil or a B-cell reduction or depletion agent such as Rituximab) to reduce the progression of the mammal to end stage kidney disease. Having the ability to administer one or more immunosuppressive agents to mammals (e.g., humans) having membranous nephropathy that was identified as being likely to progress to end stage kidney disease can allow clinicians and patients to treat membranous nephropathy effectively.
In general, one aspect of this document features a method for identifying a mammal as having membranous nephropathy that is likely or unlikely to progress to end stage kidney disease. The method comprises (or consists essentially of or consists of) (a) determining if kidney tissue from the mammal expresses a polypeptide, wherein the polypeptide is an exostosin 1 (EXT1) polypeptide or an exostosin 2 (EXT2) polypeptide; (b) classifying the mammal as having the membranous nephropathy that is unlikely to progress to the end stage kidney disease if the kidney tissue expresses the polypeptide; and (c) classifying the mammal as having the membranous nephropathy that is likely to progress to the end stage kidney disease if the kidney tissue does not express the polypeptide. The mammal can be a human. The polypeptide can be the EXT1 polypeptide. The polypeptide can be the EXT2 polypeptide. The method can comprise classifying the mammal as having the membranous nephropathy that is unlikely to progress to the end stage kidney disease. The method can comprise classifying the mammal as having the membranous nephropathy that is likely to progress to the end stage kidney disease. The method can comprise using immunohistochemistry to determine if the kidney tissue expresses the polypeptide. The method can comprise using laser microdissection and mass spectrometry to determine if the kidney tissue expresses the polypeptide. The method can comprise determining that the kidney tissue expresses the EXT1 polypeptide and the EXT2 polypeptide. The method can comprise determining that the kidney tissue does not express the EXT1 polypeptide and the EXT2 polypeptide.
In another aspect, this document features a method for treating a mammal having membranous nephropathy likely to progress to end stage kidney disease. The method comprises (or consists essentially of or consists of) (a) determining that kidney tissue from the mammal does not express a polypeptide, wherein the polypeptide is an exostosin 1 (EXT1) polypeptide or an exostosin 2 (EXT2) polypeptide; and (b) administering an immunosuppressant to the mammal to reduce the rate of progression to the end stage kidney disease. The mammal can be a human. The polypeptide can be the EXT1 polypeptide. The polypeptide can be the EXT2 polypeptide. The method can comprise using immunohistochemistry to determine that the kidney tissue does not express the polypeptide. The method can comprise using laser microdissection and mass spectrometry to determine that the kidney tissue does not express the polypeptide. The method can comprise determining that the kidney tissue does not express the EXT1 polypeptide and the EXT2 polypeptide.
In another aspect, this document features a method for treating a mammal having membranous nephropathy likely to progress to end stage kidney disease. The method comprises (or consists essentially of or consists of) administering an immunosuppressant to a mammal identified as having kidney tissue that does not express a polypeptide, wherein the polypeptide is an exostosin 1 (EXT1) polypeptide or an exostosin 2 (EXT2) polypeptide. The mammal can be a human. The polypeptide can be the EXT1 polypeptide. The polypeptide can be the EXT2 polypeptide. The method can comprise using immunohistochemistry to determine that the kidney tissue does not express the polypeptide. The method can comprise using laser microdissection and mass spectrometry to determine that the kidney tissue does not express the polypeptide. The method can comprise determining that the kidney tissue does not express the EXT1 polypeptide and the EXT2 polypeptide.
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 pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
This document provides methods and materials for identifying and/or treating mammals (e.g., humans) having membranous nephropathy (e.g., lupus membranous nephritis). For example, this document provides methods and materials for identifying a mammal (e.g., a human) having membranous nephropathy (e.g., lupus membranous nephritis) as being likely or unlikely to progress to end stage kidney disease.
