One antigen closer to understanding membranous nephropathy.

Membranous nephropathy is an immune-mediated kidney disease characterized by subepithelial immune deposits. Through the utilization of advanced proteomic techniques, multiple target antigens have been identified, including those associated with primary and secondary etiologies. Nonsteroidal anti-inflammatory drugs represent an important secondary cause of membranous nephropathy. In this study by Sethi et al., advanced proteomic techniques identify proprotein convertase subtilisin/kexin type 6 as a target antigen in nonsteroidal anti-inflammatory drug-associated membranous nephropathy.

Quantitative Mass Spectrometry Normalization in Urine Biomarker Analysis in Nephrotic Syndrome.

Chronic kidney disease (CKD) affects 30 million adults, costs ~$79 billion dollars (2016) in Medicare expenditures, and is the ninth leading cause of death in the United States. The disease is silent or undiagnosed in almost half of people with severely reduced kidney function. Urine provides an ideal biofluid that is accessible to high-sensitivity mass spectrometry-based proteomic interrogation and is an indicator of renal homeostasis. While the accurate and precise diagnosis and better disease management of CKD can be aided using urine biomarkers, their discovery in excessive protein or nephrotic urine samples can present challenges. In this work we present a mass spectrometry-based method utilizing multiplex tandem mass tag (TMT) quantification and improved protein quantification using reporter ion normalization to urinary creatinine to analyze urinary proteins from patients with a form of nephrotic syndrome (FSGS). A comparative analysis was performed for urine from patients in remission versus active disease flare. Two-dimensional LC-MS/MS TMT quantitative analysis identified over 1058 urine proteins, 580 proteins with 2 peptides or greater and quantifiable. Normalization of TMT abundance values to creatinine per ml of urine concentrated reduced variability in 2D-TMT-LC-MS/MS experiments. Univariate and multivariate analyses showed that 27 proteins were significantly increased in proteinuric disease flare. Hierarchical heatmap clustering showed that SERPINA1 and ORM1 were >1.5 fold increased in active disease versus remission urine samples. ELISA validation of SERPINA1 and ORM1 abundance agreed with our quantitative TMT proteomics analysis. These findings provide support for the utility of this method for identification of novel diagnostic markers of CKD and identify SERPINA1 and ORM1 as promising candidate diagnostic markers for FSGS.

Serum and Urine Interferon Gamma-Induced Protein 10 (IP-10) Levels in Lupus Nephritis.

Background: Lupus nephritis (LN) is a prevalent and severe complication of systemic lupus erythematosus (SLE). Non-invasive diagnostics are limited, and current therapies have inadequate response rates. Expression of the chemokine Interferon-γ-induced protein 10 (IP-10) is regulated by Interferon-γ signaling and NF-κB, and its molecular activity and enhanced urine concentrations are implicated in LN, but its utility as a diagnostic marker and association with demographic, clinical, or pathologic features is not defined. Methods: 38 LN patients and 11 patients with non-LN glomerular diseases (GD) with active disease were included. Eighteen of the LN patients had achieved remission at one follow-up during the study time. Serum and urine were obtained from these samples, and the IP-10 levels were measured. Results: Serum and urine IP-10 levels are significantly enhanced in LN patients with active disease as compared with normal individuals (serum average 179.7 pg/mL vs. 7.2 pg/mL, p < 0.0001; urine average 28.7 pg/mg vs. 1.6 pg/mg, p = 0.0019) and patients with other forms of glomerular disease (serum average 179.7 pg/mL vs. 84.9 pg/mL, p = 0.0176; urine average 28.7 pg/mg vs. 0.18 pg/mg, p = 0.0011). Urine IP-10 levels are significantly higher in patients with proliferative LN (PLN) than those with membranous LN (MLN) (average 32.8 pg/mg vs. 7.6 pg/mg, p = 0.0155). Urine IP-10 levels are also higher in MLN versus primary membranous nephropathy (MN) (average 7.6 pg/mg vs. 0.2 pg/mg, p = 0.0193). Importantly, serum IP-10 levels remain elevated during active LN and LN remission, but urine IP-10 levels are decreased from active LN to remission in 72% of our patients. Lastly, serum, but not urine IP-10 levels are significantly higher in African American than White American LN patients in active LN (average 227.8 pg/mL vs. 103.4 pg/mL, p = 0.0309) and during LN remission (average 254.6 pg/mL vs. 89.2 pg/mL, p = 0.0399). Conclusions: Our findings suggest that serum and urine IP-10 measurements provide promising tests for monitoring LN activity, differentiation between classifications of LN, and differentiation between LN and other forms of glomerular disease. We also conclude that further assessment of elevated IP-10 levels in the serum and urine of high-risk populations (i.e., African American) could be beneficial in determining why many of these patients have worse outcomes and are non-responsive to standard therapeutics.

