KLF2 enhancer variant rs4808485 increases lupus risk by modulating inflammasome machinery and cellular homoeostasis.

OBJECTIVE: A recent genome-wide association study linked KLF2 as a novel Asian-specific locus for systemic lupus erythematosus (SLE) susceptibility. However, the underlying causal functional variant(s), cognate target gene(s) and genetic mechanisms associated with SLE risk are unknown. METHODS: We used bioinformatics to prioritise likely functional variants and validated the best candidate with diverse experimental techniques, including genome editing. Gene expression was compared between healthy controls (HCs) and patients with SLE with or without lupus nephritis (LN+, LN-). RESULTS: Through bioinformatics and expression quantitative trait locus analyses, we prioritised rs4808485 in active chromatin, predicted to modulate KLF2 expression. Luciferase reporter assays and chromatin immunoprecipitation-qPCR demonstrated differential allele-specific enhancer activity and binding of active histone marks (H3K27ac, H3K4me3 and H3K4me1), Pol II, CTCF, P300 and the transcription factor PARP1. Chromosome conformation capture-qPCR revealed long-range chromatin interactions between rs4808485 and the KLF2 promoter. These were directly validated by CRISPR-based genetic and epigenetic editing in Jurkat and lymphoblastoid cells. Deleting the rs4808485 enhancer in Jurkat (KO) cells disrupted NLRP3 inflammasome machinery by reducing KLF2 and increasing CASPASE1, IL-1ß and GSDMD levels. Knockout cells also exhibited higher proliferation and cell-cycle progression than wild type. RNA-seq validated interplay between KLF2 and inflammasome machinery in HC, LN+ and LN-. CONCLUSIONS: We demonstrate how rs4808485 modulates the inflammasome and cellular homoeostasis through regulating KLF2 expression. This establishes mechanistic connections between rs4808485 and SLE susceptibility.

Time-Varying Proteinuria and Progression of IgA Nephropathy: A Cohort Study.

RATIONALE & OBJECTIVE: Proteinuria is a surrogate end point for predicting long-term kidney outcomes in IgA nephropathy (IgAN) with levels<1g/day identified as a therapeutic target. However, this threshold has not been sufficiently studied. We quantified the associations of progression of IgAN with various levels of proteinuria. STUDY DESIGN: Observational cohort study. SETTING & PARTICIPANTS: 1,530 patients with IgAN and at least 12 months of follow-up at Peking University First Hospital. EXPOSURE: Proteinuria levels updated over time (time-varying proteinuria, TVP). OUTCOME: A composite kidney outcome of a 50% reduction in the estimated glomerular filtration rate or end-stage kidney disease. ANALYTICAL APPROACH: Marginal structural models. RESULTS: After a median follow-up period of 43.5 (IQR, 27.2-72.8) months, 254 patients (16.6%) developed the composite kidney outcome. A graded association was observed between TVP and composite kidney outcomes with higher risk among those with proteinuria of≥0.5g/day. Compared with TVP<0.3g/day, the HRs for proteinuria levels of 0.3 to<0.5g/day, 0.5 to<1.0g/day, 1.0 to<2.0g/day, and≥2.0g/day were 2.22 (95% CI, 0.88-5.58), 4.04 (95% CI, 1.93-8.46), 8.46 (95% CI, 3.80-18.83), and 38.00 (95% CI, 17.62-81.95), respectively. The trend was more pronounced in patients with baseline proteinuria of≥1.0g/day, among whom a higher risk was observed with TVP of 0.3 to<0.5g/day compared with TVP<0.3g/day (HR, 3.26 [95% CI, 1.07-9.92], P=0.04). However, in patients with baseline proteinuria levels of<1g/day, the risk of composite kidney outcome only began to increase when TVP was≥1.0g/day (HR, 3.25 [95% CI, 1.06-9.90]). LIMITATIONS: Single-center observational study, selection bias, and unmeasured confounders. CONCLUSIONS: This study showed that patients with IgAN and proteinuria levels of>0.5g/day, have an elevated risk of kidney failure especially among patients with proteinuria levels≥1.0g/day before initiating treatment. These data may serve to inform the selection of proteinuria targets in the treatment of IgAN. PLAIN-LANGUAGE SUMMARY: The presence of proteinuria has often been considered a surrogate end point and a possible therapeutic target in clinical trials in IgA nephropathy (IgAN). Some guidelines recommend a reduction in proteinuria to<1g/day as a treatment goal based on the results of previous longitudinal studies. However, these findings may have been biased because they did not properly adjust for time-dependent confounders. Using marginal structural models to appropriately account for these confounding influences, we observed that patients with IgAN and proteinuria levels≥0.5g/day have an elevated risk of kidney failure, especially among patients who had proteinuria levels of≥1.0g/day before initiating treatment. These data may serve to inform the selection of proteinuria targets in the treatment of IgAN.

