Background: Interstitial fibrosis in lupus nephritis (LN) is often infiltrated by immune cells. However, this is typically regarded as nonspecific “scar reaction” rather than active disease requiring treatment. In contrast, scar inflammation in kidney allografts is associated with chronic rejection and treated with immunosuppression. Objectives: To investigate the relationship between inflammatory fibrosis and kidney disease progression in LN. Methods: Interstitial fibrosis and tubular atrophy (IFTA) were scored in 124 kidney biopsies from patients classified as LN according to the 2018 International Society of Nephrology/Renal Pathology Society criteria. Inflammation in areas of IFTA (i-IFTA) was graded 0-3 by 2 operators and reviewed by a senior renal pathologist based on extent according to the Banff Classification of Allograft Pathology (<10%, 10-25%, 26-50%, >50%, respectively). Glomerular filtration rate (GFR) was estimated using the CKD-EPI equation. Significant GFR loss was defined as a decline of >15 ml/min at 3 years from biopsy or end-stage kidney disease (ESKD) by year 3 requiring dialysis or transplant. Results: The clinical and demographic characteristics of the cohort are summarized in Table 1. IFTA was observed in 88/124 (71%) biopsies, and i-IFTA was identified in 76/88 (86%) cases. The distribution of i-IFTA grades according to the degree of IFTA is illustrated in Figure 1A. Of the 53 IFTA cases with 3-year follow-up data available, significant GFR loss was observed in 22/53 (42%) cases. As expected, IFTA was associated with GFR loss (p for trend = 0.03). In patients with moderate-to-severe IFTA (grade 2 or 3), the degree of i-IFTA was associated with higher risk of significant GFR loss (Figure 1B). The risk of significant GFR loss in this subgroup was 0/2 (0%), 1/2 (50%), 3/4 (75%), and 7/8 (87.5%) for i-IFTA grades 0, 1, 2, and 3, respectively (p for trend = 0.018). Conclusion: Inflammation in areas of IFTA is frequently observed in LN and exhibits substantial heterogeneity in its severity. For patients with baseline IFTA grades >1, the degree of i-IFTA emerged as a strong predictor of poor renal outcomes. These data support the routine scoring of i-IFTA in LN due to its prognostic implications and nominate i-IFTA as a potential therapeutic target. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Andrea Fava Sanofi, AnnexonBio, AstraZeneca, UCB., Silvia Malvica: None declared, Paride Fenaroli: None declared, Serena Bagnasco: None declared, Jeffrey Hodgin AstraZeneca, Eli Lilly, Gilead, Janssen, Moderna, Novo Nordisk, Regeneron, Peter Izmirly: None declared, H. Michael Belmont Aurinia, Alexion, KAtherine Preisinger: None declared, Jill P Buyon Bristol-Myers Squibb(BMS), GlaxoSmithKlein(GSK), Related Sciences, Laurence Magder: None declared, Michelle Petri Arthros-FocusMedEd, Aurinia, Amgen, AnaptysBio, Annexon Bio, Argenx, AstraZeneca, Axdev, Boxer Capital, Cabaletto Bio, Caribou Biosciences Inc, CVS Health, Escient Pharmaceuticals, Exo Therapeutics, Gentibio, GSK, Horizon Therapeutics, iCell Gene Therapeutics, Idorsia Pharmaceuticals, Kira Pharmaceuticals, Eli Lilly, MedShr, Momenta Pharmaceuticals, Nexstone Immunology, Nimbus Lakshmi, Proviant, Regeneron Pharmaceuticals, Sanofi, Seismic Therapeutic, Sinomab Biosciences, Takeda, Tenet Medicines Inc, TG Therapeutics, UCB, Zydus. DSBM: CTI Clinical Trial and Consulting Services, Emergent Biosolutions, IQVIA, Merck EMD Serono., AstraZeneca, Aurinia, Eli Lilly, Exagen, GSK, Janssen., Avi Rosenberg: None declared.Figure 1Prevalence of i-IFTA and association with significant GFR loss. (A) Distribution of i-IFTA groups according to the respective IFTA score (n=124). (B) Significant GFR loss according to i-IFTA score. Bars display the frequency of GFR loss in patients with IFTA 0 or 1 (n=37) and IFTA >1 (n=16). In patients with IFTA >1 (right panel), the risk of significant GFR loss was 0%, 50%, 75%, and 87.5% for i-IFTA 0, 1, 2, and 3, respectively. Chi-square for trends. Table 1Clinical and demographic characteristics.Overalln124Age, years (mean (SD))36.4 (12.7)Female (%)113 (84.9)Race/Ethnicity (%)Asian9 (12.3)Black38 (52.1)White21 (28.8)Other5 (6.8)Hispanic15 (20.5)eGFR, ml/min (mean (SD))82.8 (28.8)UPCR, g/g (mean (SD))2.07 (2.46)ISN class (%)II4 (4.9)III14 (17.3)III+V17 (21.0)IV6 (7.4)IV+V9 (11.1)V31 (38.3)NIH Activity Index (mean (SD))3.84 (4.36)NIH Chronicity Index (mean (SD))3.17 (2.50)

