Serum C3 Levels Research Articles

P0097IDENTIFICATION OF TWO NOVELS SINGLE NUCLEOTIDE VARIANTS OF THE COMPLEMENT GENES IN A PATIENT WITH THE ASSOCIATION OF PREGNANCY ATYPICAL HEMOLYTIC UREMIC SYNDROME AND C3 GLOMERULOPATHY

Abstract Background and Aims Advances in the past 2 decades have shown atypical hemolytic uremic syndrome (aHUS) to be a disorder of the alternative pathway of complement. Most aHUS cases involve sequence variations in genes encoding complement proteins The term pregnancy associated aHUS (P-aHUS) refer to the thrombotic microangiopathy (TMA) that result from uncontrolled complement activation during pregnancy or the postpartum period. P-aHUS is a devastating systemic disease, with high maternal mortality and morbidity rates, in the pre-eculizumab era. The term ‘C3 glomerulopathy’ (C3GN) encompasses a heterogeneous spectrum of immune-mediated nephropathies that share a common pathological feature, glomerular deposition of C3. This entity may progress to advanced stages of chronic kidney disease and shares a common genetic risk factors with aHUS. Even more, some authors even suggest that C3GN and aHUS represent two forms of a disease spectrum with a common pathogenic principle. Here, we report this rare association and describe the family genetic variants that could cause it Method We describe the clinical and laboratoy data of a patient with the association of aHUS and C3 Glomerulopathy. A genetic study was performed and her available relatives were also screened for mutations/polymorphisms in aHUS-associated complement genes. After extracting gDNA from whole blood (Wizard Genomic DNA Purification Kit, Promega), PCR products of coding sequences and intronic flanking regions of complement genes were sequenced by ABI PRISM 310 Genetic Analyzer (Applied Biosystems). In silico analysis for pathogenicity was completed with Polyphen2-HDIV, PhyloP/Phastcons (MutationTaster), SIFT and PANTHER. All the participants provided informed written consent Results In 3/2012 a 27-year-old patient, with no family history, started his current illness one month after his first natural birth, with acute renal failure and microangiopathic hemolytic anemia, demonstrating a severe TMA in a renal biopsy. Laboratory results showed low C3 serum levels, but C4 were normal, haptoglobine was undetectably low, and all ADAMTS13 parameters were normal. Her urine tests showed also glomerular hematuria and proteinuria in the nephrotic range. She was treated with plasmapheresis and fresh frozen plasma with hematological improvement, but hemodialysis was required for more than 3 months. Despite partial recovery of renal function, six months later reappeared anemia and developed severe arterial hypertension, congestive heart failure and progressive renal insufficiency. Diagnosis of aHUS was made and start treatment with eculizumab with progressive recovery of renal function in the following months. Nevertheless the C3 serum levels persisted low and the proteinuria and hematuria did not change even after long term treatment with eculizumab. Retrospectively, her urynalisis before the pregnant showed proteinuria and hematuria, and a revision of a renal biopsy revealed the presence of dominant C3 deposits in the immunofluorescence, and electrondense deposits in the electronic microscopy, suggesting the diagnostic of C3GN. In a genetic study two novel single nucleotide variants were founded (CFH c.575G>A, p.C192Y (exon 5) (NM_000186), predicted to be pathogenic by 4 of 5 available pathogenicity prediction programs; and CFI c.1189G>T, p.V397L (exon 11) (NM_000204), predicted pathogenic by 0 of 6 available pathogenicity prediction programs). (Figure 1) Conclusion We present here the family genetic bases of a patient who developed a C3GN and a aHUS with a different response to treatment with eculizumab. In this case we identified two novel genetic variants in the CFH and CFI genes in a patient with aHUS, who inherited one variant from each parent. Although the CFI variant is predicted to be benign, the CFH variant is predicted to be damaging. It is located in exon 5, which encodes a portion of the factor H protein implicated in binding to C3b

P0461BASELINE SERUM C3 LEVELS IN PATIENTS WITH ANCA ASSOCIATED VASCULITIS AND ITS CLINICAL IMPLICATION

Abstract Background and Aims The evolution of renal disease and its global impairment in patients diagnosed with ANCA-associated vasculitis (AAV) has been related to baseline C3 serum levels in several studies. The objective of this study is to evaluate in patients diagnosed with AAV and renal impairment the association between baseline serum C3 levels and the clinical outcomes. Method Our cohort included 56 patients diagnosed with AAV diagnosed from January 1986 to January 2018 in Hospital La Paz. According to C3 serum level measurements, patients were divided into low and normal concentration groups at onset. Histological, clinical and laboratory data were compared between groups. Results Among the 56 AAV cases, 58.9% were women and 96.5% Caucasian with an age mean and SD of 61.08 ± 15.11 years. Mean follow-up was 10.03 ± 7.02 years. MPO ANCAs were found in 69.8% of the patients and PR3 ANCAs in 22.64%. AAV diagnosis was earlier in PR3 AVV (47.59 ± 13.62 years) compared to MPO AVV (65.66 ± 11.53 years) (p=0.001). At diagnosis, baseline serum C3 was measured in 53 patients (94.6%) being lower in MPO AAV (112.79 ± 33.98) vs PR3 AAV (132.41 ± 20.69) (p=0.023). Low C3 was way more frequent in patients with higher percentage of glomeruli showing >50% of sclerosis (100% vs 32.5%) (p=0.007). Staphylococcus aureus nasal carriers were more frequent in patients with low C3 (27.2% vs 3.1%, p=0.045). 15 patients (27.77%) died during follow-up and patient survival median was 21.75 years (IC 95%: 15.41-28.08). 17 patients (31.48%) reached end-stage renal disease (ESRD) during follow-up and renal survival median was 20.1 years (IC 95%: 19.27-21.05). There were no statistical differences in the levels of baseline serum C3 according to sex, relapses, extrarenal manifestations, treatment, malignancy, cardiovascular events and time to ESRD or death. Conclusion Baseline serum C3 was lower in MPO AV, in Staphylococcus aureus nasal carriers and higher severity histological damage (glomerular sclerosis >50%). Despite previous evidence, in our cohort, time to ESRD and time to death seemed not to be affected.

