How should we measure and interpret glomerular inflammation and what is the best anti-inflammatory approach in IgA nephropathy?

IgA Nephropathy

Background and Aims The clinical presentation and disease course of IgAN, the most common primary glomerulonephritis globally, vary considerably among patients. IgAN is diagnosed by kidney biopsy; histopathology findings of prognostic value are summarized and scored using the Oxford MEST-C classification system (mesangial [M] and endocapillary [E] hypercellularity, segmental glomerulosclerosis [S], interstitial fibrosis/tubular atrophy [T], and cellular crescents [C]). Studies show that higher M, E, and C scores are associated with increased glomerular inflammation. Overactivation of the complement system, especially the alternative pathway, plays an important role in the pathogenesis of IgAN. Studies have demonstrated an association between the intensity of glomerular C3 staining on kidney biopsy, and poor clinical outcomes in patients with IgAN. Despite this, the MEST-C classification system does not incorporate complement activity. This study evaluated the association of glomerular C3 staining intensity with demographics, proteinuria, hematuria, markers of glomerular inflammation, and MEST-C scores, in a US cohort of patients with IgAN. Method This retrospective cross-sectional analysis of adult patients (≥18 years of age) with biopsy-confirmed IgAN, used biopsy data (January 2016–December 2016) from Arkana Laboratories, a national and international nephropathology reference laboratory. Patient demographic and clinical characteristics at the time of kidney biopsy were stratified by intensity of C3 deposition, assessed by immunofluorescence staining (from 0 [negative staining] to 3+ [high-intensity staining]). Glomerular inflammation was defined as patients with either M1 or E1 or C1/C2 scores. Between-group differences were assessed using Kruskal–Wallis tests for continuous variables and chi-square tests for categorical variables. The association between glomerular C3 deposition intensity and MEST-C scores was assessed using multivariable ordinal logistic regression models and reported as odds ratios (OR) and 95% confidence intervals (CI). Results Kidney biopsy data from 470 patients with IgAN were included; 63.8% were male, and the mean age ± standard deviation was 46.9 ± 16.0 years. Patient demographics and clinical characteristics stratified by C3 deposition are presented in Table 1. A C3 deposition intensity score of ≥1 was present in 77.7% of patients and there was an association between C3 deposition intensity and hematuria (P = 0.01). There was evidence of some association between C3 deposition intensity and individual MEST-C scores: M1 (P = 0.05), E1 (P = 0.45), and C1/C2 (P = 0.08). C3 deposition intensity was associated with the presence of any MEST-C score M1, E1, or C1/C2, indicative of glomerular inflammation (P = 0.04) (Table 2). When adjusted for patient age and sex, patients with M1 or E1 or C1/C2 scores had 55% higher odds of greater C3 deposition intensity compared with patients without M1 or E1 or C1/C2 scores (OR: 1.55, 95% CI: 1.11–2.15; P = 0.009). Conclusion In this study, >75% of patients with IgAN had C3 staining intensity ≥1. Furthermore, in our model, glomerular inflammation (M1 or E1 or C1/C2 score) was associated with higher odds of C3 deposition intensity when adjusted for patient age and sex. Further research is needed to confirm these findings and assess whether including the degree of glomerular C3 deposition, as indicated by staining intensity, can be of additional prognostic value.

POS-109 INTERIM RESULTS OF PHASE 1 AND 2 TRIALS TO INVESTIGATE THE SAFETY, TOLERABILITY, PHARMACOKINETICS, PHARMACODYNAMICS, AND CLINICAL ACTIVITY OF BION-1301 IN PATIENTS WITH IgA NEPHROPATHY

Development of Immunoglobulin A Nephropathy- Like Disease in β-1,4-Galactosyltransferase-I-Deficient Mice