Association between urinary C4d levels and disease progression in IgA nephropathy.

BACKGROUND: C4d mesangial deposition, a hallmark of lectin pathway activation in IgA nephropathy (IgAN), has been shown to be associated with risk of kidney failure. To date, the relationship between urinary C4d and renal outcome remain unelucidated. METHODS: A total of 508 patients with biopsy-proven IgAN were enrolled in this study, whose baseline urine samples at the time of biopsy were collected and the levels of urinary C4d were quantified by enzyme-linked immunosorbent assay. The time-averaged C4d (TA-C4d) and the change in proteinuria were measured in sequential urine samples obtained from IgAN patients. The kidney progression event was defined as a 50% estimated glomerular filtration rate (eGFR) decline or end-stage kidney disease (ESKD) or death. RESULTS: After a median follow-up of 36 months, 70 (13.8%) of the participants reached the kidney progression event. Higher levels of urinary C4d/creatinine were found to be associated with decreased eGFR, massive proteinuria, lower serum albumin levels, hypertension, and severe Oxford E and T scores. Upon adjusting for traditional risk factors (including demographics, eGFR, proteinuria, hypertension, Oxford pathologic score, and immunosuppressive therapy), elevated levels of urinary C4d/creatinine were independently associated with an increased risk of CKD progression (adjusted HR per standard deviation increment of log-transformed C4d/creatinine: 1.46; 95% CI: 1.04 to 2.06; P=0.030). In reference to the low C4d group, the risk of poor renal outcome increased for the high C4d group (adjusted HR: 1.93; 95% CI: 1.05 to 3.54; P=0.033). Additionally, a low baseline C4d level was independently assosicated with a favorable proteinuria response to immunosuppressive therapy at three months (adjusted relative risk: 2.20; 95% CI: 1.04-4.63, P=0.038). CONCLUSION: The urinary C4d, serving as a non-invasive biomarker, is associated with the progression of IgAN and holds the potential to predict proteinuria response in this disease.

Genetics of IgA nephrology: risks, mechanisms, and therapeutic targets.

IgA nephropathy (IgAN) is a genetically complex multifactorial trait. Over the past decade, population-based genome-wide association studies (GWAS) have identified more than 30 IgAN risk loci, providing novel perspectives on both the epidemiology of the disease and its underlying molecular mechanisms. In addition, the association between IgAN and galactose-deficient IgA1 (Gd-IgA1) presented another avenue for genetic exploration due to the heritability of the elevated serum Gd-IgA1 levels. These endeavors also yielded and enabled refinement of polygenic risk scores, which may help identify specific groups of individuals at significantly increased risks, leading to stratifications of medical treatments. In this review, we aim to explore the existing evidence for genetic causation in IgAN. We summarize the state of genetic research in IgAN and how it has led to the reformulation of the new pathogenesis model and novel therapeutic targets.

ADAMTS7-Mediated Complement Factor H Degradation Potentiates Complement Activation to Contributing to Renal Injuries.

BACKGROUND: The dysfunction of complement factor H (CFH), the main soluble complement negative regulator, potentiates various complement-induced renal injuries. However, insights into the underlying mechanism of CFH dysfunction remain limited. In this study, we investigated whether extracellular protease-mediated degradation accounts for CFH dysfunction in complement-mediated renal injuries. METHODS: An unbiased interactome of lupus mice kidneys identified CFH-binding protease. In vitro cleavage assay clarified CFH degradation. Pristane-induced SLE or renal ischemia-reperfusion (I/R) injury models were used in wild-type and ADAMTS7-/- mice. RESULTS: We identified the metalloprotease ADAMTS7 as a CFH-binding protein in lupus kidneys. Moreover, the upregulation of ADAMTS7 correlated with CFH reduction in both lupus mice and patients. Mechanistically, ADAMTS7 is directly bound to CFH complement control protein (CCP) 1-4 domain and degraded CCP 1-7 domain through multiple cleavages. In mice with lupus nephritis or renal I/R injury, ADAMTS7 deficiency alleviated complement activation and related renal pathologies, but without affecting complement-mediated bactericidal activity. Adeno-associated virus-mediated CFH silencing compromised these protective effects of ADAMTS7 knockout against complement-mediated renal injuries in vivo. CONCLUSION: ADAMTS7-mediated CFH degradation potentiates complement activation and related renal injuries. ADAMTS7 would be a promising anticomplement therapeutic target that does not increase bacterial infection risk.

