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Comprehensive single-cell transcriptional profiling defines shared and unique epithelial injury responses during kidney fibrosis
全面的单细胞转录谱分析确定了肾脏纤维化过程中共同和独特的上皮损伤反应

Graphical abstract  图形摘要

Highlights  亮点

  • sci-RNA-seq3 transcriptionally profiles 309,666 cells from 24 kidneys without batch effects
    sci-RNA-seq3 对来自 24 个肾脏的 309 666 个细胞进行转录分析,无批次效应
  • Two injured proximal tubule cell states with distinct metabolic profiles revealed
    两种受伤的近端肾小管细胞状态具有不同的代谢特征
  • Transiently activated lipid metabolism and PLIN2+ lipid droplets appear in early IRI
    瞬时激活的脂质代谢和 PLIN2+ 脂滴出现在早期 IRI 中
  • Nephron epithelia possess both shared and segmentspecific injury and repair responses
    肾小球上皮既有共同的损伤和修复反应,也有特定节段的损伤和修复反应

Authors  作者

Haikuo Li, Eryn E. Dixon, Haojia Wu, Benjamin D. Humphreys
Haikuo Li、Eryn E. Dixon、Haojia Wu、Benjamin D. Humphreys

Correspondence  通信

In brief  简而言之

Li et al. profile the full time courses of mouse kidney fibrogenesis using singlecell combinatorial indexing RNA sequencing. They describe diverse injury states of proximal tubular cells, including one cell state with enhanced lipid metabolism at an early phase of ischemia-induced injury. This single-cell atlas defines kidney epithelial injury responses in fibrosis.
Li等人利用单细胞组合索引RNA测序技术描绘了小鼠肾脏纤维化的全过程。他们描述了近端肾小管细胞的多种损伤状态,包括缺血诱导损伤早期脂质代谢增强的一种细胞状态。该单细胞图谱定义了纤维化过程中肾脏上皮细胞的损伤反应。

Comprehensive single-cell transcriptional profiling defines shared and unique epithelial injury responses during kidney fibrosis
全面的单细胞转录谱分析确定了肾脏纤维化过程中共同和独特的上皮损伤反应

Haikuo Li, 1 1 ^(1){ }^{1} Eryn E. Dixon, 1 1 ^(1){ }^{1} Haojia Wu, 1 1 ^(1){ }^{1} and Benjamin D. Humphreys 1 , 2 , 3 , 1 , 2 , 3 , ^(1,2,3,^(**)){ }^{1,2,3,{ }^{*}}
Haikuo Li、 1 1 ^(1){ }^{1} Eryn E. Dixon、 1 1 ^(1){ }^{1} Haojia Wu、 1 1 ^(1){ }^{1} and Benjamin D. Humphreys 1 , 2 , 3 , 1 , 2 , 3 , ^(1,2,3,^(**)){ }^{1,2,3,{ }^{*}}
1 1 ^(1){ }^{1} Division of Nephrology, Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA
1 1 ^(1){ }^{1} 美国密苏里州圣路易斯市华盛顿大学医学系肾脏病科
2 2 ^(2){ }^{2} Department of Developmental Biology, Washington University in St. Louis, St. Louis, MO, USA
2 2 ^(2){ }^{2} 美国密苏里州圣路易斯市华盛顿大学发育生物学系
3 3 ^(3){ }^{3} Lead contact   3 3 ^(3){ }^{3} 牵头联络人*Correspondence: humphreysbd@wustl.edu
*通信:humphreysbd@wustl.edu
https://doi.org/10.1016/j.cmet.2022.09.026

Abstract  摘要

SUMMARY The underlying cellular events driving kidney fibrogenesis and metabolic dysfunction are incompletely understood. Here, we employed single-cell combinatorial indexing RNA sequencing to analyze 24 mouse kidneys from two fibrosis models. We profiled 309,666 cells in one experiment, representing 50 cell types/states encompassing epithelial, endothelial, immune, and stromal populations. Single-cell analysis identified diverse injury states of the proximal tubule, including two distinct early-phase populations with dysregulated lipid and amino acid metabolism, respectively. Lipid metabolism was defective in the chronic phase but was transiently activated in the very early stages of ischemia-induced injury, where we discovered increased lipid deposition and increased fatty acid β β beta\beta-oxidation. Perilipin 2 was identified as a surface marker of intracellular lipid droplets, and its knockdown in vitro disrupted cell energy state maintenance during lipid accumulation. Surveying epithelial cells across nephron segments identified shared and unique injury responses. Stromal cells exhibited high heterogeneity and contributed to fibrogenesis by epithelial-stromal crosstalk.
摘要 驱动肾脏纤维化和代谢功能障碍的潜在细胞事件尚不完全清楚。在这里,我们采用单细胞组合索引 RNA 测序技术分析了来自两种纤维化模型的 24 个小鼠肾脏。我们在一次实验中分析了 309,666 个细胞,代表了 50 种细胞类型/状态,包括上皮细胞、内皮细胞、免疫细胞和基质细胞。单细胞分析确定了近端肾小管的多种损伤状态,包括两种不同的早期阶段细胞群,它们分别存在脂质和氨基酸代谢失调。脂质代谢在慢性阶段存在缺陷,但在缺血诱导损伤的早期阶段被短暂激活,我们发现脂质沉积增加,脂肪酸 β β beta\beta 氧化增加。Perilipin 2被鉴定为细胞内脂滴的表面标志物,体外敲除它将破坏脂质积累过程中细胞能量状态的维持。对各肾节段上皮细胞的调查发现了共同和独特的损伤反应。基质细胞表现出高度的异质性,并通过上皮细胞与基质细胞之间的串联促进了纤维形成。

INTRODUCTION  引言

Chronic kidney disease (CKD) affects 10 % 10 % ∼10%\sim 10 \% of the population worldwide and ultimately can lead to kidney failure (Hill et al., 2016; Kalantar-Zadeh et al., 2021). With no cure and relatively few therapies that slow progression, people with CKD suffer considerable morbidity and mortality. Across all CKDs, regardless of the underlying cause, dysregulated epithelial metabolism is increasingly recognized as an important pathological feature that drives interstitial fibrosis (Kang et al., 2014; Slee, 2012; Tran et al., 2016; Zhu et al., 2021). Understanding the earliest cellular events driving kidney fibrogenesis will improve our knowledge of CKD pathophysiology and may identify new, effective therapeutic targets.
慢性肾脏病(CKD)影响着全球 10 % 10 % ∼10%\sim 10 \% 的人口,并最终导致肾衰竭(Hill 等人,2016 年;Kalantar-Zadeh 等人,2021 年)。由于无法治愈,而减缓病情恶化的疗法又相对较少,因此慢性肾功能衰竭患者的发病率和死亡率都相当高。在所有慢性肾脏病中,无论其根本原因如何,上皮代谢失调越来越被认为是导致间质纤维化的一个重要病理特征(Kang 等人,2014 年;Slee,2012 年;Tran 等人,2016 年;Zhu 等人,2021 年)。了解驱动肾脏纤维化的最早细胞事件将提高我们对慢性肾脏病病理生理学的认识,并可能确定新的、有效的治疗靶点。
Single-cell RNA sequencing (scRNA-seq) allows for the unbiased characterization of cell transcriptomics and has been widely applied to decipher cell fate dynamics and metabolic heterogeneity (Evers et al., 2019; Kuppe et al., 2021; Park et al., 2018; Stuart and Satija, 2019). The most commonly used platform for scRNA-seq is based upon droplet microfluidics, but due to several technical limitations including low throughput, difficulty in analyzing multiple samples or time points, batch effects, and incompatibility with fixed samples, many studies analyze a limited number of samples, providing only a “snapshot” of a specific biological condition (Li and Humphreys, 2021; Stoeckius et al., 2018; Weinreb et al., 2018). Previous work has character-
单细胞 RNA 测序(scRNA-seq)可对细胞转录组学进行无偏见表征,并已广泛应用于破译细胞命运动力学和代谢异质性(Evers 等,2019 年;Kuppe 等,2021 年;Park 等,2018 年;Stuart 和 Satija,2019 年)。最常用的 scRNA-seq 平台基于液滴微流控技术,但由于一些技术限制,包括通量低、难以分析多个样本或时间点、批次效应以及与固定样本不兼容等,许多研究分析的样本数量有限,只能提供特定生物条件的 "快照"(Li 和 Humphreys,2021;Stoeckius 等人,2018;Weinreb 等人,2018)。以前的工作已经

ized CKD and kidney fibrosis at single-cell resolution (Dhillon et al., 2021; Lu et al., 2021; Wu et al., 2019; Zhang et al., 2021), but these studies typically lack multiple time points especially in early stages. Even though sample-multiplexing approaches exist (e.g., CITE-seq; Stoeckius et al., 2017), this method does not easily scale up, which is detrimental for analysis of the kidney due to diversity of cell types and states that arise during injury and progression of fibrosis (Balzer et al., 2022; Gerhardt et al., 2021; Kirita et al., 2020; Wu et al., 2019). As an example, proximal tubule (PT) cells constitute 50 % 50 % ∼50%\sim 50 \% of the total kidney cell number, so rare cell types and states may be underrepresented in a lower complexity scRNA-seq dataset. While multiple scRNA-seq experiments and large-scale data integration could resolve these limitations, batch effect correction would be required (Tran et al., 2020).
等,2021;Wu 等,2019;Zhang 等,2021),但这些研究通常缺乏多个时间点,尤其是在早期阶段。尽管存在样本多路复用方法(如 CITE-seq;Stoeckius 等人,2017 年),但这种方法不容易扩展,这对分析肾脏不利,因为在损伤和纤维化进展过程中会出现多种细胞类型和状态(Balzer 等人,2022 年;Gerhardt 等人,2021 年;Kirita 等人,2020 年;Wu 等人,2019 年)。例如,近端肾小管(PT)细胞占肾细胞总数的 50 % 50 % ∼50%\sim 50 \% ,因此稀有细胞类型和状态在复杂度较低的scRNA-seq数据集中可能代表性不足。虽然多个 scRNA-seq 实验和大规模数据整合可以解决这些局限性,但需要进行批次效应校正(Tran 等人,2020 年)。
Here, we optimized single-cell combinatorial indexing RNAseq (sci-RNA-seq) (Cao et al., 2020, 2019) in order to decipher the molecular events driving kidney fibrogenesis. We leveraged the high-throughput, high sample-multiplexing capacity, and low costs of sci-RNA-seq to characterize two mouse models of kidney injury and fibrosis, unilateral ischemia-reperfusion injury (uni-IRI) and unilateral ureteral obstruction (UUO), at multiple time points. sci-RNA-seq is compatible with tissue fixation, which stabilizes RNA immediately after tissue collection preventing degradation, allowing multi-site sample acquisition, and facilitating storage of samples from multiple time points prior
在这里,我们优化了单细胞组合索引RNAseq(sci-RNA-seq)(Cao等人,2020年,2019年),以破译驱动肾脏纤维化的分子事件。我们利用sci-RNA-seq的高通量、高样本复用能力和低成本特点,在多个时间点对单侧缺血再灌注损伤(uni-IRI)和单侧输尿管梗阻(UUO)这两种小鼠肾脏损伤和纤维化模型进行了表征。sci-RNA-seq与组织固定兼容,它能在组织采集后立即稳定RNA,防止降解,允许多点样本采集,便于在采集前储存多个时间点的样本。
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Figure 1. A single-cell transcriptomics landscape of mouse kidney fibrogenesis profiled with sci-RNA-seq3
图 1.用 sci-RNA-seq 分析的小鼠肾脏纤维化的单细胞转录组学图谱3

(A) Summary of experimental methodology. n = 2 n = 2 n=2\mathrm{n}=2 per time point. Nuclei were extracted from all kidney samples and profiled with a three-level combinatorial indexing sequencing strategy. Cells were demultiplexed based on 1 st 1 st  1^("st ")1^{\text {st }} indexing barcodes to identify sample origins in data analysis. Figure created with BioRender.com.
(A) 实验方法概要。 n = 2 n = 2 n=2\mathrm{n}=2 每个时间点。从所有肾脏样本中提取细胞核,采用三级组合索引测序策略进行分析。根据 1 st 1 st  1^("st ")1^{\text {st }} 索引条形码对细胞进行解复用,以便在数据分析中识别样本来源。图由 BioRender.com 绘制。

(B) Immunofluorescence staining of HAVCR1 (red), collagen type I (green), lotus tetragonolobus lectin (LTL; white), and DAPI (blue) on tissue sections collected from all healthy and diseased conditions of our study cohort. Scale bars, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}.
(B) 从我们的研究队列的所有健康和患病情况中采集的组织切片上的 HAVCR1(红色)、I 型胶原(绿色)、莲四叶凝集素(LTL;白色)和 DAPI(蓝色)的免疫荧光染色。比例尺, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}

© Pseudobulk trajectory projection (using Monocle2) of all sample conditions in the study cohort revealing distinct transcriptomic signature of uni-IRI and UUO. Each dot represents a sample ( n = 24 n = 24 n=24\mathrm{n}=24 in total).
研究队列中所有样本条件的假体轨迹投影(使用 Monocle2)揭示了 uni-IRI 和 UUO 不同的转录组特征。每个点代表一个样本(共 n = 24 n = 24 n=24\mathrm{n}=24 个)。

(D) An atlas of mouse kidney fibrogenesis. A UMAP presentation (center) shows 309,666 cells profiled from 24 individual mouse kidneys of 11 healthy or diseased conditions. The surrounding circular layouts indicate the cell number of each population ( log 10 log 10 log_(10)\log _{10}-transformed scale bar), 19 major cell types (outer layout), and
(D) 小鼠肾脏纤维化图谱。UMAP 图示(中心)显示了 11 种健康或患病情况下 24 个小鼠肾脏中的 309,666 个细胞。周围的圆形布局显示了每个群体的细胞数( log 10 log 10 log_(10)\log _{10} -转化比例条)、19 种主要细胞类型(外部布局)和

Article  文章

to processing. We have generated an atlas of kidney fibrogenesis (data visualizer available at http://humphreyslab.com/ SingleCell/) from a single experiment with 11 biological conditions and 24 samples. This approach enabled the elimination of batch effects and profiling of 309,666 cells. We report that uni-IRI and UUO induced two distinct PT cell states after injury with unique transcriptomic signatures and fate outcomes. Further investigation of these two cell states highlighted their distinct mechanisms of metabolic regulation, including activated lipid metabolism in the earliest stages of uni-IRI where we identified PLIN2+ lipid droplets. Additionally, we describe both shared and unique epithelial responses to injury and repair across nephron segments, as well as kidney stromal heterogeneity and intercellular communication dynamics during kidney fibrosis. This atlas of kidney fibrogenesis serves as a unique resource and reveals previously unappreciated epithelial cell states.
到处理。我们从包含 11 种生物条件和 24 个样本的单一实验中生成了肾脏纤维化图谱(数据可视化器可在 http://humphreyslab.com/ SingleCell/ 上获取)。这种方法消除了批次效应,对 309,666 个细胞进行了分析。我们报告说,uni-IRI 和 UUO 在损伤后诱导了两种不同的 PT 细胞状态,它们具有独特的转录组特征和转归结果。对这两种细胞状态的进一步研究突显了它们不同的代谢调控机制,包括在单IRI的最早阶段激活脂质代谢,我们在该阶段发现了PLIN2+脂滴。此外,我们还描述了各肾节段上皮细胞对损伤和修复的共同和独特反应,以及肾脏纤维化过程中肾脏基质的异质性和细胞间的通讯动态。该肾脏纤维化图谱是一种独特的资源,揭示了以前未被认识的上皮细胞状态。

RESULTS  结果

Generation of two mouse models of kidney fibrogenesis We performed uni-IRI and UUO surgeries on 8- to 9 -week-old adult male C57BL6/J mice and collected samples at multiple time points during disease progression ( 0 and 6 h and 2 , 7 , 14 2 , 7 , 14 2,7,142,7,14, and 28 days post uni-IRI or 0 , 2 , 4 , 6 , 10 0 , 2 , 4 , 6 , 10 0,2,4,6,100,2,4,6,10, and 14 days post UUO; n = 2 n = 2 n=2n=2 for each time point) (Figure 1A). To validate each sample prior to scRNA-seq, we first stained kidney injury and fibrosis markers by immunofluorescence (Figure 1B). In mice with uniIRI, the kidney injury marker HAVCR1 was strongly upregulated after 2 days post injury (uni-IRI D2), and its expression gradually decreased during the repair phase after uni-IRI D7. The fibrosis marker collagen type I (COL1) started to accumulate at uni-IRI D2 and was highly abundant at uni-IRI D14. By uni-IRI D28, the expression of HAVCR1 was close to baseline while COL1 expression was only partially resolved, suggesting an acute kidney injury (AKI) to CKD transition (Figure 1B). By contrast, UUO kidneys had sustained HAVCR1 expression and increased upregulation of COL1 over the full time course (Figure 1B), reflecting the more aggressive fibrotic burden in this model. Successful induction of injury and fibrogenesis on mouse kidneys was also confirmed by qPCR where we measured Havcr1 and myofibroblast marker genes Acta2 and Col1a1 and observed similar expression patterns (Figure S1A).
建立两种肾脏纤维化小鼠模型 我们对 8 到 9 周大的成年雄性 C57BL6/J 小鼠进行了单侧肾脏造影(uni-IRI)和单侧肾脏造影(UUO)手术,并在疾病进展的多个时间点收集样本(单侧肾脏造影(uni-IRI)后的 0 和 6 h 以及 2 , 7 , 14 2 , 7 , 14 2,7,142,7,14 和 28 天,或单侧肾脏造影(UUO)后的 0 , 2 , 4 , 6 , 10 0 , 2 , 4 , 6 , 10 0,2,4,6,100,2,4,6,10 和 14 天;每个时间点的 n = 2 n = 2 n=2n=2 )(图 1A)。为了在 scRNA-seq 之前验证每个样本,我们首先用免疫荧光染色了肾损伤和纤维化标记物(图 1B)。在uni-IRI小鼠中,肾损伤标志物HAVCR1在损伤后2天(uni-IRI D2)强烈上调,在uni-IRI D7后的修复阶段其表达逐渐下降。纤维化标志物 I 型胶原蛋白(COL1)从损伤后 2 天(uni-IRI D2)开始积累,到损伤后 14 天时达到高表达水平。到单IRI D28时,HAVCR1的表达接近基线,而COL1的表达仅部分缓解,这表明急性肾损伤(AKI)向CKD过渡(图1B)。相比之下,UUO 肾脏在整个过程中 HAVCR1 表达持续,COL1 上调增加(图 1B),这反映了该模型中纤维化负担更具侵袭性。我们通过 qPCR 检测了 Havcr1 和肌成纤维细胞标记基因 Acta2 和 Col1a1,并观察到了类似的表达模式(图 S1A)。
Characterization of kidney fibrogenesis with sci-RNA-seq3
利用 sci-RNA-seq 分析肾脏纤维形成的特征3

