Sox9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys [RNA-Seq 2]. Sox9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys [RNA-Seq 2]

The steps governing healing with or without fibrosis within the same microenvironment are unclear. After acute kidney injury (AKI), the injured proximal tubular epithelial cells activate Sox9 for self-restoration. Using head-to-head comparison of injury- induced Sox9-lineages via spatiotemporal mapping, single-cell sequencing, and single-nuclei chromatin accessibility profiling, we identified a dynamic SOX9 switch. Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). In contrast, lineages that maintained Sox9 activity in attempt to regenerate, demarcated by SOX9-induced Cadherin 6 (SOX9on-on CDH6pos ) cell state, generated single-cell Wnt activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease. Transplanted human kidneys displayed similar SOX9/CDH6/WNT2B responses post-injury. Thus, we uncovered a mechanism linking fibrosis to sustained efforts to regenerate the injured tissue. Overall design: RNAseq datasets from FACS-sorted (enriched) control uninjured Slc34a1 expressing proximal tubular epithelial cells (PTECs), cells that activate Sox9 at 48h after kidney ischemia reperfusion injury (IRI), and cells the express Sox9 at d14 after kidney IRI. Each group containing 3 biological replicates.

同一微环境下伴或不伴纤维化的肾脏修复调控机制目前尚不明确。急性肾损伤（acute kidney injury, AKI）发生后，受损的近端肾小管上皮细胞会激活Sox9以实现自我修复。我们通过时空图谱绘制、单细胞测序及单核染色质开放性谱分析，对头对头比较损伤诱导的Sox9谱系，鉴定出一个动态的SOX9转换开关。完成上皮再生的谱系会沉默SOX9表达并实现无纤维化修复（记为SOX9on-off）；与之相反，那些为完成再生而持续维持Sox9活性的谱系，以SOX9诱导的钙粘蛋白6（cadherin 6, CDH6）阳性细胞状态（SOX9on-on CDH6pos）为特征，会产生单细胞水平的Wnt活性，进而在邻近成纤维细胞中引发纤维增生反应，推动急性肾损伤进展为慢性肾病。移植的人类肾脏在损伤后也表现出类似的SOX9/CDH6/WNT2B应答模式。据此，我们揭示了一条将纤维化与受损组织持续再生尝试相关联的分子机制。
整体实验设计：本研究的RNA测序数据集来源于经荧光活化细胞分选（fluorescence-activated cell sorting, FACS）富集的三类样本：未损伤的表达Slc34a1的近端肾小管上皮细胞（proximal tubular epithelial cells, PTECs）、肾脏缺血再灌注损伤（ischemia reperfusion injury, IRI）后48小时激活Sox9的细胞，以及肾脏缺血再灌注损伤后14天表达Sox9的细胞。每组包含3个生物学重复。