Genome-Wide Association Study in Acute Tubulointerstitial Nephritis.

SIGNIFICANCE STATEMENT: Polymorphisms of HLA genes may confer susceptibility to acute tubulointerstitial nephritis (ATIN), but small sample sizes and candidate gene design have hindered their investigation. The first genome-wide association study of ATIN identified two significant loci, risk haplotype DRB1*14-DQA1*0101-DQB1*0503 (DR14 serotype) and protective haplotype DRB1*1501-DQA1*0102-DQB1*0602 (DR15 serotype), with amino acid position 60 in the peptide-binding groove P10 of HLA-DR ß 1 key. Risk alleles were shared among different causes of ATIN and HLA genotypes associated with kidney injury and immune therapy response. HLA alleles showed the strongest association. The findings suggest that a genetically conferred risk of immune dysregulation is part of the pathogenesis of ATIN. BACKGROUND: Acute tubulointerstitial nephritis (ATIN) is a rare immune-related disease, accounting for approximately 10% of patients with unexplained AKI. Previous elucidation of the relationship between genetic factors that contribute to its pathogenesis was hampered because of small sample sizes and candidate gene design. METHODS: We undertook the first two-stage genome-wide association study and meta-analysis involving 544 kidney biopsy-defined patients with ATIN and 2346 controls of Chinese ancestry. We conducted statistical fine-mapping analysis, provided functional annotations of significant variants, estimated single nucleotide polymorphism (SNP)-based heritability, and checked genotype and subphenotype correlations. RESULTS: Two genome-wide significant loci, rs35087390 of HLA-DQA1 ( P =3.01×10 -39 ) on 6p21.32 and rs2417771 of PLEKHA5 on 12p12.3 ( P =2.14×10 -8 ), emerged from the analysis. HLA imputation using two reference panels suggested that HLA-DRB1*14 mainly drives the HLA risk association . HLA-DRB1 residue 60 belonging to pocket P10 was the key amino acid position. The SNP-based heritability estimates with and without the HLA locus were 20.43% and 10.35%, respectively. Different clinical subphenotypes (drug-related or tubulointerstitial nephritis and uveitis syndrome) seemed to share the same risk alleles. However, the HLA risk genotype was associated with disease severity and response rate to immunosuppressive therapy. CONCLUSIONS: We identified two candidate genome regions associated with susceptibility to ATIN. The findings suggest that a genetically conferred risk of immune dysregulation is involved in the pathogenesis of ATIN.

MTMR3 risk alleles enhance Toll Like Receptor 9-induced IgA immunity in IgA nephropathy.

Multiple genome-wide association studies (GWASs) have reproducibly identified the MTMR3/HORMAD2/LIF/OSM locus to be associated with IgA nephropathy (IgAN). However, the causal variant(s), implicated gene(s), and altered mechanisms remain poorly understood. Here, we performed fine-mapping analyses based on GWAS datasets encompassing 2762 IgAN cases and 5803 control individuals, and identified rs4823074 as the candidate causal variant that intersects the MTMR3 promoter in B-lymphoblastoid cells. Mendelian randomization studies suggested the risk allele may modulate disease susceptibility by affecting serum IgA levels through increased MTMR3 expression. Consistently, elevated MTMR3 expression in peripheral blood mononuclear cells was observed in patients with IgAN. Further mechanistic studies in vitro demonstrated that MTMR3 increased IgA production dependent upon its phosphatidylinositol 3-phosphate binding domain. Moreover, our study provided the in vivo functional evidence that Mtmr3-/- mice exhibited defective Toll Like Receptor 9-induced IgA production, glomerular IgA deposition, as well as mesangial cell proliferation. RNA-seq and pathway analyses showed that MTMR3 deficiency resulted in an impaired intestinal immune network for IgA production. Thus, our results support the role of MTMR3 in IgAN pathogenesis by enhancing Toll Like Receptor 9-induced IgA immunity.