Patients with Proliferative Lupus Nephritis Have Autoantibodies That React to Moesin and Demonstrate Increased Glomerular Moesin Expression.

Kidney involvement in systemic lupus erythematosus (SLE)-termed lupus nephritis (LN)-is a severe manifestation of SLE that can lead to end-stage kidney disease (ESKD). LN is characterized by immune complex deposition and inflammation in the glomerulus. We tested the hypothesis that autoantibodies targeting podocyte and glomerular cell proteins contribute to the development of immune complex formation in LN. We used Western blotting with SLE sera from patients with and without LN to identify target antigens in human glomerular and cultured human-derived podocyte membrane proteins. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified the proteins in the gel regions corresponding to reactive bands observed with sera from LN patients. We identified 102 proteins that were present in both the podocyte and glomerular samples. We identified 10 high-probability candidates, including moesin, using bioinformatic analysis. Confirmation of moesin as a target antigen was conducted using immunohistochemical analysis (IHC) of kidney biopsy tissue and enzyme-linked immunosorbent assay (ELISA) to detect circulating antibodies. By IHC, biopsies from patients with proliferative lupus nephritis (PLN, class III/IV) demonstrated significantly increased glomerular expression of moesin (p < 0.01). By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against moesin (p < 0.01). This suggests that moesin is a target glomerular antigen in lupus nephritis.

TNIP1/ABIN1 and lupus nephritis: review.

SLE is a complex autoimmune disease with genetic, epigenetic, immune-regulatory, environmental and hormonal factors. Kidney inflammation and injury, termed lupus nephritis (LN), occurs in over half of patients with SLE and is a leading cause of disability and death. There is a high degree of short-term and long-term side effects associated with current LN therapies and they are not effective for many patients. Thus, novel therapies with reduced toxicity and improved efficacy are drastically needed. Many of the known LN susceptibility genes have functions that mediate inflammation via cytokine/chemokine production and activation of myeloid and B cells. Understanding the cellular and molecular mechanisms mediated by these variant gene products provides valuable insight for the development of improved and personalised diagnostics and therapeutics. This review describes variants in the TNIP1 (tumour necrosis factor α-induced protein 3-interacting protein 1) gene associated with risks for SLE and LN and potential roles for loss of function of its protein product ABIN1 in the activation of myeloid and B-cell-mediated injury in LN.

Precision Targeting of NF-κB Signaling in Lupus Nephritis.

Lupus Nephritis (LN) is the leading cause of morbidity and mortality from Systemic Lupus Erythematosus (SLE) and enhanced activation of the transcription regulator nuclear factor kappa B (NF-κB) is implicated as a central player in the development and progression of LN. SLE and LN are proposed to develop through a "two-hit" process of genetic mutation or variants providing susceptibility to disease provoking molecular events in response to environmental triggers (viral infection, medication, etc.). Many of the susceptibility genes identified in association with LN are involved in NF-κB regulation and loss of function of some of the protein products in animal's results in protection from or development of SLE and LN phenotypes. This short commentary will discuss these factors and implications in precision treatment of LN.

Utilization of Biomarkers in Lupus Nephritis.

Lupus nephritis (LN) occurs in up to 60% of SLE patients, and is a leading cause of disability and death. Current treatment of LN consists of a combination of high dose corticosteroids that non-specifically decrease inflammation and cytotoxic medications that reduce auto-antibody production. That combination of therapy is associated with significant side effects while remission rates remain inadequate. Since the introduction of biologics into the pharmacological armamentarium, there has been hope for less toxic and more effective therapies for LN. Unfortunately, after multiple clinical trials, no biologic has improved efficacy over standard of care therapies for LN. This is likely, in part, due to disease heterogeneity. The utilization of biomarkers in LN may provide a way to stratify patients and guide therapeutic options. In this review, we summarize traditional and novel LN biomarkers and discuss how they may be used to diagnose, stratify, and guide therapy in patients with LN, bringing precision medicine to the forefront of LN therapy.