P0387PATHOLOGICAL FEATURES AND CLINICAL COURSE OF LATE-ONSET LUPUS NEPHRITIS

Abstract Background and Aims It has been described that the early onset of lupus nephritis (LN) at a young age can determine the clinical course and outcome of the disease, being more aggressive with a higher mortality rate. However, no studies have explored the influence of age at onset of the disease in European cohorts of LN patients, neither have they analyzed treatment outcomes depending on age at presentation. Therefore, the aim of our study was to compare the clinical and histological characteristics of patients with late-onset LN compared to those with an onset at a younger age. Method We performed a retrospective observational study of biopsy-proven LN diagnosed between 1981 and 2018. Patients were stratified according to their age at presentation of LN, considering an onset with >50 years old as late-onset. Demographic, clinical and serological data at presentation of systemic lupus and LN, during and at the end of follow-up, as well as extrarenal manifestations, histological characteristics of the kidney biopsy, clinical course, received treatment regimens and related adverse effects were registered. Results 85 LN patients were included in the study, 10 of whom had late-onset LN (11.7%). Late-onset LN patients presented significantly higher serum C3 and C4 levels at the time of kidney biopsy (92.8 ± 22.4 mg/dl vs 62.5 ± 25.7 mg/dl, p=0.002, and 18.6 ± 5.0 vs. 9.3±5.8 mg/dl, p<0.001 respectively), and had lower eGFR measured by CKD-EPI (58.50±19.79 vs 90.41±30.53,p=0.003), a higher frequency of serositis manifestations (53.45% vs 17.46%, p=0.034), predominantly pleuritis (79.37% vs 51.75% p=0.013). Early-onset LN had predominantly Class IV LN (50% vs 10%, p=0.017), whereas in late-onset LN the most frequent type was Class V LN (50% vs 21.3%, p=0.048). Tubular atrophy was more common in late-onset LN (75% vs 34.9%, p=0.035). The group of patients with late-onset LN achieved complete clinical remission more frequently (87.5% vs 57.1%), but there were no differences in relapses between the groups. No differences were found in treatment regimens among the groups. Late-onset LN patients were more seronegative (100% vs 61.3%, p=0.015) at the end of follow-up. Severe infectious complications related to immunosuppression were significantly higher in late-onset LN patients (62.5% vs 22.2%, p=0.027) Conclusion Late-onset LN has a milder complement consumption at presentation compared to early-onset, and Class V LN is its most frequent form of presentation. Complete renal remission is more frequently achieved in late-onset LN compared to those with an early-onset, but present more adverse events associated with immunosuppression, particularly infectious complications.

AB0051 SERUM AMYLOID A AND PENTRAXIN 3: INNATE IMMUNE RESPONSE AND DISEASE ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

Background:Systemic Lupus Erythematosus (SLE) is an autoimmune disease that involves several molecular patterns with a wide spectrum of clinical manifestations and symptoms. Inflammation and related pathway play a role in SLE pathogenesis. The pentraxin superfamily including long and short pentraxin, C Reactive Protein CRP, Serum amyloid A (SAA), Pentraxin 3 (PTX3) are key components of innate immune system and induce a variety of inflammation associated pathway. However Literature provides several evidences that CRP serum levels not correlated with clinical and immunological manifestations. This situation affected clinical practice and the patient follow up. PTX3 have been identified as a component of inflammatory status in several autoimmune conditions. SAA is an acute phase protein secreted in large quantity during inflammation.Objectives:We want to evaluated SAA, PTX3 and CRP concentrations, their correlation between SLE Disease Activity Index (SLEDAI), that including complement fractions C3, C4.Methods:We enrolled fifty patients that fulfilled the SLE American College of Rheumatology criteria and fifty healthy subjects. The SLE disease activity was classified with the SLEDAI (0 to 12). Patients were divided into two groups according to SLEDAI score: inactive group (Group 1, 25 patients, 50%: SLEDAI < 4) and active group (Group 2, 25 patients, 50%: SLEDAI 5 to 12). PTX3 concentration was measured by a sandwich ELISA kit (Hycult) with 2.8 ng/mL cut-off point. SAA concentration was detected by nephelometry performed on a BN ProSpec System (Siemens, Germany), with assay kit based on polyclonal antibodies (Siemens Healthcare Diagnostics Products, Germany, 6.5 mg/L cut-off point). High sensitive CRP concentrations were determined using the ci8200 platform (Abbott Laboratories Chicago, Illinois).Results:Plasma PTX3 and serum SAA levels was significantly higher in SLE patients than in the healthy subjects (PTX311.5 ± 7.3 ng/mL vs 2.3 ± 1.1; p < 0.001; SAA: 87 ±77 mg/L vs 2.6±2.5; p < 0.001). These differences were not evident in CRP levels (8.5 ± 7.8 mg/L vs 6.2± 2.5). Considering two groups, there were statistical differences in PTX3 level (Group 2: 14.9 ± 12 ng/mL vs Group 1: 2.16 ±0.5 ng/mL, p<0,05) and SAA concentration (Group 2: 114 ± 89 ng/mL vs Group 1: 3.6 ±1.7 ng/mL, p<0,05) but not in CRP concentration (Group 2: 11.5 ± 8.4 mg/L vs Group 1: 9.5 ±3.5). There was a significantly negative correlation between C3, C4 fractions, PTX3 and SSA levels (respectively r = −0.74, p=<0.05, and r = −0.79, p<0.05). No statistical correlation were appeared between C3, C4 fractions and CRP serum levels (r= −0,12., p= 0.82, and r= −0.18, p= 0,21). We noted a positive significant correlation between SLEDAI, PTX3 and SAA concentration (r = 0.79, p < 0.05, 0.83, p < 0.05, respectively) an increase in PTX3 and SAA levels followed the lupus flare and symptoms. No significant correlation appeared between SLEDAI and CRP (r= 0.15, p=0.89)Conclusion:PTX3 and SAA concentration was significantly higher in SLE patients than the healthy control subjects and their levels reflected disease activity. We showed a direct correlation between PTX3 and SAA. In SLE patients PTX3 and SAA concentrations were correlated with SLEDAI. We suggest an integrate viewpoint in witch SAA and PTX3 may play a role as a biomarker of disease activity, with synergic work during SLE events. Evidences suggested that PTX3 and SAA could trigger the same molecular pathway, by TLR4, via NF-kB.