Interleukin 4-driven reversal of self-reactive B cell anergy contributes to the pathogenesis of systemic lupus erythematosus.

OBJECTIVES: Reactivation of anergic autoreactive B cells (BND cells) is a key aetiological process in systemic lupus erythematosus (SLE), yet the underlying mechanism remains largely elusive. This study aimed to investigate how BND cells participate in the pathogenesis of SLE and the underlying mechanism. METHODS: A combination of phenotypical, large-scale transcriptome and B cell receptor (BCR) repertoire profiling were employed at molecular and single cell level on samples from healthy donors and patients with SLE. Isolated naïve B cells from human periphery blood were treated with anti-CD79b mAb in vitro to induce anergy. IgM internalisation was tracked by confocal microscopy and was qualified by flow cytometer. RESULTS: We characterised the decrease and disruption of BND cells in SLE patients and demonstrated IL-4 as an important cytokine to drive such pathological changes. We then elucidated that IL-4 reversed B cell anergy by promoting BCR recycling to the cell surface via STAT6 signalling. CONCLUSIONS: We demonstrated the significance of IL-4 in reversing B cell anergy and established the scientific rationale to treat SLE via blocking IL-4 signalling, also providing diagnostic and prognostic biomarkers to identify patients who are most likely going to benefit from such treatments.

Discontinuation of Renin-Angiotensin System Inhibitors and Clinical Outcomes in Chronic Kidney Disease: A Systemic Review and Meta-Analysis.

BACKGROUND: Discontinuation of renin-angiotensin system (RAS) inhibitors is common in patients with chronic kidney disease (CKD), and the potential danger has been reported in several studies. However, a comprehensive analysis has not been conducted. OBJECTIVES: This study sought to evaluate the effects of discontinuation of RAS inhibitors in CKD. METHOD: Relevant studies up to November 30, 2022, were identified in the PubMed, Embase, Web of Science, and Cochrane Library databases. Efficacy outcomes included the composite of all-cause mortality, cardiovascular events, and end-stage kidney disease (ESKD). Results were combined using a random-effects or fixed-effects model, and sensitivity analysis used the leave-one-out method. RESULTS: Six observational studies and one randomized clinical trial including 244,979 patients met the inclusion criteria. Pooled data demonstrated that discontinuation of RAS inhibitors was associated with an increased risk of all-cause mortality (HR 1.42, 95% CI 1.23-1.63), cardiovascular event risk (HR 1.25, 95% CI 1.17-1.22), and ESKD (HR 1.23, 95% CI 1.02-1.49). In sensitivity analyses, the risk for ESKD was reduced. Subgroup analysis showed that the risk of mortality was more pronounced in patients with eGFR above 30 mL/min/m2 and in patients with hyperkalemia-related discontinuation. In contrast, patients with eGFR below 30 mL/min/m2 were at great risk of cardiovascular events. CONCLUSIONS: The discontinuation of RAS inhibitors in patients with CKD was associated with a significantly increased risk of all-cause mortality and cardiovascular events. These data suggest that RAS inhibitors should be continued in CKD if the clinical situation allows.

Intensive blood pressure control and the progression of IgA nephropathy: a cohort study using marginal structural models.

BACKGROUND: In chronic kidney disease, current guidelines recommend systolic blood pressure (SBP) below 120 mmHg. However, the renoprotective effect of intensive blood-pressure (BP) lowering on immunoglobulin A nephropathy (IgAN) remains undetermined. We aimed to determine the effect of intensive BP control on the progression of IgAN. METHODS: At Peking University First Hospital, 1530 patients with IgAN were enrolled. An examination of the relationship between baseline and time-updated BP and composite kidney outcomes, defined as development of end-stage kidney disease (ESKD) or a 30% decline in estimated glomerular filtration rate (eGFR), was conducted. Baseline and time-updated BPs were modeled using multivariate causal hazards models and marginal structural models (MSMs). RESULTS: In a median follow-up of 43.5 (interquartile range 27.2, 72.7) months, 367 (24.0%) patients experienced the composite kidney outcomes. No significant associations were found between baseline BP and the composite outcomes. Using MSMs with time-updated SBP for analysis, a U-shaped association was found. In reference to SBP 110-119 mmHg, hazard ratios (95% confidence intervals) for the SBP categories <110, 120-129, 130-139 and ≥140 mmHg were 1.48 (1.02-2.17), 1.13 (0.80-1.60), 2.21 (1.54-3.16) and 2.91 (1.94-4.35), respectively. The trend was more prominent in patients with proteinuria ≥1 g/day and eGFR ≥60 mL/min/1.73 m2. After analyzing time-updated diastolic BP, no similar trend was observed. CONCLUSIONS: In patients with IgAN, intensive BP control during the treatment period may retard the kidney disease progression, but the potential risk of hypotension still needs to be considered.