Precision medicine in lupus nephritis: can biomarkers get us there?

Patients with systemic lupus erythematosus frequently develop lupus nephritis (LN), a condition that can lead to end-stage kidney disease. Multiple serum and urine biomarkers for LN have been proposed in recent years, yet none have become incorporated into clinical use. The majority of studies have been single center with significant variability in cohorts, assays, and sample storage, leading to inconclusive results. It has become clear that no single biomarker is likely to be sufficient to diagnose LN, identify flares, and define the response to therapy and prognosis. A more likely scenario is a panel of urine, serum, tissue, and genetic biomarkers. In this review, we summarize traditional and novel biomarkers and discuss how they may be utilized in order to bring precision medicine to clinical practice in LN.

Neutrophil exocytosis induces podocyte cytoskeletal reorganization and proteinuria in experimental glomerulonephritis.

Acute glomerulonephritis is characterized by rapid glomerular neutrophil recruitment, proteinuria, and glomerular hypercellularity. The current study tested the hypothesis that the release of neutrophil granule contents plays a role in both the loss of filtration barrier leading to proteinuria and the increase in glomerular cells. Inhibition of neutrophil exocytosis with a peptide inhibitor prevented proteinuria and attenuated podocyte and endothelial cell injury but had no effect on glomerular hypercellularity in an experimental acute glomerulonephritis model in mice. Cultivation of podocytes with neutrophil granule contents disrupted cytoskeletal organization, an in vitro model for podocyte effacement and loss of filtration barrier. Activated, cultured podocytes released cytokines that stimulated neutrophil chemotaxis, primed respiratory burst activity, and stimulated neutrophil exocytosis. We conclude that crosstalk between podocytes and neutrophils contributes to disruption of the glomerular filtration barrier in acute glomerulonephritis. Neutrophil granule products induce podocyte injury but do not participate in the proliferative response of intrinsic glomerular cells.

Plasma Cell Depletion Attenuates Hypertension in an Experimental Model of Autoimmune Disease.

Numerous studies show a direct relation between circulating autoantibodies, characteristic of systemic autoimmune disorders, and primary hypertension in humans. Whether these autoantibodies mechanistically contribute to the development of hypertension remains unclear. Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by aberrant immunoglobulin production, notably pathogenic autoantibodies, and is associated with prevalent hypertension, renal injury, and cardiovascular disease. Because plasma cells produce the majority of serum immunoglobulins and are the primary source of autoantibodies in SLE, we hypothesized that plasma cell depletion using the proteasome inhibitor bortezomib would lower autoantibody production and attenuate hypertension. Thirty-week-old female SLE (NZBWF1) and control (NZW [New Zealand White]) mice were injected IV with vehicle (0.9% saline) or bortezomib (0.75 mg/kg) twice weekly for 4 weeks. Bortezomib treatment significantly lowered the percentage of bone marrow plasma cells in SLE mice. Total plasma IgG and anti-dsDNA IgG levels were higher in SLE mice compared with control mice but were lowered by bortezomib treatment. Mean arterial pressure (mm Hg) measured in conscious mice by carotid artery catheter was higher in SLE mice than in control mice, but mean arterial pressure was significantly lower in bortezomib-treated SLE mice. Bortezomib also attenuated renal injury, as assessed by albuminuria and glomerulosclerosis, and reduced glomerular immunoglobulin deposition and B and T lymphocytes infiltration into the kidneys. Taken together, these data show that the production of autoantibodies by plasma cells mechanistically contributes to autoimmune-associated hypertension and suggests a potential role for patients with primary hypertension who have increased circulating immunoglobulins.

ABIN1 Determines Severity of Glomerulonephritis via Activation of Intrinsic Glomerular Inflammation.