Nuclear suspensions were prepared from each sample, fixed, and snap-frozen. This enabled us to process all 24 samples simultaneously in a single experiment to achieve sample-multiplexing using the sci-RNA-seq3 protocol (Cao et al., 2019, 2020), which employed a combinatorial indexing strategy based
从每个样本中制备核悬浮液,固定并速冻。这样,我们就能在一次实验中同时处理所有 24 个样本,利用 sci-RNA-seq3 协议(Cao 等人,2019 年,2020 年)实现样本复用。

on reverse transcription (RT), hairpin ligation, and indexed PCR. In sci-RNA-seq3, the nuclei from each sample were divided into several wells of four 96 -well plates, and thus, the first barcode introduced by RT allowed sample identification (Figure 1A). In addition to the multiplexing capacity, high-throughput, and relatively low cost of sci-RNA-seq3, common laboratory supplies could be used, and the protocol was modifiable. Early results revealed several challenges in applying the original sci-RNA-seq3 protocol to kidney, including low nuclei extraction yield, nuclei aggregation in the suspension, reduced library quality due to non-uniform transposase activity and incomplete purification. We therefore separately optimized each of these steps in our modified protocol, including performing a Tn5 transposase activity test which significantly improved library yield and quality. The changes to the original protocol are summarized in Table S1 and in more detail in the STAR Methods. We included a species-mixing control with nuclei harvested from human HEK293T and mouse C3H/10T1/2 cultured cells in order to evaluate doublet frequency.
在 sci-RNA-seq3 中,每个样本的细胞核都被分装在四个 96 孔板的多个孔中,因此,RT 引入的第一个条形码可用于样本识别(图 1A)。在 sci-RNA-seq3 中,每个样本的细胞核都被分成 4 个 96 孔板中的几个孔,因此 RT 引入的第一个条形码可用于样本识别(图 1A)。sci-RNA-seq3 除了具有复用能力、高通量和相对较低的成本外,还可以使用普通的实验室用品,而且方案可以修改。早期结果显示,在肾脏中应用原始的 sci-RNA-seq3 方案存在一些挑战,包括细胞核提取率低、悬浮液中细胞核聚集、转座酶活性不均匀导致文库质量下降以及纯化不完全。因此,我们在修改后的方案中分别对这些步骤进行了优化,包括进行 Tn5 转座酶活性测试,从而显著提高了文库的产量和质量。表 S1 总结了对原始方案的改动,更多细节见 STAR 方法。我们使用从人类 HEK293T 和小鼠 C3H/10T1/2 培养细胞中提取的细胞核进行物种混合对照,以评估双顶体频率。
We sequenced the entire sci-RNA-seq3 library on one NovaSeq 6000 flow cell. Over half of the reads (60.4%) mapped to intronic regions, as expected for single-nucleus sequencing (Wu et al., 2019). After demultiplexing, we first assessed doublets by analyzing the species-mixing samples. This revealed a very low cell collision rate of 1.3% (Figure S1B). For the remaining mouse kidney samples, we generated a total of 413,681 raw cell transcriptomes at a minimum threshold of 200 uniform molecular identifiers (UMIs) per cell. We detected an average of 1,165 UMIs/cell (Table S1). After quality control procedures including removal of predicted doublets and artifacts (STAR Methods), we proceeded to analyze 309,666 high-quality cells. We first projected the pseudobulk transcriptomes of all 24 samples into two dimensions in an unsupervised fashion. This revealed clearly distinct trajectories between the uni-IRI and UUO samples and low variation between biological replicates. The uni-IRI 6 h and UUO D2 samples were similar, but later samples diverged substantially (Figure 1C). Even though uni-IRI and UUO are both models of kidney fibrosis, the distinct trajectories suggested quite different cellular mechanisms.
我们在一个 NovaSeq 6000 流式细胞上对整个 sci-RNA-seq3 文库进行了测序。超过一半的读数(60.4%)映射到了内含子区域,这也是单核测序的预期结果(Wu 等人,2019 年)。解复用后,我们首先通过分析物种混合样本来评估双倍性。结果显示,细胞碰撞率非常低,仅为 1.3%(图 S1B)。对于其余的小鼠肾脏样本,我们以每个细胞 200 个统一分子识别码(UMI)为最低阈值,共生成了 413,681 个原始细胞转录组。我们平均检测到 1,165 个 UMIs/细胞(表 S1)。经过质量控制程序,包括去除预测的双倍和伪影(STAR 方法)后,我们对 309,666 个高质量细胞进行了分析。我们首先以无监督的方式将所有 24 个样本的假体转录组投影到两个维度。结果显示,uni-IRI 样本和 UUO 样本之间的轨迹明显不同,生物重复之间的差异也很小。单IRI 6小时样本和UUO D2样本相似,但之后的样本差异很大(图1C)。尽管 uni-IRI 和 UUO 都是肾脏纤维化的模型,但其不同的轨迹表明它们的细胞机制截然不同。
The large size of this scRNA-seq dataset allowed for a detailed characterization of cellular heterogeneity in healthy and fibrotic kidneys. Cell clustering of the 309,666 cells revealed 19 major cell clusters, including cells of the PT, loop of Henle (LoH), and podocytes (Figure 1D). We performed subclustering analysis on all major cell clusters, which identified a total of 50 cell types or states (summarized in Table S2) including low abundance cell types such as juxtaglomerular apparatus (JGA), dendritic cell subtypes (Figure S1C), and vascular cells (Figure S1D). The 19 major clusters were annotated based on expression of known marker genes (Figure 1E) and data integration with previous cell atlas resources such as our bilateral IRI (bi-IRI) scRNA-seq
该 scRNA-seq 数据集规模庞大,可以详细描述健康肾脏和纤维化肾脏的细胞异质性。对 309,666 个细胞进行的细胞聚类分析发现了 19 个主要细胞群,包括 PT 细胞、亨列环(LoH)细胞和荚膜细胞(图 1D)。我们对所有主要细胞簇进行了亚聚类分析,共鉴定出 50 种细胞类型或状态(摘要见表 S2),包括并肾小球器(JGA)、树突状细胞亚型(图 S1C)和血管细胞(图 S1D)等低丰度细胞类型。根据已知标记基因的表达(图 1E)以及与以前的细胞图谱资源(如我们的双侧 IRI(bi-IRI)scRNA-seq)的数据整合,对 19 个主要细胞群进行了注释。
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UUO PT cells  UUO PT 电池


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Figure 2. Diverse cell states of injured proximal tubule
图 2.损伤的近端小管的多种细胞状态

(A) UMAP plot of all PT cells after quality control in subclustering analysis. S1, S2, and S3 indicate the three anatomical segments of PT.
(A) 亚聚类分析中经过质量控制的所有 PT 细胞的 UMAP 图。S1、S2 和 S3 表示 PT 的三个解剖区段。

(B) Dot plot showing expression of marker genes of each PT cell clusters, including 3 clusters in healthy states and 7 injured cell states expressing Havcr1.
(B) 点阵图显示各 PT 细胞群标记基因的表达情况,包括 3 个健康细胞群和 7 个表达 Havcr1 的受伤细胞群。

© Heatmap showing cluster-specific transcription factor activity predicted by gene regulatory network analysis. Color density corresponds to average activity of the indicated gene relative to all PT cells.
© 热图显示基因调控网络分析预测的集群特异性转录因子活性。颜色密度对应于所示基因相对于所有 PT 细胞的平均活性。

(D) Transcription factor activity and single-cell pathway analysis showing activities of Smad1 and NF-кB/TNF- α α alpha\alpha pathways are enriched in the FR-PTC cluster.
(D) 转录因子活性和单细胞通路分析显示,Smad1 和 NF-кB/TNF- α α alpha\alpha 通路的活性在 FR-PTC 簇中富集。

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dataset (Kirita et al., 2020; Figure S1E). Correlation analysis across cell types indicated high transcriptomic similarity between fibroblasts (Prkg1/Gpc6 high) and myofibroblasts (Col1a1/Col1a2 high) and across distal nephron epithelia (thick ascending limb [TAL], distal convoluted tubule [DCT], connecting tubule [CNT], principal cell [PC], type A intercalated cell of collecting duct [ICA], and type B intercalated cell of collecting duct [ICB]) (Figure S1F). We next identified cells according to sample condition (health, uni-IRI or UUO), which revealed that nearly all cells from disease time points distributed distinctly from the healthy cells-reflecting that fibrosis affects the entire organ (Figures S1G and S1H). Myofibroblasts and immune cells were quite sparse in health but underwent proliferative expansion during disease (Figures S1I and S1J).
数据集(Kirita 等人,2020 年;图 S1E)。跨细胞类型的相关性分析表明,成纤维细胞(Prkg1/Gpc6 高)和肌成纤维细胞(Col1a1/Col1a2 高)之间以及远端肾小管上皮(粗升支[TAL]、远端曲小管[DCT]、连接小管[CNT]、主细胞[PC]、A 型肾小管[PC])之间的转录组高度相似、PC]、集合管 A 型夹层细胞[ICA]和集合管 B 型夹层细胞[ICB])(图 S1F)。接下来,我们根据样本条件(健康、uni-IRI 或 UUO)对细胞进行了鉴定,结果显示,几乎所有来自疾病时间点的细胞的分布都与健康细胞截然不同--这反映出纤维化影响了整个器官(图 S1G 和 S1H)。肌成纤维细胞和免疫细胞在健康时非常稀少,但在疾病期间却发生了增殖扩张(图 S1I 和 S1J)。

Diverse PT injury states
不同的 PT 损伤状态

Our initial clustering suggested considerable heterogeneity in PT cell states during fibrosis (Figure 1D). We therefore performed unsupervised subclustering on PT cells alone ( 130,503 cells after quality control; Figure 2A). This revealed the expected healthy S1/S2/S3 PT clusters, as well as multiple injury states (high expression of Havcr1 and Nrg1), including acute injury (PTAclnj), repairing (PT-R), a state we have previously characterized as failed repair PT cells (FR-PTCs) (Kirita et al., 2020; Muto et al., 2021) and two apparent intermediate PT injury states, located between healthy cells and FR-PTC in the UMAP space, which we annotated as Type1 and Type2 injured PT cells. All injured PT cell states were also characterized by downregulation of healthy PT marker genes such as solute-linked carriers (e.g., S/c34a1, S/c5a12, and S/c7a13), suggesting cell dedifferentiation. The significances of PT-AcInj, PT-R, and FR-PTC have been described in previous scRNA-seq studies (Gerhardt et al., 2021; Kirita et al., 2020; Lu et al., 2021; Rudman-Melnick et al., 2020) and were benchmarked in our large-scale dataset. Specifically, PT-AcInj cells expressed genes encoding heatshock proteins (HSPs) (e.g., Hspa1b and Hsp90aa1) and showed high activity of Hsf 1 in the transcription factor activity analysis (Figures 2B, 2C, and S2A). The PT-R cluster strongly expressed genes associated with cell proliferation (e.g., Top2a, Mki67, and Lmnb1) and scored highly by cell-cycle scoring analysis (Figure S2B). FR-PTC were characterized by expression of known marker genes Vcam1 and Kcnip4. Smad1, an essential component of TGF- β β beta\beta signaling (Zhang et al., 2015), exhibited high transcription factor activity in FR-PTC (Figures 2C and 2D) and sin-gle-cell pathway activity analysis revealed that NF-кB and TNF- α α alpha\alpha pathways were also highly active (Figure 2D), indicating that these cells were proinflammatory and profibrotic, confirming prior results (Markó et al., 2016; Ramseyer and Garvin, 2013; Shimizu et al., 2011; Zager et al., 2005).
我们最初的聚类结果表明,纤维化过程中 PT 细胞的状态存在相当大的异质性(图 1D)。因此,我们仅对 PT 细胞进行了无监督子聚类(质控后为 130,503 个细胞;图 2A)。这揭示了预期的健康 S1/S2/S3 PT 聚类,以及多种损伤状态(Havcr1 和 Nrg1 高表达),包括急性损伤(PTAclnj)、修复状态(PT-R)、一种我们之前表征为修复失败 PT 细胞(FR-PTCs)的状态(Kirita 等人,2020 年;Muto 等人,2021 年),以及两种明显的中间 PT 损伤状态,它们位于 UMAP 空间中的健康细胞和 FR-PTC 之间,我们将其注释为 1 型和 2 型损伤 PT 细胞。所有损伤的 PT 细胞状态都具有健康 PT 标记基因下调的特征,如溶质连接载体(如 S/c34a1、S/c5a12 和 S/c7a13),这表明细胞发生了去分化。之前的 scRNA-seq 研究(Gerhardt 等人,2021 年;Kirita 等人,2020 年;Lu 等人,2021 年;Rudman-Melnick 等人,2020 年)已经描述了 PT-AcInj、PT-R 和 FR-PTC 的重要性,我们的大规模数据集也对其进行了基准测试。具体而言,PT-AcInj 细胞表达了编码热休克蛋白(HSPs)(如 Hspa1b 和 Hsp90aa1)的基因,并在转录因子活性分析中显示出 Hsf 1 的高活性(图 2B、2C 和 S2A)。PT-R 簇强烈表达与细胞增殖相关的基因(如 Top2a、Mki67 和 Lmnb1),在细胞周期评分分析中得分很高(图 S2B)。已知标记基因 Vcam1 和 Kcnip4 的表达是 FR-PTC 的特征。 TGF- β β beta\beta 信号转导的重要组成部分Smad1(Zhang等人,2015年)在FR-PTC中表现出很高的转录因子活性(图2C和2D),单细胞通路活性分析表明,NF-кB和TNF- α α alpha\alpha 通路也高度活跃(图2D),表明这些细胞具有促炎和促组织坏死的特性,证实了之前的研究结果(Markó等人,2016年;Ramseyer和Garvin,2013年;Shizu等人,2011年;Zager等人,2005年)、2016;Ramseyer 和 Garvin,2013;Shimizu 等人,2011;Zager 等人,2005)。
By contrast, we struggled to annotate the Type1 and Type2 PT cell clusters to previously published work, possibly because these cells were enriched in early and middle stages of kidney fibrogenesis (i.e., IRI 6 h, UUO D2-D4) that have not been previ-
相比之下,我们很难将 1 型和 2 型 PT 细胞集群注释为以前发表的研究成果,这可能是因为这些细胞富集于肾脏纤维化的早期和中期阶段(即 IRI 6 h、UUO D2-D4),而这些阶段尚未被研究。

ously analyzed in scRNA-seq studies. Surveying the proportion of each PT cell type across conditions (Figures 2E and S2C) revealed that Type1 injured PT was primarily found in uniIRI (occurrence frequency in uni-IRI: UUO 11 : 1 11 : 1 ∼11:1\sim 11: 1 ) and Type2 injured PT was more specific to UUO samples (abundance of Type1:Type2 injured PT ~ 10:1). More specifically, in uni-IRI, Type1 injured cells comprised 80 % 80 % 80%80 \% of all PT cells at 6 h after injury, with this proportion falling rapidly to 5 % 5 % 5%5 \% of the total by D2. In UUO, Type2 injured cells also appeared in the early time point (D2) and dominated the PT population (62% of the total cells), but their frequency did not fall as quickly as Type1 injured cells in uni-IRI (Figure 2E). This analysis also highlighted distinct outcomes of PT successful repair versus failed repair in the two mouse models: the frequency of healthy PT cells was reduced remarkably by both uni-IRI or UUO surgeries, but only in uni-IRI did these cells return to their prior uninjured state (Figure 2E). While a small percentage of FR-PTC ( 4 % 4 % ∼4%\sim 4 \% ) remained at -IRI D28, FR-PTC constituted a large and increasing proportion of all cells as the time course proceeded in UUO ( 55 % 55 % ∼55%\sim 55 \% at UUO D14) (Figure 2E).
在scRNA-seq研究中被广泛分析。调查不同条件下每种 PT 细胞类型的比例(图 2E 和 S2C)发现,1 型损伤 PT 主要存在于 uniIRI 中(uni-IRI:UUO 中的出现频率 11 : 1 11 : 1 ∼11:1\sim 11: 1 ),而 2 型损伤 PT 在 UUO 样本中更具特异性(1 型:2 型损伤 PT 的丰度约为 10:1)。更具体地说,在单IRI中,1型损伤细胞在损伤后6小时占所有PT细胞的 80 % 80 % 80%80 \% ,到D2时,这一比例迅速下降到 5 % 5 % 5%5 \% 。在 UUO 中,2 型损伤细胞也出现在早期时间点(D2),并在 PT 群体中占主导地位(占细胞总数的 62%),但其频率下降的速度不如在 uni-IRI 中的 1 型损伤细胞(图 2E)。这项分析还突显了两种小鼠模型中PT成功修复与失败修复的不同结果:健康PT细胞的频率在uni-IRI或UUO手术中都显著降低,但只有在uni-IRI中这些细胞才恢复到之前的未损伤状态(图2E)。虽然在 -IRI D28 时仍有一小部分 FR-PTC ( 4 % 4 % ∼4%\sim 4 \% ),但随着 UUO 的时间进程(UUO D14 时为 55 % 55 % ∼55%\sim 55 \% ),FR-PTC 在所有细胞中所占的比例越来越大(图 2E)。