P0276SERUM C3 LEVELS AND THE PROGNOSIS OF ANCA-ASSOCIATED VASCULITIS: A SINGLE-CENTER RETROSPECTIVE STUDY

Figure: Background and Aims Despite improved survival, patients affected by ANCA-associated vasculitis (AAV) presenting with rapidly progressive glomerulonephritis (RPGN) still remain at a higher risk of death relative to the general population. Recent findings suggest a role for the activation of the complement alternative pathway in the pathogenesis of the disease. We aimed to retrospectively evaluate the presentation and outcome of a cohort of patients with AAV and RPGN according to ANCA specificity, investigating the role of complement pathways. Method We retrospectively examined 1,547 biopsies performed between 1996 and 2019 in our center, which included 124 patients presenting with paucimmune RPGN. Patients without ANCA serology and/or with missing data (n=44), ANCA negative RPGN (n=15) and anti-GBM disease (n=2) were excluded from our analysis. Seventy-eight patients with AAV and RPGN (63 biopsy proven) were analysed and compared according to ANCA serotype (n=30 MPO, n=21 PR3). Furthermore, data were analysed on the basis of median levels of serum C3 (1.05 g/l). Statistical analysis Data are mean ± SD or median and interquartile range for variables not normally distributed. Groups were compared using ANOVA after logarithmic transformation of skewed variables. Primary endpoint was renal survival defined as need for renal replacement therapy (NRRT); secondary outcome was patient survival. Cumulative and renal survivals were calculated using Kaplan-Meier method and differences between groups were analysed with the log-rank test. Multivariate Cox proportional hazard analysis for time to NRRT was performed to determine the significance of different risk factors in renal outcome. A p value of < 0.05 was considered statistically significant. Results Patients had a mean age of 64± yrs and eGFR ±15 ml/min at diagnosis. Patients with MPO-AAV were older (69±11 vs. 61±14 yrs, p=0.02) and showed lower levels of C3 (1.0±0.22 vs 1.25±0.2 g/l, p=0.009) as compared to PR3 patients. Moreover, those presenting with C3 levels lower than median value had more advanced renal damage at diagnosis (eGFR 7± ml/min vs 15.5± ml/min, p= 0.04). Serum C3, but not C4 levels, significantly correlated with eGFR, proteinuria and serum uric acid at diagnosis. Twenty-seven (39.7%) reached ESRD requiring RRT. The 1-, 5- and 10- yrs patients renal survivals were 81%, 62.9 % and 52.4%. Renal outcome was neither influenced by ANCA serotype nor gender but patients with C3 levels below median value exhibited a worse renal prognosis (Log rank p=0.02) and a 5 times higher risk of NRRT (HR 5.1, 95% CI 1.4-18.7, p=0.01) independently by potential confounding factors. Conclusion Referral to the nephrologist was late, at an advanced stage of disease, indicating the need to improve the awareness of AAV and RPGN in order to favour early treatment. In this cohort, low C3 indicated a more severe renal damage and predicted poorer renal outcome thus suggesting a role of complement activation in the pathogenesis and progression of these nephritides.

