Supplementary Material for: Integrative analysis by mendelian randomization and large-scale single-cell transcriptomics reveals causal links between B cell subtypes and diabetic kidney disease

Introduction: The growing incidence of diabetic kidney disease (DKD) and the difficulties in its management underscore the need for a more comprehensive understanding of its pathogenesis. Recent studies have emphasized the significant impact of circulating immunity on the development of diabetic microvascular complications including retinopathy and neuropathy, research on circulating immunity in DKD remains limited. Methods: This study utilized mendelian randomization (MR) analysis to explore the potential independent causal relationships between circulating immune cells and DKD pathogenesis. Additionally, a combination of single-cell disease relevance score (scDRS) and immune cell infiltration analysis was employed to map the circulating immunity landscape in DKD patients. Results: Ten immune traits, including 5 of B cells, 2 of T cells, 2 of granulocytes and one of monocytes, were defined to be associated with the pathogenesis of DKD. Notably, IgD-CD27- B cell Absolute Count [IVW: OR, 1.102 (1.023 to 1.189), p=0.011] and IgD-CD24- B cell Absolute Count [IVW: OR, 1.106 (1.030 to 1.188), p=0.005] were associated with promoting DKD pathogenesis, while CD24+CD27+ B cell %B cell [IVW: OR, 0.943 (0.898 to 0.989), p=0.016] demonstrated a protective effect against DKD onset. The presence of B cell activating factor receptor (BAFF-R) on CD20-CD38- B cell [IVW: OR, 0.946 (0.904 to 0.989), p=0.015] and BAFF-R on IgD-CD38+ B cell [IVW: OR, 0.902 (0.834 to 0.975), p=0.009] also indicated a potential role in preventing DKD. Single-cell disease relevance score (scDRS) analysis revealed that two main subsets of B cells, naïve B and memory B cells, had a higher proportion of DKD-related cells or a higher scDRS score of DKD phenotype, indicating their strong association with DKD. Furthermore, immune infiltrate deconvolution analysis showed a notable decrease in the circulating memory B cells and class-switched memory B cells in DKD patients compared to those of DM patients without DKD. Conclusion: Our study revealed the causal relations between circulating immunity and DKD susceptibility, particularly highlighted the potential roles of B cell subtypes in DKD development. Further studies addressing the related mechanisms would broaden the current understanding of DKD pathogenesis.

引言：糖尿病肾病（diabetic kidney disease, DKD）的发病率日益攀升，加之其临床诊疗面临诸多困境，凸显了对其发病机制开展更全面研究的必要性。已有研究强调，循环免疫对糖尿病微血管并发症（包括视网膜病变与神经病变）的发生发展具有重要影响，但针对DKD中循环免疫的相关研究仍较为匮乏。方法：本研究采用孟德尔随机化（mendelian randomization, MR）分析，探讨循环免疫细胞与DKD发病机制之间潜在的独立因果关联。此外，本研究联合运用单细胞疾病相关性评分（single-cell disease relevance score, scDRS）与免疫细胞浸润分析，构建了DKD患者的循环免疫图谱。结果：本研究鉴定出10种与DKD发病机制相关的免疫特征，其中B细胞相关5种、T细胞相关2种、粒细胞相关2种、单核细胞相关1种。值得注意的是，IgD-CD27- B细胞绝对计数[逆方差加权（inverse variance weighted, IVW）：比值比（odds ratio, OR）=1.102，95%置信区间：1.023~1.189，p=0.011]与IgD-CD24- B细胞绝对计数[IVW: OR=1.106，95%置信区间：1.030~1.188，p=0.005]与DKD的发病风险升高相关；而CD24+CD27+ B细胞占B细胞总数的比例[IVW: OR=0.943，95%置信区间：0.898~0.989，p=0.016]则对DKD的发生具有保护作用。CD20-CD38- B细胞表面的B细胞活化因子受体（B cell activating factor receptor, BAFF-R）表达[IVW: OR=0.946，95%置信区间：0.904~0.989，p=0.015]以及IgD-CD38+ B细胞表面的BAFF-R表达[IVW: OR=0.902，95%置信区间：0.834~0.975，p=0.009]，同样提示其在DKD的预防中具有潜在作用。单细胞疾病相关性评分（scDRS）分析显示，B细胞的两个主要亚群——初始B细胞与记忆B细胞——中，DKD相关细胞的占比更高，或其DKD表型的scDRS评分更高，表明这两类细胞与DKD存在强关联。此外，免疫浸润反卷积分析显示，与无DKD的糖尿病患者相比，DKD患者的循环记忆B细胞与类别转换记忆B细胞数量显著降低。结论：本研究揭示了循环免疫与DKD易感性之间的因果关联，尤其强调了B细胞亚群在DKD发生发展中的潜在作用。后续针对相关机制开展的研究，将进一步深化我们对DKD发病机制的认知。