Any appropriate mammal having membranous nephropathy can be identified as being likely or unlikely to progress to end stage kidney disease. For example, humans and other primates such as monkeys having membranous nephropathy can be identified as being likely or unlikely to progress to end stage kidney disease. In some cases, dogs, cats, horses, cows, pigs, sheep, mice, or rats having membranous nephropathy can be identified as being likely or unlikely to progress to end stage kidney disease as described herein.
As described herein, mammals (e.g., humans) having membranous nephropathy that have kidney tissue (e.g., GBM) that does not express EXT1 or EXT2 polypeptides can be identified as being likely to progress to end stage kidney disease, and mammals (e.g., humans) having membranous nephropathy (e.g., lupus membranous nephritis) that contain kidney tissue (e.g., GBM) that expresses EXT1 or EXT2 polypeptides (e.g., kidney tissue that is EXT1-positive and EXT2-positive) can be identified as being unlikely to progress to end stage kidney disease.
Any appropriate method can be used to determine if a mammal (e.g., a human) having membranous nephropathy expresses or does not express EXT1 or EXT2 polypeptides. For example, immunological techniques such as immunohistochemistry (IHC) techniques, immunofluorescence (IF) techniques, or Western blot techniques can be used to determine if a mammal (e.g., a human) has kidney tissue (e.g., GBM) that expresses or does not express EXT1 or EXT2 polypeptides. In some cases, a kidney tissue sample obtained from a mammal to be tested can be stained using an anti-EXT1 antibody to determine if the mammal has kidney tissue (e.g., GBM) that expresses or does not express EXT1 polypeptides. In some cases, a kidney tissue sample obtained from a mammal to be tested can be stained using an anti-EXT2 antibody to determine if the mammal has kidney tissue (e.g., GBM) that expresses or does not express EXT2 polypeptides. Any appropriate sample can be used to determine if a mammal (e.g., a human) has kidney tissue (e.g., GBM) that expresses or does not express EXT1 or EXT2 polypeptides. For example, kidney tissue biopsies can be obtained from a mammal (e.g., a human) being tested and used to determine if a mammal (e.g., a human) has kidney tissue (e.g., GBM) that expresses or does not express EXT1 or EXT2 polypeptides.
As used herein, kidney tissue is considered to express a polypeptide (e.g., an EXT1 polypeptide or an EXT2 polypeptide) when the GBM shows granular staining for the polypeptide (e.g., an EXT1 polypeptide or an EXT2 polypeptide) on immunohistochemistry or immunofluorescence microscopy using an anti-EXT1 antibody or an anti-EXT2 antibody. As used herein, kidney tissue is considered to not express a polypeptide (e.g., an EXT1 polypeptide or an EXT2 polypeptide) when the GBM shows no granular staining for the polypeptide (e.g., an EXT1 polypeptide or an EXT2 polypeptide) on immunohistochemistry or immunofluorescence microscopy using an anti-EXT1 antibody or an anti-EXT2 antibody.
A human EXT1 polypeptide can have the amino acid sequence set forth in
Once a mammal (e.g., a human) having membranous nephropathy is identified as having kidney tissue that does not express an EXT1 polypeptide and/or an EXT2 polypeptide (e.g., EXT1-negative and EXT2-negative kidney tissue) as described herein, the mammal can be classified as having membranous nephropathy likely to progress to end stage kidney disease. Once a mammal (e.g., a human) having membranous nephropathy is identified as having kidney tissue that expresses an EXT1 polypeptide and/or an EXT2 polypeptide (e.g., EXT1-positive and EXT2-positive kidney tissue) as described herein, the mammal can be classified as having membranous nephropathy unlikely to progress to end stage kidney disease.
As described herein, this document also provides methods and materials for treating a mammal having membranous nephropathy likely to progress to end stage kidney disease. For example, a mammal (e.g., a human) having membranous nephropathy that was identified as likely to progress to end stage kidney disease as described herein can be treated with one or more immunosuppressants. In some cases, a mammal (e.g., a human) having membranous nephropathy that was identified as being likely to progress to end stage kidney disease as described herein can be administered, or instructed to self-administer, one or more immunosuppressants (e.g., anti-CD20 antibodies such as rituximab) to treat membranous nephropathy and/or slow the progression to end stage kidney disease.