P0455CLINICAL PROFILE OF C3 GLOMERULOPATHY AT A TERTIARY CARE CENTRE

Abstract Background and Aims To study the clinico-pathological profile of patients with C3 glomerulonephritis at a tertiary care entre. Method A prospective observational study of all cases of C3 glomerulonephritis was done between August 2016 and December 2017. C3 glomerulonephritis was defined by predominant glomerular C3 fragment deposition with electron-dense deposits on EM Results Total of the 1190 patients under went biopsy during the study period. C3 glomerulonephritis was present in 32 cases (2.68%). The mean age was 35±2 years. Out of the 32 patients, 18 were male ( 56.25% ) and 14 were female (43.75% ). Hypertension was present in 30 out of 32 patients (93.7%) while diabetes was present in 7 patients (21.8%). Most common presentation was with RPGN (72%). 100% patients had active urinary sediments. The mean serum creatinine at admission was 5.15mg/dl while it was 3.18mg/dl at discharge. The mean serum C3 was 83.8 mg/dl with 18 patients (56.25%) having low serum C3 levels in our study. All patients had normal serum C4 levels with mean being 29.34 mg/dl. Anti CFH antibody titres were measured in 6 patients of with it was elevated in 2 patients (33.3%). On LM, 14 out of 32 had crescentic GN (43.75%), 12 had diffuse exudative pattern (37.5%), 4 had diffuse mesangioproliferative pattern (12.5%), 3 had MPGN pattern (9.37%), 3 had focal exudative pattern (9.37%), 1 patient had glomerular TMA (3.15%) and 1had chronic sclerosing pattern ( 3.15%). On IF, all patients had dominant C3 staining. Also 3 (9.37%) patients had IgG staining of 1+ intensity. EM study was done in two patients and was suggestive of DDD in one patient (3.15%). All patients were managed with pulse steroids followed by oral steroids. IV Cyclophosphamide was used monthly for 6 doses in 14 patients ( 43.7%) while oral cyclophosphamide was used in 2 patients (6.25%). Plasmapharesis was done in 11 patients (34.37%) and 11 patients required haemodialysis (34.37%) Conclusion C3 glomerulonephritis has emerged as a new entity in the recent past with significant percentage of our patients presenting with RPGN. Hypertension was present in most patients. On light microscopy, the most common presentation was crescentic glomerulonephritis, One third of our patients had advanced renal failure requiring haemodialysis. Plasmapharesis was done in one third of patients.

Effects of single or conjoint administration of lactic acid bacteria as potential probiotics on growth, immune response and disease resistance of snakehead fish (Channa argus)

Lactic acid bacteria (LAB) has been documented to promoting growth, enhancing immunity and disease resistance. In this study, we aimed to evaluate the single or conjoint effects of Lactococcus lactis L19 (Genbank: MT102745.1) and Enterococcus faecalis W24 (Genbank: MT102746.1) isolated from the intestine of Channa argus (C. argus) on growth performance, immune response and disease resistance of C. argus. A total of 720 apparently healthy C. argus (9.50 ± 0.03 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with L. lactis (L19), E. faecalis (W24), and L. lactis L19 + E. faecalis W24 (L + W) at 1.0 × 108 cfu/g basal diet for 56 days. After feeding, the final body weight (FBW), weight gain (WG), feed efficiency ratio (FER), specific growth rate (SGR) and protein efficiency ratio (PER) had significantly increased (p < 0.05), especially with L19. The results indicated that single or conjoint administration of LAB as potential probiotics can induce high levels of IgM, ACP, AKP, LZM, C3 and C4 activity in serum, which may effectively induce humoral immunity, and L19 induce even higher levels. Meanwhile, when compared to CK group, the results of qPCR showed that LAB administration significantly up-regulated (p < 0.05) the expression of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, HSP70, HSP90, TGF-β in the spleen, head kidney, gill, liver and intestine of C. argus. After challenge with Aeromonas veronii, the survival rates in all LAB-fed groups were significantly higher (p < 0.05) than that of the CK group, and the L19 group showed the highest (63.3%) disease resistance. Our data indicated that L. lactis L19 and E. faecalis W24, as a feed additive at 1.0 × 108 cfu/g feed, could promote growth performance, enhance immune response and disease resistance of C. argus, with greatest effects in fish fed L. lactis L19 for 56 days. Hence, these LAB additives could be used as promising probiotics for C. argus. L19 was more effective than W24 or the mixture of the two for promoting growth performance, enhancing immune response and disease resistance of C. argus.

C3(H2O) prevents rescue of complement-mediated C3 glomerulopathy in Cfh-/- Cfd-/- mice.

Therapeutic complement inhibition is a major focus for novel drug development. Of upstream targets, factor D (FD) is appealing because it circulates in plasma at low concentrations and has a single function: to cleave factor B to generate C3 convertase of the alternative pathway (AP). Mice with a targeted deletion of factor H (FH; Cfh-/- mice) develop C3 glomerulopathy (C3G) due to uncontrolled AP activity. To assess the impact of FD inhibition, we studied Cfh-/- Cfd-/- mice. We show that C3G in Cfh-/- mice is not rescued by removing FD. We used serum from Cfh-/- Cfd-/- mice to demonstrate that residual AP function occurs even when both FD and FH are missing and that hemolytic activity is present due to the action of C3(H2O). We propose that uncontrolled tick-over leads to slow activation of the AP in Cfh-/- Cfd-/- mice and that a minimal threshold of FH is necessary if tissue deposition of C3 is to be prevented. The FD/FH ratio dictates serum C3 level and renal C3b deposition. In C3G patients with chronic renal disease, the FD/FH ratio correlates inversely with C3 and C5 serum levels, suggesting that continuous AP control may be difficult to achieve by targeting FD.