Pathological and clinical characteristics of late-onset oligomeganephronia based on a histomorphometric study.

BACKGROUND: Late-onset oligomeganephronia (OMN) is a rare chronic kidney disease and has no quantitative criteria for diagnosis yet. The current study aimed to explore its clinicopathological features by histomorphometric analysis. METHODS: We retrospectively re-reviewed all patients with enlarged and sparse glomeruli by light microscopy at Peking University First Hospital from 2012 to 2021, excluding those with any factor known to contribute to similar changes. Age- and sex-matched patients with thin basement membrane nephropathy were selected as control to establish the cut-off values for glomerulomegaly and rarity. Late-onset OMN cases were then confirmed and the clinicopathological characteristics were summarized. RESULTS: Mean diameter and density of cortical glomeruli in control was 156.53 ± 27.50 µm and 4.07 ± 0.63 /mm2, giving a lower limit of 211.53 µm for glomerulomegaly and an upper of 2.81 /mm2 for rarity. Seven adults of three females and four males were finally diagnosed as late-onset OMN with a mean age of 26.57 years. They showed mild to moderate proteinuria and/or renal dysfunction at biopsy with the mean proteinuria, serum creatinine (Scr) level, and estimated glomerular filtration rate of 0.50 g/d (0.10-0.95 g/d), 140.9 µmol/L (95.1-227.1 µmol/L), and 58.7 mL/min/1.73m2 (21.3-98.0 mL/min/1.73m2), respectively. Four patients (57.1%) had normal Scr at diagnosis. Six patients with available data showed renal tubular injury with increased urinary microalbumin in all, elevated N-acetyl-ß-glucosaminidase in two, and elevated α1 microglobulin in five. Kidney size was normal or slightly reduced. The mean density and glomerular diameter of the seven cases was 0.86 mm2 (0.55-1.41 /mm2) and 229.73 µm (211.88-260.66 µm). Segmental glomerular sclerosis was observed in six (85.7%) with four (66.7%) of perihilar type. Proximal tubule dilation was observed in all, focal to diffuse, lining with enlarged epithelial cells. The mean foot process width was 634.02 nm, wider than 472.54 nm of the control (P = 0.0002). CONCLUSION: Late-onset OMN should be considered a special entity with relatively slow clinical progress characterized by hypertrophy of the sparsely distributed nephron.

Comparison between hydroxychloroquine and systemic corticosteroids in IgA nephropathy: a two-year follow-up study.

BACKGROUND: Hydroxychloroquine (HCQ) is recommended as a treatment for IgA nephropathy (IgAN) to control proteinuria. The long-term effects of HCQ compared to systemic corticosteroid therapy remain unclear. METHODS: We conducted a retrospective case‒control study at Peking University First Hospital. Thirty-nine patients with IgAN who received HCQ for at least 24 months without corticosteroids (CSs) or other immunosuppressive agents were included. Thirty-nine matched patients who received systemic CS therapy were selected using propensity score matching. Clinical data over a 24-month period were compared. RESULTS: In the HCQ group, the level of proteinuria decreased from 1.72 [1.44, 2.35] to 0.97 [0.51, 1.37] g/d (-50.5 [-74.0, -3.4] %, P < 0.001) at 24 months. A significant decline in proteinuria was also found in the CS group, but no significant differences were found between the HCQ group and CS group in the levels of proteinuria (0.97 [0.51, 1.37] vs. 0.53 [0.25, 1.81] g/d, P = 0.707) and change rates (-50.5% [-74.0%, -3.4%] vs. -63.7% [-78.5%, -24.2%], P = 0.385) at 24 months. In addition, the decline rates of eGFR between the HCQ and CS groups were comparable (-7.9% [-16.1%, 5.8%] vs. -6.6% [-14.9%, 5.3%], P = 0.758). More adverse events were observed in the CS group. CONCLUSIONS: Long-term use of HCQ can maintain stable renal function with minimal side effects. In patients who cannot tolerate corticosteroids, HCQ might be an effective and safe supportive therapy for IgAN.