Any appropriate immunosuppressant can be administered to a mammal (e.g., a mammal that was identified as being likely to progress to end stage kidney disease and/or that was identified as having EXT1-negative and/or EXT2-negative kidney tissue) to treat membranous nephropathy and/or slow the progression to end stage kidney disease. In some cases, an immunosuppressant used as described herein to treat membranous nephropathy and/or slow the progression to end stage kidney disease can reduce inflammation and/or reduce B-cell autoantibody production within a mammal. Examples of immunosuppressants that can be used as described herein to treat membranous nephropathy and/or slow the progression to end stage kidney disease include, without limitation, mycophenolate mofetil (e.g., Cellcept); steroids such as prednisone; B-cell inhibitors such as anti-CD20 antibodies (e.g., rituximab); calcineurin inhibitors such as cyclosporine and tacrolimus; and alkylating agents/chemotherapeutic drugs such as cyclophosphamide.
In some cases, two or more (e.g., two, three, four, five, six, or more) immunosuppressants can be administered to a mammal having membranous nephropathy that was identified as being likely to progress to end stage kidney disease as described herein. For example, two immunosuppressants (e.g., prednisone and Cellcept) can be administered to a human having membranous nephropathy that was identified as being likely to progress to end stage kidney disease as described herein.
In some cases, one or more immunosuppressants can be administered to a mammal once or multiple times over a period of time ranging from days to months. In some cases, one or more immunosuppressive drugs can be given to achieve remission of membranous nephropathy, and then given during follow up periods to prevent relapse of the membranous nephropathy. In some cases, one or more immunosuppressants can be formulated into a pharmaceutically acceptable composition for administration to a mammal (e.g., a human) having membranous nephropathy that was identified as being likely to progress to end stage kidney disease as described herein to reduce inflammation and/or to reduce B-cell autoantibody production within that mammal. For example, a therapeutically effective amount of an immunosuppressant can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, in the form of sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, or granules.
Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in a pharmaceutical composition described herein can include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
A pharmaceutical composition containing one or more immunosuppressants can be designed for oral or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration. When being administered orally, a pharmaceutical composition can be in the form of a pill, tablet, or capsule. Compositions suitable for parenteral administration can include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze dried (lyophilized) condition requiring the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
In some cases, a pharmaceutically acceptable composition including one or more immunosuppressants can be administered locally or systemically. For example, a composition provided herein can be administered locally by intravenous injection or blood infusion. In some cases, a composition provided herein can be administered systemically, orally, or by injection to a mammal (e.g., a human).
Effective doses can vary depending on the severity of the nephropathy, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating physician.
An effective amount of a composition containing one or more immunosuppressants can be any amount that slows the progression to end stage kidney disease and/or reduces inflammation or B-cell autoantibody production (e.g., B-cell antibody production inhibition or reduction in the number of B-cells) within a mammal having membranous nephropathy without producing significant toxicity to the mammal. For example, an effective amount of rituximab to treat membranous nephropathy that was identified as being likely to progress to end stage kidney disease as described herein can be from about 500 mg to about 1.5 g (e.g., from about 500 mg to about 1.25 g, from about 500 mg to about 1.0 g, from about 500 mg to about 750 mg, from about 750 mg to about 1.5 g, from about 1 g to about 1.5 g, or from about 1.25 g to about 1.5 g) administered intravenously about two weeks apart. In some cases, an effective amount of rituximab to treat membranous nephropathy that was identified as being likely to progress to end stage kidney disease as described herein can be from about 200 mg/m2 to about 500 mg/m2 (e.g., from about 200 mg/m2 to about 450 mg/m2, from about 200 mg/m2 to about 400 mg/m2, from about 200 mg/m2 to about 375 mg/m2, from about 250 mg/m2 to about 500 mg/m2, from about 300 mg/m2 to about 500 mg/m2, from about 350 mg/m2 to about 500 mg/m2, or from about 350 mg/m2 to about 400 mg/m2) administered weekly for about four weeks. If a particular mammal fails to respond to a particular amount, then the amount of an immunosuppressant can be increased by, for example, two fold. After receiving this higher amount, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. In some cases, the effective amount of a composition containing one or more immunosuppressants can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition can require an increase or decrease in the actual effective amount administered.