BACKGROUND:Interstitial fibrosis in lupus nephritis (LN) is often infiltrated by immune cells but typically regarded as non-specific 'scar reaction'. This study aimed to investigate the relationship between inflammatory fibrosis and kidney disease progression in LN. METHODS:Interstitial fibrosis and tubular atrophy (IFTA) were scored in 124 LN kidney biopsies. Inflammation in areas of IFTA (i-IFTA) was graded 0-3 according to the Banff Classification of Allograft Pathology. Significant glomerular filtration rate (GFR) loss was defined as a decline of >15 mL/min at 3 years from biopsy. Immune cell phenotype was defined by serial immunohistochemistry (13-plex). RESULTS:IFTA was observed in 88/124 (71%) biopsies, and i-IFTA was identified in 76/88 (86%) cases. The distribution of i-IFTA grades was heterogeneous across all IFTA grades. In patients with moderate-to-severe IFTA (>25%), the degree of i-IFTA was associated with a higher risk of significant GFR loss: 0/1 (0%), 0/3 (%), 3/4 (75%) and 7/9 (78%) for i-IFTA grades 0, 1, 2 and 3, respectively (p=0.015). Multiplexed histology revealed that i-IFTA was mostly composed of CD163+ macrophages and CD4 T cells, followed by CD8 T cells and granulocytes. CONCLUSION:I-IFTA is frequently observed in LN and is dominated by macrophages and T cells. For patients with baseline IFTA >25%, the degree of i-IFTA emerged as a predictor of GFR loss. These data support the routine scoring of i-IFTA in LN due to its prognostic implications and nominate i-IFTA as a potential therapeutic target.

Deposition of autoantibodies in glomeruli is a key factor in the development of lupus nephritis (LN). For a long time, anti-dsDNA and anti-C1q antibodies were thought to be the main cause of the kidney damage. However, recent studies have shown that the list of autoantibidies that have renal tropism and deposit in the kidney in LN is increasing and the link between anti-dsDNA and renal pathology is weak due to potential confounders. Aspecific bindings of dsDNA with cationic antibodies and of anti-dsDNA with several renal antigens such as actinin, laminin, entactin, and annexinA2 raised doubts about the specific target of these antibodies in the kidney. Moreover, the isotype of anti-dsDNA in SLE and LN has never received adequate interest until the recent observation that IgG2 are preponderant over IgG1, IgG3 and IgG4. Based on the above background, recent studies investigated the involvement of anti-dsDNA IgG2 and of other antibodies in LN. It was concluded that circulating anti-dsDNA IgG2 levels do not distinguish between LN versus non-renal SLE, and, in patients with LN, their levels do not change over time. Circulating levels of other antibodies such as anti-ENO1 and anti-H2 IgG2 were, instead, higher in LN vs non-renal SLE at the time of diagnosis and decreased following therapies. Finally, new classes of renal antibodies that potentially modify the anti-inflammatory response in the kidney are emerging as new co-actors in the pathogenetic scenario. They have been defined as 'second wave antibodies' for the link with detoxifying mechanisms limiting the oxidative stress in glomeruli that are classically stimulated in a second phase of inflammation. These findings have important clinical implications that may modify the laboratory approach to LN. Serum levels of anti-ENO1 and anti-H2 IgG2 should be measured in the follow up of patients for designing the length of therapies and identify those patients who respond to treatments. Anti-SOD2 could help to monitor and potentiate the anti-inflammatory response in the kidney.