Background and Aims IgA nephropathy (IgAN) is a common primary glomerulonephritis, often leading to kidney failure. Pathogenesis of IgAN is associated with immune complexes formed in the circulation from aberrantly glycosylated IgA1 that is bound by IgG autoantibodies. Additional serum components, such as complement C3 (C3), are also associated with these immune complexes. Some of these circulating immune complexes deposit in the glomeruli and induce kidney injury with varying manifestations of glomerulonephritis. IgAN is diagnosed by evaluation of kidney biopsy specimens. Routine immunofluorescence microscopy reveals glomerular immune-complex deposits containing (co)dominant IgA with variable presence of IgG and/or IgM and usually C3. Light microscopy features describe the degree of kidney injury based on the Oxford classification MEST-C scores: Mesangial hypercellularity (M0 or M1), endocapillary hypercellularity (E0 or E1), segmental glomerulosclerosis (S0 or S1), interstitial fibrosis/tubular atrophy (T0, T1, or T2), and cellular/fibrocellular crescents (C0, C1, or C2). In this study, we used high-resolution confocal microscopy and multi-fluorochrome staining to assess colocalization of C3-IgA, C3-IgG, and IgA-IgG in glomerular immune-complex deposits of kidney-biopsy specimens from patients with IgAN and correlated the data with the Oxford classification MEST-C scores. Method Frozen-tissue sections (4 μm) of remnant kidney-biopsy specimens from 17 IgAN patients were fixed, blocked, and stained with fluorochrome-labeled reagents specific for human C3 (polyclonal goat IgG), IgA (polyclonal goat IgG), and IgG (Fc-specific nanobody). DAPI was used to stain nuclei. Using a high-resolution confocal microscope (Nikon A1R/SIM), z-stacks of glomerular images with four fluorochromes from multiple optical planes were acquired (objective 60×). For each slide, 60 regions of interest (ROIs) were selected in multiple optical planes of glomeruli in areas with C3 staining. Pearson's correlation coefficient (PCC) colocalization data were collected for C3-IgA, C3-IgG, and IgA-IgG pairwise comparisons in 60 ROIs. Mean values of PCC for each pairwise comparison were calculated. To correlate the results with Oxford MEST-C scores, patients were divided in paired groups based on the absence or presence of each Oxford MEST-C score variable: M0 vs. M1, E0 vs. E1, S0 vs. S1, T0 vs. T1+T2, C0 vs. C1+C2. Differences in PCC in C3-IgA, C3-IgG, and IgA-IgG for each paired group were evaluated. Furthermore, proteins from the glomerular immune-complex deposits of additional pooled remnant kidney-biopsy specimens were extracted and analyzed by SDS-PAGE immunoblotting with C3-specific antibody. Results All 17 samples showed staining for C3, IgA, and IgG, including those that had been reported negative for IgG (n = 14) or C3 (n = 1) by routine immunofluorescence microscopy. Immunoblotting confirmed the presence of C3 in the immune-complex deposits, revealing α chain and β chain of C3. Pairwise comparison of Pearson correlation coefficient values showed greater colocalization of C3-IgA than that of C3-IgG (P < 0.0001) and IgA-IgG (P < 0.0001), whereas colocalizations for C3-IgG and IgA-IgG were similar (P = 0.063). Only colocalization of C3-IgA correlated with MEST-C scores. Specifically, colocalization of C3-IgA was higher in IgAN patients with M1 (P = 0.0185), E1 (P = 0.0042), and C1+C2 (P = 0.0043) scores compared to IgAN patients with M0, E0, and C0 scores, respectively. C3-IgA colocalization for S0 vs. S1 and T0 vs. T1+T2 did not differ. Conclusion Our results support the role of C3-IgA-IgG immune complexes in the pathogenesis of IgAN. Greater C3-IgA colocalization was associated with mesangial hypercellularity, endocapillary hypercellularity, and cellular/fibrocellular crescents, underscoring a role of complement for inducing acute kidney damage in IgAN.

Cytokine gene polymorphisms: Can these differentiate renal disease entities?