Two types of cell states of injured PT
受伤 PT 的两种细胞状态

We next asked whether the ability of PT to successfully repair in uni-IRI but not in UUO might be related to Type1 versus Type2 injury. Gene ontology (GO) enrichment analysis on differentially expressed genes (DEGs) for the two populations highlighted wound healing, cell junction organization, and cell-cell adhesion in Type1 injured PT and epithelial morphogenesis and MAPK signaling in the Type2 group (Figure S2D). Regulation of cell motility was a shared term in both groups (Figure S2D). Type1 injured PT cells were primarily observed early-at 6 h post uniIRI with defining DEGs such as Plin2 and Col27a1 (Figures 2B and S2E). We found that Elf3, a transcription factor that has been reported to be upregulated in both mouse and human AKI samples (Famulski et al., 2012; Rudman-Melnick et al., 2020), showed high gene activity in Type1 injured PT cells (Figures 2C and S2F). This PT subpopulation also exhibited activated EGFR signaling (Figure S2F), a pathway known to promote PT recovery after AKI (Tang et al., 2013). For Type2 injured PT, we observed some cluster-specific DEGs including S/c6a6, Bcat1, and S/c7a12, but others that were in common with the FR-PTC cluster (e.g., Sema5a, Dcdc2a, and Ypel2) (Figure S2E), hinting at a lineage relationship between Type2 PT and FR-PTC. Correlation analysis confirmed high similarity between Type2 PT and FR-PTC compared with the other cell states (Figure S2G). Mapping the Type1 and Type2 subclusters back onto the entire dataset revealed that they constituted the major cluster annotated as “PT-Inj” (Figure S2H). Collectively, these results led us to hypothesize that the Type1 PT injury state is protective while the Type2 state leads to FR-PTC driving fibrogenesis.
我们接下来要问的是,PT 在单IRI 而非UUO 中成功修复的能力是否可能与1型损伤和2型损伤有关。对两组人群差异表达基因(DEGs)的基因本体论(GO)富集分析显示,在1型损伤的PT中,伤口愈合、细胞连接组织和细胞-细胞粘附;而在2型组中,上皮形态发生和MAPK信号转导(图S2D)。细胞运动调节是两组的共同术语(图 S2D)。1型受伤的PT细胞主要是在单IRI后6小时早期观察到的,其中有Plin2和Col27a1等确定的DEGs(图2B和S2E)。我们发现,Elf3--一种已被报道在小鼠和人类 AKI 样本中上调的转录因子(Famulski 等人,2012 年;Rudman-Melnick 等人,2020 年)--在 1 型损伤 PT 细胞中显示出较高的基因活性(图 2C 和 S2F)。这种 PT 亚群还表现出活化的表皮生长因子受体(EGFR)信号传导(图 S2F),这是一种已知能促进 AKI 后 PT 恢复的途径(Tang 等人,2013 年)。对于2型损伤的PT,我们观察到了一些簇特异的DEGs,包括S/c6a6、Bcat1和S/c7a12,但也观察到了其他与FR-PTC簇相同的DEGs(如Sema5a、Dcdc2a和Ypel2)(图S2E),这暗示了2型PT与FR-PTC之间的血缘关系。相关性分析证实,与其他细胞状态相比,2 型 PT 和 FR-PTC 之间具有高度相似性(图 S2G)。将 Type1 和 Type2 亚簇映射回整个数据集后发现,它们构成了注释为 "PT-Inj "的主要簇(图 S2H)。综合这些结果,我们推测类型 1 PT 损伤状态是保护性的,而类型 2 状态会导致 FR-PTC 驱动纤维化。
To better characterize potential lineage relationships between Type1/Type2 injured PT and other PT subpopulations, we leveraged single-cell trajectory inference analysis (Qiu et al., 2017)
为了更好地描述 1 型/2 型受伤 PT 与其他 PT 亚群之间的潜在血统关系,我们利用了单细胞轨迹推断分析(Qiu 等人,2017 年)。
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Figure 3. Dysregulated lipid metabolism in proximal tubule cells during fibrogenesis and activated fatty acid oxidation after short-term lipid deposition
图 3纤维化过程中近端小管细胞的脂质代谢失调以及短期脂质沉积后脂肪酸氧化被激活

(A) Protein-protein interaction (PPI) enrichment analysis on upregulated differentially expressed genes of Type1 injured PT cells showing terms associated with lipid metabolism.
(A) 蛋白质-蛋白质相互作用(PPI)富集分析表明,1 型损伤 PT 细胞上调的差异表达基因与脂质代谢相关。

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and identified two major trajectories starting from Type1 injured PT in uni-IRI (Figure 2F, left panel): these cells first became repairing cells and then either differentiated into healthy PT cells (successful repair trajectory) or FR-PTC (failed repair trajectory). Consistent with this result, the successful repair trajectory upregulated healthy PT marker genes, but the FR-PTC lineage failed to do so (Figure S2I). By contrast only one major trajectory was observed in UUO: it started from healthy cells and ended at FR-PTC with Type2 injured PT cells located in between (Figure 2F, right panel). In this UUO trajectory, expression of FRPTC markers gradually increased over pseudotime (Figure S2I). This analysis suggested that the Type1 injury state was a bipotential one with the ability to become either healthy cells or FRPTC, whereas the Type2 category was unipotential with the ability only to differentiate into FR-PTC. We also performed trajectory analysis on dataset combining uni-IRI and UUO cells, which presented consistent results (Figure S2J). To further support this hypothesis, we conducted a computational cell fate mapping analysis, which simulated a birth-death process based on the Markov chains model (Lange et al., 2022) (STAR Methods). We observed that in uni-IRI, Type1 injured PT cells showed the highest probability of contributing to the repairing cell lineage (Figure 2G, left panel), which could further develop into either healthy cells or FR-PTC (Figure S2K). By contrast, the majority of Type2 injured PT cells in UUO differentiated into FR-PTC (Figure 2G, right panel). Even though a small proportion of Type2 injured PT cells were predicted to acquire a repairing cell state, most of these ultimately adapted the FR-PTC phenotype (Figure S2K).
图 2F 左侧面板):这些细胞首先成为修复细胞,然后分化为健康 PT 细胞(成功修复轨迹)或 FR-PTC(失败修复轨迹)。与这一结果一致的是,成功的修复轨迹上调了健康 PT 的标记基因,但 FR-PTC 系则没有上调(图 S2I)。相比之下,在 UUO 中只观察到一条主要的轨迹:它从健康细胞开始,到 FR-PTC 结束,2 型损伤 PT 细胞位于两者之间(图 2F 右面板)。在这一 UUO 轨迹中,FRPTC 标记的表达随着假时间逐渐增加(图 S2I)。这一分析表明,1 型损伤状态是一种双潜能状态,既能成为健康细胞,也能成为 FRPTC,而 2 型损伤状态是单潜能状态,只能分化为 FR-PTC。我们还对单IRI和UUO细胞的数据集进行了轨迹分析,结果一致(图S2J)。为了进一步支持这一假设,我们进行了计算细胞命运图谱分析,根据马尔可夫链模型模拟了出生-死亡过程(Lange 等人,2022 年)(STAR 方法)。我们观察到,在uni-IRI中,1型受伤的PT细胞对修复细胞系的贡献概率最高(图2G,左侧面板),可进一步发育成健康细胞或FR-PTC(图S2K)。相比之下,UUO 中大多数 2 型损伤 PT 细胞分化为 FR-PTC(图 2G 右图)。尽管预测有一小部分2型损伤的PT细胞会获得修复细胞状态,但这些细胞中的大多数最终都适应了FR-PTC表型(图S2K)。

Dysregulated PT lipid metabolisms during fibrogenesis
纤维形成过程中 PT 脂质代谢失调

Next, we aimed to explore metabolic mechanisms underlying the distinct fate outcomes of Type1 and Type2 injured PT cells. Pro-tein-protein interaction (PPI) enrichment analysis on the top DEGs for Type1 injured PT cells highlighted terms associated with lipid metabolism, including mitochondrial long-chain fatty acid β β beta\beta-oxidation (FAO), regulation of lipid transport, and lipid localization (Figure 3A). Previous studies have demonstrated defective FAO metabolism of PT cells in CKD, which could be reversed by restoring the capacity of FAO (Kang et al., 2014;
接下来,我们旨在探索1型和2型损伤PT细胞不同命运结果的代谢机制。对1型损伤的PT细胞的顶级DEGs进行的前蛋白-蛋白相互作用(PPI)富集分析强调了与脂质代谢相关的术语,包括线粒体长链脂肪酸 β β beta\beta -氧化(FAO)、脂质转运调控和脂质定位(图3A)。先前的研究表明,CKD 中 PT 细胞的 FAO 代谢存在缺陷,而这种缺陷可以通过恢复 FAO 的能力来逆转(Kang 等,2014;
Pei et al., 2020; Stadler et al., 2015; Wu et al., 2020). Consistently, by scoring genes involved in FAO across all PT cells, we observed a reduced FAO activity in the middle stages of uniIRI (i.e., D2/7/14) and all UUO samples (Figures 3B and S3A). FR-PTC exhibited the lowest FAO score when compared with healthy PT (Figure 3B), and the proportion of FR-PTC correlated negatively with FAO activity (Figure S3B), highlighting the central role of this population in CKD.
Pei等人,2020;Stadler等人,2015;Wu等人,2020)。同样,通过对所有 PT 细胞中参与 FAO 的基因进行评分,我们观察到在 uniIRI 的中期(即 D2/7/14)和所有 UUO 样本中 FAO 活性降低(图 3B 和 S3A)。与健康 PT 相比,FR-PTC 的 FAO 得分最低(图 3B),FR-PTC 的比例与 FAO 活性呈负相关(图 S3B),突显了这一群体在 CKD 中的核心作用。
By contrast, we noticed an unexpected increase in the FAO score at uni-IRI 6 h (Figure 3B), which implied activated lipid metabolism in Type1 injured PT cells at this early phase. In addition, we observed activated peroxisome proliferator-activated receptor signaling, reflected by significantly increased expression of FAO rate-limiting genes such as Cpt1a, Acox1, Hadha, and Hadhb (Figure S3A) (Mann-Whitney U U UU test with the Benjamini-Hochberg correction).
相比之下,我们注意到,在单IRI 6小时时,FAO评分意外增加(图3B),这意味着1型损伤的PT细胞在这一早期阶段激活了脂质代谢。此外,我们还观察到过氧化物酶体增殖激活受体信号的激活,这反映在 FAO 限速基因如 Cpt1a、Acox1、Hadha 和 Hadhb 的表达显著增加(图 S3A)(经本杰明-霍奇伯格校正的 Mann-Whitney U U UU 检验)。
We hypothesized that the upregulated FAO gene expression at uni-IRI 6 h would be accompanied by increased lipid deposition. A variety of proteins are required for maintenance of cytoplasmic lipid droplets, so we examined the expression of genes involved in formation and maintenance of lipid droplets. Consistent with this hypothesis, the lipid droplet score was significantly increased at uni-IRI 6 h but returned to baseline for all subsequent time points (Figure 3C). The expression of lipid droplet genes Plin2, Fabp4, Acs/4, and Ehd1 were upregulated in the Type1 injured PT cells compared with the healthy (Figure S3A).
我们假设,在单IRI 6小时内,FAO基因表达的上调将伴随着脂质沉积的增加。维持细胞质脂滴需要多种蛋白质,因此我们检测了参与脂滴形成和维持的基因的表达。与这一假设一致的是,脂滴得分在单IRI 6小时时显著增加,但在随后的所有时间点都恢复到基线(图3C)。与健康细胞相比,脂滴基因 Plin2、Fabp4、Acs/4 和 Ehd1 在 1 型损伤的 PT 细胞中表达上调(图 S3A)。
Next, we analyzed lipid content at multiple time points identifying a striking increase in Oil Red O-positive lipid droplets throughout both cortical and medullary tubules at uni-IRI 6 h compared with healthy kidney (Figure 3D). Interestingly, at uniIRI D2, most lipids were cleared from cortical tubular cells, though we also observed mild persistence of intraluminal, extracellular lipids in outer medullary casts (Figure 3D). Oil Red O-positive lipid droplets were undetectable at uni-IRI D7 and later time points (Figures 3D and S3C). On the other hand, UUO lacked the early lipid droplet accumulation but gradually accumulated tubular lipids over time (Figures 3D and S3C).
接下来,我们分析了多个时间点的脂质含量,发现与健康肾脏相比,在单IRI 6小时时,皮质和髓质肾小管中油红O阳性脂滴显著增加(图3D)。有趣的是,在 uniIRI D2 时,大多数脂质从皮质肾小管细胞中清除,但我们也观察到在髓质外铸型中轻度持续存在管腔内、细胞外脂质(图 3D)。油红 O 阳性脂滴在 uni-IRI D7 及以后的时间点检测不到(图 3D 和 S3C)。另一方面,UUO 缺乏早期脂滴积累,但随着时间的推移逐渐积累管状脂质(图 3D 和 S3C)。
To quantitatively determine this transient lipid accumulation, we measured the abundance of triglycerides (TAGs), free fatty acids (FFAs), and cholesterol in uni-IRI mouse kidney tissues
为了定量测定这种瞬时脂质积累,我们测量了单IRI小鼠肾组织中甘油三酯(TAG)、游离脂肪酸(FFA)和胆固醇的丰度。
from multiple time points with mass spectrometry. With a total of 47 TAG species analyzed, this lipidomics analysis revealed an 6 6 ∼6\sim 6-fold increased abundance of total TAGs at uni-IRI 6 h compared with healthy tissues (Figure 3E). 70 % 70 % ∼70%\sim 70 \% of the accumulated TAGs were a combination of palmitate (16:0), oleate (18:1), and linoleate (18:2). Consistent with our Oil Red O staining, TAG was still abundant at uni-IRI D2, but it decreased nearly to baseline by uni-IRI D7 and D14 (Figure 3E). We also observed a 1.8 -fold increased abundance of total FFAs at uni-IRI 6 h compared with those of healthy tissues (Figure 3F). Among the 16 FFA species analyzed, palmitic acid (16:0) and oleic acid (18:1) were the two major FFAs in both healthy and diseased mouse kidneys, constituting ~50% of total FFAs (Figure 3F). Lipidomic analysis was also performed on UUO D10 and UUO D14 samples, where we identified 2 2 ∼2\sim 2-fold increased TAG abundance but almost no differences in FFA abundance (Figure S3D). In addition, we did not identify obvious changes in cholesterol abundance across samples (Figure S3E).
通过质谱法分析了多个时间点的总 TAG。这项脂质组学分析共分析了 47 种 TAG,结果显示,与健康组织相比,单IRI 6 h 时总 TAG 丰度增加了 6 6 ∼6\sim 6 -倍(图 3E)。 70 % 70 % ∼70%\sim 70 \% 累积的 TAG 是棕榈酸酯(16:0)、油酸酯(18:1)和亚油酸酯(18:2)的组合。与我们的油红 O 染色结果一致,TAG 在单内切酶切 D2 时仍很丰富,但在单内切酶切 D7 和 D14 时几乎降至基线(图 3E)。我们还观察到,与健康组织相比,单IRI 6 h时总FFA的丰度增加了1.8倍(图3F)。在分析的 16 种 FFA 中,棕榈酸(16:0)和油酸(18:1)是健康和患病小鼠肾脏中的两种主要 FFA,占总 FFA 的约 50%(图 3F)。我们还对UUO D10和UUO D14样本进行了脂质体分析,发现TAG丰度增加了 2 2 ∼2\sim 2 倍,但FFA丰度几乎没有差异(图S3D)。此外,我们没有发现不同样本中胆固醇丰度的明显变化(图 S3E)。
Therefore, our results suggested a transient upregulation of genes involved in FAO and lipid metabolism, accompanied by cytoplasmic lipid accumulation, at the earliest time points after uni-IRI.
因此,我们的研究结果表明,在单IRI后的最早时间点,参与粮农组织和脂质代谢的基因出现了短暂的上调,同时伴有细胞质脂质的积累。
Fatty acid exposure in vitro leads to lipid accumulation and FAO burst
脂肪酸在体外暴露会导致脂质积累和 FAO 爆发

To study how PT cells respond to short-term lipid accumulation, we established an in vitro model by treating primary human renal PT epithelial cells (RPTECs) with oleic or palmitic acids for 6 h . Oil Red O and BODIPY 493/503 staining confirmed a striking increase in intracellular lipid deposition after 6-h exposure of oleic or palmitic acids (Figure 3G). A longer exposure (2-6 days) to fatty acids resulted in an increased size of Oil Red O+ lipid aggregates (Figure S3H). We also treated RPTECs with fluorescently labeled palmitic acid (BODIPY C 16 C 16 C_(16)\mathrm{C}_{16} ) for 6 h and validated that RPTECs actively transported fatty acids leading to intracellular lipid accumulation (Figure S3F). Both 6-h oleic and palmitic acid exposure led to significant upregulation of CD36, which encodes a plasma membrane receptor for long-chain fatty acid transport and CPT1A, which encodes a mitochondrial membrane enzyme for long-chain fatty acyl-coenzyme A (CoA) transport (Figure S3G).
为了研究 PT 细胞如何应对短期脂质积累,我们建立了一个体外模型,用油酸或棕榈酸处理原代人肾 PT 上皮细胞(RPTECs)6 小时。油红 O 和 BODIPY 493/503 染色证实,接触油酸或棕榈酸 6 小时后,细胞内脂质沉积显著增加(图 3G)。接触脂肪酸的时间越长(2-6 天),油红 O+ 脂质聚集体的体积就越大(图 S3H)。我们还用荧光标记的棕榈酸(BODIPY C 16 C 16 C_(16)\mathrm{C}_{16} )处理 RPTECs 6 小时,验证了 RPTECs 能主动转运脂肪酸,导致细胞内脂质积累(图 S3F)。暴露 6 小时的油酸和棕榈酸都会导致 CD36(编码长链脂肪酸转运的质膜受体)和 CPT1A(编码长链脂肪酸酰辅酶 A(CoA)转运的线粒体膜酶)的显著上调(图 S3G)。
Previous studies reported mitochondrial dysregulation in PT cells in kidney diseases (Chung et al., 2019; Mori et al., 2021; Zhan et al., 2013). We stained for mitochondria and observed that most mitochondrion had a thread-like appearance in the steady state, but an increased fraction of mitochondria became fragmented into a sphere-like appearance (i.e., mitochondrial fission) after 6-h oleic or palmitic acid treatment (Figure S3I). On the other hand, we did not observe significant changes in reactive oxygen species, a feature of mitochondrial damage, after the 6-h fatty acid treatment (Figure S3J).
之前的研究报道了肾脏疾病中PT细胞线粒体的失调(Chung等人,2019年;Mori等人,2021年;Zhan等人,2013年)。我们对线粒体进行了染色,观察到大多数线粒体在稳定状态下呈线状外观,但在油酸或棕榈酸处理6小时后,有越来越多的线粒体碎裂成球状外观(即线粒体裂变)(图S3I)。另一方面,在脂肪酸处理 6 小时后,我们没有观察到作为线粒体损伤特征的活性氧发生显著变化(图 S3J)。
Next, to answer whether the accumulated lipid droplets could be oxidized later, we exposed RPTECs to lipids for 6 h then washed them away and replaced with normal culture medium (without fatty acid supplements) for 2 days. We observed very little Oil Red O staining at 2 days post culture medium renewal (Figure 3H), indicating that the deposited lipids induced by 6-h fatty acid treatment were largely cleared from cells by this time point. We asked whether this lipid clearance was the consequence of
接下来,为了回答积累的脂滴日后是否会被氧化,我们将 RPTEC 暴露于脂质中 6 小时,然后将其洗去,换上正常培养基(不含脂肪酸补充剂)培养 2 天。我们观察到,在培养基更新后的第 2 天,油红 O 染色非常少(图 3H),这表明经 6 小时脂肪酸处理诱导的沉积脂质在这个时间点基本上已从细胞中清除。我们询问这种脂质清除是否是由于

