Abstract：
OBJECTIVE: Diabetic kidney disease (DKD) is the leading cause of chronic and end-stage kidney disease in the USA and worldwide. Animal models have taught us much about DKD mechanisms, but translation of this knowledge into treatments for human disease has been slowed by the lag in our molecular understanding of human DKD.
METHODS: Using our Spatial TissuE Proteomics (STEP) pipeline (comprising curated human kidney tissues, multiplexed immunofluorescence and powerful analysis tools), we imaged and analysed the expression of 21 proteins in 23 tissue sections from individuals with diabetes and healthy kidneys (n=5), compared to those with DKDIIA, IIA-B and IIB (n=2 each) and DKDIII (n=1).
RESULTS: These analyses revealed the existence of 11 cellular clusters (kidney compartments/cell types): podocytes, glomerular endothelial cells, proximal tubules, distal nephron, peritubular capillaries, blood vessels (endothelial cells and vascular smooth muscle cells), macrophages, myeloid cells, other CD45+ inflammatory cells, basement membrane and the interstitium. DKD progression was associated with co-localised increases in inflammatory cells and collagen IV deposition, with concomitant loss of native proteins of each nephron segment. Cell-type frequency and neighbourhood analyses highlighted a significant increase in inflammatory cells and their adjacency to tubular and αSMA+ (α-smooth muscle actin-positive) cells in DKD. Finally, DKD progression showed marked regional variability within single tissue sections, as well as inter-individual variability within each DKD class.
CONCLUSIONS: Using the STEP pipeline, we found alterations in protein expression, cellular phenotypic composition and microenvironment structure with DKD progression, demonstrating the power of this pipeline to reveal the pathophysiology of human DKD.
METHODS: Using our Spatial TissuE Proteomics (STEP) pipeline (comprising curated human kidney tissues, multiplexed immunofluorescence and powerful analysis tools), we imaged and analysed the expression of 21 proteins in 23 tissue sections from individuals with diabetes and healthy kidneys (n=5), compared to those with DKDIIA, IIA-B and IIB (n=2 each) and DKDIII (n=1).
RESULTS: These analyses revealed the existence of 11 cellular clusters (kidney compartments/cell types): podocytes, glomerular endothelial cells, proximal tubules, distal nephron, peritubular capillaries, blood vessels (endothelial cells and vascular smooth muscle cells), macrophages, myeloid cells, other CD45+ inflammatory cells, basement membrane and the interstitium. DKD progression was associated with co-localised increases in inflammatory cells and collagen IV deposition, with concomitant loss of native proteins of each nephron segment. Cell-type frequency and neighbourhood analyses highlighted a significant increase in inflammatory cells and their adjacency to tubular and αSMA+ (α-smooth muscle actin-positive) cells in DKD. Finally, DKD progression showed marked regional variability within single tissue sections, as well as inter-individual variability within each DKD class.
CONCLUSIONS: Using the STEP pipeline, we found alterations in protein expression, cellular phenotypic composition and microenvironment structure with DKD progression, demonstrating the power of this pipeline to reveal the pathophysiology of human DKD.
摘要：
目的：糖尿病肾病（DKD）是美国乃至全世界慢性和终末期肾病的主要病因。动物模型教会了我们很多关于DKD机制的知识，但是，由于我们对人类DKD的分子理解滞后，因此将这些知识转化为人类疾病的治疗方法。
方法：使用我们的空间组织蛋白质组学（STEP）管道（包括经过筛选的人类肾脏组织，多重免疫荧光和强大的分析工具)，我们成像并分析了来自糖尿病和健康肾脏的23个组织切片中21种蛋白质的表达（n=5），与DKDIIA相比，IIA-B和IIB(各n=2)和DKDIII(n=1)。
结果：这些分析显示存在11个细胞簇（肾脏区室/细胞类型）：足细胞，肾小球内皮细胞，近端小管，远端肾单位,肾小管周围毛细血管,血管（内皮细胞和血管平滑肌细胞），巨噬细胞，骨髓细胞，其他CD45+炎症细胞，基底膜和间质。DKD进展与炎症细胞和胶原IV沉积的共定位增加相关，伴随着每个肾单位段的天然蛋白质的损失。细胞类型频率和邻域分析强调了DKD中炎性细胞的显着增加及其与肾小管和αSMA（α-平滑肌肌动蛋白阳性）细胞的邻接。最后,DKD进展在单个组织切片中显示出明显的区域变异性，以及每个DKD类别内的个体差异。
结论：使用STEP管道，我们发现了蛋白质表达的改变，细胞表型组成和微环境结构与DKD进展，展示了这一管道揭示人类DKD病理生理学的力量。
方法：使用我们的空间组织蛋白质组学（STEP）管道（包括经过筛选的人类肾脏组织，多重免疫荧光和强大的分析工具)，我们成像并分析了来自糖尿病和健康肾脏的23个组织切片中21种蛋白质的表达（n=5），与DKDIIA相比，IIA-B和IIB(各n=2)和DKDIII(n=1)。
结果：这些分析显示存在11个细胞簇（肾脏区室/细胞类型）：足细胞，肾小球内皮细胞，近端小管，远端肾单位,肾小管周围毛细血管,血管（内皮细胞和血管平滑肌细胞），巨噬细胞，骨髓细胞，其他CD45+炎症细胞，基底膜和间质。DKD进展与炎症细胞和胶原IV沉积的共定位增加相关，伴随着每个肾单位段的天然蛋白质的损失。细胞类型频率和邻域分析强调了DKD中炎性细胞的显着增加及其与肾小管和αSMA（α-平滑肌肌动蛋白阳性）细胞的邻接。最后,DKD进展在单个组织切片中显示出明显的区域变异性，以及每个DKD类别内的个体差异。
结论：使用STEP管道，我们发现了蛋白质表达的改变，细胞表型组成和微环境结构与DKD进展，展示了这一管道揭示人类DKD病理生理学的力量。