Advances in our understanding of the pathogenesis of immunoglobulin A nephropathy (IgAN) have led to the identification of novel therapeutic targets and potential disease-specific treatments. Specifically, a proliferation-inducing ligand (APRIL) has been implicated in the pathogenesis of IgAN, mediating B-cell dysregulation and overproduction of pathogenic galactose-deficient IgA1 (Gd-IgA1). Animal and clinical studies support the involvement of APRIL in the pathogenesis and progression of IgAN. An elevated level of APRIL is found in IgAN when compared with controls, which correlates with the level of Gd-IgA1 and associates with more severe disease presentation and worse outcomes. Conversely, anti-APRIL therapy reduces pathogenic Gd-IgA1 and IgA immune complex formation and ameliorates the severity of kidney inflammation and injury. Genome-wide association studies in IgAN have identified TNFSF13 and TNFRSF13B, a cytokine ligand-receptor gene pair encoding APRIL and its receptor, respectively, as risk susceptibility loci in IgAN, further supporting the causal role of the APRIL signalling pathway in IgAN. Several novel experimental agents targeting APRIL, including atacicept, telitacicept, zigakibart and sibeprenlimab, are currently under investigation as potential therapies in IgAN. Preliminary results suggest that these agents are well-tolerated, and reduce levels of Gd-IgA1, with corresponding improvement in proteinuria. Further studies are ongoing to confirm the safety and efficacy of anti-APRIL approaches as an effective therapeutic strategy in IgAN.

Background and Aims Atrasentan is a potent and highly selective endothelin A receptor antagonist. In the prespecified 36-week interim analysis (IA) of the Phase 3 global ALIGN clinical trial, atrasentan demonstrated superiority versus placebo with a clinically meaningful and statistically significant reduction in proteinuria (primary endpoint) in patients with IgA Nephropathy (IgAN) receiving supportive care. Here, we report on additional post-hoc analyses from the ALIGN trial. Method ALIGN is an ongoing Phase 3, randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of atrasentan vs placebo in adults with IgAN at risk of progressive loss of renal function. The main stratum of the ALIGN study included patients with biopsy-proven IgAN and proteinuria of ≥1g/day while on maximum tolerated dose of renin-angiotensin inhibitors. Patients were randomized to receive atrasentan 0.75 mg or placebo orally once daily for 132 weeks while continuing supportive care. These post-hoc analyses assessed the change from baseline in 24-hour UPCR at Week 36 based on baseline proteinuria (<1 or ≥1 g/g), MEST-C scores and baseline hematuria. The analyses were performed on the IA data set which included the first 270 of 340 patients randomized to the main stratum. For the analysis by MEST-C score, 160 of 270 patients from the IA data set were included (i.e. patients with MEST-C scores available at baseline). Results Atrasentan showed a statistically significant and clinically meaningful proteinuria reduction of 36.1% (95% CI: 26.4%, 44.6%; P < 0.0001) relative to placebo, after 36 weeks of treatment. The reduction in proteinuria in patients with baseline UPCR <1 g/g and ≥1 g/g (LS mean % UPCR change from baseline relative to placebo at Week 36) was −28.7 (95% CI: −47.5, −3.2)​ and −38.3 (95% CI: −47.4, −27.5), respectively (Fig.). In addition, efficacy benefit favoring atrasentan was shown irrespective of MEST-C score and baseline hematuria level (Fig.). Conclusion Consistent with the primary endpoint findings, atrasentan shows favorable efficacy versus placebo in patients with IgAN regardless of baseline UPCR (<1 g/g and ≥ 1g/g), MEST-C score, and baseline hematuria. These results add to previously presented subgroup analysis data, and further support the efficacy of atrasentan across a broad range of IgAN patients.