oxidation or due to other mechanisms such as lipid secretion. The presence of the lipolysis inhibitor Atglistatin during the 2-day chase prevented clearance of lipid droplets for both palmitic and oleic acids (Figure 3H), while 2-day treatment of Atglistatin alone did not induce significant lipid accumulation (Figure S3K). These results strongly suggest that RPTEC clearance of lipid accumulation occurs through FAO.
在 2 天的追逐过程中,脂肪分解抑制剂阿司司他丁的存在阻止了棕榈酸和油酸脂滴的清除(图 3H)。在 2 天的追逐过程中,脂肪分解抑制剂 Atglistatin 的存在阻止了棕榈酸和油酸脂滴的清除(图 3H),而单独使用 Atglistatin 的 2 天处理并未诱发显著的脂质积累(图 S3K)。这些结果有力地表明,RPTEC 是通过 FAO 清除脂质积累的。
To directly measure FAO and determine whether a dysregulation of glucose metabolism might also be involved, we measured the real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on RPTECs after 6-h fatty acid treatment, in a similar approach as described before (Kang et al., 2014). We identified a significantly higher OCR in cells pretreated with oleic or palmitic acids than control cells (Figures 3I and S3L), suggesting enhanced FAO activity. Injection of etomoxir, a CPT1 inhibitor, and oligomycin, an ATP synthase inhibitor, both reduced OCR, confirming that the increased OCR observed was the consequence of increased FAO (Figure S3L). In addition, an increased ECAR was also identified in cells pretreated with fatty acids (Figures 31 and S 3 L ). These results suggest that 6-h fatty acid exposure increases both FAO and glycolysis activity, characteristics of an energetically active cell state (Hocaoglu et al., 2021).
为了直接测量 FAO 并确定葡萄糖代谢失调是否也可能参与其中,我们采用与之前描述的类似方法(Kang 等,2014 年)测量了脂肪酸处理 6 小时后 RPTECs 的实时耗氧率(OCR)和细胞外酸化率(ECAR)。我们发现用油酸或棕榈酸预处理的细胞的 OCR 明显高于对照细胞(图 3I 和 S3L),这表明 FAO 活性增强。注射 CPT1 抑制剂 etomoxir 和 ATP 合酶抑制剂 oligomycin 都会降低 OCR,这证实了观察到的 OCR 增加是 FAO 增加的结果(图 S3L)。此外,在用脂肪酸预处理的细胞中也发现了 ECAR 的增加(图 31 和图 S3L)。这些结果表明,暴露于脂肪酸 6 小时会增加 FAO 和糖酵解活性,这是细胞能量活跃状态的特征(Hocaoglu 等人,2021 年)。
Next, to study cellular responses to lipid accumulation at the gene expression level, we performed bulk RNA-seq on RPTECs treated with oleic acids for 6 h (Ole_6 h). We also sequenced cells that were exposed to medium without fatty acid supplements for 2 days after the 6-h treatment (Ole_6 h + 2 days) to study the long-term effect of lipid accumulation. GO analysis indicated that the upregulated DEGs of the Ole_6 h group compared with control cells were enriched in intracellular lipid droplets (FDR = 3.84 × 10 3 = 3.84 × 10 3 =3.84 xx10^(-3)=3.84 \times 10^{-3} ). Consistently, genes associated with FAO and lipid metabolism were significantly upregulated in the Ole_6 h group (GO term FDR = 7.94 × 10 5 = 7.94 × 10 5 =7.94 xx10^(-5)=7.94 \times 10^{-5} ), including CPT1A and genes encoding long-chain acyl-CoA synthetases (ACSLs) (Figure 3J). Glucose metabolic process was also an upregulated GO term in the enrichment analysis (FDR = 5.88 × 10 3 5.88 × 10 3 5.88 xx10^(-3)5.88 \times 10^{-3} ), supporting our ECAR measurement mentioned above. Interestingly, we found that genes involved in DNA replication and cell-cycle regulation were significantly upregulated in the Ole_6 h + 2 h + 2 h+2\mathrm{h}+2 days group (GO term FDR = 6.36 × 10 14 = 6.36 × 10 14 =6.36 xx10^(-14)=6.36 \times 10^{-14} ), including MKI67, TOP2A, and genes encoding minichromosome maintenance (MCM) proteins (Figure 3J). Thus, our results suggested that fatty acid exposure and lipid accumulation in RPTECs promotes cell proliferation, consistent with our singlecell fate mapping analysis identifying that Type1 injured PT cells (enriched at uni-IRI 6 h) were precursors of Mki67-expressing PT-R cells (enriched at uni-IRI D2) (Figures 2F and 2G).
接下来,为了在基因表达水平上研究细胞对脂质积累的反应,我们对用油酸处理 6 小时(Ole_6 h)的 RPTEC 进行了大量 RNA-seq。我们还对在 6 小时处理后暴露于不含脂肪酸补充剂的培养基中 2 天(Ole_6 h + 2 天)的细胞进行了测序,以研究脂质积累的长期影响。GO分析表明,与对照组相比,Ole_6 h组上调的DEGs富集在细胞内脂滴中(FDR = 3.84 × 10 3 = 3.84 × 10 3 =3.84 xx10^(-3)=3.84 \times 10^{-3} )。同样,与 FAO 和脂质代谢相关的基因在 Ole_6 h 组显著上调(GO 项 FDR = 7.94 × 10 5 = 7.94 × 10 5 =7.94 xx10^(-5)=7.94 \times 10^{-5} ),包括 CPT1A 和编码长链酰基-CoA 合成酶(ACSLs)的基因(图 3J)。在富集分析中,葡萄糖代谢过程也是一个上调的 GO 项(FDR = 5.88 × 10 3 5.88 × 10 3 5.88 xx10^(-3)5.88 \times 10^{-3} ),这支持了我们上述的 ECAR 测量。有趣的是,我们发现参与 DNA 复制和细胞周期调控的基因在 Ole_6 h + 2 h + 2 h+2\mathrm{h}+2 天组显著上调(GO 项 FDR = 6.36 × 10 14 = 6.36 × 10 14 =6.36 xx10^(-14)=6.36 \times 10^{-14} ),包括 MKI67、TOP2A 和编码迷你染色体维护(MCM)蛋白的基因(图 3J)。因此,我们的结果表明,RPTECs 中的脂肪酸暴露和脂质积累会促进细胞增殖,这与我们的单细胞命运图谱分析一致,即 1 型损伤的 PT 细胞(富集于 uni-IRI 6 h)是表达 Mki67 的 PT-R 细胞(富集于 uni-IRI D2)的前体(图 2F 和 2G)。
PLIN2 marks lipid droplets in Type1 injured proximal tubular cells and maintains cell energy state PLIN2, also known as perilipin 2 or adipose differentiationrelated protein, is a lipid droplet surface protein and an essential component of the PPAR signaling pathway (Kimmel and Sztalryd, 2016). Our scRNA-seq data identified Plin2 as a marker gene of Type 1 injured PT cells (Figures 2B and 4A). Reanalyzing a recently published spatial transcriptomic analysis of mouse kidney bi-IRI (Dixon et al., 2022) revealed the transiently increased expression of Plin2 throughout the kidney cortex at 12 h post-surgery (Figure 4B). To further validate Plin2 as a
PLIN2标记1型损伤近端肾小管细胞中的脂滴并维持细胞能量状态 PLIN2,又称perilipin 2或脂肪分化相关蛋白,是一种脂滴表面蛋白,也是PPAR信号通路的重要组成部分(Kimmel和Sztalryd,2016年)。我们的 scRNA-seq 数据将 Plin2 鉴定为 1 型损伤 PT 细胞的标记基因(图 2B 和 4A)。重新分析最近发表的小鼠肾脏双IRI空间转录组分析(Dixon等人,2022年)发现,在手术后12小时,Plin2在整个肾皮质中的表达瞬时增加(图4B)。为了进一步验证
A
B
Plin2 expression (spatial transcriptomics)
Plin2 的表达(空间转录组学)


E DAPI BODIPY493/503 PLIN2
I

Figure 4. PLIN2 marks lipid droplets in Type1 injured proximal tubule and maintains cell energy state
图 4.PLIN2 标记 1 型损伤近端小管中的脂滴并维持细胞能量状态

(A) Specific expression of Plin2 in Type1 injured PT cells.
(A) Plin2 在 1 型损伤 PT 细胞中的特异性表达。

(B) Revisiting a spatial transcriptomics dataset on female bi-IRI kidneys identifying transient upregulation of Plin2 at kidney cortex in early stages. Each spot of a tissue section is colored by gene expression.
(B) 重新审视雌性双IRI肾脏的空间转录组学数据集,发现Plin2在早期阶段在肾皮质短暂上调。组织切片上的每个点都根据基因表达情况着色。

© Specific upregulation of PLIN2 in PT at uni-IRI 6 h validated by immunofluorescence staining of PLIN2 (red), LTL (green), and DAPI (blue) on multiple group conditions. Scale bars, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}.
在多组条件下,PLIN2(红色)、LTL(绿色)和 DAPI(蓝色)的免疫荧光染色验证了单 IRI 6 h PT 中 PLIN2 的特异性上调。比例尺, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}

(D) Immunofluorescence staining of PLIN2 (red), LTL (green), and DAPI (blue) on a tissue section collected from uni-IRI 6 h showing presence of intracellular PLIN2+ droplets. Scale bars, 10 μ m 10 μ m 10 mum10 \mu \mathrm{~m}.
(D) PLIN2(红色)、LTL(绿色)和 DAPI(蓝色)的免疫荧光染色显示细胞内存在 PLIN2+ 小滴。比例尺, 10 μ m 10 μ m 10 mum10 \mu \mathrm{~m}

(E) Immunofluorescence staining of PLIN2 (red), BODIPY493/503 (green), and DAPI (blue) on a uni-IRI 6 h tissue section showing localization of PLIN2 at the surface of lipid droplets. Scale bars, 10 μ m 10 μ m 10 mum10 \mu \mathrm{~m}. A single droplet was encircled and presented in the top-right panel.
(E) PLIN2(红色)、BODIPY493/503(绿色)和 DAPI(蓝色)在 uni-IRI 6 h 组织切片上的免疫荧光染色显示 PLIN2 定位于脂滴表面。比例尺, 10 μ m 10 μ m 10 mum10 \mu \mathrm{~m} 。单个液滴被包围并显示在右上角面板中。

marker gene for Type1 injured PT cell state, we performed immunofluorescence and identified that PLIN2 localized to intracellular basolateral droplets at uni-IRI 6 h but no other time points (Figures 4C and 4D). PLIN2 expression was absent in the time course of UUO, including at UUO 6 h (Figure 4C). To further validate that PLIN2 expression co-localized with lipid compounds, we stained PLIN2 with lipid probe BODIPY 493/503 on the uniIRI 6 h tissue and found that PLIN2 coated the surface of BODIPY+ lipid particles (Figure 4E). PLIN2 also co-localized with oxidized low-density lipoprotein (oxLDL) (Figure 4F). Together, our results identified PLIN2 as a marker Type1 injured PT cells and a surface protein of intracellular lipid droplets. Surveying a previous RNA-seq study on folic acid-induced mouse nephropathy (Craciun et al., 2016) identified upregulation of Plin2 at 1 day post injury and then gradual decreased expression (Figure S4A), suggesting that increased Plin2 expression could be induced by other types of kidney injury.
我们用免疫荧光法检测了PLIN2,发现在uni-IRI 6 h时,PLIN2定位于细胞内基底液滴,而其他时间点则没有(图4C和4D)。在 UUO 的时间过程中,包括在 UUO 6 小时,PLIN2 没有表达(图 4C)。为了进一步验证 PLIN2 的表达与脂质化合物共定位,我们用脂质探针 BODIPY 493/503 对 uniIRI 6 h 组织上的 PLIN2 进行染色,发现 PLIN2 包覆在 BODIPY+ 脂质颗粒表面(图 4E)。PLIN2 还与氧化低密度脂蛋白(oxLDL)共定位(图 4F)。总之,我们的研究结果确定 PLIN2 是 1 型损伤 PT 细胞的标志物,也是细胞内脂滴的表面蛋白。此前一项关于叶酸诱导的小鼠肾病的RNA-seq研究(Craciun等人,2016年)发现,Plin2在损伤后1天出现上调,随后表达逐渐下降(图S4A),这表明其他类型的肾损伤也可能诱导Plin2表达增加。
Next, we sought to investigate the mechanism of Plin2 upregulation in Type1 injured PT cells. Previous studies have reported that PLIN2 expression can be increased either by cellular uptake of fatty acids or endoplasmic reticulum (ER) stress (Chen et al., 2017; Dalen et al., 2006; Gao and Serrero, 1999). Therefore, we exposed FFAs or chemical ER stress inducers on RPTECs. We observed 10 10 ∼10\sim 10-fold increased expression of PLIN2 after a 6-h treatment of oleic or palmitic fatty acids by qPCR analysis (Figure 4G). PLIN2 expression was significantly reduced when cells were exposed to culture medium without fatty acid supplements for 2 days (Figure 4G), suggesting a positive correlation between PLIN2 expression level and abundance of lipids. 6-h treatment of ER stress inducers including Tunicamycin and Thapsigargin could also increase PLIN2 expression, but the fold change ( 1.5 1.5 ∼1.5-\sim 1.5- fold) was much lower than observed with fatty acid treatments (Figure S4B). We also performed immunofluorescence on fatty-acid-treated RPTECs and confirmed strongly upregulated PLIN2 protein expression in almost all cells (Figure 4H). With the bulk RNA-seq data on RPTECs mentioned above, we surveyed the gene expression of all PLIN family members (Figure S4C) and found that PLIN4 was another significantly upregulated gene ( 1.9 -fold) after 6-h oleic acid treatment, but it was much more lowly expressed compared with PLIN2 (average transcript per million [TPM] of PLIN2, 1,104.90; average TPM
接下来,我们试图研究Plin2在1型损伤的PT细胞中上调的机制。之前的研究报道,PLIN2的表达可通过细胞摄取脂肪酸或内质网(ER)应激而增加(Chen等人,2017;Dalen等人,2006;Gao和Serrero,1999)。因此,我们将脂肪酸或化学ER应激诱导剂暴露于RPTECs上。通过qPCR分析,我们观察到油酸或棕榈脂肪酸处理6小时后,PLIN2的表达增加了 10 10 ∼10\sim 10 -倍(图4G)。当细胞暴露在不含脂肪酸补充剂的培养基中2天时,PLIN2的表达明显降低(图4G),这表明PLIN2的表达水平与脂质的丰度呈正相关。6小时的ER应激诱导剂(包括Tunicamycin和Thapsigargin)处理也能增加PLIN2的表达,但其折叠变化( 1.5 1.5 ∼1.5-\sim 1.5- 折叠)远低于脂肪酸处理所观察到的(图S4B)。我们还对脂肪酸处理过的 RPTEC 进行了免疫荧光检测,结果证实几乎所有细胞中的 PLIN2 蛋白表达都强烈上调(图 4H)。利用上述 RPTECs 的大量 RNA-seq 数据,我们调查了所有 PLIN 家族成员的基因表达情况(图 S4C),发现 PLIN4 是另一个在油酸处理 6 小时后显著上调的基因(1.9 倍),但与 PLIN2 相比,它的表达量要低得多(PLIN2 的平均百万转录本 [TPM],1,104.90;PLIN4 的平均百万转录本 [TPM],1,104.90;PLIN4 的平均百万转录本 [TPM],1,104.90)。