Low dose interleukin-2 decreases P-glycoprotein activity and exerts synergistic anti-inflammatory effects with immunosuppressants in the treatment of lupus nephritis with steroid and immunosuppressive drug resistance

Abstract Objective P-glycoprotein (P-gp) is capable of transporting glucocorticoids and immunosuppressants out of lymphocytes, resulting in drug resistance in lupus nephritis (LN). IL-2 decreases P-gp expression in cultured cells from human colon carcinoma. Here, we investigated the effects of low-dose IL-2 on the functionality of P-gp in PBMCs of steroid resistant LN patients and in kidneys in MRL/lpr mice. Methods 30 drug resistant LN patients were treated with glucocorticoids and immunosuppressants with or without low-dose IL-2 for 12 weeks. Serum and urine collected before and after treatment were analyzed for anti-dsDNA, C3, C4, renal and liver function. P-gp expression and activity were detected in PBMCs by flow cytometry and rhodamine 123. MRL/lpr mice were treated with methylprednisolone (MP) with or without IL-2 for 4 weeks. Pathological changes as well as protein and mRNA expression of P-gp were assessed in kidneys. Results Compared to patients without IL-2 treatment, the patients treated with IL-2 had higher serum C3 (p=0.01) and C4 levels (p=0.04), while lower proteinuria(p=0.04), serum creatinine (p=0.03), anti-dsDNA levels (p&amp;lt; 0.001), and accordingly lower SLEDAI (p=0.03). These modifications were associated with reduced P-gp expression (p&amp;lt; 0.01) and activity (p=0.02) in PBMCs. Moreover, compared with controls, MRL/lpr mice treated with IL-2 and MP showed reduced proteinuria (p=0.002) and fewer acute and chronic renal pathological lesions. IL-2 attenuated renal deposition of IgG/C3, decreased renal expression of P-gp. Conclusion Low-dose IL-2 decreases P-gp activity and exerts synergistic anti-inflammatory effects with immunosuppressants in the treatment of LN with steroid and immunosuppressants resistance.

Influence of air pollution on renal activity in patients with childhood-onset systemic lupus erythematosus.

Childhood-onset systemic lupus erythematosus (cSLE) is an autoimmune and multifactorial disease that can affect the renal system. Exposure to air pollution can trigger systemic inflammation in cSLE patients and increase risk of disease activity. We evaluated effects of individual real-time exposure to air pollutants on renal activity in cSLE patients using the Systemic Lupus Erythematosus Disease Activity Index 2000. Longitudinal panel study of 108 repetitive measures from 9 pediatric lupus patients. Over three consecutive weeks, daily individual levels of fine particulate matter (PM2.5) and nitrogen dioxide (NO2) were measured, as well as weekly clinical evaluation and laboratory tests. This was repeated every 10weeks over a 1-year period. Specific generalized estimating equation models were used to evaluate the impact of these pollutants on risk of nephritis and anti-dsDNA > 20UI/mL and on 24-h urine protein and serum complement (C3) levels. An interquartile range (IQR) increase of 18.12μg/m3 in PM2.5 daily concentration was associated with increased risk of nephritis and positive results for anti-dsDNA. Moreover, increase in 24-h urine protein and decrease in C3 serum levels also associated with exposure to pollutants. An IQR increase in PM2.57-day moving average was associated with increased risks of leukocyturia (3.4; 95% CI 2.6:4.3), positive anti-dsDNA (3.1; 95% CI 2.1:4.0), and 36.3-mg increase (95% IC 20.2:52.3) in 24-h urine protein. An IQR increase (63.1μg/m3) in 7-day cumulative NO2 levels was associated with decreased serum C3 levels. This prospective study suggests exposure to air pollution can trigger renal activity in cSLE patients.

A Validation of the 2018 Revision of International Society of Nephrology/Renal Pathology Society Classification for Lupus Nephritis: A Cohort Study from China

Background: A revision of the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification for lupus nephritis has been published in 2018. The current study aimed to verify the utility of this system. Materials and Methods: A total of 101 lupus nephritis patients from a large Chinese cohort who underwent renal biopsy in Peking University First Hospital were reevaluated by 2 renal pathologists, who had no knowledge of the clinical findings. The association between clinical data at the time of initial renal biopsy and follow-up and pathological features were further analyzed on all patients selected. Results: The mean age of the cohort was 33 years with a male/female ratio of 1:9, and a median follow-up period of 128 months. The presence and extent of mesangial hypercellularity, endocapillary hypercellularity, global and segmental glomerulosclerosis, neutrophil exudation/karyorrhexis, glomerular hyaline deposits, extracapillary proliferation (crescents), tubular atrophy/interstitial fibrosis, and interstitial inflammation were significantly correlated with several clinical renal injury indices (systemic lupus erythematosus disease activity index, serum creatinine value, proteinuria, and C3 level) at the time of biopsy. By multivariable Cox hazard analysis, fibrous crescents, tubular atrophy/interstitial fibrosis, and the modified National Institutes of Health chronicity index were independent risk factors for patients’ composite renal outcomes (hazard ratio [HR] 4.100 [95% CI 1.544–10.890], p = 0.005; HR 8.584 [95% CI 2.509–29.367], p = 0.001; and HR 3.218 [95% CI 1.138–9.099], p = 0.028; respectively). Conclusions: The 2018 revision of the ISN/RPS classification for lupus nephritis has utility for prediction of clinical renal outcomes.

The association of neutrophil-to-lymphocyte ratio with all-cause mortality in Chinese patients with MPO-ANCA associated vasculitis.

