Amado Nathalia, Mathews Jeremy, Henry Gervaise, Fusco Alexandria, Egeland Thomas, Malewska Alicia, Cantarel Brandi, Strand Douglas, Baker Linda
J Urol. 2021 Sep;206(Suppl 3):e796. doi: 10.1097/JU.0000000000002065.09. Epub 2021 Aug 4.
Prune Belly Syndrome (PBS) is characterized by bladder dysmyogenesis, yielding a dysfunctional compliant thick wall with excess collagen deposition. To dissect the cellular heterogeneity and gene expression networks altered in PBS, we report the cell type composition and transcriptional activity of PBS human bladder by using single cell RNA sequencing (scRNA-seq).
Using IRB-approved methods, bladder dome from 2 PBS and 6 non-PBS control (CO) males underwent fresh single-cell digestion. scRNA-seq was performed and 5277 and 31828 bladder cells from PBS and CO patients was detected, respectively. Cell type clusters were graphically displayed by Uniform Manifold Approximation and Projection (UMap) plot and differentially expressed genes (DEGs) were generated to assign each cluster identity. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed for PBS affected genes.
We identified 17 distinct bladder cell clusters, including 6 fibroblast (1, 2, 3, and 4, immunofibroblast, myofibroblast), 1 smooth muscle (SM), and 2 urothelial (umbrella and basal+intermediate) clusters (Fig 1A-B). Counts of individual cell types were expressed as relative proportions, identifying significant PBS fibroblast enrichment, (67% PBS vs 40% CO). Five of 6 PBS fibroblast sub-types are proportionately fewer in number than in CO. The exception is a dominant fibroblast sub-type we label as fibroblast 4, (61% of all PBS fibroblasts vs <10% CO fibroblast subtypes). SM and urothelial cell populations are dramatically reduced in PBS (SM: 5% PBS vs 11% CO and urothelial: <1% PBS vs 7% CO) (Fig 1C-E). PBS fibroblast DEGs, but not SM cells, are enriched in collagen genes. Fibroblast markers (DCN and PLA2G2A) and SM genes (DES, TPM2 and TAGLN) are reduced (by 4, 13, 2, 4, and 2x respectively) in PBS cells (Fig 1G). KEGG pathways analysis for fibroblasts and SM showed a highly similar enrichment for neurodegenerative disease pathways (Fig 1H-I).
Using scRNA-seq, we identified and characterized the disarrayed cell type populations in PBS bladders, generating their unbiased transcriptomic signatures which highlight commonality with neurodegenerative diseases. This PBS transcriptomic map is a step toward potential markers for diagnosis and therapeutic intervention.[Figure: see text]Source of Funding:NIH DK100483, DK127589 PI: Baker, L.
梅干腹综合征（PBS）的特征是膀胱肌发育异常，导致功能失调的顺应性厚壁，并伴有过量胶原沉积。为了剖析PBS中细胞异质性和基因表达网络的改变，我们通过单细胞RNA测序（scRNA-seq）报告了PBS患者膀胱的细胞类型组成和转录活性。
采用经机构审查委员会批准的方法，对2例PBS男性患者和6例非PBS对照（CO）男性患者的膀胱穹窿进行新鲜单细胞消化。进行scRNA-seq检测，分别从PBS和CO患者中检测到5277个和31828个膀胱细胞。通过均匀流形近似和投影（UMap）图以图形方式显示细胞类型簇，并生成差异表达基因（DEG）以确定每个簇的特征。对PBS相关基因进行京都基因与基因组百科全书（KEGG）途径分析。
我们鉴定出17个不同的膀胱细胞簇，包括6个成纤维细胞簇（1、2、3和4，免疫成纤维细胞、肌成纤维细胞）、1个平滑肌（SM）簇和2个尿路上皮簇（伞状和基底+中间）（图1A - B）。将各细胞类型的计数表示为相对比例，发现PBS患者的成纤维细胞显著富集（PBS为67%，CO为40%）。6种PBS成纤维细胞亚型中有5种在数量上比CO患者中的比例少。例外的是一种占主导地位的成纤维细胞亚型，我们将其标记为成纤维细胞4（占所有PBS成纤维细胞的61%，而CO成纤维细胞亚型中该比例<10%）。PBS患者的SM和尿路上皮细胞群体显著减少（SM：PBS为5%，CO为11%；尿路上皮：PBS<1%，CO为7%）（图1C - E）。PBS成纤维细胞的DEG在胶原基因中富集，但SM细胞中未富集。PBS细胞中的成纤维细胞标记物（DCN和PLA2G2A）和SM基因（DES、TPM2和TAGLN）减少（分别减少4、13、2、4和2倍）（图1G）。对成纤维细胞和SM的KEGG途径分析显示，神经退行性疾病途径的富集高度相似（图1H - I）。
通过scRNA-seq，我们鉴定并表征了PBS膀胱中紊乱的细胞类型群体，生成了它们无偏倚的转录组特征，突出了与神经退行性疾病的共性。这张PBS转录组图谱是朝着诊断和治疗干预的潜在标志物迈出的一步。[图：见正文]资金来源：美国国立卫生研究院DK100483、DK127589 项目负责人：贝克，L.