Chronic kidney disease (CKD) progresses through the replacement of functional tissue compartments with fibrosis, a maladaptive repair process. Shifting kidney repair towards a physiologically intact architecture, rather than fibrosis, is key to blocking CKD progression. Much research into the mechanisms of fibrosis is performed in rodent models with less attention to the human genetic context. Recently, human induced pluripotent stem cell (iPSC)-derived organoids have shown promise in overcoming the limitation. In this study, we developed a fibrosis model that uses human iPSC-based three-dimensional renal organoids, in which exogenous Transforming growth factor- β1 (TGF-β1) induced the production of extracellular matrix. TGF-β1-treated organoids showed tubulocentric collagen 1α1 production by regulating downstream transcriptional regulators, Farnesoid X receptor, phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3), and transcriptional coactivator with PDZ-binding motif (TAZ). Increased nuclear TAZ expression was confirmed in the tubular epithelium in human kidney biopsies with tubular injury and early fibrosis. A dual bile acid receptors agonist (INT-767) increased Farnesoid X receptor and reduced p-SMAD3 and TAZ, attenuating TGF-β1-induced fibrosis in kidney organoids. Finally, we show that TAZ interacted with TEA-domain transcription factors and p-SMAD3 with TAZ and TEA-domain transcription factor 4 co-regulating collagen 1α1 gene transcription. In summary, we establish a novel, readily manipulable fibrogenesis model and posit a role for bile acid receptor agonism early in renal parenchymal fibrosis.

Background Apolipoprotein L1 (APOL1) high-risk variants contribute to kidney disease among individuals with African ancestry. We sought to describe cell-specific APOL1 variant-induced pathways using two mouse models. Methods We characterized bacterial artificial chromosome/APOL1 transgenic mice crossed with HIV-associated nephropathy (HIVAN) Tg26 mice and bacterial artificial chromosome/APOL1 transgenic mice given IFN-gamma. Results Both mouse models showed more severe glomerular disease in APOL1-G1 compared with APOL1-G0 mice. Synergistic podocyte-damaging pathways activated by APOL1-G1 and by the HIV transgene were identified by glomerular bulk RNA sequencing (RNA-seq) of HIVAN model. Single-nuclear RNA-seq revealed podocyte-specific patterns of differentially expressed genes as a function of APOL1 alleles. Shared activated pathways, for example, mammalian target of rapamycin, and differentially expressed genes, for example, Ccn2, in podocytes in both models suggest novel markers of APOL1-associated kidney disease. HIVAN mouse-model podocyte single-nuclear RNA-seq data showed similarity to human focal segmental glomerulosclerosis glomerular RNA-seq data. Differential effects of the APOL1-G1 variant on the eukaryotic initiation factor 2 pathway highlighted differences between the two models. Conclusions These findings in two mouse models demonstrated both shared and distinct cell type-specific transcriptomic signatures induced by APOL1 variants. These findings suggest novel therapeutic opportunities for APOL1 glomerulopathies.

Chronic kidney disease progresses through the replacement of functional tissue compartments with fibrosis, a maladaptive repair process. Shifting kidney repair toward a physiologically intact architecture, rather than fibrosis, is key to blocking chronic kidney disease progression. Much research into the mechanisms of fibrosis is performed in rodent models with less attention to the human genetic context. Recently, human induced pluripotent stem cell (iPSC)-derived organoids have shown promise in overcoming the limitation. In this study, we developed a fibrosis model that uses human iPSC-based 3-dimensional renal organoids, in which exogenous transforming growth factor-I31 (TGF-I31) induced the production of extracellular matrix. TGF-I31-treated organoids showed tubulocentric collagen 1a1 production by regulating downstream transcriptional regulators, Farnesoid X receptor, phosphorylated mothers against decapentaplegic homolog 3 (pSMAD3), and transcriptional coactivator with PDZ-binding motif (TAZ). Increased nuclear TAZ expression was confirmed in the tubular epithelium in human kidney biopsies with tubular injury and early fibrosis. A dual bile acid receptor agonist (INT-767) increased Farnesoid X receptor and reduced p-SMAD3 and TAZ, attenuating TGF-I31-induced fibrosis in kidney organoids. Finally, we show that TAZ interacted with TEA-domain transcription factors and p-SMAD3 with TAZ and TEAdomain transcription factor 4 coregulating collagen 1a1 gene transcription. In summary, we establish a novel, readily manipulable fibrogenesis model and posit a role for bile acid receptor agonism early in renal parenchymal fibrosis. (c) 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.