The frequency of administration of one or more immunosuppressants can be any amount that slows the progression to end stage kidney disease and/or reduces inflammation or B-cell autoantibody production (e.g., B-cell antibody production inhibition or reduction in the number of B-cells) within a mammal having membranous nephropathy without producing significant toxicity to the mammal. For example, the frequency of administration of an immunosuppressant can be from about once a day to about once a month (e.g., from about once a week to about once every other week). The frequency of administration of one or more immunosuppressants can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing one or more immunosuppressants can include rest periods. For example, a composition containing one or more immunosuppressants can be administered daily over a two-week period followed by a two-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in administration frequency.
An effective duration for administering a composition containing one or more immunosuppressants can be any duration that slows the progression to end stage kidney disease and/or reduces inflammation or B-cell autoantibody production (e.g., B-cell antibody production inhibition or reduction in the number of B-cells) within a mammal having membranous nephropathy without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several days to several months. In general, the effective duration for administering a composition containing one or more immunosuppressants to treat membranous nephropathy can range in duration from about one month to about five years (e.g., from about two months to about five years, from about three months to about five years, from about six months to about five years, from about eight months to about five years, from about one year to about five years, from about one month to about four years, from about one month to about three years, from about one month to about two years, from about six months to about four years, from about six months to about three years, or from about six months to about two years). In some cases, the effective duration for administering a composition containing one or more immunosuppressants to treat membranous nephropathy can be for as long as the mammal is alive. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.
In some cases, a course of treatment and/or the severity of one or more symptoms related to membranous nephropathy can be monitored. Any appropriate method can be used to determine whether or not membranous nephropathy is being treated and/or to determine whether or not progression to end stage kidney disease is being slowed. For example, remission and relapse of the disease can be monitored by testing for one or more markers for membranous nephropathy. In some cases, remission can be ascertained by detecting the disappearance or reduction of autoantibodies to THS7DA, NELL1, Sema3B, and/or PLA2R in the sera. In some cases, relapse of membranous nephropathy can be ascertained by a reappearance or elevation of autoantibodies to THS7DA, NELL1, Sema3B, and/or PLA2R in the sera.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Patients with a diagnosis of LMN on kidney biopsies were screened. The clinical information was obtained from the electronic medical records for the in-house biopsies. For other biopsies received from outside institutions, clinical information was limited and therefore, medical records with pertinent clinical information were requested.
Patients with clinical history of systemic lupus erythematosus (SLE) and biopsy-proven LMN were further analyzed. Light microscopy, immunofluorescence microscopy, and electron microscopy were performed in each case. Lupus nephritis (LN, Class III and IV) was classified according to the ISN/RPS classification (Bajema et al., Kidney Int., 93:789-796 (2018)). All these patients had paraffin-embedded tissue blocks available. Immunohistochemistry (IHC) studies were performed on 4 micron tissue sections using antibodies against EXT1/EXT2 as described elsewhere (Sethi et al., J. Am. Soc. Nephrol., 30:1123-1136 (2019)). Laser microdissection and mass spectrometry (MS/MS) were performed on a subset of these cases. Details of MS/MS are described elsewhere (Tomas et al., N. Engl. J. Med., 371:2277-2287 (2014)).