Transcription factor NF-κB regulates expression of numerous genes that control inflammation and is activated in glomerular cells in glomerulonephritis (GN). We previously identified genetic variants for a NF-κB regulatory, ubiquitin-binding protein ABIN1 as risk factors for GN in systemic autoimmunity. The goal was to define glomerular inflammatory events controlled by ABIN1 function in GN. Nephrotoxic serum nephritis was induced in wild-type (WT) and ubiquitin-binding deficient ABIN1[D485N] mice, and renal pathophysiology and glomerular inflammatory phenotypes were assessed. Proteinuria was also measured in ABIN1[D485N] mice transplanted with WT mouse bone marrow. Inflammatory activation of ABIN1[D472N] (D485N homolog) cultured human-derived podocytes, and interaction with primary human neutrophils were also assessed. Disruption of ABIN1 function exacerbated proteinuria, podocyte injury, glomerular NF-κB activity, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in antibody-mediated nephritis. Transplantation of WT bone marrow did not prevent the increased proteinuria in ABIN1[D845N] mice. Tumor necrosis factor-stimulated enhanced expression and secretion of NF-κB-targeted proinflammatory mediators in ABIN1[D472N] cultured podocytes compared with WT cells. Supernatants from ABIN1[D472N] podocytes accelerated chemotaxis of human neutrophils, and ABIN1[D472N] podocytes displayed a greater susceptibility to injurious morphologic findings induced by neutrophil granule contents. These studies define a novel role for ABIN1 dysfunction and NF-κB in mediating GN through proinflammatory activation of podocytes.

Re-Examining Neutrophil Participation in GN.

Significant advances in understanding the pathogenesis of GN have occurred in recent decades. Among those advances is the finding that both innate and adaptive immune cells contribute to the development of GN. Neutrophils were recognized as key contributors in early animal models of GN, at a time when the prevailing view considered neutrophils to function as nonspecific effector cells that die quickly after performing antimicrobial functions. However, advances over the past two decades have shown that neutrophil functions are more complex and sophisticated. Specifically, research has revealed that neutrophil survival is regulated by the inflammatory milieu and that neutrophils demonstrate plasticity, mediate microbial killing through previously unrecognized mechanisms, demonstrate transcriptional activity leading to the release of cytokines and chemokines, interact with and regulate cells of the innate and adaptive immune systems, and contribute to the resolution of inflammation. Therefore, neutrophil participation in glomerular diseases deserves re-evaluation. In this review, we describe advances in understanding classic neutrophil functions, review the expanded roles of neutrophils in innate and adaptive immune responses, and summarize current knowledge of neutrophil contributions to GN.

Renal Protection by Genetic Deletion of the Atypical Chemokine Receptor ACKR2 in Diabetic OVE Mice.

In diabetic nephropathy (DN) proinflammatory chemokines and leukocyte infiltration correlate with tubulointerstitial injury and declining renal function. The atypical chemokine receptor ACKR2 is a chemokine scavenger receptor which binds and sequesters many inflammatory CC chemokines but does not transduce typical G-protein mediated signaling events. ACKR2 is known to regulate diverse inflammatory diseases but its role in DN has not been tested. In this study, we utilized ACKR2(-/-) mice to test whether ACKR2 elimination alters progression of diabetic kidney disease. Elimination of ACKR2 greatly reduced DN in OVE26 mice, an established DN model. Albuminuria was significantly lower at 2, 4, and 6 months of age. ACKR2 deletion did not affect diabetic blood glucose levels but significantly decreased parameters of renal inflammation including leukocyte infiltration and fibrosis. Activation of pathways that increase inflammatory gene expression was attenuated. Human biopsies stained with ACKR2 antibody revealed increased staining in diabetic kidney, especially in some tubule and interstitial cells. The results demonstrate a significant interaction between diabetes and ACKR2 protein in the kidney. Unexpectedly, ACKR2 deletion reduced renal inflammation in diabetes and the ultimate response was a high degree of protection from diabetic nephropathy.

Differential expression of endoplasmic reticulum stress-response proteins in different renal tubule subtypes of OVE26 diabetic mice.

Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-responseinduced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced proapoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.

Changing the concepts of immune-mediated glomerular diseases through proteomics.

Standard classification of glomerular diseases is based on histopathologic abnormalities. The recent application of proteomic technologies has resulted in paradigm changes in the understanding and classification of idiopathic membranous nephropathy and membranoproliferative glomerulonephritis. Those examples provide evidence that proteomics will lead to advances in understanding of the molecular basis of other glomerular diseases, such as lupus nephritis. Proof of principle experiments show that proteomics can be applied to patient renal biopsy specimens. This viewpoint summarizes the advances in immune-mediated glomerular diseases that have relied on proteomics, and potential future applications are discussed.

Autoantibodies targeting glomerular annexin A2 identify patients with proliferative lupus nephritis.