IgA nephropathy (IgAN) is a common cause of primary glomerulonephritis worldwide. It is characterised by the deposition of poorly galactosylated IgA1 immune complexes in the renal mesangium, leading to end stage renal damage in 30% of patients. At present, the pathogenesis of IgAN is not well understood. IgAN GWAS studies revealed an association between IgAN and a SNP within the coding sequence of the CARD9 gene. CARD9 is an essential mediator of the innate immune system, however its place in the pathogenesis of IgAN has yet to be defined. The aim of this study was to investigate the role of CARD9 in IgAN.In this project, the expression of CARD9 mRNA was higher in; peripheral blood mononuclear cells (PBMCs) from IgAN patients compared to healthy controls, and in IgAN renal biopsy tissue compared to other renal diseases. rs4077515-T was associated with increased synthesis of IgA and poorly O-galactosylated IgA1 by activated PMBCs from both IgAN patients and healthy subjects. Immunohistochemistry demonstrated CARD9 protein in both the glomerular and tubulointerstitial compartments in IgAN and western blotting revealed the presence of CARD9 in cell lysates from human renal cells. Human mesangial cells with the rs4077515-T allele synthesised significantly more of the pro-inflammatory cytokine IL-6 following exposure to IgA1. Similar increases were seen in mRNA coding for IL-6, IL-6 signal transducer (IL-6ST) and monocyte chemoattractant protein 1(MCP-1), suggesting that the presence of rs4077515-T could result in a greater inflammatory response to mesangial IgA deposition in IgAN. A significant association between rs4077515-T and the likelihood of renal function decline in Chinese IgAN patients was observed.This study provides an insight into the contribution of CARD9 in; the generation of poorly O-galactosylated IgA1 by human PBMCs, the response of mesangial cells to IgA deposition in IgAN, the likelihood of renal function decline in IgAN.

BACKGROUND AND AIMSSeveral models have been proposed to describe the pathogenesis of Immunoglobulin A nephropathy (IgAN) and, among them, the multihit model where the gut-microbiota may play an important role. These models explain the pathogenesis of IgAN caused by the production of aberrant IgA, but it is believed that further predisposing factors are present, including immunological, genetic, environmental or nutritional factors.Recently, the role of IL-6 in IgAN pathogenesis is becoming increasingly important. It is essential for the deposition of glomerular immunoglobulin A and the development of renal disease in Cd37-deficient mice, although the pathogenetic mechanisms that determine its increase are not well known.A possible hypothesis emerges from our recent work on genome-wide DNA methylation screening in patients with IgAN, which identified, among other findings, a hypermethylated region comprising Vault 2–1 RNA (VTRNA2-1), a non-RNA coding also known as a precursor of miR-886 (pre-mi-RNA). Consistently, VTRNA2-1 expression was found downregulated in IgAN patients.Here we studied the involvement of the VTRNA2-1/PKR/CREB/IL-6 pathway in IgAN.METHODTotal RNA were isolated from PBMCs of IgAN patients, transplanted IgAN patients (TP-IgAN), non-IgAN transplanted patients (TP) and healthy subjects (HS). VTRNA2-1, CREB and PKR transcripts were evaluated by RT-PCR. Total and phosphorylated PKR, CREB and Il-6 proteins were evaluated by ELISA. Poly (I: C), a synthetic analogue of dsRNA and Pfizer-BioNTech COVID-19 COMIRNATY vaccine were used to transfect patient PBMCs. PKR inhibitor imoxin (C16) 1 µM was used to stimulate patient PBMCs.RESULTSHere we confirm that VTRNA2-1 transcript was down-regulated in native and transplanted IgAN subjects compared to HS and non IgAN transplanted patients, with a decrease of 30- and 100-folds, respectively (P < 0.05, and P < 0.0001). IgAN patients with downregulated VTRNA2-1 showed a PKR overactivation (fold increase of phosphorilation of 2.6- in IgAN and 2-folds in TP-IgAN patients; P < 0.05), coherently with the role played by VTRNA2-1 that binds to PKR and inhibits its phosphorylation. Then, we found that PKR causes the activation of CREB, a classical cAMP-inducible CRE-binding factor (fold increase of phosphorilation of 3- in IgAN and 2.67-folds in TP-IgAN patients; P < 0.01). CREB, interacting with a region of the IL-6 promoter, led to IL-6 production. Indeed, in IgAN patients we showed a IL-6 mean increase to 120 pg/mL compared to the respective controls (P < 0.05). Moreover, the IL-6 levels correlated with CREB and PKR phosphorylation (r = 0.97; P = 0.0006 and r = 0.89; P = 0.0064, respectively, for IgAN and TP-IgAN patients).Since PKR is normally activated by bacterial and viral RNA, we hypothesized that these microorganisms can further activate the PKR/CREB/IL-6 pathway leading to an excess of IL-6 production. This may explain both the high levels of IL-6, and infection involvement in the disease, and cases of IgAN associated with COVID-19 infection or with COVID-19 RNA-vaccination, and recent data showing microbiota involvement in IgAN. Effectively, we found that IgAN PMBCs stimulated with RNA poly(I: C) or the COVID-19 RNA-vaccine showed a significant increase in IL-6 levels compared to not-stimulated PBMCs (P < 0.05), supporting the pathogentic role played by viral RNA in IgAN pathogenesis and explaining the cases of IgAN patients developing episodes of macrohematuria after a COVID-19 infection or vaccination.Finally, we showed that the IL-6 secretion can be reduced by the PKR inhibitor imoxin (fold decrease of 5-folds in IgAN and TP-IgAN patients; P < 0.05).CONCLUSIONIn conclusion, the discovery of the upregulated VTRNA2-1/PKR/CREB/IL-6 pathway in IgAN patients may provide a new pathogenic mechanism in IgAN and may be useful for the development of novel therapeutic approaches, likely by modulating the VTRNA2-1 methylation level in IgAN patients.