INTRODUCTION:IgG4-related disease (IgG4-RD) is a rare fibro-inflammatory disease affecting multiple organs. In recent years basic and translational research has unveiled the role of different cellular subtypes and cytokines in inducing and perpetuating the pathological process, eventually leading to fibrosis of affected tissues. Hopefully, the growing knowledge of the disease pathogenesis will lead to patient-tailored treatments in the near future.AREAS COVERED:This review focuses on the most recent discoveries concerning the pathogenic mechanisms underlying IgG4-RD and highlights their potential role as specific therapeutic targets.EXPERT OPINION:IgG4-RD is a systemic and multifaceted disease. Its sensitivity to glucocorticoids is well known, however new targeted therapies are emerging that can reduce glucocorticoid exposure and achieve sustained clinical responses. Clinicians managing patients with such a rare and heterogeneous disease must therefore be aware of its varied phenotype and traditional and novel therapeutic strategies.

Topic: 16. Myeloproliferative neoplasms - Clinical Background: Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis in which somatic mutations involving MAPK and PI3K-AKT pathways play a crucial pathogenic role. The geoepidemiology of the disease is still unknown. Aims: Our study aimed at assessing the geographic origin of adult ECD patients in Italy and France. Methods:Methods (Italian cohort) - Data of 139 patients diagnosed from 1996 to 2022 were obtained from clinical charts of Italian ECD Referral Care Centres (i.e., IRCCS Meyer, Florence, and IRCCS San Raffaele, Milan) and of other Centres belonging to the Italian ECD Network (ItalECD). Patients with childhood-onset ECD (n=5), born in other countries (n=13), or lacking data (n=4) were excluded (N=117). Metrics regarding Italian Region demographics (i.e., resident adult population) were obtained from public reports produced by the Italian national institute of statistics (ISTAT) and used to normalise the number of ECD diagnoses. Methods (French cohort) - Data of 354 patients diagnosed from 1982 to 2022 were obtained from clinical charts of the French ECD Referral Care Centre (i.e., Hôpital Universitaire Pitié Salpêtrière - Charles Foix, Paris). Patients with childhood-onset ECD (n=4), born in overseas French Regions (n=5), born in other countries (n=96), or lacking data (n=11) were excluded (N=238). Metrics regarding French Region demographics (i.e., resident adult population) were obtained from public reports produced by the French national institute of statistics (INSEE) and used to normalise the number of ECD diagnoses. Methods (statistical analysis) - The test described and validated by Ohno Y et al. to assess significance for geographic clusters of disease (Int J Epidemiol. 1979;8(3):273-80) was used to analyse observed clustering of ECD diagnoses in Italian and French Regions. The number of diagnoses per 1’000’000 adult residents for each Region was classified according to their ratio over national average, which was taken as 100: (1) above national average ratio (>120), (2) in line with national average ratio (80-119.9), (3) below national average ratio (<80). Results: The Italian Regions showing a frequency of ECD diagnoses over national average (i.e., 2.35/1’000’000 adult residents) clustered in Southern Italy (Fig. 1A), whereas French Regions exhibiting a rate of diagnosis over national average (i.e., 4.68/1’000’000 adult residents) clustered in the central area of the country (Fig. 1B). Statistical analysis demonstrated that such geographic aggregations were statistically significant (P-value < 0.01 for Italy and < 0.05 for France). Summary/Conclusion: The significant geographic clustering of ECD diagnoses, with higher incidences in Southern Italy and central Regions of France, suggests the potential role of genetics and/or environmental exposures in the development of the disease. Further epidemiological studies are required to highlight specific risk factors.Keywords: Myeloid malignancies, Risk factor, Macrophage, Epidemiology