of PLIN4, 0.93). Therefore, in vitro modeling of PLIN2 activation was consistent with our in vivo observation in uni-IRI mouse surgery, indicating that fatty acid exposure is sufficient to induce PLIN2 upregulation in PT cells.
PLIN4,0.93)。因此,PLIN2激活的体外模型与我们在单IRI小鼠手术中的体内观察结果一致,表明脂肪酸暴露足以诱导PT细胞中的PLIN2上调。
To further investigate the functional significance of PLIN2 in response to lipid accumulation, we performed PLIN2 gene knockdown with small interfering RNA (siRNA) on RPTECs. Successful gene knockdown was validated with qPCR analysis, revealing 15 15 ∼15\sim 15 - to 20 -fold decreased expression of PLIN2 in cells treated with PLIN2 siRNA (siPLIN2) both with and without fatty acid exposure compared with corresponding controls (Figure S4D). Importantly, siPLIN2 treatment did not significantly alter fatty acid uptake and lipid accumulation as demonstrated by Oil Red O staining (Figure S4E). Next, to ask whether PLIN2 was important for cellular metabolic activities, we imposed 6-h fatty acid treatment on RPTECs with or without siPLIN2 and measured real-time OCR and ECAR. In the absence of fatty acid pretreatment, we observed a significantly reduced OCR and ECAR in cells treated with siPLIN2 compared with cells treated with non-targeting control siRNA (siNT) (Figures 4 I and S4F). With 6-h oleic or palmitic acid pretreatment, both OCR and ECAR were significantly increased in siNT-treated cells (Figures 41 and S4F), consistent with our results presented in Figure 31 . By contrast, for cells treated with siPLIN2, the decreased OCR was only partially reversed by 6-h palmitic acid pretreatment and could not be increased by oleic acid exposure (Figures 4 I and S4F), implying defective lipid metabolism after PLIN2 knockdown. Both decreased OCR and ECAR suggested that siPLIN2 knockdown drove a metabolically quiescent cell state.
为了进一步研究 PLIN2 在脂质积累中的功能意义,我们用小干扰 RNA(siRNA)对 RPTECs 进行了 PLIN2 基因敲除。通过 qPCR 分析验证了基因敲除的成功,结果显示,与相应的对照组相比,用 PLIN2 siRNA(siPLIN2)处理过和没有接触过脂肪酸的细胞中 PLIN2 的表达都下降了 15 15 ∼15\sim 15 - 到 20 - 倍(图 S4D)。重要的是,油红 O 染色显示,siPLIN2 处理并没有明显改变脂肪酸摄取和脂质积累(图 S4E)。接下来,为了弄清PLIN2对细胞代谢活动是否重要,我们在有或没有siPLIN2的RPTECs上施加了6小时脂肪酸处理,并测量了实时OCR和ECAR。在没有脂肪酸预处理的情况下,我们观察到与非靶向对照 siRNA(siNT)处理的细胞相比,siPLIN2 处理的细胞的 OCR 和 ECAR 明显降低(图 4 I 和 S4F)。经 6 小时油酸或棕榈酸预处理后,siNT 处理的细胞的 OCR 和 ECAR 均明显增加(图 41 和 S4F),这与图 31 中的结果一致。相比之下,用 siPLIN2 处理的细胞,OCR 的降低仅在棕榈酸预处理 6 小时后被部分逆转,并且不能通过油酸暴露而增加(图 4 I 和 S4F),这意味着 PLIN2 敲除后的脂质代谢缺陷。OCR和ECAR的降低都表明,siPLIN2敲除导致细胞处于代谢静止状态。
Next, we exposed siPLIN2-treated RPTECs to oleic acids for 6 h (siPLIN2 + Ole6 h) and performed RNA-seq to determine the transcriptomic variations caused by gene knockdown. Compared with the siNT + Ole6 h group, the siPLIN2-treated cells upregulated genes associated with autophagy and reticulophagy (GO term FDR = 4.28 × 10 3 = 4.28 × 10 3 =4.28 xx10^(-3)=4.28 \times 10^{-3} ) such as genes encoding autophagy activating kinases ULK1/2 (Figure 4J). Previous studies have reported autophagy can induced by cell stress and nutrient deprivation in kidney and persistent activation of autophagy after kidney injury leads to maladaptive repair (Tang et al., 2020), implying that normal PLIN2 function could be essential for successful repair of Type1 injured PT cells. We also
接下来,我们将 siPLIN2 处理的 RPTECs 暴露于油酸 6 h(siPLIN2 + Ole6 h),并进行 RNA-seq 分析,以确定基因敲除引起的转录组变化。与 siNT + Ole6 h 组相比,siPLIN2 处理的细胞上调了与自噬和网状吞噬相关的基因(GO term FDR = 4.28 × 10 3 = 4.28 × 10 3 =4.28 xx10^(-3)=4.28 \times 10^{-3} ),如编码自噬激活激酶 ULK1/2 的基因(图 4J)。先前的研究表明,肾脏细胞应激和营养剥夺可诱导自噬,肾脏损伤后自噬的持续激活会导致不适应性修复(Tang 等,2020),这意味着 PLIN2 的正常功能可能对 1 型损伤 PT 细胞的成功修复至关重要。我们还
identified a decreased glucose metabolism gene profile and increased expression of genes involved in amino acid transport in siPLIN2 + Ole 6 h cells compared with siNT + Ole6 h (Figure 4J). The reduced expression of genes responsible for glycolysis (GO term FDR = 4.22 × 10 3 = 4.22 × 10 3 =4.22 xx10^(-3)=4.22 \times 10^{-3} ) such as ENO1/2 and HK1/2 was consistent with our observation of decreased ECAR after PLIN2 knockdown (Figure 4I).
与 siNT + Ole6 h 相比,siPLIN2 + Ole 6 h 细胞中葡萄糖代谢基因谱减少,参与氨基酸转运的基因表达增加(图 4J)。负责糖酵解的基因(GO term FDR = 4.22 × 10 3 = 4.22 × 10 3 =4.22 xx10^(-3)=4.22 \times 10^{-3} )如 ENO1/2 和 HK1/2 的表达减少与我们观察到的 PLIN2 敲除后 ECAR 的减少一致(图 4I)。
In the above analysis, we found that genes associated with DNA replication and cell-cycle regulation were upregulated in Ole_6 h + 2 days cells compared with control cells (Figure 3J). We wondered whether PLIN2 knockdown undermined this cellular event as it disrupted the metabolic cellular response after 6-h fatty acid exposure. Thus, cells exposed to normal culture medium for 2 days after the 6-h oleic acid treatment (siPLIN2 + Ole6 h + 2 h + 2 h+2\mathrm{h}+2 days) were analyzed by RNA-seq. Compared with siNT + Ole6 h + 2 days, we found that DNA replication was a downregulated GO term in siPLIN2+Ole6 h + 2 h + 2 h+2\mathrm{h}+2 days cells (FDR = 7.72 × 10 5 = 7.72 × 10 5 =7.72 xx10^(-5)=7.72 \times 10^{-5} ), reflected by decreased expression of genes associated with DNA primase activity (e.g., GINS1/2 and PRIM2), DNA polymerase regulation (e.g., RFC2/5 and PCNA) and genes encoding MCM proteins (Figure S4G). Therefore, this RNA-seq analysis indicated that PLIN2 knockdown reduced activities of DNA replication and cell proliferation after 6-h fatty acid uptake.
在上述分析中,我们发现与对照细胞相比,与DNA复制和细胞周期调控相关的基因在Ole_6 h + 2天细胞中上调(图3J)。我们想知道,PLIN2 基因敲除是否会破坏这一细胞事件,因为它破坏了脂肪酸暴露 6 小时后的细胞代谢反应。因此,在6小时油酸处理后暴露于正常培养基2天(siPLIN2 + Ole6 h + 2 h + 2 h+2\mathrm{h}+2 天)的细胞进行了RNA-seq分析。与 siNT + Ole6 h + 2 天相比,我们发现在 siPLIN2+Ole6 h + 2 h + 2 h+2\mathrm{h}+2 天细胞中,DNA 复制是一个下调的 GO 项(FDR = 7.72 × 10 5 = 7.72 × 10 5 =7.72 xx10^(-5)=7.72 \times 10^{-5} ),这反映在与 DNA 引物酶活性(如 GINS1/2 和 PRIM2)、DNA 聚合酶调控(如 RFC2/5 和 PCNA)和编码 MCM 蛋白的基因的表达减少上(图 S4G)。因此,RNA-seq分析表明,PLIN2基因敲除会降低6小时脂肪酸摄取后的DNA复制和细胞增殖活性。
Taken together, our results highlight PLIN2 as a marker of intracellular lipid droplets in Type1 injured PT cells and knockdown studies show that PLIN2 regulates energy homeostasis in PT cells.
综上所述,我们的研究结果表明,PLIN2 是 1 型损伤 PT 细胞中细胞内脂滴的标记物,而基因敲除研究表明 PLIN2 调节 PT 细胞的能量平衡。

Metabolic variations in Type2 injured PT cells
2 型受伤 PT 细胞的代谢变化

To better characterize the metabolic consequences of Type2 injured PT, we performed PPI enrichment analysis and identified amino acid metabolism as a downregulated pathway compared with healthy PT (Figure S5A), consistent with previous studies reporting defective amino acid metabolisms in CKD (Garibotto et al., 2010; Kang et al., 2014). We also identified several genes associated with amino acid transport and catalysis that were among top upregulated markers of S3 segment cells of Type2 injured PT, including Bcat1, S/c6a6, and Slc7a12 (Figure 5A). A survey of the Human Protein Atlas (Uhlén et al., 2015) confirmed that the proteins encoded by these genes were expressed in the renal tubule. Revisiting a published RNA-seq work on PT-enriched transcripts of UUO mice (Wu et al., 2020) further validated increased expression of Bcat1, S/c6a6, and S/c7a12, as well as other DEGs of Type2 injured PT, in UUO D5/10 mouse kidneys than contralateral control kidneys (Figure S5B).
为了更好地描述 2 型损伤 PT 的代谢后果,我们进行了 PPI 富集分析,发现与健康 PT 相比,氨基酸代谢是一个下调途径(图 S5A),这与之前报告 CKD 中氨基酸代谢缺陷的研究一致(Garibotto 等,2010 年;Kang 等,2014 年)。我们还发现了几个与氨基酸转运和催化相关的基因,这些基因是2型损伤PT的S3节段细胞的最高上调标志物,包括Bcat1、S/c6a6和Slc7a12(图5A)。对人类蛋白质图谱(Human Protein Atlas)的调查(Uhlén 等人,2015 年)证实,这些基因编码的蛋白质在肾小管中表达。对已发表的关于 UUO 小鼠 PT 富集转录本的 RNA-seq 研究(Wu 等人,2020 年)进行重访,进一步验证了 Bcat1、S/c6a6 和 S/c7a12 以及其他 2 型损伤 PT 的 DEGs 在 UUO D5/10 小鼠肾脏中的表达量比对侧对照肾脏高(图 S5B)。
Bcat1 is branched-chain amino acid (BCAA) transaminase 1 and it is responsible for transamination of BCAAs (including leucine, isoleucine, and valine) resulting in production of branched chain keto acids (BCKAs) and glutamate (Adeva et al., 2011). Immunostaining results verified that the expression of BCAT1 was specific to PT cells and mostly limited to UUO (Figure 5B), where Type2 injured cells were highly abundant. The upregulation of BCAT1, compared with healthy controls, was also observed in UUO D10/14 (Figures 5B and S5C), in which the Type2 state still existed. In addition, we found that genes that are responsible for BCKA catalysis, including Bckdha, Bckdhb, and Ppm1k, were downregulated in injured PT
Bcat1是支链氨基酸(BCAA)转氨酶1,负责BCAA(包括亮氨酸、异亮氨酸和缬氨酸)的转氨作用,从而产生支链酮酸(BCKAs)和谷氨酸(Adeva等人,2011年)。免疫染色结果证实,BCAT1的表达特异于PT细胞,且主要局限于UUO(图5B),在UUO中,2型损伤细胞大量存在。与健康对照组相比,在UUO D10/14中也观察到了BCAT1的上调(图5B和S5C),其中2型状态仍然存在。此外,我们还发现,负责 BCKA 催化的基因,包括 Bckdha、Bckdhb 和 Ppm1k,在损伤的 PT

compared with healthy PT (Figure S5D), confirming a recent report (Piret et al., 2021). Next, we measured the concentration of BCAAs in mouse kidney cortical tissues across multiple time points. We identified an increased BCAA accumulation during the UUO time course, and the concentration in the uni-IRI time course was not changed too much (Figure 5C). Re-analyzing a previous human dataset (Nakagawa et al., 2015) confirmed a significantly increased BCAT1 expression in patients with CKD than controls (Figure 5D).
与健康 PT 相比(图 S5D),这证实了最近的一份报告(Piret 等人,2021 年)。接下来,我们测量了多个时间点小鼠肾皮质组织中 BCAAs 的浓度。我们发现在 UUO 时间过程中 BCAA 积累增加,而在 uni-IRI 时间过程中浓度变化不大(图 5C)。重新分析之前的人类数据集(Nakagawa 等人,2015 年)证实,与对照组相比,CKD 患者的 BCAT1 表达量显著增加(图 5D)。
SLC6A6, also known as TauT, is a transporter of the sulfurcontaining amino acid taurine, and the accumulation of taurine has been described in patients with kidney failure (Mozaffari, 2003; Suliman et al., 2002). We found the expression of SLC6A6 was also significantly elevated in patients with CKD than healthy controls (Figure 5E). Next, we validated the increased expression of S/c6a6 in Type2 injured PT by RNA in situ hybridization (RNA ISH), in which the expression of S/c6a6 was not observed in healthy and uni-IRI samples, but was upregulated as early as at UUO D2 in outer stripe of the outer medulla (OSOM) of kidney, where S3 segment of PT cells are supposed to locate, (Figure 5F). We also co-stained SIc6a6 with Havcr1 and validated the expression of S/c6a6 in Havcr1-expressing injured PT (Figure S5E). S/c7a12 encodes a transporter for cationic amino acids and recent work demonstrated that S/c7a12 was present in kidney PT in disease and the upregulation was accompanied by emergence of Vcam1-expressing FR-PTC (Gerhardt et al., 2021), concordant with our analysis identifying the high probability of Type2 injured PT differentiating into FR-PTC (Figures 2F and 2G). Taken together, these results suggest that Type2 PT cells are characterized by dysregulated amino acid metabolisms.
SLC6A6 又称 TauT,是含硫氨基酸牛磺酸的转运体,已有肾衰竭患者出现牛磺酸蓄积的描述(Mozaffari,2003;Suliman 等人,2002)。我们发现,与健康对照组相比,SLC6A6 在 CKD 患者中的表达也明显升高(图 5E)。接下来,我们通过 RNA 原位杂交(RNA ISH)验证了 S/c6a6 在 2 型损伤 PT 中表达的增加,其中 S/c6a6 的表达在健康样本和单 IRI 样本中均未观察到,但早在 UUO D2 阶段,S/c6a6 的表达就在肾脏外髓质的外侧条纹(OSOM)中上调,而 S3 段 PT 细胞应该位于外侧条纹(图 5F)。我们还将 SIc6a6 与 Havcr1 共同染色,验证了 S/c6a6 在表达 Havcr1 的损伤 PT 中的表达(图 S5E)。S/c7a12 编码阳离子氨基酸转运体,最近的研究表明,S/c7a12 存在于患病的肾脏 PT 中,其上调伴随着表达 Vcam1 的 FR-PTC 的出现(Gerhardt 等人,2021 年),这与我们的分析结果一致,即 2 型损伤 PT 极有可能分化为 FR-PTC(图 2F 和 2G)。综上所述,这些结果表明,2 型 PT 细胞的特点是氨基酸代谢失调。

Shared and unique cellular response of tubular epithelia in fibrogenesis
纤维形成过程中肾小管上皮细胞共同和独特的细胞反应

Our analysis revealed that most injured PT cells in uni-IRI repair (Figure 2E, left panel), whereas they do not in UUO (Figure 2E, right panel). We next sought to compare shared and unique responses to injury across tubular epithelial cell types. We identified multiple subtypes of LoH (Figure 6A) and cells of the distal nephron, including DCT, CNT, and PCs of collecting duct (Figure 6B) and Type A and Type B intercalated cells of collecting duct (Figure S6A). Subclustering of LoH showed TAL to be the abundant population, and it expressed marker genes such as SIc12a1 and Umod (Figure 6C). TAL cells could also be stratified by their cortical (Kng2/Thsd4 high) or medullary (Mrps6/ Tmem207 high), as could PCs of the collecting duct (medullary PC2, Pcdh7 high; cortical PC3, Mgat4c high) (Figure S6B). Interestingly, we found another group of TAL-expressing healthy marker genes at a lower level and showing enhanced expression of a well-known injury marker Lcn2 (also known as Ngal) that we annotated as injured TAL (TAL-inj). TAL-inj also showed upregulation of Kctd1, a gene that regulates reabsorption of paracellular urinary Ca 2 + / Mg 2 + Ca 2 + / Mg 2 + Ca^(2+)//Mg^(2+)\mathrm{Ca}^{2+} / \mathrm{Mg}^{2+} and performs a protective role in kidney fibrosis (Marneros, 2020, 2021). Compared with healthy TAL, cells of TAL-inj showed increased expression of genes associated with profibrotic and proinflammatory signaling, such as Tgfbr1, Map3k1, Stat3, and Myh9 (Figure S6C). GO enrichment analysis presented terms that also appeared in injured PT, such as cell junction organization, actin cytoskeleton regulation,
我们的分析表明,在单IRI修复过程中,大多数受伤的PT细胞会修复(图2E,左侧面板),而在UUO中则不会(图2E,右侧面板)。接下来,我们试图比较不同类型的肾小管上皮细胞对损伤的共同和独特反应。我们确定了 LoH 的多种亚型(图 6A)和远端肾小管的细胞,包括集合管的 DCT、CNT 和 PCs(图 6B)以及集合管的 A 型和 B 型夹层细胞(图 S6A)。LoH的亚聚类显示TAL是大量的细胞群,它表达SIc12a1和Umod等标记基因(图6C)。TAL 细胞也可按皮质(Kng2/Thsd4 高)或髓质(Mrps6/ Tmem207 高)分层,集合管的 PCs 也是如此(髓质 PC2,Pcdh7 高;皮质 PC3,Mgat4c 高)(图 S6B)。有趣的是,我们发现另一组 TAL 表达健康标记基因的水平较低,而著名的损伤标记基因 Lcn2(又称 Ngal)表达增强,我们将其注释为损伤 TAL(TAL-inj)。TAL-inj还显示出Kctd1的上调,Kctd1是一种调节尿液旁 Ca 2 + / Mg 2 + Ca 2 + / Mg 2 + Ca^(2+)//Mg^(2+)\mathrm{Ca}^{2+} / \mathrm{Mg}^{2+} 重吸收的基因,在肾脏纤维化中起保护作用(Marneros,2020,2021)。与健康的TAL相比,TAL-inj细胞中与促纤维化和促炎症信号转导相关的基因,如Tgfbr1、Map3k1、Stat3和Myh9的表达增加了(图S6C)。GO富集分析显示的术语也出现在受伤的PT中,如细胞连接组织、肌动蛋白细胞骨架调控等、

Figure 5. Dysregulation of genes involved in amino acid metabolisms in Type2 injured PT cells
图 5.2 型损伤 PT 细胞中涉及氨基酸代谢的基因失调

(A) Dot plot showing that three amino acid metabolism-associated genes, Bcat1, S/c6a6, and S/c7a12, were specifically upregulated in Type2 injured PT.
(A) 点阵图显示,Bcat1、S/c6a6 和 S/c7a12 这三个氨基酸代谢相关基因在 2 型损伤 PT 中特异性上调。

(B) Specific upregulation of BCAT1 in PT after UUO validated by immunofluorescence staining of BCAT1 (red), LTL (green), and DAPI (blue) on multiple group conditions. Outer medulla regions are presented. Scale bars, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}.
(B) 通过对 BCAT1(红色)、LTL(绿色)和 DAPI(蓝色)进行免疫荧光染色,验证了 UUO 后 PT 中 BCAT1 的特异性上调。图示为外髓区域。比例尺, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}