Categorical variables were presented as counts (percent) while continuous variables were presented as mean (standard deviation) if they were normally distributed as determined by Shapiro-Wilk test, or as median (interquartile range [IQR]) if non-normal. For comparisons of categorical variables between groups, the Pearson's chi-square test was used if the number of elements in each cell was ≥5; Fisher's exact test was used otherwise. For comparison of continuous variables between groups, an unpaired Student's t-test for independent samples was used for distributions consistent with normality as above, and the Mann-Whitney U test was used otherwise.
The Kaplan Meier method was used to assess cumulative incidence of end-stage kidney disease (ESKD) during the course of the follow-up. ESKD was defined as dependence of renal replacement therapy. Cox proportional hazards regression models were used to determine predictive factors of the ESKD. The hazard ratio (HR) was report with a 95% confidence intervals CI when appropriate.
P-values less than 0.05 (2-sided) were considered significant. IBM® SPSS® Statistics for MacOS, version 25 (IBM, Armonk, NY, USA) was used for data analysis.
A total of 374 cases with adequate tissue for IHC were included in the study. All patients had a clinical history of SLE. The kidney biopsy specimens of all LMN cases showed the characteristic findings of thickened GBM on light microscopy, bright IgG and C3 staining along the capillary wall on immunofluorescence microscopy, and subepithelial deposits on electron microscopy.
Of 374 biopsies, 122 (32.6%) biopsies had positive EXT1/EXT2 staining on IHC, and 252 (67.4%) were negative for EXT1/EXT2 (
Of the 122 patients, the median age at the time of kidney biopsy was 32 years (IQR: 25-41) (Table 1A). The majority were female (n=106; 86.9%) patients. At presentation, the median serum creatinine (S Cr) and proteinuria were 0.8 mg/dL (IQR: 0.6-1.2; n=112) and 3.5 g/24 hours (IQR: 2-6.6; n=95), respectively. Fifty-seven (60%, n=95) patients presented with proteinuria≥3.5 g/day at the time of kidney biopsy. Forty-one (33.6%) patients had hematuria.
Thirty-two (26.2%) patients had other associated autoimmune disorders including Grave's disease, antiphospholipid antibody syndrome, mixed connective tissue disease, rheumatoid arthritis, Sjögren syndrome, CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) and/or tested positive for other autoimmune serologies including anti-Smith, anti-SSA, anti-SSB, anti-ribonucleoprotein, anti-nuclear cytoplasmic antibodies, anti-Scl 70, anti-histone and/or anti-chromatin antibodies, lupus anticoagulant, respectively. Complement data were available in 62 patients, of which thirty-one (50%) had low C3 and C4, 4 (6.5%) low C3 only, 3 (4.8%) low C4 only, and the remaining 24 (38.7%) had normal complement levels.
Ninety-two (75.4%) biopsies showed pure class V LMN and remaining 30 (24.6%) biopsies had mixed class V with proliferative features (19 Class III and 11 Class IV LN, Table 1A). The kidney biopsy specimens of all cases of EXT1/EXT2-positive LMN showed the characteristic findings of thickened GBM on light microscopy (
EXT1/EXT2-Negative LMN
Of the 252 patients, the median age at the time of kidney biopsy was 35 years (IQR: 27-45) (Table 1A). The majority were female (n=205; 81.3%). At the time of kidney biopsy, the median SCr and urine proteinuria were 0.9 mg/dL (IQR: 0.7-1.4; n=227) and 3 g/24 hours (IQR: 1.8-4.8; n=199), respectively. Eighty-seven (43.7%, n=199) patients presented with proteinuria≥3.5 g/day at the time of kidney biopsy. Eighty-seven (34.5%) patients had hematuria.
Forty-seven (18.7%) patients had other associated autoimmune disorders (rheumatoid arthritis, Sjögren syndrome, Graves' disease, mixed connective tissue disease, immune thrombocytopenic purpura) and/or tested positive for other autoimmune serologies including anti-Smith, rheumatoid factor, anti-SSA, anti-SSB, anti-ribonucleoprotein, anti-nuclear cytoplasmic antibodies, anti-Scl 70, and anti-histone antibodies, respectively. Complement data were available in 126 patients, of which 62 (49.2%) had low C3 and C4, 20 (15.9%) low C3 only, 11 (8.7%) low C4 only, and the remaining 33 (26.2%) had normal complement levels.