Abstract Background and Aims ANCA-associated vasculitis (AAV) represent a group of systemic autoimmune diseases characterized by necrotizing inflammation of small to medium-size blood vessels frequently associated with anti-neutrophil cytoplasmic antibody (ANCA) positivity. Another term they are commonly referred with is “paucimmune vasculitis”, reflecting the lack of immune complex deposition on histological samples, in contrast with other small vessel vasculitis, e.g. cryoglobulinemic vasculitis. The absence or the paucity of deposition of immunoglobulins and complement components was interpreted in the past as a lack of complement cascade involvement in the pathogenesis of AAV. Only in relatively recent times the possible role of complement in AAV was investigated. A few studies proved the relationship between C3 hypocomplementemia and worse renal outcome[1]. Moreover, the overactivation of the alternative complement pathway (ACP) in AAV could be responsible for thrombotic microangiopathy (TMA). A strong association between low serum C3 and histologic signs of thrombotic microangiopathy on renal biopsy was shown in patients with AAV, with a worse renal prognosis. Currently, there are no clear early histological markers of endothelial damage that allow to identify high-risk patients, other than the seldom not identified or misidentified presence of TMA. The association between renal arteriolar C4d deposition (as a vicariate of TMA) has been already described in several glomerulonephritis, including IgA nephropathy [2], but its prognostic meaning has not yet been explored [3]. Our study aims to explore the predictive role of arteriolar C4d in ANCA-associated GN. Method All patients with ANCA-associated GN who underwent a kidney biopsy between January 1990 and June 2021 at the Renal Unit of Parma University Hospital, Parma, Italy with at least 6 months follow-up data were included. All histology slides were retrieved and reviewed by experienced nephropathologists. Percent IFTA, percent global glomerulosclerosis, Brix score, the presence or absence of TMA, the positivity or negativity of C4d (immunohistochemical staining on paraffin-embedded formalin-fixed slides) were taken note of. C4d positivity was correlated with the presence of TMA and patients outcome (Fig. 1). Results We identified 58 patients who met our criteria. 14 patients were excluded because biopsy specimens were not available for histologic review. Clinical and laboratory baseline and follow-up data for the remaining 44 patients were thus collected. Conclusion C4d and TMA were independently associated (p = 0.003). A tendency towards association between C4d and death-censored ESRD was observed (Fig. 2), although not statistically significant (p = 0.434), likely due to the limited sample size. In order to test our hypothesis, validation on a larger cohort is currently underway.

This geoepidemiological study, performed in Italy and France, shows that Erdheim-Chester disease is increasingly diagnosed and cases cluster in specific geographic areas, namely southern Italy and central France. Disease frequency inversely correlates with the Human Development Index.