© Concentrations of branched chain amino acids (BCAAs) measured in mouse cortical tissues across all group conditions of this study cohort showing significantly increased BCAA concentration in UUO kidneys. Data are shown as the mean ± ± +-\pm SEM. p < 0.0001 p < 0.0001 ^(********)p < 0.0001{ }^{* * * *} \mathrm{p}<0.0001 by Student’s t test.
本研究中所有组别小鼠皮质组织中测定的支链氨基酸(BCAAs)浓度显示,UUO 肾脏中的 BCAA 浓度显著增加。数据以平均值 ± ± +-\pm SEM表示。 p < 0.0001 p < 0.0001 ^(********)p < 0.0001{ }^{* * * *} \mathrm{p}<0.0001 采用学生 t 检验。

( D and E) Violin plots showing increased expression of BCAT1 (D) and SLC6A6 (E) in biopsy samples of patients with CKD than controls.
( D 和 E) Violin 图显示,与对照组相比,CKD 患者活检样本中 BCAT1(D)和 SLC6A6(E)的表达量增加。

(F) Representative images of RNA in situ hybridization staining of S/c6a6 on multiple group conditions revealing gene upregulation in UUO. Scale bars, 200 μ m 200 μ m 200 mum200 \mu \mathrm{~m} for the upper panel and 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m} for the lower panel. Cortical ©, outer stripe of the outer medulla (OSOM), and medullary (M) regions were highlighted. See also Figure S5E.
(F) 多组条件下 S/c6a6 RNA 原位杂交染色的代表性图像,显示 UUO 中的基因上调。上图的比例尺为 200 μ m 200 μ m 200 mum200 \mu \mathrm{~m} ,下图的比例尺为 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m} 。皮质©、外髓质外侧条纹(OSOM)和髓质(M)区域突出显示。另见图 S5E。

Figure 6. Shared and unique injury responses of renal tubular epithelial cells
图 6.肾小管上皮细胞共同和独特的损伤反应

(A and B) UMAP plots of cells of LoH (A) and DCT, CNT, and PC (B) in subclustering analysis. Abbreviations of cell types have been described in Figure 1D. ( C C CC and D D DD ) Dot plots showing expression of genes specific to cell clusters identified in (A) and (B). Visualization was performed on dataset combining both uni-IRI and UUO subsets.
(A 和 B)亚聚类分析中 LoH 细胞(A)和 DCT、CNT 和 PC 细胞(B)的 UMAP 图。细胞类型缩写见图 1D。( C C CC D D DD ) 点阵图显示了(A)和(B)中确定的细胞簇特异基因的表达。可视化是在结合了 uni-IRI 和 UUO 子集的数据集上进行的。

(E) Connected bar plots displaying the proportional abundance of healthy and injured TECs (including TAL, DCT, and CNT) in each group condition, which identifies a shared injury response of TECs in an insult-dependent manner.
(E)连接的柱状图显示了各组条件下健康和损伤的 TEC(包括 TAL、DCT 和 CNT)的丰度比例,这表明 TEC 以损伤依赖的方式做出了共同的损伤反应。

(F) Heatmaps presenting expression of genes that are either co-varied across all injured TECs or dysregulated in a cell-type-specific manner compared with the healthy state of each TEC.
(F) 热图显示了与健康状态相比,所有受伤的 TEC 基因表达共变或以细胞类型特异性的方式表达失调的基因。

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cell migration, and epithelial cell differentiation (Figure S6D). Using a similar approach, we identified injured DCT (DCT-inj) and CNT (CNT-inj) both of which showed downregulated healthy marker genes (e.g., Slc12a3 for DCT and S/c8a1 for CNT) and increased expression of fibrotic genes (Figures 6D and S6E). We identified Trpv5, a gene encoding a calcium channel essential for Ca 2 + Ca 2 + Ca^(2+)\mathrm{Ca}^{2+} reabsorption in kidney (De Groot et al., 2008), as upregulated in both DCT-inj and CNT-inj.
细胞迁移和上皮细胞分化(图 S6D)。使用类似的方法,我们鉴定了损伤的DCT(DCT-inj)和CNT(CNT-inj),这两种基因都显示健康标记基因下调(如DCT的Slc12a3和CNT的S/c8a1)和纤维化基因表达增加(图6D和S6E)。我们发现,编码肾脏 Ca 2 + Ca 2 + Ca^(2+)\mathrm{Ca}^{2+} 重吸收所必需的钙通道的基因Trpv5(De Groot等人,2008年)在DCT-inj和CNT-inj中均上调。
We next surveyed the transition between these epithelial cells in health and disease across the full time course of either uni-IRI and UUO. We found that injury cell states (i.e., TAL-inj, DCT-inj, and CNT-inj) were largely absent in healthy kidneys (Figure 6E), but as expected, their numbers increased after either insult. Similar to PT, injured TAL, DCT, and CNT took on a transient injury state but then repaired at later time points, whereas these same cell types remained injured through the UUO time course (Figure 6E).
接下来,我们调查了这些上皮细胞在健康和疾病状态下,在单IRI和UUO的整个过程中的转变情况。我们发现,健康肾脏中基本不存在损伤细胞状态(即 TAL-inj、DCT-inj 和 CNT-inj)(图 6E),但正如预期的那样,它们的数量在两种损伤后都有所增加。与 PT 相似,受伤的 TAL、DCT 和 CNT 呈短暂的损伤状态,但随后会在较后的时间点修复,而这些相同类型的细胞在整个 UUO 时间过程中仍处于损伤状态(图 6E)。
We examined the DEGs for each injured subtype compared with its healthy state and identified those that were common to all injury states (Figure 6F, left panel). For example, Spp1 encodes osteopontin, a pleiotropic glycoprotein, which is induced in both AKI and CKD and is important for tubulogenesis (Kaleta, 2019; Khamissi et al., 2022; Wu et al., 2022). Here, its upregulation was observed not only in TAL/DCT/ CNT, but also in PT, though the expression is more increased in Type2 injured PT than the Type1 state. We also identified Nrg1, which modifies EGFR signaling, as a gene that co-varied across injury states (Harskamp et al., 2016). Some identified genes have poorly understood functions in epithelia, such as Syne2, which contributes to maintenance of the nuclear envelope structure, though its role in cell proliferation in skin wound healing has also been noted (Rashmi et al., 2012). We also identified Wwc1 as an injury-associated gene and its protein product (also known as KIBRA, kidney- and brain-expressed protein) is an upstream regulator of Hippo pathway and KIBRA overexpression can disrupt cytoskeleton of podocytes via inhibiting YAP signaling (Meliambro et al., 2017). Each injured nephron segment also expressed transcripts unique to that segment (Figure 6F, right panel), such as upregulation of Rbms3 (encoding a c-Myc single-strand-binding protein; Penkov et al., 2000) in TAL-inj and decreased expression of Plcl1 (encoding a regulator of GABA ( A ) GABA ( A ) GABA(A)\operatorname{GABA}(\mathrm{A}) receptors; Kanematsu et al., 2007) in CNT-inj.
我们研究了每种损伤亚型的 DEGs 与其健康状态的 DEGs,并确定了所有损伤状态下的共同 DEGs(图 6F,左侧面板)。例如,Spp1编码骨蛋白,这是一种多向糖蛋白,在AKI和CKD中都会被诱导,对肾小管生成很重要(Kaleta,2019;Khamissi等人,2022;Wu等人,2022)。在这里,不仅在 TAL/DCT/ CNT 中观察到其上调,在 PT 中也观察到其上调,但在 2 型损伤 PT 中的表达比 1 型状态下更高。我们还发现,改变表皮生长因子受体(EGFR)信号转导的 Nrg1 也是一个在不同损伤状态下共同变化的基因(Harskamp 等人,2016 年)。一些已发现的基因在上皮细胞中的功能尚不清楚,如 Syne2,它有助于维持核膜结构,但它在皮肤伤口愈合的细胞增殖中的作用也已被注意到(Rashmi 等人,2012 年)。我们还发现 Wwc1 是一种损伤相关基因,其蛋白产物(又称 KIBRA,肾和脑表达蛋白)是 Hippo 通路的上游调节因子,KIBRA 过表达可通过抑制 YAP 信号转导破坏荚膜细胞的细胞骨架(Meliambro 等人,2017 年)。每个受伤的肾小管节段也表达该节段特有的转录本(图 6F 右侧面板),如 TAL-inj 中 Rbms3(编码一种 c-Myc 单链结合蛋白;Penkov 等人,2000 年)上调,CNT-inj 中 Plcl1(编码一种 GABA ( A ) GABA ( A ) GABA(A)\operatorname{GABA}(\mathrm{A}) 受体调节因子;Kanematsu 等人,2007 年)表达减少。

Heterogeneity of kidney stroma
肾脏基质的异质性

In response to injury, kidney resident pericytes and fibroblasts proliferate and differentiate into myofibroblasts with increased cell motility and extracellular matrix (ECM) deposition, contributing to kidney fibrosis (Kuppe et al., 2021; Sato and Yanagita, 2017). However, it remains unclear whether the fibroblasts or myofibroblasts are homogeneous populations or heterogeneous groups with subtypes performing distinct functions (Humphreys, 2018). Therefore, we next aimed to characterize kidney stromal cell heterogeneity.
为应对损伤,肾脏常驻周细胞和成纤维细胞增殖并分化为肌成纤维细胞,细胞运动性和细胞外基质(ECM)沉积增加,导致肾脏纤维化(Kuppe 等人,2021 年;Sato 和 Yanagita,2017 年)。然而,目前仍不清楚成纤维细胞或肌成纤维细胞是同质群体还是具有不同功能亚型的异质群体(Humphreys,2018)。因此,我们接下来的目标是鉴定肾脏基质细胞的异质性。
Subclustering of fibroblasts and myofibroblasts led to identification of multiple subtypes of kidney stroma including Ren1-expressing JGA cells (Figures 7A and S7A). Three clusters showed elevated expression of Acta2 and Col1a1, classic myofibroblast marker genes (Myo-2/3/4) (Figures 7A and S7A). One subpopulation (Myo-1) exhibited high transcriptomic similarity with Myo 2 / 3 / 4 2 / 3 / 4 2//3//42 / 3 / 4 and showed increased expression of multiple myosin genes (Figure S7B), suggesting an enhanced capacity for cell migration and contraction, so we considered this as a myofibroblast subtype as well. We annotated the remaining clusters as fibroblasts (Fib-1/2/3) due to the presence of fibroblast marker genes and their high abundance in healthy kidneys (Figure 7B). Time course analysis revealed that in uni-IRI, the total number of (myo)fibroblasts peaked at day 2 and was then decreased moderately with time (Figure S7C, left panel), whereas in UUO, myofibroblasts accumulated across the time course accounting for over 30% of the total kidney cells at D14 (Figure S7C, right panel).
对成纤维细胞和肌成纤维细胞进行亚群分类,发现了肾基质的多种亚型,包括表达 Ren1 的 JGA 细胞(图 7A 和 S7A)。有三个细胞群显示典型的肌成纤维细胞标记基因(Myo-2/3/4)Acta2 和 Col1a1 表达升高(图 7A 和 S7A)。一个亚群(Myo-1)表现出与 Myo 2 / 3 / 4 2 / 3 / 4 2//3//42 / 3 / 4 高度的转录组相似性,并显示多个肌球蛋白基因的表达增加(图 S7B),表明细胞迁移和收缩能力增强,因此我们将其也视为肌成纤维细胞亚型。由于成纤维细胞标记基因的存在及其在健康肾脏中的高丰度(图 7B),我们将其余的集群注释为成纤维细胞(Fib-1/2/3)。时间进程分析表明,在 uni-IRI 中,(肌)成纤维细胞的总数在第 2 天达到峰值,然后随着时间的推移适度减少(图 S7C,左侧面板),而在 UUO 中,肌成纤维细胞在整个时间进程中不断积累,在第 14 天时占肾脏细胞总数的 30% 以上(图 S7C,右侧面板)。
Kidney stromal heterogeneity included differences in regional localization. We identified Fib-1/2 as cortical fibroblasts (Itih5 high) and Fib-3 and Myo-4 as medullary (Spon1/Bmpr1b high) (Figure S7A). We found that Myo-4 specifically expressed Prickle1 (Figure S7A), which encodes a nuclear receptor that regulates cell polarity and is involved in Wnt signaling (Yang et al., 2013). Immunofluorescence analysis on UUO D10 tissues confirmed that PRICKLE1 was specifically expressed on nuclear membranes of α α alpha\alpha-SMA+ myofibroblasts in the inner medulla, but not in cortical regions (Figure 7C). The regional heterogeneity of stromal cells was further confirmed by spatial transcriptomic analysis of an existing dataset (Dixon et al., 2022), which indicated a higher expression of Itih5 in cortex than medulla and me-dulla-specific expression of Spon1 and Bmpr1b (Figures 7D and S7D).
肾脏基质异质性包括区域定位的差异。我们确定 Fib-1/2 为皮质成纤维细胞(Itih5 高),Fib-3 和 Myo-4 为髓质成纤维细胞(Spon1/Bmpr1b 高)(图 S7A)。我们发现Myo-4特异性表达Prickle1(图S7A),Prickle1编码一种调节细胞极性并参与Wnt信号转导的核受体(Yang等人,2013年)。UUO D10组织的免疫荧光分析证实,PRICKLE1在内侧髓质的 α α alpha\alpha -SMA+肌成纤维细胞的核膜上特异表达,而在皮质区域则没有(图7C)。对现有数据集(Dixon 等人,2022 年)进行的空间转录组学分析进一步证实了基质细胞的区域异质性,该分析表明皮质中 Itih5 的表达高于髓质,Spon1 和 Bmpr1b 的表达也具有髓质特异性(图 7D 和 S7D)。
Figure 7. Heterogeneity of kidney stromal cells and cell-cell communications in kidney fibrogenesis
图 7:肾脏纤维形成过程中肾脏基质细胞的异质性和细胞间的通讯肾脏纤维形成过程中肾脏基质细胞的异质性和细胞间的交流

(A) UMAP plot of all stromal subtypes identified in subclustering analysis. Fib, fibroblast; Myo, myofibroblast; JGA, juxtaglomerular apparatus. See also Figure S7A.
(A) 亚聚类分析中确定的所有基质亚型的 UMAP 图。Fib:成纤维细胞;Myo:肌成纤维细胞;JGA:并集器。另见图 S7A。

(B) Condition map showing unique distribution of stromal cells in different experimental groups.
(B) 显示不同实验组基质细胞独特分布的条件图。

© Immunofluorescence staining of DAPI (blue), α α alpha\alpha-SMA (i.e., ACTA2) (green), PRICKLE1 (red), and LTL (white) on a tissue section collected from UUO D10 identifying PRICKLE1 expression on nuclear membranes of myofibroblasts in kidney medulla. Scale bars, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}.
从 UUO D10 采集的组织切片上的 DAPI(蓝色)、 α α alpha\alpha -SMA(即 ACTA2)(绿色)、PRICKLE1(红色)和 LTL(白色)免疫荧光染色显示 PRICKLE1 在肾髓质肌成纤维细胞核膜上的表达。比例尺, 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m}

(D) Expression of two region-specific genes in a spatial transcriptomics dataset on female bi-IRI kidneys. Each spot of a tissue section is colored by gene expression. See also Figure S7D.
(D) 雌性双 IRI 肾脏空间转录组学数据集中两个区域特异性基因的表达。组织切片上的每个点都根据基因表达情况着色。另见图 S7D。

(E) Gene module activities on myosin, mitochondrial respiratory chain reactions, extracellular matrix (ECM) and heat-shock proteins (HSPs) in each stromal subtype. Gene module scores are shown as means. For the convenience of data visualization, normalization is performed by adjusting the lowest score of each module as 0 .
(E) 各基质亚型中肌球蛋白、线粒体呼吸链反应、细胞外基质(ECM)和热休克蛋白(HSPs)的基因模块活动。基因模块得分以平均值表示。为便于数据可视化,将每个模块的最低分值调整为 0,从而进行归一化处理。

(F) Heatmap showing the number of significant ligand-receptor pairs in cell-cell interaction (CCI) analysis, predicted by CellPhoneDB, between major kidney cell types. Log-transformed data are shown. Populations with similar transcriptomics are combined for the convenience of data visualization.
(F) 热图显示 CellPhoneDB 预测的主要肾细胞类型之间细胞-细胞相互作用(CCI)分析中重要配体-受体对的数量。显示的是对数转换数据。为便于数据可视化,合并了具有相似转录组学的群体。

(G) Numbers of significant CCIs identified by CellPhoneDB across the time courses of uni-IRI and UUO.
(G) CellPhoneDB 在 uni-IRI 和 UUO 的时间进程中识别出的重要 CCIs 数量。