Primary membranous nephropathy in two siblings with one combined with anti-glomerular basement membrane disease: a case report.

BACKGROUND: The phospholipase A2 receptor (PLA2R) associated with membranous nephropathy (MN) is an organ-specific autoimmune disease associated with PLA2R and human leukocyte antigen (HLA) genes. Familial PLA2R-related MN is rarely reported. The combination of anti-GBM disease and MN has been well documented, though the mechanism behind it remains unclear. CASE PRESENTATION: We describe two siblings diagnosed with pathology-confirmed PLA2R-related MN 1 year apart. And one of the two siblings developed an anti-GBM disease. The high-resolution HLA typing showed identical alleles in both siblings, specifically heterozygotes of DRB1*15:01/*03:01. CONCLUSION: We describe a familial case of PLA2R-related MN supporting the role of genetic factors that HLA-DRB1*15:01 and DRB1*03:01 predispose patients in the development of PLA2R-related MN in the Han Chinese population. The combination of MN and anti-GBM disease may also partially be associated with the same susceptible HLA allele DRB1*15:01.

MNX1 Promotes Anti-HER2 Therapy Sensitivity via Transcriptional Regulation of CD-M6PR in HER2-Positive Breast Cancer.

Although targeted therapy for human epidermal growth factor receptor 2 (HER2)-positive breast cancer has significantly prolonged survival time and improved patients' quality of life, drug resistance has gradually emerged. This study explored the mechanisms underlying the effect of the motor neuron and pancreatic homeobox 1 (MNX1) genes on drug sensitivity in HER2-positive breast cancer. From July 2017 to 2018, core needle biopsies of HER2-positive breast cancer were collected from patients who received paclitaxel, carboplatin, and trastuzumab neoadjuvant therapy at our center. Based on treatment efficacy, 81 patients were divided into pathological complete response (pCR) and non-pCR groups. High-throughput RNA sequencing results were analyzed along with the GSE181574 dataset. MNX1 was significantly upregulated in the pCR group compared with the non-pCR group in both sequencing datasets, suggesting that MNX1 might be correlated with drug sensitivity in HER2-positive breast cancer. Meanwhile, tissue array results revealed that high MNX1 expression corresponded to a good prognosis. In vitro functional tests showed that upregulation of MNX1 significantly increased the sensitivity of HER2-positive breast cancer cells to lapatinib and pyrotinib. In conclusion, MNX1 may serve as a prognostic marker for patients with HER2-positive breast cancer, and its expression may facilitate clinical screening of patients sensitive to anti-HER2-targeted therapy.

A Lightweight Trust Mechanism with Attack Detection for IoT.

In this paper, we propose a lightweight and adaptable trust mechanism for the issue of trust evaluation among Internet of Things devices, considering challenges such as limited device resources and trust attacks. Firstly, we propose a trust evaluation approach based on Bayesian statistics and Jøsang's belief model to quantify a device's trustworthiness, where evaluators can freely initialize and update trust data with feedback from multiple sources, avoiding the bias of a single message source. It balances the accuracy of estimations and algorithm complexity. Secondly, considering that a trust estimation should reflect a device's latest status, we propose a forgetting algorithm to ensure that trust estimations can sensitively perceive changes in device status. Compared with conventional methods, it can automatically set its parameters to gain good performance. Finally, to prevent trust attacks from misleading evaluators, we propose a tango algorithm to curb trust attacks and a hypothesis testing-based trust attack detection mechanism. We corroborate the proposed trust mechanism's performance with simulation, whose results indicate that even if challenged by many colluding attackers that can exploit different trust attacks in combination, it can produce relatively accurate trust estimations, gradually exclude attackers, and quickly restore trust estimations for normal devices.

Expanding the Scope of Targeted Metabolomics by One-pot Microscale Synthesis and Tailored Metabolite Profiling: Investigation of Bile Acid-Amino Acid Conjugates.