APOL1 high-risk variants partially explain the high kidney disease prevalence among African ancestry individuals. Many mechanisms have been reported in cell culture models, but few have been demonstrated in mouse models. Here we characterize two models: (1) HIV- associated nephropathy (HIVAN) Tg26 mice crossed with bacterial artificial chromosome (BAC)/APOL1 transgenic mice and (2) interferon-ψ administered to BAC/APOL1 mice. Both models showed exacerbated glomerular disease in APOL1-G1 compared to APOL1-G0 mice. HIVAN model glomerular bulk RNA-seq identified synergistic podocyte-damaging pathways activated by the APOL1-G1 allele and by HIV transgenes. Single-nuclear RNA-seq revealed podocyte-specific patterns of differentially-expressed genes as a function of APOL1 alleles. Eukaryotic Initiation factor-2 pathway was the most activated pathway in the interferon-ψ model and the most deactivated pathway in the HIVAN model. HIVAN mouse model podocyte single-nuclear RNA-seq data showed similarity to human focal segmental glomerulosclerosis (FSGS) glomerular bulk RNA-seq data. Furthermore, single-nuclear RNA-seq data from interferon-ψ mouse model podocytes ( in vivo ) showed similarity to human FSGS single-cell RNA- seq data from urine podocytes ( ex vivo ) and from human podocyte cell lines ( in vitro ) using bulk RNA-seq. These data highlight differences in the transcriptional effects of the APOL1 -G1 risk variant in a model specific manner. Shared differentially expressed genes in podocytes in both mouse models suggest possible novel glomerular damage markers in APOL1 variant-induced diseases. Transcription factor Zbtb16 was downregulated in podocytes and endothelial cells in both models, possibly contributing to glucocorticoid-resistance. In summary, these findings in two mouse models suggest both shared and distinct therapeutic opportunities for APOL1 glomerulopathies. Significance statement Coding variants in APOL1, encoding apolipoprotein L1, contribute to kidney disease in individuals with African ancestry. The mechanisms for glomerular injury remain incompletely understood. We studied two transgenic mouse models, HIV-associated nephropathy and interferon-ψ administration. Using glomerular and single-nuclear RNA sequencing, we identified genes differentially expressed among mice with kidney risk alleles (G1) and the common variant (G0). Both models exhibited up-regulation of genes that indicated podocyte damage with risk alleles compared to the common variant. One gene down-regulated in both models was Zbtb16, encoding a transcription factor, that may contribute to glucocorticoid-resistance. Overall, the findings suggest both shared and distinct alterations in the two disease models.

Mechanisms for the generation of anti-dsDNA autoantibodies are still not completely elucidated. One theory states that dsDNA interacts for mimicry with antibodies raised versus other antigens but molecular features for mimicry are unknown. Here we show that, at physiological acid-base balance, anti-Annexin A1 binds IgG2 dsDNA in a competitive and dose-dependent way with Annexin A1 and that the competition between the two molecules is null at pH 9. On the other hand, these findings also show that dsDNA and Annexin A1 interact with their respective antibodies on a strictly pH-dependent basis: in both cases, the binding was minimal at pH 4 and maximal at pH9-10. The anionic charge of dsDNA is mainly conferred by the numerous phosphatidic residues. The epitope binding site of Annexin A1 for anti-Annexin A1 IgG2 was here characterized as a string of 34 amino acids at the NH2 terminus, 10 of which are anionic.Circulating levels of anti-dsDNA and anti-Annexin A1 IgG2 antibodies were strongly correlated in patients with systemic lupus erythematosus (n 496) and lupus nephritis (n 425) stratified for age, sex, etc. These results show that dsDNA competes with Annexin A1 for the binding with anti-Annexin A1 IgG2 on a dose and charged mediated base, being able to display an inhibition up to 75%. This study provides the first demonstration that dsDNA may interact with antibodies raised versus other anionic molecules (anti-Annexin A1 IgG2) because of charge mimicry and this interaction may contribute to anti-dsDNA antibodies generation.

Background Jagged ends of plasma DNA are a recently recognized class of fragmentomic markers for cell-free DNA, reflecting the activity of nucleases. A number of recent studies have also highlighted the importance of jagged ends in the context of pregnancy and oncology. However, knowledge regarding the generation of jagged ends is incomplete. Methods Jaggedness of plasma DNA was analyzed based on Jag-seq, which utilized the differential methylation signals introduced by the DNA end-repair process. We investigated the jagged ends in plasma DNA using mouse models by deleting the deoxyribonuclease 1 (Dnase1), DNA fragmentation factor subunit beta (Dffb), or deoxyribonuclease 1 like 3 (Dnase1l3) gene. Results Aberrations in the profile of plasma DNA jagged ends correlated with the type of nuclease that had been genetically deleted, depending on nucleosomal structures. The deletion of Dnase1l3 led to a significant reduction of jaggedness for those plasma DNA molecules involving more than 1 nucleosome (e.g., size ranges 240-290 bp, 330-380 bp, and 420-470 bp). However, less significant effects of Dnase1 and Dffb deletions were observed regarding different sizes of DNA fragments. Interestingly, the aberration in plasma DNA jagged ends related to multinucleosomes was observed in human subjects with familial systemic lupus erythematosus with Dnase1l3 deficiency and human subjects with sporadic systemic lupus erythematosus. Conclusions Detailed understanding of the relationship between nuclease and plasma DNA jaggedness has opened up avenues for biomarker development.