(H) Connected bar plots displaying the number of significant CCls between (myo)fibroblasts and PT cells in each group condition. Fibroblast and myofibroblast are combined to increase robustness of data analysis. PT_injury combines PT-AcInj, PT-R and Type1/2 injured PT cells.
(H)连线条形图显示各组条件下(肌)成纤维细胞和 PT 细胞之间显著的 CCls 数量。成纤维细胞和肌成纤维细胞合并在一起,以提高数据分析的稳健性。PT_injury 结合了 PT-AcInj、PT-R 和 Type1/2 损伤 PT 细胞。
Next, we assessed functional differences between myofibroblast subtypes. In addition to high expression of myosin genes (Figures 7E and S7B), Myo-1 cells showed increased expression of / / 34 / / 34 ////34/ / 34 and components of the ERK/MAPK pathway (Figures S7A and S7E), indicating their inflammatory properties (Boström and Lundberg, 2013; Pat et al., 2003; Shoji et al., 2016). For Myo-2 cells, we observed upregulation of l/31ra (Figure S7A), a gene that is crucial for IL-31 signaling and has been found overexpressed in dermal fibroblasts of patients with systemic sclerosis (Kuzumi et al., 2021). Importantly, Myo-2 cells exhibited significantly enhanced activity of the mitochondrial respiratory chain as indicated by gene module scoring analysis (Mann-Whitney U test) (Figure 7E), including subunits of NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome c (CYC) reductase, CYC oxidase, and ATP synthase (Figure S7F). A majority of heat-shock-protein-encoding genes were also upregulated in Myo-2 (Figures 7E and S7G), indicating that these cells performed highly active metabolic activities and stress response. In addition, even though all these myofibroblast subtypes had increased expression of Acta2 and Col1a1 compared with the other kidney cell types, we noticed that one myofibroblast cluster, Myo-3, exhibited the highest expression of these genes and ECM deposition score (Figure 7E), including elevated expression of various glycoproteins, collagens, and proteoglycans (Figure S 7 H ). Therefore, we annotated myofibroblast (Myo-3) as the major population responsible for ECM synthesis in kidney fibrosis. Although highly abundant collagens (i.e., COL1 and COL3) were mostly detected in Myo-3, we found that genes encoding rare collagens, such as Col6a3, Col6a4, Col7a1, and Col9a1, were produced in myofibroblast group Myo-4 (Figure S71), highlighting potential functional differences within myofibroblast subtypes.
接下来,我们评估了肌成纤维细胞亚型之间的功能差异。除了肌球蛋白基因的高表达(图 7E 和 S7B)外,Myo-1 细胞还显示出 / / 34 / / 34 ////34/ / 34 和 ERK/MAPK 通路成分的表达增加(图 S7A 和 S7E),这表明它们具有炎症特性(Boström 和 Lundberg,2013 年;Pat 等人,2003 年;Shoji 等人,2016 年)。对于 Myo-2 细胞,我们观察到了 l/31ra 的上调(图 S7A),该基因对 IL-31 信号转导至关重要,在系统性硬化症患者的真皮成纤维细胞中发现了该基因的过度表达(Kuzumi 等人,2021 年)。重要的是,通过基因模块评分分析(曼-惠特尼U检验),Myo-2细胞的线粒体呼吸链活性明显增强(图7E),包括NADH:泛醌氧化还原酶、泛醌-细胞色素c(CYC)还原酶、CYC氧化酶和ATP合成酶的亚基(图S7F)。大多数热休克蛋白编码基因也在Myo-2中上调(图7E和S7G),表明这些细胞进行着高度活跃的代谢活动和应激反应。此外,尽管与其他肾脏细胞类型相比,所有这些肌成纤维细胞亚型的 Acta2 和 Col1a1 表达量都有所增加,但我们注意到,其中一个肌成纤维细胞集群 Myo-3 的这些基因表达量和 ECM 沉积得分最高(图 7E),包括各种糖蛋白、胶原和蛋白多糖的表达量升高(图 S 7H)。因此,我们将肌成纤维细胞(Myo-3)命名为肾脏纤维化中负责 ECM 合成的主要群体。 虽然高含量胶原(即 COL1 和 COL3)大多在 Myo-3 中检测到,但我们发现编码罕见胶原(如 Col6a3、Col6a4、Col7a1 和 Col9a1)的基因在肌成纤维细胞组 Myo-4 中产生(图 S71),这突显了肌成纤维细胞亚型内部潜在的功能差异。
Dynamics of cell-cell interactions in kidney fibrogenesis Intercellular communication drives kidney fibrosis. We analyzed cell-cell interaction (CCI) activity across all major cell types based on their ligand-receptor transcriptomic signature and identified that fibroblast and myofibroblast displayed the strongest capacity to interact with other cell types (Figure 7F). We also observed higher CCI activity in diseased PT cells (e.g., Type1/2 injured PT and FR-PTC) compared with healthy PT (Figure 7F). We calculated CCI scores across the uni-IRI and UUO time courses and found that the total number of significant CCls was low in health but increased after injury (Figure 7G). Specifically, in uni-IRI, the number of interactions peaked at day 2 and then gradually decreased, and in UUO, we observed an increasing activity of CCl which reached highest level at around day 10 (Figure 7G).
肾脏纤维化过程中的细胞-细胞相互作用动力学 细胞间通信驱动肾脏纤维化。我们根据配体-受体转录组特征分析了所有主要细胞类型的细胞-细胞相互作用(CCI)活性,发现成纤维细胞和肌成纤维细胞与其他细胞类型相互作用的能力最强(图 7F)。我们还观察到,与健康 PT 相比,患病 PT 细胞(如 1/2 型损伤 PT 和 FR-PTC)的 CCI 活性更高(图 7F)。我们计算了单IRI和UUO时间进程中的CCI评分,发现健康时有意义的CCl总数较少,但损伤后有所增加(图7G)。具体而言,在单IRI中,相互作用的数量在第2天达到峰值,然后逐渐减少;在UUO中,我们观察到CCl的活性不断增加,在第10天左右达到最高水平(图7G)。
We next characterized PT and myofibroblast crosstalk. Fibroblasts and myofibroblasts were grouped together and CCI scores were calculated with healthy PT, FR-PTC, and injured PT. We found that interactions between FR-PTC and fibroblasts (FR-fibroblast) had the most robust CCl score (Figure 7H). In uniIRI, we observed a strong FR-fibroblast interaction beginning at day 2 when FR-PTC started to expand, and interestingly, the interaction was still active at day 28 (Figure 7H), even though FR-PTC only constituted < 5 % < 5 % < 5%<5 \% of the total PT cells at this point (Figure 2E). In UUO, a similar pattern was evident beginning at day 6 (Figure 7H). The importance of FR-fibroblast interactions
我们接下来描述了 PT 和肌成纤维细胞串扰的特征。我们将成纤维细胞和肌成纤维细胞分组,并计算了健康 PT、FR-PTC 和损伤 PT 的 CCI 分数。我们发现,FR-PTC 与成纤维细胞之间的相互作用(FR-成纤维细胞)具有最稳健的 CCl 评分(图 7H)。在 uniIRI 中,我们观察到从第 2 天开始,当 FR-PTC 开始扩张时,FR-成纤维细胞之间的相互作用很强,有趣的是,尽管此时 FR-PTC 只占 PT 细胞总数的 < 5 % < 5 % < 5%<5 \% ,但这种相互作用在第 28 天仍很活跃(图 7H)(图 2E)。在 UUO 中,类似的模式从第 6 天开始就很明显(图 7H)。FR-成纤维细胞相互作用的重要性

encouraged us to identify molecule pairs responsible for the communication between the two cell types. In addition to strong interactions between fibroblast integrins and VCAM1/COL18A1/ SPP1 expressed by FR-PTC (Figure S7J), we identified CD44FGFR2 as a significantly dysregulated receptor-receptor pair in both uni-IRI and UUO (Figure S7J). The CD44-FGFR2 interaction was highly specific to the FR-fibroblast CCl as its activity was not statistically significant in interactions between fibroblasts and other PT subtypes (Figure S7K). CD44 is a receptor for hyaluronic acid, and its upregulation in injured PT cells has been well characterized (Lewington et al., 2000; Schiessl et al., 2018), and FGFR2 is known to be essential for kidney development and its ablation ameliorates kidney fibrosis (Hains et al., 2008; Xu and Dai, 2017). In our dataset, Cd44 was specifically expressed in FR-PTC and Fgfr2 could be detected in multiple (myo)fibroblast subtypes with highest expression in Myo-1 (Figure S7L), which reinforced the critical role of CD44-FGFR2 interaction in FR-fibroblast intercellular communication. In addition, we also examined communications between fibroblasts and LoH cells, identifying enhanced activity of EPHB2-EFNA5 interaction in kidney fibrogenesisFigure S7. The expression of Ephb2 (encoding ephrin type-B receptor 2) was specific to LoH cells and was upregulated in TAL-inj compared with its healthy state (Figure S7M), which was supported by several previous studies (Huang et al., 2021; Ogawa et al., 2006), suggesting that Eph/Ephrin signaling axis may be a mediator of kidney fibrogenesis.
这促使我们确定了负责两种细胞类型之间交流的分子对。除了成纤维细胞整合素与 FR-PTC 表达的 VCAM1/COL18A1/ SPP1 之间的强相互作用(图 S7J)外,我们还发现 CD44FGFR2 是 uni-IRI 和 UUO 中显著失调的受体-受体对(图 S7J)。CD44-FGFR2相互作用对FR-成纤维细胞CCl具有高度特异性,因为其活性在成纤维细胞和其他PT亚型之间的相互作用中没有统计学意义(图S7K)。CD44 是透明质酸的受体,其在损伤的 PT 细胞中的上调已被充分表征(Lewington 等,2000;Schiessl 等,2018),已知 FGFR2 对肾脏发育至关重要,其消融可改善肾脏纤维化(Hains 等,2008;Xu 和 Dai,2017)。在我们的数据集中,Cd44在FR-PTC中特异性表达,Fgfr2可在多种(成纤维细胞)亚型中检测到,其中Myo-1的表达量最高(图S7L),这加强了CD44-FGFR2相互作用在FR-成纤维细胞细胞间通讯中的关键作用。此外,我们还研究了成纤维细胞与 LoH 细胞之间的通讯,发现 EPHB2-EFNA5 相互作用在肾脏纤维化中的活性增强图 S7。Ephb2(编码ephrin B型受体2)是LoH细胞的特异性表达,与健康状态相比,在TAL-inj中表达上调(图S7M),这得到了之前一些研究的支持(Huang等人,2021年;Ogawa等人,2006年),表明Eph/Ephrin信号轴可能是肾脏纤维化的介质。

DISCUSSION  讨论

Our dataset has been deposited into an online interactive scRNA-seq data analyzer (http://humphreyslab.com/SingleCell/ ), which allows researchers to visualize expression of any gene of interest among different cell types or disease groups. We specifically profiled samples of uni-IRI and UUO, two well-characterized models of kidney injury and fibrosis, and present a computational workflow (STAR Methods) for integrating our dataset with other scRNA-seq atlases so comparative and joint analysis can be performed with batch effects removed. For example, we integrated our previous scRNA-seq dataset on bi-IRI mouse kidneys (Kirita et al., 2020) with this uni-IRI subset and found that all major cell states could be identified in both models (Figure S1E).
我们的数据集已存入在线交互式 scRNA-seq 数据分析器 ( http://humphreyslab.com/SingleCell/ ),研究人员可以通过它直观地看到不同细胞类型或疾病组中任何感兴趣基因的表达情况。我们特别分析了uni-IRI和UUO样本,这是两种表征良好的肾损伤和肾纤维化模型,并介绍了将我们的数据集与其他scRNA-seq图谱集整合的计算工作流程(STAR方法),这样就可以在去除批次效应的情况下进行比较和联合分析。例如,我们将之前关于双IRI小鼠肾脏的scRNA-seq数据集(Kirita等人,2020年)与这一uni-IRI子集整合在一起,发现两种模型中的所有主要细胞状态都能被识别出来(图S1E)。
Our scRNA-seq library was generated with the sci-RNA-seq3 protocol, a technology based on sci (also known as split-pool barcoding). sci-RNA-seq3 differs from widely adopted droplet microfluidic solutions, such as 10X Chromium, by marking each cell with a unique combination of several barcodes (instead of one barcode). Though still early in development, sci-based approaches have been applied to a growing number of studies in recent years, due to its high-throughput capabilities, samplemultiplexing capacity and utilization of common laboratory equipment (Li and Humphreys, 2021). Here, we demonstrated its applicability in solid tissues collected from disease models. The high-throughput and highly multiplexed dataset enables the identification of rare cell types in the time course of disease progression, such as the Type1/2 injured PT cells described here. Sci-based methods also provide a cost-effective solution to constructing comprehensive human cell atlases by profiling multiple samples in parallel to minimize batch effects, and recent
我们的 scRNA-seq 文库是用 sci-RNA-seq3 协议生成的,这是一种基于 sci(也称作分割池条形码)的技术。sci-RNA-seq3 与广泛采用的液滴微流控解决方案(如 10X Chromium)不同,它是用多个条形码的独特组合(而不是一个条形码)标记每个细胞。基于 sci 的方法虽然仍处于发展初期,但由于其高通量能力、样本复用能力和对普通实验室设备的利用,近年来已被越来越多的研究采用(Li 和 Humphreys,2021 年)。在此,我们展示了其在疾病模型实体组织中的适用性。高通量和高度多路复用的数据集能够在疾病进展的时间过程中识别罕见的细胞类型,如本文所述的1/2型受伤的PT细胞。基于科学的方法还为构建全面的人类细胞图谱提供了一种具有成本效益的解决方案,它可以并行剖析多个样本,最大程度地减少批次效应。

improvements have been made to achieve higher gene detection sensitivity and co-measurement of multiple modalities (Ma et al., 2020; Martin et al., 2021).
为了实现更高的基因检测灵敏度和多种模式的共同测量,已经进行了改进(Ma 等人,2020 年;Martin 等人,2021 年)。
Our results have been comprehensively validated through reanalyzing existing mouse and human datasets on relevant disease models. For example, we observed upregulation of Plin2 (or human PLIN2) in the folic-acid-induced mouse nephropathy model (Craciun et al., 2016; Figure S4A) and in a human renal IRI model (Park et al., 2020) (Mendeley Data). Our characterization of Type2 injured PT cells was supported by a previous dataset, which profiled PT-enriched transcripts in UUO mice (Wu et al., 2020; Figure S5B). We also surveyed a prior work on biopsy samples of patients with CKD (Nakagawa et al., 2015) and validated increased expression of Type2, but not Type1, injured PT marker genes in patients with CKD compared with control (Figures 4D and 4E; Mendeley Data). An interesting and open question is whether the abundance of either injured PT state in the early stages of human kidney disease correlates with long-term patient outcomes.
通过重新分析相关疾病模型的现有小鼠和人类数据集,我们的结果得到了全面验证。例如,我们在叶酸诱导的小鼠肾病模型(Craciun 等人,2016 年;图 S4A)和人类肾脏 IRI 模型(Park 等人,2020 年)中观察到了 Plin2(或人类 PLIN2)的上调(Mendeley 数据)。我们对 2 型损伤 PT 细胞的特征描述得到了先前数据集的支持,该数据集分析了 UUO 小鼠中 PT 丰富的转录本(Wu 等人,2020 年;图 S5B)。我们还调查了之前对 CKD 患者活检样本的研究(Nakagawa 等人,2015 年),并验证了与对照组相比,CKD 患者中 2 型(而非 1 型)损伤 PT 标记基因的表达增加(图 4D 和 4E;Mendeley 数据)。一个有趣而悬而未决的问题是,在人类肾脏疾病的早期阶段,两种损伤性 PT 状态的丰富程度是否与患者的长期预后相关。
PT cells have high baseline energy demands and preferentially utilize lipids to generate ATP. Accumulation of lipids in PT is dependent on uptake of serum-FFAs (Zeng et al., 2017) and defects in lipid metabolism are a well-recognized defect of CKD (Kang et al., 2014; Stadler et al., 2015; Tran et al., 2016). A recent study demonstrated that long-term fatty acid uptake (10-day palmitic acid administration) promoted inflammation and fibrogenesis of mouse PT cells (Mori et al., 2021). Here, we identified an unexpected, transient lipid accumulation and enhanced expression of FAO-related genes in Type1 injured PT cells (Figures 3A-3F). Three experimental observations led to the conclusion that the increased expression of FAO genes contributed to an increased FAO phenotype: (1) cells had very low content intracellular lipids at uni-IRI D2 (Figure 3D), implying the deposited lipids in the first 6 h were utilized over the next day; (2) in vitro modeling of lipid accumulation, in combination with the use of a lipolysis inhibitor revealed that clearance of lipid droplets was through FAO (Figure 3H); and (3) direct metabolic measurement identified an increased OCR after 6-h fatty acid treatment (Figures 31 and S3L). Interestingly, in our bulk RNAseq analysis of cells harvested at 2 days after 6-h oleic acid treatment, we found upregulation of genes involved in DNA replication, cell-cycle regulation, and cell proliferation (Figure 3J), which are high-energy-demand cellular events. This was also consistent with our observation that Mki67-expressing PT-R cells were most abundant at uni-IRI D2 (Figure 2E). Therefore, the deposited lipids in 6 h may serve as an essential energy source for injured epithelia following injury, promoting tubular repair through proliferative expansion.
PT 细胞具有较高的基线能量需求,并优先利用脂质产生 ATP。PT 中脂质的积累依赖于对血清-FFA 的吸收(Zeng 等人,2017 年),脂质代谢缺陷是公认的 CKD 缺陷(Kang 等人,2014 年;Stadler 等人,2015 年;Tran 等人,2016 年)。最近的一项研究表明,长期摄入脂肪酸(10 天棕榈酸给药)会促进小鼠 PT 细胞的炎症和纤维化(Mori 等人,2021 年)。在这里,我们在 1 型损伤的 PT 细胞中发现了意想不到的瞬时脂质积累和 FAO 相关基因的表达增强(图 3A-3F)。通过三项实验观察,我们得出结论:FAO 基因表达的增加导致了 FAO 表型的增加:(1)细胞在单IRI D2时细胞内脂质含量很低(图3D),这意味着前6小时沉积的脂质在第二天被利用;(2)脂质积累的体外建模,结合使用脂肪分解抑制剂,发现脂滴的清除是通过FAO进行的(图3H);(3)直接代谢测量发现脂肪酸处理6小时后OCR增加(图31和S3L)。有趣的是,在对 6 小时油酸处理后 2 天收获的细胞进行大量 RNAseq 分析时,我们发现参与 DNA 复制、细胞周期调控和细胞增殖的基因上调(图 3J),这些都是高能量需求的细胞事件。这也与我们的观察结果一致,即表达 Mki67 的 PT-R 细胞在 uni-IRI D2 处最为丰富(图 2E)。因此,6 小时内沉积的脂质可能是损伤后受伤上皮的重要能量来源,通过增殖扩张促进肾小管修复。
Lipid droplets, also known as lipid vacuoles, are organelles whose phospholipid monolayer is decorated with lipid binding proteins and containing a hydrophobic core consisting of neutral lipids. Here, we identified an 10 10 ∼10\sim 10-fold increased PLIN2 expression after a 6-h fatty acid stimulus in vitro, with resolution of expression 2 days after removal of fatty acids from the media (Figure 4G), implying that PLIN2 plays a key role in how the cell responds to intracellular lipid accumulation. Further, PLIN2 gene knockdown caused a decrease in OCR and ECAR activities (Figures 4 I and S4F), suggesting that PLIN2 regulates cellular metabolism. Although a reduced OCR may be expected to
脂滴又称脂质空泡,是一种细胞器,其磷脂单层由脂质结合蛋白装饰,并含有由中性脂质组成的疏水核心。在这里,我们发现在体外6小时的脂肪酸刺激后,PLIN2的表达量增加了 10 10 ∼10\sim 10 -倍,从培养基中去除脂肪酸2天后,PLIN2的表达量消失(图4G),这意味着PLIN2在细胞如何应对细胞内脂质积累方面起着关键作用。此外,PLIN2 基因敲除导致 OCR 和 ECAR 活性降低(图 4 I 和 S4F),表明 PLIN2 调节细胞代谢。虽然 OCR 的降低可能会

reflect a downregulation of FAO genes, we did not identify decreased expression of genes encoding mitochondrial FAO components such as CPT1A and CPT2. Instead, we observed significantly decreased expression of ACSL3, ACSL4, and ACSL5, which encode cytosolic proteins that convert lipolysisderived FFAs into fatty acyl-CoA (Li et al., 2010). These results indicate that PLIN2 regulates acyl-CoA generation by lipolysis but does not directly affect mitochondrial β β beta\beta-oxidation. Overall, we propose the model presented in Figure 4K: after IRI, Type1 injured PT cells rapidly accumulate lipid droplets, inducing PLIN2 expression, leading to enhanced PLIN2-dependent FAO activity with subsequent consumption of these lipids, promoting epithelial proliferation and tubule regeneration. Why this lipid accumulation and consumption process does not occur in Type2 injured PT (or in UUO), and whether lack of lipid acquisition in early stages is responsible for the poor fate outcome of kidney fibrogenesis, requires further investigation.
我们没有发现编码线粒体 FAO 组成部分(如 CPT1A 和 CPT2)的基因表达下降,这反映了 FAO 基因的下调。相反,我们观察到 ACSL3、ACSL4 和 ACSL5 的表达明显下降,它们编码的细胞膜蛋白能将脂肪分解产生的 FFA 转化为脂肪酰基-CoA(Li 等人,2010 年)。这些结果表明,PLIN2 可调节脂肪分解产生的酰基-CoA,但不会直接影响线粒体 β β beta\beta 氧化。总之,我们提出了图 4K 所示的模型:IRI 后,1 型损伤 PT 细胞迅速积聚脂滴,诱导 PLIN2 表达,导致 PLIN2 依赖性 FAO 活性增强,随后消耗这些脂质,促进上皮细胞增殖和肾小管再生。为什么这种脂质积累和消耗过程不会发生在2型损伤的PT(或UUO)中,早期缺乏脂质获取是否是肾脏纤维化的不良命运结局的原因,还需要进一步研究。