A total of 171 (67.9%) cases showed pure class V MN, and the remaining 81 (32.1%) cases had mixed class V with proliferative features (29 Class III and 52 Class IV lupus nephritis). The kidney biopsy specimens of all cases of EXT1/EXT2-negative LMN showed the characteristic findings of thickened GBM on light microscopy (
Subendothelial and mesangial/paramesangial deposits were present in 146 (57.9%) and in 239 (94.8%) of 252 cases, respectively. Tubuloreticular inclusions were present in 199 (79%). Laser microdissection and mass spectrometry was performed in a subset of cases (n=7) and was negative for EXT 1 and EXT2, thus confirming the IHC findings (data not shown).
The clinical and laboratory features comparing EXT1/EXT2-positive and EXT1/EXT-2 negative LMN patients are described in Table 1A. In summary, compared to EXT1/EXT2-negative cohort (n=252), EXT1/EXT2-positive LMN (n=122) patients were slightly younger (P=0.013), have lower serum creatinine levels (P=0.022), tend to present with proteinuria≥3.5 gms/24 hours (P=0.009), and have less chronicity (glomerulosclerosis: P=0.001; interstitial fibrosis and tubular atrophy: P<0.0001). Based on Ehrenreich and Churg classification, a higher proportion of EXT1/EXT2-negative LMN have class III lesions compared to EXT1/EXT2-positive LMN (66.3% vs 45.9%, P<0.0001). On the other hand, a higher proportion of EXT1/EXT2-positive LMN have class II lesions compared to EXT1/EXT2-negative LMN (47.5% vs 28.6%, P<0.0001).
EXT1/EXT2 in Proliferative Lupus Nephritis (LN) without LMN
38 cases of class I-IV (class I: n=1; class II: n=7; class III: n=6; class IV; n=24) lupus nephritis with no component of LMN also were stained. All cases were negative for EXT1/EXT2 on IHC.
During the course of follow-up, four LMN cases had re-biopsy after the initial diagnosis of LMN; three of which were EXT1/EXT2-positive and one was EXT1/EXT2-negative LMN. All three biopsies of EXT-positive LMN did not show significant progression of chronicity indexes, while the single EXT1/EXT2-negative LMN showed increased global glomerulosclerosis and IFTA (Table 1C). EXT1 staining done in re-biopsies of four cases showed bright (3+) EXT1 staining in two cases (case 1 and 2) and weak (1+) staining in one case (case 3) of EXT1/EXT2-positive LMN, while it was negative in one EXT1/EXT2-negative LMN. In two EXT1/EXT2-positive LMN cases (case 1 and 2), re-biopsies were done after follow-up of 2 years. In one EXT1/EXT2-positive LMN case, re-biopsy was done after 10 years of follow-up, and in the one EXT1/EXT2-negative LMN case, re-biopsy was done after 7 years of follow-up.
Clinical follow-up data were available in a total of 160 patients. Clinical characteristics, follow-up, and outcome data are summarized in Tables 2-4 and
Of the 160 patients with follow up, 64 (40%) biopsies were EXT1/EXT2-positive, and 96 (60%) biopsies were EXT1/EXT2-negative LMN (Table 2). The proportion of patients with proliferative features was not statistically significantly different between groups: 16 (25%) of 64 in the EXT1/EXT2-positive group versus 31 (32.3%) of 96 patients in the EXT1/EXT2-negative group showed proliferative features (P=0.321). At presentation, the median SCr was statistically significantly higher in EXT1/EXT2-negative patients (0.90 vs. 0.80 mg/dL, P=0.004), but there were no differences in the presence of hematuria, or proteinuria≥3.5 g/day. Kidney biopsies of EXT1/EXT2-negative patients showed a statistically significantly higher median of global glomerulosclerosis and IFTA (P<0.0001). The treatment was variable and included the following: i) EXT1/EXT2-positive LMN: five received steroids only, 14 received immunosuppressants, and 22 received a combination of steroids and immunosuppressants, and for the remaining, treatment details were not available; ii) EXT1/EXT2-negative LMN: three were managed conservatively, four received steroids only, 18 received immunosuppressants, and 51 received a combination of steroids and immunosuppressants, and for the remaining, treatment details were not available. The most common immunosuppressants included plaquenil, mycophenolate mofetil, cyclophosphamide, and azathioprine.