Accurate metabolite characterization plays a vital role in targeted metabolomics. Nonetheless, the library of metabolites is still limited, especially for downstream conjugates, and it is time-consuming to synthesize each of these compounds due to high structural diversity. Herein, a green and smart strategy was developed to expand the scope of targeted metabolomics. The reference standards were synthesized in a one-pot microscale reaction, and the analytical method was tailored using the synthetic products. A group of new metabolites, namely bile acid-amino acid conjugates (BA-AAs), was studied as a proof-of-concept. First, in total 160 BA-AAs were synthesized using a small amount (2 mg each) of bile acids and low-toxic reagents within 4 h. Then, an ultra-high-performance liquid chromatography /Orbitrap-MS method was established to comprehensively profile 202 bile acid derivatives in 20 min. Finally, the method was applied to mice with inflammatory bowel disease (IBD) to discover the accumulation of 70 rare BA-AAs in small intestine and liver, where 55 were first reported from biosamples. These BA-AAs are farnesoid X receptor modulators and might contribute to the development of IBD. Our study demonstrated a feasible approach for the broad-spectrum targeted metabolomics of bile acids.

The level of urinary C4d is associated with disease progression in IgA nephropathy with glomerular crescentic lesions: a cohort study.

BACKGROUND: Positive glomerular C4d staining, representative of lectin pathway activation, has been proven to be associated with unfavorable outcomes in immunoglobulin A nephropathy (IgAN). Our previous study suggested that urinary C4d correlated positively with an increase in crescents while the relationship between urinary C4d and disease severity and progression remains unelucidated. METHODS: In this study we enrolled 168 patients diagnosed with IgAN with varying proportions of crescent formation at the time of biopsy. An independent cohort of 107 IgAN patients was enrolled for validation. Kidney biopsy specimens were stained using immunohistochemistry. Urinary C4d levels at renal biopsy were measured by enzyme-linked immunosorbent assay. The primary endpoint was end-stage kidney disease (ESKD). RESULTS: Higher urinary C4d/creatinine levels were associated with a lower estimated glomerular filtration rate (eGFR); massive proteinuria; hypertension and severe Oxford M, E, T and C scores. After a median follow-up of 19 months (interquartile range 9-27), 53 (31.5%) participants reached ESKD. High urinary C4d/creatinine levels were independently and significantly associated with a risk of developing ESKD [hazard ratio per standard deviation increment of log-transformed C4d/creatinine 7.623 (95% confidence interval 4.117-14.113)]. CONCLUSIONS: The urinary C4d/creatinine level is a potential useful biomarker that was associated with disease severity and progression in patients with IgAN and crescents.

Rapidly progressive IgA nephropathy: clinicopathological characteristics and outcomes assessed according to the revised definition of the KDIGO 2021 Guideline.

BACKGROUND: Rapidly progressive immunoglobulin A nephropathy (RPIgAN) is a severe clinical phenotype of IgAN associated with a poor outcome. The recently published Kidney Disease: Improving Global Outcomes (KDIGO) 2021 Guideline for the Management of Glomerular Diseases has proposed a new definition for RPIgAN that is based simply on a ≥50% decline in the estimated glomerular filtration rate (eGFR) over ≤3 months. METHODS: In 1677 IgAN patients followed at a single centre in China, we evaluated the utility of this new definition to identify the highest-risk IgAN patients who might be suitable for combination immunosuppressive therapy. RESULTS: The proportion of a ≥50% decline in eGFR over ≤3 months was 5.2%. The majority of these patients had reversible causes, with only 2.3% (39/1677) meeting the KDIGO 2021 criteria for RPIgAN. These patients had a significantly higher risk for end-stage kidney disease (ESKD) than non-RPIgAN patients (logrank P < 0.001). RPIgAN was an independent risk factor for ESKD [hazard ratio 3.99 (95% confidence interval 2.25-7.09); P <0.001]. A minority of the RPIgAN patients (25.6%) had ≥50% crescents. There was no significant difference in the risk for ESKD between patients in the RPIgAN group with ≥50% crescents and ˂50% crescents (logrank P = 0.27). Patients with RPIgAN and ≥50% crescents had a higher risk for ESKD than patients with non-RPIgAN and ≥50% crescents (logrank P = 0.04). CONCLUSIONS: These data support the validity of the KDIGO 2021 definition but require independent validation in other non-Chinese cohorts.

Amino acid signatures of HLA Class-I and II molecules are strongly associated with SLE susceptibility and autoantibody production in Eastern Asians.

