IgA肾病与膜性肾病患者肠道微生物菌群结构分析

Intestinal microflora changes in patients with immunoglobulin A nephropathy and membranous nephropathy

摘要:

【背景】越来越多的证据表明肠道失衡与免疫介导的疾病相关，但肠道菌群和免疫介导的肾脏疾病之间的关系仍不清楚。【目的】通过Illumina高通量测序方法对IgA肾病(immunoglobulin A nephropathy, IgAN)、膜性肾病(membranous nephropathy, MN)患者和健康人群的肠道菌群进行比较。【方法】回顾性选择2020年9月-2021年12月期间，在甘肃省人民医院肾内科行肾穿刺活检并诊断为IgAN及MN患者的新鲜粪便标本，分别编号为IgAN组和MN组，收集体检中心健康人群粪便标本作为健康对照组，每组样本为10例。采用高通量测序技术对粪便样本中所有细菌的16S rrna基因V3-V4区进行DNA测序，然后进行分类操作单元(operational taxonomic units, OTU)、物种分类、α多样性、β多样性等分析，比较3组之间的肠道菌群差异。【结果】与健康对照组相比，门水平上IgAN组的变形菌门(Proteobacteria)和放线菌门(Actinobacteria)比例明显增高，分别为18% vs. 4%和18.3% vs. 5%；属水平上IgAN组大肠杆菌-志贺氏菌和双歧杆菌属丰度显著增加(14.1% vs. 2.1%和17.5% vs. 4.7%)，而粪杆菌属(11% vs. 20.5%)、拟杆菌属(8.0% vs. 21%)、巨单胞菌属(1.8% vs. 8.0%)丰度降低。与健康对照组相比，门水平上MN组的变形菌门丰度增加(20% vs. 4%)；属水平上MN组的埃舍里奇-志贺氏菌(13.8% vs. 2.1%)丰度增加，而双歧杆菌属(3.2% vs. 4.7%)、粪杆菌属(18% vs. 20.5%)、拟杆菌属(14.3% vs. 21%)、巨单胞菌属(1% vs. 8%)丰度降低。α多样性分析结果显示，肾病组丰富度指数低于健康对照组，多样性指数高于健康对照组。通过相似性分析(analysis of similarities, ANOISM)发现IgAN组与健康对照组之间肠道菌群构成的差异具有统计学意义(R=0.19,P=0.013)，MN患者和健康对照组肠道菌群构成存在差异，但结果不具有统计学意义(R=0.08, P=0.08)。线性判别分析(linear discriminant analysis, LDA)差异贡献分析发现，3组之间共有14个物种存在显著差异。【结论】IgAN和MN患者的肠道微生物特征与健康对照组不同。

关键词:IgA肾病;膜性肾病;肠道菌群;16S rRNA基因

Abstract:

[Background] There is increasing evidence that intestinal imbalance is associated with immune-mediated disease. However, the mechanistic link between intestinal flora and immune-mediated kidney disease remains unclear. [Objective] To compare the intestinal flora by using high throughput 16S ribosomal RNA (rRNA) gene sequencing between patients with immunoglobulin A nephropathy (IgAN) and membranous nephropathy (MN) and healthy people. [Methods] Fresh fecal samples from patients with IgAN and MN who underwent renal biopsy in the Department of Nephrology of Gansu Provincial Hospital from September 2020 to December 2021 were retrospectively selected and divided into an IgAN group and a MN group, and the fecal samples from healthy people in the physical examination center were collected as a healthy control group, with 10 cases in each group. The 16S rRNA gene V3−V4 region of all bacteria in fecal samples was sequenced by high-throughput sequencing technology, and then biodiversity analysis was performed, including operational taxonomic units (OTU) analysis, species classification analysis, alpha diversity analysis, beta diversity analysis, etc., to compare the intestinal flora differences among the three groups. [Results] As compared with the healthy control group, Proteobacteria and Actinobacteria at phylum level in the IgAN group were significantly increased (18% vs. 4% and 18.3% vs. 5%, respectively). The abundance of Escherichia-Shigella and Bifidobacterium at genus level was significantly higher in the IgAN group (14.1% vs. 2.1% and 17.5% vs. 4.7%, respectively), while the abundance of Faecalibacterium (11% vs. 20.5%), Bacteroides (8.0% vs. 21%), and Megomonas (1.8% vs. 8.0%) was significantly lower. The abundance of Proteobacteria at phylum level increased in the MN group as compared with the healthy control group (20% vs. 4%). At genus level, the abundance of Escherich-Shigella increased in the MN group (13.8% vs 2.1%), the abundance of Bifidobacteria (3.2% vs. 4.7%), Faecalibacterium (18% vs. 20.5%), Bacteroides (14.3% vs. 21%), and Megomonas (1% vs. 8%) decreased. Alpha diversity analysis showed that the richness index of the IgAN and MN groups was lower than that of the healthy control group, and the diversity index was higher than that of the healthy control group. Principal coordinates analysis (PCoA) showed statistically significant differences in the composition of intestinal flora between the IgAN group and the healthy control group (ANOISM, R=0.19, P=0.013). There were differences in the composition of intestinal flora between the MN group and the healthy control group, but the results were not statistically significant (ANOISM, R=0.08, P=0.08). Linear discriminant analysis (LDA) for differential contribution revealed that 14 species had significant differences among the three groups. [Conclusion] The intestinal microbiome characteristics of patients with IgAN and MN are different from those of healthy people.

Key words:immunoglobulin A nephropathy;membranous nephropathy;intestinal flora;16S rrna gene