Both groups were followed for a similar period of time (48.6 vs 50.6 months, P=0.848). At the end of the follow up, patients who were EXT1/EXT2-negative presented with statistically significant higher values of SCr (1.10 vs. 0.90 mg/dL, P=0.049). During the course of the disease, patients who were EXT1/EXT2-negative evolved more frequently to ESKD (18.8% vs. 3.1%, P=0.003), and the time to this event was statistically significantly shorter in this group when compared to EXT1/EXT2-positive patients (101 vs. 116 months, P=0.007,
Only Class V LMN (without Class III/IV LN)
Forty-eight (75%) of 64 EXT1/EXT2-positive cases and 65 (67.7%) of 96 EXT1/EXT2-negative cases had pure class V with no class III/IV LN (Table 3). Similarly to what was observed in the combined cohort, EXT1/EXT2-negative cases had statistically significantly higher values of SCr at diagnosis when compared with EXT1/EXT2-positive patients (0.80 vs. 0.70 mg/dL, P=0.010). The EXT1/EXT2 positive patients presented more frequently with proteinuria≥3.5 g/day at diagnosis (57.5% vs. 35.3%, P=0.035). Kidney biopsies of EXT1/EXT2-negative patients showed a statistically significantly higher median of global glomerulosclerosis and IFTA (P<0.0001). At the end of the follow up, there were no differences between groups on the median SCr or proteinuria≥3.5 g/day. During the course of the disease, patients who were EXT1/EXT2-negative evolved more frequently to ESKD (16.9% vs. 4.2%, P=0.036) but the mean time to ESKD was not statistically significantly different between groups (104 vs. 115 months, P=0.079,
Class V LMN with Class III/IV LN
Sixteen (25%) of 64 EXT1/EXT2-positive cases and 31 (32.3%) of 96 EXT1/EXT2-negative cases were class V LMN with proliferative features (class III or IV) (Table 4). At the time of diagnosis of LMN, there was no statistically significant difference between the two groups regarding SCr, hematuria, proteinuria, or presence of proteinuria≥3.5 g/day. However, in EXT1/EXT2-negative patients, kidney biopsies showed a statistically significantly higher median of global glomerulosclerosis and IFTA (P=0.013 and P=0.003, respectively). At the end of the follow up, there were no differences between groups on the median SCr or proteinuria≥3.5 g/day. EXT1/EXT2-positive patients with LN did not evolve to ESKD during the course of the disease (0.0% vs. 22.6%, P=0.039), whereas EXT1/EXT2-negative patients developed ESKD in a mean time of 93 months (P=0.031,
Exostosins (EXT1/EXT2) are glycosyltransferases that exist as heterodimeric co-polymerase complex responsible for synthesis of heparin sulfate chain in the glomerular basement membrane (Rops et al., Kidney Int., 86:932-942 (2014)). In this study of 374 patients of LMN, a subset (32.6%) of LMN were positive for EXT1/EXT2. EXT1/EXT2-positive LMN have less chronicity on kidney biopsy, have lower serum creatinine but a higher proportion of patients with proteinuria≥3.5 g/day at presentation, and on follow-up develop markedly decreased incidence of ESKD compared to EXT1/EXT2-negative LMN. The difference in chronicity between the EXT1/EXT2-positive and EXT1/EXT2-negative LMN was noted in all groups, i.e. overall LMN with or without class III/IV LN, pure LMN, and LMN with class III/IV LN. Similarly, ESKD developed more frequently in EXT1/EXT2-negative LMN in all groups, i.e. overall LMN with or without class III/IV LN, pure LMN, and LMN with class III/IV LN, compared to EXT1/EXT2-positive LMN. These findings have significant bearing when considering the high burden of SLE in worldwide and in particular North America where the prevalence of SLE is 241/100,000 (Stojan et al., Curr. Opin. Rheumatol., 30:144-150 (2018)). Furthermore, the mortality rate is increased to ˜2.6 fold in SLE, and the risk of mortality is the highest in patients with renal disease (Lee et al., Lupus, 25:727-734 (2016)). LN is the most common renal manifestation in SLE, almost 10% of the patients will develop ESKD, and 20% of the LN biopsies show LMN with or without concurrent class III/IV LN (Almaani et al., Clin. J. Am. Soc. Nephrol., 12:825-835 (2017); Alarcon, Reumatol. Clin., 7:3-6 (2011); Huong et al., Medicine (Baltimore), 78:148-166 (1999)).