Neutrophil-to-lymphocyte ratio (NLR) has been recently reported to be a promising inflammatory marker to assess systemic inflammation in many disorders. However, there are only a few studies looking at NLR in patients with myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study was thus undertaken to explore the relationship between NLR at diagnosis with inflammatory response and disease activity among MPO-AAV patients in a single Chinese center. Furthermore, we evaluated whether NLR could predict the renal prognosis and patient outcome. 188 patients with MPO-AAV were included in this study. Baseline NLR was positively correlated with CRP (r = 0.404, P < 0.001) and negatively with serum levels of C3 (r = - 0.163, P = 0.035), but it had no obvious correlation with Birmingham Vasculitis Activity Score (BVAS). Patients with MPO-AAV having NLR ≥ 9.53 exhibited higher risk for all-cause mortality than those having NLR < 9.53 (P < 0.0001). However, no significant difference was found in the kidney survival between patients having NLR ≥ 9.53 and those NLR < 9.53 at diagnosis. In multivariate analysis, NLR was positively associated with all-cause mortality (P = 0.037, HR = 1.98, 95% CI 1.04-3.78). There was no association between NLR with ESRD observed using univariate analysis or multivariate analysis. This large retrospective study of MPO-AAV patients in a single Chinese center demonstrates that NLR positively correlates with CRP and negatively correlates with serum levels of C3 in Chinese patients with MPO-AAV. Importantly, higher NLR predicts increased mortality and is, therefore, a useful independent prognostic in MPO-AAV.

Complement profile in microscopic polyangiitis and granulomatosis with polyangiitis: analysis using sera from a nationwide prospective cohort study

Objective The complement cascade, especially the alternative pathway of complement, has been shown in basic research to be associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). We aimed to elucidate relationships between serum complement components and clinical characteristics in AAV. Method In a nationwide prospective cohort study (RemIT-JAV-RPGN), we measured the serum levels of C1q, C2, C3, C3b/iC3b, C4, C4b, C5, C5a, C9, factor B, factor D, factor H, factor I, mannose-binding lectin, and properdin in 52 patients with microscopic polyangiitis (MPA) and 39 patients with granulomatosis with polyangiitis (GPA). Results The properdin level of MPA and GPA was significantly lower than that of healthy donors. The properdin level was negatively correlated with the Birmingham Vasculitis Activity Score (BVAS) (ρ = −0.2148, p = 0.0409). The factor D level at 6 months was significantly positively correlated with the Vasculitis Damage Index (VDI) at 6, 12, and 24 months (ρ = 0.4207, 0.4132, and 0.3115, respectively). Patients with a higher ratio of C5a to C5 had higher neutrophil percentage and serum immunoglobulin G levels, and significantly lower creatinine levels. Cluster analysis divided the MPA and GPA patients into three subgroups. A principal component (PC) analysis aggregated 15 types of complements into alternative pathway-related PC 1 and complement classical pathway and common pathway-related PC 2. Conclusions The serum levels of properdin and factor D were correlated with the BVAS and the VDI in MPA and GPA, respectively. Our analyses suggested the pathological heterogeneity of MPA and GPA from the aspect of complement components.

Total Serum Calcium Level Is Negatively Correlated With Systemic Lupus Erythematosus Activity.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease and hypocomplementemia signifies disease activity. Several studies have shown that calcium may help maintain optimum function of immune system and metabolism in SLE. The aim of our study was to analyze the relationship between total serum calcium level and SLE activity. A total of 66 patients with SLE and 214 healthy controls were included in this study. Our results showed lower serum levels of calcium (P < .001), complement C3 (P < .001), complement C4 (P < .001), and albumin (P < .001) in patients with SLE. A negative correlation was found between serum calcium level and systemic lupus erythematosus disease activity index (SLEDAI) rating (r = −0.394, P = .001). Additionally, serum level of calcium was positively correlated with serum complement C3 level (r = 0.366, P = .003) in patients with SLE, while no such correlation was found between serum calcium level and complement C4 (r = −0.190, P = .126). Likewise, patients with SLE with normal serum calcium level showed higher complement C3 level (P < .01) than that of patients with low serum calcium level. Overall, the results displayed that patients with SLE have lower serum calcium level compared to healthy controls, and the serum calcium level is positively correlated with SLEDAI rating and serum complement C3 level in patients with SLE. In conclusion, the total serum calcium level is negatively correlated with SLE disease activity.

Dietary montmorillonite as growth promoter and immunomodulator in rainbow trout (Oncorhynchus mykiss)

A 98-day research was conducted to assess the influence of dietary montmorillonite (MMT) on growth and immune competence in rainbow trout (Oncorhynchus mykiss). Fish fry (mean weight: 500 ± 35 mg) were fed with four experimental diets contained MMT at levels of 0 (as control group), 1, 2 and 4%. At the end of the experiment, the survival rate and growth parameters and serum total protein and albumin levels in MMT experimental groups was higher than the control group. Regarding humoral immune responses, fish being fed with 2 and 4% MMT diets showed the greatest and the lowest serum C3, C4 and lysozyme levels, respectively. Serum alternative complement pathway activity (ACH50) in groups fed with 1 and 2% MMT supplemented diets was higher than the other groups. At the end of the challenge test with VHSV, the mortality rate reached to 28 and 20% in the control group and 4% MMT supplemented groups, respectively, whereas fish fed with 1 and 2% MMT supplemented diets showed the lowest mortality rate (2.5%) Fish fed with 1 and 2% MMT supplemented groups showed higher head kidney IL-1β and TNF-α genes expression compared to the other treatment groups. In addition, fish fed with 2% MMT-supplemented diet had the highest head kidney Lyz-2 gene expression. In summary, the results of this research suggests that supplementing diet with MMT at 1 or 2% improve growth and non-specific humoral immune responses and induce head kidney immune relate genes expression and disease resistance against VHS in O. mykiss.