Introduction:Proliferative lupus nephritis (LN) progresses to end-stage kidney disease (ESKD) in roughly 10% of the cases despite treatment. Other than achieving <0.8 g/24h proteinuria at 12 months after treatment, early biomarkers predicting ESKD or death are lacking. Recent studies encompassing not only LN have highlighted the central role of the alternative complement pathway (ACP), with or without histological evidence of thrombotic microangiopathy (TMA), as a key promotor of renal death. Methods:We assessed whether persistent isolated C3 hypocomplementemia (PI-LowC3), that is not accompanied by C4 hypocomplementemia, 6 months after kidney biopsy, is associated with an increased risk of death or ESKD in proliferative LN. Results:We retrospectively followed-up 197 patients with proliferative LN (51 with PI-LowC3) for a median of 4.5 years (interquartile-range: 1.9-9.0), 11 of whom died and 22 reached ESKD. After adjusting for age, gender, ethnicity, hypertension, mycophenolate, or cyclophosphamide use, PI-LowC3 was associated with a hazard ratio [HR] of the composite outcome ESKD or death of 2.46 (95% confidence interval [CI]: 1.22-4.99, P = 0.012). These results were confirmed even after controlling for time-varying estimated glomerular filtration rate (eGFR) measurements in joint longitudinal-survival multiple regression models. After accounting for the competing risk of death, PI-LowC3 patients showed a strikingly increased risk of ESKD (adjusted HR 3.41, 95% CI: 1.31-8.88, P = 0.012). Conclusion:Our findings support the use of PI-LowC3 as a low-cost readily available biomarker, allowing clinicians to modify treatment strategies early in the course of disease and offering a rationale for complement blockade trials in this particularly at-risk subgroup of LN patients.

OBJECTIVE:Current lupus nephritis (LN) treatments are effective in only 30% of patients, emphasizing the need for novel therapeutic strategies. We undertook this study to develop mechanistic hypotheses and explore novel biomarkers by analyzing the longitudinal urinary proteomic profiles in LN patients undergoing treatment. METHODS:We quantified 1,000 urinary proteins in 30 patients with LN at the time of the diagnostic renal biopsy and after 3, 6, and 12 months. The proteins and molecular pathways detected in the urine proteome were then analyzed with respect to baseline clinical features and longitudinal trajectories. The intrarenal expression of candidate biomarkers was evaluated using single-cell transcriptomics of renal biopsy sections from LN patients. RESULTS:Our analysis revealed multiple biologic pathways, including chemotaxis, neutrophil activation, platelet degranulation, and extracellular matrix organization, which could be noninvasively quantified and monitored in the urine. We identified 237 urinary biomarkers associated with LN, as compared to controls without systemic lupus erythematosus. Interleukin-16 (IL-16), CD163, and transforming growth factor β mirrored intrarenal nephritis activity. Response to treatment was paralleled by a reduction in urinary IL-16, a CD4 ligand with proinflammatory and chemotactic properties. Single-cell RNA sequencing independently demonstrated that IL16 is the second most expressed cytokine by most infiltrating immune cells in LN kidneys. IL-16-producing cells were found at key sites of kidney injury. CONCLUSION:Urine proteomics may profoundly change the diagnosis and management of LN by noninvasively monitoring active intrarenal biologic pathways. These findings implicate IL-16 in LN pathogenesis, designating it as a potentially treatable target and biomarker.