Human leukocyte antigen (HLA) is a key genetic factor conferring risk of systemic lupus erythematosus (SLE), but precise independent localization of HLA effects is extremely challenging. As a result, the contribution of specific HLA alleles and amino-acid residues to the overall risk of SLE and to risk of specific autoantibodies are far from completely understood. Here, we dissected (a) overall SLE association signals across HLA, (b) HLA-peptide interaction, and (c) residue-autoantibody association. Classical alleles, SNPs, and amino-acid residues of eight HLA genes were imputed across 4,915 SLE cases and 13,513 controls from Eastern Asia. We performed association followed by conditional analysis across HLA, assessing both overall SLE risk and risk of autoantibody production. DR15 alleles HLA-DRB1*15:01 (P = 1.4x10-27, odds ratio (OR) = 1.57) and HLA-DQB1*06:02 (P = 7.4x10-23, OR = 1.55) formed the most significant haplotype (OR = 2.33). Conditioned protein-residue signals were stronger than allele signals and mapped predominantly to HLA-DRB1 residue 13 (P = 2.2x10-75) and its proxy position 11 (P = 1.1x10-67), followed by HLA-DRB1-37 (P = 4.5x10-24). After conditioning on HLA-DRB1, novel associations at HLA-A-70 (P = 1.4x10-8), HLA-DPB1-35 (P = 9.0x10-16), HLA-DQB1-37 (P = 2.7x10-14), and HLA-B-9 (P = 6.5x10-15) emerged. Together, these seven residues increased the proportion of explained heritability due to HLA to 2.6%. Risk residues for both overall disease and hallmark autoantibodies (i.e., nRNP: DRB1-11, P = 2.0x10-14; DRB1-13, P = 2.9x10-13; DRB1-30, P = 3.9x10-14) localized to the peptide-binding groove of HLA-DRB1. Enrichment for specific amino-acid characteristics in the peptide-binding groove correlated with overall SLE risk and with autoantibody presence. Risk residues were in primarily negatively charged side-chains, in contrast with rheumatoid arthritis. We identified novel SLE signals in HLA Class I loci (HLA-A, HLA-B), and localized primary Class II signals to five residues in HLA-DRB1, HLA-DPB1, and HLA-DQB1. These findings provide insights about the mechanisms by which the risk residues interact with each other to produce autoantibodies and are involved in SLE pathophysiology.

Interaction between GALNT12 and C1GALT1 Associates with Galactose-Deficient IgA1 and IgA Nephropathy.

BACKGROUND: Galactose-deficient IgA1 plays a key role in the pathogenesis of IgA nephropathy, the most common primary GN worldwide. Although serum levels of galactose-deficient IgA1 have a strong genetic component, the genetic link between this molecule and IgA nephropathy has not yet been clearly established. METHODS: To identify novel loci associated with galactose-deficient IgA1, we performed a quantitative genome-wide association study for serum galactose-deficient IgA1 levels, on the basis of two different genome-wide association study panels conducted in 1127 patients with IgA nephropathy. To test genetic associations with susceptibility to IgA nephropathy, we also enrolled 2352 patients with biopsy-diagnosed IgA nephropathy and 2632 healthy controls. Peripheral blood samples from 59 patients and 27 healthy controls were also collected for gene expression analysis. RESULTS: We discovered two loci, in C1GALT1 and GALNT12, that achieved genome-wide significance, explaining about 3.7% and 3.4% of variance in serum galactose-deficient IgA1 levels, respectively. We confirmed the previously reported association of C1GALT1 with serum galactose-deficient IgA1 levels, but with a different lead single-nucleotide polymorphism (rs10238682; ß=0.26, P=1.20×10-9); the locus we identified at GALNT12 (rs7856182; ß=0.73, P=2.38×10-9) was novel. Of more interest, we found that GALNT12 exhibits genetic interactions with C1GALT1 in both galactose-deficient IgA1 levels (P=1.40×10-2) and disease risk (P=6.55×10-3). GALNT12 mRNA expression in patients with IgA nephropathy was significantly lower compared with healthy controls. CONCLUSIONS: Our data identify GALNT12 as a novel gene associated with galactose-deficient IgA1 and suggest novel genetic interactions. These findings support a key role of genetically conferred dysregulation of galactose-deficient IgA1 in the development of IgA nephropathy.