SUN-460 RECURRENT ACUTE KIDNEY INJURY AND GROSS HEMATURIA IN A CHILD WITH DENSE DEPOSIT DISEASE (DDD) AND PRESERVED KIDNEY FUNCTIONS ON LONG TERM FOLLOW-UP

Glomerulonephritis (GN) in children are in most cases post infectious GN (PIGN), characterized by complement activation with reduction of serum C3 levels that usually normalizes within four to eight weeks. When serum C3 levels are persistently low, other GN needs to be ruled out such as membranoproliferative GN (MPGN). Cases with MPGN and reduction of C3 levels in the absence of Ig on biopsy are now classified as “C3 GN” a member of “C3 glomerulopathies” (C3Gs); this also includes dense deposit disease (DDD). DDD is a rare disorder that most commonly affects children. The rarity of DDD, which afflicts 2 - 3 only two to three individuals per million populations, has impeded studies into its clinical course and optimal treatment. Its natural history is variable, but approximately 50% of patients progress to ESRD within 8 to 10 year. Case Report A previously healthy 11-year-old child was admitted at age of 8 years with history of fever, vomiting and red color urine since 4 days before admission. He had history of Upper Respiratory Tract Infection (URTI) 2 weeks before presentation without any history of skin rash or bleeding tendency or trauma or any drug ingestion. Family history was negative for any renal disease or bleeding tendency. At the presentation, he had oliguria, mild generalized body edema with acceptable blood pressure readings. Urinalysis showed proteinuria (2+) and dysmorphic erythrocytes in the urinary sediment. Laboratory tests showed picture of acute kidney injury(AKI) serum creatinine was 463 umol/l, hypocomplementemia C3 (0.51 mg/dL; normal rang value 0.9-1.8 g/l), high C-reactive protein (113 mg/l ; normal range <3 mg/l), High microalbuminuria (1088.4 mg/l ;, normal range 4-25 mg/l) & Microalbumine/creatinine ratio of 194 mg/mmol( N.R:0-2.5 mg/mmol) with low serum albumin of 29 g/l. The other immunological workup was normal. The initial diagnosis was acute post-streptococcal GN. Three days later, his urine output was improving with trending down of renal profile on conservative management till normalization on follow up. However, due to persistent hypocomplementemia C3 (0.6 mg/dl) after twelve weeks of onset. His Complement analysis revealed alternate pathway activation, persistent low levels of C3, low CH50 but normal levels of C5b-C9 and negative C3 nephritic factor (C3Nef) auto-antibodies. To exclude secondary G.N, a renal biopsy was performed and the result was consistent with C3glomuralopathy-DDD. Subsequently, genetic evaluation was performed for complement genes which were negative. On follow up, he was doing well with persistent microscopic hematuria and low C3. Then, he developed another episode of AKI with gross hematuria which was 2 years from the first presentation and resolved spontaneously. We repeated renal biopsy to determine disease activity and exclude other causes of GN. Again, the biopsy result was consistent with DDD. During his long term follow-up (over 36 months), he was doing well with persistent low C3, microscopic hematuria and non-significant proteinuria (24hr urine for protein was 0.1g/l) with preserved renal functions (creatinine 59 0-60 umol/l).View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) We report a unique case of DDD in a child with preserved renal functions who had persistent microscopic hematuria and with hypocomplementemia (C3) on long term follow-up.

Association of Apolipoprotein C3 Levels and Hepatic Steatosis: A Follow-Up Study.

Apolipoprotein C3 is a lipid-binding protein with a pivotal role in triglyceride metabolism and inflammation. This 11-year follow-up study aimed to evaluate apolipoprotein C3 levels and other parameters as markers of hepatic steatosis, in a random, population-based cohort in southern Germany. In 2013, we selected and re-examined 406 study participants (193 women, 213 men; average age 58.1±11.3 years) from the original "Echinococcus multilocularis and other internal diseases in Leutkirch I" (EMIL I) cohort studied in 2002. All participants received upper abdominal sonography to grade potential hepatic steatosis, and blood tests to determine apolipoprotein C3 levels and other laboratory parameters. Body mass index, waist-to-hip ratio, and anthropometric measures were documented. The follow-up study conducted in 2013 included a partial correlation analysis. We found an association between hepatic steatosis and elevated apolipoprotein C3 levels (p<0.0001). Study participants with a novel diagnosis of hepatic steatosis had the highest apolipoprotein C3 serum levels (p=0.0002). Hepatic steatosis was associated with low levels of high density lipoprotein cholesterol (p=0.0374), high levels of total cholesterol (p=0.0117), increased homeostasis model assessment of insulin resistance (p=0.0002), elevated alanine transaminase (p<0.0001), elevated aspartate transaminase (p=0.0003), and elevated C-reactive protein (p=0.0446). Apolipoprotein C3 serum levels were associated with the presence, disease grade, and new development of hepatic steatosis likewise to biomarkers of the metabolic syndrome.