Cell-free extrachromosomal circular DNA (eccDNA) as a distinct topological form from linear DNA has recently gained increasing research interest, with possible clinical applications as a class of biomarkers. In this study, we aimed to explore the relationship between nucleases and eccDNA characteristics in plasma. By using knockout mouse models with deficiencies in deoxyribonuclease 1 (DNASE1) or deoxyribonuclease 1 like 3 (DNASE1L3), we found that cell-free eccDNA in Dnase1l3-/- mice exhibited larger size distributions than that in wild-type mice. Such size alterations were not found in tissue eccDNA of either Dnase1-/- or Dnase1l3-/- mice, suggesting that DNASE1L3 could digest eccDNA extracellularly but did not seem to affect intracellular eccDNA. Using a mouse pregnancy model, we observed that in Dnase1l3-/- mice pregnant with Dnase1l3+/- fetuses, the eccDNA in the maternal plasma was shorter compared with that of Dnase1l3-/- mice carrying Dnase1l3-/- fetuses, highlighting the systemic effects of circulating fetal DNASE1L3 degrading the maternal eccDNA extracellularly. Furthermore, plasma eccDNA in patients with DNASE1L3 mutations also exhibited longer size distributions than that in healthy controls. Taken together, this study provided a hitherto missing link between nuclease activity and the biological manifestations of eccDNA in plasma, paving the way for future biomarker development of this special form of DNA molecules.

Chronic kidney disease-associated pruritus (CKD-aP) has been recognized for over a century. The complex pathophysiology of CKD-aP makes it challenging to find an effective treatment; the proposed therapeutic options come from anecdotal reports and small clinical trials, which at best compare the test drug against placebo. Gabapentinoids have shown relevant efficacy but there are serious safety concerns about their possible central nervous system toxicity. Recently difelikefalin, a κ-opioid receptor agonist, has been the first Food and Drug Administration (FDA)-approved drug for moderate-severe CKD-aP treatment. Approval from other regulatory agencies is expected in 2022. In this article, preclinical, pharmacokinetic and safety studies on difelikefalin are reported, but a great part of the data derive from meeting abstracts and non-peer-review communications and this is a possible cause for concern regarding bias in publication. A review of published and unpublished studies about difelikefalin in CKD-aP treatment is provided. Currently, two published large trials show that difelikefalin offers a new therapeutic opportunity to treat CKD-aP, a condition that leads to both worse survival and quality of life in hemodialysis patients.

Background: Nucleic acid binding proteins are frequently targeted as autoantigens in systemic lupus erythematosus (SLE) and other interferon (IFN)-linked rheumatic diseases. The AIM-like receptors (ALRs) are IFN-inducible innate sensors that form supramolecular assemblies along double-stranded (ds)DNA of various origins. Here, we investigate the ALR absent in melanoma 2 (AIM2) as a novel autoantigen in SLE, with similar properties to the established ALR autoantigen interferon-inducible protein 16 (IFI16). We examined neutrophil extracellular traps (NETs) as DNA scaffolds on which these antigens might interact in a pro-immune context. Methods: AIM2 autoantibodies were measured by immunoprecipitation in SLE and control subjects. Neutrophil extracellular traps were induced in control neutrophils and combined with purified ALR proteins in immunofluorescence and DNase protection assays. SLE renal tissues were examined for ALR-containing NETs by confocal microscopy. Results: AIM2 autoantibodies were detected in 41/131 (31.3%) SLE patients and 2/49 (4.1%) controls. Our SLE cohort revealed a frequent co-occurrence of anti-AIM2, anti-IFI16, and anti-DNA antibodies, and higher clinical measures of disease activity in patients positive for antibodies against these ALRs. We found that both ALRs bind NETs in vitro and in SLE renal tissues. We demonstrate that ALR binding causes NETs to resist degradation by DNase I, suggesting a mechanism whereby extracellular ALR-NET interactions may promote sustained IFN signaling. Conclusions: Our work suggests that extracellular ALRs bind NETs, leading to DNase resistant nucleoprotein fibers that are targeted as autoantigens in SLE. Funding: These studies were funded by NIH R01 DE12354 (AR), P30 AR070254, R01 GM 129342 (JS), K23AR075898 (CM), K08AR077100 (BA), the Jerome L. Greene Foundation and the Rheumatology Research Foundation. Dr. Antiochos and Dr. Mecoli are Jerome L. Greene Scholars. The Hopkins Lupus Cohort is supported by NIH grant R01 AR069572. Confocal imaging performed at the Johns Hopkins Microscopy Facility was supported by NIH Grant S10 OD016374.