We highlighted two dysregulated pathways, lipid and amino acid metabolism, in diseased PT cells, but we also acknowledge that kidney fibrogenesis affects many other metabolic networks. For example, SLC5A2 (the target of SGLT2 inhibitors) is responsible for 90 % 90 % ∼90%\sim 90 \% of tubular glucose transport (Wen et al., 2021). As a consequence of cell dedifferentiation in both uni-IRI and UUO, we observed a remarkable reduction in expression of S/c5a2 in PT. We also identified decreased expression of genes encoding phosphofructokinase, glucose-6-phosphatase, and isocitrate dehydrogenase, which could be recovered in late time points of uni-IRI but remained at low levels in UUO, suggesting disrupted glucose metabolism in kidney fibrogenesis. In addition, we found that the two genes encoding subunits of lactate dehydrogenase, Ldha and Ldhb, were dysregulated with patterns consistent with a recent report (Osis et al., 2021) (Mendeley Data), indicating off-balance interconversion between lactate and pyruvate. Interestingly, we identified Hmox1, which encodes heme oxygenase-1 (HO-1, an essential modulator of glucose metabolism), and its transcription factor Nfe2l2 as two upregulated markers of early IRI injury (i.e., uni-IRI 6 h) distributed in Type1 injury and acute injury PT cells (Mendeley Data). Previous studies have demonstrated a protective role of HO 1 HO 1 HO-1\mathrm{HO}-1 against kidney injury and exhibited significant elimination of tubular injury and interstitial fibrosis in UUO mice following treatment with an HO-1 inducer (Bolisetty et al., 2017; Kim et al., 2006). Understanding the specific role of HO 1 HO 1 HO-1\mathrm{HO}-1 in maintaining renal glucose metabolism in disease states will require further investigation.
我们强调了病变 PT 细胞中脂质和氨基酸代谢这两条失调的途径,但我们也承认肾脏纤维化会影响许多其他代谢网络。例如,SLC5A2(SGLT2 抑制剂的靶点)负责 90 % 90 % ∼90%\sim 90 \% 肾小管葡萄糖转运(Wen 等,2021 年)。由于单IRI和UUO中细胞的去分化,我们观察到PT中S/c5a2的表达显著减少。我们还发现编码磷酸果糖激酶、葡萄糖-6-磷酸酶和异柠檬酸脱氢酶的基因表达减少,这些基因在单IRI的晚期可以恢复,但在UUO中仍处于低水平,这表明肾脏纤维化过程中糖代谢紊乱。此外,我们还发现编码乳酸脱氢酶亚基的两个基因 Ldha 和 Ldhb 发生了失调,其模式与最近的一份报告(Osis 等人,2021 年)(Mendeley Data)一致,表明乳酸和丙酮酸之间的相互转化失衡。有趣的是,我们发现编码血红素加氧酶-1(HO-1,葡萄糖代谢的重要调节因子)的 Hmox1 及其转录因子 Nfe2l2 是分布在 1 型损伤和急性损伤 PT 细胞中的早期 IRI 损伤(即 6 小时的单 IRI)的两个上调标志物(Mendeley 数据)。先前的研究表明, HO 1 HO 1 HO-1\mathrm{HO}-1 对肾脏损伤具有保护作用,在使用HO-1诱导剂治疗后,UUO小鼠的肾小管损伤和间质纤维化明显消除(Bolisetty等人,2017年;Kim等人,2006年)。了解 HO 1 HO 1 HO-1\mathrm{HO}-1 在疾病状态下维持肾脏葡萄糖代谢的具体作用还需要进一步研究。

This high-throughput dataset enabled us to discover a shared injury response of all major TEC structures including PT, TAL, DCT, and CNT. Although TAL-inj, DCT-inj, and CNT-inj were described as populations covering injured cells from both uniIRI and UUO in this analysis, we could not exclude the possibility that they were heterogeneous groups composed of multiple injury states, as characterized in injured PT. For example, we found that Gcnt2, whose deficiency can cause abnormal morphology of tubule epithelium (Chen et al., 2005), was upregulated in TAL-inj specifically at uni-IRI 6 h , but not in UUO (Figure S6F). Higher detection resolution will be needed for this additional subclustering analysis.
这一高通量数据集让我们发现了所有主要 TEC 结构(包括 PT、TAL、DCT 和 CNT)的共同损伤反应。虽然在这项分析中,TAL-inj、DCT-inj 和 CNT-inj 被描述为涵盖了 uniIRI 和 UUO 的损伤细胞群,但我们不能排除它们是由多种损伤状态组成的异质群的可能性,正如损伤的 PT 所描述的那样。例如,我们发现 Gcnt2(其缺乏可导致肾小管上皮细胞形态异常(Chen 等,2005 年))在 TAL-inj 中的上调特异性地发生在 uni-IRI 6 h 时,而在 UUO 中则没有(图 S6F)。这种额外的亚聚类分析需要更高的检测分辨率。
This single-cell atlas of kidney fibrogenesis also serves as a unique resource to study fibrotic responses of other non-epithelial cells such as stromal cells (Figures 7A-7E), immune cells
这一肾脏纤维形成的单细胞图谱也是研究其他非上皮细胞(如基质细胞(图 7A-7E)、免疫细胞)纤维化反应的独特资源。

(Figure S1C), and endothelial cells (ECs) (Figure S1D). We have performed subclustering analysis on all these populations to illustrate the complexity of the dataset. For example, we identified a group of macrophages ( M φ 2 M φ 2 Mvarphi-2\mathrm{M} \varphi-2 ) marked by elevated expression of a lysozyme gene Lyz2, Tgfbi, and various genes encoding HSPs (Figure S1C; Mendeley Data). M φ 2 φ 2 varphi-2\varphi-2 showed high abundance at uni-IRI 6 h/D2 but was low abundance in UUO (Mendeley Data). It has been reported that TGFBI+ macrophages can capture apoptotic cells and induce fibrotic responses (Nacu et al., 2008), and our results indicate that M φ 2 M φ 2 Mvarphi-2\mathrm{M} \varphi-2 could be an essential population initiating immune response against kidney injury. In the subclustering of ECs, we found that a subgroup of EC (Activated EC) exhibited upregulated expression of Rapgef5 and Magi1, genes involved in abnormal angiogenesis and endothelial activation (Abe et al., 2019; Hong et al., 2007; Figure S1D). This cell type was rarely observed in healthy tissues but could proliferate rapidly in disease, particularly after UUO D6 (Mendeley Data), and it would be interesting to learn its lineage progenitors and functional importance in kidney fibrogenesis in future studies.
(图 S1C)和内皮细胞(ECs)(图 S1D)。我们对所有这些群体进行了亚聚类分析,以说明数据集的复杂性。例如,我们发现了一组巨噬细胞( M φ 2 M φ 2 Mvarphi-2\mathrm{M} \varphi-2 ),其特征是溶菌酶基因Lyz2、Tgfbi和各种编码HSP的基因表达量升高(图S1C;Mendeley数据)。M φ 2 φ 2 varphi-2\varphi-2 在 uni-IRI 6 h/D2 中的丰度较高,但在 UUO 中的丰度较低(Mendeley 数据)。据报道,TGFBI+巨噬细胞可捕获凋亡细胞并诱导纤维化反应(Nacu 等,2008 年),我们的结果表明, M φ 2 M φ 2 Mvarphi-2\mathrm{M} \varphi-2 可能是启动肾损伤免疫反应的一个重要群体。在EC亚群中,我们发现一个EC亚群(激活EC)表现出Rapgef5和Magi1的表达上调,这些基因参与异常血管生成和内皮激活(Abe等人,2019年;Hong等人,2007年;图S1D)。这种细胞类型在健康组织中很少被观察到,但在疾病中却能迅速增殖,尤其是在 UUO D6 之后(Mendeley 数据),在未来的研究中了解其系祖细胞及其在肾脏纤维化中的功能重要性将是非常有趣的。
In summary, we leveraged sci-RNA-seq3 to generate a highthroughput single-cell transcriptomic landscape of kidney fibrogenesis. PT cell dedifferentiation was a shared injury response in both uni-IRI and UUO models, but unique cell states existed in each model, such as the Type1 and Type2 injured PT, characterized by dysregulated lipid and amino acid metabolism, respectively. We also identified both shared and unique injury and repair responses in epithelial cells across nephron segments and demonstrated the heterogeneity of kidney stromal cells. Since kidney fibrosis affects nearly all renal cell types encompassing epithelia, stroma, endothelia, and the immune system, it is critical to construct a comprehensive network of cell-cell communications for translational studies. Our work highlights the utility of analyzing detailed time courses of kidney fibrogenesis and validates sci-RNA-seq3 as a powerful method for analyzing multiple samples at once.
总之,我们利用 sci-RNA-seq3 生成了肾脏纤维化的高通量单细胞转录组图谱。在uni-IRI和UUO模型中,PT细胞去分化是一种共同的损伤反应,但每种模型中都存在独特的细胞状态,如1型和2型损伤PT,其特征分别是脂质和氨基酸代谢失调。我们还确定了各肾节段上皮细胞共同和独特的损伤和修复反应,并证明了肾脏基质细胞的异质性。由于肾脏纤维化影响到几乎所有肾细胞类型,包括上皮细胞、基质细胞、内皮细胞和免疫系统,因此构建一个全面的细胞-细胞通讯网络对于转化研究至关重要。我们的工作凸显了分析肾脏纤维化的详细时间过程的实用性,并验证了 sci-RNA-seq3 是同时分析多个样本的强大方法。

Limitations of study  研究的局限性

Our work employs two widely adopted mouse kidney fibrogenesis models, uni-IRI and UUO, but how generalizable our findings are to other forms of kidney injury is unresolved. This version of sci-RNA-seq3 is technically limited in gene detection sensitivity. Also, it is challenging to assess the significance of Type1 injured PT in humans because this cell state only transiently appears 6 h 6 h ∼6h\sim 6 \mathrm{~h} after AKI, and few if any such early AKI human samples are available. Finally, our transcriptomic characterization is unimodal. Future multi-modality measurements such as combined transcriptomic and epigenomic readouts will be needed to depict a complete cell atlas of kidney fibrosis.
我们的研究采用了两种被广泛采用的小鼠肾脏纤维化模型--uni-IRI 和 UUO,但我们的发现对其他形式的肾脏损伤有多大的普适性还没有解决。这一版本的 sci-RNA-seq3 在基因检测灵敏度方面存在技术限制。此外,评估 1 型损伤 PT 在人体中的意义也很有挑战性,因为这种细胞状态只在 AKI 后短暂出现 6 h 6 h ∼6h\sim 6 \mathrm{~h} ,而这种早期 AKI 人体样本即使有也很少。最后,我们的转录组特征描述是单模态的。未来需要进行多模态测量,如结合转录组学和表观基因组学读数,以描绘肾脏纤维化的完整细胞图谱。

STAR ***\star METHODS  星级 ***\star 方法

Detailed methods are provided in the online version of this paper and include the following:
本文的在线版本提供了详细的方法,包括以下内容:
  • KEY RESOURCES TABLE  关键资源表
  • RESOURCE AVAILABILITY  资源可用性
  • Lead contact  主要联系人
  • Materials availability  材料供应
  • Data and code availability
    数据和代码的可用性
  • EXPERIMENTAL MODEL AND SUBJECT DETAILS
    实验模型和受试者详情
  • METHOD DETAILS  方法细节
  • Mouse kidney sample processing
    小鼠肾脏样本处理
  • Cell culture  细胞培养
  • Quantitative polymerase chain reaction (qPCR) analysis
    定量聚合酶链反应(qPCR)分析
  • Immunofluorescence  免疫荧光
  • Lipid staining  脂质染色
  • RNA in situ hybridization (ISH)
    RNA 原位杂交 (ISH)
O In vitro fatty acid exposure, ER stress induction and lipolysis inhibition
O 体外脂肪酸暴露、ER 应激诱导和脂肪分解抑制

Fatty acid internalization assay
脂肪酸内化试验

Mitochondria staining on cultured cells
对培养细胞进行线粒体染色

In vitro gene knockdown
体外基因敲除

ROS staining  ROS 染色
Metabolic measurement  代谢测量
Nuclei isolation and fixation (mouse kidney)
细胞核分离和固定(小鼠肾脏)

Nuclei isolation and fixation (cultured cells)
细胞核分离和固定(培养细胞)

sci-RNA-seq3 library generation
sci-RNA-seq3 文库生成

Next-generation Sequencing for sci-RNA-seq3
用于 sci-RNA-seq 的新一代测序3

sci-RNA-seq3 data pre-processing
sci-RNA-seq3 数据预处理

Pseudobulk trajectory ordering
伪舱轨迹排序

Doublet estimation, quality control and cell clustering
双音估计、质量控制和细胞聚类

Gene module scoring  基因模块评分
Single-cell trajectory inference
单细胞轨迹推断

Single-cell fate mapping on time-series datasets
在时间序列数据集上绘制单细胞命运图谱

Gene enrichment analysis
基因富集分析

Single-cell pathway and transcription factor (TF) activity prediction
单细胞通路和转录因子 (TF) 活性预测
  • Cell-cell interaction analysis
    细胞-细胞相互作用分析
Comparison and integration with other datasets
与其他数据集的比较和整合

Bulk RNA-seq  批量 RNA-seq
Lipidomics analysis  脂质组学分析
Measurement of BCAA concentration
测量 BCAA 浓度
  • QUANTIFICATION AND STATISTICAL ANALYSIS
    量化和统计分析
SUPPLEMENTAL INFORMATION  补充资料
Supplemental information can be found online at https://doi.org/10.1016/j. cmet.2022.09.026.
补充信息可在线查阅:https://doi.org/10.1016/j. cmet.2022.09.026。

ACKNOWLEDGMENTS  致谢

These experiments were funded by NIH grants DK103740 and UC2DK126024 to B.D.H. The authors acknowledge the Washington University Diabetes Research Center for providing training for Seahorse Analyzer applications. The authors also acknowledge the Washington University Genome Technology Access Center and Center for Genome Sciences & Systems Biology for sequencing support.
这些实验由美国国立卫生研究院资助 DK103740 和 UC2DK126024 给 B.D.H。作者感谢华盛顿大学糖尿病研究中心提供的海马分析仪应用培训。作者还感谢华盛顿大学基因组技术访问中心和基因组科学与系统生物学中心提供的测序支持。

AUTHOR CONTRIBUTIONS  作者贡献

H.L. and B.D.H. conceived, coordinated, and designed the study. H.L. performed experiments with contributions from E.E.D. and H.W.; H.L. and B.D.H. analyzed data. H.L. and B.D.H. wrote the manuscript. All authors read and approved the final manuscript.
H.L.和B.D.H.构思、协调和设计了这项研究。H.L.进行了实验,E.E.D.和H.W.提供了帮助;H.L.和B.D.H.分析了数据。H.L.和B.D.H.撰写了手稿。所有作者阅读并批准了最终手稿。

DECLARATION OF INTERESTS
利益申报

B.D.H. is a consultant for Janssen Research & Development, LLC, Pfizer, and Chinook Therapeutics and holds equity in Chinook Therapeutics and grant
B.D.H. 是 Janssen Research & Development, LLC、辉瑞公司和 Chinook Therapeutics 的顾问,并持有 Chinook Therapeutics 的股权和赠款。

funding from Chinook Therapeutics, Janssen Research & Development, LLC, and Pfizer; all interests are unrelated to the current work.
资金来自 Chinook Therapeutics、Janssen Research & Development, LLC 和辉瑞公司;所有利益均与当前工作无关。

INCLUSION AND DIVERSITY  包容性和多样性

We support inclusive, diverse, and equitable conduct of research.
我们支持以包容、多元和公平的方式开展研究。

Received: March 3, 2022
收到:2022 年 3 月 3 日

Revised: July 19, 2022
修订:2022 年 7 月 19 日

Accepted: September 28, 2022
接受:2022 年 9 月 28 日

Published: October 19, 2022
出版日期2022 年 10 月 19 日

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