Atypical presentation of acute post-infectious glomerulonephritis in patients with sickle cell disease: report of two cases

BackgroundSickle cell disease (SCD) is a highly prevalent genetic disease worldwide. In the natural evolution of SCD, glomerular lesions can develop, presenting histopathological patterns of segmental or focal membranoproliferative glomerulosclerosis, with or without thrombotic microangiopathy. We report two cases of acute post-infectious glomerulonephritis (APIGN), with atypical presentations, in patients with SCD.Case presentationCase 1: An 18-year-old female with SCD presented with a 21-day history of progressive oedema, accompanied by dyspnoea, productive cough, fever, and chest pain. Blood tests showed the following: haemoglobin 6.1 g/dl; leucocytes 18,820 cells/mm3; and creatinine 0.49 mg/dl. A urine sample evidenced leucocyturia and haematuria. The 24-h proteinuria was 8.99 g, serum albumin level was 1.2 g/dl, low serum C3 levels and high levels of anti-streptolysin O. Renal biopsy was consistent with APIGN. The patient was treated with diuretic and anti-proteinuric agents, subsequently evolving to reversal of the renal alterations. Case 2: A 12-year-old male with SCD presented with a 20-day history of a non-productive cough and progressive oedema, together with hypertension. The serum creatinine concentration was 0.48 mg/dl. A urine sample evidenced leukocyturia and haematuria. The 24-h proteinuria was 12.5 g, and the serum albumin level was 2.6 g/dl. The levels of C3 and C4 were normal. Renal biopsy revealed APIGN. The patient was treated with diuretic and anti-proteinuric agents, subsequently evolving reversal of the renal alterations.ConclusionsThe presentation of the two cases reported here are not typical of SCD-related kidney injury. Analysis of the renal biopsy specimens elucidated the diagnosis, affecting the prognosis, because that of APIGN is highly favourable, unlike that of nephrotic syndrome associated with SCD glomerulopathy.

Effects of Colesevelam on Bowel Symptoms, Biomarkers, and Colonic Mucosal Gene Expression in Patients With Bile Acid Diarrhea in a Randomized Trial

Approximately one-third of patients with IBS-diarrhea (IBS-D) have increased bile acid (BA) synthesis or excretion. An open-label study showed benefits of colesevelam on bowel functions, consistent with luminal BA sequestration by colesevelam. We compared the effects of colesevelam vs placebo on symptoms and gene expression patterns in the sigmoid colon mucosa in patients with BA diarrhea associated with IBS-D. We performed a double-blind, parallel-group study of 30 adults with IBS-D and evidence of increased BA synthesis or fecal excretion, from December 2017 through December 2018 at a single center. Patients were randomly assigned (1:1) to groups given colesevelam (3 tablets, 625 mg each) or matching placebo, orally twice daily for 4 weeks. Stool diaries documented bowel functions for 8 days before and 28 days during colesevelam or placebo. Stool and fasting serum samples were collected for analyses of fecal BAs and serum levels of C4 and FGF19. We measured colonic transit by scintigraphy, mucosal permeability by invivo excretion of saccharide probes, and mRNA levels in rectosigmoid biopsies. All measurements were made at baseline and on the last days of treatment. The primary endpoints were change in total fecal BA concentration and stool consistency. Compared with placebo, colesevelam was associated with significant changes in sequestered fecal total BA excretion (P < .001) and serum levels of C4 and FGF19 (both P < .001), and with a mean increase in fecal level of deoxycholic acid (10%; P = .07) compared to placebo. Colesevelam decreased colon mucosal expression of NR1H4 and P2RY4 and increased expression of GPBAR1, compared with baseline. Stool frequency and consistency, colonic transit, and permeability did not differ significantly between groups. Colesevelam was well tolerated. In a randomized trial, we found that colesevelam increases delivery of total and secondary BAs to stool, hepatic BA synthesis, and colonic mucosal expression of genes that regulate BA, farnesoid X, and GPBAR1 receptors. Larger studies are needed to determine the effects on clinical responses. ClinicalTrials.gov no: NCT03270085.

Risk factors for treatment resistance and relapse of Chinese patients with MPO-ANCA-associated vasculitis.

Identification of risk factors for treatment resistance and relapse would be crucial to personalization therapy in patients with myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (MPO-AAV). Current evidence with regard to the risk factors for treatment resistance and relapse remains limited and inconclusive. We aimed to assess the predictors for treatment resistance and relapse in a single-center cohort of Chinese patients with MPO-AAV in this study. In total, 184 patients with MPO-AAV were included. Treatment resistance occurred in 64 (34.9%) of 184 patients and was positively associated with lung involvement (odds ratio [OR] 3.581, 95% CI 1.137-11.278, p = 0.029) and the initial serum creatinine level (OR 1.004, 95% CI 1.001-1.007, p = 0.010) and was negatively associated with platelet (OR 0.992, 95% CI 0.987-0.998, p = 0.007) and serum C3 levels (OR 0.998, 95% CI 0.996-0.999, p = 0.004). Relapse occurred in 29 (24.17%) of 120 patients in whom remission was achieved and was independently associated with lung involvement (hazard ratio [HR] 4.595, 95% CI 1.272-16.599, p = 0.020) and cardiovascular involvement (HR 3.689, 95% CI 1.237-11, p = 0.019,). The serum globulin was demonstrated to be negatively associated with relapse independently (HR 0.876; 95% CI 0.806-0.953; p = 0.002). This retrospective study of MPO-AAV patients in a single Chinese center suggests that treatment resistance was positively associated with lung involvement and the initial serum creatinine level and was negatively associated with platelet and serum C3 levels. Lung involvement and cardiovascular involvement were associated with an increased risk of relapse, while the higher serum globulin was demonstrated to be in association with a decreased risk of relapse.