PURPOSE: Patients with systemic lupus erythematosus (SLE) frequently develop lupus nephritis (LN), a complication frequently leading to end stage kidney disease. Immune complex deposition in the glomerulus is central to the development of LN. Using a targeted proteomic approach, we tested the hypothesis that autoantibodies targeting glomerular antigens contribute to the development of LN. EXPERIMENTAL DESIGN: Human podocyte and glomerular proteins were separated by SDS-PAGE and immunoblotted with sera from SLE patients with and without LN. The regions of those gels corresponding to reactive bands observed with sera from LN patients were analyzed using LC-MS/MS. RESULTS: LN reactive bands were seen at approximately 50 kDa in podocyte extracts and between 36 and 50 kDa in glomerular extracts. Those bands were analyzed by LC-MS/MS and 102 overlapping proteins were identified. Bioinformatic analysis determined that 36 of those proteins were membrane associated, including a protein previously suggested to contribute to glomerulonephritis and LN, annexin A2. By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against annexin A2. CONCLUSION AND CLINICAL RELEVANCE: Proteomic approaches identified multiple candidate antigens for autoantibodies in patients with LN. Serum antibodies against annexin A2 were significantly elevated in subjects with proliferative LN, validating those antibodies as potential biomarkers.

Human MCS5A1 candidate breast cancer susceptibility gene FBXO10 is induced by cellular stress and correlated with lens epithelium-derived growth factor (LEDGF).

Genetic variation and candidate genes associated with breast cancer susceptibility have been identified. Identifying molecular interactions between associated genetic variation and cellular proteins may help to better understand environmental risk. Human MCS5A1 breast cancer susceptibility associated SNP rs7042509 is located in F-box protein 10 (FBXO10). An orthologous Rattus norvegicus DNA-sequence that contains SNV ss262858675 is located in rat Mcs5a1, which is part of a mammary carcinoma susceptibility locus controlling tumor development in a non-mammary cell-autonomous manner via an immune cell-mediated mechanism. Higher Fbxo10 expression in T cells is associated with Mcs5a increased susceptibility alleles. A common DNA-protein complex bound human and rat sequences containing MCS5A1/Mcs5a1 rs7042509/ss262858675 in electrophoretic mobility shift assays (EMSAs). Lens epithelium-derived growth factor (LEDGF), a stress-response protein, was identified as a candidate to bind both human and rat sequences using DNA-pulldown and mass spectrometry. LEDGF binding was confirmed by LEDGF-antibody EMSA and chromatin immunoprecipitation (ChIP). Ectopic expression of LEDGF/p75 increased luciferase activities of co-transfected reporters containing both human and rat orthologs. Over-expressed LEDGF/p75 increased endogenous FBXO10 mRNA levels in Jurkat cells, a human T-cell line, implying LEDGF may be involved in increasing FBXO10 transcript levels. Oxidative and thermal stress of Jurkat cells increased FBXO10 and LEDGF expression, further supporting a hypothesis that LEDGF binds to a regulatory region of FBXO10 and increases expression during conditions favoring carcinogenesis. We conclude that FBXO10, a candidate breast cancer susceptibility associated gene, is induced by cellular stress and LEDGF may play a role in expression of this gene.

ABIN1 dysfunction as a genetic basis for lupus nephritis.

The genetic factors underlying the pathogenesis of lupus nephritis associated with systemic lupus erythematosus are largely unknown, although animal studies indicate that nuclear factor (NF)-κB is involved. We reported previously that a knockin mouse expressing an inactive form of ABIN1 (ABIN1[D485N]) develops lupus-like autoimmune disease and demonstrates enhanced activation of NF-κB and mitogen-activated protein kinases in immune cells after toll-like receptor stimulation. In the current study, we show that ABIN1[D485N] mice develop progressive GN similar to class III and IV lupus nephritis in humans. To investigate the clinical relevance of ABIN1 dysfunction, we genotyped five single-nucleotide polymorphisms in the gene encoding ABIN1, TNIP1, in samples from European-American, African American, Asian, Gullah, and Hispanic participants in the Large Lupus Association Study 2. Comparing cases of systemic lupus erythematosus with nephritis and cases of systemic lupus erythematosus without nephritis revealed strong associations with lupus nephritis at rs7708392 in European Americans and rs4958881 in African Americans. Comparing cases of systemic lupus erythematosus with nephritis and healthy controls revealed a stronger association at rs7708392 in European Americans but not at rs4958881 in African Americans. Our data suggest that variants in the TNIP1 gene are associated with the risk for lupus nephritis and could be mechanistically involved in disease development via aberrant regulation of NF-κB and mitogen-activated protein kinase activity.