The results provided herein demonstrate that EXT1/EXT2-positive LMN is a subgroup in LMN that is less likely to develop ESKD, and identification of this subgroup would be helpful to the management and prognosis of LMN. One question that arises is why do patients that are EXT1/EXT2-positive have better outcomes, despite higher proteinuria at presentation? Also, how does being positive for EXT1/EXT2 protect LMN from developing more progressive disease? Exostosins are present in the podocyte Golgi apparatus where they are responsible for the glycosylation of heparan sulfates that are eventually transported to the GBM (Busse-Wicher et al., Matrix Biology, 35:25-33 (2014); and Ahn et al., Nature Genetics, 11:137-143 (1995)). The exostosins have a short N-terminal cytoplasmic tail, a single transmembrane domain, a stem/stalk region, and a long globular catalytic C terminal domain (Duncan et al., J. Clin. Inv., 108:511-516 (2001); and McCormick et al., Proc. Natl. Acad. Sci. USA, 97:668-673 (2000)). EXT like other glycosyltransferases are secreted into the extracellular medium including the GBM in a truncated form (Paulson et al., J. Biol. Chem., 264:17615-17618 (1989)). It is hypothesize herein that in patients with EXT1/EXT2-positive LMN, the increased secretion of catalytic domain EXT1/EXT2 into the GBM results in increased synthesis of the heparan sulfates which in turn may offer protection from damaging events such as leukocyte infiltration, complement activation, cytokine production, etc (
The results provided herein indicate that cases of LN and LMN should be stained for EXT1/EXT2. The EXT1/EXT2-positive cases in LN may help detect an unknown component of LMN. In addition, it is reasonable to just stain for EXT1 instead of staining for both EXT1 and EXT2 since no single case showed isolated positivity and EXT1 appears brighter than EXT2. Finally, most cases of LMN were not re-biopsied. In this cohort, only four LMN cases were re-biopsied of which three were EXT1/EXT2-positive and one was EXT1/EXT2-negative. None of the EXT1/EXT2-positive LMN showed progression of the chronicity indexes while the single EXT1/EXT2-negative LMN case showed progression of chronicity indexes.
This was a retrospective study of LMN biopsies, and detailed follow-up was available in 160 (42.8%) patients of the 374 patients of LMN. Specific treatment details along with length of treatment were not available in many patients.
To summarize, 32.6% of LMN patients were positive for EXT1/EXT2. The extent of chronic changes including global glomerulosclerosis and tubular atrophy and interstitial fibrosis in EXT1/EXT2-positive LMN are less compared to EXT1/EXT2-negative LMN. EXT1/EXT2-positive LMN rarely develop ESKD compared to EXT1/EXT2-negative LMN.
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. Patent Application Ser. No. 63/111,982, filed on Nov. 10, 2020. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/057259 | 10/29/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63111982 | Nov 2020 | US |