IgA nephropathy (IgAN) is the most common chronic glomerulonephritis of unknown cause. Two factors are important in the pathogenesis of IgAN; one is IgA immune complex (IgA-IC) formation and the other is IgA-IC deposition to mesangium. For the formation of IgA-IC, antibody of IgA class, T-cells which help the generation of IgA antibody, and antigen are necessary. The serum IgA level in patients with IgAN is elevated probably due not only to the activation of B-cell but also to the hyperactivity of alpha beta chain positive T-cells. Analysis of the immunoglobulin gene of IgAN patients indicated that the genetic polymorphism could be correlated with the production of IgA antibody. The gamma delta chain positive T-cell may play some role for the formation of IgA-IC. Food, bacteria, viral proteins are reported to be the antigens of IgA-IC. Regarding to IgA-IC deposition, cytokines from T-cells and the molecular weight of IgA-IC are also indispensable factors. Clinically, long-term prognosis of IgAN are not unanimously the same. Approximately 15% patients progress to renal failure over a period of 10 years, and 25% to renal failure within 20 years. Roughly, patients can be classified into three groups in view of clinical course. First group maintains stable renal function for more than 20 years, second exhibits progressive course in more than 20 years and third progresses more rapidly than the second. Although the exact mechanism(s) governing the fate of renal function in IgAN is unclear, non-immunological factors, such as intraglomerular hypertension, are presumed to participate in its progression.(ABSTRACT TRUNCATED AT 250 WORDS)

Background and Aims Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are associated with better cardiovascular outcomes and with nephroprotection independent of diabetes status. Recent data suggest that SGLT2i significantly reduce proteinuria in patients with IgA nephropathy (IgAN). Data available for the use of the MEST-C score to determine if treatment would benefit IgAN patients is scarce. The aim of our study was to assess the degree of proteinuria reduction after SGLT2i treatment in IgAN and its possible association with the MEST-C score. Method 63 adult patients with biopsy-proven IgA nephropathy, eGFR ≥ 30 mL/min/1.73 m2 (CKD-epi), and total urine protein of &amp;gt;0.5 grams/day at screening were included. Patients with secondary IgAN and crescents were excluded. All receiving maximally tolerated RASi for at least 12 weeks. All patients started dapagliflozin 10 mg once per day after baseline data were registered. The total follow-up was 12 months. The main outcome was the decline in eGFR and the reduction in 24-hour proteinuria from SGLT2i initiation to 1,3, 6, 9, 12 months, end-stage renal disease, or kidney or cardiovascular death. Three repeated measurements of proteinuria (mg/24H) were performed, 90 days before SGLT2i treatment initiation, and only patients with stable proteinuria were enrolled in the study. Results 49 patients (35/14 M/F), with mean age of 41,9 ± 19,6 years, completed follow up. 8 patients discontinued treatment due to adverse events attributed to dapagliflozin. At baseline eGFR was 68.21±18,1 ml/min/1.73 m2 and Upr: 2196±1091 mg/24H. The MEST-C scores were correlated with the serum creatinine level, proteinuria, and hematuria. The mean serum creatinine level was higher in M1, E1, S1, and T1 compared with score “0”. It was statistically significant (p = 0.03) only in the T-score. The mean proteinuria level was again higher in M1, S1, and T1 but reached statistical significance (p = 0.037) in only the T1-score. E1 has higher hematuria. The Upr/24H difference for dapagliflozin was −24.7% (95% CI −27.5, −23.2; p &amp;lt; 0.001) at the end of follow-up (month 12). Proteinuria reduction was similar across all eGFRs, age, and baseline proteinuria. Among MEST-C score parameters, T1 had a negative impact on the antiproteinuric effect of dapagliflozin (p = 0.01) No hypoglycemic events were reported and no deaths occurred. Conclusion In the present study, the use of SGLT2i was associated with a significant reduction of proteinuria in patients with IgA nephropathy and maximally tolerated RASi. We analyzed renal outcomes in association with pathologic features. We established that the presence of the T1 score has a negative impact on proteinuria reduction after SGLT2i treatment. Further studies are needed to investigate the impact of the use of the Oxford Classification score for developing individual treatment therapies.

There is controversy over whether IgA nephropathy (IgAN) and Henoch-Schönlein purpura nephritis (HSPN) are the same diseases. This study focuses on the clinicopathological comparison between HSPN and IgAN in children. Children with IgAN and HSPN who had a diagnostic renal biopsy were enrolled. This study collected the clinical data of patients at biopsy, re-evaluated the pathological lesions of patients according to the Oxford Classification (MEST-C), and made a retrospective comparison between IgAN and HSPN on different stratifications of the course (Tc) and proteinuria. A total of 142 children with IgAN and 57 children with HSPN were enrolled. Various stratification showed the same result, which suggested that IgAN showed more mesangial proliferation (M). HSPN showed more segmental glomerulosclerosis in the Tc > 12m group than IgAN (S 60.0% vs. 9.10%, P = 0.008). In the non-nephrotic-range and nephrotic-range proteinuria group, there were no significant differences in MEST-C scores between IgAN and HSPN. M is more common in IgAN. HSPN had more S than IgAN over the course of more than 12months. These results indicate the differences in the pathogenesis in IgAN and HSPN. We propose early biopsy and active treatment of HSPN within 12months to delay the development of chronic lesions.

IgA nephropathy, described in 1968 as IgA-IgG immune-complex disease, is an autoimmune disease. Galactose-deficient IgA1 is recognized by unique autoantibodies, resulting in the formation of pathogenic immune complexes that ultimately induce glomerular injury. Thus, formation of the galactose-deficient IgA1-containing immune complexes is a critical factor in the pathogenesis of IgA nephropathy. Studies of molecular defects of IgA1 can define new biomarkers specific for IgA nephropathy that can be developed into clinical assays to aid in the diagnosis, assessment of prognosis, and monitoring of disease progression.

Abnormal IgA1 O-Glycosylation in a Multi-ethnic population of IgA Nephropathy Patients in KwaZulu Natal, South Africa.